Use of bafilomycin a1 in the preparation of a medicament for treating cataracts

Abstract

The application discloses a use of bafilomycin A1 in preparation of a drug for treating cataract; the drug for treating cataract comprises bafilomycin A1 and pharmaceutically acceptable adjuvants. The application proves through experiments that bafilomycin A1 has an effect of intervening or inhibiting formation of a lens vacuole in a zebrafish model of cataract caused by loss of function of PIKfyve, and indicates that bafilomycin A1 has a rescuing effect on lens opacity of the zebrafish cataract model.

Description

Technical Field

[0001] This invention relates to cataract treatment drugs, and particularly to the use of bafloxacin A1 in the preparation of cataract treatment drugs. Background Technology

[0002] Cataracts are the leading cause of blindness worldwide, seriously endangering the health of the Chinese people. Statistics show that cataracts account for 41.06% of blindness in my country and 49.38% of low vision cases are caused by cataracts. Cataracts refer to the clouding of the lens inside the eye, changing it from transparent to opaque, obstructing light from entering the eye and thus affecting vision. In the early stages, when the clouding is mild or small, it does not affect vision, but it gradually worsens until it affects vision or even causes blindness. Vision loss caused by cataracts cannot be corrected with glasses. Cataracts are classified into several types according to their cause, including age-related, complication, developmental, traumatic, toxic, metabolic, radiation-induced, and secondary cataracts. They are also classified into two main categories according to the time of onset: congenital cataracts and acquired cataracts. The main symptom of cataracts is decreased vision. Clinically, in the early stages, this may manifest as blurred vision, fixed black spots in front of the eyes, and double vision; in more severe cases, only light perception may be available.

[0003] Currently, surgery is the primary treatment for cataracts. However, surgery carries postoperative risks such as endophthalmitis and corneal endothelial decompensation. Furthermore, some patients may be unable to tolerate surgery due to poor overall health, and some may refuse treatment out of fear, leading to delayed diagnosis and even blindness. There are also cases where patients cannot afford the surgery. Therefore, developing effective cataract prevention and treatment drugs has significant and long-term practical value.

[0004] Bafilomycin A1 (Baf-A1) is a macrolide antibiotic derived from Streptomyces griseus, and is also a potent vacuolar H1N1 antibiotic. + -ATPase (vacuolar-type H) + Baf-A1 is a specific inhibitor of ATPase (V-ATPase), which inhibits the binding of autophagosomes and lysosomes, thereby suppressing the autophagy pathway. V-ATPase is widely distributed in the intracellular membrane systems of eukaryotes, including the Golgi apparatus, secretory granules, lysosomes, and endosomes. Baf-A1 inhibits the fusion between autophagosomes and lysosomes by blocking the proton pump activity, thus preventing the maturation of autophagosomes. The half-inhibitory concentration is in the range of 4-400 nM.

[0005] Baf-A1 plays a role in the endosome-lysosome system, including inhibiting membrane transport of early to late endosomes by preventing the formation of endosome carrier vesicle intermediates, suppressing homoendosome fusion by affecting membrane potential, and interfering with the acidification of endosomes and lysosomes. In COS-7 cells, PIKfyve dysfunction induces vacuolation; the addition of Baf-A1 can rescue cytoplasmic vacuolation by inhibiting abnormal endosome fusion, restoring normal cell morphology; and even early pretreatment with Baf-A1 can prevent significant early endosome expansion.

[0006] Low concentrations (1 nM) of Baf-A1 effectively and specifically inhibited and killed pediatric B-cell acute lymphoblastic leukemia cells. In xenograft mouse models, Baf-A1 primarily inhibited early and late autophagy pathways through multiple targets, suppressing mitochondrial-induced caspase-independent apoptosis; it weakened protective autophagy and induced apoptosis, thereby delaying leukemia onset and inhibiting and killing primary leukemia cells; it also promoted the formation of the Beclin1-Bcl-2 complex, ultimately leading to the inhibition of autophagy and activation of apoptosis. In the macrophage system, Baf-A1 also prevented cholesterol transport within macrophage lysosomes and was used to distinguish different types of ATPase; it inhibited macrophage phagocytosis by blocking autophagic flux and promoted apoptosis in colon cancer cells.

[0007] Baf-A1, as a known autophagy inhibitor, is widely used and recognized in anti-tumor therapy, and also possesses other pharmacological effects such as antibacterial and immunosuppressive properties. However, there are currently no reports on its application in ophthalmology. Summary of the Invention

[0008] In order to overcome the above-mentioned shortcomings and deficiencies of the prior art, the purpose of this invention is to provide the use of bafloxacin A1 in the preparation of cataract treatment drugs.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] Use of bafloxacin A1 in the preparation of drugs for treating cataracts.

[0011] Specifically, the cataract treatment drug includes bafloxacin A1 and pharmaceutically acceptable excipients.

[0012] Preferably, the cataract treatment drug is an eye drop, a gel, or a subconjunctival implant.

[0013] Specifically, the molecular formula of bafloxacin A1 is C 35 H 58 O9.

[0014] Bafilomycin A1 (Baf-A1) is a macrolide antibiotic derived from *Streptomyces griseus*. This invention provides the use of bafilomycin A1 in the preparation of drugs for treating cataracts. This invention builds upon the established pikfyve Δ8 Based on a zebrafish cataract model, Baf-A1 was used to intervene in lens opacity in a zebrafish cataract model with PIKfyve dysfunction. Results showed that after Baf-A1 treatment, the PIKfyve... Δ8 The zebrafish lens opacity was significantly treated; compared with the dimethyl sulfoxide (DMSO) group and the blank control group, the number of lens vacuoles in the Baf-A1 group was significantly reduced. Statistical analysis showed a significant difference between the Baf-A1 group and the DMSO group, indicating that Baf-A1 has a rescue effect on lens opacity in a zebrafish model of cataracts. Attached Figure Description

[0015] Figure 1(a) shows the pikfyve of the blank control group. Δ8 First imaging image of the lens phenotype of a zebrafish.

[0016] Figure 1(b) shows the pikfyve data for the blank control group. Δ8 Second imaging image of the lens phenotype of a zebrafish.

[0017] Figure 2(a) shows the pikfyve of the DMSO group. Δ8 First imaging image of the lens phenotype of a zebrafish.

[0018] Figure 2(b) shows the pikfyve of the DMSO group. Δ8 Second imaging image of the lens phenotype of a zebrafish.

[0019] Figure 3(a) shows the pikfyve of the Baf-A1 group. Δ8 First imaging image of the lens phenotype of a zebrafish.

[0020] Figure 3(b) shows the pikfyve of the Baf-A1 group. Δ8 Second imaging image of the lens phenotype of a zebrafish.

[0021] Figure 4 The results of quantitative analysis of the number of lens vacuoles after 4.5 hours in the blank control group without any treatment.

[0022] Figure 5 The results of quantitative analysis of the number of vacuoles in the lens before and after treatment with 1‰ DMSO in the DMSO group.

[0023] Figure 6The results of quantitative analysis of the number of lens vacuoles in the Baf-A1 group before and after treatment with 1 μM Baf-A1.

[0024] Figure 7 The results of quantitative comparative analysis of the number of lens vacuoles after treatment with 1‰ DMSO in the DMSO group and after treatment with 1 μM Baf-A1 in the Baf-A1 group. Detailed Implementation

[0025] This invention discloses the use of bafloxacin A1 in the preparation of drugs for treating cataracts. Those skilled in the art can draw upon this content and make appropriate modifications. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The application of this invention has been described through preferred embodiments, and those skilled in the art will clearly be able to modify or appropriately change and combine the applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0026] Example

[0027] This embodiment discloses the use of bafloxacin A1 in the preparation of a cataract treatment drug, which can be formulated as eye drops, gels, subconjunctival implants, or other dosage forms; the cataract treatment drug includes bafloxacin A1 and pharmaceutically acceptable excipients.

[0028] This embodiment is based on the established pikfyve Δ8 Based on the zebrafish model, this embodiment utilizes Baf-A1 to intervene in lens opacity in a zebrafish cataract model with impaired PIKfyve function. The specific experiment is as follows:

[0029] 1. Materials:

[0030] 1.1 Reagents:

[0031] Baf-A1 was purchased from MedChemExpress in the United States.

[0032] DMSO was purchased from Sigma-Aldrich, USA.

[0033] Low gel temperature agarose was purchased from Sigma-Aldrich, USA.

[0034] 1.2 Preparation of main reagents:

[0035] 1uM Baf-A1: Add 100ug of Baf-A1 to 160.6ul of DMSO and mix well to obtain 1mM baf-A1. Then add 5ul of 1mM baf-A1 to 5ml of egg water and mix well.

[0036] 1‰ DMSO: Add 5ul of DMSO to 5ml of egg water and mix well.

[0037] 1.3 Main experimental instruments:

[0038] Illuminated incubator (DAIHAN, South Korea)

[0039] Laser confocal microscope (Zeiss AG, Germany)

[0040] Stereoscopic dissecting microscope (Olympus Corporation, Japan)

[0041] High-temperature sterilizer (Zealway, USA)

[0042] 2. Experimental Methods:

[0043] Baf-A1 was dissolved in DMSO solution at a concentration of 1 mM, and diluted with egg water to a final concentration of 1 μM. Pikfyve, 4 days after fertilization... Δ8 Zebrafish embryos were retrieved and randomly divided into a blank control group, a DMSO group, and a Baf-A1 group. Imaging was performed under a confocal microscope, and the number of vacuoles in the lenses of the three groups was counted. After the first imaging, the DMSO group embryos were treated with egg water containing 1‰ DMSO for 4.5 hours in a 28.5℃ light-controlled incubator; the Baf-A1 group embryos were treated with a diluted 1 μM Baf-A1 solution; the blank control group received no treatment. After 4.5 hours, the DMSO and Baf-A1 group embryos were repeatedly rinsed with egg water; subsequently, a second imaging and vacuole count were performed on all three groups under a confocal microscope.

[0044] This experiment used Graphpad Prism software for statistical processing and analysis of the data. The data are expressed as (Mean±SD). The T-test was used to analyze and compare the data of each group (P<0.05 was considered statistically significant, and P<0.01 was considered highly statistically significant).

[0045] 3. Experimental Results:

[0046] pikfyve, the blank control group in this experiment Δ8 The first and second imaging images of the zebrafish lens phenotype are shown in Figure 1(a) and Figure 1(b), respectively; pikfyve of the DMSO group Δ8 The first and second imaging images of the zebrafish lens phenotype are shown in Figure 2(a) and Figure 2(b), respectively; pikfyve of the Baf-A1 group Δ8The first and second imaging images of the zebrafish lens phenotype are shown in Figure 3(a) and Figure 3(b), respectively; the quantitative analysis results of the number of lens vacuoles in the blank control group after 4.5 hours without any treatment are shown in Figure 3(b). Figure 4 As shown; the quantitative analysis results of the number of vacuoles in the lens before and after treatment with 1‰ DMSO are as follows. Figure 5 As shown; the quantitative analysis results of the number of lens vacuoles before and after treatment with 1 μM Baf-A1 in the Baf-A1 group are as follows. Figure 6 As shown; the quantitative comparison analysis results of the number of lens vacuoles after treatment with 1‰ DMSO in the DMSO group and after treatment with 1 μM Baf-A1 in the Baf-A1 group are as follows. Figure 7 As shown.

[0047] This experiment found that pikfyve treated with Baf-A1... Δ8 In zebrafish, the number of vacuoles in the lens was significantly reduced compared to before treatment with Baf-A1 (Fig. 3(a) and Fig. 3(b)), and the difference was statistically significant (P = 0.0106). Figure 6 ); while pikfyve after DMSO treatment Δ8 Zebrafish showed an increase in the number of vacuoles in their lens (Fig. 2(a) and Fig. 2(b)), and the number of vacuoles before and after treatment was statistically significant (P < 0.0001). Figure 5 In the untreated blank control group, the number of lens vacuoles increased after 4.5 hours. Statistical analysis showed that the number of vacuoles before and after 4.5 hours was statistically significant (P = 0.023). Figure 4 The number of lens vacuoles after drug treatment in the DMSO group and the Baf-A1 group was compared and analyzed, and the results showed a significant statistical difference (P = 0.0004). Figure 7 The above data demonstrate that Baf-A1 has a reducing or rescuing effect on lens vacuoles and a definite therapeutic effect on lens opacity, i.e., cataract models. The DMSO group, on the other hand, considered pikfyve... Δ8 The zebrafish's lens opacity is in a developing stage (increased vacuoles). DMSO has no inhibitory effect on vacuoles, hence the increased number of vacuoles. The blank control group consisted of pikfyves. Δ8 The number of vacuoles in the lens of zebrafish also increased, which is likely related to the lens opacity being in a developmental stage.

[0048] In summary, this embodiment is based on the established pikfyve Δ8Based on the zebrafish model, Baf-A1 was used to intervene in the lens opacity of a zebrafish cataract model with impaired PIKfyve function. The results showed that after Baf-A1 treatment, the PIKfyve... Δ8 The zebrafish lens showed significant treatment; compared to the DMSO group and the blank control group, the number of lens vacuoles in the Baf-A1 group was significantly reduced. Statistical analysis showed a significant difference between the Baf-A1 group and the DMSO group, indicating that Baf-A1 has a rescue effect on lens opacity in the zebrafish cataract model.

[0049] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of bafloxacin A1 in the preparation of drugs for treating cataracts caused by PIKfyve dysfunction.

2. The use according to claim 1, characterized in that, The drug includes bafloxacin A1 and pharmaceutically acceptable excipients.

3. The use according to claim 1, characterized in that, The medication is an eye drop, gel, or subconjunctival implant.

4. The use according to claim 3, characterized in that, The molecular formula of the bafloxacin A1 is C 35 H 58 O9.

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