Application of caerin1.1 / 1.9 in preparation of antiviral drugs

Abstract

The invention relates to the technical field of biological medicines, relates to applications of caerin1.1, caerin1.9 and caerin1.1 / 1.9 in preparation of medicines for preventing / treating viruses, and in particular relates to applications of caerin1.1, caerin1.9 and caerin1.1 / 1.9 in preparation of medicines for preventing / treating herpes simplex viruses. The herpes simplex virus disclosed by the invention is one or two of HSV-1 (Herpes Simplex Virus) or HSV-2 (Herpes Simplex Virus). The caerin1.1, the caerin1.9 or the composition of the caerin1.1 and the caerin1.9 disclosed by the invention has different degrees of prevention and treatment effects on HSV-1 and / or HSV-2, and has a more obvious effect on the HSV-2. Compared with an existing antiviral drug, the compound has a better effect of preventing and treating viruses, the prevention effect is obviously better than that of acyclovir, the application range is wide, and potential toxic and side effects are low.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of caerin 1.1, caerin 1.9, and caerin 1.1 / 1.9 in the preparation of drugs for the prevention / treatment of viral infections, and more specifically to the application of caerin 1.1, caerin 1.9, and caerin 1.1 / 1.9 in the preparation of drugs for the prevention / treatment of herpes simplex virus. Background Technology

[0002] Herpes simplex (HS) is a common viral infection, known in Traditional Chinese Medicine as "heat sores." It is primarily caused by two viruses: herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2). HSV-1 typically causes infection on the face, brain, and upper body, while HSV-2 mainly causes infection on the genitals and lower body. Herpes simplex can be classified as primary infection or recurrent infection based on the infection status.

[0003] Current treatments for herpes simplex primarily include antiviral medication and symptomatic treatment. Nucleoside antiviral drugs (such as acyclovir) are commonly used clinically, but long-term use can lead to drug resistance and significant side effects. Therefore, developing antiviral drugs with proven efficacy and fewer side effects is a focus of pharmaceutical research.

[0004] The Caerin1 peptide family consists of antimicrobial peptides secreted by the skin of the Australian tree frog, including F1 (Caerin1.1). F3 (Caerin 1.9), F1 (Caerin 1.1), and F3 (Caerin 1.9) were originally isolated from the skin secretions of the Australian tree frog (Litoria); they have antibacterial and antitumor growth activities.

[0005] There are no publicly available reports on the use of caerin 1.1, caerin 1.9, or caerin 1.1 / 1.9 in the prevention / treatment of HSV-1 and HSV-2 viruses. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the technical problem solved by the present invention is to provide the application of caerin 1.1, caerin 1.9, and caerin 1.1 / 1.9 in the preparation of antiviral drugs.

[0007] This invention is achieved through the following technical solution: Application of caerin 1.1, caerin 1.9, and caerin 1.1 / 1.9 in the preparation of drugs for the prevention / treatment of viruses.

[0008] Application of caerin 1.1, caerin 1.9, and caerin 1.1 / 1.9 in the preparation of drugs for the prevention / treatment of diseases caused by viruses.

[0009] Furthermore, the virus in question is herpes simplex virus.

[0010] Furthermore, the virus is one or both of HSV-1 or HSV-2.

[0011] Furthermore, the present invention provides the use of caerin 1.1, caerin 1.9, and caerin 1.1 / 1.9 in the preparation of medicaments for the prevention / treatment of skin diseases caused by herpes simplex virus.

[0012] The skin diseases mentioned include: herpes simplex, herpes vaginitis, and genital warts.

[0013] The caerin 1.1 / 1.9 is a composition of caerin 1.1 and caerin 1.9, wherein the mass ratio of caerin 1.1 to caerin 1.9 is 1:0.5-1:3, preferably 1:1-1:2.

[0014] When caerin 1.1 or caerin 1.9 is used alone, the concentration of caerin 1.1 is 0.2~1μM and the concentration of caerin 1.9 is 0.1~2μM. Preferably, the concentration of caerin 1.1 is 0.2~1μM and the concentration of caerin 1.9 is 0.5~2μM. At this concentration, the preventive effect is better.

[0015] Furthermore, when caerin 1.1 and caerin 1.9 are used in combination, the concentration of caerin 1.1 is 0.1~0.8 μM and the concentration of caerin 1.9 is 0.05~1.5 μM.

[0016] When caerin 1.1 at a concentration of 0.4–0.8 μM and caerin 1.9 at a concentration of 0.5–1.5 μM are used in combination, the inhibitory effect on HSV-1 is optimal and the therapeutic effect is strongest. When caerin 1.1 at a concentration of 0.8 μM and caerin 1.9 at a concentration of 1.5 μM are used in combination, the viral level drops to the level consistent with that of normal cells.

[0017] When caerin 1.1 at a concentration of 0.6–0.8 μM and caerin 1.9 at a concentration of 1–1.5 μM are used in combination, the inhibitory effect on HSV-2 is optimal, the therapeutic effect is strongest, and the viral level is reduced to the level consistent with that of normal cells.

[0018] The amino acid sequence of caerin1.1 is shown in SEQ ID No.1.

[0019] The amino acid sequence of caerin1.9 is shown in SEQ ID No.2.

[0020] SEQ ID No.1: GLLSVLGSVAKHVLPHVLPHVVPVIAEHL-NH2; SEQ ID No.2: GLFGVLGSIAKHVLPHVVPVIAEKL-NH2; The caerin 1.1, caerin 1.9, or combinations thereof (caerin 1.1 / 1.9) of the present invention can be prepared into pharmaceutical compositions with pharmaceutically acceptable carriers or excipients.

[0021] The pharmaceutical composition can be a clinically acceptable topical preparation, such as an ointment, gel, or patch.

[0022] This invention provides the use of caerin 1.1, caerin 1.9, or combinations thereof (caerin 1.1 / 1.9) in the preparation of drugs for the prevention / treatment of viruses, especially HSV-1 or HSV-2. In vitro experimental results show that these two peptides or combinations thereof have significant inhibitory effects on HSV-1 or HSV-2 viruses, especially under prophylactic administration conditions (1 hour and 2 hours before infection), where they outperform acyclovir control drug.

[0023] Compared with existing antiviral drugs, this polypeptide has the following advantages: 1. It exhibits good inhibitory effects on both HSV-1 and HSV-2, and can be used to prepare preparations for various skin diseases caused by HSV-1 or HSV-2; 2. In experimental designs for infection prevention, its inhibitory effect was significantly better than that of acyclovir; 3. It can be used for both prevention and treatment, and has a wide range of applications; 4. The peptides have a clear source and low potential toxicity. Attached Figure Description

[0024] Figure 1 The killing effect of F1 and F3 on Vero cells.

[0025] Figure 2 This refers to the cytotoxic effect of TeA on Vero cells.

[0026] Figure 3 To detect viral load in HSV-1 and HSV-2 infected cells by PCR after treatment administration, F1, F3, and F1F3 were used.

[0027] Compared with the normal control group, # indicates P<0.05, ## indicates P<0.01; compared with the virus control group, * indicates P<0.05, ** indicates P<0.01; compared with the TeA group, & indicates P<0.05, && indicates P<0.01.

[0028] Figure 4 To prevent the inhibition rate of HSV-1 and HSV-2 infected cells after administration, F1, F3, and F1F3 were measured.

[0029] Compared with the TeA group, & indicates P<0.05, and && indicates P<0.01.

[0030] Figure 5 This is a dynamic diagram showing the lesion development of herpes simplex skin disease in HSV-1 infected guinea pigs treated with F1F3 ointment in Example 5.

[0031] Figure 6 This is a dynamic graph showing the development of skin lesions in HSV-2-infected guinea pigs with herpes simplex vaginitis treated with F1F3 ointment in Example 6.

[0032] Figure 7 The histopathological examination results of F1F3 ointment in HSV-2-infected guinea pigs with herpes simplex vaginitis in Example 6 are shown.

[0033] A: Blank control group A♀08 stained with HE at 100×; B: Model control group B♀06 stained with HE at 100×; C: Positive control group C♀03, HE staining 100×; D: F1F3 low-dose group D♀07, HE staining 100×; E: F1F3 medium-dose group E♀05 HE stained 100×; F: F1F3 high-dose group F♀08 HE stained 100×. Detailed Implementation

[0034] The raw materials used in the following examples are as follows: F1: caerin1.1, Sino-Austrian Biomedical Technology (Guangdong) Co., Ltd.

[0035] F3: caerin 1.9, Sino-Austrian Biopharmaceutical Technology (Guangdong) Co., Ltd.

[0036] F1F3: A composition of caerin 1.1 and caerin 1.9, wherein the mass ratio of caerin 1.1 to caerin 1.9 is 1:1.

[0037] P3: Sino-Austrian Biomedical Technology (Guangdong) Co., Ltd.

[0038] Temporin A (TeA): Sino-Austrian Biomedical Technology (Guangdong) Co., Ltd.

[0039] Acyclovir: Guangdong Linen Pharmaceutical Research Institute Co., Ltd.

[0040] Cell line: Vero (African green monkey kidney cells); Source: Purchased from ATCC, LOT: 62488537.

[0041] HSV-1: Source: Purchased from ATCC, LOT: 60286834; HSV-2: ATCC (American Standard Biological Collection Center).

[0042] Preparation of F1 stock solution: Weigh 0.0112g (peptide content 89.5%) of F1 powder, dissolve it in 1mL of PBS to make a 10mg / mL LF1 peptide solution, and let it stand overnight at 4℃ to allow it to fully dissolve.

[0043] Preparation of F3 stock solution: Weigh 0.0111g (peptide content 89.8%) of F2 powder, dissolve it in 1mL of PBS to make a 10mg / mL LF3 peptide solution, and let it stand overnight at 4℃ to allow it to fully dissolve.

[0044] Example 1: Cytotoxicity test of F1 and F3 against Vero After the cells in the 96-well plates grew into a monolayer, a cytotoxicity assay was performed. The wells included a control group, F1 group, F3 group, P3 control group, and Temporin A group. The control group received only complete culture medium to eliminate the influence of the culture medium on absorbance measurement. The cell seeding density was 5×10⁻⁶ cells / well. 4 Cells / mL were added to each well of all groups except the control group. After overnight incubation, 100 mL of drug solution and 100 mL of complete culture medium were added according to the group design, and the cells were incubated for 24 hours. CCK-8 assay was then performed, with absorbance (OD) measured at 450 nm using a microplate reader. Experimental groups and dosages are shown in Table 1.

[0045] The concentrations of F1 and F3 were 0.039–20 μM, the concentration of TeA was 0.49–250 μM, and the concentration of P3 was 50 μg / mL.

[0046] Cell viability (%) = (mean OD value of the drug treatment group - mean OD value of the blank group) / (mean OD value of the normal cell control group - mean OD value of the blank group) × 100%, inhibition rate (%) = 1 - cell viability (%).

[0047] Reed-Muench method for calculating half-maximal inhibitory concentration (IC50) 50 ).

[0048] Distance ratio = (inhibition rate above 50% - 50%) / (inhibition rate above 50% - inhibition rate below 50%) IC 50 Logarithm = Log(concentration with an inhibition rate higher than 50%) + distance ratio × logarithm of dilution factor.

[0049] The cytotoxicity of F1, F3, and TeA to Vero is shown in Table 2. Figure 1 and Figure 2 As shown.

[0050] Table 1. Grouping and Dosage of Cytotoxicity Tests

[0051] Table 2. Killing effects of F1, F3 and TeA on Vero cells.

[0052] The results showed that the cytotoxicity of F1, F3, and TeA was manifested by cell shrinkage, rounding, and detachment, and their absorbance values ​​also decreased significantly. The absorbance values ​​measured by the CCK8 assay showed that F1, F3, and TeA had low cytotoxicity to Vero cells. 50 The values ​​were 1.135 μM, 1.723 μM, and 39.30 μM, respectively.

[0053] Example 2: In vitro antiviral assays of F1, F3, and F1F3—therapeutic administration After the cells in the 96-well plates had grown into a monolayer, an in vitro antiviral assay was performed. The viral challenge dose was 30-100 TCID. 50 After the virus adsorbed onto monolayer cells for 1-2 hours, the virus solution was aspirated, and 100 μL of drug solution and 100 μL of maintenance solution were added to each well. A virus control group, a positive drug control group, and a blank cell control group were also included, with three replicates for each group. The cells were incubated at 35-37℃ in a 5% CO2 incubator. The lesions were observed daily under an inverted microscope for several days, and the cell pathogenesis (CPE) of each well was recorded. When the lesions in the virus control group reached "++++" and the normal cell control group showed no lesions, a photograph was taken to determine the optimal time point for CCK-8 assay. CCK-8 assay was performed, and the absorbance (OD value) was measured at 450 nm using a microplate reader. The inhibition rate of the drug group against the virus was calculated from the absorbance value. Simultaneously, PCR-fluorescent probe method was used to detect the virus content. Experimental groups and dosages are shown in Table 3.

[0054] Table 3 Antiviral test groupings and dosages

[0055] The results of PCR fluorescent probe detection to determine the viral load of samples after HSV-1 and HSV-2 cell therapy are shown in Table 4.

[0056] Table 4. Viral content after cell therapy for HSV-1 and HSV-2 infections using F1, F3, and combination therapy.

[0057] The results showed that 0.2-1 μM F1, 0.1-2 μM F3, and the combined use of F1 (0.1-0.8 μM) and F3 (0.05-1.5 μM) (in ratios of 8:15, 3:5, 4:5, and 2:1, respectively) at final concentrations were effective against HSV-1 virus. PCR-fluorescent probe assays showed that 2 hours after HSV-1 virus infection of cells, different concentrations of F1, F3, the combined use of F1 and F3, and Temporin A were added for treatment. The Ct value of the virus control group was 19.67. The Ct values ​​of the 1μM F1, 2μM F3, and combined F1 (0.4–0.8μM) and F3 (0.5–1.5μM) treatment groups were 25.79, 27.93, 30.35, 27.25, and 25.12, respectively, representing increases of approximately 6.12, 8.26, 10.68, 7.58, and 5.45. The viral level in the combined F1 (0.8μM) and F3 (1.5μM) treatment group decreased to levels consistent with the normal cell control. PCR detection of Temporin A at concentrations ranging from 0.1μM to 2μM showed no significant increase in viral Ct values ​​compared to the virus control group. These results indicate that F1, F3, and their combined use can all significantly inhibit HSV-1 viral replication.

[0058] In addition, 0.2~1μM F1, 0.1~2μM F3, and a combination of F1 (0.1~0.8μM) and F3 (0.05~1.5μM) (in ratios of 8:15, 3:5, 4:5, and 2:1) were used to treat HSV-2 virus. PCR-fluorescent probe results showed that 2 hours after HSV-2 virus infection of cells, different concentrations of F1, F3, a combination of F1 and F3, and Temporin A were added for treatment. The viral load in the control group was 8.27 μg copies / mL. The viral loads in the 1 μM F1, 2 μM F3, and combined F1 (0.6–0.8 μM) and F3 (1–1.5 μM) groups were 6.60, 6.44, 5.96, and 6.02 μg copies / mL, respectively, representing decreases of approximately 1.67, 1.83, 2.31, and 2.25 μg copies / mL. The combined F1 (0.6–0.8 μM) and F3 (1–1.5 μM) group showed a viral level consistent with the normal cell control. PCR detection of Temporin A at concentrations of 0.1–2 μM showed no decrease in viral load compared to the control group. These results indicate that F1, F3, and their combined use can significantly inhibit HSV-2 viral replication.

[0059] Example 3: In vitro antiviral assays of F1, F3, and F1 / F3 – prophylactic administration After the cells in the 96-well plate have grown into a monolayer, the test substance or positive control is mixed with a certain amount of virus solution to achieve the set final concentration of the test substance, and the viral challenge dose is 30-100 TCID. 50 The cells were then infected. Culture medium served as a blank control (normal cell control group), a negative control (virus control group), and a positive control group, with three replicates per group. One to two hours after the virus adsorbed onto the monolayer of cells, the virus solution was aspirated. When more than half of the cells in the virus control group showed CPE (i.e., lesions reached "+++"), photographs were taken, and cell viability was detected using the CCK-8 assay. Experimental groups and dosages were as shown in Table 3.

[0060] Viral inhibition rate = (Drug group OD) 450 -Virus control group OD 450 ) / (OD of normal cell control group) 450 -Virus control group OD 450 ) × 100%.

[0061] The results of prophylactic administration at 1 hour and 2 hours are shown in Table 5 (1 hour after prophylactic administration) and Table 6 (2 hours after prophylactic administration), respectively. Table 5. Inhibition rates of F1, F3 and combined medications against HSV-1 and HSV-2 viruses (1 hour after prophylactic administration)

[0062] Table 6. Inhibition rates of F1, F3 and combined medications against HSV-1 and HSV-2 viruses (2 hours after prophylactic administration)

[0063] The results showed that the inhibitory effects of F1, F3, and F1F3 on HSV-1 were as follows: 1 hour or 2 hours after prophylactic administration. Prophylactic administration (1 h): F1 at concentrations ranging from 0.2 μM to 1 μM showed inhibitory effects on HSV-1 virus according to CPE and CCK-8 results, with inhibition rates of 29.1%–55.9%. F3 at concentrations ranging from 0.5 μM to 2 μM showed inhibitory effects on HSV-1 virus according to CPE and CCK-8 results, with inhibition rates of 29.7%–69.3%, both higher than the equivalent dose Temporin A group. Combined administration of F1 (0.1–0.8 μM) and F3 (0.05–1.5 μM) showed inhibitory effects on HSV-1 virus according to CPE and CCK-8 results, with inhibition rates of 34.6%–60.9%. Temporin A at concentrations ranging from 0.1 μM to 1 μM showed inhibitory effects on HSV-1 virus according to CPE and MTT results, with inhibition rates of 19.8%–51.9%.

[0064] Prophylactic administration (2h): F1 showed inhibitory effects on HSV-1 virus in the CPE and CCK-8 results within the concentration range of 0.2μM–1μM, with an inhibition rate of 37.1%–55.8%. F3 showed inhibitory effects on HSV-1 virus in the CPE and CCK-8 results within the concentration range of 0.1μM–2μM, with an inhibition rate of 27.6%–55.8%, and the virus inhibition rate was higher than that of the equal-dose Temporin A group in the 1μM–2μM concentration range. Combined administration of F1 (0.1–0.8μM) and F3 (0.05–1.5μM) showed inhibitory effects on HSV-1 virus in the CPE and CCK-8 results, with an inhibition rate of 46.2%–73.4%, higher than the single-dose group and the Temporin A group. Temporin A showed inhibitory effects on HSV-1 virus in the CPE and MTT results within the concentration range of 0.1μM–2μM, with an inhibition rate of 9.5%–45.8%.

[0065] When F1 and F3 are used in combination, a good inhibition rate can be achieved even at low concentrations, such as F1+F3 (0.1+0.05μM).

[0066] The inhibitory effects of F1, F3, and F1F3 on HSV-2 after 1 or 2 hours of prophylactic administration are as follows: Prophylactic administration (1 h): F1 showed inhibitory effects on HSV-2 virus in the CPE and CCK-8 results within the concentration range of 0.2 μM–1 μM, with inhibition rates of 19.6%–43.3%. F3 showed inhibitory effects on HSV-2 virus in the CPE and CCK-8 results within the concentration range of 0.5 μM–2 μM, with inhibition rates of 18.5%–63.4%, significantly higher than the equivalent dose Temporin A group in the 1 μM–2 μM concentration range. Combined administration of F1 (0.1–0.8 μM) and F3 (0.05–1.5 μM) showed inhibitory effects on HSV-2 virus in the CPE and CCK-8 results, with inhibition rates of 22.3%–64.9%. Temporin A showed some inhibitory effects on HSV-2 virus in the CPE and MTT results within the concentration range of 0.1 μM–2 μM, with inhibition rates of 17.2%–24.9%.

[0067] Prophylactic administration (2h): F1 showed inhibitory effects on HSV-2 virus in the CPE and CCK-8 results within the concentration range of 0.2μM–1μM, with inhibition rates of 28.0%–50.9%. F3 showed inhibitory effects on HSV-2 virus in the CPE and CCK-8 results within the concentration range of 0.5μM–2μM, with inhibition rates of 23.6%–66.4%, and was higher than the equivalent dose Temporin A group in the 1.5μM–2μM concentration range. Combined administration of F1 (0.1–0.8μM) and F3 (0.05–1.5μM) showed inhibitory effects on HSV-2 virus in the CPE and CCK-8 results, with inhibition rates of 52.0%–69.9%. Temporin A showed inhibitory effects on HSV-2 virus in the CPE and MTT results within the concentration range of 0.1μM–2μM, with inhibition rates of 31.5%–54.1%.

[0068] When F1 and F3 are used in combination, a good inhibition rate can be achieved even at low concentrations, such as F1+F3 (0.1+0.05μM) and (0.2+0.1μM).

[0069] Example 5: Experimental Study on the Effect of F1F3 Ointment on Herpes Simplex Virus-1 in Guinea Pigs Preparation of F1F3 ointment: Using light liquid paraffin, white petrolatum, solid paraffin, white beeswax, isopropyl myristate, and glyceryl monostearate and glyceryl distearate as the matrix, and a combination of F1 and F3 as the main pharmaceutical ingredient, the content of both F1 and F3 is above 95%. The mass ratio of F1 to F3 is 1:1. F1F3 ointment is prepared according to conventional methods to obtain F1F3 ointment 1-3 (concentrations of 1%, 2%, and 3%, respectively). In F1F3 ointment 1, each 10 grams of ointment contains 0.1g of F1 and 0.2g of F1 and 0.2g of F3; in F1F3 ointment 3, each 10 grams of ointment contains 0.3g of F1 and 0.3g of F3.

[0070] Imiquimod cream: Specifications: 250mg: 12.5mg.

[0071] Blank ointment: A blank ointment was prepared using light liquid paraffin, white petrolatum, solid paraffin, white beeswax, isopropyl myristate, and glyceryl mono- and di-stearate as a base, according to conventional methods.

[0072] Guinea pig: SPF grade (Guangdong Provincial Medical Laboratory Animal Center).

[0073] Experimental grouping: Sixty qualified guinea pigs for adaptation observation were randomly divided into 6 groups according to body weight, with 10 guinea pigs in each group (half male and half female). The groups were: blank control group, model control group, positive control group, low-dose F1F3 ointment group (ointment 1), medium-dose F1F3 ointment group (ointment 2), and high-dose F1F3 ointment group (ointment 3). The grouping and dosage of each experimental group are shown in Table 7.

[0074] Table 7. Summary of Groups and Dosage Design

[0075] Modeling: Except for the blank control group, all other groups used HSV-1 herpes simplex virus to establish a model. Hair was removed from the backs of guinea pigs using a razor or depilatory cream, covering an area of ​​approximately 4cm × 4cm. Residual hair or depilatory cream was washed away with 0.9% sodium chloride injection. Local anesthesia was achieved by applying 2%–5% lidocaine hydrochloride injection to the areas to be punctured. After disinfecting the punctured skin with povidone-iodine, sterile No. 7 needles were used to create punctures on both sides of the punctured skin, resulting in slight bleeding, with lesions approximately 2cm × 2cm in size. 50μL of HSV-1 virus solution was applied to each punctured area, and the infection was induced by friction, allowing the virus to penetrate the skin.

[0076] Administration and observation: On the second day after modeling, each group was given local administration according to the dosage set in Table 7.

[0077] Administration route and dosage: Starting on the second day of modeling, apply the drug topically to the skin lesion area on the back of the guinea pig. The corresponding treatment area is approximately 2cm x 2cm. Apply once daily, covering with weighing paper, then with sterile gauze and secured with a bandage. Remove the medication after 6 hours of contact, unbandage, and gently wipe with sterile saline. Continue treatment for 14 consecutive days. Observe and record skin lesions daily.

[0078] Group A (blank control group) received no medication.

[0079] Group B (model control group) animals were given blank ointment.

[0080] Group C (positive control group) received 5% imiquimod emulsion at a dose of 0.625 mg / cm³. 2 (Based on imiquimod), approximately equivalent to the intended adult clinical dosage (3g of cream per tube, covering an area of ​​240 cm²). 2 The number of warts is 1 times that of the lesions.

[0081] Groups D, E, and F (low, medium, and high doses of the test substance) were given the corresponding doses of F1F3 ointment 1-3.

[0082] Skin lesion scoring criteria: Redness of the skin is 0.5 points; 1-3 blisters are 1 point; 3-5 blisters are 2 points; 6-10 blisters are 3 points; more than 10 blisters are 4 points; 20%-40% crusting deducts 1 point; 50%-100% crusting deducts 2 points; 10%-50% crust removal is 0.5 points; 60%-100% crust removal is grade 0. Clinical scoring results are shown in Table 8 and... Figure 5 .

[0083] Table 8. Effects on clinical skin scores in guinea pigs ( )

[0084] Table 8 (continued) Effects on clinical skin scores in guinea pigs ( )

[0085] Table 8 (continued) Effects on clinical skin scores in guinea pigs ( )

[0086] Clinical scoring results showed that no abnormalities were observed in the blank control group animals during the modeling period (D1-D14). After HSV-1 virus inoculation via puncture wounds, the modeled guinea pigs all developed typical skin herpes lesions: on D1, skin infection and redness / swelling were visible; on D3, vesicles of varying sizes began to appear in the model control group and all drug-treated groups; from D3 to D9, the clinical scores of the model control animals were significantly higher than those of the blank control group (P < 0.05 or 0.01). With prolonged observation, the clinical scores of the infected animals showed a trend of first increasing and then decreasing, reaching a peak in skin herpes lesions on D6-D7. Later, the animals entered a self-limiting phase, with the redness and swelling at the lesions beginning to subside, and some vesicles forming crusts.

[0087] The clinical scores of the test drug and positive control groups showed the same trend as the model group, initially increasing and then decreasing. The clinical scores of the low (1%), medium (2%), and high (3%) dose groups of the test drug peaked on day 7, slightly delaying the development of herpes symptoms compared to the model control group (peaking on day 6). From the onset of vesicle emergence in the model animals to the peak of herpes symptoms, the clinical scores of the low, medium, and high dose groups of the test drug were all lower than those of the model control group, indicating milder symptoms and a certain dose-response relationship. Specifically, the clinical scores of the medium dose group (days 4 and 5 after administration) and the high dose group (days 3, 4, and 8 after administration) showed significant improvement compared to the model control group, with statistically significant differences (P < 0.05). The clinical scores of the positive control group on days 3-5, 7, and 8 after administration were also significantly improved (P<0.05 or 0.01), and the clinical score on day 9 was higher than that of the model control group (P<0.01). This may be related to the adverse reactions of imiquimod to broken skin, including mild to moderate local skin inflammation reactions such as erythema, edema, erosion, and ulceration.

[0088] The experimental results above indicate that, under the conditions of this experiment, F1F3 ointment at concentrations of 2% and 3% (calculated as F1F3) significantly improved the blistering symptoms of HSV-1-infected guinea pigs.

[0089] Example 6: Experimental Study on the Effect of F1F3 Ointment on Herpes Simplex Vaginitis of the Skin in HSV-2 Infected Guinea Pigs The preparation of blank ointment and F1F3 ointment is the same as in Example 5.

[0090] Laboratory animals: Guinea pig: SPF grade (Guangdong Provincial Medical Laboratory Animal Center).

[0091] Experimental grouping: Sixty female guinea pigs that passed the adaptation observation were randomly divided into 6 groups of 10 each: a positive control group, a model control group, a blank control group, a low-dose F1F3 ointment group (Ointment 1), a medium-dose F1F3 ointment group (Ointment 2), and a high-dose F1F3 ointment group (Ointment 3). The grouping and dosage of each experimental group are shown in Table 9.

[0092] Table 9. Summary of Groups and Dosage Design

[0093] Modeling: Except for the blank control group, the treatment group used HSV-2 herpes simplex virus to establish the model. First, the vulva was cleaned with physiological saline, and the vagina was rubbed several times with a sterile cotton swab. Then, the vulva of the guinea pig was gently tapped with a plum blossom needle to induce vulvar hyperemia and slight bleeding, which was wiped clean with a sterile cotton swab. 0.15 ml of HSV-2 virus solution was drawn into a 1 ml syringe, fitted with a gavage needle, and inserted into the guinea pig's vagina about 2-3 cm (near the vaginal fornix). The virus was injected into the vaginal fornix and then slowly withdrawn. A gelatin sponge was inserted into the vagina to maintain the virulence. A small amount of virulence was dripped from the needle tip onto the vulva and rubbed with a glass rod to allow the virus to penetrate the skin.

[0094] Administration and Observation: On the second day after modeling, each group was administered medication according to the dosages set in Table 9. The blank control group received no medication, the model control group received blank ointment, and the positive control group and the F1F3 ointment low, medium, and high dose groups were administered once daily for 13 consecutive days. The systemic and vulvar symptoms of the guinea pigs were observed and scored daily. Clinical scoring results are shown in Table 10 and... Figure 6 .

[0095] The scoring method followed Kern's method. The development of lesions on the external genitalia was observed for 14 consecutive days after viral inoculation. The severity of the lesions was scored as follows: 0, asymptomatic; 0.5, redness and swelling without injury; 1.5, single small vesicle (<2mm); 2.0, single large vesicle (>2mm); 2.5, multiple small vesicles and / or vaginal ulcers (bleeding); 3.0, multiple large vesicles; 3.5, severe vulvar swelling; 4.0, multiple small / large vesicles fused together; 4.5, hind limb paralysis; 5.0, vulvar ulcer.

[0096] Table 10. Vulvar symptom scores of guinea pigs infected with HSV-2 virus in each group (x±S, n=10)

[0097] Simultaneously, vaginal secretions were collected on days 1, 3, 5, 7, 9, 11, and 14 after viral inoculation to determine the virulence of the virus in the vaginal secretions. Weighing was performed every 3 days. The day after the last administration, vaginal mucosal tissue was taken for fixation, embedding, serial paraffin sectioning, hematoxylin-eosin (HE) staining, and microscopic observation of the pathological condition of the vaginal mucosal tissue.

[0098] Clinical scoring results of guinea pig vulva showed that no abnormalities were observed in the blank control group animals during clinical observation from D1 to D14 after modeling. In the model control group, guinea pigs developed significant redness, swelling, vesicles, and ulcers on the vulva after modeling, with clinical scores peaking on D6-D7, indicating successful establishment of the guinea pig herpes simplex vaginitis model. In the positive control group, clinical scores from D7 to D10 were significantly lower than those of the model control group. Clinical scores in the low, medium, and high dose groups of F1F3 ointment peaked on D7. Compared with the model control group (peaking on D6), the development of herpes symptoms was slightly delayed. From the onset of vesicles to the peak of herpes symptoms, the clinical scores of all dose groups of the test substance were lower than those of the model control group, indicating milder symptoms. Clinical scores from D7 to D11 were significantly lower than those of the model control group, shortening the symptom healing time.

[0099] The viral load results of guinea pig vaginal secretions showed that the highest viral load was observed in the D1 group of animals in the model control group, and the viral load decreased over time. The viral load gradually decreased. In the low-dose F1F3 ointment group, the viral load reached its peak on day 3, while in the medium-dose F1F3 ointment group, the viral load decreased on day 5. The viral load reached its peak in the high-dose F1F3 ointment group, with the highest viral load in D1. As time progressed, the viral load decreased to the detection limit (Table 11).

[0100]

[0101] Histopathological examination results are shown in Figure 7 The results showed that the vaginal skin cell layer of the negative control group guinea pigs was intact, without edema or inflammatory cell infiltration. After injecting HSV-2 herpes simplex virus into the vagina of guinea pigs to establish a guinea pig herpes simplex vaginitis model, the vaginas of the model control group animals showed varying degrees of edema in the submucosal lamina propria, and inflammatory cell infiltration in the mucosal layer and submucosal lamina propria. The inflammatory cells were mainly granulocytes and lymphocytes, indicating that the model was successfully established.

[0102] The vaginal lesions in the model control group and each treatment group are as follows: Comparison of the degree of inflammatory cell infiltration in the vaginal mucosa and submucosal lamina propria among the groups: Positive control group > F1F3 ointment medium-dose group > F1F3 ointment low-dose group > F1F3 ointment high-dose group > Model control group. Compared with the model control group, the degree of mucosal inflammatory cell infiltration in the positive imiquimod control group was the most severe, which is consistent with the local skin inflammation adverse reaction that occurred clinically after several doses. The degree of inflammatory cell infiltration in the F1F3 ointment group was milder than that in the imiquimod group, suggesting that it has a certain degree of irritation to the vaginal mucosa tissue with prolonged administration. Comparison of the degree of edema in the vaginal submucosal lamina propria among the groups: Except for the negative control group, no significant differences were observed among the groups.

[0103] Based on clinical observation results, the herpes symptoms in all groups were in the healing stage at the end of the administration period. The healing time of the symptoms in each group was different in the early stage of the model development. The efficacy of the test substance was evaluated by combining the herpes symptom score.

[0104] In summary, under the conditions of this study, F1F3 ointment at concentrations of 1%, 2%, and 3% (calculated as F1F3) significantly improved the herpes symptoms of HSV-2-infected guinea pigs with herpetic vaginitis and shortened the healing time. Furthermore, it showed fewer adverse reactions compared to the positive control group.

[0105] Example 7: The therapeutic effect of F1F3 ointment on condyloma acuminata (1) Using the guinea pig herpes simplex animal model as an alternative test for the pharmacodynamics of condyloma acuminata. Genital warts (CA) are a sexually transmitted disease caused by the human papillomavirus (HPV). They commonly occur in the anus and external genitalia, and are one of the most common STIs. HPV can be classified into two types based on the site of infection: dermatotropic and mucotropic. The former primarily infects the skin, causing warts and verrucous epidermal dysplasia, while the latter mainly affects the mucous membranes of the reproductive and respiratory tracts, causing genital warts, laryngeal papillomas, and cervical ectropion. Typical early CA often presents as light brown, skin-colored, or light red hemispherical papules with a smooth surface. As it progresses, it gradually increases to the size of a grain of rice, becoming soft, and its number gradually increases from single to multiple. The surface becomes uneven, resembling papillary, cauliflower-like, cockscomb-like, or granular shapes. There may be a small amount of exudate on the surface, with an odor, and it bleeds upon touch, appearing white or grayish-white.

[0106] Herpes simplex virus (HSV) infection is a common viral infection in humans, frequently affecting the skin and mucous membranes, most commonly the face and genitals. Clinical manifestations include erythema, papules, papulovesicles, vesicles, and erosions, which may be accompanied by itching or pain. The course of the disease is self-limiting, typically resolving spontaneously within 1-2 weeks. The lesions such as erythema, swelling, and vesicles are caused by epidermal cell damage due to infection and the body's immune response to the virus, while crusting is the healing process following these lesions. Therefore, when evaluating the efficacy of anti-HSV drugs, the primary criterion should be the appearance of papulovesicles and vesicles, while crusting can be used as an auxiliary criterion for assessing prognosis.

[0107] Based on the rash characteristics and symptoms of condyloma acuminata and herpes simplex virus, the guinea pig herpes simplex animal model described above can be used as an alternative model for the pharmacodynamics of condyloma acuminata in this experiment to evaluate the therapeutic effect of F1F3 ointment on condyloma acuminata. The experimental methods and results are as described in Example 5.

[0108] Experimental results showed that F1F3 ointment at concentrations of 2% and 3% (calculated as F1F3) significantly improved the blistering symptoms of HSV-1-infected guinea pigs. Based on these results, it can be inferred that F1F3 ointment has an ameliorative effect on skin lesions with similar characteristics to condyloma acuminata.

[0109] (2) Using the guinea pig herpes simplex vaginitis animal model as an alternative test for the pharmacodynamics of condyloma acuminata. Genital warts commonly occur around the genitals in both women and men, such as the inner labia minora, urethral opening, vagina, glans penis, and crown. Typical changes associated with condyloma acuminata are usually seen on the mucous membranes or moist areas such as the sulcus and perianal region. Early cervical lesions are bright red. Small papules initially appear, then gradually develop into rough, uneven, grayish-white flat papules, with exudate, bleeding, and erosion on the surface. Clinical symptoms. In a guinea pig vaginal herpes model, redness, swelling, and herpes also appear at the site of vaginal infection.

[0110] Therefore, based on the characteristics and symptoms of rashes of condyloma acuminata and herpes simplex virus, the guinea pig herpes simplex vaginitis animal model can be used as an alternative model for the pharmacodynamics of condyloma acuminata to evaluate the therapeutic effect of F1F3 ointment on condyloma acuminata. The experimental methods and results are shown in Example 6.

[0111] F1F3 ointment at concentrations of 1%, 2%, and 3% (calculated as F1F3) significantly improved the herpes symptoms of HSV-2-infected guinea pigs with herpes simplex vaginitis and shortened the healing time. Based on these results, it can be inferred that F1F3 ointment has an ameliorative effect on skin lesions with similar characteristics to condyloma acuminata.

Claims

1. Application of caerin 1.1, caerin 1.9, and caerin 1.1 / 1.9 in the preparation of drugs for the prevention / treatment of viruses.

2. The application according to claim 1, characterized in that, In the caerin1.1 / 1.9 mixture, the mass ratio of caerin1.1 to caerin1.9 is 1:0.5 - 1:3, preferably 1:1 - 1:

2.

3. The application according to claim 1 or 2, characterized in that, The virus in question is herpes simplex virus.

4. The application according to claim 3, characterized in that, The herpes simplex virus mentioned is one or both of HSV-1 or HSV-2.

5. Application of caerin 1.1, caerin 1.9, and caerin 1.1 / 1.9 in the preparation of drugs for the prevention / treatment of skin diseases caused by herpes simplex virus.

6. The application according to claim 5, characterized in that, The skin diseases mentioned are herpes simplex, herpes vaginitis, and genital warts.

7. The application according to claim 1, 2, or 5, characterized in that, The amino acid sequence of caerin1.1 is shown in SEQ ID No.

1.

8. The application according to claim 1, 2 or 5, characterized in that, The amino acid sequence of caerin1.9 is shown in SEQ ID No.

2.

9. The application according to any one of claims 1-8, characterized in that, The caerin 1.1, caerin 1.9, or caerin 1.1 / 1.9 are prepared into a pharmaceutical composition with a pharmaceutically acceptable carrier or excipient.

10. The application according to claim 9, characterized in that, The pharmaceutical composition is a topical preparation, preferably an ointment, patch, or gel.

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