Application of tofacitinib in preparation of medicine for treating dry age-related macular degeneration

Abstract

The invention discloses application of tofacitinib in preparation of a medicine for treating dry age-related macular degeneration, and belongs to the technical field of medicine application. The preclinical research proves that the tofacitinib can be used for effectively treating the dry AMD, and the effects of the tofacitinib are reflected in protecting the retina-choroid structure, maintaining the normal thickness of the retina-choroid, protecting the retinal pigment epithelium (RPE) structure, protecting the photoreceptor cell structure and protecting the choroid structure. In addition, tofacitinib can also play a therapeutic role by inhibiting expression of inflammatory factors in eye tissues. The tofacitinib disclosed by the invention has a remarkable treatment potential on the dry AMD, and a new drug choice and strategy are provided for clinical treatment of the dry AMD.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical applications, and particularly to the application of tofacitinib in the preparation of a medicament for treating dry age-related macular degeneration. Background Art

[0002] Age-related macular degeneration (AMD) is the leading cause of vision loss in developed countries, and its pathogenesis is an active research topic. Clinically, AMD mainly includes two types: atrophic (dry) AMD and exudative (wet) AMD. Dry AMD develops slowly, mainly manifested by drusen, atrophy of the retinal pigment epithelium (RPE) and photoreceptor cells, accompanied by macular dysfunction and degenerative changes, which will also seriously affect the quality of life of patients. The incidence of dry AMD is high and usually progresses slowly, but it may develop into a more severe type, that is, wet AMD, resulting in more severe central vision loss. The AREDS study in the United States shows that some patients with advanced dry AMD may also develop choroidal neovascularization, thus transforming from dry AMD to wet AMD. Therefore, early prevention or timely diagnosis and treatment of AMD can reduce or delay vision loss.

[0003] However, the current treatment methods for dry AMD are very limited. For example, Patent CN107406479A discloses the use of a selective adenosine A1 agonist compound for treating, alleviating or preventing age-related macular degeneration. However, more of them are mainly intervention methods of supplementing antioxidant vitamins and minerals. For example, Patent CN101262855A discloses a method for treating and / or preventing age-related macular degeneration, including providing an effective amount of carotenoids and / or vitamin C, vitamin E, β-carotene, zinc and / or copper, and / or a mixture thereof to a subject. The interpretation of the "Clinical Diagnosis and Treatment Path of Senile Macular Degeneration in China" discloses the treatment of senile macular degeneration with antioxidant vitamins and minerals, as well as laser photocoagulation and anti-VEGF. However, there is currently no obvious evidence to support these drugs in the treatment of AMD before the middle stage. It can be seen that the current treatment options for dry AMD are severely limited and most of them have limited effects. The complexity of the pathogenesis of AMD makes the development of treatment methods complex, which makes it crucial to determine effective treatment targets.

[0004] Tofacitinib, chemically named [(3R,4R)-1-cyanoacetyl-4-methylpiperidin-3-yl]-N-methyl-7H-pyrrolo[2,3d]pyrimidin-4-amine, can block the signal transduction of a variety of inflammatory cytokines. There is no report in the prior art on its application in the treatment of AMD. In a large number of drug screening works carried out by the inventor in the early stage, it was unexpectedly found that tofacitinib showed a significant therapeutic effect on the dry AMD model. Summary of the Invention

[0005] The object of the present invention is to provide the use of tofacitinib in the preparation of a drug for treating dry age-related macular degeneration, so as to solve the problems existing in the above-mentioned prior art. The present invention discovers that tofacitinib has a good therapeutic effect on dry AMD, providing a new idea for the clinical treatment of dry AMD.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides the use of tofacitinib in the preparation of a drug for treating dry age-related macular degeneration.

[0008] Optionally, the tofacitinib is used to protect the retina-choroid structure and / or maintain the normal thickness of the retina-choroid.

[0009] Optionally, the tofacitinib is used to protect the retinal pigment epithelium structure.

[0010] Optionally, the tofacitinib is used to protect the photoreceptor cell structure.

[0011] Optionally, the tofacitinib is used to protect the choroid structure.

[0012] Optionally, the tofacitinib is used to inhibit the expression of inflammatory factors;

[0013] The dry age-related macular degeneration includes dry age-related macular degeneration caused by sodium iodate.

[0014] The present invention also provides a drug for treating dry age-related macular degeneration, and the active ingredient of the drug is tofacitinib;

[0015] The drug further comprises a pharmaceutically acceptable excipient;

[0016] The excipient includes at least one of a diluent, a filler, a shaping agent, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, a lubricant, and a flavoring agent.

[0017] Optionally, the dry age-related macular degeneration includes dry age-related macular degeneration caused by sodium iodate.

[0018] Optionally, the dosage form of the drug includes a liquid dosage form.

[0019] Optionally, the administration mode of the drug includes administration by an ocular administration route.

[0020] The present invention discloses the following technical effects:

[0021] Preclinical animal experiments have confirmed that tofacitinib can significantly improve the pathological manifestations and visual function indicators in a dry age-related macular degeneration model. Specifically, tofacitinib treatment can:

[0022] (1) Protect the overall structure of the retina and choroid, slow down or prevent its destruction under pathological conditions, and help maintain the normal thickness of the retina (especially the outer nuclear layer) and choroid.

[0023] (2) Protect the structural integrity of retinal pigment epithelial (RPE) cells and reduce their damage and atrophy.

[0024] (3) Protect the structure of photoreceptor cells (including rod cells and cone cells) and reduce their loss.

[0025] (4) Protect the structure of the choroid and reduce its damage and disorder in the pathological model.

[0026] (5) Inhibit the expression levels of various inflammatory factors in retinal and choroidal tissues.

[0027] In summary, the present invention first reveals the potential of tofacitinib in the treatment of dry AMD, providing experimental evidence and new treatment ideas for the development of new drugs for the treatment of dry AMD. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0029] Figure 1 are the electroretinogram and optical coherence tomography results for each group; A: electroretinogram detection results; B: quantitative results of electroretinogram detection; C: optical coherence tomography results, the area where the retinal structure is damaged in the control group is indicated by the white arrow, and the scale bar is 100 μm; D: the left figure is the statistical result of the relative thickness of the outer nuclear layer, and the right figure is the statistical result of the relative thickness of the retina-choroid;

[0030] Figure 2 are the fundus examination results for each group; among them, the area of fundus lesions is indicated by the white arrow;

[0031] Figure 3 are the detection results of retinal pigment epithelial structure damage; A: retinal pigment epithelial structures in each group; B: RPE65 immunofluorescence staining of retinal frozen sections;

[0032] Figure 4For detecting the structural damage of photoreceptor cells (A - C) and the choroid structure (D);

[0033] Figure 5 It is the relative expression levels of genes CCL2, CSF1, TNFα, CXCL9, and CXCL10 in the retina and genes IL6, CCL2, CXCL9, and CXCL10 in the choroid in Example 2. Detailed implementation manners

[0034] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation to the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0036] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0037] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are also obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0038] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open - ended terms, meaning including but not limited to.

[0039] For dry AMD, the present invention selects wild - type C57 mice to establish a dry AMD disease model and uses the small - molecule drug Tofacitinib to treat the model mice. For specific content, refer to the following examples.

[0040] Example 1

[0041] 1. Establishment and treatment of animal models

[0042] Male C57 wild-type mice aged 2 - 3 months were selected, and sodium iodate was injected to establish a dry AMD model mouse. In the process of establishing this model, sodium iodate was intraperitoneally injected, and the injection dose for each mouse was 15 mg / kg.

[0043] On the 7th day after model establishment, the mice were anesthetized with a mixture of ketamine (dosage: 50 mg / kg) and xylazine (dosage: 20 mg / kg) by intraperitoneal injection. Compound tropicamide eye drops were used for mydriasis, and drug injection was performed by intravitreal injection (a hole was punched at the inferior equatorial edge of the mouse eyeball with a needle, and a micro syringe was used for drug injection. After injection, the needle was left in the eye for 10 seconds and then removed). The concentration of tofacitinib was: 0.4 mg / mL, and the dosage was 1.5 μL / eye. At the same time, normal saline with the same dose was used as a control.

[0044] On the 14th day after model establishment, the mice were anesthetized with a mixture of ketamine (dosage: 50 mg / kg) and xylazine (dosage: 20 mg / kg) by intraperitoneal injection. Compound tropicamide eye drops were used for mydriasis, and drug injection was performed by intravitreal injection (a hole was punched at the inferior equatorial edge of the mouse eyeball with a needle, and a micro syringe was used for drug injection. After injection, the needle was left in the eye for 10 seconds and then removed.). The concentration of tofacitinib was: 0.4 mg / mL, and the dosage was 1.5 μL / eye. At the same time, normal saline with the same dose was used as a control.

[0045] Starting from the 21st day after model establishment, electroretinography (ERG), optical coherence tomography (OCT) and fundus examination were performed on the treated model mice.

[0046] 2. Detection

[0047] Starting from the 21st day after model establishment, histopathological detection was performed on the treated model mice.

[0048] 2.1 Retinal pigment epithelial cell (RPE) spreading: Take the eyeballs of the model mice after treatment, rinse them with PBS, remove the conjunctival tissue outside the eyeballs under a dissecting microscope, cut the eyeballs along the corneal equator, carefully clamp and remove the lens, cut the optic cup into an average petal shape, carefully peel off the retinal layer with forceps, fix the RPE-choroid in 4% PFA for 2 h, and rinse it 3 times with PBS. Add the blocking solution (5% serum, 0.25% TritonX-100, 0.06% sodium azide), and incubate at room temperature for 2 h. Add ActinRed (rhodamine phalloidin, KeyGEN BioTECH, China, #KGMP0012, 1:100), and incubate in the dark at room temperature for 2 h. Take it out and wash it 3 times with PBS, 5 min each time. Observe and take pictures under the microscope.

[0049] 2.2 Immunofluorescence staining: Take the eyeballs of the model mice after treatment, rinse them with PBS, fix the eyeballs in 4% PFA for 5 min, then transfer the eyeballs to a dissecting microscope, make a small incision on the cornea with an ophthalmic scissors, transfer the eyeballs after the incision to 4% PFA fixative, and continue to fix on ice for 2 h, wash 3 times with PBS. Place the fixed eyeballs in 30% sucrose solution for dehydration overnight until the eyeballs sink to the bottom. Cut the eyeballs along the corneal equator under a dissecting microscope, remove the lens, place the eyeballs in an embedding cassette containing embedding medium, adjust the position of the eyeballs and then place them at -80 °C for freezing for 30 min. Section the embedded eyeballs on a cryostat, select a thickness of 12 μm, and adhere the eyeball sections to glass slides. Place the glass slides with the adhered eyeball sections in a 37 °C constant temperature oven and bake for 30 minutes. After baking, wash 3 times with PBS, 5 min each time. Add the blocking solution (5% serum, 0.25% TritonX-100, 0.06% sodium azide), and incubate at room temperature for 2 h. Add the primary antibodies of RPE65, Rodopsin, Blue opsin, Red / Green opsin, and ELN respectively, and incubate overnight at 4 °C. Remove the primary antibodies and wash 3 times with PBS, 5 min each time. Add diluted goat anti-rabbit IgG (H+L) secondary antibodies, DyLight TM 488 and DAPI, and incubate in the dark at room temperature for 1 h, wash 3 times with PBS, 5 min each time. Mount the slides and observe and take pictures under a laser confocal microscope system.

[0050] 3. Results

[0051] 3.1 ERG detection

[0052] The ERG data results are as Figure 1As shown in A, the a-waves of each group were statistically analyzed. The results showed that after treatment with Tofacitinib, the a-waves of the treatment group (NaIO3 + Tofacitinib) were significantly higher than those of the control group (NaIO3 + NaCl) ( Figure 1 left panel of B), and the b-waves of the treatment group were significantly higher than those of the control group ( Figure 1 right panel of B). This indicates that Tofacitinib treatment improved the retinal function of NaIO3-induced dry AMD model mice, especially the functions of photoreceptor cells (reflected by a-waves) and inner retinal cells (reflected by b-waves).

[0053] 3.2 OCT examination

[0054] After drug treatment, OCT examination was performed. The detection results are shown in Figure 1 C. The results showed that the retinal structure of the control group mice was disordered, the layers were unclear, obvious retinal thinning and structural damage areas were visible. After treatment with Tofacitinib, the retina-choroid structure of the treatment group was more complete than that of the control group. Quantitative analysis showed that the statistical results of the relative thickness of the outer nuclear layer (ONL) showed that the ONL thickness of the treatment group was significantly higher than that of the control group ( Figure 1 left panel of D), and the statistical results of the relative thickness of the retina-choroid (Retina + Choroid) showed that the Retina + Choroid thickness of the treatment group was significantly higher than that of the control group ( Figure 1 right panel of D). This indicates that Tofacitinib treatment helps to maintain the normal thickness of the retina (especially the ONL layer where photoreceptor cells are located) and choroid, and protects the overall structure of the retina-choroid.

[0055] 3.3 Fundus examination

[0056] After drug treatment, fundus examination was performed. The detection results are shown in Figure 2 . The results showed that extensive pigment disorders, depigmentation patches and atrophy areas were visible in the fundus of the control group mice. After treatment with Tofacitinib, the fundus lesion areas of the treatment group were significantly less than those of the control group (indicated by white arrows).

[0057] 3.4 Detection of retinal pigment epithelium (RPE) structural damage

[0058] After drug treatment, RPE structural damage detection was performed. The detection results are shown in Figure 3 A - B. The results showed that the RPE cells in the control group had irregular morphology, disordered arrangement, disrupted cell - cell junctions, and even cell shedding and atrophy. Compared with the control group, the RPE structure of the treatment group was more complete, indicating that Tofacitinib can protect the RPE structure of dry AMD model mice.

[0059] 3.5 Detection of photoreceptor cell structure damage

[0060] After drug treatment, the detection of photoreceptor cell structure damage was carried out, and the detection results are as shown in Figure 4 A - C below. Photoreceptor cells were detected using photoreceptor marker antibodies (Rodopsin, Blue opsin, and Red / green opsin). The results showed that in the control group, significant damage and a decrease in cell number were observed in the outer segments and inner nuclear layers of rod cells (positive for Rodopsin), short - wavelength cone cells (positive for Blue opsin), and middle - and long - wavelength cone cells (positive for Red / green opsin). After treatment with Tofacitinib, compared with the control group, the structures of rod cells, short - wavelength cone cells, and middle - and long - wavelength cone cells in the treatment group were more intact, indicating that Tofacitinib can protect the photoreceptor cell structure in dry AMD model mice.

[0061] 3.6 Detection of choroid structure

[0062] After drug treatment, the detection of choroid structure was carried out, and the detection results are as shown in Figure 4 D below. Elastin ELN was used to indicate the choroid structure. The results showed that in the control group, the choroid structure was disordered, and elastic fibers were broken and reduced. After treatment with Tofacitinib, compared with the control group, the choroid structure in the treatment group was more intact than that in the control group, indicating that Tofacitinib can protect the choroid structure in dry AMD model mice.

[0063] The above results consistently show that in a dry AMD mouse model induced by sodium iodate, intravitreal injection of Tofacitinib can significantly improve retinal function (ERG), protect the structure integrity of the retina (including RPE and photoreceptor cells) and choroid, maintain their normal thickness, and reduce fundus lesions. These results confirm the significant therapeutic effect of Tofacitinib on dry AMD.

[0064] Example 2 Effects on inflammatory factors

[0065] 1. Experimental method

[0066] Male C57 wild - type mice at 2 - 3 months old were selected, and a dry AMD mouse model was established by injecting sodium iodate. In this model establishment process, sodium iodate was injected intraperitoneally, and the injection dose for each mouse was 15 mg / kg.

[0067] On the 7th day of model establishment, mice were anesthetized by intraperitoneal injection of a mixture of ketamine (dosage: 50 mg / kg) and xylazine (dosage: 20 mg / kg). Compound tropicamide eye drops were used for mydriasis. Drug injection was performed by intravitreal injection (a hole was punched at the inferior equatorial edge of the mouse eyeball with a needle, and a microinjector was used for drug injection. After injection, the needle was left in the eye for 10 seconds and then removed). The concentration of tofacitinib was 0.4 mg / mL, and the drug dosage was 1.5 μL / eye. At the same time, an equal amount of normal saline was used as a control.

[0068] 48 hours after tofacitinib injection, mouse eyeballs were taken, and the retina and choroid were dissected and separated, and RNA was extracted. The extraction procedure is as follows.

[0069] RNA extraction:

[0070] 1. The dissected retina and choroid were separately transferred to RNase-free 1.5 mL centrifuge tubes. The retina and choroid were ground on ice using a tissue grinder, 1 mL of Trizol RNA extraction solution was added, and the mixture was vortexed for 20 seconds on a vortex mixer.

[0071] 2. 200 μL of chloroform solution was added to the centrifuge tube. After thorough shaking, the mixture was allowed to stand for 10 min, and then centrifuged at 12000 rpm at 4 °C for 15 min.

[0072] 3. The supernatant after centrifugation was transferred to a new RNase-free 1.5 mL centrifuge tube, an equal volume of isopropanol was added, and the mixture was inverted and mixed well. After making a mark, it was allowed to stand at -20 °C for 2 h.

[0073] 4. Centrifuge at 12000 rpm at 4 °C for 5 min, and discard the supernatant.

[0074] 5. 1 mL of 75% ethanol pre-cooled on ice was added to the centrifuge tube. The mixture was inverted and mixed well, then centrifuged at 12000 rpm at 4 °C for 5 min, and the supernatant was discarded.

[0075] 6. 1 mL of pre-cooled 75% ethanol was added to the centrifuge tube. After inverting and mixing well, it was centrifuged at 12000 rpm at 4 °C for 5 min. The supernatant was aspirated dry with a pipette gun, and the tube was opened and dried until the white precipitate turned transparent.

[0076] cDNA synthesis:

[0077] 1. The dried RNA was dissolved with an appropriate amount of DEPC water (pre-cooled on ice in advance), and the RNA concentration was measured.

[0078] 2. 1 μg of RNA was taken for reverse transcription to synthesize cDNA. Novizan reverse transcription kit was used, and cDNA synthesis was carried out according to the instructions.

[0079] RT-qPCR

[0080] According to the known ORF sequence of the target gene, specific primers for the target gene were designed using Primer Premier 5.0 software, and the length of the amplified product was 100 - 250 bp.

[0081] The qPCR reaction system is shown in Table 1, and the reaction program is shown in Table 2.

[0082] Table 1 Reaction System

[0083]

[0084] Table 2 Reaction Program

[0085] Temperature Time Pre-denaturation at 95°C 30s Denaturation at 95°C 3s Annealing / Extension at 60°C 30s 95℃ 15s 60℃ 60s 95℃ 30s 60℃ 15s

[0086] After the reaction, melting curve analysis was performed, and the critical cycle number Ct value was automatically obtained by the software. The 2 -ΔΔCt algorithm was used to analyze the obtained data in Excel software, and the data results were plotted as a bar graph in Graphpad Prism 5 software. Each reaction was repeated three times.

[0087] 2. Experimental Results

[0088] The mRNA expression levels of multiple inflammatory factors in the retina and choroid of dry AMD model mice 48 hours after tofacitinib treatment were detected by RT-qPCR. The results are as Figure 5 shown.

[0089] In the retina tissue, compared with the control group, tofacitinib treatment significantly reduced the mRNA expression levels of chemokine CCL2, colony-stimulating factor CSF1, tumor necrosis factor TNFα, and chemokines CXCL9 and CXCL10.

[0090] In the choroid tissue, compared with the control group, tofacitinib treatment also significantly reduced the mRNA expression levels of interleukin IL6, chemokines CCL2, CXCL9, and CXCL10.

[0091] These results indicate that tofacitinib treatment can effectively inhibit the up-regulation of the expression of key inflammatory factors in the eyes (including the retina and choroid) of sodium iodate-induced dry AMD model mice. This suggests that the therapeutic effect of tofacitinib is at least partially achieved by inhibiting the intraocular inflammatory response.

[0092] Combining the results of Example 1 and Example 2, the present invention confirms that tofacitinib has significant therapeutic effects in a preclinical dry AMD animal model. Its mechanism of action may involve multiple aspects, including directly or indirectly protecting the structure and function of the retina (RPE, photoreceptor cells) and choroid, maintaining tissue thickness, and inhibiting intraocular inflammatory responses. These findings provide strong experimental support for tofacitinib as a potential new drug for the treatment of dry age-related macular degeneration and open up new avenues for the clinical treatment of this disease.

[0093] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of tofacitinib in the preparation of drugs for treating dry age-related macular degeneration.

2. The use according to claim 1, characterized in that The tofacitinib is used to protect the retinal-choroid structure and / or maintain the normal thickness of the retina-choroid.

3. The use according to claim 1, characterized in that The tofacitinib is used to protect the retinal pigment epithelium structure.

4. The use according to claim 1, characterized in that The tofacitinib is used to protect the photoreceptor cell structure.

5. The use according to claim 1, characterized in that The tofacitinib is used to protect the choroidal structure.

6. The use according to claim 1, characterized in that The tofacitinib is used to inhibit the expression of inflammatory factors in ocular tissues; The dry age-related macular degeneration includes dry age-related macular degeneration caused by sodium iodate.

7. A drug for treating dry age-related macular degeneration, characterized in that: The active ingredient of the drug is tofacitinib; The drug further comprises a pharmaceutically acceptable excipient; The auxiliary materials include at least one of diluents, fillers, excipients, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and flavoring agents.

8. The drug according to claim 7, characterized in that The dry age-related macular degeneration includes dry age-related macular degeneration caused by sodium iodate.

9. The drug according to claim 7, characterized in that The dosage form of the drug includes a liquid dosage form.

10. The drug according to claim 7, characterized in that The administration method of the drug includes administration via the ocular route.

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