CN114099515A - Application of methotrexate in preparation of medicine for treating retinitis pigmentosa - Google Patents
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Images
Classifications
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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
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to application of methotrexate in preparation of a medicine for treating retinitis pigmentosa. Methotrexate can inhibit activation of NFkB signal channel and expression of interleukin 1 beta (IL1 beta), interleukin 6(IL6) and chemokine ligand 2(CCL2) at the downstream of the NFkB signal channel in retinal cells, reduce proinflammatory immune cell aggregation and photoreceptor cell death in retina, thereby playing roles in resisting inflammation and improving activity of photoreceptor cells and realizing immune regulation and treatment effects on retinal degeneration diseases.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of methotrexate in preparation of a medicine for treating retinitis pigmentosa.
Background
Retinitis Pigmentosa (Retinitis Pigmentinosa) is an inherited retinal disease caused by mutations in more than 150 genes. Retinitis pigmentosa is the leading cause of blindness, with a prevalence of about one in three thousandths, with over 150 million patients worldwide.
Retinal pigment degeneration is usually chronic and progressive. The initial stage of the disease manifests as nyctalopia, then progresses to a diminished visual field, eventually leading to blindness. In the early stage of pathological changes, the rod cells distributed around the rod cells responsible for vision under dim light gradually denature, and the vision of a patient under dim light gradually weakens to show night blindness; as the lesion progresses to the metaphase, the peripheral visual field is gradually lost due to the massive deletion of the rod cells, and the visual field is reduced; when the pathological changes reach the later stage, cone cells which are enriched in the fovea and are responsible for color vision perception are further apoptotic, the central visual field is gradually lost, and finally, the rod cells and the cone cells disappear to cause blindness.
Retinitis pigmentosa is listed in 120 rare disease lists in China and is a hereditary disease, and no effective treatment means exists at present because retinitis pigmentosa involves too many mutant genes and the death mechanism of photoreceptors in the disease process is not clear.
Disclosure of Invention
The invention aims to provide the application of methotrexate in preparing a medicament for treating retinitis pigmentosa, and realize the immunoregulation and treatment effects on retinal degeneration diseases.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides application of methotrexate in preparing a medicine for treating retinitis pigmentosa.
The invention also provides application of methotrexate in preparing a medicine for inhibiting intraocular proinflammatory factors and/or chemokines.
Preferably, the proinflammatory factors comprise interleukin 1 β and/or interleukin 6; the chemokine includes chemokine ligand 2.
The invention also provides application of methotrexate in preparing a medicament for inhibiting the accumulation of intraocular proinflammatory immune cells.
Preferably, the proinflammatory immune cells comprise macrophages and microglia.
The invention also provides application of methotrexate in preparation of a medicine for improving the activity of retinal cells.
The invention also provides application of methotrexate in preparation of a medicine for regulating immune signal pathways.
Preferably, the immune signaling pathway comprises the NFkB signaling pathway.
Preferably, the dosage concentration of the medicine is preferably 5-40 mg/70 kg/week/person based on the mass of methotrexate in the medicine.
The invention also provides a medicament for treating retinitis pigmentosa, and the effective component of the medicament comprises methotrexate.
The invention provides application of methotrexate in preparing a medicine for treating retinitis pigmentosa. Methotrexate can inhibit activation of NFkB signal channel and expression of interleukin 1 beta (IL1 beta), interleukin 6(IL6) and chemokine ligand 2(CCL2) at the downstream of the NFkB signal channel in retinal cells, reduce proinflammatory immune cell aggregation and photoreceptor cell death in retina, thereby playing roles in resisting inflammation and improving activity of photoreceptor cells and realizing immune regulation and treatment effects on retinal degeneration diseases.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments will be briefly described below.
FIG. 1 is the result of fundus image of mouse;
FIG. 2 is a mouse retinal pigment epithelium slide FITC (green) labeled actin (F-actin) at a 20 micron scale;
FIG. 3 shows the nuclear DNA staining of mouse retinal photoreceptor cells and all cells, wherein the DAPI-labeled blue fluorescence signal is the nuclear DNA staining of mouse retinal photoreceptor cells and all cells, the TUNEL-labeled red fluorescence is the DNA staining of dead cells, and the scale bar is 100 μm;
FIG. 4 is a graph of immunohistochemistry results for macrophages/microglia in mouse retina, scaled at 100 microns;
FIG. 5 is an immunofluorescence image of mouse retinal epithelial slides, scaled to 100 microns;
FIG. 6 shows the results of measuring the expression levels of different proteins in the retinas of the control group and the sodium iodate-treated group of mice in example 2;
FIG. 7 shows the expression levels of different proteins in retinas of control and sodium iodate-treated mice in example 2;
FIG. 8 is a graph showing the results of measuring the expression levels of various proteins in the retinas of mice treated with sodium iodate and with sodium iodate plus methotrexate in example 2;
FIG. 9 shows the expression levels of different proteins in the retinas of mice treated with sodium iodate and with sodium iodate plus methotrexate in example 2;
FIG. 10 shows the results of the mouse retinal RNA extraction and real-time fluorescent quantitative PCR assay;
FIG. 11 is an immunofluorescence image of a human primary retinal pigment epithelium slab at a scale bar of 10 microns.
Detailed Description
The invention provides application of methotrexate in preparing a medicine for treating retinitis pigmentosa.
In the invention, the butterfly carbaminate can reduce the retinal aggregation of proinflammatory immune cells, reduce the expression of proinflammatory factors and chemotactic factors, and inhibit the expression of proinflammatory immune signal paths and downstream inflammatory factors thereof, thereby playing roles in immunoregulation and anti-inflammation, improving the activity of retinal cells and further playing a role in treating retinal pigment degeneration. The proinflammatory factors of the invention preferably include interleukin 1 beta and/or interleukin 6; the chemokine preferably comprises chemokine ligand 2; the proinflammatory immune cells comprise macrophages and microglia; the immune signaling pathway comprises the NFkB signaling pathway.
In the present invention, the structural formula of methotrexate is as follows:
in the invention, the dosage concentration of the medicine is preferably 5-40 mg/70 kg/week/person, more preferably 5.7-35 mg/70 kg/week/person, and even more preferably 10-20 mg/70 kg/week/person, based on the mass of methotrexate in the medicine. The source of the methotrexate is not critical in the present invention, and can be obtained commercially or prepared by itself. In the specific implementation process of the invention, the methotrexate is preferably purchased from selelck, cat number: # S1210.
The invention also provides application of methotrexate in preparing a medicine for inhibiting intraocular proinflammatory factors and/or chemokines. The proinflammatory factors of the invention comprise interleukin 1 beta and/or interleukin 6; the chemokine includes chemokine ligand 2.
The invention also provides application of methotrexate in preparing a medicament for inhibiting the accumulation of intraocular proinflammatory immune cells. The proinflammatory immune cells of the invention include macrophages and microglia.
The invention also provides application of methotrexate in preparation of a medicine for improving the activity of retinal cells.
The invention also provides application of methotrexate in preparation of a medicine for regulating immune signal pathways. The immune signaling pathway of the present invention comprises the NFkB signaling pathway.
The concentration of methotrexate and the source of methotrexate used in the preparation of drugs for inhibiting intraocular proinflammatory factors and/or chemokines, drugs for promoting intraocular proinflammatory immune cell aggregation, drugs for increasing retinal cell activity or drugs for regulating immune signal pathways are the same as above, and are not described herein again.
Animal experiment results show that in a mouse disease model of retinitis pigmentosa induced by sodium iodate injection, low-dose methotrexate (1 mg/kg in mice, which is equivalent to 5.7mg/70kg in human) can inhibit proinflammatory nuclear factor-kB signal activation and expression of downstream inflammatory factors, thereby playing roles in immune regulation and anti-inflammation. The experimental result of human retinal pigment epithelial cells shows that in a human disease model of retinal pigment degeneration induced by glucose oxidase, methotrexate can inhibit activation of NFkB signal pathway in retinal cells and expression of interleukin 1 beta (IL1 beta), interleukin 6(IL6) and chemokine ligand 2(CCL2) at the downstream of the NFkB signal pathway, reduce proinflammatory immune cell aggregation and photoreceptor cell death in the retina, thereby playing the roles of resisting inflammation and improving photoreceptor cell activity and realizing immune regulation and treatment effects on retinal degeneration diseases.
The invention also provides a medicament for treating retinitis pigmentosa, and the effective component of the medicament comprises methotrexate. The methotrexate can be used alone or matched with other medicines for treating retinitis pigmentosa, so that the effect of treating retinitis pigmentosa is achieved. The medicament for treating retinitis pigmentosa provided by the invention preferably further comprises auxiliary materials. The invention does not specially limit the types and the dosage of the auxiliary materials, and the auxiliary materials are added according to the requirements of different formulations. In the present invention, the dosage form of the drug preferably includes an injectable drug or an orally administered drug, and more preferably includes an injectable drug. In the drug for treating retinitis pigmentosa, the concentration of methotrexate in the drug is preferably the same as that described above, based on the mass of methotrexate in the drug, and thus the details are not repeated herein.
In order to further illustrate the present invention, the following detailed description of the technical solutions provided by the present invention is made with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Experimental Material
1.1 reagents and materials
Sodium iodate (Sodium iodide, SI) (Sigma-Aldrich, S4007), 1ML syringe (KDL), 30 total 5-8 weeks of C57BL/6 mice (Central laboratory animal university of Zhongshan), sterile Phosphate Buffer (PBS), methotrexate (select, S1210), DMSO (MP Biomedicals, 196055), Polyoxy ethylene300 (select, S6704), Tween80(BioFrox, 1716ML100), Sodium fluorescein (Alcon laboratories, TX, USA), BCA protein concentration determination Reagent (Biyun, P0012), reverse transcription kit (Novonopraz, R223-01), Superl Premix Plus (SYBR Green) (Tiangen, InvFP 205), trinogen Reagent (Thermo Fis, 15596026), and protein antibodies and real-time fluorescent quantitative PCR primers are shown in tables 1-2.
TABLE 1 protein antibodies used in the assay procedure
TABLE 2 PCR primers used in the course of the experiments
1.2 instruments
The electrophoresis apparatus (Bio-Rad,
tetra), gel imaging system (Tanon, Tanon5200), bulk microscope (ZEISS, SteREO Discovery), PCR instrument (ABI, VERITI PCR) fluorescent quantitative PCR instrument (Roche, LC480), Micron IV retinal imaging system (Phoenix Research Laboratories, Pleasanton, CA, USA), New Ganoderma ultrasound instrument (SCIENTZ-IID), centrifuge (Eppendorf, 5424R).
Example 2
Animal experiments
1 animal experiment group
A total of 30C 57BL/6 mice (Experimental animal center, university of Zhongshan) at 5-8 weeks were divided into 10 groups each of a control group (PBS), a sodium iodate-treated group (SI), and a sodium iodate-plus-methotrexate-administered group (SI + MTX). Specific grouping and administration modes are shown in table 3.
TABLE 3 animal Experimental groups and modes of administration
TABLE 4 methotrexate liquid compositions
| Composition (I) | Methotrexate (MTX) | DMSO | Polyoxyethylene300 | Tween80 | Sterilized water |
| Dosage of | 0.2g | 20mL | 300mL | 50mL | 630mL |
2. Test procedure
After 3 days of treatment according to the dosing regime of table 3, the following steps were carried out:
2.1 photographing fundus of mouse
Mouse fundus photography was performed before the sodium iodate injection and three days after the injection in step 2.2, respectively, and the specific operations were as follows: before the fundus photography of the mice, the mice were anesthetized with 4% chloral hydrate (100. mu.l/10 g), 1-2 drops of compound tropicamide eye drops dilate the pupils, and hypromellose gel lubricates the cornea. Thereafter, a photograph of the fundus was taken using the Micron IV retinal imaging system (mice could continue to be raised to the experimental end after taking the photograph.
2.2 dissecting and taking the mouse retina
Three days after injection according to the administration method in table 3, all mice were sacrificed by cervical dislocation, the eyelids of the mice were opened with one hand to protrude the eyeball, and the other hand-held forceps were inserted into the orbit, and the eyeball was taken out together with the optic nerve by applying force from below the eyeball to above. The cells were placed in a 10cm cell culture dish containing PBS and dissected under a stereomicroscope in the following general procedure: the extraocular muscles and connective tissue attached to the eyeball floating in PBS were removed with ophthalmic scissors. A small incision is made at the scleral edge, left hand forceps are inserted through the incision to clamp the cornea, and right hand ophthalmic scissors are used to cut the eyeball along the scleral edge and divide the eyeball into two parts, namely, the anterior pole of the eyeball (cornea, iris, crystalline lens) and the posterior cup (sclera, choroid, retina). And (3) peeling the retina from the rearview cup by using tweezers, putting the retina into a centrifuge tube with the information of the sample name, the material taking date and the like marked in advance, putting the centrifuge tube into liquid nitrogen for quick freezing, and performing subsequent operation after the same batch of materials are taken out.
2.3 fluorescence microscopy of mouse retina
The primary mouse retinal epithelial cells obtained in step 2.2 were cultured in a DMEM high-sugar medium, 10% fetal bovine serum, and 1% penicillin-streptomycin solution. The culture conditions were 37 ℃ and 5% CO2. Cells were passaged in six-well plates containing coverslips and, after confluency, were evenly divided into control, glucose oxidase-treated, and glucose oxidase plus methotrexate-administered groups, each set in 3 replicates.
After the treatment, the cells were washed three times with room temperature PBS, and then fixed with 1.5ml of 4% paraformaldehyde per well for 20 minutes at room temperature. After fixation was completed, the cells were washed three times with PBS for 5 minutes each. The cell-bearing slides were then blocked with 2.5% BSA at room temperature for 20 min. After blocking was complete, hybridization was performed using 2.5% BSA containing anti-IL 1 β (1:100 vol/vol configuration). Hybridization was performed in a four-degree wet box overnight. After the primary anti-hybridization was completed, the cells were washed three times with PBS for 5 minutes each. Slides were then stained with secondary antibodies TUNEL and IBA1 containing red fluorescent labels, as well as green labeled FITC and blue fluorescent DAPI. Room temperature for 1 hour. After staining was completed, the cells were washed three times with PBS for 5 minutes each. And finally, sealing by using an anti-quenching sealing sheet, and observing and photographing under a fluorescence microscope.
2.4 mouse retinal protein extraction and Western immunoblotting experiments
Adding 200 μ l RIPA lysate containing protease inhibitor into centrifuge tube containing mouse retina sample, placing histon into Xinzhi ultrasonic instrument, connecting with ice water circulation, setting ultrasonic energy at 60%, total time 1min, 2 s on, 2 s off. After fully cracking the tissue cells, centrifuging the tissue cells for 10min at 12000rpm by a 4 ℃ centrifuge, separating protein supernatant from other substance precipitates, taking the supernatant part, and adding the supernatant part into a centrifuge tube with marked information. Protein supernatant was subjected to concentration measurement using the BCA protein concentration measurement kit, and a 50. mu.g protein sample was taken for a Western blot experiment.
2.5 mouse retina RNA extraction and real-time fluorescent quantitative PCR experiment
1ml of Trizol reagent was added to the centrifuge tube containing the mouse retina sample and ground with a RNAse-free grind bar to allow sufficient lysis of the tissue cells and RNA extraction according to Trizol reagent instructions.
Mu.g of RNA was taken and cDNA was synthesized using a reverse transcription kit. The SuperReal Premix Plus (SYBR Green) and a fluorescent quantitative PCR instrument are used for carrying out real-time fluorescent quantitative PCR experiments, and the program is set as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 10sec, annealing at 58 ℃ for 20sec, extension at 72 ℃ for 20sec, and single fluorescence signal acquisition during the extension phase. ACTB (beta actin) as an internal reference gene, 2-ΔΔCtThe method calculates the ratio of the target gene mRNA expression quantity to the reference gene mRNA expression quantity, and carries out relative quantification.
3. Test results
(1) The photographing result of the mouse fundus is shown in figure 1, and the staining result of the mouse retinal pigment epithelial cell slide is shown in figures 2-5. Wherein, the detection results corresponding to the control group (PBS), the sodium iodate treatment group (SI) and the sodium iodate and methotrexate administration treatment group (SI + MTX) are shown in the figures 1 to 5 from left to right,
from fig. 1-5, it can be seen that three days after the sodium iodate group (SI) was injected, the retinas of mice in the sodium iodate-treated group were significantly damaged compared with those in the control group (PBS), and the model construction was successful. Specifically, in fig. 2 is FITC-labeled actin, and in fig. 3 is nuclear DNA of TUNEL-labeled dead retinal photoreceptor cells and DAPI-labeled all cells. As can be seen in figures 2 and 3, methotrexate treatment inhibited retinal deformity and photoreceptor cell death.
Black arrows in fig. 4 indicate pro-inflammatory macrophages/microglia in the aggregated retinal photoreceptor layer and subretinal space, fig. 5 FITC-labeled actin, nuclear DNA of DAPI-labeled all cells, and IBA 1-labeled macrophages and microglia. As can be seen in fig. 4 and 5, methotrexate inhibits the aggregation of pro-inflammatory immune cells of the retina in a mouse disease model of retinal degeneration.
(2) The results of western blotting experiments are shown in fig. 6-9, and the results of real-time fluorescence quantitative PCR experiments are shown in table 5 and fig. 10 below, wherein P values in fig. 7, 9 and 10 are calculated from independent sample t tests: p <0.05, x: p <0.01, x: p <0.0001, error bars are standard deviations, test sample is 5.
TABLE 5 real-time fluorescent quantitative PCR experiment results
From fig. 7-10 and table 5, it can be seen that the sodium iodate plus methotrexate treatment group (SI + MTX) reduced sodium iodate-induced upregulation of RNA expression of the proinflammatory autoimmune genes cGAS, STING and proinflammatory factors interleukin 1 β, interleukin 6 and chemokine ligand 2 in the retinal pigment epithelium, as well as elevated levels of cGAS, STING proteins, activation of proinflammatory NFkB signals (elevated phosphorylation levels), cleavage/activation of proinflammatory interleukin protein IL1 β. Methotrexate can inhibit the expression of pro-inflammatory genes in mouse models of retinal degeneration.
Example 3
Human retinal pigment epithelial cell assay
1. The primary human retinal pigment epithelial cells were cultured in DMEM high-glucose medium, 10% fetal bovine serum, and 1% penicillin-streptomycin solution. The culture conditions were 37 ℃ and 5% CO2. Cells were passaged in six-well plates containing coverslips and, after confluency, were evenly divided into control, glucose oxidase-treated, and glucose oxidase plus methotrexate-administered groups, each set in 3 replicates.
2. The Glucose oxidase-treated group and the Glucose oxidase plus methotrexate-treated group induced cell damage denaturation by treatment with 20mU/ml Glucose oxidase (Glucose oxidase) in complete medium for 3 hours, the control-treated group was treated with PBS for 3 hours, and after Glucose oxidase for 3 hours, the Glucose oxidase plus methotrexate-treated group was treated with 1 μ M methotrexate (configured in DMSO) for 24 hours; adding DMSO with the same volume as that of the glucose oxidase and methotrexate administration treatment group into the glucose oxidase treatment group, and treating for 24 h; the control group was treated for 24 hours with the addition of sterile Phosphate Buffered Saline (PBS) in an equal volume to the glucose oxidase plus methotrexate-administered group. The above treatment process is carried out in a cell culture incubator.
After the glucose oxidase-treated group and the glucose oxidase plus methotrexate-treated group were completed, the cells were washed three times with room-temperature PBS, and then fixed with 1.5ml of 4% paraformaldehyde for 20 minutes at room temperature per well. After fixation was completed, the cells were washed three times with PBS for 5 minutes each. The cell-bearing slides were then blocked with 2.5% BSA for 20 min at room temperature. After blocking was complete, hybridization was performed using 2.5% BSA containing anti-IL 1 β (1:100 vol/vol configuration). Hybridization was performed in a four-degree wet box overnight. After the primary anti-hybridization was completed, the cells were washed three times with PBS for 5 minutes each. Slides were then stained with secondary antibodies TUNEL and IBA1 containing red fluorescent labels, as well as green labeled FITC and blue fluorescent DAPI. Room temperature for 1 hour. After staining was completed, the cells were washed three times with PBS for 5 minutes each. Finally, the solution is blocked by an anti-quenching sealing piece and is observed and photographed under a fluorescence microscope, and the result is shown in figure 11.
Fig. 11 is a glucose oxidase-treated group and a glucose oxidase plus methotrexate-treated group in this order from top to bottom, in fig. 11 the inflammatory protein interleukin 1 β (IL1 β) is labeled with red fluorescence, the actin cell morphology is labeled with FITC (green), and the nuclear DNA of all cells is labeled with DAPI. As can be seen from FIG. 11, methotrexate (1. mu.M, 24 hours) significantly reduced the up-regulation of the expression of the proinflammatory factor interleukin 1. beta. in cells treated with the oxidant glucose oxidase (20mU/ml, 3 hours), as well as cell degeneration.
From the above examples, it can be seen that methotrexate of the present invention can inhibit activation of NFkB signaling pathway in retinal cells and expression of interleukin 1 β (IL1 β), interleukin 6(IL6) and chemokine ligand 2(CCL2) downstream thereof, reduce aggregation of proinflammatory immune cells and death of photoreceptor cells in retina, thereby exerting anti-inflammatory and photoreceptor cell activity enhancing effects, and realizing immune regulation and therapeutic effects on retinal degenerative diseases.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
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Claims (10)
1. Use of methotrexate in the manufacture of a medicament for the treatment of retinitis pigmentosa.
2. The application of methotrexate in preparing medicine for inhibiting intraocular proinflammatory factor and/or chemotactic factor.
3. The use according to claim 2, wherein the proinflammatory factors comprise interleukin 1 β and/or interleukin 6; the chemokine includes chemokine ligand 2.
4. The application of methotrexate in preparing medicine for inhibiting proinflammatory immune cell aggregation in eyes.
5. The use according to claim 3, wherein the proinflammatory immune cells comprise macrophages and microglia.
6. Use of methotrexate for the manufacture of a medicament for increasing the activity of retinal cells.
7. The application of methotrexate in preparing medicine for regulating immune signal path.
8. The use of claim 7, wherein said immune signaling pathway comprises the NFkB signaling pathway.
9. The use according to any one of claims 1 to 8, wherein the medicament is preferably used in a dose concentration of 5 to 40mg/70 kg/week/person, based on the mass of methotrexate in the medicament.
10. A medicine for treating retinitis pigmentosa is characterized in that the effective component comprises methotrexate.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1735400A (en) * | 2002-12-04 | 2006-02-15 | 参天制药株式会社 | Drug delivery system using subconjunctival depot |
| CN104334173A (en) * | 2012-05-01 | 2015-02-04 | 特兰斯拉图姆医学公司 | Methods for treating and diagnosing blinding eye diseases |
| CN104884049A (en) * | 2012-11-08 | 2015-09-02 | 克莱尔塞德生物医学股份有限公司 | Methods and devices for the treatment of ocular diseases in human subjects |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1735400A (en) * | 2002-12-04 | 2006-02-15 | 参天制药株式会社 | Drug delivery system using subconjunctival depot |
| CN101336887A (en) * | 2002-12-04 | 2009-01-07 | 参天制药株式会社 | Drug delivery system using subconjunctival depot |
| CN104334173A (en) * | 2012-05-01 | 2015-02-04 | 特兰斯拉图姆医学公司 | Methods for treating and diagnosing blinding eye diseases |
| CN104884049A (en) * | 2012-11-08 | 2015-09-02 | 克莱尔塞德生物医学股份有限公司 | Methods and devices for the treatment of ocular diseases in human subjects |
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