Application of antisense oligonucleotides in the preparation of drugs for treating diseases caused by thyroid abnormalities

Abstract

The present invention relates to the field of gene therapy for thyroid-related eye diseases, and specifically to the use of antisense oligonucleotides in the preparation of drugs for treating diseases caused by thyroid abnormalities. The experimental results of the present invention show that the antisense oligonucleotide S-ASODN-1 with a specific sequence targeting the insulin-like growth factor-1 receptor IGF-1R can effectively reduce the concentration of IGF-1R and anti-thyroid peroxidase antibodies (TPO-Ab), increase the level of thyroid stimulating hormone (TSH), and is used to treat TED.

Description

Technical Field

[0001] The present invention relates to the field of gene therapy for thyroid-related eye diseases, and in particular to use of a fully phosphorothioated antisense oligonucleotide targeting the insulin-like growth factor-1 receptor IGF1R gene or a composition thereof in a thyroid-related eye disease model and in the treatment of thyroid-related eye diseases. Background Art

[0002] Thyroid-associated eye disease (TAO) (also known as thyroid eye disease (TED), Graves' ophthalmopathy or orbitopathy (GO), thyrotoxic exophthalmos, thyroid dysfunction eye disease and several other terms) is an orbital disease related to thyroid dysfunction, ranking first in the incidence of adult orbital diseases and the most common extrathyroidal manifestation of diffuse toxic goiter (GD), with an incidence of 25-40% of GD. It can also be seen in 2% of patients with chronic lymphocytic thyroiditis, a small number of patients with hypothyroidism (hypothyroidism) and people with normal thyroid function. According to an article from the Chinese Center for Disease Control, the overall incidence of GD in the population is about 1%, and the incidence in women is much higher than that 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 its own orbital fibroblasts that overexpress IGF-1R, causing inflammation of the muscles and fat tissue behind the eye, causing the eye to push forward and bulge outward. Long-term accumulation of this irreversible damage can even lead to blindness.

[0004] TAO is divided into two types, active TAO, which usually lasts for 1 to 3 years, is characterized by a persistent autoimmune / inflammatory reaction in the orbital soft tissues. Active TAO causes expansion and remodeling of the ocular soft tissues. The autoimmune / inflammatory reaction of active TAO resolves spontaneously and the condition transforms into inactive TAO. Inactive TAO is a term used to describe the long-term / permanent sequelae of active TAO. The cause of TAO is unknown. TAO is usually associated with Graves' hyperthyroidism, but can also occur as part of other autoimmune conditions that affect the thyroid gland and produce pathology in the orbit and periorbital tissues and rarely in the pretibial skin (pretibial myxedema) or fingers (thyroid clubbing). TAO is an autoimmune orbital disease in which the orbit and periocular soft tissues are primarily affected, with secondary effects on the eyeball and vision. In TAO, inflammation and expansion of the orbital soft tissues (primarily eye muscles and fat) forces the eyeball forward (bulges) out of its socket - a phenomenon known as proptosis or exophthalmos. Although most cases of TAO do not cause vision loss, the condition can cause vision-threatening exposure keratopathy, troublesome diplopia (doublevision), and compressive dysthyroid optic neuropathy. TAO may precede, coincide with, or follow systemic complications of thyroid dysfunction. Ocular manifestations of TAO include upper eyelid retraction, eyelid lag, swelling, redness (erythema), conjunctivitis and bulging eyes (proptosis or exophthalmos), chemosis, periorbital edema, and altered eye motility, with significant functional, social, and cosmetic consequences. Many of the signs and symptoms of TAO, including proptosis and ocular hyperemia, are caused by expansion of the orbital fat tissue and periorbital muscles. The increase in fat tissue volume is due in part to the development of new fat cells (adipogenesis) within the orbital fat. The accumulation of hydrophilic glycosaminoglycans (mainly hyaluronic acid) in the perimuscular connective tissue between the orbital fat tissue and the extraocular muscle fibers further expands the fat compartment and enlarges the extraocular muscle body. Hyaluronic acid is produced by fibroblasts resident in the orbital fat and extraocular muscles, and its synthesis in vitro 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 moderate to severe active TED. The first-line treatment is intravenous corticosteroid pulse therapy, which has problems such as unsatisfactory improvement of exophthalmos and hormone-related systemic side effects, and there is still a large unmet clinical need. Second-line treatment includes another hormone pulse or combined orbital radiotherapy or other immunomodulators. Biological agents such as teprotumumab, tocilizumab, and rituximab are also recommended as second-line treatment options for moderate to severe active TED by the EUGOGO Guidelines and the Chinese Guidelines for the Clinical Diagnosis and Treatment of Thyroid Eye Disease (2022) and the American Thyroid Association and the European Thyroid Association's consensus on thyroid eye disease. Especially for TED with significant exophthalmos, teprotumumab can be used as the first choice.

[0006] Infiltrating CD34+ fibrocytes, derived from the myelomonocytic lineage, enter from the circulation and set the stage for disease. They express and present autoantigens that may be similar to those found in the thyroid gland. These include low levels of TSHR, thyroglobulin, and other thyroid antigens. Fibrocytes present antigens to antigen-specific T cells, which in turn 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. Resident orbital localized fibroblasts are CD34-, and CD34+ fibroblasts entering the orbit encounter resident CD34 fibroblasts. When activated, these cells produce a number of pro- and anti-inflammatory factors, including cytokines, among which are: interleukin-1β, -6, -8, -10, -12, and -16; tumor necrosis factor-α; a chemokine called "regulated activation, 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 display IGF-IR. Therefore, therapeutic approaches based on inhibition of IGF-IR may reach all of these cells. Cytokine-activated fibroblasts synthesize hyaluronic acid (HA) and other glycosaminoglycans, which can expand orbital tissue volume and may cause proptosis.

[0008] In addition, autoantibodies to the thyroid stimulating hormone receptor (TSHR) act as TSHR agonists, inducing excessive secretion of thyroid hormones, resulting in the thyroid gland being free from pituitary control. TSHR autoantibodies also underlie Graves' ophthalmopathy and pretibial myxedema. The synergistic effects of IGF-1R and TSHR autoantibodies lead to expansion and inflammation of retroorbital tissue.

[0009] Table 1 introduces some of the drugs currently entering the clinical stage for the treatment of thyroid-related eye diseases.

[0010] Table 1 Drugs under development for thyroid eye disease

[0011]

[0012] Insulin-like growth factors (IGFs), also known as somatomedins, include IGF-I and IGF-II. These growth factors can only exert their biological effects when bound to their receptor IGF1R. The insulin-like growth factor-1 receptor IGF1R belongs to the receptor tyrosine kinase family and is located on the cell membrane. After binding to IGFs, it dimerizes, allowing the tyrosine domains to approach and trigger autophosphorylation, which then activates intracellular RAS-RAF-MAPK, PI3K-PKB / AKT and other cell proliferation-related signaling pathways. The activation of IGF1R is crucial to stimulating the growth and survival of tumor cells.

[0013] Table 2 shows some of the indications for drugs currently under development that target the IGF1R receptor, none of which involve thyroid-related eye disease.

[0014] Table 2 IGF-1R target drugs under development

[0015]

[0016]

[0017] IGF1-1R inhibitors can theoretically prevent IGF-1R from binding to TSHR, blocking downstream signal transduction mediated by the TSHR / IGF-1R complex, thereby improving TED. In recent years, the newly developed GIP-1R monoclonal antibodies have focused on TED. Teprotumumab is the only marketed drug in the TED field.

[0018] Antisense oligonucleotide (ASODN) is a type of artificially synthesized oligonucleotide fragment with a length of 15 to 30 nucleotides. It mainly interferes with the transcription and translation of target genes through the principle of base complementary pairing, thereby achieving targeted gene therapy. ASODN has the advantages of abundant candidate targets, rational directional design, high efficiency in vitro and in vivo, and large-scale artificial synthesis, and is considered to be a highly promising gene therapy drug. In recent years, with the breakthroughs in nucleotide chemical modification technology and delivery technology, ASODN drug research and development has set off a new wave. Some well-known pharmaceutical companies at home and abroad have made antisense drugs one of the key directions of their new drug research and development. Currently, there are 9 antisense oligonucleotide drugs on the market (Table 3), none of which involve thyroid-related eye diseases.

[0019] Table 3 Marketed antisense oligonucleotide drugs

[0020]

[0021]

[0022] Therefore, providing antisense oligonucleotides for treating thyroid-related eye diseases has important practical significance. Summary of the invention

[0023] In view of this, the present invention treats TED by designing an antisense oligonucleotide S-ASODN-1 with a unique sequence targeting insulin-like growth factor-1 receptor IGF1R.

[0024] The present application first discovered that an antisense oligonucleotide S-ASODN-1 with a specific sequence targeting the insulin-like growth factor-1 receptor IGF-1R can effectively reduce the concentration of IGF-1R and anti-thyroid peroxidase antibodies (TPO-Ab), increase the level of thyroid stimulating hormone (TSH), and be used to treat TED. In fact, there is no antisense oligonucleotide in the existing drugs for the treatment of TED that have been approved or entered the clinical stage. Moreover, not any antisense oligonucleotide targeting any sequence of IGF-1R can treat TED. For example, the other antisense oligonucleotide sequences S-ASODN-2, S-ASODN-3, S-ASODN-4, and S-ASODN-5 of the present application cannot effectively regulate the levels of TPO-Ab and TSH, and cannot be used to treat TED.

[0025] The present invention provides a therapeutically effective amount of a thiocyanate antisense oligonucleotide targeting the IGF-1R gene or a composition thereof for use in preparing a drug for treating a disease caused by abnormal thyroid function. The thiocyanate antisense oligonucleotide targeting the IGF-1R gene

[0026] (I), having the nucleotide sequence shown in SEQ ID No.1; or

[0027] (II) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences of the nucleotide sequence as shown in (I), and having the same or similar function as the nucleotide sequence shown in (I); or

[0028] (III), a nucleotide sequence having at least 80% nucleotide identity with the nucleotide sequence shown in (I) or (II);

[0029] Preferably, the phosphorothioate antisense oligonucleotide targeting the IGF-1R gene has a nucleotide sequence with 85%, 90%, 95%, 96%, 97%, 98% or more 99% nucleotide identity with the nucleotide sequence shown in (I) or (II).

[0030] In some preferred embodiments, the sequence of the thiolated antisense oligonucleotide targeting the IGF-1R gene is a nucleotide sequence having more than 80% nucleotide identity with the 5'-TCCTCCGGAGCCAGACTTCA-3' sequence, preferably a nucleotide sequence having more than 85%, 90%, 95%, 96%, 97%, 98%, or 99% nucleotide identity; more preferably, the sequence of the thiolated antisense oligonucleotide targeting the ICFIR gene is 5'-TCCTCCGGAGCCAGACTTCA-3'.

[0031] In some preferred embodiments, the disease caused by abnormal thyroid function includes thyroid-related eye disease.

[0032] In some preferred embodiments, the phosphorothioate antisense oligonucleotide targeting IGF-1R gene further has other chemical modifications.

[0033] In some preferred embodiments, the other chemical modifications are selected from one or more of the following: locked nucleic acid modification, 2-methoxyethyl modification and 2-oxymethyl modification.

[0034] In some preferred embodiments, the composition further comprises at least one additional active agent.

[0035] In some preferred embodiments, the at least one additional active agent is a therapeutic agent.

[0036] In some preferred embodiments, the therapeutic agent is selected from one or more of the following: corticosteroids, biologics, traditional immunosuppressants.

[0037] In some preferred embodiments, the corticosteroid is selected from glucocorticoid ivGC.

[0038] In some preferred embodiments, patent 202180081949.7 describes that intravenous glucocorticoids (ivGC) and oral glucocorticoids are used to treat patients with moderate to severe active TAO.

[0039] In some preferred embodiments, the biological agent is selected from one or more of the following: CD20+B cell inhibitors, IL-6R antibodies, IL-17A antagonists, FcRn antagonists, IL-11R blocking antibodies, anti-TNFα antibodies, and thyroid stimulating hormone receptor TSHR inhibitors.

[0040] In some preferred embodiments, the traditional immunosuppressant is selected from one or more of the following: mycophenolate mofetil, cyclosporine, methotrexate and azathioprine.

[0041] Guidelines for the diagnosis and treatment of thyroid-related eye diseases in China (2022) Chin J Ophthalmol, September 2022, Vol.58, No.9

[0042] In some preferred embodiments, the therapeutic agent is selected from one or more of the following: secukinumab, tocilizumab, satralizumab, vunakizumab, batoclimab, TOUR006, LASN01, infliximab, rituximab, and selenium.

[0043] In some preferred embodiments, patent 202180081949.7 records that the pharmaceutical composition for treating the thyroid-related eye disease further includes corticosteroids; rituximab or other anti-CD20 antibodies; tocilizumab or other anti-IL-6 antibodies; or selenium, infliximab or other anti-TNFα antibodies or thyroid stimulating hormone receptor (TSHR) inhibitors.

[0044] In some preferred embodiments, the thyroid-related eye disease manifests as one or more of the following symptoms: eyelid signs, proptosis, diplopia, ocular motility disorders, conjunctival edema, optic neuropathy, corneal lesions, conjunctival keratopathy, and systemic symptoms associated with patients with hyperthyroidism.

[0045] In some preferred embodiments, the diplopia includes constant diplopia, non-constant diplopia, and intermittent diplopia.

[0046] 1) Eyelid sign: It is an important sign of thyroid-related eye disease. The main manifestation is a wide opening of the palpebral fissure, exposing part of the sclera, which is called eyelid retraction. When the eyeball turns downward, the upper eyelid cannot fall with it, which is called upper eyelid hysteresis.

[0047] 2) Protrusion of eyeballs: Thyroid-related eye diseases are mostly caused by protrusion of both eyes at the same time, but they can occur one after another. In the early stage, it is mostly axial protrusion of eyeballs. In the later stage, due to fibrosis and contracture of the extraocular muscles, the protrusion of eyeballs is fixed in a certain eye position, affecting the appearance. The symptoms of protrusion of eyeballs develop faster in patients with hyperthyroidism. In some patients, the protrusion of eyeballs becomes more obvious after the hyperthyroidism is controlled;

[0048] 3) Diplopia and ocular motility disorders: Thyroid-related eye diseases inevitably affect the extraocular muscles, with edema and inflammatory infiltration in the early stage and fibrosis in the later stage. Multiple extraocular muscles may be affected successively and the degree of involvement varies among patients. Diplopia may manifest as multi-ocular diplopia, and in the late stage, diplopia is constant. The frequency of extraocular muscle involvement is in the order of inferior rectus, medial rectus, superior rectus, and lateral rectus. Rectus muscle fibrosis can cause restrictive extraocular muscle lesions;

[0049] 4) Conjunctival edema: The conjunctiva is congested and the blood vessels are tortuous and dilated, mostly at the end of the horizontal muscle. Severe conjunctival edema can protrude beyond the palpebral fissure, appearing translucent or turbid, leading to incomplete closure of the palpebral fissure and secondary corneal lesions;

[0050] 5) Optic neuropathy: According to foreign reports, 5% to 10% of patients with Graves' ophthalmopathy develop optic neuropathy, of which 30% may suffer from irreversible loss of visual function, which is more common in the elderly, men and smokers. The reason is that the increased intraorbital pressure caused by edema and hypertrophy of the orbital tissue and muscles compresses the optic nerve at the orbital apex, causing decreased vision and visual field defects (especially at the lower part). Some patients may experience increased intraocular pressure;

[0051] 6) Corneal lesions: Exposure keratitis occurs due to incomplete closure of the palpebral fissure caused by eyelid changes and severe protrusion of the eyeball. In the early stage, there is punctate epithelial detachment under the cornea. In severe cases, corneal ulcers or even perforations may occur, which seriously damages visual function.

[0052] 7) Conjunctival and corneal lesions, edema of the soft tissue in the orbit, and increased pressure in the orbit can lead to conjunctival congestion and edema, and severe conjunctiva protrudes outside the palpebral fissure;

[0053] 8) Patients with hyperthyroidism also have systemic symptoms, such as irritability, increased basal metabolic rate, accelerated pulse, weight loss, increased appetite, hand tremors, etc.

[0054] As used herein, "thyroid associated eye disease" (TAO), "thyroid eye disease" (TED), "Graves' ophthalmopathy" or "Graves' orbitopathy" (GO) refer to the same disorder or condition and are used interchangeably. They all refer to inflammatory orbital pathology associated with some autoimmune thyroid disorders, most commonly "Graves' disease" (GD), but sometimes with other diseases (e.g., Hashimoto's thyroiditis).

[0055] The terms "proptosis" and "exophthalmos" (also known as exophthalmos / exophthalmia / exorbitism) refer to a forward protrusion, displacement, bulging, or protrusion of an organ. As used herein, the terms refer to a forward protrusion, displacement, bulging, or protrusion of the eye forward away from the orbit. Some technicians in the field consider proptosis and exophthalmos to have the same meaning and often use them interchangeably, while others believe that there is a subtle difference in their meanings. Some people use proptosis to refer to severe bulging eyes; or to refer to endocrine-related bulging eyes. Others use the term proptosis when describing bulging eyes associated with, for example, the eyes of subjects with TAO (TED or GO).

[0056] As used herein, the terms "exophthalmos" and "proptosis" are used interchangeably and refer to a forward protrusion, displacement, bulging or protrusion of the eye forward away from the orbit. Due to the rigid bony structure of the orbit with only an anterior opening for expansion, any increase in the soft tissue contents of the orbit, either laterally or posteriorly, will displace the eyeball forward. Exophthalmos or proptosis may be the result of several disease processes, including infection, inflammation, tumors, trauma, cancer metastasis, endocrine disorders, vascular disease, and extraorbital lesions. TAO (TED or GO) is currently recognized as the most common cause of exophthalmos in adults. Protrusion may be bilateral, as is common in TAO (TED or GO); or unilateral, as is common in orbital tumors.

[0057] In some preferred embodiments, the phosphorothioate antisense oligonucleotide targeting IGF-1R gene or the composition thereof is formulated as a lyophilized agent or an injection.

[0058] In some preferred embodiments, the phosphorothioate antisense oligonucleotides targeting the IGF-1R gene or compositions thereof are administered in combination with one or more thyroid eye disease treatments.

[0059] In some preferred embodiments, the thyroid eye disease treatment is radiation therapy and / or surgical treatment.

[0060] In some preferred embodiments, the dosage of the phosphorothioate antisense oligonucleotide targeting the IGFIR gene comprises 2.5 mg / kg to 10 mg / kg.

[0061] The present invention finds that the antisense oligonucleotide S-ASODN-1 with a specific sequence targeting the insulin-like growth factor-1 receptor IGF-1R can effectively reduce the concentration of IGF-1R and anti-thyroid peroxidase antibody (TPO-Ab), increase the level of thyroid stimulating hormone (TSH), and is used to treat TED. In fact, there is no antisense oligonucleotide in the existing drugs for treating TED that have been approved or entered the clinical stage. Moreover, not any antisense oligonucleotide targeting any sequence of IGF-1R can treat TED. For example, the other antisense oligonucleotide sequences S-ASODN-2, S-ASODN-3, S-ASODN-4, and S-ASODN-5 of the present application cannot effectively regulate the levels of TPO-Ab and TSH, and cannot be used to treat TED. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0063] Figure 1 This is the curve of animal body weight changes during the drug administration period of rats;

[0064] Figure 2 The changes of TPOAB concentration after S-ASODN-1 acted on the rat thyroid-associated eye disease model (IU / mL, Mean±SEM);

[0065] Figure 3 The changes of TSH concentration after S-ASODN-1 acted on the rat thyroid-associated eye disease model (mU / L, Mean±SEM);

[0066] Figure 4 The change of IGF-1R concentration after S-ASODN-1 acted on the rat thyroid-associated eye disease model (ng / mL, Mean±SEM);

[0067] Figure 5-1 to Figure 5-10 Pathological photographs of extraocular muscle lesions after the rat thyroid-related eye disease model was acted on, respectively: 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 (magnification 200 times). DETAILED DESCRIPTION

[0068] The present invention discloses the use of antisense oligonucleotides in the preparation of drugs for treating diseases caused by abnormal thyroid glands. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired effect. It should be particularly noted that 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 methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0069] The antisense oligonucleotide provided by the present invention is used in the preparation of drugs for treating diseases caused by thyroid abnormalities, and the raw materials and reagents used can all be purchased from the market.

[0070] The present invention will be further described below in conjunction with embodiments:

[0071] Example 1: Synthesis of all-phosphorothioate antisense oligonucleotides

[0072] 1. S-ASODN-1 (sequence: 5'-TCCTCCGGAGCCAGACTTCA-3' (SEQ ID NO: 1))

[0073] The solid phase synthesis method was used for synthesis. The instrument used was the OligoPilot 400 synthesizer of GE Company of the United States. The synthesis steps were as follows:

[0074] 1) Deprotection

[0075] The toluene solution of dichloroacetic acid is used as a deprotection agent to remove the 5'-DMT protecting group of the starting nucleoside dA(bz) on the carrier, thereby releasing the 5'-hydroxyl group.

[0076] 2) Coupling

[0077] Acetonitrile is used as solvent and 5-ethylthiotetrazolyl is used as activating agent to activate the dC(bz) phosphoramidite monomer to form an active intermediate, which then undergoes a condensation reaction with the 5'-hydroxyl group of the nucleoside dA(bz) to perform coupling.

[0078] 3) Thio

[0079] Hydrogenated xanthan gum is a thioagent that oxidizes phosphites into stable thiophosphates.

[0080] 4) Hydroxyl protection

[0081] Acetic anhydride is used as a protecting agent to protect the 5'-hydroxyl group of the nucleoside that has not undergone coupling reaction.

[0082] According to the nucleotide sequence of S-ASODN-1, the above steps 1)-4) are repeated to sequentially couple the corresponding nucleosides until the coupling of the S-ASODN sequence is completed.

[0083] 5) Deprotection

[0084] Dichloroacetic acid is used as a deprotection reagent to remove the DMT protecting group of the last nucleoside dT to obtain S-ASODN-1 connected to the carrier.

[0085] 6) Aminolysis

[0086] Add concentrated ammonia water to carry out aminolysis reaction, hydrolyze the ester bond between the carrier and the nucleotide, and remove the protecting groups on phosphate, adenine, guanine and cytosine. Filter, rinse with ethanol aqueous solution, and collect the filtrate.

[0087] 7) Purification

[0088] The filtrate was subjected to reverse phase column chromatography and freeze-dried to obtain the product with a purity of 93.2%.

[0089] The sequence of S-ASODN-2 is: 5'-TTCATTCCTTTTATTTGGGA-3' (SEQ ID NO: 2), the sequence of S-ASODN-3 is: 5'-GGACCCTCCTCCGGAGCC-3' (SEQ ID NO: 3),

[0090] The sequence of S-ASODN-4 is: 5'-GAGAAACAGGAGCCCCCACA-3' (SEQ ID NO: 4),

[0091] The sequence of S-ASODN-5 is: 5'-GCGCGGCTGGAAAGCGCGTT-3' (SEQ ID NO: 5), and the synthesis method is the same as above, and the purities are 91.1%, 93.5%, 92.4% and 92.8% respectively.

[0092] Example 2: Drug efficacy experiment in rat thyroid-related eye disease model

[0093] 1. Experimental Materials

[0094] Test sample: antisense oligonucleotide S-ASODN-1 (sequence:

[0095] 5'-TCCTCCGGAGCCAGACTTCA-3'), S-ASODN-2 (sequence:

[0096] 5'-TTCATTCCTTTTATTTGGGA-3'), S-ASODN-3 (sequence:

[0097] 5'-GGACCCTCCTCCGGAGCC-3'), S-ASODN-4 (sequence:

[0098] 5'-GAGAAACAGGAGCCCCCACA-3') and S-ASODN-5 (sequence: 5'-GCGCGGCTGGAAAGCGCGTT-3')

[0099] Positive drugs: Teprotumumab (500 mg / bottle, Horizon Therapeutics USA, Inc.);

[0100] Drug solvent: sodium chloride injection;

[0101] Reagents used: thyroglobulin (catalog number: T885815-100 mg; Shanghai McLean Biochemical Technology Co., Ltd.), isoflurane (batch number: 20221201, 100 mL, Jiangsu Hengfengqiang Biotechnology Co., Ltd.), Freund's complete adjuvant (5 mL / bottle, Chondrex), TSH Elisa kit (96T, Wuhan Huamei), TPO-Ab Elisa kit (96T, Wuhan Huamei), IGF-1R Elisa kit (48T, Wuhan Huamei).

[0102] Experimental animals: 60 7-week-old female SPF Wistar rats weighing 164.7 g to 179.9 g were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. The test animals were kept in sterile, independently ventilated IVC cages, with 6 rats per cage. The bedding was sterilized corn cob bedding. The rats were fed with sterilized feed specially formulated for rats and had free access to purified water. The temperature in the animal laboratory was maintained at around 25°C, the relative humidity was maintained at 40-70%, and the light was on for 12 hours per day.

[0103] 2. Experimental Methods

[0104] 2.1 Animal grouping and model establishment

[0105] Sixty 7-week-old female SPF Wistar rats (164.7 g to 179.9 g) were randomly divided into a control group (0.9% NaCl, 50 μL / eye, qw×4), a model group (0.9% NaCl, 50μL / eye, qw×4), S-ASODN-1 low-dose group (2.5mg / kg, 50μL / eye, qw×4), S-ASODN-1 medium-dose group (5mg / kg, 50μL / eye, qw×4), S-ASODN-1 high-dose group (10mg / kg, 50μL / eye, qw×4), S-ASODN-2 high-dose group (10mg / kg, 50μL / eye, qw×4), S-ASODN-3 high-dose group (10mg / kg, 50μL / eye, qw×4), S-ASODN-4 high-dose group (10mg / kg, 50μL / eye, qw×4), S-ASODN-5 high-dose group (10mg / kg, 50μL / eye, qw×4), positive drug group (Teprotumumab, 10mg / kg, tail vein injection), 6 rats / group. The information of animal groups is shown in Table 4.

[0106] Table 1 Animal grouping information

[0107]

[0108] Remark:

[0109] [1] The first digit of the animal number represents the group, the second letter represents the sex (F for female, M for male), and the third, fourth, and fifth digits represent the individual animal number;

[0110] [2] qw×4, once a week, for a total of 4 doses.

[0111] 2.2 Experimental treatment plan

[0112] Except the control group, the thyroid eye disease model of Wistar rats was established in each group by feeding with 0.6% NaI aqueous solution combined with intraperitoneal injection of thyroglobulin and incomplete / complete Freund's adjuvant emulsion. The drugs were injected retrobulbarly or intravenously into both eyes (OU) starting from the 27th day of modeling, recorded as D1 (or M27), once a week for 4 consecutive weeks, recorded as D2 (M28), D3 (M29), D4 (M30) ...; D1 before drug administration was recorded as D0.

[0113] 2.3 Evaluation indicators

[0114] Clinical observation, body weight, serum thyroid peroxidase antibody (TPO-AB) and thyroid stimulating hormone (TSH) concentrations at D0 and D23, insulin-like growth factor 1 receptor (IGF-1R) concentrations in intact right eye (OD) tissue, and histopathological examination of OU extraocular muscles.

[0115] Body weight measurement: on the day of animal receipt, the day of quarantine completion, the day of grouping, and once a week after grouping. Animals will be weighed when they are found dead or dying.

[0116] Protein detection: On D0 and D23, serum of all animals in the group was collected and ELISA was used to measure TPOAb and TSH. On D23, OD eyeballs of animals in each group were collected and ELISA was used to measure IGF-1R.

[0117] Pathological examination: M49 (D23), all surviving animals were collected for macroscopic observation of the lesions. After the tissues were fixed for at least 48 hours, the extraocular muscle tissues and thyroid tissues of all planned and unplanned dissected animals were collected, dehydrated, embedded, sliced, stained with hematoxylin-eosin, and read.

[0118] 2.4 Data Processing

[0119] General condition, histopathology and other data were analyzed descriptively. Quantitative indicators such as body weight were calculated as mean ± standard error (Mean ± SEM) by group. Among them, the comparison between the two groups was performed by t-test, and P < 0.05 was considered statistically significant. For comparison between multiple groups, the data of each indicator were analyzed according to the following procedures: Levene's test for homogeneity of variance. If the variance was homogeneous (P> 0.05), one-way analysis of variance (ANOVA) was used for statistical analysis. If ANOVA was statistically significant (P ≤ 0.05), a comprehensive comparison was performed using the LSD method. If the variance was unequal (P ≤ 0.05), Dunnett's T3 test was used for statistical analysis.

[0120] SPSS25.0 was used for the above statistical operations.

[0121] 3. Experimental results

[0122] 3.1 Observation of weight and general condition

[0123] During the experiment, animal No. 2F003 in the model group developed abdominal distension at M27 (D1) and died at M29 (D3); animal No. 4F005 in the S-ASODN-1 medium-dose group died at M46 (D20); no abnormal signs were observed in the animals in the other groups during the experiment.

[0124] On D23, the average 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 experiment, compared with the normal control group, the body weight of Wistar rats in the model group increased slowly, and the body weight at M19, D0, and D7 decreased significantly (P<0.05); compared with the model group, the body weight growth trend 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 was consistent during the administration period, and there was no statistical difference among the groups (P>0.05). Figure 1 .

[0125] 3.2 TPO-Ab protein detection

[0126] When thyroid peroxidase antibodies (TPO-Ab) are high, it means that the body's immune system has attacked the thyroid gland. This condition is often associated with autoimmune thyroid diseases, such as thyroid-associated eye disease.

[0127] 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 before and after administration on D0 and D23 are shown in the table below and Figure 2 .

[0128] Table 5

[0129]

[0130]

[0131] During the experiment, compared with the control group, the serum TPO-Ab concentrations of Wistar rats in the other groups before administration on D0 were significantly increased (P<0.001), indicating that the mouse thyroid-associated eye disease model was successfully established in each group.

[0132] Compared with D0 before administration, the serum TPO-Ab concentration of Wistar rats in the S-ASODN-1 low-dose group, S-ASODN-1 medium-dose group, S-ASODN-1 high-dose group, and positive drug group decreased by about 70 to 170 IU / mL after the last administration on D23; the TPO-Ab concentration decrease was about 10 to 30 times that of the model group, which was significantly reduced (P<0.001), showing a good therapeutic effect. Among them, the TPO-Ab concentration of the S-ASODN-1 high-dose group was only 48.57 IU / mL after the last administration on D23, and the TPO-Ab concentration decrease was about 27.97 times that of the model group, which showed the best therapeutic effect.

[0133] Compared with D0 before administration, there was no statistical difference in 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 last administration on D23 (P>0.05). The TPO-Ab concentration 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 did not change significantly, and the treatment effect was poor or no treatment effect.

[0134] In the positive drug group, the decrease in TPO-Ab concentration was about 22.50 times that of the model group. From the above data, it can be seen that the order of treatment effect of each group is: S-ASODN-1 high-dose group > positive drug group >

[0135] 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.

[0136] 3.3 TSH protein detection

[0137] Under normal circumstances, the thyroid stimulating hormone (TSH), also known as thyroid-stimulating hormone, secreted by the pituitary gland regulates the secretion of thyroxine, while the thyrotropin-releasing hormone (TRH) of the hypothalamus regulates the secretion of TSH. There is a typical negative feedback inhibition between thyroid hormone and TSH, that is, when the thyroid hormone level increases, the secretion of TSH is inhibited, and when the thyroid hormone level is low, the pituitary gland is stimulated to increase TSH secretion. The level of thyroxine (T2 or T6) in the serum decreases, causing the serum FSH level to increase and the TSH response to TRH stimulation to increase. Thyroid dysfunction changes the above regulation and response. The state of thyroid function is closely related to thyroid-related eye diseases, so understanding the state of thyroid function is of great significance for the diagnosis of thyroid-related eye diseases.

[0138] 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 are shown in the following table and Figure 3 .

[0139] Table 6

[0140]

[0141]

[0142] During the experiment, compared with the control group, the serum TSH concentrations of Wistar rats in the other groups before administration on D0 were significantly reduced (P<0.001), which proved that the mouse thyroid-related eye disease model was successfully established in each group.

[0143] Compared with D0 before administration of each group, the serum TSH concentration of Wistar rats in the S-ASODN-1 low-dose group increased significantly after the last administration on D23 (P<0.05), and the serum TSH concentration of Wistar rats in the S-ASODN-1 medium-dose group, S-ASODN-1 high-dose group, and positive drug group increased significantly (P<0.001). In the above three groups, the THS concentration increased to 0.64-1.70 IU / mL, which was about 3-15 times that of the model group, showing a good therapeutic effect. Among them, the THS concentration in the S-ASODN-1 high-dose group increased to 2.18 IU / mL after the last administration on D23, which was about 15.45 times that of the model group, and the therapeutic effect was the best.

[0144] There was no statistical difference among 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 (P>0.05). The 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 did not change significantly before and after administration, and the therapeutic effect was poor or no therapeutic effect.

[0145] In the positive drug group, the increase in THS concentration was about 11.45 times that of the model group. From the above data, it can be seen that the order of treatment effect of each group is: 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.

[0146] 3.4IGF-1R detection

[0147] The results of the detection of insulin-like growth factor 1 receptor (IGF-1R) concentration in D23 intact OD (right eye) tissue are shown in the following table and Figure 4 :

[0148] Table 7

[0149]

[0150]

[0151] During the experiment, compared with the control group, the IGF-1R concentration 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 was significantly increased (P<0.001); the IGF-1R concentration in the right eye tissue of Wistar rats in the S-ASODN-1 medium-dose and positive drug groups was significantly increased (P<0.05); there was no statistical difference in the IGF-1R concentration in the right eye tissue of Wistar rats in the S-ASODN-1 high-dose group (P>0.05).

[0152] Compared with the model group, the IGF-1R concentration in the right eye tissue of Wistar rats in the positive drug group and S-ASODN-1 high-dose group was significantly decreased (P<0.001); the IGF-1R concentration 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 was significantly decreased (P<0.05); the IGF-1R concentration in the right eye tissue of Wistar rats in the S-ASODN-1 medium-dose group was significantly decreased (P<0.01).

[0153] 3.5 Pathological testing

[0154] Depend on Figure 5-1 to Figure 5-10 The pathological photos of extraocular muscle lesions after the rat thyroid-associated eye disease model (magnification 200 times) show that:

[0155] Control group: No obvious abnormalities were found in the extraocular muscles of the left and right eyes of 6 / 6 animals.

[0156] Model group: 1 / 5 animals showed slight swelling of the extraocular muscles in the left eye; 4 / 5 animals showed slight degeneration and necrosis of the extraocular muscles in the left eye; 5 / 5 animals showed slight degeneration and necrosis of the extraocular muscles in the right eye.

[0157] S-ASODN-1 low-dose group: 3 / 6 animals had slight swelling of the extraocular muscle fibers in the left eye; 3 / 6 animals had slight degeneration and necrosis of the extraocular muscle fibers in the left eye; 4 / 6 animals had slight swelling of the extraocular muscle fibers in the right eye; 2 / 6 animals had slight degeneration and necrosis of the extraocular muscle fibers in the right eye.

[0158] In the medium-dose S-ASODN-1 group, slight swelling of the extraocular muscle fibers was observed in the left eye of 4 / 5 animals; slight degeneration and necrosis of the extraocular muscle fibers were observed in the left eye of 1 / 5 animals; slight swelling of the extraocular muscle fibers was observed in the right eye of 3 / 5 animals; slight degeneration and necrosis of the extraocular muscle fibers were observed in the right eye of 2 / 5 animals.

[0159] S-ASODN-1 high-dose group: No obvious abnormalities were found in the extraocular muscles of the left and right eyes of 6 / 6 animals.

[0160] S-ASODN-2 high-dose group: 6 / 6 animals showed slight muscle fiber degeneration and necrosis in the left eye extraocular muscles; 1 / 6 animals showed slight swelling of the extraocular muscles in the right eye extraocular muscles; 5 / 6 animals showed slight muscle fiber degeneration and necrosis in the right eye extraocular muscles.

[0161] S-ASODN-3 high-dose group: 1 / 6 animals had slight swelling of the extraocular muscle fibers in the left eye; 5 / 6 animals had slight degeneration and necrosis of the extraocular muscles in the left eye; 3 / 6 animals had slight swelling of the extraocular muscle fibers in the right eye; 3 / 6 animals had slight degeneration and necrosis of the extraocular muscles in the right eye.

[0162] S-ASODN-4 high-dose group: 1 / 6 animals showed slight swelling of the extraocular muscle fibers in the left eye; 5 / 6 animals showed slight degeneration and necrosis of the extraocular muscle fibers in the left eye; 6 / 6 animals showed slight degeneration and necrosis of the extraocular muscle fibers in the right eye.

[0163] S-ASODN-5 high-dose group: 1 / 6 animals showed slight swelling of the extraocular muscle fibers in the left eye; 5 / 6 animals showed slight degeneration and necrosis of the extraocular muscle fibers in the left eye; 2 / 6 animals showed slight swelling of the extraocular muscle fibers in the right eye; 4 / 6 animals showed slight degeneration and necrosis of the extraocular muscle fibers in the right eye.

[0164] No obvious abnormalities were found in the extraocular muscles of the left and right eyes of 6 / 6 animals in the high-dose group of the positive drug group.

[0165] The number of lesions is summarized in Table 8.

[0166] Table 8 Summary of cases of extraocular muscle lesions

[0167]

[0168] in conclusion:

[0169] Under the experimental conditions, Wistar rats were induced to have orbital tissue lesions by repeated intraperitoneal injections of bovine thyroglobulin (Tg), and the serum TPOAB of the animals increased significantly. The course of the disease in both eyes was consistent, indicating that the rat model was successfully established. By injecting 50 μL / eye behind the eyeball, the drug was administered once a week for four consecutive weeks. The high-dose S-ASODN-1 group was superior to the positive drug group in regulating the expression of rat serum TPO-Ab protein, rat serum TSH protein, and rat right eyeball IGF-1R expression. It also showed the effect of reducing tissue lesions in the extraocular muscle pathological sections, showing a good effect in treating thyroid eye disease.

[0170] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a therapeutically effective amount of a phosphorothioate antisense oligonucleotide targeting the IGFIR gene or a composition thereof in the preparation of a medicament for treating a disease caused by abnormal thyroid function, characterized in that: The nucleotide sequence of the phosphorothioate antisense oligonucleotide targeting the IGF-1R gene is shown in SEQ ID No. 1; the disease caused by abnormal thyroid function is thyroid-related eye disease.

2. The use according to claim 1, wherein the composition further comprises at least one additional active agent; wherein the at least one additional active agent is a therapeutic agent.

3. The use according to claim 2, wherein the therapeutic agent is selected from one or more of the following: biological agents or traditional immunosuppressants.

4. The method of claim 2, wherein the therapeutic agent is selected from corticosteroids.

5. The use according to claim 4, wherein The corticosteroid is selected from glucocorticoid ivGC.

6. The use according to claim 3, wherein The biological agent is selected from one or more of the following: CD20+B cell inhibitors, IL-6R antibodies, IL-17A antagonists, FcRn antagonists, IL-11R blocking antibodies, anti-TNF α antibodies, and thyroid stimulating hormone receptor TSHR inhibitors.

7. The use according to claim 3, wherein The traditional immunosuppressant is selected from one or more of the following: mycophenolate mofetil, cyclosporine, methotrexate and azathioprine.

8. The use according to claim 2, wherein The therapeutic agent is selected from one or more of the following: secukinumab, tocilizumab, satralizumab, vunakizumab, batoclimab, TOUR006, LASN01, infliximab, rituximab, and selenium.

9. The use according to any one of claims 1 to 8, wherein the thyroid-related eye disease manifests itself as one or more of the following symptoms: eyelid signs, proptosis, diplopia, ocular motility disorders, conjunctival edema, optic neuropathy or corneal lesions.

10. The use according to any one of claims 1 to 8, wherein the thyroid-related eye disease manifests itself in the following symptoms: conjunctival keratopathy.

11. The use according to claim 9, wherein the diplopia comprises constant diplopia, non-constant diplopia, and intermittent diplopia. 12 . The use according to claim 1 , wherein the phosphorothioate antisense oligonucleotide targeting IGF-1R gene or the composition thereof is formulated as a lyophilized agent or an injection.

13. The use according to any one of claims 1 to 8, wherein the phosphorothioate antisense oligonucleotide targeting IGF-1R gene or a composition thereof is administered in combination with one or more thyroid eye disease treatments.

14. The use according to claim 13, wherein the treatment of thyroid eye disease is radiotherapy and / or surgical treatment.

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