Composition, eye drops and use thereof

Abstract

The present invention relates to the field of biotechnology, and in particular to a composition, eye drops and their use. The present invention provides a composition, eye drops and their use in preparing a product for inhibiting corneal neovascularization. The present invention prepares an eye drop containing recombinant human IL-18 that has an inhibitory effect on corneal neovascularization, which is effective, easy to use and can be mass-produced. It not only has social benefits, but also will produce huge economic benefits.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a composition, eye drops and applications thereof. Background Art

[0002] Corneal tissue is an important component of the ocular refractive matrix, and a transparent cornea is very important for vision. Normal corneal tissue has no blood vessels to maintain its transparency. However, when the ocular surface tissue is stimulated by external factors, such as infection, trauma, and immune response, new capillaries will grow from the corneal limbus into the cornea. When the new blood vessels enter the corneal periphery for more than 1 mm, it is pathological corneal neovascularization. Corneal neovascularization not only seriously affects vision, but is also a high-risk factor for rejection after allogeneic corneal transplantation, leading to surgical failure. Therefore, the development of eye drops that inhibit the development of corneal neovascularization has always been a hot topic and difficulty in ophthalmology.

[0003] There are many factors that cause corneal neovascularization, the main reasons of which are: (1) Microbial infection, such as various viruses, bacteria, chlamydia and other pathogenic microorganisms. When the pathogen infection enters the chronic stage, corneal neovascularization may occur. (2) Physical trauma to the cornea, such as surgery, mechanical damage, etc., can cause corneal neovascularization. (3) Chemical damage to the cornea, such as acid and alkali burns, burns, are often accompanied by a large number of corneal neovascularization, and even granuloma-like vascular tissue. (4) Other factors, such as immune allergic reactions, corneal malnutrition, late diabetes, wearing contact lenses, etc., can also cause corneal neovascularization.

[0004] Corneal neovascularization is a complex physiological or pathological process, including three stages: (1) Under the stimulation of external factors (hypoxia, inflammation, trauma, etc.), the secretion of cytokines that promote angiogenesis increases, the local vascular basement membrane degrades, and endothelial cells have space to migrate and proliferate; (2) Endothelial cells migrate to the place where the inducer exists and divide and proliferate to form hollow primitive blood vessels composed only of endothelial cells. (3) The primitive blood vessels interact with smooth muscle cells, peripheral cells and extracellular matrix and gradually mature. This reaction process involves a large number of cytokines, including: (1) vascular endothelial growth factor (VEGF) family and angiopoietin (Ang) family, which specifically act on vascular endothelial cells; (2) certain growth factors and chemokines, such as FGF-2, PDGF, GM-CGF, etc., which act not only on endothelial cells, but also on other types of cells; (3) Cytokines that have indirect effects on endothelial cells, such as TNFα and TGFβ. The former can induce the expression of VEGF and its receptors; the latter can induce the expression of TNFα, VEGF and FGF-2. Inhibiting the effects of various cytokines in the process of corneal neovascularization has become a therapeutic target.

[0005] Studies have shown that systemic injection of interleukin 18 (IL-18) into tumor-bearing mice not only inhibits tumor angiogenesis, but also significantly inhibits FGF-induced corneal capillary angiogenesis. Compared with wild-type mice, IL-18 knockout mice showed obvious vascular proliferation, disorder and exudation in the fundus vessels within 7 days after birth; 14 days after birth, histopathology confirmed abnormal vascular morphology and exudation, and RT-PCR showed that cytokines closely related to angiogenesis: VEGF, bFGF, PDGF and PEDF were overexpressed, indicating that IL-18 has a regulatory effect on the growth of new blood vessels in mice. The retinal cells of wild-type mice (C57BL / 6) continuously express IL-18. When the wild-type mice at 7 days old were placed under 75% oxygen conditions for 5 days (between 7 and 12 days), there was a transient decrease in IL-18 expression on the 17th day. Along with the decrease in IL-18, VEGF expression appeared in the retina and oxygen-induced retinopathy (OIR) occurred, but by the 24th day, the retinal neovascularization had regressed to normal levels. In contrast, IL-18 gene knockout mice still developed severe OIR on the 17th day even in a higher oxygen concentration environment, and it lasted for up to 24 days, indicating that IL-18 has an inhibitory effect on retinal neovascularization (CNV). Although subcutaneous injection of recombinant IL-18 into mice during OIR did not significantly inhibit the occurrence of fundus lesions, OIR was still obvious at 24 days when endogenous IL-18 was neutralized by subcutaneous injection of IL-18BP, indicating that IL-18 regulates retinal pathological CNV by promoting CNV regression rather than inhibiting its generation; intraocular or systemic injection of mouse-derived IL-18 can prevent the occurrence of experimental CNV induced by hypoxia or laser.

[0006] Animal experiments have shown that CNV mouse models, intraocular injection of VEGF, and VEGF transgenic mice all have obvious fundus vascular exudation. Injection of recombinant IL-18 into these mice significantly reduced the area of ​​new blood vessels and the area of ​​blood exudation, indicating that IL-18 has an inhibitory effect on VEGF-induced new blood vessels and vascular leakage. Vascular endothelial cells fuse with each other through specific transmembrane proteins to form tight junctions, among which claudin-5 is the structural basis of normal tight junctions between cells. Reduced or absent expression of Claudin-5 leads to the destruction of the structure and function of tight junctions, causing leakage between cells. The expression of Claudin-5 in retinal vascular cells of ischemic fundus lesions and VEGF transgenic mice is significantly reduced. When these mice are injected with IL-18 intraocularly, the expression of Claudin-5 in their retinal vascular cells is significantly increased, which is significantly different from the control group; indicating that IL-18 has multiple regulatory targets in inhibiting angiogenesis and blood leakage.

[0007] In recent years, the research on the effect of IL-18 on neovascularization has mainly focused on retinal and choroidal neovascularization, and the dosage forms used are mostly liquid dosage forms, and the method of use is mostly injection; there are no reports on research and application of corneal neovascularization.

[0008] Corneal neovascularization is often accompanied by stromal edema, fat deposition, scar formation and other causes of vision loss. It is one of the most common causes of blindness and the most difficult medical problem to treat. Therefore, for many years, people have been developing effective methods to treat corneal neovascularization. The ideal anti-corneal neovascularization treatment should be able to effectively inhibit the growth of neovascularization, be cheap and have no side effects. The current methods for treating corneal neovascularization include (1) removing the cause, and treating according to different causes of the disease, such as anti-infection treatment. (2) Drug treatment, mainly targeting inhibitors of angiogenic factor expression. Many studies have confirmed that vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) and others are all related to corneal neovascularization, and therefore have become new targets for the treatment of corneal neovascularization. (3) Antibodies, through the neutralization of monoclonal antibodies, block the effects of endogenous cytokines, such as anti-VEGF monoclonal antibodies and anti-bFGF monoclonal antibodies, which are still in the basic research stage. (4) Spironolactone, which works by inhibiting VEGF and bFGF. (5) Curcumin works by inhibiting bFGF. (6) Heparin sulfate works by inhibiting bFGF and its receptor. All these therapeutic drugs have certain therapeutic effects on corneal neovascularization, but they cannot completely solve the problem of corneal neovascularization treatment. Therefore, it is very necessary to develop new and effective anti-corneal neovascularization preparations. Summary of the invention

[0009] In view of this, the present invention provides a composition, eye drops and their use. The present invention provides a composition, eye drops and their use in preparing a product for inhibiting corneal neovascularization. The present invention prepares an eye drop containing recombinant human IL-18 that has an inhibitory effect on corneal neovascularization, which is effective, easy to use and can be mass-produced. It not only has social benefits, but also will generate huge economic benefits.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0011] The present invention provides a composition, comprising: 1 μg / mL to 5 mg / mL of recombinant human IL-18, 0.1 to 5% by mass volume of a viscosity lubricant, 0.01 to 3% by mass volume of a Zeta potential regulator, 1 to 8% by mass volume of a viscosity increasing matrix, 0.1 to 4% by mass volume of a drug carrier, and 0.1 to 0.9% by mass volume of an osmotic pressure regulator;

[0012] The viscosity lubricant is selected from at least one of hyaluronic acid, gelatin, HPMC, polyethylene glycol or propylene glycol;

[0013] The zeta potential regulator is selected from at least one of chitosan, modified chitosan, polylactic acid or povidone K;

[0014] The viscosity-increasing matrix is ​​selected from at least one of Ploxamer 407, Ploxamer 124, Ploxamer 188, Ploxamer 237, or Ploxamer 338;

[0015] The drug carrier includes cyclodextrin;

[0016] The osmotic pressure regulator includes sodium chloride or mannitol;

[0017] In some specific embodiments of the present invention, the Zeta potential of the composition comprises +20 to 55 mV.

[0018] In some specific embodiments of the present invention, the composition comprises the following components:

[0019] .

[0020] In some specific embodiments of the present invention, the composition comprises the following components:

[0021] .

[0022] In some specific embodiments of the present invention, the composition comprises the following components:

[0023] .

[0024] In some specific embodiments of the present invention, the composition comprises the following components:

[0025] .

[0026] In some specific embodiments of the present invention, the composition comprises the following components:

[0027] .

[0028] In some specific embodiments of the present invention, the composition comprises the following components:

[0029] .

[0030] The invention also provides application of the composition in preparing a product for inhibiting corneal neovascularization.

[0031] In some specific embodiments of the present invention, the method of inhibiting corneal neovascularization includes any of the following:

[0032] (I) inhibiting microtubule formation in human vascular endothelial cells; and / or

[0033] (II), inhibiting VEGF expression; and / or

[0034] (III) Inhibit the area of ​​new blood vessel formation.

[0035] The present invention also provides eye drops comprising the composition.

[0036] The present invention also provides the use of the eye drops in preparing medicines for treating and / or improving vision loss.

[0037] The present invention also provides the use of the eye drops in preparing a medicine for reducing the rejection reaction occurring after allogeneic corneal transplantation.

[0038] The present invention provides the following beneficial effects:

[0039] The invention prepares eye drops containing recombinant human IL-18 and having an inhibitory effect on corneal neovascularization, which is effective, easy to use, and can be mass-produced. It not only has social benefits, but also will generate huge economic benefits. The output value is expected to be more than hundreds of millions of yuan. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0041] Figure 1 Shows the purification results of recombinant human IL-18 protein;

[0042] Figure 2 The results showed that recombinant human IL-18 eye drops can inhibit the microtubule formation of human vascular endothelial cells;

[0043] Figure 3 It shows the inhibitory effect of recombinant human IL-18 eye drops on VEGF expression;

[0044] Figure 4 Shows the area of ​​corneal neovascularization on the 21st day. DETAILED DESCRIPTION

[0045] The present invention discloses compositions, eye drops and their applications. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve them. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art and are deemed to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0046] In view of the special physiological and pathological characteristics of the eye, macromolecular drugs are more likely to penetrate the cornea and increase the permeability of corneal epithelial cells. We increase drug adhesion, improve retention time, targeting ability and permeability, accurately control the molecular shape through the ratio of excipients, increase the effectiveness of functional groups, and give the nano delivery system precise size, potential, and morphological characteristics, thereby achieving drug sustained and controlled release, forming a new protein sustained-release drug delivery system with good biocompatibility, low toxicity and high bioavailability.

[0047] The present invention prepares recombinant human IL-18 into eye drops for inhibiting corneal neovascularization, and provides a new preparation for the treatment of corneal neovascularization. The eye drops are effective, easy to use, and can be mass-produced.

[0048] In some specific embodiments of the present invention, the eye drops are composed of the following components in the following concentrations:

[0049] Recombinant human IL-18 10μg / mL

[0050] Gelatin: 1mg / mL

[0051] Modified chitosan: 0.1mg / mL

[0052] Ploxamer 407: 10mg / mL

[0053] Lubricating cyclodextrin: 1mg / mL

[0054] Sodium chloride: 9mg / mL.

[0055] In some specific embodiments of the present invention, the eye drops are composed of the following components in the following concentrations:

[0056] Recombinant human IL-18 100 μg / mL

[0057] Hyaluronic acid: 1mg / mL

[0058] Polylactic acid: 0.1mg / mL

[0059] Ploxamer 124: 10mg / mL

[0060] Lubricating cyclodextrin: 1mg / mL

[0061] Sodium chloride: 9mg / mL.

[0062] In some specific embodiments of the present invention, the eye drops are composed of the following components in the following concentrations:

[0063] Recombinant human IL-18 100 μg / mL

[0064] Gelatin: 2mg / mL

[0065] Modified chitosan: 0.2mg / mL

[0066] Ploxamer 188: 20mg / mL

[0067] Lubricating cyclodextrin: 1mg / mL

[0068] Sodium chloride: 9mg / mL.

[0069] In some specific embodiments of the present invention, the eye drops are composed of the following components in the following concentrations:

[0070] Recombinant human IL-18 0.5mg / mL

[0071] Hyaluronic acid: 2mg / mL

[0072] Polylactic acid: 0.2mg / mL

[0073] Ploxamer 407: 20mg / mL

[0074] Lubricating cyclodextrin: 1mg / mL

[0075] Sodium chloride: 9mg / mL.

[0076] In some specific embodiments of the present invention, the eye drops are composed of the following components in the following concentrations:

[0077] Recombinant human IL-18 0.5mg / mL

[0078] Polyethylene glycol: 1mg / mL

[0079] Chitosan: 0.1mg / mL

[0080] Ploxamer 407: 10mg / mL

[0081] Lubricating cyclodextrin: 1mg / mL

[0082] Sodium chloride: 9mg / mL.

[0083] In some specific embodiments of the present invention, the eye drops are composed of the following components in the following concentrations:

[0084] Recombinant human IL-18 100 μg / mL

[0085] Hyaluronic acid: 5mg / mL

[0086] Polylactic acid: 0.1mg / mL

[0087] Ploxamer 188: 10mg / mL

[0088] Lubricating cyclodextrin: 1mg / mL

[0089] Sodium chloride: 9mg / mL.

[0090] In some specific embodiments of the present invention, the eye drops are composed of the following components in the following concentrations:

[0091] Recombinant human IL-18 1mg / mL

[0092] Hyaluronic acid: 2mg / mL

[0093] Modified chitosan: 0.2mg / mL

[0094] Ploxamer 407: 10mg / mL

[0095] Lubricating cyclodextrin: 1mg / mL

[0096] Sodium chloride: 9mg / mL.

[0097] In some specific embodiments of the present invention, the eye drops are composed of the following components in the following concentrations:

[0098] Recombinant human IL-18 1mg / mL

[0099] Hyaluronic acid: 1mg / mL

[0100] Povidone K: 0.2mg / mL

[0101] Ploxamer 338: 10mg / mL

[0102] Lubricating cyclodextrin: 1mg / mL

[0103] Sodium chloride: 9mg / mL.

[0104] The raw material information used in the present invention is as follows:

[0105]

[0106] Unless otherwise specified, the composition, eye drops and raw materials and reagents used in the application thereof provided by the present invention can be purchased from the market.

[0107] The present invention will be further described below in conjunction with embodiments:

[0108] Example 1 Purity determination of recombinant human IL-18

[0109] The purity of the recombinant human IL-18 protein raw material was detected by SDS-PAGE method with a concentration of 5% for the concentrated gel and a concentration of 12% for the separated gel, and a constant current of 25 mA for 90 minutes. The results showed that the purity of the recombinant human IL-18 protein was more than 95% ( Figure 1 ).

[0110] The amino acid sequence of the recombinant human IL-18 protein is shown in SEQ ID NO: 1: YFGKLESKLPVIRNLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGMAVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGHDNKMQFESSSYEGYFLACEKERDLPKLILKKEDELGDRSIMFTVQNED

[0111] Example 2 Preparation of recombinant human IL-18 eye drops

[0112] Recombinant human IL-18 eye drops were prepared according to the recombinant human IL-18 eye drops prescription shown in Table 1.

[0113] Table 1 Prescription of recombinant human IL-18 eye drops

[0114]

[0115] Example 3 Preparation of recombinant human IL-18 eye drops

[0116] Recombinant human IL-18 eye drops were prepared according to the recombinant human IL-18 eye drops prescription shown in Table 2.

[0117] Table 2 Prescription of recombinant human IL-18 eye drops

[0118]

[0119] Example 4 Preparation of recombinant human IL-18 eye drops

[0120] Recombinant human IL-18 eye drops were prepared according to the recombinant human IL-18 eye drops prescription shown in Table 3.

[0121] Table 3 Prescription of recombinant human IL-18 eye drops

[0122]

[0123] Effect Example 1 Inhibitory effect of recombinant IL-18 eye drops on microtubule formation in human vascular endothelial cells

[0124] Take the HUVEC cells in normal culture, remove the original culture medium, add PBS to wash, and then add trypsin to digest for 3-5 minutes to terminate the digestion. Use a pipette to blow into single cells, take 20 μL of cell suspension and add 20 μL of trypan blue for counting. Centrifuge the remaining cell suspension at 1000 rpm for 5 minutes, resuspend in culture medium, and count at 3×10 5 10 cells / well (6-well plate) were plated and cultured overnight at 5% CO2 and 37°C. The recombinant human IL-18 eye drops prepared in Examples 2 to 4 and the culture medium were added to the 6-well plate at a ratio of culture medium: eye drops (volume ratio) = 9:1. PBS was used instead of eye drops as a blank control and cultured for 48 hours. The treated human vascular endothelial cells were taken and the concentration was adjusted to 1.2×10 5 / mL was inoculated into a 24-well plate covered with Matrigel artificial matrix gel, 1mL per well, and routinely cultured for 16 h. The formation of microtubule-like structures was observed every 4 h, and 5 fields of view were randomly selected for photography under an optical microscope (100×). The images were analyzed using Microvision Saisam software, and the microtubule-like structures in 5 fields of view in each well were counted and statistically analyzed. Statistical analysis of the number of microtubules formed showed that the number of microtubules formed in the experimental group was significantly lower than that in the control group.

[0125] Effect Example 2 Inhibitory effect of recombinant human IL-18 eye drops on VEGF

[0126] Human vascular endothelial cell suspension was inoculated into 6-well culture plates (2×10 5 / well), incubated in a 37°C 5% CO2 incubator overnight. The recombinant human IL-18 eye drops prepared in Examples 2 to 4 were added to a 6-well plate at a medium: eye drops (volume ratio) of 9:1, and PBS was used instead of eye drops as a blank control for 24 hours. The culture supernatants of the control group and the experimental group were collected respectively, and the expression of VEGF protein in the cell culture supernatant was detected using a VEGF cytokine ELISA detection kit (provided by MLBIO, catalog number M1023364-2). The results showed that ( Figure 3 ), the expression of VEGF in the experimental group was significantly decreased compared with that in the control group.

[0127] Effect Example 3: Inhibition of new blood vessels in rabbit cornea after alkali burning by recombinant human IL-18 eye drops

[0128] New Zealand white rabbits were selected as experimental subjects, and a filter paper with a diameter of 6 mm soaked in 1 mol / L NaOH was used for modeling. Each eye was treated for 30 seconds. After modeling, the ocular surface was cleaned with normal saline, and levofloxacin eye drops were applied to the eyes. A circular white color in the center of the cornea indicated a successful burn. The rabbits with successful modeling were randomly divided into a model control group and an experimental group. The experimental groups were given the recombinant human IL-18 eye drops prepared in Examples 2 to 4, twice a day, 50 μL each time, for 21 consecutive days. The area of ​​corneal neovascularization on the 21st day was statistically analyzed. The results showed that ( Figure 4 ), the eye drops of Examples 2 to 4 have a significant inhibitory effect on corneal neovascularization caused by alkali burning.

[0129] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A composition, characterized in that include: 1μg / ml~5mg / ml recombinant human IL-18, 0.1~5% by mass volume of viscosity lubricant, 0.01~3% by mass volume of Zeta potential regulator, 1~8% by mass volume of viscosity increasing matrix, 0.1~4% by mass volume of drug carrier, 0.1~0.9% by mass volume of osmotic pressure regulator; The viscosity lubricant is selected from at least one of hyaluronic acid, gelatin, HPMC, polyethylene glycol or propylene glycol; The zeta potential regulator is selected from at least one of chitosan, modified chitosan, polylactic acid or povidone K; The viscosity-increasing matrix is ​​selected from at least one of Ploxamer 407, Ploxamer 124, Ploxamer 188, Ploxamer 237, or Ploxamer 338; The drug carrier includes cyclodextrin; The osmotic pressure regulator includes sodium chloride or mannitol.

2. The composition according to claim 1, characterized in that The zeta potential of the composition includes +20 to 55 mV.

3. The composition according to claim 1 or 2, characterized in that The components include:

4. The composition according to any one of claims 1 to 3, characterized in that The components include:

5. The composition according to any one of claims 1 to 4, characterized in that The components include:

6. Use of the composition according to any one of claims 1 to 5 in the preparation of a product for inhibiting corneal neovascularization.

7. The use according to claim 6, characterized in that The inhibition of corneal neovascularization growth includes any of the following: (I) inhibiting microtubule formation in human vascular endothelial cells; and / or (II), inhibiting VEGF expression; and / or (III) Inhibit the area of ​​new blood vessel formation.

8. Eye drops, characterized in that The method comprises the composition according to any one of claims 1 to 5.

9. Use of the eye drops according to claim 8 in the preparation of a medicament for treating and / or improving vision loss.

10. Use of the eye drops according to claim 8 in the preparation of a medicament for reducing rejection reaction occurring after allogeneic corneal transplantation.

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