Use of bone morphogenetic protein-4 for the preparation of a medicament for the treatment of corneal diseases

Abstract

This invention belongs to the field of biopharmaceuticals, specifically relating to the use of bone morphogenetic protein-4 (BMP4) in the preparation of drugs for treating corneal diseases. BMP4 has a mild growth-promoting effect on corneal epithelium and stroma, and can treat CNV caused by epithelial damage. BMP4 can also be used as a drug to repair epithelial damage. BMP4 is used to inhibit corneal inflammatory response and plays an important role in the corneal damage repair process. Its administration method is convenient and non-invasive, and it is easy to promote.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceuticals, specifically relating to the use of bone morphogenetic protein-4 in the preparation of drugs for treating corneal diseases. Background Technology

[0002] Corneal diseases are common in clinical practice and are among the most important causes of blindness worldwide. Under normal circumstances, the cornea is avascular, transparent connective tissue, serving as the first line of defense on the ocular surface and a crucial component of the refractive system. Exposed to the external environment, the cornea is susceptible to damage from microorganisms, trauma, and chemical and physical stimuli. Furthermore, the lack of blood vessels in the cornea limits its nutrient supply, making it prone to infection once microorganisms invade. The corneal epithelium, located on the outermost layer of the cornea, is particularly vulnerable to attack by pathogenic microorganisms.

[0003] The cornea, under normal circumstances, is an avascular, transparent connective tissue, serving as the first line of defense on the ocular surface and a crucial component of the anterior segment's refractive system. Corneal neovascularization (CNV) occurs pathologically, with newly formed blood vessels branching from the capillaries and venules of the peripheral corneal plexus. These vessels can block light from entering the eye, affecting visual sensitivity. CNV has a very high rate of blindness; in the United States, the blindness rate among ophthalmology patients is as high as 4.14%, and in my country, a large number of patients lose their sight due to it each year.

[0004] Corneal neovascularization (CNV) is a common complication of various eye diseases. Clinically, CNV can be classified into three types: deep neovascularization involving the Descemet's membrane, stromal neovascularization, and pannus-like neovascularization. CNV can be secondary to chemical burns, ischemia, infection, trauma, and inflammation, leading to further exacerbation of corneal inflammation, corneal edema, and corneal scarring. Other causes of CNV include contact lens exposure, limbal stem cell loss, and corneal and conjunctival degeneration, such as pterygium and limbal degeneration. CNV is also a major cause of corneal transplant rejection, severely affecting the prognosis of penetrating keratoplasty. Current treatments for CNV include corticosteroid eye drops, nonsteroidal anti-inflammatory drugs (NSAIDs), photodynamic therapy, laser photocoagulation, fine-needle thermal therapy, conjunctival and limbal stem cell transplantation, amniotic membrane transplantation, and anti-angiogenic drug therapy. However, all of these methods have certain limitations in clinical treatment and can lead to related side effects. In particular, long-term use of hormones can cause complications such as high intraocular pressure and cataracts. Because the pathogenesis of corneal neovascularization is not yet fully understood, there is no highly effective treatment. How to effectively inhibit the occurrence and development of CNV has become a hot topic in corneal disease research.

[0005] Existing research indicates that anti-angiogenic therapy cannot completely eliminate neovascularization. One reason is that cytokines / growth factors, such as VEGF, can cause CNV. Other factors involved include PDGF, MMPs, angiostatin, endostatin, pigment epithelial-derived factor, thromboretin, and insulin-like growth factor.

[0006] Currently, there is no definitive mechanism for CNV formation. CNV is the result of multiple factors working together, and these factors are intricately interconnected. VEGF is a key factor currently being studied for CNV, and VEGF inhibitors are the most commonly used treatments. Some drugs have already been applied in clinical practice, and these research results will provide important theoretical basis for anti-CNV treatment. VEGF-A is the most important member of the VEGF family that promotes pathological angiogenesis. Studies have shown that instilling, subconjunctival, and intraocular injections of VEGF-A antibodies can partially inhibit CNV and improve corneal transparency by accelerating basement membrane reconstruction after corneal alkali burns. However, it is only effective in the early stages of CNV. When VEGF expression is downregulated, the drug is ineffective against chronic CNV, which involves mature neovascularization with pericytes and smooth muscle cells. Moreover, this antibody only inhibits macrophage infiltration in the early stages of CNV and has no significant effect in the later stages. It is more effective on superficial vessels than on deeper vessels.

[0007] Treatment of CNV with anti-VEGF antibodies has certain limitations; it only addresses the symptoms and does not treat the underlying cause. In some cases, repeated treatment is necessary over a period of time. The mechanisms of angiogenesis are complex, with various pathogenic factors interacting, providing numerous sites and opportunities for drugs to inhibit CNV formation.

[0008] The corneal tissue expresses a variety of cytokines and receptors, such as insulin-like growth factor, platelet-derived growth factor, fibroblast growth factor, and tumor necrosis factor, forming a corneal cytokine network that regulates corneal cell metabolism, maintains the dynamic homeostasis of corneal physiology, and participates in corneal immune regulation. The repair process of corneal epithelial damage is also accomplished through a high degree of temporal and spatial coordination among various cells and numerous cytokines. In recent years, immunohistochemistry, in situ hybridization, and other methods have revealed that cytokines (BMPs) are widely expressed in the ocular tissues of rodents, including corneal epithelium, conjunctival epithelium, corneal endothelium, retinal pigment epithelium, ciliary body, iris, and ganglion cells. BMPs can work synergistically with other cytokines to form the corneal cytokine network. These cytokines induce biological changes in target cells through autocrine, paracrine, and distal secretion, producing specific biological effects, thereby maintaining the normal function of corneal tissue or playing a role after its injury. Numerous cytokines are involved in the occurrence, development, and progression of corneal diseases. Studying the roles of these cytokines can lay the foundation for future clinical treatment of corneal diseases. Clinical application of drugs such as epidermal growth factor to promote corneal epithelial repair has achieved certain therapeutic effects, but carries risks such as neovascularization and CNV (corneal vascular disease). Interestingly, the currently recognized most effective treatment for CNV is bevacizumab eye drops, but its side effect is to exacerbate corneal epithelial defects. This indicates that bevacizumab is not suitable for CNV concurrent with corneal epithelial defects. Therefore, finding the optimal drug to promote corneal epithelial repair while minimizing side effects is essential.

[0009] Bone morphogenetic protein (BMP) is an extracellular protein that functions by binding to protein receptors on the cell surface. BMP4 can inhibit tumor cell growth, plays an important role in lung stem cell differentiation, and regulates the proliferation and shedding of pseudostratified ciliated columnar epithelium in the lung. BMP4 can mediate vascular endothelial cell apoptosis and inhibit angiogenesis. Current research shows that BMP4 is expressed in all layers of the normal adult cornea, suggesting that members of the bone morphogenetic protein family may participate in maintaining the physiological function and metabolic homeostasis of the human cornea. Moreover, the expression of BMP4 in epithelial cells of bullous keratosis and keratoconus is significantly increased compared to normal cornea. During migration and proliferation of damaged epithelial cells, MMP-9 is synthesized. When MMP-9 is neutralized with corresponding antibodies, epithelial migration is significantly accelerated, suggesting that MMP-9 is related to the degradation and remodeling of the basement membrane and stroma behind the leading edge of epithelial migration. The expression of MMP-9 increases with the large-scale growth of epithelial cells, and the level of MMP-9 is significantly downregulated when the basement membrane is fully synthesized. Summary of the Invention

[0010] The purpose of this invention is to provide the use of BMP4 in the preparation of a drug for inhibiting corneal neovascularization, which has the effect of inhibiting corneal neovascularization and mildly promoting the growth of corneal epithelium and stroma.

[0011] Another object of the present invention is to provide an eye drop with BMP4 as the active ingredient.

[0012] A better technical solution: The concentration of the active ingredient BMP4 in the eye drops is 1ug / ml-50ug / ml.

[0013] A better technical solution: The concentration of the active ingredient BMP4 in the eye drops is 20ug / ml.

[0014] This invention reveals that after BMP4 binds to BMP receptor-II, it phosphorylates BMP receptor-I to form a complex, activating the downstream Smad family of receptors. This complex then phosphorylates with Smad1 and / or Smad5 to form R-Smads. Smad4, together with R-Smads, forms the activated Smad complex, which enters the cell nucleus. Following a series of reactions, it participates in regulating the expression of VEGF, TNFα, and MMP9. Smad ubiquitination regulatory factor (Smurf) 1 is a common Smad antagonist that can block the binding of BMP4 to Smad, thereby interrupting subsequent reactions. BMP4 has a significant inhibitory effect on CNV and a mild growth-promoting effect on corneal epithelium and stroma. In treating CNV caused by epithelial damage, BMP4 can be used as a drug to repair epithelial damage. BMP4 plays an important role in the corneal injury repair process by inhibiting corneal inflammation. Its administration method is convenient, non-invasive, and easy to promote. Attached Figure Description

[0015] Figure 1 Ink perfusion patch for suture-induced corneal neovascularization in rats;

[0016] Figure 2 BMP-4 inhibits suture-induced CNV in rats via ink perfusion mounts. Figure 2 In the middle: A is the untreated group, B is the control group, C is the drug-treated group, and D and E illustrate the effect of BMP4 in inhibiting CNV by measuring CNV length and density, respectively.

[0017] Figure 3 ELISA results show that BMP-4 effectively inhibits corneal angiogenesis factors;

[0018] Figure 4 Line graph showing the relationship between the proliferation promotion rate of three cell types and BMP4 concentration in the MTT assay. Detailed Implementation

[0019] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0020] Example 1: A suture-induced rat CNV model was established, and the changes in CNV were observed.

[0021] Animals were anesthetized preoperatively with an intraperitoneal injection of 8% chloral hydrate. Tropicamide eye drops were applied topically (Santen, Osaka, Japan). Two 10-0 corneal sutures (Johnson & Johnson Medical Ltd., St. Stevens-Woluwe, Belgium) were placed 1.5 mm from the limbus, passing through the epithelium and stroma but not the endothelium. The distance between the two sutures was 1 mm. Postoperatively, 0.3% ofloxacin eye ointment was applied to prevent infection. Over time, if the sutures were removed, the naturally occurring blood vessels would recede; therefore, the sutures remained in place until the end of treatment.

[0022] like Figure 1 As shown, CNV growth was most active on day 7 after suture induction, forming a large and dense capillary network. Figure 1 In the diagram: A. Normal cornea; B. Day 1 after suture induction: corneal edema near the suture, limbal vascular dilation; C. Day 3 after suture induction: limbal vessels near the suture extend towards the suture; D. Day 5 after suture induction: limbal vessels near the suture grow significantly towards the suture; E. Day 7 after suture induction: a large and dense neovascular network grows from the nearby limbus towards the suture; F. Day 14 after suture induction: a large and dense neovascular network grows around the suture, with some regression of the vessels compared to before.

[0023] Example 2: Preparation and application of eye drops with BMP4 as the active ingredient.

[0024] A 20 μg / ml BMP4 solution was prepared by dissolving BMP4 in 4 mM HCl. This solution was then used to instill 20 μg / ml BMP4 solution into the external eye of the suture-induced CNV model in Example 1, 1 drop per application, 3 times daily for 7 consecutive days (the corneal sutures were preserved during this process). A 4 mM HCl solution was used as the control group. Figure 2 As shown, after 7 days of application, the area of ​​CNV in the drip group was significantly smaller than that in the control group. Figure 3As shown, ELISA kits were used to detect the protein levels of VEGF, TNFα, and MMP-9 in the cornea. A shows the ELISA results for corneal VEGF and BMP4, revealing a significant decrease in VEGF protein levels in the treatment group, while no difference in BMP4 levels was observed between groups during the treatment process. B shows the ELISA results for corneal MMP-9 and TNF-α, showing significant decreases in both in the treatment group. The experimental group showed significantly lower levels than the control group, which was statistically significant (*P<0.01).

[0025] Because corneal epithelial cells consist of only 4-6 layers of cells and are very thin, the blade scraping method makes it easier to control the extent and depth of damage, and it is also an ideal model for studying corneal epithelial repair after injury. This model was chosen to study the comparative effects of drugs. After intraperitoneal and ocular surface anesthesia, rats were gently etched with a 6mm diameter circular epithelial area in the center of the cornea using a corneal trephine under an operating microscope. A sterile cotton swab dipped in 20% alcohol was placed in the center of the etched area for 30 seconds, followed by rinsing with copious amounts of sterile water. Then, the entire epithelial cell layer in the etched area was scraped away with a corneal epithelial scalpel. The model was established in both eyes, and the rats were randomly divided into a BMP4 group and a VEGF antibody group, with 7 rats in each group. In the BMP4 group, on the day the model was established, 20ug / ml BMP4 protein was instilled into the left eye, 1 drop / time, 3 times / day, for 2 days. The corresponding solvent was instilled into the right eye, using the same method. VEGF antibody administration method: On the day the model was established, 5ug / ml VEGF antibody was instilled into the left eye, 1 drop / time, 3 times / day, for 2 days. The right eye was instilled with the corresponding solvent, using the same method. Fluorescein sodium staining was used to detect corneal epithelial damage in rats. Subsequently, paraffin sections were stained with HE and TUNEL to observe changes in corneal epithelial cells. It was found that after 7 days of instillation, the CNV area in the instillation group was significantly smaller than that in the control group.

[0026] Example 3: Study on the interaction between BMP4, VEGF and MMP-9 during the corneal repair process after injury.

[0027] MTT assays were performed on rat corneal epithelial cells, corneal stromal cells, and umbilical vein endothelial cells. A series of BMP4 concentration gradients were designed to act on the three cell types. Finally, the formazan crystals generated by MTT were reduced by succinate dehydrogenase, and the absorbance was measured using an ELISA reader to obtain experimental data. A corneal epithelial injury model was established using the epithelial scraping method. The steps were as follows: Wistar rats were used as experimental animals. The experiment was divided into normal tissue detection and epithelial injury tissue detection. Paraffin sections were prepared and TUNEL and HE staining was performed. Corneal epithelial cells were cultured, and BMP4 protein and VEGF antibody were added to the culture medium, with solvent controls and blank controls set up. After one day of culture, the expression levels of BMP4, VEGF, MMP-9, and Capase-3 in each group and the epithelial cell viability were detected, and TUNEL staining was performed. Figure 4 As shown, when the mass concentration of BMP4 solution is between 1ug / ml and 20ug / ml, the optimal BMP4 concentration is 20ug / ml. It has a mild promoting effect on corneal epithelial cells and corneal stromal cells, but a significant inhibitory effect on umbilical vein endothelial cells. Based on the above experimental data, to achieve a BMP4 concentration that significantly inhibits umbilical vein endothelial cells without inhibiting corneal stromal cells and corneal epithelial cells, further research was conducted to elucidate the role of BMP4, VEGF, and MMP-9 in the formation and growth of corneal epithelial repair. The expression of Capase-3 protein and TUNEL staining were used to assess epithelial cell apoptosis. Comparative analysis was used to clarify more effective treatment methods for epithelial damage, laying a theoretical foundation for future clinical applications. The above experimental results elucidate the relationships among BMP4, VEGF, and MMP-9 in the corneal repair process after injury, comparing their different roles in corneal epithelial repair. Cellular experiments show that BMP4 can enhance epithelial cell activity. Further investigation into the effects of different reagents on various proteins in the corneal epithelium further confirms the phase-regulatory relationship of BMP4, VEGF, and MMP-9 in corneal repair, laying a theoretical foundation for their clinical application.

[0028] As can be seen from the above examples, in the rat suture-induced CNV model, BMP4 instillation on both the day of modeling and 7 days after modeling effectively inhibited CNV, which was statistically significant. Instillation of eye drops with BMP4 as the active ingredient 7 days after modeling significantly reduced the protein levels of VEGF, TNF-α, and MMP9 compared to the control group, which was statistically significant. There was no statistically significant difference in efficacy between the BMP4-instilled group and the dexamethasone-instilled group on the day of CNV modeling. This invention uses suture-induced rat CNV as a model to reveal the inhibitory effect and possible mechanism of the BMP4-Smad signaling pathway on CNV, and provides eye drops with BMP4 as the active ingredient as a drug for treating corneal neovascularization.

Claims

1. Use of bone morphogenetic protein-4 (BMP-4) in the preparation of drugs for the treatment of corneal neovascularization.

2. The use according to claim 1, characterized in that, The medication is an eye drop.

3. The use according to claim 2, characterized in that, In the eye drops, the concentration of bone morphogenetic protein-4 is from 1 µg / ml to 50 µg / ml.

4. The use according to claim 3, characterized in that, In the eye drops, the concentration of bone morphogenetic protein-4 is 20 µg / ml.

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