CN113425728A - Use of ZD6474 in the manufacture of a medicament for use in the treatment of pterygium followed by recurrence - Google Patents
Use of ZD6474 in the manufacture of a medicament for use in the treatment of pterygium followed by recurrence Download PDFInfo
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- UHTHHESEBZOYNR-UHFFFAOYSA-N vandetanib Chemical compound COC1=CC(C(/N=CN2)=N/C=3C(=CC(Br)=CC=3)F)=C2C=C1OCC1CCN(C)CC1 UHTHHESEBZOYNR-UHFFFAOYSA-N 0.000 title claims abstract description 70
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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- 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
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Abstract
本发明公开了一种ZD6474在翼状胬肉术后抗复发的治疗药物制备中的应用，旨在解决现有技术中治疗胬肉术后复发的抗代谢类及细胞毒性类药物效果有限，副作用及眼表损伤大的技术问题。本发明将ZD6474在翼状胬肉术后抗复发的治疗药物制备中应用，对于正常人体组织及细胞的损伤较小，安全性更佳，可行性好。ZD6474对于HPFs可以特异性地减少其迁移及纤维活化、对于并HUVEC可以减少其迁移、小管形成及诱导其凋亡增加；在一定程度上弥补现有翼状胬肉术中使用抗代谢药物的明显眼表毒性的不足，可在保证疗效的情况下提高安全性，为翼状胬肉术后抗复发的治疗提供了更为安全有效的新选择。
The invention discloses the application of ZD6474 in the preparation of an anti-recurrence therapeutic drug after pterygium surgery, and aims to solve the problem that the anti-metabolite and cytotoxic drugs in the prior art have limited effects, side effects and adverse effects on the postoperative recurrence of pterygium. Technical problems with large damage to the ocular surface. The invention uses ZD6474 in the preparation of anti-recurrence therapeutic drugs after pterygium surgery, and has less damage to normal human tissues and cells, better safety and good feasibility. ZD6474 can specifically reduce the migration and fiber activation of HPFs, and can reduce the migration, tubule formation and induce increased apoptosis of HUVECs. The lack of surface toxicity can improve the safety while ensuring the efficacy, and provide a safer and more effective new option for the anti-recurrence treatment after pterygium surgery.
Description
Technical Field
The invention relates to the technical field of a medicament for treating recurrence after pterygium surgery, in particular to an application of ZD6474 in preparation of a medicament for treating recurrence after pterygium surgery.
Background
Pterygium is a chronic proliferative disease of conjunctiva, which is typically manifested by abnormal hyperplastic bulbar conjunctiva and fibrous tissue under the bulbar conjunctiva, which cross the limbus and grow into the cornea to form a typical triangular morphology. Pterygium is one of the most common diseases in ophthalmology, and can cause eye symptoms and signs such as eye redness, corneal astigmatism and visual deterioration in addition to aesthetic problems of patients, and influence the daily life and the work quality of the patients. In view of the fact that the occurrence and development mechanism of pterygium involves various factors, including damaged limbal stem cell barrier, ultraviolet irradiation, up-regulation of growth factors, fibroblast hyperproliferation, immune disorders and inflammatory responses related to cytokines and inflammatory factors, viral infection, windy geographic environment factors and genetic factors, etc., pterygium lacks effective drug therapy, and the treatment mode is mainly surgical resection. Pterygium excision in combination with autologous conjunctival transplantation is considered a suitable surgical procedure, but even with autologous conjunctival transplantation, the problem of recurrence of the pterygium after surgery is unavoidable.
Given the more aggressive nature of the recurrent pterygium following surgical treatment, adhesion of the underlying tissue can also significantly increase the difficulty of re-surgery.
In recent years, research on reduction of recurrence after pterygium has focused on the application of novel anti-cell proliferation drugs and improvement of surgical methods (e.g., reduction of suture use, fixation with fibrin glue, etc.), and has made progress. The main clinical uses of antimetabolites and cytotoxic drugs include mitomycin C (MMC) and 5-fluorouracil (5-fluorouracil,5-FU), and the action mechanism of the antimetabolites and cytotoxic drugs is to inhibit cell DNA replication and protein synthesis, thereby achieving the effect of inhibiting cell growth and proliferation. Therefore, antimetabolite and cytotoxic drugs have been tried to be applied during or at an early stage after surgery to reduce postoperative recurrence. The combined treatment of antimetabolites and the like can reduce the recurrence rate of pterygium, but has serious complications threatening vision, including corneal tissue and scleral tissue lysis, microbial infection, hair loss, gastrointestinal tract reaction, bone marrow suppression and other adverse reactions. In addition, the eye surface tissue is damaged to different degrees, so that the clinical application of the eye surface tissue is limited.
Therefore, finding safer and more effective therapeutic drugs or means for preventing recurrence after pterygium has clear clinical value and significance.
Disclosure of Invention
The invention aims to solve the technical problem of providing the application of ZD6474 in the preparation of anti-recurrence medicaments after pterygium surgery, so as to solve the technical problems of limited effect, large side effect and large ocular surface injury of anti-metabolism medicaments and cytotoxic medicaments for treating recurrence after pterygium surgery in the prior art.
In order to solve the technical problems, the invention adopts the following technical scheme:
ZD6474 for use in the manufacture of a medicament for use in the treatment of pterygium followed by recurrence resistance.
Preferably: the concentration of ZD6474 in the medicament is controlled to be between 0.1uM/mL and 5 uM/mL.
Preferably: the therapeutic agent is an agent that inhibits excessive activation of the fiber cells.
Preferably: the therapeutic agent is an agent that inhibits the formation of new blood vessels.
Compared with the prior art, the invention has the main beneficial technical effects that:
the ZD6474 is applied to the treatment of recurrence resistance after pterygium surgery, and has less damage to normal human tissues and cells, better safety and good feasibility. ZD6474 may specifically reduce its migration and fibre activation for HPFs, and reduce its migration, tubule formation and induce its increased apoptosis for HUVECs; compared with the traditional antimetabolite, ZD6474 is more in line with the current precise and individualized treatment concept, overcomes the defect of obvious ocular surface toxicity of the existing antimetabolite in pterygium to a certain extent, can improve the safety under the condition of ensuring the curative effect, and provides a safer and more effective new choice for the treatment of recurrence resistance after pterygium.
Drawings
FIG. 1 is a primary cell of HPFs;
FIG. 2 shows 3 generation cells of HPFs;
FIG. 3 is a primary cell of HTFs;
FIG. 4 shows generation 3 cells of HTFs;
FIG. 5 is an immunohistochemical identification of HPFs cells;
FIG. 6 is an immunohistochemical identification of HTFs cells;
figure 7 is a statistical plot of the drug safety and concentration range of ZD6474 against HPFs;
figure 8 is a statistical plot of the drug safety and concentration range of ZD6474 against HTFs;
FIG. 9 is a cell scratch experiment of HPFs;
FIG. 10 is a cell scratch experiment for HTFs;
FIG. 11 is an electron micrograph of vimentin expression from HPFs;
FIG. 12 is an electron micrograph of the expression of the α -SMA proteins of the HPFs;
figure 13 is a statistical plot of the drug safety and concentration range of ZD6474 against HUVEC;
FIG. 14 is a cell scratch experiment of HUVEC.
Detailed Description
The following examples are intended to illustrate the present invention in detail and should not be construed as limiting the scope of the present invention in any way.
The instruments and devices referred to in the following examples are conventional instruments and devices unless otherwise specified; the related reagents are all conventional reagents in the market, if not specifically indicated; the related experimental cell strains are all conventional commercial cell strains; the test methods involved are conventional methods unless otherwise specified.
ZD6474, Vandetanib, referred to in the examples below, is a multiple kinase inhibitor, a tyrosine kinase inhibitor, a synthetic small molecule anilinoquinazoline compound whose molecular structure is as follows:
example 1: culture and characterization of HPFs and HTFs cells
Primary Human Pterygium Fibroblasts (HPFs) and human Tenon's bursa fibroblasts (HTFs) were cultured by tissue block adherence method and immunohistochemical identification was performed in parallel.
(1) Primary culture of HPFs
After 5-7 days of primary adherent culture of pterygium tissue blocks, a microscope (multiplied by 4) observes that a small amount of fusiform cells climb out around the block, the conventional solution change is carried out, the culture is continued, the primary cells gradually divide and proliferate, and the cells are merged into pieces (see figure 1), and the shapes of the merged cells are a few epithelial cell types and most fibroblast types. After about 15-18 days, the cells around the tissue block are dense, digestion passage is carried out, the culture is continued, after 3 passages of subculture, the cultured cells are observed by a microscope (multiplied by 4 times) to be completely composed of single-layer fusiform fibroblasts and accord with the morphological characteristics of HPFs (see figure 2).
(2) Primary culture of HTFs:
after the primary adherent culture of the Tenons capsule tissue blocks is carried out for 5-7 days, a microscope (multiplied by 4 times) observes that a small amount of fusiform cells climb out from the periphery of the blocks, the conventional liquid change is carried out, the culture is continued, the primary cells gradually divide and proliferate, and are converged into sheets (shown in figure 3), and the cell morphology after the confluence is a few epithelial cell types and most fibroblast types. After about 20-25 days, the cells around the tissue block are dense, digestion passage is carried out, the culture is continued, after 3 passages of subculture, the cultured cells are observed by a microscope (multiplied by 4 times) to be completely composed of single-layer fusiform fibroblasts and accord with the morphological characteristics of HTFs (see figure 4).
(3) Cultured 3-generation HPFs cells were slide-plated and immunofluorescent stained. The results are shown in FIG. 5.
The results show that: when observing HPFs cell by a microscope (multiplied by 10 times), the staining of green fluorescent Vimentin marked by a visible marker is positioned in the whole cytoplasm and under the cell membrane, the cell nucleus can see DAPI marked light blue fluorescence, the Vimentin antibody shows positive immunofluorescence staining, meanwhile, the staining of cytokeratin 12(keraton 12) antibody does not show fluorescent staining, the cell nucleus can see DAPI marked light blue fluorescence, and the K12 antibody shows negative immunofluorescence staining. The vimentin is a mesenchymal cell specific marker, the K12 is an epithelial cell specific marker, and the cultured primary cells are determined to be human pterygium fibroblasts according to the characteristics of HPFs by combining morphology and immunohistochemical staining results.
(4) The cultured 3-generation HTFs were subjected to cell slide and immunofluorescent staining, and the results are shown in fig. 6.
The results show that: and (3) observing the wavy protein staining of the visible marker green fluorescence of the HTFs cells by a microscope (multiplied by 10 times), locating the HTFs cells in the whole cytoplasm and under the cell membrane, wherein the cell nucleus can see the light blue fluorescence of the DAPI marker, the wavy antibody is positive in immunofluorescence staining, meanwhile, the cell keratin 12(keraton 12) antibody staining is not stained with the fluorescence, the cell nucleus can see the light blue fluorescence of the DAPI marker, and the K12 antibody is negative in immunofluorescence staining. And (3) combining the morphological and immunohistochemical staining results, according with the characteristics of HTFs, and determining the cultured primary cells as human tenon's bursa fibroblasts.
Example 2: detection of the cytotoxicity and drug concentration Range of ZD6474 against HPFs and HTFs
The HPFs and HTFs cells obtained in example 1 were cultured, and the HPFs and HTFs cells were divided into 3 groups:
control group: incubation with 0.1% DMSO under normal conditions; group ZD 6474: the culture medium is respectively added with 4 ZD6474(100nM/mL, 500nM/mL, 1uM/mL, 5uM/mL) with different concentrations; MMC group: the medium was supplemented with 2 different concentrations of mitomycin (MMC) (50ug/ml and 200ug/ml), respectively.
The CCK8 test was performed on each of the HPFs and HTFs separately to observe the cytotoxicity and safety of the drugs and to determine the drug concentrations of ZD6474 and MMC.
The specific results are as follows:
(1) culturing HPFs cells to the 3 rd generation, uniformly inoculating the HPFs to a 96-well plate according to the density of 10 x 4 after digestion until the cells grow uniformly to be 80% fused, detecting the drug cytotoxicity by a CCK8 method, and determining the drug safety concentration range.
The CCK8 results for HPFs are shown in FIG. 7:
the absorbance value of the control group was 0.418. + -. 0.047. After ZD6474 at each concentration of 100nM/ml-5uM/ml was exposed to HPFs24 hours, the absorbance values of CCK8 were stable between 0.401 and 0.439 compared with the control group, no decrease in significant absorbance values was observed, and no statistical difference was detected in the absorbance values of each group compared with the control group (P >0.05), indicating that ZD6474 showed no significant cytotoxicity in the range of 100nM/ml-5uM/ml and no significant cytotoxicity in the range of 10-50 ug/ml.
(2) Conventionally culturing HTFs to the 3 rd generation, uniformly inoculating the HTFs into a 96-well plate according to the density of 10 x 4 after digestion until the cells grow to 80% and fuse, detecting the cytotoxicity of the drug by a CCK8 method, and determining the safe concentration range of the drug.
The CCK8 results for HTFs are shown in fig. 8:
the absorbance value of the control group was 0.418. + -. 0.047. After ZD6474 at each concentration of 100nM/ml-1uM/ml was exposed to HTFs24 for hours, the absorbance values for CCK8 were stable between 0.401 and 0.439 compared with the control group, no decrease in significant absorbance values was observed, and no statistical difference was observed in the absorbance values for each group compared with the control group (P >0.05), indicating that ZD6474 showed no significant cytotoxicity in the range of 100nM/ml-1uM/ml, and that MMC showed significant cytotoxicity in each concentration range.
Example 3: testing the Effect of ZD6474 on cell migration in HPFs and HTFs
The cells and experimental groups used in the experiments were the same as in example 2.
(1) And (3) culturing HPFs conventional cells to 4-6 th generation, uniformly inoculating the cells into a scribed 6-hole plate after digestion, carrying out a cell scratching experiment after the cells are completely fused, and comparing the scratching area after 24 hours of drug intervention with the scratching area before the intervention.
The scratch results for HPFs are shown in fig. 9:
the scratch area after 24 hours of the control group was significantly reduced compared to that before the intervention, and the reduction ratio of the scratch area of the control group was 83.46%. ZD6474 has a significantly greater 24-hour reduction ratio of scratch area from 100nM/ml to 5uM/ml than the control, wherein the reduction ratios of scratch area in 1uM and 5uM groups are 30.11% and 18.34%, respectively, and the difference from the control is statistically significant (P <0.05), and the effect of inhibiting migration is drug concentration dependent. The 24-hour scratch area of MMC50ug/ml and MMC200ug/ml is also widened compared with that of the control group, the scratch area reduction rate is 44.34% and 32.75%, respectively, which shows that MMC can inhibit the migration of HPFs, the migration inhibition effect is weaker than that of ZD6474, and the difference between the two groups has statistical significance (P is less than 0.05).
The scratch test shows that ZD6474 can obviously inhibit the migration of HPFs, and the effect is increased along with the increasing concentration of the drug, and the effect of inhibiting the migration is stronger than that of MMC.
(2) Culturing HTFs conventional cells to 4-6 th generation, uniformly inoculating the cells into a streaked 6-well plate after digestion, carrying out a cell scratching experiment after the cells are completely fused, and comparing the scratching area after drug intervention for 24 hours with the scratching area before the intervention.
The scratch results for HTFs are shown in fig. 10:
the scratch area after 24 hours of the control group was slightly smaller than that before the intervention, and the scratch area reduction rate of the control group was 21.82%. ZD6474 has no significant change in the scratch area at 24 hours from 100nM/ml to 5uM/ml compared to the control, and the scratch area reduction ratio of ZD1 to 5uM/ml is slightly greater than that of the control, where the difference in scratch area of the 5uM group compared to the control is statistically significant (P < 0.05). The 24-hour scratch area reduction ratios of MMC50ug/ml and MMC200ug/ml are slightly increased compared with the control group, the scratch areas are 14.53% and 14.58%, respectively, which shows that the MMC can inhibit the migration of HTFs.
The scratch test shows that ZD6474 has a certain migration-inhibiting effect on HTFs.
By using Image J software and comparing scratch areas of HPFs and HTFs, ZD6474 has the effect of inhibiting migration of HPFs and HTFs, but the effect of ZD6474 on inhibiting migration of HPFs is obviously stronger than that on inhibiting migration of HTFs, which shows that ZD6474 can act on HPFs with stronger proliferative activity more selectively to inhibit migration and proliferation of HPFs, and has weaker effect on HTFs of normal cells to show better selection specificity.
Example 4: assays for the expression of ZD6474 on HPFs vimentin and alpha-SMA the cells and experimental groups used were as in example 2.
(1) After cell-plating on HPFs, expression of vimentin was detected by immunofluorescence staining after hours of intervention of HPFs24 with ZD 6474.
The results are shown in FIG. 11:
the control group shows that HPFs cells are full in shape, arranged in a feather shape, narrow in cell gap, obvious in intracellular wavy protein staining and rich in protein expression quantity. The ZD6474 with the concentration of 500nM/ml-5uM/ml is used for the stem prognosis, so that the HPFs cell shape loses the original full shape and becomes slender, the intercellular space is obviously widened, and the expression level of the intracellular vimentin is obviously reduced compared with a control group, which shows that the ZD6474 can obviously reduce the expression of the HPFs intracellular vimentin. The MMC50ug/ml is used for carrying out dry prognosis, the cell morphology and the intracellular vimentin expression level of the HPFs are not obviously changed compared with a control group, the MMC concentration is increased to 200ug/ml for carrying out dry prognosis, the cell morphology of the HPFs is slightly shrunk compared with the control group, and the vimentin expression level is also reduced to a certain extent, which indicates that the expression of the vimentin of the HPFs can be reduced by applying the MMC of 200ug/ml, but the inhibition effect of the MMC on the cell morphology and the vimentin expression of the HPFs is weaker than that of ZD 6474.
(2) After cell climbing of HPFs, ZD6474 was used to intervene in HPFs24 hours before immunofluorescence staining was performed to detect the expression of alpha-SMA protein.
The results are shown in FIG. 12:
the control group shows that HPFs cells are full in shape, arranged in a feather shape, narrow in cell gap, obvious in intracellular alpha-SMA protein staining and rich in protein expression quantity. ZD6474 at 500nM/ml-5uM/ml is used for dry prognosis, it can be seen that the HPFs cell shape loses the original full shape and becomes slender, the cell gap is obviously widened, and the expression level of the alpha-SMA protein in the cell is obviously reduced compared with the control group, which shows that ZD6474 can obviously reduce the expression of the alpha-SMA protein in the HPFs cell. The MMC50ug/ml is used for carrying out dry prognosis, the cell morphology of the HPFs and the expression quantity of the alpha-SMA protein in the cell are not obviously changed compared with a control group, the MMC concentration is increased to 200ug/ml for carrying out the dry prognosis, the cell morphology of the HPFs is slightly shrunk compared with the control group, and the expression quantity of the alpha-SMA protein is also reduced to a certain extent, which indicates that the expression of the alpha-SMA protein of the HPFs can be reduced by applying the MMC of 200ug/ml, but the inhibition effect of the MMC on the cell morphology of the HPFs and the expression of the alpha-SMA protein is weaker than that of ZD 6474.
Example 5: detection of ZD6474 cytotoxicity against HUVEC and drug concentration Range
Human umbilical vein vascular endothelial cells (HUVEC cells) were routinely cultured, and the HUVEC cells were divided into 3 groups: control group: incubation with 0.1% DMSO under normal conditions; group ZD 6474: the medium was supplemented with 3 different concentrations of ZD6474(500nM/mL, 1uM/mL, 5 uM/mL); MMC group: the medium was supplemented with 2 different concentrations of MMC (50ug, 200 ug/ml).
Culturing HUVEC until it is in logarithmic growth phase, digesting, uniformly inoculating to 96-well plate according to 10 × 4 density, detecting drug cytotoxicity by CCK8 method after cell growth is 80% fusion, and determining drug safety concentration range.
The results of the HUVEC CCK8 experiment are shown in FIG. 13:
the absorbance value of the control group was 0.418. + -. 0.047. After ZD6474 at each concentration of 100nM/ml-1uM/ml was allowed to act on HUVEC24 for hours, the absorbance values for CCK8 were stable between 0.401 and 0.439 compared with the control group, no decrease in significant absorbance values was observed, and no statistical difference was observed in the absorbance values for each group compared with the control group (P >0.05), indicating that ZD6474 showed no significant cytotoxicity in the range of 100nM/ml-1uM/ml and that MMC showed significant cytotoxicity in each concentration range.
Example 6: testing the Effect of ZD6474 on cell migration of HUVEC
The cells and experimental groups used in the experiments were the same as in example 5.
Culturing HUVEC until the HUVEC is in logarithmic growth phase, uniformly inoculating the HUVEC in a streaked 6-hole plate after digestion, performing a cell scratching experiment after the cells are completely fused, and comparing the scratching area after the medicine intervention for 24 hours with the scratching area before the intervention.
The scratching results for HUVEC are shown in fig. 14:
the control group had a significantly smaller scratch area after 24 hours than before the intervention, and the control group had a dry-prognosis scratch area reduction ratio of 39.26%. ZD6474 has a 24 hour scratch area significantly wider than that of 27.15% and 19.09% from each of the 500nM/ml-5uM/ml groups than that of the control group, respectively, wherein the rate of scratch area reduction between the 1uM and 5uM groups is statistically significant (P <0.05), and the effect of inhibiting migration is drug concentration dependent. The 24-hour scratch area of MMC50ug/ml and MMC200ug/ml is also widened compared with that of the control group, the scratch area is 32.28 percent and 22.31 percent, which shows that MMC can also inhibit the migration of HUVEC, the migration inhibition is weaker than that of ZD6474, and the difference between the two groups has statistical significance (P < 0.05).
The scratch test proves that ZD6474 can obviously inhibit the migration of HUVEC, the effect is increased along with the increasing concentration of the drug, and the effect of inhibiting the migration is stronger than that of MMC.
In conclusion, the absorbance values of CCK8 of ZD6474 at concentrations below 5uM/ml were not statistically different from the control group, ZD6474 at concentrations below 1uM/ml was also safe for use in HTFs without significant cytotoxicity, while the absorbance values of MMC at concentrations below 50ug/ml were significantly reduced from the control group, the difference being statistically significant. The scratch area of ZD6474 after the intervention of HPFs24 hours is obviously widened compared with that of the control group, and the difference has statistical significance, namely ZD6474 can obviously inhibit the migration of HPFs, the effect is gradually increased along with the increase of concentration gradient, the migration inhibition effect is more obvious compared with HTFs, and the effect on HPFs is more selective. The expression level of vimentin and alpha-SMA protein of HPFs is also obviously reduced under the intervention of ZD6474, and the effect is concentration-related, which indicates that ZD6474 can effectively reduce the fiber activity of HPFs.
It is known that ZD6474 at local therapeutic concentrations is less cytotoxic for HPFs and HTFs and that ZD6474 is safer for normal cells than the MMC is significantly more cytotoxic for HTFs. ZD6474 acts more specifically on HPFs than normal HTFs, inhibits their proliferative migration and reduces fibroblast activation, and may play a therapeutic role in preventing pterygium postoperative recurrence.
ZD6474 at concentrations below 1uM/ml may be safely applied to HUVEC without significant cytotoxicity. The scratch area after ZD6474 intervenes HUVEC24 for a short time is significantly broadened compared to the scratch area of the control group.
It is known that ZD6474 acts safely on HUVEC, inhibits cell functions such as proliferation, migration, and tubule formation of HUVEC, effectively inhibits neovascularization, and exerts its therapeutic effect on recurrence after pterygium resistance.
In conclusion, ZD6474 may specifically and selectively reduce the fibre activity of HPFs; can reduce the migration of HUVEC and effectively inhibit the tube forming effect of HUVEC.
The invention is explained in detail above with reference to the drawings and the embodiments; however, it will be understood by those skilled in the art that various changes in the specific parameters of the embodiments described above may be made or equivalents of the related materials and method steps may be substituted without departing from the spirit of the invention, thereby forming a plurality of specific embodiments, all of which are within the scope of the invention and will not be described in detail herein.
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| 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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| 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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