CN113244236A - Application of ceritinib in preparation of medicine for treating thyroid-associated ophthalmopathy - Google Patents
Application of ceritinib in preparation of medicine for treating thyroid-associated ophthalmopathy Download PDFInfo
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- CN113244236A CN113244236A CN202110610004.1A CN202110610004A CN113244236A CN 113244236 A CN113244236 A CN 113244236A CN 202110610004 A CN202110610004 A CN 202110610004A CN 113244236 A CN113244236 A CN 113244236A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/14—Drugs for disorders of the endocrine system of the thyroid hormones, e.g. T3, T4
- A61P5/16—Drugs for disorders of the endocrine system of the thyroid hormones, e.g. T3, T4 for decreasing, blocking or antagonising the activity of the thyroid hormones
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Abstract
The invention discloses an application of ceritinib in preparation of a medicine for treating thyroid-related ophthalmopathy, and relates to the field of medicines. The pathogenesis of thyroid-associated eye diseases is complex, in which differentiation of fat precursor cells in the orbit is enhanced and a large amount of inflammatory factors are secreted, etc., eventually leading to clinical phenotypes such as eyeball protrusion and increased intraocular pressure. According to the invention, lipid accumulation condition of cells with complete fat induced differentiation is evaluated in a Bodipy staining and high content instrument analysis mode, so that drugs capable of effectively inhibiting fat differentiation and lipid accumulation thereof are screened, and the result shows that ceritinib has an obvious inhibition effect on the fat differentiation of human-derived orbital fibroblasts, can inhibit the expression of IL-17A induced inflammatory factors, and verifies the therapeutic effect of ceritinib on thyroid-related eye diseases.
Description
Technical Field
The invention belongs to the field of medicines, relates to a new application of ceritinib, and particularly relates to an application of ceritinib in preparation of a medicine for treating thyroid-related ophthalmopathy.
Background
Thyroid-associated ophthalmopathy (TAO) is an organ-specific autoimmune Disease with exophthalmos as an important sign, mainly caused by hyperthyroidism (GD). The disease can lead to orbital distortion, diplopia and even vision loss. Thus, Graves' eye disease has a great negative impact on the quality of life, mental well-being and socioeconomic status of patients. The incidence of TAO is high, and is the most prominent orbital disease. Global GD prevalence is between 0.2-1.3%, with proportions that cause mild and moderate-severe TAO of about 20-30% and 5%, respectively.
The pathogenesis of TAO is complex, and is related to various factors such as genetic, environmental and immune dysfunction. Thyroid Stimulating Hormone Receptor (TSHR) is the currently recognized TAO pathogenic autoantigen. Inflammatory cells that accumulate in ocular tissues are activated after binding to the TSHR of orbital fibroblasts, secreting some cytokines and inflammatory factors. These factors stimulate fibroblasts to secrete more inflammatory factors and to secrete large amounts of extracellular matrix components. Meanwhile, the differentiation of fat precursor cells in the orbit is enhanced, the swelling of extraocular muscles caused by inflammation, edema caused by the hydrophilic property of extracellular matrix components and the enhancement of fat differentiation are enhanced, and finally, clinical phenotypes such as eyeball protrusion, intraocular pressure increase and the like are caused together.
Studies have found that the proportion of IL-17A-producing T cells in Graves eye disease patients is significantly higher than that in healthy controls (P)<0.01) and recruits CD4 in the orbital tissues of Graves eye disease patients+And CD8+T cells. In addition, orbital tissues of Graves eye disease patients express more IL-17A receptor, IL-17A and its associated cytokines.
Retrobulbar lipodysplasia is an important pathological feature in TAO. The differentiation process of adipocytes is regulated by a series of signals: the fat precursor cells firstly express C/EBP delta and C/EBP beta after being stimulated by differentiation, the C/EBP delta and the C/EBP beta jointly activate the expression of PPAR gamma and C/EBP alpha, and then the PPAR gamma and the C/EBP alpha are mutually activated to form a forward regulation loop to activate and express most of fat development related genes to complete the differentiation process. Currently, there is no FDA-approved small molecule drug for TAO that inhibits lipohypertrophy.
Disclosure of Invention
The invention provides an application of ceritinib in preparation of a drug for treating thyroid-related ophthalmopathy, aiming at the problem that the existing TAO has no FDA-approved small-molecule drug for inhibiting lipohypertrophy. Ceritinib (Ceritinib) is a potent ALK inhibitor approved by the FDA for the treatment of Anaplastic Lymphoma Kinase (ALK) positive metastatic non-small cell lung cancer (NSCLC) following crizotinib treatment failure (secondary to drug resistance or intolerance) in adults. However, no report about the therapeutic effect of Ceritinib on the thyroidism is found by examining relevant documents at home and abroad.
Preferably, the symptoms of thyroid-related eye disease include exophthalmos, swelling of soft eye tissue, and increased intraocular pressure.
Preferably, ceritinib is used alone.
Preferably, the ceritinib is used in the form of a pharmaceutical composition.
Preferably, the medicine is a medicinal preparation prepared from ceritinib and medicinal auxiliary materials.
The ceritinib is used for inhibiting fat differentiation and lipid accumulation of orbital fibroblasts.
The ceritinib is used for inhibiting the inflammatory response of orbital fibroblasts.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, lipid accumulation condition of cells with complete fat-induced differentiation is evaluated by means of Bodipy staining and high content instrument analysis to screen drugs capable of effectively inhibiting fat differentiation and lipid accumulation thereof, and the result shows that Ceritinib has an inhibition effect on fat differentiation of human-derived orbital fibroblasts, can inhibit the expression of IL-17A-induced inflammatory factors, and verifies the therapeutic effect of Ceritinib on thyroid-related eye diseases.
Drawings
FIG. 1 is a flow chart of the high content screening of drugs for regulating adipocyte differentiation according to the present invention.
FIG. 2 is a graph of the effect of high content screening drugs of the present invention on adipocyte differentiation; wherein:
udiff indicates an uninduced differentiation group; Diff/DMSO represents a differentiation-inducing control group; Diff/(A-K) 1. mu.M represents the differentiation inducing + drug (A-K) group, and A-K represent different types of drugs.
Figure 3 is a graph of the effect of Ceritinib on adipocyte differentiation, where:
a is Bodipy imaging lipid drop and fluorescence intensity diagram;
b is a diagram of relative degree of adipocyte differentiation;
udiff indicates an uninduced differentiation group; Diff/DMSO represents a differentiation-inducing control group; Diff/Ceritinib-1. mu.M represents the differentiation inducing + drug (1. mu.M Ceritinib) group, i.e., Ceritinib intervention group.
FIG. 4 is a graph showing the results of detecting the expression of genes involved in the differentiation of Ceritinib stem preadipocytes, wherein:
a is the expression detection result of the C/EBP delta gene when the differentiation is carried out for 1 h;
b is the expression detection result of the PPAR gamma 2 gene at the differentiation time of 144 h;
DMSO represents the uninduced differentiation group; ceritinib-1. mu.M represents the differentiation inducing + drug (1. mu.M) group.
Fig. 5 is a graph showing the results of detecting the expression of inflammatory factors associated with Ceritinib intervention group, in which:
a is the expression detection result of the inflammatory factor IL-1 beta;
b is the expression detection result of the inflammatory factor IL-6;
c is an expression detection result of an inflammatory factor IL-8;
DMSO represents the non-inflammatory response group; IL-17A represents a control group that induces an inflammatory response; IL-17A + Ceritinib-1. mu.M represents the group inducing inflammatory response + drug (1. mu.M Ceritinib).
Detailed Description
The technical solution of the present invention is further described below with reference to the accompanying drawings and examples.
The invention provides a new application of Ceritinib, namely the application of the Ceritinib in preparing a medicament for treating thyroid-related eye diseases, wherein symptoms of the thyroid-related eye diseases comprise eyeball protrusion, eye soft tissue swelling and intraocular pressure increase.
The Ceritinib can be independently used when preparing a medicine for treating thyroid-related eye diseases; can also be used in the form of pharmaceutical compositions; or mixing with medicinal adjuvants, and making into medicinal preparation, such as tablet, capsule, granule, pill, suppository, membrane, colloid, unguent, powder, injection, mixture, oral liquid, syrup, medicated liquor, sol, emulsion or eye drop.
The pharmaceutical preparation contains effective dose of Ceritinib, and the rest is pharmaceutically acceptable carriers and excipients which are nontoxic and inert to human and animals. The pharmaceutically acceptable carrier or excipient is one or more selected from solid, semi-solid and liquid diluents, and nontoxic and inert pharmaceutically acceptable carriers and excipients for fillers.
The medicine can inhibit fat differentiation and lipid accumulation of the orbital fibroblasts of patients with thyroid-associated eye diseases; also can inhibit inflammatory reaction of ocular fibroblast of thyroid gland related ophthalmopathy patients.
According to the invention, the lipid accumulation condition of cells with complete fat-induced differentiation is evaluated by Bodipy staining and high content instrument analysis to screen drugs capable of effectively inhibiting lipid accumulation, and the result shows that the Ceritinib has an inhibition effect on the fat differentiation of human-derived orbital fibroblasts, can slow down the expression of IL-17A-induced inflammatory factors, and verifies the therapeutic effect of the Ceritinib on thyroid-related eye diseases.
The experimental procedures and experimental results of the present invention are explained in detail below.
High content screening medicine for regulating and controlling adipocyte differentiation
1. Experimental methods
FIG. 1 shows the process of high content screening of drugs that regulate adipocyte differentiation. The method specifically comprises the following steps:
TAO lipo precursor cells were inoculated in a 96-well plate or a 384-well plate, and after the cells were confluent, fresh DMEM/F12 medium containing 10% FBS and 1% streptomycin was changed, and the culture was continued for 2 days.
The following inducers were added for fat differentiation, including 5. mu.g/ml insulin, 1. mu.M dexamethasone, 0.5mM Isoxobutylmethyxanthine (IBMX) and 1. mu.M rosiglitazone, while one test drug was added to each well (test drug using DMSO as a solvent). Fresh culture medium (including 10% FBS, 5. mu.g/ml insulin, 1. mu.M rosiglitazone and corresponding test drug) was changed at days 2, 4, 6, 8, 10 and 12 after induction. After inducing differentiation for 14 days, the culture medium was discarded, and fixed with 4% paraformaldehyde for 30 minutes.
The fixed cells were washed 3 times with PBS, followed by addition of PBS containing Bodipy 493/503 and DPAI for 10min at room temperature, followed by 3 washes with PBS, and the cells were soaked in PBS. And analyzing the whole Bodipy fluorescence intensity of the cells by using a high content instrument, and representing the differentiation degree of the fat cells by using the fluorescence intensity.
2. Results of the experiment
The result of screening drugs from a small molecule drug library by using the high content drug screening method in the in vitro cell model is shown in fig. 2, and most of the screened drugs have no inhibitory effect on adipocyte differentiation and lipid accumulation thereof in the differentiation process of TAO adipocyte precursor cells at a concentration of 1 μ M.
However, as shown in FIG. 3, the Ceritinib intervention group showed a significant inhibitory effect. The number of lipid droplets visible in the bright field was significantly lower than in the control group (a of fig. 3); semiquantitative analysis of fluorescently labeled Bodipy lipid droplets showed that lipid accumulation in the Ceritinib dried group was reduced by about 60% compared to the control group (fig. 3B).
Molecular mechanism of (di) Ceritinib for inhibiting adipocyte differentiation
1. Experimental methods
In order to research the molecular mechanism of the Ceritinib for inhibiting the adipocyte differentiation, cells at different differentiation time are collected, RNA is extracted, and qRT-PCR is used for detecting the expressions of adipocyte differentiation related genes such as C/EBP delta, C/EBP beta, PPAR gamma, C/EBP alpha and the like.
2. Results of the experiment
As shown in FIG. 4, Ceritinib inhibits the expression of PPAR γ 2 and C/EBP δ, which are important genes for adipocyte differentiation, i.e., C/EBP δ is inhibited by Ceritinib in the early stage of adipocyte differentiation induction, and PPAR γ 2 is inhibited in the later stage. This is consistent with the results of Ceritinib inhibiting adipocyte differentiation.
(III) Ceritinib inhibits inflammatory response of fibroblasts
1. Experimental methods
Orbital adipocyte precursor cells were cultured in DMEM/F12 medium containing 10% FBS and 1% streptomycin, and inflammatory response was induced by the inflammatory factor IL-17A, with the addition of Ceritinib. After the reaction was completed, cells were collected and RNA was extracted, and the expression of the relevant inflammatory factors was detected by qRT-PCR.
2. Results of the experiment
As shown in FIG. 5, the results show that Ceritinib significantly reduces the expression of inflammatory factors IL-1 beta, IL-6 and IL-8, indicating that Ceritinib is effective in slowing the inflammatory response of TAO, thereby treating thyroid-related eye diseases.
In conclusion, Ceritinib (Ceritinib) can effectively inhibit fat differentiation of lipo progenitor cells and lipid accumulation thereof, can effectively slow down inflammatory reaction of TAO, and can be used for preparing medicines for treating thyroid-related eye diseases.
While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.
Claims (7)
1. Application of ceritinib in preparation of medicines for treating thyroid-associated ophthalmopathy.
2. The use as claimed in claim 1, wherein the symptoms of thyroid-related eye disease include exophthalmos, swelling of soft tissues of the eye, or increased intraocular pressure.
3. The use of claim 1, wherein ceritinib is used alone.
4. The use of claim 1, wherein ceritinib is used in the form of a pharmaceutical composition.
5. The use of claim 1, wherein the medicament is a pharmaceutical preparation prepared from ceritinib and pharmaceutical excipients.
6. The use of any one of claims 1 to 5, wherein ceritinib is used to inhibit adipose differentiation and lipid accumulation of orbital fibroblasts.
7. The use of any one of claims 1 to 5, wherein ceritinib is used to inhibit the inflammatory response of orbital fibroblasts.
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Citations (5)
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| CN105061397A (en) * | 2015-08-07 | 2015-11-18 | 武汉英普瑞医药科技有限公司 | C-type crystal form of ceritinib, preparation method therefor and application thereof |
| CN106456640A (en) * | 2014-05-29 | 2017-02-22 | 诺华股份有限公司 | Ceritinib formulation |
| SG11201804440XA (en) * | 2015-12-04 | 2018-06-28 | Penn State Res Found | Chemical reprogramming of human glial cells into neurons with small molecule cocktail |
| CN108939055A (en) * | 2017-05-27 | 2018-12-07 | 复旦大学附属眼耳鼻喉科医院 | Purposes of the recombined human PH20 in preparation treatment thyroid-associated ophthalmopathy drug |
| CN112165934A (en) * | 2018-05-25 | 2021-01-01 | 克劳德布雷克医疗有限责任公司 | Compositions and methods for treating ocular congestion |
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- 2021-06-01 CN CN202110610004.1A patent/CN113244236B/en active Active
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| CN106456640A (en) * | 2014-05-29 | 2017-02-22 | 诺华股份有限公司 | Ceritinib formulation |
| CN105061397A (en) * | 2015-08-07 | 2015-11-18 | 武汉英普瑞医药科技有限公司 | C-type crystal form of ceritinib, preparation method therefor and application thereof |
| SG11201804440XA (en) * | 2015-12-04 | 2018-06-28 | Penn State Res Found | Chemical reprogramming of human glial cells into neurons with small molecule cocktail |
| CN108939055A (en) * | 2017-05-27 | 2018-12-07 | 复旦大学附属眼耳鼻喉科医院 | Purposes of the recombined human PH20 in preparation treatment thyroid-associated ophthalmopathy drug |
| CN112165934A (en) * | 2018-05-25 | 2021-01-01 | 克劳德布雷克医疗有限责任公司 | Compositions and methods for treating ocular congestion |
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