Use of bioactive protein in preparation of drug for treating herpes virus infection and pharmaceutical composition for treating herpes virus infection

Abstract

Provided are use of a bioactive protein in the preparation of a drug for treating herpes virus infection and a pharmaceutical composition for treating herpes virus infection. The pharmaceutical composition mainly comprises a bioactive protein, a matrix, a humectant, a pH regulator, and a preservative. The present invention has the following technical effects: 1) By means of a large amount of research and experimental verification, the inventors have discovered for the first time that anhydridized lactoferrin, a modified bioactive protein, has an inhibitory effect on herpes zoster virus (VZV). 2) In the pharmaceutical composition, the type and amount of excipient and the proportion of the main ingredient are reasonable. The pharmaceutical composition is administered in ointment form, in spray form, or in other dosage forms suitable for external use, has a significant therapeutic effect, has small local toxic side effects, has very low acute dermal toxicity, causes no irritation or allergy, does not interfere with normal skin functions, and is safe and effective.

Description

Use of bioactive protein in preparation of medicine for treating herpes virus infection and medicine composition for treating herpes virus infection TECHNICAL FIELD

[0001] The present application relates to use of bioactive protein in preparation of medicine for treating herpes virus infection and medicine composition for treating herpes virus infection, and belongs to the technical field of medicine. BACKGROUND

[0002] Herpes viruses are a kind of viruses with envelope and double-stranded DNA genome. Currently, eight kinds of herpes viruses that can infect human beings have been found, including herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), varicella zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpes virus 6 (HHV-6), human herpes virus 7 (HHV-7) and Kaposi's sarcoma-associated herpesvirus (KSHV). Patients and healthy carriers are the sources of infection, and the infection is mainly transmitted through direct close contact and sexual contact. HSV invades the body through various routes such as oral cavity, respiratory tract, genital mucosa and damaged skin. Human infection is very common, and the infection rate is as high as 80-90%. The common clinical manifestations are local aggregation of herpes on mucosa or skin, and occasionally severe systemic diseases involving internal organs.

[0003] Currently, since there is no medicine that can directly kill herpes viruses, the treatment principle for herpes virus HSV infection is to take oral antiviral drugs, such as acyclovir, to take mecobalamin to relieve pain and prevent complications such as post-herpes neuralgia. Long-term use of antiviral drug acyclovir may cause damage to liver or kidney function. Symptoms such as joint pain, diarrhea, headache, nausea, vomiting, dizziness, etc. may occur.

[0004] HSV is a virus belonging to the herpes virus family, subfamily a, and has a plasmid size of about 180 nm. In a sexually disease hospital, it is clinically classified into type 1 and type 2 according to antigenicity. Type 1 is mainly obtained from lip lesions, and type 2 can be isolated from genital lesions. Infection is caused by human-to-human contact. The number of infected people from four months after the onset to several years can reach 50-90% of the total population, and it is the most vulnerable virus to humans, but only a part of the clinical patients are affected. This disease can be divided into: herpes labialis, herpes keratitis, herpes dermatitis, genital herpes, Kaposi's disease, and sometimes is the cause of meningitis and encephalitis.

[0005] Shingles is an acute infectious skin disease caused by varicella-zoster virus. Children without immunity to the virus are infected and develop chickenpox. Some patients become virus carriers without symptoms after infection. Due to the neurotropic nature of the virus, it can remain latent in neurons of the spinal cord nerve ganglion after infection, and when resistance is low or tired, infected, cold, the virus can grow and reproduce again, and move along the nerve fibers to the skin, causing strong inflammation of the affected nerves and skin.

[0006] The main function of the lovir class is to interfere with viral DNA polymerase, thereby inhibiting viral replication, and under the action of DNA polymerase, it can also bind to the growing DNA chain, causing the interruption of DNA chain extension, and has an antiviral effect, but the drug is difficult to penetrate the capsid protein to act on DNA, so the treatment effect is poor.

[0007] The principle of action of interferon is to induce cells to synthesize antiviral proteins, thereby exerting an antiviral effect, but when the virus is in a high replication state, the effect of interferon is small.

[0008] Therefore, it is necessary to find a drug that can directly kill herpes virus and has good treatment effect and small side effects.

[0009] SUMMARY

[0010] The present application aims to overcome the shortcomings of the prior art, and provides the use of bioactive proteins in the preparation of a drug for treating herpes virus infection and a drug composition for treating herpes virus infection.

[0011] The technical solution of the present application to solve the technical problem is as follows:

[0012] In the first aspect of the present application, the use of bioactive proteins in the preparation of a drug for treating herpes virus infection is provided, the herpes virus is varicella-zoster virus VZV, and the bioactive protein is selected from anhydridized lactoferrin, anhydridized lactoglobulin, anhydridized whey protein or anhydridized histone.

[0013] Preferably, in the application, the bioactive protein is 4-hydroxyphthalic anhydride modified lactoferrin. The 4-hydroxyphthalic anhydride modified lactoferrin of the present application is the 4-hydroxyphthalic anhydride modified lactoferrin disclosed in Chinese Patent "202110238692.3", and the preparation method also refers to the method in Chinese Patent "202110238692.3".

[0014] Through a large amount of research work, the inventors accidentally found that the modified bioactive protein, such as anhydride lactoferrin, has an inhibitory effect on varicella-zoster virus (VZV). The anhydride lactoferrin is derived from milk protein, and the lactoferrin obtained after anhydride modification has a large number of negative charges on the surface, which can combine with the positive charge region of herpes virus capsid protein, block the process of virus invasion into host cells, and thus achieve the purpose of blocking herpes virus infection.

[0015] In the second aspect of the present application, a pharmaceutical composition capable of preventing and treating herpes virus infection is provided. The herpes virus is varicella-zoster virus VZV, and the pharmaceutical composition is prepared from 0.01-25% bioactive protein, 0.01-5% matrix, 0.1-5% humectant, 0.1-4% pH regulator, 0.01-3% preservative, and 58-99.77% solvent, calculated by weight percentage. The bioactive protein is selected from anhydride lactoferrin, anhydride lactoglobulin, anhydride whey protein or anhydride histone.

[0016] The anhydride lactoferrin, anhydride lactoglobulin, anhydride whey protein and anhydride histone are derived from milk protein, and the anhydride lactoferrin, anhydride lactoglobulin, anhydride whey protein or anhydride histone obtained after modification have a large number of negative charges, which can combine with the positive charge region of herpes virus capsid protein, block the process of virus invasion into host cells, and thus achieve the purpose of blocking herpes virus infection.

[0017] Preferably, the pharmaceutical composition is prepared from 0.01-20% bioactive protein, 0.02-2% matrix, 0.5-3% humectant, 0.2-2% pH regulator, 0.05-1% preservative, and 72-99.22% solvent, calculated by weight percentage.

[0018] Preferably, in the pharmaceutical composition, the matrix includes one or more of carbomer, sodium alginate, poloxamer and hydroxypropyl methyl cellulose.

[0019] The matrix is a stable, non-irritating and non-sensitizing medical polymer material, which can cover the damaged tissue, isolate and prevent wound infection, repair mucosal damage, and promote wound healing.

[0020] Preferably, the humectant in the pharmaceutical composition is selected from one or more of glycerin, propylene glycol, sodium hyaluronate.

[0021] The humectant has very good moisture absorption, is relatively mild and non-irritating, can form a protective film on the skin, isolate the invasion of air, well moisturize the skin, and absorb the moisture in the air, effectively preventing the skin from cracking.

[0022] Preferably, the pH regulator in the pharmaceutical composition is selected from one or more of triethanolamine, NaOH, hydrochloric acid, phosphoric acid, and lactic acid.

[0023] The normal pH value of the skin surface ranges from 4.0 to 7.0, and the addition of a pH regulator can reasonably design the pH value of the drug, which is beneficial to the health of the skin and is good for the skin.

[0024] Preferably, the preservative in the pharmaceutical composition is selected from one or more of phenoxyethanol, benzoic acid and sodium benzoate, nipagin, and sorbic acid.

[0025] The preservatives all have broad-spectrum antibacterial properties, especially very strong inhibitory effect on gram-positive and gram-negative bacteria. The preservatives disturb the protein activity of the bacterial cell membrane, cause cell death and block the growth and reproduction, thereby achieving the antibacterial effect.

[0026] Preferably, the solvent in the pharmaceutical composition is purified water.

[0027] Preferably, the dosage form of the pharmaceutical composition is an external use dosage form, and the mass of each component in the prescription per 100 g is as follows:

[0028] The preparation process of the external use dosage form is as follows:

[0029] 1) Take the prescription amount of the base, add it to part of the solvent and stir, then add the prescription amount of the bioactive protein, the humectant, the pH regulator, and the preservative, stir to dissolve, and finally supplement the solvent and stir uniformly.

[0030] 2) If the above drug is filled into a packaging container of an ointment, an external use ointment is obtained; if the above drug is filled into a packaging container of a spray, an external use spray is obtained; if the above drug is filled into a packaging container of other external use dosage forms, other external use dosage forms are obtained.

[0031] In one preferred embodiment of the present application, the bioactive protein in the pharmaceutical composition is 4-hydroxyphthalic anhydride modified lactoferrin; the amount of anhydridized lactoferrin is 5-10% by weight; the amount of the matrix is 0.02-2%; the amount of the humectant is 1.0-2.0%; the amount of the pH regulator is 0.1-0.5%; the amount of the preservative is 0.1-1%, and the amount of the solvent is the balance.

[0032] In one preferred embodiment of the present application, the pharmaceutical composition is a bioactive protein external ointment, and the mass of each component in 100 g of the prescription is as follows:

[0033] The preparation method of the external ointment is as follows: each component is weighed according to the above prescription of the bioactive protein external ointment, and then poloxamer is first taken in the prescription amount, added to purified water and stirred, and then anhydridized lactoferrin, glycerol, NaOH and nipagin in the prescription amount are added and stirred to dissolve, and finally purified water is added and stirred until uniform, to obtain the bioactive protein external ointment. The above pharmaceutical composition is filled into a packaging container of the ointment to obtain the bioactive protein external ointment.

[0034] In one preferred embodiment of the present application, the pharmaceutical composition is a bioactive protein external spray, and the mass of each component in 100 g of the prescription is as follows:

[0035] The preparation method of the bioactive protein external spray is as follows:

[0036] Each component is weighed according to the above prescription of the bioactive protein external ointment, and then carbomer is first taken in the prescription amount, added to purified water and stirred, and then anhydridized lactoferrin, glycerol, NaOH and phenoxyethanol in the prescription amount are added and stirred to dissolve, and finally purified water is added and stirred until uniform, to obtain the bioactive protein external spray. The above pharmaceutical composition is filled into a packaging container of the spray to obtain the bioactive protein external spray.

[0037] The present application has the following technical effects:

[0038] 1) The inventor first discovered that the modified bioactive protein, i.e., anhydridized lactoferrin, has an inhibitory effect on varicella-zoster virus (VZV) through a large amount of research and test verification work, which is verified in Examples 12-13.

[0039] 2) The pharmaceutical composition of the present application, the type and amount of adjuvant and the main component ratio are reasonable, and the drug is administered in the form of ointment, spray or other suitable external preparation, which has significant curative effect, small local toxic and side effects, very low acute toxicity to the skin, no irritation and sensitization, does not hinder the normal function of the skin, and is safe and effective. The appearance of the preparation prepared according to the prescription of the present application is good, the viscosity is moderate, and it is easy to be applied and sprayed. The preparation process of the present application is feasible, the quality is controllable, the preparation process is simple, and it is suitable for industrialized mass production. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 is an in vitro transdermal test.

[0041] Figure 2 is the tissue penetration rate of the antiviral ointment and spray in Example 13. DETAILED DESCRIPTION

[0042] The present application will be further described below in conjunction with specific embodiments, which are not limitations of the present application. The embodiments of the present application are not limited to the present application according to the prior art known in the art, and therefore any equivalent replacement in the art according to the disclosure of the present application belongs to the protection scope of the present application.

[0043] The anhydride-modified lactoferrin used in all examples of the present application is 4-hydroxyphthalic anhydride-modified lactoferrin, i.e. the 4-hydroxyphthalic anhydride-modified lactoferrin disclosed in Chinese Patent "202110238692.3", and the preparation method also refers to the method in Chinese Patent "202110238692.3".

[0044] The specific preparation method is as follows: under the condition of temperature 25℃, 4-hydroxyphthalic anhydride is dissolved in dimethyl sulfoxide to obtain a saturated 4-hydroxyphthalic anhydride solution; lactoferrin is dissolved in 0.1M trisodium phosphate solution to obtain a protein solution with a final concentration of 30mg / mL; the above-mentioned 4-hydroxyphthalic anhydride solution is added to the protein solution, stirred and mixed uniformly, the final concentration of 4-hydroxyphthalic anhydride in the reaction system is 60mM, and the reaction is allowed to stand for 2 hours; anhydride-modified lactoferrin is obtained; after filtration with 0.45uM filter membrane to remove bacteria, pH 7.2 phosphate buffer is used for ultrafiltration replacement purification, the ultrafiltration membrane used has a molecular weight cut-off of 10KD; then the bacteria are removed by filtration with 0.45uM microporous filter membrane, mannitol is added as a protective agent, and freeze-drying machine is used for freeze-drying, and the freeze-dried powder is collected, i.e. anhydride-modified lactoferrin freeze-dried powder is obtained. The protein freeze-drying step includes the following stages: pre-freezing stage: the temperature of the partition plate is lowered to -70℃, and the holding time is 6h; first sublimation stage: 12h; second sublimation stage: 7h.

[0045] Example 1 Determination of the bacteriostatic activity of anhydride-modified lactoferrin

[0046] The slant strain was inoculated into a broth peptone culture solution for 16-20 h, and then the number of colonies was counted on a nutrient agar medium plate. The concentration of the bacterial suspension was adjusted to 106-108 CFU / ml. The young bacterial suspension of the above strain was inoculated on the nutrient agar medium, and was uniformly coated. A sterile Oxford cup was placed in the center of the culture dish, and 0.1 ml of sterile microfiltrated lactoferritin and the anhydridized modified lactoferritin solution of Example 1 (pH 7.0) of different dilutions were added into the cup, with sterile water as a control. After the culture dish was placed in a 37°C constant temperature incubator for 24 h, the diameter of the inhibition zone (mm) was measured. The lowest sample concentration greater than the inhibition zone diameter (8 mm) of the control group was the minimum inhibitory concentration (MIC, μg / ml).

[0047] Table 1 Diameter of the inhibition zone (mm) produced by the antibacterial effect of anhydridized lactoferritin on different microorganisms

[0048] As can be seen from Table 1, it is found that the anhydridized lactoferritin has a broad-spectrum antibacterial and antioxidant effect, and has different degrees of inhibition and killing effect on many microorganisms, including Gram-positive and Gram-negative aerobic bacteria and anaerobic bacteria and some fungi.

[0049] Example 2 Detection of the inhibitory activity of anhydridized lactoferritin on varicella-zoster virus (VZV)

[0050] 1. Construction of VZV pseudovirus

[0051] The VZV-related gene (Genbank: E36506.1) was codon-optimized, amplified by polymerase chain reaction (PCR), cloned into a eukaryotic cell expression vector, and then subjected to enzyme digestion and sequencing identification to form a stable expression pVZV expression vector. The three expression vector plasmids and the reporter plasmid pCMV-Gluc that were correctly identified by sequencing were amplified in large quantities for standby use. The expression vector plasmid and the reporter plasmid used were purchased from Shengong Bioengineering (Shanghai) Co., Ltd.

[0052] The 293FT cells (purchased from Beinaicunlian Biotechnology Co., Ltd.) were plated in a 96-well cell culture plate, and after 12 h, the VZV expression plasmid was added to a 0.25 mol / L CaCl2 solution in different amounts. The CaCl2 solution of the plasmid was added dropwise to an equal volume of 2×HeBS (pH 6.97) solution, mixed uniformly, and then left to stand for 5 min. The mixed solution was then added to the pre-plated 293FT cell wells, and the culture medium was replaced after 12 h. After 48 h of transfection, the cells in each well were collected, lysed with the same volume of lysis solution at 37°C for 16 h, and the supernatant was collected as the pseudovirus suspension, which was stored at -80°C for standby use.

[0053] 2. Virus titer determination

[0054] The VZV pseudovirus suspension was diluted by 10 times successively for 10 times, from 10 -1 to 10 -6 The culture medium in the 96-well plate was removed, 50 microliters of the pseudovirus dilution was added, 5 microliters of normal culture medium was added, and the culture was continued at 37°C, 5% CO2 for 72 hours. 50 microliters of the culture supernatant was taken, and the Merck alkaline phosphatase detection kit was used for detection, so as to calculate the TCID50.

[0055] 3. Detection of the inhibitory activity of acid anhydride lactoferrin on VZV

[0056] The 293FT cells were passaged into a 96-well plate, and the number of cells per well was 1X105, and the culture was carried out at 37°C, 5% CO2 for 8 hours. 100 TCID50 of VZV pseudovirus (50M) was incubated with different concentrations of acid anhydride lactoferrin freeze-dried powder solution, and the incubation was carried out at 37°C for 30 minutes. The culture medium in the 96-well plate was removed, the above-mentioned incubated mixture was added, and the culture was continued at 37°C, 5% CO2 for 12 hours. Fresh culture medium was replaced, and after 72 hours of culture, 50M of the culture supernatant was taken, and the Merck alkaline phosphatase detection kit was used for detection. The IC50 (half inhibitory concentration) of acid anhydride lactoferrin was calculated by using SPSS25. The results are shown in Table 2.

[0057] Table 2 Detection of the inhibitory activity of acid anhydride lactoferrin on VZV

[0058] As can be seen from Table 2, the acid anhydride lactoferrin has an inhibitory effect on VZV.

[0059] Example 3: Biological active protein external ointment

[0060] I. Prescription: The mass of each component in 100g of the prescription is:

[0061] II. Preparation method:

[0062] According to the above prescription of the biological active protein external ointment, each component was weighed for use. First, the prescription amount of carbomer was taken, purified water was added and stirred, then the prescription amount of acid anhydride lactoferrin, glycerol, NaOH and nipagin ester was added and stirred to dissolve, and finally purified water was added and stirred uniformly to obtain the product. The above-mentioned medicine was filled into the packaging container of the ointment to obtain the biological active protein external ointment.

[0063] Example 4: Biological active protein external ointment

[0064] I. Prescription: The mass of each component in 100g of the prescription is:

[0065] II. Preparation method:

[0066] According to the above prescription of the external ointment of bioactive protein, each component is weighed for standby use. First, the poloxamer in the prescription amount is taken and stirred in purified water, then the lactoferrin anhydride, glycerol, NaOH and nipagin ester in the prescription amount are added and stirred to dissolve, and finally purified water is added and stirred uniformly. The above medicine is filled into the packaging container of the ointment to obtain the external ointment of bioactive protein.

[0067] Example 5: External ointment of bioactive protein

[0068] I. Prescription: The mass of each component in 100 g of the prescription is:

[0069] II. Preparation method:

[0070] According to the above prescription of the external ointment of bioactive protein, each component is weighed for standby use. First, the sodium alginate in the prescription amount is taken and stirred in purified water, then the lactoferrin anhydride, glycerol, NaOH and nipagin ester in the prescription amount are added and stirred to dissolve, and finally purified water is added and stirred uniformly. The above medicine is filled into the packaging container of the ointment to obtain the external ointment of bioactive protein.

[0071] Example 6: External ointment of bioactive protein

[0072] I. Prescription: The mass of each component in 100 g of the prescription is:

[0073] II. Preparation method:

[0074] According to the above prescription of the external ointment of bioactive protein, each component is weighed for standby use. First, the sodium alginate in the prescription amount is taken and stirred in purified water, then the lactoferrin anhydride, glycerol, NaOH and nipagin ester in the prescription amount are added and stirred to dissolve, and finally purified water is added and stirred uniformly. The above medicine is filled into the packaging container of the ointment to obtain the external ointment of bioactive protein.

[0075] Example 7: External spray of bioactive protein

[0076] I. Prescription: The mass of each component in 100 g of the prescription is:

[0077] II. Preparation method:

[0078] According to the above prescription of the external ointment of bioactive protein, each component is weighed for standby use. First, the sodium alginate in the prescription amount is taken and stirred in purified water, then the lactoferrin anhydride, glycerol, NaOH and nipagin ester in the prescription amount are added and stirred to dissolve, and finally purified water is added and stirred uniformly. The above medicine is filled into the packaging container of the ointment to obtain the external ointment of bioactive protein.

[0079] Example 8: Bioactive protein external use spray

[0080] I. Prescription: The mass of each component in 100 g of the prescription is as follows:

[0081] II. Preparation method:

[0082] According to the above prescription of bioactive protein external use ointment, each component is weighed for use. First, the prescription amount of poloxamer is taken and stirred with purified water. Then the prescription amount of acid anhydride lactoferrin, glycerol, NaOH and phenoxy ethanol is added and stirred to dissolve. Finally, purified water is added and stirred uniformly to obtain the product. The above drug is filled into the packaging container of the spray to obtain the bioactive protein external use spray.

[0083] Example 9: Bioactive protein external use spray

[0084] I. Prescription: The mass of each component in 100 g of the prescription is as follows:

[0085] II. Preparation method:

[0086] According to the above prescription of bioactive protein external use ointment, each component is weighed for use. First, the prescription amount of sodium alginate is taken and stirred with purified water. Then the prescription amount of acid anhydride lactoferrin, glycerol, NaOH and phenoxy ethanol is added and stirred to dissolve. Finally, purified water is added and stirred uniformly to obtain the product. The above drug is filled into the packaging container of the spray to obtain the bioactive protein external use spray.

[0087] Example 10: Bioactive protein external use spray

[0088] I. Prescription: The mass of each component in 100 g of the prescription is as follows:

[0089] II. Preparation method:

[0090] According to the above prescription of bioactive protein external use ointment, each component is weighed for use. First, the prescription amount of hydroxypropyl methyl new cellulose is taken and stirred with purified water. Then the prescription amount of acid anhydride lactoferrin, glycerol, NaOH and phenoxy ethanol is added and stirred to dissolve. Finally, purified water is added and stirred uniformly to obtain the product. The above drug is filled into the packaging container of the spray to obtain the bioactive protein external use spray.

[0091] Example 11: Biological evaluation test of the sample of Example 4

[0092] 1. In vitro cytotoxicity test (MTT method)

[0093] After the sample was extracted at 0.1 g / mL, 4 concentrations (100%, 50%, 25%, 12.5%) of the test solution were added to the 96-well culture plate (the wells contained cells cultured for 24 hours), and placed in a CO2 incubator at 37±1°C for 24 hours. The absorbance was measured at 570 nm wavelength (reference wavelength 650 nm) by using the tetrazolium salt (MTT) colorimetric method, and the relative survival rate was calculated. Cell survival rate (%) = test group D value / blank control group D value x 100%, and the results are shown in Table 3.

[0094] Table 3 Results of cytotoxicity test

[0095] As can be seen from Table 3, the relative survival rate of the negative control group was 99.99%, and there was no potential cytotoxicity. The relative survival rate of the positive control group was 3.06%, and there was potential cytotoxicity. The relative survival rate of the test group 100% extract was 90.31%, and there was no potential cytotoxicity. The relative survival rate of the test group 50% extract was 91.28%, and there was no potential cytotoxicity.

[0096] 2. Skin irritation

[0097] The sample was applied to the back skin of the experimental rabbit, and the application was removed after 4 hours of contact. The negative control was operated in the same way. At 1h, 24h, 48h and 72h after the application was removed, the skin tissue reaction of the test site was observed and scored. The skin irritation test was repeated, and after 72h of observation in the acute skin irritation test, the sample and the negative control were applied in the same way every day for 14 consecutive days, and the skin tissue reaction of the test site was observed and scored in time. The primary irritation index of the sample was calculated in the acute skin irritation test and the repeated skin irritation test, respectively. The results are shown in Tables 4-5.

[0098] Table 4 Results of rabbit skin irritation reaction score (single contact test)

[0099] Table 5 Results of rabbit skin irritation reaction score (repeated contact test)

[0100] As can be seen from Tables 4-5, the primary irritation index of the sample in the acute skin irritation test of the experimental rabbit was 0; the primary irritation index of the sample in the repeated contact skin irritation test of the experimental rabbit was 0, and the skin irritation reaction type was very slight irritation reaction.

[0101] 3. Skin sensitization reaction (closed patch test)

[0102] The guinea pigs were repeatedly exposed to the samples for a period of time for induction, and then the test group animals were challenged with the samples on the unshaved non-test sites. The skin reactions of all animals were graded 24 h and 48 h after the patch was removed. The negative control group animals were also operated in the same way. The results are shown in Table 6.

[0103] Table 6 Observation results of sensitization reaction test

[0104] As can be seen from Table 6, the skin of the test group animals and the negative control group animals did not show a patch test reaction at the challenge site, no obvious symptoms of sensitization were observed, the score was 0, and the incidence of sensitization was 0, indicating that the sample did not cause a guinea pig skin sensitization reaction.

[0105] Example 12: Comparison of the inhibitory effect of the sample of Example 4 and acyclovir cream on herpes zoster virus

[0106] Example 4 sample, usage method: apply to the affected area, three times a day, use for 4-7 days. After the use is completed, the varicella-zoster IgG antibody detection kit (enzyme-linked immunoassay) (Wuhan Biosciences Co., Ltd.) is used for detection.

[0107] Acyclovir cream (Fujian Pacific Pharmaceutical Co., Ltd.), specification: 3%, usage method: topical use. Take an appropriate amount of the product and apply it to the affected area, 4-6 times a day, for a total of 7 days. After the use is completed, the varicella-zoster IgG antibody detection kit (enzyme-linked immunoassay) (Wuhan Biosciences Co., Ltd.) is used for detection.

[0108] Table 7 Detection results Note: "-" represents negative (P / N < 1.5); "+" represents positive (P / N ≥ 2.1); P and N are the values after removing the blank control.

[0109] Detection results: the detection results of the sample of Example 4 are negative, and the detection results of acyclovir cream are positive, indicating that the sample of Example 4 has an inhibitory effect on VZV virus, and the inhibitory effect of acyclovir cream on VZV virus is weaker than that of the sample of Example 4.

[0110] Example 13. In vitro penetration and permeation of antiviral drugs and acyclovir cream in pig skin tissue

[0111] 1. In vitro transdermal test

[0112] Preparation of pig skin tissue: 2-month-old male Landrace pigs, in good health, take the abdominal skin, remove fat and hair tissue, soak in physiological saline, and store at room temperature.

[0113] Pig skin tissue was mounted in a Franz diffusion cell: a circular portion of the tissue was sandwiched between the two chambers of a Franz diffusion cell with a 1 cm 2 active diffusion area, and a wave generator was used to measure the impedance of the pig skin tissue to ensure the integrity of the tissue segment for the penetration study. Pig skin tissue with an impedance > 3 KQ.cm 2 was suitable for the study. The receptor chamber was filled with 8 ml of 5% pH 7.4 PBS solution, stirred with a 3 mm magnetic stir bar at 600 rpm, and the temperature was maintained at 37 °C with a circulating water bath.

[0114] Loading of ointment in the donor chamber: About 200 mg of antiviral drug (Example 4 ointment, Example 7 spray) was filled into a tared 1 mL syringe with acyclovir cream, respectively, and the sample was dispensed into the donor chamber. The drug and cream were spread onto the skin surface with a pre-tared applicator. After the drug and cream were loaded and spread onto the skin surface, the weight of the 1 mL syringe and the applicator was noted to determine the exact amount of drug and cream loaded into the donor chamber.

[0115] Penetration and permeation study: Time course (2 h, 4 h, and 8 h) pig vaginal penetration studies were performed. After the drug and cream were loaded, 500 pL samples were withdrawn from the receptor chamber at different time intervals and an equal volume of fresh receptor medium was used to replace the withdrawn sample each time. The samples withdrawn at each time interval were immediately stored at -20 °C until analysis. After 2, 4, and 8 h, the formulation was removed from the donor chamber by means of a syringe and cleaned with a cotton swab. The tissue was removed and gently washed 5 times with a washing solution (50% methanol water) or cleaned with a cotton swab.

[0116] Table 8 Time course IVPT study design

[0117] Preparation of sample drug solution for analysis: The samples stored at -20 °C were removed and thawed at room temperature for 30 min. The samples were centrifuged at 13000 rpm for 5 min, and an equal volume of extraction solvent was added to the 200 pL supernatant. These samples were centrifuged at 13000 rpm for 5 min and the supernatant was transferred to a vial for analysis.

[0118] The analysis detection method was detected according to the protein content determination method of Chinese Pharmacopoeia 2020 edition four general rules 0731 fourth method BCA method and the method of “acyclovir” detection item under

determination of content

[0119] Figure 1 is an in vitro tissue penetration test in skin tissue comparing the antiviral drug ointment to acyclovir cream. Bar A represents the tissue penetration of the antiviral drug ointment in skin tissue. Bar B represents the tissue penetration of acyclovir cream in skin tissue. The results show that the antiviral drug ointment has a higher tissue penetration in skin tissue than acyclovir cream, exhibits a high tissue penetration, and can produce a high anti-VZV activity due to the addition of dimethyl sulfoxide transdermal agent during the preparation of the acid anhydride lactoferrin original material.

[0120] 2. Penetration Test

[0121] The experimental procedures for the penetration test are the same as above (i.e., the specific operations of “Preparation of pig skin tissue”, “Loading of ointment in donor chamber”, “Penetration and penetration study” are the same as in the in vitro transdermal test of Experiment 1).

[0122] Chopping of pig skin tissue (active diffusion area) after IVPT: The 8mm punch of the washed pig skin tissue active diffusion area was removed, weighed and transferred to a tube. Immediately after this, the tube was placed in dry ice for about 15 min. After the specified time, the tissue was removed and placed in a pre-cooled petri dish. The tissue was chopped into smaller pieces on the petri dish with a pre-cooled scalpel. The chopped tissue was transferred to a sample tube, and the petri dish was rinsed with 1 ml of 5% pH 7.4 PBS solution and transferred to the same tissue sample tube. The tubes were stored at -70°C until analysis.

[0123] Extraction of drug from pig skin tissue: The chopped tissue sample stored at -70°C was removed and thawed at room temperature for about 90 min. The sample was kept shaking at room temperature in a BioShaker for 4 h. After 4 h, the sample was centrifuged at 13000 rpm for 5 min. To 100 μL of supernatant, 400 μL of extraction solvent was added and vortexed for 2 min. The samples were centrifuged at 13000 rpm for 5 min and the supernatant was transferred to a vial for analysis.

[0124] The analysis detection method was detected according to the protein content determination method of Chinese Pharmacopoeia 2020 Edition Vol. 4 General Rules 0731 Fourth Method BCA Method and the “Acyclovir” detection item under

Content Determination

[0125] Figure 2 is the tissue penetration of the antiviral drug ointment of Example 4 and the spray of Example 7. The spray formulation produces a similar tissue penetration to the ointment formulation, with an average of 58 ng / mg of compound in the tissue, both with high tissue penetration and both can produce high anti-VZV activity.

[0126] The above merely illustrates the embodiments of the present application, and does not limit the patent scope of the present application, and any equivalent transformation or direct or indirect application in other related technical fields based on the content of the present application is also included in the patent protection scope of the present application.

Claims

1. Use of a biologically active protein for the preparation of a medicament for the treatment of a herpes virus infection, characterized in that, The herpes virus is varicella-zoster virus (VZV), and the bioactive protein is selected from anhydrous lactoferrin, anhydrous lactalbumin, anhydrous lactalbumin or anhydrous histone.

2. The use according to claim 1, wherein the bioactive protein is 4-hydroxy anhydride modified lactoferrin.

3. A pharmaceutical composition capable of preventing the treatment of herpes virus infection, the herpes virus being varicella zoster virus (VZV), characterized in that, The pharmaceutical composition is prepared from 0.01-25% bioactive protein, 0.01-5% matrix, 0.1-5% humectant, 0.1-4% pH regulator, 0.01-3% preservative and 58-99.77% solvent.

4. The pharmaceutical composition capable of preventing and treating herpes virus infection according to claim 3, wherein The pharmaceutical composition is prepared from 0.01-20% bioactive protein, 0.02-2% matrix, 0.5-3% humectant, 0.2-2% pH regulator, 0.05-1% preservative and 72-99.22% solvent.

5. The pharmaceutical composition capable of preventing and treating herpes virus infection according to claim 3, wherein The matrix in the pharmaceutical composition comprises one or more than one of carbomer, sodium alginate, poloxamer and hydroxypropyl methyl cellulose.

6. The pharmaceutical composition capable of preventing and treating herpes virus infection according to claim 3, wherein The humectant in the pharmaceutical composition is selected from one or more of glycerol, propylene glycol and sodium hyaluronate.

7. The pharmaceutical composition capable of preventing and treating herpes virus infection according to claim 3, wherein The pH regulator in the pharmaceutical composition is selected from one or more of triethanolamine, NaOH, hydrochloric acid, phosphoric acid and lactic acid.

8. The pharmaceutical composition for preventing and treating herpes virus infection according to claim 3, characterized in that, The preservative in the pharmaceutical composition is selected from one or more of phenoxyethanol, benzoic acid and sodium benzoate, nipagin and sorbic acid.

9. The pharmaceutical composition capable of preventing and treating herpes virus infection according to claim 3, wherein The solvent in the pharmaceutical composition is purified water.

10. The pharmaceutical composition capable of preventing and treating herpes virus infection according to claim 3, wherein The dosage form of the pharmaceutical composition is an external use dosage form, and the mass of each component in 100 g of the prescription is: The preparation process of the external preparation is as follows: 1) Take the prescribed amount of matrix, add part of the solvent and stir, then add the prescribed amount of bioactive protein, humectant, pH regulator and preservative, stir to dissolve, and finally supplement the solvent and stir uniformly. 2) Fill the above-mentioned medicine into the packaging container of the external preparation, and the external preparation is obtained, such as filling the above-mentioned medicine into the packaging container of the ointment to obtain the external ointment, or filling the above-mentioned medicine into the packaging container of the spray to obtain the external spray.

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