Application of cyclic adenosine monophosphate and metal salt thereof in preparation of external product for preventing or treating fungal keratitis

Abstract

The invention belongs to the technical field of fungal keratitis medicines. The invention provides application of cyclic adenosine monophosphate and metal salt thereof in preparation of an external product for preventing or treating fungal keratitis, aiming at the problems of poor permeability, low solubility, low bioavailability and possibility of generating toxic and side effects on cornea after long-term use of a clinically common local antifungal drug for treating fungal keratitis. As an antifungal active molecule for external administration, the cAMP has good water solubility, corneal permeability, broad-spectrum antifungal activity and biological safety. Compared with a traditional antifungal drug, the cAMP can be efficiently delivered to a cornea infection part and directly acts on pathogenic fungi by virtue of excellent water solubility and cornea permeability, so that the toxic and side effects on cornea tissues are reduced, and meanwhile, the bioavailability is improved. The cAMP shows good antifungal activity when the pH (Potential of Hydrogen) value is in a range of 4 to 7.5.

Description

Technical Field

[0001] The present invention belongs to the technical field of drugs for fungal keratitis, and particularly relates to the application of cyclic adenosine monophosphate and its metal salts in the preparation of external products for preventing or treating fungal keratitis. Background Art

[0002] Fungal keratitis (FK) is a corneal infection caused by pathogenic fungi, and the main pathogenic bacteria include Fusarium, Aspergillus, Candida, etc. The course of FK progresses rapidly, and in severe cases, it can lead to corneal perforation and even blindness. The incidence of FK disease shows an increasing trend year by year. Globally, especially in developing countries, FK has become one of the main causes of blinding eye diseases. At present, local antifungal drugs are mostly used for treatment. The drugs act directly on the infected site, can be administered frequently, and have few systemic side effects. Commonly used clinical antifungal drugs include natamycin, amphotericin B, voriconazole, etc. However, these drugs often have problems such as poor permeability, low solubility, and low bioavailability, and long-term use may cause toxic and side effects on the cornea.

[0003] Cyclic adenosine monophosphate (cAMP) is an important cellular messenger molecule that plays a key regulatory role in various biological processes. The clinical applications of cAMP mainly focus on the fields of cardiovascular, respiratory, metabolic, and immune diseases, and indirectly regulate the activity of pathways by targeting its synthase (AC), degrading enzyme (PDE), or receptor protein (such as PKA). In recent years, the role of the cAMP signaling pathway in fungal infections such as Candida albicans has gradually become a research hotspot. These studies are still based on the function of cAMP as an endogenous molecule in cell signal transduction, and there are few reports on its research as an exogenous antifungal active molecule in fungal infections, especially Fusarium infections. Summary of the Invention

[0004] Aiming at the above problems that the commonly used local antifungal drugs for treating fungal keratitis in clinic have poor permeability, low solubility, low bioavailability, and may cause toxic and side effects on the cornea after long-term use, the present invention provides the application of cyclic adenosine monophosphate and its metal salts in the preparation of external products for preventing or treating fungal keratitis. Experiments have proved that cAMP has both corneal permeability, water solubility, antifungal activity, and biological safety, providing a new option for the research and development of antifungal drugs.

[0005] The present invention provides the application of cyclic adenosine monophosphate and its metal salts as fungicides in the preparation of external products for preventing or treating fungal keratitis.

[0006] Furthermore, the pathogenic bacteria of the fungal keratitis are at least one of Fusarium, Aspergillus, Candida, and Staphylococcus.

[0007] Further, the content of cyclic adenosine monophosphate and its metal salts in the topical product is 0.01 - 10 mg / mL; more preferably 0.1 - 1 mg / mL.

[0008] Further, the topical product includes topical cleansing products and topical medications; more specifically, the forms of the topical product include but are not limited to eye washes, eye drops, eye ointments, and corneal repair gels.

[0009] Further, cyclic adenosine monophosphate and its metal salts are used in the preparation of topical products for preventing or treating fungal keratitis in the form of cyclic adenosine monophosphate nanoenzymes.

[0010] Further, the cyclic adenosine monophosphate metal salt is preferably sodium cyclic adenosine monophosphate.

[0011] Further, the preparation method of the cyclic adenosine monophosphate nanoenzyme is as follows: After mixing the metal precursor solution with the cyclic adenosine monophosphate solution, add a stabilizer and / or a reducing agent, react at room temperature, then centrifuge, and freeze-dry the upper precipitate to obtain the cyclic adenosine monophosphate nanoenzyme.

[0012] Further, the metal precursor solution is a soluble Fe salt, Co salt, Mn salt, Cu salt, or Sr salt; more specifically, it is a nitrate, acetate, or sulfate, preferably Sr(NO3)2, Mn(CH3COO)2, or CuSO4.

[0013] Further, a sodium citrate solution is used as the stabilizer and / or the reducing agent.

[0014] Further, slowly drop the metal precursor solution into the cyclic adenosine monophosphate solution, stir while dropping, and after dropping, obtain a mixed solution. Then slowly drop the stabilizer and / or the reducing agent into the obtained mixed solution, and continue to stir for 6 - 10 h for the reaction.

[0015] Further, the concentration of the cyclic adenosine monophosphate solution is 3 - 8 mg / mL; the concentration of the Sr(NO3)2 solution is 130 - 300 mg / mL; the concentration of the sodium citrate solution is 10 - 37 mg / mL.

[0016] Further, the mass ratio of cyclic adenosine monophosphate, Sr(NO3)2, and sodium citrate is (5 - 7):(80 - 120):(5 - 11); more preferably, the mass ratio is 3:50:5.

[0017] Further, centrifuge at a speed of 4500 - 5500 rpm for 5 - 15 minutes.

[0018] Further, dissolve the cyclic adenosine monophosphate in deionized water and adjust the pH to neutral to prepare eye drops; preferably, use NaOH or Na2CO3 to adjust the pH.

[0019] Or dissolve the obtained cyclic adenosine monophosphate nanozyme in deionized water to prepare a nanozyme eye drop.

[0020] The present invention has the following beneficial effects:

[0021] As an antifungal active molecule for external administration, cAMP has good water solubility, corneal permeability, broad-spectrum antifungal activity and biosafety. Compared with traditional antifungal drugs, cAMP can be efficiently delivered to the corneal infection site by virtue of its excellent water solubility and corneal permeability, directly act on pathogenic fungi, reduce the toxic and side effects on corneal tissue, and improve the bioavailability at the same time. cAMP shows good antifungal activity in the pH range of 4 - 7.5. The preparation method of cAMP nanozyme is simple and the preparation time is short. The cAMP nanozyme eye drop shows a broad-spectrum antifungal effect and has good preventive and therapeutic effects on corneal fungal infections in mice. Description of the Drawings

[0022] Figure 1 For the safety of cAMP, Figure A is a double staining image of live and dead cells of cAMP co-cultured with corneal epithelial cells (HCECs) and corneal stromal cells (HTKs); Figure B is the cell viability detected by a CCK-8 kit after treatment of HCECs and HTKs with low-concentration cAMP; Figure C is a comparison of cell viability after treatment of HCECs and HTKs with high-concentration cAMP and VOR; Figure D is a slit lamp photograph and fluorescein sodium staining image at different time points after instilling cAMP (10 mg / mL, 4 times a day, for 7 consecutive days) into the eyes of normal mice. Figure E is an H&E staining image of the cornea, retina, heart, liver, spleen, lung, kidney and brain of mice on the 7th day.

[0023] Figure 2 For the broad-spectrum antibacterial activity of cAMP, Figure A is the antibacterial effect of cAMP on Fusarium solani, Candida albicans, Staphylococcus aureus and Staphylococcus epidermidis, and Figure B is the result statistics of Figure A.

[0024] Figure 3 For the in vitro and in vivo corneal permeability of cAMP.

[0025] Figure 4 For the stability of cAMP.

[0026] Figure 5 For the ROS scavenging and antioxidant properties of cAMP.

[0027] Figure 6 For the corneal fungal infection situation in the cAMP group at 7D.

[0028] Figure 7 For the corneal OCT and H&E staining images at 7D.

[0029] Figure 8 It is a PAS staining map of corneal sections at 7D.

[0030] Figure 9 It is a transmission electron microscope and EDS element mapping image photo of cAMP-Sr nanozyme.

[0031] Figure 10 It is the in vitro antibacterial activity of cAMP-Sr nanozyme.

[0032] Figure 11 It is the ROS scavenging activity of cAMP-Sr nanozyme.

[0033] Figure 12 It is the transmission electron microscope images of cAMP-Mn nanozyme and cAMP-Cu nanozyme.

[0034] Figure 13 It is the in vitro antibacterial images of cAMP-Mn nanozyme and cAMP-Cu nanozyme against drug-resistant Staphylococcus aureus. Detailed implementation manners

[0035] The present invention will be further described in detail below in conjunction with specific embodiments and the accompanying drawings.

[0036] For those not specifying specific techniques or conditions in the embodiments, they shall be in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product specifications. For those reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through regular commercial channels.

[0037] (I) Preparation part

[0038] Product information of cAMP: The chemical formula is C 10 H 12 N5O6P, the molecular weight is about 329.21, the CAS registration number is 60-92-4, and it is in white or off-white powder form.

[0039] Example 1

[0040] Dissolve cAMP in deionized water and ultrasonically dissolve it until completely dissolved to obtain cAMP solutions with concentrations of 0.01 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 2 mg / mL, 4 mg / mL, 8 mg / mL, and 10 mg / mL respectively. Among them, the pH value of the 10 mg / mL cAMP solution is measured with test paper to be 5.

[0041] Example 2

[0042] Dissolve cAMP in deionized water, and adjust the pH to neutral with NaOH or Na2CO3 to obtain a cAMP solution with the desired concentration. Or prepare a salt solution with a neutral pH value using cAMP sodium salt and deionized water.

[0043] Example 3

[0044] (1) Add 60 mg of cAMP powder to a 50 mL round-bottom flask;

[0045] (2) Add 10 mL of water to the round-bottom flask in step (1) and stir until completely dissolved;

[0046] (3) Add 1000 mg of Sr(NO3)2 to a centrifuge tube;

[0047] (4) Add 8 mL of water to the centrifuge tube in step (3) and mix well until completely dissolved;

[0048] (5) Slowly add the solution in step (4) to the solution in step (2);

[0049] (6) Add 107 mg of sodium citrate to a centrifuge tube;

[0050] (7) Add 2 mL of water to the centrifuge tube in step (6) and mix well until completely dissolved;

[0051] (8) Slowly add the solution in step (7) to the solution in step (5) and continue stirring for 10 h;

[0052] (9) Centrifuge the mixture at 5000 rpm for 10 minutes;

[0053] (10) Freeze-dry the precipitate for 72 h to obtain cAMP-Sr nanozyme.

[0054] Example 4

[0055] (1) Add 25 mg of cAMP powder to a 50 mL round-bottom flask;

[0056] (2) Add 20 mL of water to the round-bottom flask in step (1) and stir until completely dissolved;

[0057] (3) Add 1300 mg of Mn(CH3COO)2·4H2O to a centrifuge tube;

[0058] (4) Add 6 mL of water to the centrifuge tube in step (3) and mix well until completely dissolved;

[0059] (5) Slowly add the solution in step (4) to the solution in step (2);

[0060] (6) Add 150 μL of 5% ammonia water to a centrifuge tube;

[0061] (7) Stir at room temperature for 10 h;

[0062] (8) Centrifuge the mixed solution at 5000 rpm for 10 minutes;

[0063] (9) After discarding the supernatant, add 30 mL of deionized water to resuspend the precipitate, and repeat step (8);

[0064] (10) Repeat step (9) twice;

[0065] (11) Freeze-dry the precipitate for 72 h to obtain cAMP-Mn nanozyme.

[0066] Example 5

[0067] (1) Add 60 mg of cAMP powder, 1750 mg of CuSO4·5H2O particles and 200 ml of deionized water to a 500 mL round-bottom flask in sequence;

[0068] (2) Stir at 600 r / min for 5 minutes until all solid particles are dissolved to form a homogeneous solution;

[0069] (3) Use 1.0 M NaOH solution to adjust the pH value of the solution in step (2) to 7.4;

[0070] (4) Heat the mixture in step (3) to 50 °C and stir for 3 h;

[0071] (5) Centrifuge the mixed solution at 5000 rpm for 10 minutes;

[0072] (6) After discarding the supernatant, add 30 mL of deionized water to resuspend the precipitate, and repeat step (5);

[0073] (7) Repeat step (6) twice;

[0074] (8) Freeze-dry the precipitate for 72 h to obtain cAMP-Cu nanozyme.

[0075] Dissolve the nanozymes prepared in Examples 3-5 with deionized water respectively to obtain corresponding nanozyme solutions.

[0076] (II) Experimental evaluation

[0077] 1. Safety of cAMP

[0078] In vitro safety: After co-culturing different concentrations of cAMP solution (Example 1) with corneal epithelial cells (HCECs) and corneal stromal cells (HTKs), perform live / dead cell double staining and CCK-8 experiments to detect cell viability.

[0079] In vivo safety: The cAMP solution (10 mg / mL, Example 2) was instilled into the eyes of healthy mice four times a day for 7 consecutive days. The anterior segment changes were observed with a slit lamp, and the changes in the eyeballs and important organs were observed by HE staining.

[0080] The results are as Figure 1 shown. In in vitro experiments, when the cAMP concentration was less than 1 mg / mL, the cell viability was not affected. And the effect of high-concentration cAMP on cell viability was significantly less than that of voriconazole (VOR) at the same concentration. In in vivo experiments, there were no changes in the anterior segment morphology within 7 days, and there was no corneal epithelial defect. The HE staining results showed that there were no abnormalities in the structures of the cornea, retina, heart, liver, spleen, lung, kidney, and brain. It shows that cAMP has good biological safety.

[0081] 2. Broad-spectrum antibacterial activity of cAMP

[0082] PBS and the cAMP solution (10 mg / mL, Example 2) were co-cultured with pathogenic bacteria for 12 - 24 h. The diluted bacterial solution was taken and spread on plates, and after culturing in an incubator for 24 h or 48 h, photos were taken for counting, and the antibacterial rate was analyzed. The antibacterial effects of cAMP against Fusarium solani (F. solani), Candida albicans (C. albicans), Staphylococcus aureus (S. aureus), and Staphylococcus epidermidis (S. epidermidis) were tested.

[0083] The results are as Figure 2 shown. In the control group, there were more or a large number of colonies growing for all 4 kinds of bacteria. The number of colonies of Fusarium solani in the cAMP group was significantly less than that in the control group, and there were no colonies growing for Candida albicans, Staphylococcus aureus, and Staphylococcus epidermidis.

[0084] 3. In vitro and in vivo corneal permeability of cAMP

[0085] In vivo corneal permeability experiment: One drop of the cAMP solution (10 mg / mL, Example 2) was instilled into the right eyes of 24 rabbits. At different time points after administration (0 h, 1 / 4 h, 1 / 2 h, 3 / 4 h, 1 h, 1.5 h, 2 h, 3 h), 3 rabbits were randomly selected, and 50.0 μL of aqueous humor was drawn from the right corneal limbus and mixed with an equal volume of methanol solution. The mixture was filtered through a 0.22 μm filter, and the cAMP concentration was detected by UPLC.

[0086] In vitro corneal permeability experiment: Fresh rabbit corneas were removed and placed into an in vitro permeation device. Solutions of different concentrations of cAMP (Example 2) were added above, and deionized water was added below. At different time points after drug administration (0 h, 1 / 4 h, 1 / 2 h, 3 / 4 h, 1 h, 1.5 h, 2 h, and then once every 1 h until 12 h), 100 μL of liquid was taken from below (supplemented with 100.0 μL of deionized water) and mixed with an equal volume of methanol solution. The mixture was filtered through a 0.22 μm filter, and the cAMP concentration was detected using UPLC.

[0087] The results are as Figure 3 shown. In the in vitro experiment, the corneal permeability of cAMP (10 mg / mL) reached 10% at 12 h; in the in vivo experiment, the drug concentration in the aqueous humor of the 1% cAMP group was significantly higher than that of the clinically commonly used antifungal drug 1% VOR. These results indicate that cAMP has good bioavailability.

[0088] 4. Stability of cAMP

[0089] The cAMP concentrations in freshly prepared cAMP solution (10 mg / mL, Example 2) and cAMP solution stored at 4 °C for 30 days were detected using UPLC. The results are as Figure 4 shown. After storing at 4 °C for 30 days, the drug concentration and in vitro antibacterial effect remained unchanged. This has a significant advantage compared with the clinical drug voriconazole, which needs to be prepared immediately before use.

[0090] 5. Scavenging of ROS and antioxidant properties

[0091] The ability of cAMP (Example 2) to scavenge ROS and its antioxidant properties were detected using a superoxide anion detection kit, a hydroxyl radical detection kit, and an ABST antioxidant kit, respectively.

[0092] The results are as Figure 5 shown. cAMP has the ability to scavenge superoxide anions and hydroxyl radicals and also has mild antioxidant properties.

[0093] 6. In vivo antibacterial activity of cAMP

[0094] A murine fungal keratitis model infected with Fusarium solani was established. On the first day of modeling, PBS and cAMP solution (10 mg / mL, Example 2) were instilled into the eyes 4 times a day for 7 days. Corneal slit-lamp photographs were taken on days 1, 3, 5, and 7 to observe the corneal inflammation; on day 3, the eyeballs were collected for PAS staining to observe the corneal fungal hyphae; on day 7, corneal OCT and HE staining were performed to observe the corneal thickness and inflammatory cell infiltration.

[0095] Figures 6 to 8 This is the in vivo antibacterial activity of cAMP. As Figure 6As shown, on the 7th day, corneal fungal infection in the cAMP group was significantly milder than that in the control group, and there was a statistical difference in clinical scores compared with the control group. As Figure 7 shown, on the 7th day, corneal OCT showed that the corneal thickness in the cAMP group was close to that of normal corneas, while the corneas in the control group were significantly thickened; H&E staining showed that the corneal thickness in the cAMP group was close to that of normal corneas and the inflammatory response was milder, while the corneal structure in the control group was disordered, edematous and thickened with more inflammatory cell infiltration. As Figure 8 shown, no fungi or hyphae were seen in the normal group. A large number of hyphae could be seen in the epithelium and stroma of the control group on the 3rd day, and a small number of hyphae could be seen in the epithelium and stroma of the cAMP group.

[0096] 8. Physical properties of cAMP-Sr nanozyme

[0097] The physical properties of the cAMP-Sr nanozyme prepared in Example 3 were characterized. As Figure 9 shown, the nanozyme had a cubic packing morphology with a diameter of about 145 μm.

[0098] 9. In vitro antibacterial activity of cAMP-Sr nanozyme

[0099] The broad-spectrum antibacterial activity detection method of cAMP was referred to.

[0100] The results were as Figure 10 shown. The colony count in the cAMP-Sr nanozyme treatment group was significantly less than that in the control group.

[0101] 10. ROS scavenging activity of cAMP-Sr nanozyme

[0102] The performance of cAMP-Sr nanozyme in scavenging ROS was detected using a superoxide anion detection kit and a hydroxyl radical detection kit, respectively.

[0103] The results were as Figure 11 shown. The ability of cAMP-Sr nanozyme to scavenge superoxide anions and hydroxyl radicals was significantly improved compared with cAMP ( Figure 5 ).

[0104] 11. Morphology and antibacterial properties of cAMP-Mn nanozyme and cAMP-Cu nanozyme

[0105] The morphology of cAMP-Mn nanozyme (Example 4) and cAMP-Cu nanozyme (Example 5) was observed by SEM. The in vitro antibacterial activity was referred to the antibacterial activity detection method of cAMP.

[0106] The results were as Figure 12 shown. The cAMP-Mn nanozyme had an irregular flake structure with a diameter of about 30 nm. The cAMP-Cu nanozyme had an irregular flake structure with a diameter of about 100 nm.

[0107] As Figure 13 shown, the antibacterial rates of cAMP-Mn and cAMP-Cu nanozymes against drug-resistant Staphylococcus aureus were both 100%.

[0108] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of cyclic adenosine monophosphate and its metal salts as fungicides in the preparation of external products for preventing or treating fungal keratitis.

2. The application according to claim 1, characterized in that The content of cyclic adenosine monophosphate and its metal salts in the external product is 1-10 mg / mL.

3. The application according to claim 1, characterized in that, The cyclic adenosine monophosphate metal salt is sodium cyclic adenosine monophosphate.

4. The application according to claim 1, characterized in that The external product is an external cleaning product or an external medicine.

5. The application according to claim 1, wherein The external product is an eye wash, eye drops, eye ointment or corneal repair gel.

6. The application according to any one of claims 1 to 5, characterized in that, The cyclic adenosine monophosphate and its metal salts are used in the form of nanozymes in the preparation of external products for preventing or treating fungal keratitis.

7. The application according to claim 6, wherein Dissolve the cyclic adenosine monophosphate in deionized water and adjust the pH to neutral to prepare eye drops, or dissolve the cyclic adenosine monophosphate nanozyme in deionized water to prepare nanozyme eye drops.

8. The application according to claim 6, wherein The preparation method of the nanozyme is: after mixing the metal precursor solution and the cyclic adenosine monophosphate solution, add a stabilizer and / or a reducing agent, react at room temperature, then centrifuge, and freeze-dry the precipitate to obtain the cyclic adenosine monophosphate nanozyme.

9. The application according to claim 8, characterized in that, The metal precursor solution is an Fe salt solution, a Co salt solution, a Mn salt solution, a Cu salt solution or an Sr salt solution; slowly drop the metal precursor solution into the cyclic adenosine monophosphate solution, stir while dropping, and obtain a mixed solution after dropping. Slowly drop the stabilizer and / or the reducing agent into the obtained mixed solution, continue to stir for 6-10 h for reaction, and centrifuge at a speed of 4500-5500 rpm for 5-15 minutes after the reaction ends.

10. The application according to claim 8, wherein, The metal precursor solution is an Sr(NO3)2 solution, a Mn(CH3COO)2 solution or a CuSO4 solution; use a sodium citrate solution as the stabilizer and / or the reducing agent; the concentration of the cyclic adenosine monophosphate solution is 3-8 mg / mL; the concentration of the Sr(NO3)2 solution is 130-300 mg / mL; the concentration of the sodium citrate solution is 10-37 mg / mL; the mass ratio of the cyclic adenosine monophosphate, Sr(NO3)2 and sodium citrate is (5-7):(80-120):(5-11).

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