Application of periorbital injection in preparation of medicine for treating thyroid eye diseases

Abstract

The invention provides application of a periorbital injection in preparation of a medicine for treating thyroid eye diseases, and relates to the field of biological medicines. And the periorbital injection contains an anti-IGF-1R (Insulin-like Growth Factor- The periorbital injection provided by the invention has the advantages that the local dosage is small, and the side effect caused by systemic administration can be reduced; the target tissue drug concentration is high, and the effect is strong; the treatment frequency is low, and the patient tolerance is good.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to the application of a periorbital injection in the preparation of a medicine for treating thyroid eye disease. Background Art

[0002] Thyroid associated ophthalmopathy (TAO), also known as endocrine exophthalmos, infiltrative exophthalmos, and thyroid eye disease, is often complicated by Graves' disease (diffuse goiter with hyperthyroidism), also known as Graves' Ophthalmopathy (GO), accounting for about 20% of orbital diseases. TAO can cause protruding eyes, eyelid contracture, extraocular muscle dysfunction, conjunctival congestion, periorbital edema, etc. In severe cases, it can lead to exposure keratitis, diplopia, and compressive optic neuropathy, the latter of which can lead to blindness and seriously affect the patient's quality of life. TAO is a site-specific autoimmune disease dominated by cell-mediated immunity, triggered by common antigens expressed by thyroid epithelial cells, orbital preadipocytes, and fibroblasts. Inflammatory reactions, increased orbital fibroblasts, and increased orbital fat cells lead to protruding eyes, triggering the symptoms of thyroid associated ophthalmopathy. The levels of insulin-like growth factor 1 receptor (IGF-1R) and thyroid-stimulating hormone receptor (TSHR) in orbital fibroblasts of patients with thyroid-associated eye disease are abnormally high, and there is a complex signaling pathway network connection between the two, which is closely related to the occurrence of the disease.

[0003] IGF-1R belongs to the tyrosine protein kinase receptor family and is a transmembrane protein on the cell surface that can be activated by IGF-1 and IGF-2 (both insulin growth factors). Its overexpression may be associated with the onset of malignant tumors and autoimmune diseases such as multiple sclerosis, Crohn's disease, and pulmonary fibrosis. Anti-IGF-1R antibodies can be detected in most patients with Graves' disease, but are rarely found in normal people. IgG (Graves-related immunoglobulin G) isolated from the serum of patients with Graves' disease can replace IGF-1R and bind to sites on the surface of orbital fibroblasts. IGF-1R or Graves' disease-related IgG can activate IGF-1R-positive orbital fibroblasts from patients with thyroid-related eye disease, thereby activating the Akt / FRAP / mTOR / P70s6k channel to induce the expression of interleukin-16 and chemokine expressed and secreted by normal activated T cells (RANTES) on orbital fibroblasts, promote the synthesis of T cell chemokines, and cause inflammatory infiltration of T lymphocytes and production of hyaluronic acid. These all indicate that IGF-1R may be involved in the development of Graves' disease as a second antigen. In addition, reports have shown that there is a large overlap between the downstream signals of IGF-1R and TSHR, and that anti-IGF-1R monoclonal antibodies can block IGF-1R signal transduction, weaken the downstream signals of the TSHR pathway, and reduce the inflammatory response caused by thyroid-stimulating hormone, suggesting that IGF-1R is involved in TSHR signal transduction, and that IGF-1R and TSHR may form a complex functional complex that plays a synergistic role in abnormal signal transduction associated with Graves' disease.

[0004] Tepezza is the first FDA-approved drug for the treatment of thyroid eye disease. Tepezza is a fully humanized insulin-like growth factor 1 receptor (IGF-1R) monoclonal antibody that reduces inflammation and tissue cell overgrowth by binding to IGF-1R and blocking its activation and signal transduction. The recommended dose of Tepezza is an initial dose of 10 mg / kg intravenous infusion, followed by 20 mg / kg intravenous infusion every three weeks for 7 infusions. Due to the widespread expression of IGF-1R in the body, anti-IGF-1R antibodies such as Tepezza have more obvious side effects after systemic administration. Among the most commonly reported adverse reactions, muscle spasms (18%), hearing impairment (10%) and hyperglycemia (8%) have the greatest risk difference compared with the placebo group.

[0005] The Chinese patent with publication number CN115175701A provides methods for treating thyroid eye disease (TED) or reducing the severity of thyroid eye disease, as well as antibodies or antigen-binding fragments thereof that can be used in these methods, and pharmaceutical compositions comprising these antibodies or antigen-binding fragments thereof. In the method, the pharmaceutical composition is administered intravenously or orally.

[0006] The Chinese patent with publication number CN112794912B provides an antibody that can bind to anti-IGF-1R, and also provides a nucleic acid molecule encoding the antibody, a vector containing the nucleic acid, a host cell transformed by the vector, and its application. The use of the antibody is not disclosed.

[0007] In view of the above problems, the present invention proposes an application of a periorbital injection in the preparation of a drug for treating thyroid eye disease. The application disclosed in the present invention can overcome the side effects caused by systemic administration, and its advantages are mainly reflected in: high drug concentration in the target area, strong effect, and obvious therapeutic effect; small dosage, small systemic side effects, and few contraindications; fewer injection times, good patient tolerance, and high safety. Summary of the invention

[0008] In order to solve the above problems, the present invention provides an application of a periorbital injection in the preparation of a medicine for treating thyroid eye disease.

[0009] Specifically, the periorbital injection comprises an anti-IGF-1R antibody.

[0010] Specifically, the anti-IGF-1R antibody comprises at least one heavy chain and at least one light chain selected from the group consisting of:

[0011] a. a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2;

[0012] b. a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4;

[0013] c. a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6;

[0014] d. a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8;

[0015] e. a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10;

[0016] f. a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12;

[0017] g. a light chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14;

[0018] h, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16;

[0019] i. a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18;

[0020] j. a light chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20;

[0021] k, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 22;

[0022] 1. A light chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 24.

[0023] Specifically, the thyroid eye disease includes one or more of proptosis, eyelid retraction, upper eyelid lag, extraocular muscle hypertrophy, conjunctival congestion, periorbital tissue edema, incomplete eyelid closure, photophobia, and decreased vision or diplopia, tearing and foreign body sensation.

[0024] Specifically, the injection dose of the anti-IGF-1R antibody in the periorbital injection includes 1-500 mg / eye.

[0025] Preferably, the injection dose of the anti-IGF-1R antibody in the periorbital injection comprises 150 mg / eye.

[0026] Specifically, the injection volume of the anti-IGF-1R antibody in the periorbital injection is no more than 5 mL / eye.

[0027] Preferably, the injection volume of the anti-IGF-1R antibody in the periorbital injection comprises 1 mL / eye.

[0028] Specifically, the periorbital injection includes one or more of periorbital injection, periorbital injection combined with intravenous injection, and periorbital injection combined with subcutaneous injection.

[0029] Preferably, the periorbital injection is injected by periorbital injection.

[0030] Specifically, the periorbital injection method of the periorbital injection includes inserting the needle with a 2mL retrobulbar syringe into the middle and outer 1 / 3 of the upper or lower eyelid near the orbital margin, first inserting the needle vertically into the skin for 1 cm, then tilting the needle toward the bottom of the nose, and continuing to insert the needle, not more than 35 mm deep into the orbit, and injecting anti-IGF-1R antibodies after aspiration without blood return.

[0031] Specifically, the injection frequency of the periorbital injection includes one or more of once a week, once every 2 weeks, once every 3 weeks, and once every 4 weeks.

[0032] Preferably, the injection frequency of the periorbital injection is once every 2 weeks.

[0033] Specifically, the periorbital injection can be applied to humans or animals.

[0034] Preferably, the periorbital injection can be used in humans.

[0035] Specifically, the periorbital injection further comprises a pharmaceutically acceptable excipient.

[0036] Preferably, the pharmaceutically acceptable excipients include solubilizers, co-solvents, suspending agents, emulsifiers, and antibacterial agents.

[0037] Beneficial effects of the present invention:

[0038] The present invention provides an application of a periorbital injection in the preparation of a drug for treating thyroid eye disease, and relates to the field of biomedicine. The periorbital injection comprises an anti-IGF-1R antibody. The periorbital injection provided by the present invention has a small local dosage, which can reduce the side effects caused by systemic medication; the target tissue drug concentration is high and the effect is strong; the number of treatments is small and the patient's tolerance is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The results show that antibody Ab1-12 inhibits adipogenic differentiation of mouse fibroblasts 3T3 cells that stably overexpress human IGF-1R.

[0040] Figure 2 HE staining of some individual extraocular muscles. DETAILED DESCRIPTION

[0041] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0042] Example 1 Cell biological activity of anti-IGF-1R antibodies in mouse fibroblasts 3T3 cells stably overexpressing human IGF-1R.

[0043] Preparation of anti-IGF-1R antibodies Ab1-12 as shown in Table 1:

[0044] Table 1 Amino acid sequences of prepared anti-IGF-1R antibodies

[0045]

[0046]

[0047]

[0048] Mouse fibroblasts 3T3 (ATCC; catalog number CL-173) stably overexpressing human IGF-1R were used, and IGF-1 (Peprotech, 100-11) was added to the culture system of 3T3 cells for pretreatment.

[0049] (1) Evaluation of the effects of different anti-IGF-1R antibodies on the proliferation of 3T3 cells

[0050] The proliferation ability of 3T3 cells was detected by CCK-8 kit (Beyotime, C0043). First, the 3T3 cell suspension was inoculated in a 96-well plate, about 100 μL per well, and 3 duplicate wells per group. The culture plate was placed in an incubator for 4 hours, and 10 μL of 20 mg / mL concentration of Ab1-12 was added to the wells. After continuing to culture for 24-48 hours, 10 μL of CCK-8 solution was added to each well, and the mixture was gently shaken. The culture plate was placed in an incubator and incubated for 1-4 hours. The OD value at 450 nm was measured by an enzyme marker, and the inhibitory efficiency IC50 value of cell proliferation was calculated. As shown in Table 2, Ab1-12 can inhibit the proliferation of 3T3 cells.

[0051] Table 2 Inhibition of 3T3 cell proliferation by different anti-IGF-1R antibodies (IC50)

[0052]

[0053]

[0054] (2) Evaluation of the effect of anti-IGF-1R antibody on adipogenic differentiation of 3T3 cells

[0055] The suspension of 3T3 cells overexpressing human IGF-1R was inoculated in a 96-well plate, and the adipogenic differentiation of 3T3 cells was induced by self-prepared adipogenic induction solution [DMEM (high glucose) + 10% calf serum + 1% P / S + 0.5mM IBMX (3-Isobutyl-1-methylxanthine) + 0.25μM dexamethasone + 1μg / mL insulin]. A total of 14 groups were set up, namely adipogenic induction group, IGF-1 pretreatment + adipogenic induction group, and IGF-1 pretreatment + Ab1-12 + adipogenic induction group. The cells were fixed with 3.5% formaldehyde solution for 10 minutes, rinsed twice with PBS, and incubated in 60% isopropanol for 1 hour with 1.5% Oil Red O stain (O0625; Sigma-Aldrich). The cells were washed with PBS three times, and the stained Oil Red O was dissolved in DMSO. The OD value was measured at 510nm. The larger the OD value, the higher the degree of adipogenic differentiation of 3T3 cells.

[0056] The results are as follows Figure 1 As shown, 3T3 cells overexpressing human IGF1R showed a certain degree of adipogenic differentiation after being induced by adipogenic induction solution; the adipogenic differentiation of 3T3 cells was the highest after IGF-1 pretreatment and then adipogenic induction; while the adipogenic differentiation of 3T3 cells in the IGF-1 pretreatment + Ab1-12 + adipogenic induction group was significantly inhibited to varying degrees.

[0057] Example 2 Pharmacodynamic evaluation of orbital injection of anti-IGF-1R antibody in model animals

[0058] In this example, a model of hyperthyroidism and thyroid-associated eye disease in rats was induced by intragastric administration of bovine thyroglobulin to observe the effect of anti-IGF-1R antibody on thyroid-associated eye disease in rats, including the following steps:

[0059] Modeling: The rats in the model group were intraperitoneally injected with bovine thyroglobulin (SigmaAldrich, 609310) at a dose of 50 μg / 100 g every 2 weeks for 4 weeks to complete the modeling.

[0060] Grouping and administration: 40 experimental animals were divided into 4 groups, 10 animals in each group:

[0061] Group 1: Sham group, all normal rats;

[0062] Group 2: Model + PBS group, all rats were completed modeling, intravenous injection of PBS, 10 mg / kg (10 mpk), injection volume 2.5 mL / rat, once a week, for 4 consecutive times;

[0063] Group 3: Model + intravenous injection of Ab1 group, all rats with completed modeling were intravenously injected with anti-IGF-1R antibody Ab1; 10 mg / kg, injection volume 2.5 mL / rat, once a week for 4 consecutive times;

[0064] Groups 4 and 5: model + periorbital injection of Ab1 group and model + periorbital injection of Ab11 group, both of which were rats with completed modeling, and were periorbitally injected with anti-IGF-1R antibodies Ab1 and Ab11, respectively; the periorbital injection dose of groups 4 and 5 was converted according to the body surface area method, the orbital injection dose of rats = human orbital injection dose * (body surface area of ​​rats / body surface area of ​​humans), the body weight of humans was 70 kg, and the body weight of rats was 300 g; the orbital injection dose of rats was calculated to be 4 mg / eye, with an injection volume of 50 μL / eye, once every 2 weeks for 2 consecutive times.

[0065] After 4 weeks of drug administration, the animals were euthanized, and the extraocular muscles were sampled, fixed with formaldehyde, embedded in paraffin, and sliced. The samples were stained with HE and observed under a fluorescence microscope (Nikon, Eclipse Ci-L). Pathological scores were performed according to the scoring criteria shown in Table 3.

[0066] Table 3 Pathological scores of extraocular muscles in rats

[0067]

[0068] Experimental results: The pathological scoring results of extraocular muscle staining are shown in Table 4.

[0069] Table 4 Pathological scoring results of extraocular muscle staining

[0070]

[0071] Note: &, compared with the Sham group, P < 0.05; *, compared with the PBS group, P < 0.05; #, compared with the intravenous injection group, P < 0.05.

[0072] The results are as follows Figure 2 shown.

[0073] In the sham group, the muscle fibers were uniformly stained red and arranged neatly, with abundant intermuscular capillaries and normal intermuscular distance;

[0074] Various extraocular muscle lesions were observed in the model+PBS group, including severe swelling, degeneration, necrosis, and dissolution of muscle fibers, narrowing or widening of the intermuscular distance, and proliferation of intermuscular fibrous connective tissue and blood vessels, indicating that the SD rat thyroid eye disease model was successfully established;

[0075] The visible lesions in the model + intravenous injection of Ab1 group basically covered all types of lesions in the model + PBS group, and the degree of myofiber necrosis and dissolution was slightly milder than that in the model + PBS group;

[0076] The lesions in the model + periorbital injection of Ab1 and Ab11 groups were mild, the muscle fibers were basically arranged neatly, and very few muscle fibers were slightly swollen or degenerated. This shows that periorbital injection of Ab1 and Ab11 has a good effect on the treatment of thyroid eye disease in SD rats, which is significantly better than the intravenous injection group, and the medication volume and injection frequency are greatly reduced.

[0077] Example 3 Effect of periorbital injection on blood sugar

[0078] In this example, normal rats were periorbitally injected with anti-IGF-1R antibodies to observe changes in blood glucose levels in the animals. The animals were specifically divided into the following groups: a normal control group (Sham group), a PBS group, an intravenous injection of Ab11 group, and a periorbital injection of Ab1-12 group. The dose of Ab1-12 in the periorbital injection group was 4 mg / eye, and the injection volume was 50 μL / eye; the administration volume and dose of the remaining groups were consistent with those in Example 2. The drug was administered once a week for 4 consecutive times, and the rats were fasted for 12 hours, blood was collected from the tail end of the rats, and dripped on a blood glucose meter (Johnson One Touch Horizon), and the blood glucose value was read.

[0079] Experimental results: The blood glucose concentrations of each group are shown in Table 5. The results show that the blood glucose of the group injected with Ab11 intravenously increased significantly, while the blood glucose of the group injected with Ab1-12 periorbitally was normal. This indicates that intravenous injection of anti-IGF-1R antibodies can induce hyperglycemia in the body, while the volume of antibodies required for periorbital injection of anti-IGF-1R antibodies is small and does not cause the side effect of hyperglycemia.

[0080] Table 5 Blood glucose concentrations in different groups after administration

[0081]

[0082]

[0083] Note: *, compared with the sham group, p < 0.05.

Claims

1. Use of a periorbital injection in the preparation of a drug for treating thyroid eye disease, characterized in that: The periorbital injection comprises an anti-IGF-1R antibody.

2. The use according to claim 1, wherein the anti-IGF-1R antibody comprises at least one heavy chain and at least one light chain selected from the group consisting of: a. a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2; b. a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4; c. a light chain variable region comprising the amino acid sequence of SEQ ID NO: 5 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6; d. a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8; e. a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10; f. a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12; g. a light chain variable region comprising the amino acid sequence of SEQ ID NO: 13 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14; h, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 16; i. a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18; j. a light chain variable region comprising the amino acid sequence of SEQ ID NO: 19 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 20; k, a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 22; L. a light chain variable region comprising the amino acid sequence of SEQ ID NO: 23 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:

24.

3. The use according to claim 1, characterized in that: The thyroid eye disease includes one or more of proptosis, eyelid retraction, upper eyelid lag, extraocular muscle hypertrophy, conjunctival congestion, periorbital tissue edema, incomplete eyelid closure, photophobia, and decreased vision or diplopia, tearing and foreign body sensation.

4. The use according to claim 1, characterized in that: The injection dose of the anti-IGF-1R antibody in the periorbital injection is 1-500 mg / eye; preferably, the injection dose of the anti-IGF-1R antibody in the periorbital injection is 150 mg / eye.

5. The use according to claim 1, characterized in that: The injection volume of the anti-IGF-1R antibody in the periorbital injection does not exceed 5 mL / eye; preferably, the injection volume of the anti-IGF-1R antibody in the periorbital injection is 1 mL.

6. The use according to claims 1-5, characterized in that: The injection method of the periorbital injection includes one or more of periorbital injection, periorbital injection combined with intravenous injection, and periorbital injection combined with subcutaneous injection; preferably, the injection method is periorbital injection.

7. The use according to claim 1, characterized in that: The injection frequency of the periorbital injection includes one or more of once a week, once every 2 weeks, once every 3 weeks, and once every 4 weeks; preferably, the injection frequency of the periorbital injection includes once every 2 weeks.

8. The use according to claim 1, characterized in that: The periorbital injection can be applied to humans or animals.

9. The use according to claim 1, characterized in that: The periorbital injection further comprises a pharmaceutically acceptable excipient.

10. The use according to claim 9, characterized in that: The pharmaceutically acceptable excipients include solubilizers, co-solvents, suspending agents, emulsifiers, and antibacterial agents.

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