Use of a corneal collagen chemical cross-linking agent in the preparation of a medicament for treating corneal ectatic diseases

Abstract

The application discloses application of a corneal collagen chemical crosslinking agent in preparation of a medicine for treating corneal dilatation diseases, the medicine is in the form of an injection, and the chemical crosslinking agent is chemically crosslinked or / and physically crosslinked with corneal collagen.The medicine provided by the application can be directly delivered to a corneal stroma layer through a minimally invasive corneal stroma injection mode, and efficient corneal stroma crosslinking can be realized under physiological conditions (without ultraviolet light activation), the corneal epithelium integrity is reserved, the effect of minimally invasive treatment is achieved, and the damage risk caused by ultraviolet light is eliminated.

Description

Technical Field

[0001] This invention belongs to the field of ophthalmology, specifically relating to the application of a corneal collagen chemical cross-linking agent in the preparation of a drug for treating corneal ectasia. Background Technology

[0002] Corneal collagen cross-linking is a core breakthrough technology for treating corneal ectasia diseases such as keratoconus over the past two decades. This technology, known as Ultraviolet-A riboflavin-induced collagen cross-linking (UVA CXL), works by activating riboflavin in the corneal stroma with ultraviolet light, prompting covalent cross-linking of collagen within the stroma. Multiple animal and human studies have shown that UVA CXL can increase corneal elastic modulus by 2-3 times, maintain a long-term 300% significant enhancement in corneal mechanical properties, and achieve corneal planarization of at least 1 diopter. The UVA CXL cross-linking steps and principles include: first, treating the cornea with a riboflavin solution for 30 minutes to fully saturate the riboflavin in the corneal stroma, followed by applying 370 nm UVA light (3 mW / cm²). 2 Irradiation for 30 minutes at this wavelength can excite riboflavin molecules to a singlet state. If the excited-state riboflavin undergoes intersystem crossing to transform into a triplet state, reactive oxygen species will be generated, which will induce covalent cross-linking between and within matrix proteins (such as collagen) and glycoproteins. If the riboflavin does not undergo intersystem crossing, the excited-state riboflavin will directly return to the ground state and release green fluorescence. Because UVA excites all riboflavin molecules distributed along the light path, its concentration decays rapidly, thereby weakening the cross-linking efficiency of the deep stroma. UVA CXL can induce dose-dependent cytotoxicity and may cause stromal opacity. Traditional procedures remove the corneal epithelium beforehand, exposing the stromal corneal cells and endothelial cells to risk—cells in the irradiated area may die. Postoperatively, corneal epithelial cells can regenerate spontaneously, and corneal stromal cells will gradually migrate back; however, if endothelial cells are damaged due to insufficient corneal thickness during treatment, this damage will be irreversible without drug intervention.

[0003] Current research also focuses on improving standard traditional UVA CXL to meet diverse clinical needs. For example, using higher irradiance (30 mW / cm²). 2Accelerating the CXL cross-linking process reduced treatment time from 30 minutes to 15 minutes, but it consistently failed to reliably replicate the efficacy of traditional methods, possibly due to accelerated oxygen depletion under high irradiation. Furthermore, to avoid the prolonged recovery period, pain, and infection risks associated with epithelial debridement, transepithelial CXL was proposed, but its effectiveness still fell short of expectations compared to traditional methods. These modified approaches failed to consistently and effectively slow disease progression, and in some cases, the riboflavin penetration process alone caused epithelial damage.

[0004] The application of chemical cross-linking agents in corneal tissue engineering has also attracted attention. Glutaraldehyde can form a cross-linked network through the reaction of aldehyde groups with the amino groups of collagen, significantly improving the mechanical strength and resistance to enzymatic degradation of the material. However, the clinical application of glutaraldehyde poses significant biosafety risks: it has high cytotoxicity, and residual aldehyde groups in the material can inhibit the growth of corneal epithelial cells and even damage corneal endothelial cells, triggering inflammatory reactions and increasing the risk of complications.

[0005] Genipin can also react with the amino groups of collagen to form chemical cross-linked structures. Genipin exhibits superior biocompatibility compared to glutaraldehyde, with less damage to corneal stromal cells and endothelial cells. However, traditional chemical cross-linking methods, such as eye drops or soaking, have limitations; chemical cross-linking agents like genipin cannot effectively penetrate the deep layers of the corneal stroma without disrupting the corneal epithelial barrier. This invention aims to provide a technique for chemically cross-linking the corneal stroma via corneal stromal injection, opening a new avenue for corneal cross-linking surgery. Summary of the Invention

[0006] The purpose of this invention is to provide an application of a corneal collagen chemical cross-linking agent in the preparation of a drug for treating corneal ectasia. The drug is in the form of an injection. The chemical cross-linking agent is delivered directly to the corneal stroma layer through a minimally invasive corneal stromal injection. Under physiological conditions (without ultraviolet activation), efficient corneal stromal cross-linking can be achieved, preserving the integrity of the corneal epithelium, achieving the effect of minimally invasive treatment, and eliminating the risk of damage caused by ultraviolet radiation.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The application of a corneal collagen chemical cross-linking agent in the preparation of a drug for treating corneal ectasia, wherein the drug is in the form of an injection, and the chemical cross-linking agent chemically cross-links and / or physically cross-links with corneal collagen.

[0008] The chemical cross-linking agent provided by this invention can covalently or non-covalently interact with groups in corneal collagen to construct a stable and controllable cross-linking network. Therefore, the chemical cross-linking agent can be directly injected into the corneal stroma via limbal stromal injection, ensuring biocompatibility, to cross-link corneal stromal collagen. This solves the problems of existing ultraviolet-induced corneal cross-linking techniques, such as invasiveness of corneal epithelial removal, ultraviolet light damage, the possibility of intracorneal herpesvirus reactivation, limited cross-linking depth, and equipment dependence.

[0009] This invention delivers chemical cross-linking agents via corneal stromal injection, which has significant advantages over traditional immersion or eye drop administration methods. Injection allows for uniform distribution of the chemical cross-linking agent within the corneal stromal layer, avoiding direct contact between the corneal epithelium and endothelial cells and high concentrations of the cross-linking agent. While ensuring the cross-linking effect, it significantly reduces the risk of cytotoxicity. Stroma injection can bypass the corneal barrier, achieving the ideal corneal cross-linking effect with a lower effective dose, and avoiding the risk of complications caused by residual cross-linking agents.

[0010] The chemical crosslinking agent binds to corneal collagen in any of the following ways: (1) The chemical crosslinking agent has one or more functional groups such as epoxy, aldehyde, mercapto, azo, N-hydroxysuccinimide, cycloenone, o-benzoquinone, carbon-carbon double bond or carbon-carbon triple bond, and chemically crosslinks with corneal collagen through one or more reaction types such as click chemistry, free radical polymerization, Michael addition, Diels-Alder reaction, nucleophilic substitution or condensation reaction; (2) The chemical crosslinking agent physically crosslinks with corneal collagen through one or more non-covalent interactions such as hydrogen bond, hydrophobic interaction or ionic interaction.

[0011] The chemical cross-linking agent provided by this invention can covalently or non-covalently interact with groups in corneal collagen, thereby constructing a stable and controllable cross-linking network. Therefore, the chemical cross-linking agent can be directly injected into the corneal stroma via limbal stromal injection, ensuring biocompatibility, to cross-link corneal stromal collagen. This solves the problems of existing ultraviolet-induced corneal cross-linking techniques, such as invasiveness of corneal epithelial removal, UV damage, the possibility of intracorneal herpesvirus reactivation, limited cross-linking depth, and equipment dependence.

[0012] The molecular weight of the corneal collagen chemical cross-linking agent is greater than 500 Da. This invention ensures that the corneal collagen chemical cross-linking agent will not easily penetrate the corneal endothelial barrier by limiting the molecular weight.

[0013] The chemical crosslinking agent is attached to a hydrophilic polymer carrier. The crosslinking groups can be further attached to the hydrophilic polymer carrier to adjust its solubility, diffusion, and biocompatibility.

[0014] Preferably, the carrier is selected from polyethylene glycol or hyaluronic acid. For example, polyethylene glycol (PEG) can be introduced as a main chain or linker. PEG has excellent hydrophilicity, biocompatibility, and anti-protein adsorption properties, effectively reducing non-specific protein adsorption and cell adhesion, thereby reducing immune responses and the risk of inflammation. Its molecular weight and chain length are adjustable, facilitating control of the diffusion rate and distribution range of the crosslinking agent in the tissue matrix. Hyaluronic acid (HA), as a natural glycosaminoglycan, is an inherent component of the extracellular matrix of tissues such as the cornea, possessing good biorecognition and cell affinity. Using it as a carrier or copolymer component can enhance the biointegration capacity of the crosslinking system and help maintain the hydration state of the tissue. This invention, by combining functional groups with different reaction kinetics, bonding stability, and dynamic characteristics, can construct a controllable cascade crosslinking network, achieving full-process crosslinking management from rapid initial fixation to long-term adjustable crosslinking, thereby enhancing the mechanical properties of the material while maintaining the tissue's bioactivity and remodeling potential.

[0015] Preferably, the corneal collagen chemical cross-linking agent is polyethylene glycol diglycidyl ether or aldehyde-modified hyaluronic acid.

[0016] When the chemical crosslinking agent is polyethylene glycol diglycidyl ether, the epoxy groups it contains can undergo ring-opening addition reactions with nucleophilic groups such as amino, hydroxyl, and carboxyl groups in proteins to form stable CN and CO covalent bonds. This reaction is mild, and the resulting crosslinked structure has high chemical stability, making it suitable for establishing durable and robust covalent crosslinked networks. When the chemical crosslinking agent is aldehyde-modified hyaluronic acid, the aldehyde groups it contains can undergo Schiff base reactions with protein amino groups. This reaction is rapid and efficient, suitable for quickly establishing initial crosslinks. Simultaneously, the Schiff base bonds exhibit a certain degree of dynamic reversibility, enabling the crosslinked network to possess adaptive adjustment and remodeling capabilities, which is beneficial for long-term biological integration.

[0017] The injectable solution includes a corneal collagen chemical cross-linking agent and a buffer solution.

[0018] The diseases mentioned include keratoconus, corneal ectasia following corneal refractive surgery, or transparent marginal keratosis.

[0019] Compared with existing technologies, the advantages of this invention are mainly reflected in the following aspects: Compared with traditional ultraviolet-induced corneal cross-linking, this invention directly provides a drug that can be directly applied to the corneal stroma in an injectable form. The drug can be delivered directly through corneal stroma injection, and the chemical cross-linking agent in the drug can efficiently cross-link under physiological conditions without ultraviolet activation. Therefore, the application provided by this invention completely avoids the trauma and damage risks caused by epithelial removal and ultraviolet irradiation in traditional procedures, overcomes the limitations of cross-linking depth and equipment, and provides a completely new concept for corneal cross-linking while preserving epithelial integrity, achieving a safer and less invasive treatment. Attached Figure Description

[0020] Figure 1 A schematic diagram illustrating the principle of cross-linking injection into the corneal stroma; Figure 2 Slit lamp observation is shown in the example; Figure 3 AS-OCT observation for the example; Figure 4 Corneal topography observation in the embodiments; Figure 5 The figures provided are intraocular pressure and corneal endothelial cell count in the examples. Figure 6 The corneal stretching experiment is shown in the example. Figure 7 The corneal collagenase degradation experiment is shown in the example. Detailed Implementation

[0021] This invention provides the application of a corneal collagen chemical cross-linking agent in the preparation of a medicament for treating corneal ectasia. The medicament is an injectable preparation, and the matrix injection cross-linking step includes: (1) Injection material: Prepare a buffer solution containing a functional chemical cross-linking agent. The concentration range is confirmed by the CCK8 assay of corneal stromal cells and is usually within 10 mg / ml.

[0022] (2) Injection device and method: A 30G special injection needle was used to puncture the corneal stroma at the 3 o'clock and 9 o'clock positions of the limbus. After the needle was inserted, the injection was not immediately pushed in, but rather advanced a distance parallel to the corneal lamellar layers to establish a temporary microtunnel within the tissue. The cross-linking agent was then delivered at the end of the tunnel. This method aims to reduce injection resistance and prevent reagent backflow. The entire procedure was performed under a surgical microscope to visualize and monitor the needle position, depth, and reagent diffusion in real time.

[0023] (3) Observation of Effects: To comprehensively evaluate the effects of corneal cross-linking, a set of observation indicators was established. Safety assessment included intraocular pressure, slit-lamp ocular surface observation, AS-OCT imaging, corneal topography morphological analysis, corneal endothelial cell density monitoring, and ultrastructural observation of the corneal stroma under transmission electron microscopy. Efficacy verification was achieved through tensile testing of ex vivo corneas and anti-collagenase degradation experiments. The overall effect and risks of the treatment were systematically evaluated.

[0024] like Figure 1 As shown, chemical cross-linking agents can be directly delivered via corneal stroma injection, achieving efficient cross-linking under physiological conditions without the need for ultraviolet activation.

[0025] The following description is based on specific embodiments and accompanying drawings.

[0026] Example 1. Material preparation (1) Injection material 1: polyethylene glycol diglycidyl ether (PEG-DE), purchased from Aladdin Reagent Company. Prepared as a 0.1% (v / v) buffer solution.

[0027] (2) Injectable material 2: Aldehyde-modified hyaluronic acid (HA-CHO), prepared by the oxidation method: 1 g of hyaluronic acid (Maclean, 10 kDa) was dissolved in 100 mL of deionized water, and 1.2 g of sodium periodate was added. Oxidation was carried out for 2 hours under pH 4.5 and in the dark. Subsequently, 5 mL of ethylene glycol was added to quench the reaction for 1 hour. The reaction solution was dialyzed (molecular weight cutoff 3000 Da, 3 days) and freeze-dried (-80°C, 12 h) to obtain HA-CHO powder. In the experiment, HA-CHO was prepared into a 1% (w / v) solution using a balanced salt solution and used immediately after preparation to avoid oxidative degradation.

[0028] (3) Injection material 3: physiological saline solution (NS), used as experimental control.

[0029] 2. Surgical procedure Four-month-old male New Zealand white rabbits, weighing 2.0-2.5 kg, were selected and given free access to food and water during the experiment. Preoperative examinations included slit-lamp examination, tonometer readings, anterior segment optical coherence tomography (AS-OCT), corneal topography, corneal endothelial microscopy, and corneal biomechanics. Animals with intact corneal epithelium, normal intraocular pressure, normal corneal endothelial cell density, and no ocular surface abnormalities were selected for the experiment.

[0030] Anesthesia was administered using a pre-prepared mixed anesthetic (Salvastatin: Xylazine: saline = 1:1:8), administered intramuscularly at a dose of 1 ml / kg for general anesthesia; 0.5% promecaine hydrochloride eye drops were instilled into the operated eye for topical anesthesia. The rabbit was placed under a Zeiss surgical microscope to obtain a clear surgical field. Using a 30 G injection needle, stromal tunnels were constructed in the anterior stromal layer at the 3 o'clock and 9 o'clock positions of the corneal limbus, and a total volume of 0.2 ml of fluid was slowly injected into the corneal stroma. Postoperatively, antibiotic eye ointment (ofloxacin eye ointment) was applied to prevent infection.

[0031] 3. Inspection process Slit-lamp examination: The ocular surface was observed before surgery and at postoperative time points D1 (day 1), D3 (day 3), D7 (day 7), W3 (week 3), W5 (week 5), and W8 (week 8) using bright field, slit light, and cobalt blue light (fluorescein sodium concentration of 0.1% w / v).

[0032] Intraocular pressure measurement: The measurement was performed using an Icare tonometer. Each eye was measured 6 times, and the average value was taken as the final intraocular pressure value.

[0033] Anterior segment optical coherence tomography (AS-OCT): An anterior segment optical coherence tomography scanner was used to measure the central corneal thickness (CCT) at time points before surgery and at postoperative time points D1, D3, D7, W3, W5 and W8.

[0034] Corneal topography: Changes in corneal topography parameters were acquired using the Pentacam system. Anterior surface curvature and corneal thickness were measured before surgery and at time points D7, W3, W5, and W8 after surgery.

[0035] Endothelial microscopy: Corneal endothelial cell density was recorded and analyzed using corneal endothelial microscopy before surgery and at W3, W5 and W8 postoperatively.

[0036] Corneal stretching experiment: The cornea was cut into strips, and the thickness, length and width of the cornea were measured using vernier calipers. Each corneal strip was placed horizontally in the clamp of the stretching device and immersed in isotonic PBS solution throughout the process. Each corneal strip was stretched to 30% deformation at a constant loading rate of 5 mm / min. The stress-strain curves were recorded simultaneously, and the Young's modulus of the cornea was calculated from the linear segments of each stress-strain curve.

[0037] Corneal collagenase degradation experiment: Each group of isolated corneas was immersed in 0.2% type II collagenase solution and incubated at 37°C in the dark. Throughout the digestion process, images of corneal tissue morphological changes were taken every 2 hours.

[0038] 4. Observe the effect like Figure 2 As shown, slit-lamp examination revealed that, through observation of the ocular surface using bright field, slit light, and cobalt blue light, chemical cross-linking did not alter corneal transparency, and no significant ocular surface irritation was caused after stromal injection. The corneal epithelium remained intact, effectively avoiding damage to the corneal epithelium compared to traditional epithelial ablation corneal cross-linking.

[0039] like Figure 5 As shown, intraocular pressure measurement: as the time after corneal stromal injection increased, the intraocular pressure in each group remained within the normal range, and the overall intraocular pressure in each group showed a decreasing trend, which proves that the corneal stromal injection method has good intraocular safety.

[0040] like Figure 3 As shown, the anterior segment optical coherence tomography (AS-OCT) results show that the corneal structure is clear, the corneal thickness is uniform, and no structural damage or injury is observed, which indicates that the chemical cross-linking agent is uniformly cross-linked in the corneal stroma.

[0041] like Figure 4As shown, corneal topography was performed using the Pentacam system. Results showed that one week after stromal injection, corneal thickness increased slightly in all groups, indicating short-term corneal edema. As time progressed, the edema subsided, and corneal thickness gradually returned to pre-crosslinking levels. However, under chemical crosslinking, corneal stromal thickness gradually decreased, even slightly falling below pre-crosslinking thickness. The anterior corneal surface curvature showed a decreasing trend in all groups, indicating that the anterior corneal surface tends to flatten after stromal crosslinking. This result is similar to the trend of corneal curvature and thickness changes after UVA CXL, demonstrating that this study can achieve corneal crosslinking effects similar to those of classic clinical corneal crosslinking to a certain extent.

[0042] like Figure 5 As shown in the figure, endothelial microscopy revealed that the corneal endothelial cell density remained stable within two months after corneal stromal injection, further verifying the safety of corneal stromal injection with chemical cross-linking agents. Compared with ultraviolet irradiation, it can better protect the corneal endothelium.

[0043] like Figure 6 As shown in the corneal stretching experiment: the results showed that the Young's modulus of the cornea increased most significantly after cross-linking in the PEG-DE group, and its Young's modulus at 10%, 20%, and 30% deformation was statistically different from that of the NS control group; the Young's modulus of the HA-CHO group at 30% deformation was also significantly higher than that of the NS control group. These results fully demonstrate the effectiveness of cross-linking, indicating that the mechanical properties of the cornea are significantly increased after corneal stromal injection, achieving the expected purpose of corneal cross-linking.

[0044] like Figure 7 As shown in the corneal collagenase degradation experiment: it can be observed that the corneal degradation rate of the PEG-DE group and the HA-CHO group is significantly lower than that of the NS group. Through the corneal collagenase degradation experiment, this result once again verifies the effectiveness of cross-linking, indicating that chemical cross-linking can effectively enhance the stability of the corneal stromal cross-linking network.

[0045] In summary, the application provided by this invention completely avoids the risks of corneal epithelial trauma and corneal endothelial damage caused by epithelial removal and ultraviolet radiation in traditional procedures. It breaks through the limitations of cross-linking depth and equipment, ensures the effectiveness of corneal cross-linking, and provides a brand-new concept for corneal cross-linking surgery to achieve a safer, less invasive, and more effective treatment.

Claims

1. The application of a corneal collagen chemical cross-linking agent in the preparation of a medicament for treating corneal ectasia, characterized in that, The drug is in the form of an injection, and the chemical cross-linking agent undergoes chemical cross-linking and / or physical cross-linking with corneal collagen.

2. The application according to claim 1, characterized in that, The chemical crosslinking agent binds to corneal collagen in any of the following ways: (1) The chemical crosslinking agent has one or more functional groups such as epoxy, aldehyde, mercapto, azo, N-hydroxysuccinimide, cycloenone, o-benzoquinone, carbon-carbon double bond or carbon-carbon triple bond, and chemically crosslinks with corneal collagen through one or more reaction types such as click chemistry, free radical polymerization, Michael addition, Diels-Alder reaction, nucleophilic substitution or condensation reaction; (2) The chemical crosslinking agent physically crosslinks with corneal collagen through one or more non-covalent interactions such as hydrogen bond, hydrophobic interaction or ionic interaction.

3. The application according to claim 1, characterized in that, The molecular weight of the corneal collagen chemical cross-linking agent is greater than 500 Da.

4. The application according to claim 1, characterized in that, The chemical crosslinking agent is attached to a hydrophilic polymer carrier.

5. The application according to claim 4, characterized in that, The carrier is selected from polyethylene glycol or hyaluronic acid.

6. The application according to claim 1, characterized in that, The corneal collagen chemical cross-linking agent is polyethylene glycol diglycidyl ether or aldehyde-modified hyaluronic acid.

7. The application according to claim 6, characterized in that, The injectable solution includes a corneal collagen chemical cross-linking agent and a buffer solution.

8. The application according to claim 1, characterized in that, The diseases mentioned are keratoconus, corneal ectasia secondary to corneal refractive surgery, or transparent marginal keratosis.

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