Isoladine ophthalmic preparation and application thereof

Abstract

The present invention discloses an irsogladine ophthalmic preparation and its application, and relates to the technical field of ophthalmic preparations. An ophthalmic preparation, whose ingredients include irsogladine or its pharmaceutically acceptable salt or derivatives thereof; the ophthalmic preparation is used for the prevention and treatment of corneal endothelial damage in ophthalmic diseases. The present invention confirms through experimental studies that irsogladine has a good protective effect on the damage of corneal endothelial cells, and finds a new treatment method that does not require surgery for treatment in the treatment and prevention of corneal endothelial cell damage, which has good application prospects.

Description

Technical Field

[0001] This invention relates to the field of ophthalmic formulation technology, specifically to an isoprazine ophthalmic formulation and its application. Background Technology

[0002] The cornea, a vital structure of the eye, is crucial for maintaining vision through its transparency and integrity. Corneal endothelial cells are a special type of cell located in the inner layer of the cornea. They are arranged in a single layer, covering the posterior surface of the corneal endothelium and directly contacting the aqueous humor. The corneal endothelial cells form a semi-permeable membrane, allowing small molecules to pass through, while the endothelial barrier they form effectively prevents aqueous humor from entering the corneal stroma. Corneal endothelial cells have important functions, including maintaining corneal transparency, regulating corneal water and electrolyte balance, and helping to maintain the optical transparency of the cornea.

[0003] When corneal endothelial cells become diseased, a series of problems can occur. One common issue is a decrease in endothelial cell density, which leads to irregular cell arrangement and affects normal corneal function. In addition, decreased endothelial cell function can also cause corneal edema, resulting in loss of corneal transparency and thus affecting vision. Severe endothelial cell disease can lead to loss of corneal endothelial function, thereby affecting the overall health of the cornea and visual quality.

[0004] Normally, corneal endothelial cells cannot divide and proliferate. After corneal endothelial cell damage, only cells from the surrounding area can migrate to maintain normal corneal endothelial function. Currently, the main treatment for corneal endothelial diseases is corneal transplantation. However, this procedure carries risks and a long recovery time. Furthermore, due to a severe shortage of corneal donors, most patients with corneal endothelial blindness cannot receive effective treatment. Compared to traditional surgery or invasive procedures, non-invasive ophthalmic medication, as a safe and effective treatment method, has advantages such as ease of operation, low risk, and minimal patient discomfort, playing an important role in ophthalmic clinical treatment.

[0005] Therefore, finding a drug that can alleviate or repair corneal endothelial damage and resolve the various surgical risks associated with corneal transplantation is a pressing technical problem that needs to be solved by professionals in this field.

[0006] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0007] To address the aforementioned technical problems, embodiments of the present invention provide an isolaridine ophthalmic formulation and its application, thereby resolving the issues raised in the background section.

[0008] An ophthalmic preparation comprising isoladine or a pharmaceutically acceptable salt thereof or a derivative thereof.

[0009] Preferably, its components include isoladine or isoladine maleate.

[0010] Preferably, isolaridine is formulated as one or more of the following: drops, hydrogel formulations, and liposome formulations.

[0011] Preferably, its components also include at least one of a pharmaceutically acceptable carrier, excipient, adjuvant, and medium.

[0012] Preferably, the ingredients also include an adjunct therapeutic agent; the adjunct therapeutic agent is at least one of a conjunctivitis treatment drug, a keratitis treatment drug, a glaucoma treatment drug, a dry eye treatment drug, and a retinal injury treatment drug.

[0013] The application of an ophthalmic preparation as described above in ophthalmic diseases.

[0014] Preferably, the ophthalmic preparation is used for the prevention or treatment of corneal endothelial injury.

[0015] Preferably, in the application of the ophthalmic preparation according to claim 7 in ophthalmic diseases, the concentration of isolaridine in the ophthalmic preparation is 10 μg / ml-80 μg / ml.

[0016] Preferably, the concentration of isolaridine in ophthalmic preparations after corneal endothelial injury is 20 μg / ml; and the concentration of isolaridine in ophthalmic preparations before corneal endothelial injury is 40 μg / ml.

[0017] Preferably, the concentration of isolaridine maleate in the ophthalmic preparation is 40 μg / ml.

[0018] The ophthalmic preparation of isolaridine and its application provided by the embodiments of the present invention have the following beneficial effects: The present invention has confirmed through experimental research that isolaridine has a good repair effect on corneal endothelial cell damage. In the treatment of corneal endothelial cell damage, a new treatment method that does not require surgery has been found, which has good application prospects. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the efficacy evaluation of isolaridine administration after cell damage.

[0020] Figure 2 This is a schematic diagram illustrating the efficacy evaluation of isolaridine administration before cell damage.

[0021] Figure 3 A schematic diagram illustrating the efficacy evaluation of simultaneous administration of isolaridine maleate and BAK.

[0022] Figure 4 This is a schematic diagram illustrating the efficacy evaluation of isolaridine maleate re-administration after BAK injury.

[0023] Figure 5 This is a schematic diagram illustrating the efficacy evaluation of cells after BAK damage and re-administration of isolaridine maleate in a culture medium without growth factors. Detailed Implementation

[0024] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] To address the aforementioned technical problems, embodiments of the present invention provide an isolaridine ophthalmic formulation and its application, thereby resolving the issues raised in the background section.

[0026] I. Experimental Materials and Sources

[0027] 1. Test materials

[0028] Human corneal endothelial cell complete culture medium (Wuhan Pronosei Life Science Technology Co., Ltd.), trypsin-EDTA digestion solution (Beijing Solarbio Science & Technology Co., Ltd.), penicillin-streptomycin mixture (100×, penicillin-streptomycin; Beijing Solarbio Science & Technology Co., Ltd.), thiazolyl blue solution (MTT; Shanghai Yuanye Biotechnology Co., Ltd.), isoladine maleate (I133615-1g, ≥98.0%, CAS: 84504-69-8), isoladine (H1525080, ≥98.0%, CAS: 57381-26-7).

[0029] 2. Instruments

[0030] Clean bench (Harbin Donglian Electronic Technology Development Co., Ltd.), CO2 incubator (Wegen Technology Co., Ltd.), HH-2 digital display constant temperature water bath (Baita Jinchang Experimental Instrument Factory), multifunctional microplate reader (TECAN, Switzerland), Olympus inverted fluorescence microscope (Olympus Corporation, Japan).

[0031] II. Establishment of a human corneal endothelial cell (B4G12) injury model

[0032] B4G12 cells (obtained from Qingdao Eye Hospital) were used at a rate of 1×10⁻⁶. 4Cells were seeded at a density of 100 cells / well in 96-well plates and cultured for 24 h in a cell culture incubator. Cells were then treated with complete culture medium, with different concentration gradients of benzalkonium chloride (BAK) solution added. After 48 hours, the culture medium was removed, and 100 μL of complete culture medium containing 10% MTT was added to each well. The plates were incubated at 37°C for 4 h, followed by incubation at 37°C for 4 h. Then, 150 μL of DMSO was added to each well, and the plates were shaken horizontally for 10 min. The absorbance of each component was measured at 490 nm using a microplate reader. Five replicates were set for each concentration, and cell viability was calculated. Each experiment was repeated three times. The experimental groups and results are shown in Table 1 below.

[0033] It should be noted that the purpose of adding benzalkonium chloride (BAK) solution is to simulate the damage to human corneal endothelial cells caused by benzalkonium chloride, thereby establishing a B4G12 cell damage model. The cell viability calculation formula is as follows:

[0034]

[0035] Table 1

[0036]

[0037] As shown in Table 1, the cell viability gradually decreased with increasing benzalkonium chloride concentration in the range of 0.05-0.50 μg / ml. Therefore, the selected concentration for invasive administration was 0.15 μg / ml, with an expected cell viability of 60%-70%.

[0038] III. Evaluation of the efficacy of isolarine (Ir) in cells and screening of dosage concentrations

[0039] Example 1: Pharmacodynamic evaluation and concentration screening of drug administration after cell damage

[0040] 1. Experimental Grouping

[0041] Blank control group, model group (0.15 μg / ml BAK solution), positive control group (0.15 μg / ml BAK solution, 430 μg / ml Coenzyme Q10 solution), solvent control group (0.15 μg / ml BAK solution, DMSO content 0.02%), and post-injury treatment group (10 μg / ml, 20 μg / ml, 40 μg / ml, 80 μg / ml, 160 μg / ml).

[0042] 2. Experimental Methods

[0043] B4G12 cells were counted using a cell counting method, with B4G12 cells counted at a rate of 1 × 10⁻⁶. 4Cells were seeded at a density of 100 cells / well in 96-well plates and cultured for 24 hours. Cells were then treated with complete culture medium. BAK solution was added to each experimental group, and after 48 hours, the culture medium was removed. Different concentrations of drug solutions were added to each experimental group, and after another 48 hours, the culture medium was removed. 100 μL of complete culture medium containing 10% MTT was added to each well, and the plates were incubated at 37°C for 4 hours. After incubation, 150 μL of DMSO was added to each well, and the plates were shaken horizontally for 10 minutes. The absorbance of each component was measured at 490 nm using a microplate reader. Five replicates were set for each concentration, and cell viability was calculated. Each experiment was repeated three times. Results are shown below. Figure 1 .

[0044] Depend on Figure 1 It is known that Ir can repair corneal endothelial cells damaged by DMSO with significant efficacy. The repair effect on corneal endothelial cells damaged by BAK is most obvious at a concentration of 20 μg / ml. At other concentrations (10 μg / ml-20 μg / ml), the efficacy is also good with the increase of the dosage, but further increases will have an inhibitory effect on cell repair, but there is still a repair effect. Moreover, Ir has a better therapeutic effect than coenzyme Q10 within a certain concentration range.

[0045] Example 2: Pharmacodynamic evaluation and concentration screening of pre-injury administration

[0046] 1. Experimental Grouping

[0047] Blank control group, model group (0.15 μg / ml BAK solution), positive control group (0.15 μg / ml BAK solution, 430 μg / ml coenzyme Q10 solution), solvent control group (0.15 μg / ml BAK solution, DMSO content 0.02%), and pre-injury drug administration treatment group (10 μg / ml, 20 μg / ml, 40 μg / ml, 80 μg / ml, 160 μg / ml);

[0048] 2. Experimental Methods

[0049] B4G12 cells were counted using a cell counting method, with B4G12 cells counted at a rate of 1 × 10⁻⁶. 4 Cells were seeded at a density of 100 cells / well in 96-well plates and cultured for 24 h in a cell culture incubator. Cells were then treated with complete culture medium, and BAK solution was added to each experimental group. Different concentrations of drug solution were added to each experimental group 30 min before injury. After 48 hours, the culture medium was removed, and 100 μL of complete culture medium containing 10% MTT was added to each well. The plates were incubated at 37°C for 4 h, followed by 150 μL of DMSO to each well. The plates were then shaken horizontally for 10 min. The absorbance of each component was measured at 490 nm using a microplate reader. Five replicates were set for each concentration, and cell viability was calculated. Each experiment was repeated three times. Results are shown below. Figure 2 .

[0050] Depend on Figure 2 Analysis shows that: by comparing the model control group and the pre-injury drug treatment group, Ir can protect the corneal endothelium to reduce damage; by comparing the treatment group and the positive control group, Ir has a better therapeutic effect than coenzyme Q10 within a certain concentration range; based on the results of different concentrations in the treatment group, Ir exhibits a concentration-dependent effect in the range of 10μg / ml-40μg / ml, with the effect of protecting the corneal endothelium and reducing damage gradually increasing with increasing concentration, and the optimal protective concentration being 40μg / ml; thereafter, the protective effect is inhibited with further increases in concentration, but a protective effect still exists.

[0051] IV. Evaluation of the efficacy of isolaridine maleate (IM) in cells and screening of drug concentrations

[0052] Example 3: Pharmacodynamic evaluation and concentration screening of concurrent administration with BAK

[0053] 1. Experimental grouping and methods

[0054] B4G12 cells were counted using a cell counting method, with B4G12 cells counted at a rate of 1 × 10⁻⁶. 4 Cells were seeded at a density of 100 cells / well in 96-well plates and cultured for 24 h in a cell culture incubator. Cells were then treated with complete culture medium. BAK solution was added to each well according to the experimental groups (see Table 2 below), along with different concentrations of drug solutions. After 48 hours, the culture medium was removed. 100 μL of complete culture medium containing 10% MTT was added to each well, and the plates were incubated at 37°C for 4 h. Afterward, 150 μL of DMSO was added to each well, and the plates were shaken horizontally for 10 min. The absorbance of each component was measured at 490 nm using a microplate reader. Five replicates were set for each concentration, and cell viability was calculated. Each experiment was repeated three times.

[0055] Table 2

[0056]

[0057] The experimental results are shown in Figure 3 ;Depend on Figure 3 The results showed that isolaridine maleate, administered as a salt of isolaridine, had a significant inhibitory effect on corneal endothelial cells. The inhibitory effect was more pronounced at a concentration of 40 μg / ml. Subsequently, as the concentration continued to increase, it had an inhibitory effect on cell proliferation.

[0058] Example 4: Efficacy evaluation and concentration screening 24 hours after BAK injury

[0059] 1. Experimental grouping and methods

[0060] B4G12 cells were counted using a cell counting method, with B4G12 cells counted at a rate of 1 × 10⁻⁶.4 Cells were seeded at a density of 100 cells / well in 96-well plates and cultured for 24 hours in a cell culture incubator. Cells were then treated with complete culture medium. BAK solution was added to each well according to the experimental groups (see Table 3 below). After 24 hours, the culture medium was removed. Different concentrations of drug solution were added to the treatment groups. After 48 hours, the culture medium was removed, and 100 μL of complete culture medium containing 10% MTT was added to each well. The plates were incubated at 37°C for 4 hours. Afterward, 150 μL of LDMSO was added to each well, and the plates were shaken horizontally for 10 minutes. The absorbance of each component was measured at 490 nm using a microplate reader. Five replicates were set for each concentration, and cell viability was calculated. Each experiment was repeated three times.

[0061] Table 3

[0062]

[0063] The experimental results are shown in Figure 4 ;Depend on Figure 4 The results showed that isoladine maleate has a significant repair effect on damaged corneal endothelial cells, and the optimal protective concentration is 40 μg / ml.

[0064] Example 5: Efficacy evaluation and concentration screening 24 hours after BAK injury

[0065] 1. Experimental grouping and methods

[0066] B4G12 cells were counted using a cell counting method, with B4G12 cells counted at a rate of 1 × 10⁻⁶. 4 Cells were seeded at a density of 100 cells / well in 96-well plates and cultured for 24 hours. Cells were then treated with complete culture medium. BAK solution was added to each well according to the experimental groups (see Table 4 below). After 24 hours, the culture medium was removed. Different concentrations of drug solution were added to the treatment groups. After 48 hours, the culture medium was removed, and 100 μL of complete culture medium containing 10% MTT was added to each well. The plates were incubated at 37°C for 4 hours. Afterward, 150 μL of LDMSO was added to each well, and the plates were shaken horizontally for 10 minutes. The absorbance of each component was measured at 490 nm using a microplate reader. Five replicates were set for each concentration, and cell viability was calculated. Each experiment was repeated three times.

[0067] Table 4

[0068]

[0069] The experimental results are shown in Figure 5 ;Depend on Figure 5 The results showed that isoladine maleate had a significant repair effect on damaged corneal endothelial cells in a culture medium without growth factors, and the optimal protective concentration was 40 μg / ml.

[0070] The results of Examples 3, 4, and 5 show that IM can enhance cell proliferation and repair damage when administered simultaneously or after injury, at concentrations of 20 μg / ml to 40 μg / ml. Among these, 40 μg / ml is more effective when administered simultaneously, and 80 μg / ml is still effective when administered after injury, but further increases in concentration inhibit cell proliferation. Finally, when the concentration of DMSO does not exceed 0.02%, the results show that this concentration causes less damage to the corneal endothelial tissue.

[0071] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Use of an ophthalmic preparation in the preparation of a drug for ophthalmic diseases, characterized in that: The ophthalmic preparation comprises irsogladine or irsogladine maleate; the ophthalmic disease is corneal endothelial injury disease; and the concentration of irsogladine in the ophthalmic preparation is 10 μg / ml-80 μg / ml.

2. Use of the ophthalmic preparation according to claim 1 in the preparation of drugs for ophthalmic diseases, characterized in that: Its components also include a pharmaceutically acceptable carrier.

3. Use of the ophthalmic preparation according to claim 1 in the preparation of medicaments for ophthalmic diseases, characterized in that: The ophthalmic preparation ingredients also include an additional therapeutic agent; the additional therapeutic agent is at least one of a conjunctivitis treatment drug, a keratitis treatment drug, a glaucoma treatment drug, a dry eye treatment drug, and a retinal damage treatment drug.

4. Use of the ophthalmic preparation according to claim 1 in preparing medicines for ophthalmic diseases, characterized in that: The concentration of irsogladine in the ophthalmic preparation after corneal endothelial injury is 20 μg / ml; the concentration of irsogladine in the ophthalmic preparation before corneal endothelial injury is 40 μg / ml.

5. Use of the ophthalmic preparation according to claim 1 in the preparation of medicaments for ophthalmic diseases, characterized in that: The concentration of irsogladine maleate in the ophthalmic preparation is 40 μg / ml.

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