Application of biologically active protein in preparing medicine for treating herpes virus infection and medicine composition for treating herpes virus infection

Abstract

The present invention relates to the application of biologically active proteins in the preparation of drugs for treating herpes virus infection and a pharmaceutical composition for treating herpes virus infection, and belongs to the field of medical technology. The pharmaceutical composition of the present invention mainly includes biologically active proteins, matrix, moisturizing agent, pH adjusting agent, and preservative. The present invention has the following technical effects: 1) The inventors have discovered for the first time through a large number of studies and experimental verification work that the modified biologically active protein, i.e., anhydride lactoferrin, has an inhibitory effect on herpes zoster virus (VZV), and this performance has been verified in Examples 12-13. 2) In the pharmaceutical composition of the present invention, the types, dosages, and proportions of the main components of the auxiliary materials are reasonable, and the ointment, spray, or other dosage forms suitable for external use are administered, and the curative effect is remarkable, the local toxic and side effects are small, the acute toxicity of the skin is very low, there is no irritation and sensitization, the normal function of the skin is not hindered, and it is safe and effective.

Description

Technical Field

[0001] The invention relates to application of biologically active proteins in preparing medicines for treating herpes virus infections and a medicine composition for treating herpes virus infections, belonging to the technical field of medicines. Background Art

[0002] Herpes viruses (HSV) are a class of viruses with envelope and double-stranded DNA genome. There are currently 8 types of herpes viruses that can infect humans, including: herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), varicellazoster 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 disease is mainly transmitted through direct close contact and sexual contact. HSV invades the body through multiple pathways such as the oral cavity, respiratory tract, genital mucosa and damaged skin. Human infection is very common, with an infection rate of 80-90%. The common clinical manifestation is localized clusters of herpes on the mucous membranes or skin. Occasionally, severe systemic diseases may occur, involving the internal organs.

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

[0004] Herpes simplex virus HSV belongs to the a virus subfamily of the herpesviridae family, and the virus plasmid size is about 180 nanometers. In the STD hospital, the virus is currently divided into type 1 and type 2 based on the difference in antigenicity. Type 1 is mainly obtained from oral and labial lesions, and type 2 can be isolated from genital lesions. Infection is caused by human contact. From four months to several years after the occurrence, the number of people infected can reach 50-90% of the total population. It is the most vulnerable virus to humans, but only a part of them develop the disease clinically. This disease can be divided into: oral herpes, herpetic keratitis, herpetic dermatitis, genital herpes, Kaposi's disease, etc., and is sometimes the cause of meningitis and encephalitis.

[0005] Herpes zoster is an acute infectious skin disease caused by the varicella-zoster virus. Children who are not immune to the virus will develop chickenpox after being infected. Some patients become carriers of the virus after being infected without symptoms. Since the virus is neurotropic, it can remain dormant in the neurons of the dorsal root ganglia of the spinal nerves for a long time after infection. When the body's resistance is low or when there is fatigue, infection, or cold, the virus can grow and reproduce again and move along the nerve fibers to the skin, causing severe inflammation of the invaded nerves and skin.

[0006] Lovir drugs mainly inhibit viral replication by interfering with viral DNA polymerase, and can also bind to growing DNA chains under the action of DNA polymerase, causing the extension of the DNA chain to be interrupted, thus playing an antiviral role. However, it is difficult for the drug to penetrate the capsid protein and act on DNA, so the therapeutic effect is poor.

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

[0008] Therefore, it is necessary to find a drug that can directly kill herpes virus and has good therapeutic effect and few side effects. Summary of the invention

[0009] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an application of a bioactive protein in the preparation of a drug for treating herpes virus infection and a pharmaceutical composition for treating herpes virus infection.

[0010] The technical solution of the present invention to solve the technical problem is as follows:

[0011] In the first aspect of the present invention, there is provided the use of a biologically active protein in the preparation of a drug for treating herpes virus infection, wherein the herpes virus is herpes zoster virus VZV, and the biologically active protein is selected from anhydride-modified lactoferrin, anhydride-modified lactoglobulin, anhydride-modified whey protein or anhydride-modified histone.

[0012] Preferably, in the application, the biologically active protein is 4-hydroxyphthalic anhydride-modified lactoferrin. The 4-hydroxyphthalic anhydride-modified lactoferrin of the present invention is the 4-hydroxyphthalic anhydride-modified lactoferrin disclosed in Chinese Patent "202110238692.3", and the preparation method is also prepared by referring to the method in Chinese Patent "202110238692.3".

[0013] Through extensive research, the inventor unexpectedly discovered that modified bioactive proteins, such as anhydride lactoferrin, have an inhibitory effect on herpes zoster virus (VZV). Anhydride lactoferrin is derived from milk protein. The surface of lactoferrin obtained after anhydride modification carries a large amount of negative charge, which combines with the positively charged region of the herpes virus capsid protein to block the process of the virus invading the host cell, thereby achieving the purpose of blocking herpes virus infection.

[0014] In the second aspect of the present invention, a pharmaceutical composition capable of preventing and treating herpes virus infection is provided. The herpes virus is herpes zoster virus VZV, and the pharmaceutical composition is prepared by 0.01-25% of biologically active protein, 0.01-5% of matrix, 0.1-5% of moisturizer, 0.1-4% of pH regulator, 0.01-3% of preservative, and 58-99.77% of solvent, calculated by weight percentage, and the biologically active protein is selected from anhydride lactoferrin, anhydride lactoglobulin, anhydride whey protein or anhydride histone.

[0015] Anhydride lactoferrin, anhydride lactoglobulin, anhydride whey protein and anhydride histone are derived from milk protein. The modified anhydride lactoferrin, anhydride lactoglobulin, anhydride whey protein or anhydride histone carries a large amount of negative charge, which can combine with the positively charged region of the herpes virus capsid protein to block the process of the virus invading the host cells, thereby achieving the purpose of blocking the herpes virus infection.

[0016] Preferably, the pharmaceutical composition is prepared by weight percentage from 0.01 to 20% of biologically active protein, 0.02 to 2% of matrix, 0.5 to 3% of moisturizer, 0.2 to 2% of pH adjuster, 0.05 to 1% of preservative, and 72 to 99.22% of solvent.

[0017] Preferably, in the pharmaceutical composition, the matrix includes one or more of carbomer, sodium alginate, poloxamer, and hypromellose.

[0018] The matrix is ​​a medical polymer material with stable properties, no irritation, and no allergenicity. It can cover the eroded and damaged tissue, isolate and prevent wound infection, repair mucosal damage, and promote wound healing.

[0019] Preferably, in the pharmaceutical composition, the moisturizer is selected from one or more of glycerol, propylene glycol, and sodium hyaluronate.

[0020] The moisturizing agent has very good hygroscopicity, is relatively mild and non-irritating, can form a protective film on the skin to isolate the invasion of air, the skin can be well moisturized, the moisture in the air can also be absorbed, and can effectively prevent the skin from drying out.

[0021] Preferably, in the pharmaceutical composition, the pH adjuster is selected from one or more of triethanolamine, NaOH, hydrochloric acid, phosphoric acid, and lactic acid.

[0022] The normal pH value of the skin surface ranges from 4.0 to 7.0. The addition of pH regulators can reasonably design the pH value of the drug, which is beneficial to skin health and beneficial to the skin.

[0023] Preferably, in the pharmaceutical composition, the preservative is selected from one or more of phenoxyethanol, benzoic acid and sodium benzoate, parabens and sorbic acid.

[0024] The preservatives all have broad-spectrum antibacterial properties, especially strong inhibitory effects on Gram-positive and Gram-negative bacteria. The preservatives achieve antibacterial effects by disrupting the protein activity of bacterial cell membranes, causing cell death and blocking their growth and reproduction.

[0025] Preferably, in the pharmaceutical composition, the solvent is purified water.

[0026] Preferably, the dosage form of the pharmaceutical composition is an external dosage form, and the mass of each component in each 100g of the prescription is:

[0027] Bioactive Protein 0.01g-10g Matrix 0.02g-2.0g Moisturizer 0.5g-3g pH Adjusters 0.2g-2g preservative 0.05g-1g Solvents margin

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

[0029] 1) Take the prescribed amount of matrix, add it to part of the solvent and stir, then add the prescribed amount of bioactive protein, moisturizer, pH adjuster and preservative, stir to dissolve, finally add more solvent, stir evenly, and the product is obtained.

[0030] 2) If the above-mentioned drugs are filled into the packaging container of ointment, an external ointment can be obtained; if the above-mentioned drugs are filled into the packaging container of spray, an external spray can be obtained; if the above-mentioned drugs are filled into the packaging container of other external dosage forms, other external dosage forms can be obtained.

[0031] In one preferred embodiment of the present invention, in the pharmaceutical composition, the biologically active protein is 4-hydroxyphthalic anhydride-modified lactoferrin; calculated by weight percentage, the amount of anhydride-modified lactoferrin is 5-10%; the amount of matrix is ​​0.02-2%; the amount of moisturizer is 1.0-2.0%; the pH adjuster is 0.1-0.5%; the amount of preservative is 0.1-1%, and the balance is solvent.

[0032] In one preferred embodiment of the present invention, the pharmaceutical composition is a bioactive protein external ointment, and the specific ingredients and the mass of each ingredient in each 100g of the prescription are:

[0033]

[0034] The preparation method of the external ointment is as follows: according to the above prescription of the biologically active protein external ointment, each component is weighed for standby use, firstly the prescribed amount of poloxamer is taken, purified water is added and stirred, then the prescribed amount of anhydride lactoferrin, glycerol, NaOH and parabens are added and stirred to dissolve, finally purified water is added and stirred evenly, and the above drugs are filled into the packaging container of the ointment to obtain the biologically active protein external ointment.

[0035] In one preferred embodiment of the present invention, the pharmaceutical composition is a bioactive protein external spray, and the specific ingredients and the mass of each ingredient in each 100g of the prescription are:

[0036]

[0037] The preparation method of the biologically active protein external spray is as follows:

[0038] According to the above prescription of the biologically active protein external ointment, each component is weighed for use, first the prescribed amount of carbomer is taken, and purified water is added and stirred, then the prescribed amount of acid anhydride lactoferrin, glycerol, NaOH, and phenoxyethanol are added and stirred to dissolve, and finally purified water is added and stirred evenly to obtain the ointment. The above drugs are filled into the packaging container of the spray to obtain the biologically active protein external spray.

[0039] The present invention has the following technical effects:

[0040] 1) Through extensive research and experimental verification, the inventors discovered for the first time that the modified bioactive protein, namely anhydride-modified lactoferrin, has an inhibitory effect on herpes zoster virus (VZV), and this property has been verified in Examples 12-13.

[0041] 2) The pharmaceutical composition of the present invention has reasonable types, dosages and proportions of main ingredients, and is administered in ointments, sprays or other dosage forms suitable for external use, with significant therapeutic effects, small local toxicity and side effects, very low acute skin toxicity, no irritation and sensitization, no obstruction of normal skin function, and safe and effective. The preparation prepared according to the prescription of the present invention has good appearance, moderate viscosity, and is easy to apply and spray. The preparation process of the present invention is feasible, the quality is controllable, the preparation process is simple, and it is suitable for industrial batch production. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 In vitro skin penetration test.

[0043] Figure 2 It is the tissue penetration rate of the antiviral drug ointment and spray in Example 13. DETAILED DESCRIPTION

[0044] The present invention is further described below in conjunction with specific implementation modes, which is not a limitation of the present invention. According to the prior art known in the art, the implementation modes of the present invention are not limited thereto. Therefore, all equivalent replacements in the art made according to the disclosure of the present invention belong to the protection scope of the present invention.

[0045] The anhydride-modified lactoferrin used in all embodiments of the present invention is lactoferrin modified with 4-hydroxyphthalic anhydride, that is, the 4-hydroxyphthalic anhydride-modified lactoferrin disclosed in Chinese Patent "202110238692.3", and the preparation method is also prepared by referring to the method in Chinese Patent "202110238692.3".

[0046] The specific preparation method is as follows: at a temperature of 25°C, 4-hydroxyphthalic anhydride is dissolved in dimethyl sulfoxide to obtain a saturated 4-hydroxyphthalic anhydride solution; lactoferrin is dissolved in a 0.1M trisodium phosphate solution to obtain a protein solution with a final concentration of 30 mg / mL; the above 4-hydroxyphthalic anhydride solution is added to the protein solution, stirred and mixed, the final concentration of 4-hydroxyphthalic anhydride in the reaction system is 60 mM, and the reaction is allowed to stand for 2 hours; anhydride lactoferrin is obtained; after sterilization by suction filtration with a 0.45uM filter membrane, ultrafiltration replacement purification is performed using a pH7.2 phosphate buffer, and the ultrafiltration membrane used has a molecular weight cutoff of 10KD; and then sterilization is performed by suction filtration with a 0.45uM microporous filter membrane, a protective agent mannitol is added, freeze drying is performed using a freeze dryer, and freeze-dried powder is collected to obtain anhydride lactoferrin freeze-dried powder. The protein freeze-drying process includes the following stages: pre-freezing stage: the temperature of the partition drops to -70°C and is maintained for 6 hours; primary sublimation stage: maintained for 12 hours; secondary sublimation stage: maintained for 7 hours.

[0047] Example 1 Determination of antibacterial activity of anhydride lactoferrin

[0048] The slant strain was inoculated with broth peptone culture medium for 16-20 hours, and the nutrient agar medium plate was counted to adjust the bacterial suspension concentration to 106-108 CFU / ml. The young bacterial suspension of the above-mentioned strain was inoculated on the nutrient agar medium and spread evenly. A sterile Oxford cup was placed in the center of the culture dish, and 0.1 ml of lactoferrin of different dilutions after sterile microfiltration and the anhydride-modified lactoferrin solution of Example 1 (pH 7.0) were added to the cup, with sterile water as a control. After the culture dish was placed in a 37°C constant temperature incubator and cultured for 24 hours, the diameter of the inhibition zone was measured (mm). The minimum sample concentration greater than the diameter of the inhibition zone of the control group (8 mm) is the minimum inhibitory concentration (MIC, μg / ml).

[0049] Table 1 Diameter of inhibition zone produced by anhydride lactoferrin against different microorganisms (mm)

[0050]

[0051] As can be seen from Table 1, it was found that the acid anhydride lactoferrin has a broad-spectrum antibacterial and antioxidant effect, and has different degrees of inhibition and killing effects on many microorganisms, including Gram-positive and Gram-negative aerobic and anaerobic bacteria and some fungi.

[0052] Example 2 Detection of the inhibitory activity of anhydride lactoferrin on herpes zoster virus (VZV)

[0053] 1. Construction of VZV pseudovirus

[0054] 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 digested and sequenced to form a stable expression vector of pVZV. The three expression vector plasmids and reporter plasmid pCMV-Gluc that were correctly sequenced were amplified in large quantities for later use. The expression vector plasmids and reporter plasmids used were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0055] 293FT cells (purchased from Beina Chuanglian Biotechnology Co., Ltd.) were plated in 96-well cell culture plates. After 12 h, the VZV expression plasmid was added with different amounts of 0.25 mol / L CaCl 2 In the solution, the CaCl 2 The solution was added dropwise to an equal volume of 2×HeBS (pH=6.97) solution, mixed evenly, and added to the pre-plated 293FT cell wells after standing for 5 minutes. The culture medium was replaced after 12 hours. 48 hours after transfection, cells from each well were collected and lysed with the same volume of lysis buffer at 37°C for 16 hours. The supernatant was collected as the pseudovirus suspension, which was packaged and placed at -80°C for later use.

[0056] 2. Virus titer determination

[0057] The VZV pseudovirus suspension was diluted 10 times in succession. -1 to 10 -6 Remove the culture medium in the 96-well wells, add 50 μl of pseudovirus dilution, and then add 5 μl of normal culture medium, at 37°C and 5% CO 2 The culture was continued for 72 h, and 50 μl of the culture supernatant was taken and tested using the Merck alkaline phosphatase assay kit to calculate the TCID50.

[0058] 3. Detection of the inhibitory activity of anhydride lactoferrin against VZV

[0059] 293FT cells were subcultured into 96-well plates with 1×105 cells per well and incubated at 37°C and 5% CO 2The culture medium in the 96-well plate was removed and the mixture after incubation was added. The culture medium was incubated at 37°C and 5% CO. 2 The culture was continued for 12 hours, and fresh culture medium was replaced. After 72 hours of culture, 50 M culture supernatant was taken and tested using Merck alkaline phosphatase detection kit, and the IC50 (half-maximal inhibitory concentration) of anhydride lactoferrin was calculated using SPSS25. The results are shown in Table 2.

[0060] Table 2 Detection of the inhibitory activity of anhydride lactoferrin on VZV

[0061]

[0062] As can be seen from Table 2, anhydride lactoferrin has an inhibitory effect on VZV.

[0063] Example 3: Bioactive protein topical ointment

[0064] 1. Prescription: The mass of each ingredient in 100g of this prescription is:

[0065] Lactoferrin 5g Carbomer 1g glycerin 2g NaOH 0.3g Parabens 0.1g Purified water 91.6g

[0066] 2. Preparation method:

[0067] According to the above prescription of the biologically active protein external ointment, each component is weighed for use, first the prescribed amount of carbomer is taken, purified water is added and stirred, then the prescribed amount of anhydride lactoferrin, glycerol, NaOH, and parabens are added and stirred to dissolve, and finally purified water is added and stirred evenly to obtain the ointment. The above drugs are filled into the packaging container of the ointment to obtain the biologically active protein external ointment.

[0068] Example 4: Bioactive protein topical ointment

[0069] 1. Prescription: The mass of each ingredient in 100g of this prescription is:

[0070]

[0071] 2. Preparation method:

[0072] According to the above prescription of the biologically active protein external ointment, each component is weighed for use, firstly the prescribed amount of poloxamer is taken, purified water is added and stirred, then the prescribed amount of anhydride lactoferrin, glycerol, NaOH, and parabens are added and stirred to dissolve, finally purified water is added and stirred evenly to obtain the ointment. The above drugs are filled into the packaging container of the ointment to obtain the biologically active protein external ointment.

[0073] Example 5: Bioactive protein topical ointment

[0074] 1. Prescription: The mass of each ingredient in 100g of this prescription is:

[0075]

[0076] 2. Preparation method:

[0077] According to the above prescription of the biologically active protein external ointment, each component is weighed for use, firstly the prescribed amount of sodium alginate is taken, purified water is added and stirred, then the prescribed amount of anhydride lactoferrin, glycerol, NaOH, and parabens are added and stirred to dissolve, finally purified water is added and stirred evenly to obtain the ointment. The above drugs are filled into the packaging container of the ointment to obtain the biologically active protein external ointment.

[0078] Example 6: Bioactive protein topical ointment

[0079] 1. Prescription: The mass of each ingredient in 100g of this prescription is:

[0080]

[0081] 2. Preparation method:

[0082] According to the above prescription of the biologically active protein external ointment, each component is weighed for use, firstly taking the prescribed amount of hydroxypropyl methylcellulose, adding purified water and stirring, then adding the prescribed amount of anhydride lactoferrin, glycerol, NaOH, and parabens and stirring to dissolve, finally adding purified water and stirring evenly to obtain the ointment. The above drugs are filled into the packaging container of the ointment to obtain the biologically active protein external ointment.

[0083] Example 7: Biologically active protein topical spray

[0084] 1. Prescription: The mass of each ingredient in 100g of this prescription is:

[0085]

[0086] 2. Preparation method:

[0087] According to the above prescription of the biologically active protein external ointment, each component is weighed for use, first the prescribed amount of carbomer is taken, and purified water is added and stirred, then the prescribed amount of acid anhydride lactoferrin, glycerol, NaOH, and phenoxyethanol are added and stirred to dissolve, and finally purified water is added and stirred evenly to obtain the ointment. The above drugs are filled into the packaging container of the spray to obtain the biologically active protein external spray.

[0088] Example 8: Biologically active protein topical spray

[0089] 1. Prescription: The mass of each ingredient in 100g of this prescription is:

[0090]

[0091] 2. Preparation method:

[0092] According to the above prescription of the biologically active protein external ointment, weigh each component for use, first take the prescribed amount of poloxamer, add purified water and stir, then add the prescribed amount of acid anhydride lactoferrin, glycerol, NaOH, phenoxyethanol and stir to dissolve, finally add purified water and stir evenly to obtain. Fill the above drugs into the packaging container of the spray to obtain the biologically active protein external spray.

[0093] Example 9: Biologically active protein topical spray

[0094] 1. Prescription: The mass of each ingredient in 100g of this prescription is:

[0095]

[0096] 2. Preparation method:

[0097] According to the above prescription of the biologically active protein external ointment, weigh each component for use, first take the prescribed amount of sodium alginate, add purified water and stir, then add the prescribed amount of acid anhydride lactoferrin, glycerol, NaOH, phenoxyethanol and stir to dissolve, finally add purified water and stir evenly to obtain. Fill the above drugs into the packaging container of the spray to obtain the biologically active protein external spray.

[0098] Example 10: Biologically active protein topical spray

[0099] 1. Prescription: The mass of each ingredient in 100g of this prescription is:

[0100]

[0101] 2. Preparation method:

[0102] According to the above prescription of the biologically active protein external ointment, weigh each component for use, first take the prescribed amount of hypromellose, add purified water and stir, then add the prescribed amount of acid anhydride lactoferrin, glycerol, NaOH, phenoxyethanol and stir to dissolve, finally add purified water and stir evenly to obtain. Fill the above drugs into the packaging container of the spray to obtain the biologically active protein external spray.

[0103] Example 11: Biological evaluation test of the sample of Example 4

[0104] 1. In vitro cytotoxicity test (MTT method)

[0105] After the sample was extracted at 0.1 g / mL, four concentrations of the extract (100%, 50%, 25%, 12.5%) were added to a 96-well culture plate (the wells contained cells cultured for 24 hours) and CO was set. 2 After culturing in a constant temperature incubator at 37±1°C for 24 hours, the absorbance was measured at a wavelength of 570nm (reference wavelength 650nm) using a tetrazolium salt (MTT) colorimetric method on an enzyme reader to calculate the relative survival rate. Cell survival rate (%) = D value of the test group / D value of the blank control group × 100%, the results are shown in Table 3.

[0106] Table 3 Cytotoxicity test results

[0107]

[0108]

[0109] As can be seen from Table 3, the relative survival rate of cells in the negative control group was 99.99%, with no potential cytotoxicity; the relative survival rate of cells in the positive control group was 3.06%, with potential cytotoxicity; the relative survival rate of cells in the 100% extract of the experimental group was 90.31%, with no potential cytotoxicity; the relative survival rate of cells in the 50% extract of the experimental group was 91.28%, with no potential cytotoxicity.

[0110] 2. Skin irritation

[0111] The sample was applied to the back skin of the experimental rabbit, and the patch was removed after 4 hours of contact. The negative control was operated in the same way. Observe the skin tissue reaction of the test site 1h, 24h, 48h and 72h after removing the patch and score it. Repeat the skin irritation test. After 72 hours of observation in the acute skin irritation test, apply the sample and negative control in the same way every day for 14 consecutive days, observe the skin tissue reaction of the test site on time and score it. Calculate the primary irritation index of the sample for the acute skin irritation test and the repeated skin irritation test respectively. The results are shown in Table 4-5.

[0112] Table 4 Rabbit skin irritation reaction scoring results (single contact test)

[0113]

[0114] Table 5 Rabbit skin irritation reaction (repeated contact test) scoring results

[0115]

[0116] It can be seen from Table 4-5 that the primary irritation index of the sample in the acute skin irritation test on experimental rabbits is 0; the primary irritation index of the sample in the repeated contact skin irritation test on experimental rabbits is 0, and the skin irritation reaction type is a very mild irritation reaction.

[0117] 3. Skin allergy (closed patch test)

[0118] After the guinea pigs were induced by repeated contact with the samples for a period of time, the test group animals were stimulated with the samples at the shaved untested parts. The skin reactions at the stimulated parts of all animals were graded again 24h and 48h after the patch was removed. The negative control group animals were operated in the same way. The results are shown in Table 6.

[0119] Table 6 Observation results of sensitization test

[0120]

[0121] As can be seen from Table 6, no patch test reaction was observed on the skin of the stimulated parts of the experimental group animals and the negative control group animals, no obvious allergic reaction symptoms were observed, the score level was 0, and the sensitization incidence rate was 0, indicating that the samples did not cause allergic reaction in the guinea pig skin in the guinea pig skin sensitization test.

[0122] Example 12: Comparison of the inhibitory effects of the sample in Example 4 and acyclovir cream on herpes zoster virus

[0123] Example 4 Sample, usage: Apply to the affected area three times a day for 4 to 7 days. After use, use the Varicella-Zoster IgG Antibody Detection Kit (ELISA) (Wuhan Biotechnology Co., Ltd.) for detection.

[0124] Acyclovir cream (Fujian Pacific Pharmaceutical Co., Ltd.), specification: 3%, usage: topical. Apply an appropriate amount of this product to the affected area, 4 to 6 times a day, for 7 days. After use, use the varicella-zoster IgG antibody detection kit (enzyme-linked immunosorbent assay) (Wuhan Biotechnology Co., Ltd.) for detection.

[0125] Table 7 Test results

[0126]

[0127] Note: “-” represents negative (P / N<1.5); “+” represents positive (P / N≥2.1); P and N are the values ​​after removing the blank control.

[0128] Test results: The test result of the sample of Example 4 was negative, and the test result of the acyclovir cream was positive, indicating that the sample of Example 4 had an inhibitory effect on the VZV virus, and the inhibitory effect of the acyclovir cream on the VZV virus was weaker than that of the sample of Example 4.

[0129] Example 13. In vitro penetration and permeation of antiviral drugs and acyclovir cream in pig skin tissue 1. In vitro permeation test

[0130] Preparation of pig skin tissue: Obtain abdominal skin from 2-month-old male Large White pigs in good health, remove fat and hair tissue, soak in physiological saline, and store at room temperature.

[0131] The porcine skin tissue was placed in a Franz diffusion trough: a circular portion of the tissue was sandwiched between a 1 cm 2 The impedance of the porcine skin tissue was measured between the two chambers of the Franz diffusion trough with the active diffusion area using a waveform generator to ensure the integrity of the tissue fragment used for penetration studies. Impedance > 3KΩ.cm 2 The pig skin tissue of was suitable for the study. The receiving chamber was filled with 8 ml of 5% pH 7.4 PBS solution, stirred at 600 rpm with a 3 mm magnetic stir bar, and the temperature was maintained at 37°C with a circulating water bath.

[0132] Loading of ointment in the donor chamber: About 200 mg of antiviral drug (Example 4 ointment, Example 7 spray) and acyclovir cream were filled into tared 1 mL syringes, respectively, and the samples were dispensed into the donor chamber. The drug and cream were spread on the skin surface with a pre-weighed applicator. After loading and spreading the drug and cream on the skin surface, the weight of the 1 mL syringe and applicator was marked to determine the exact amount of drug and cream loaded into the donor chamber.

[0133] Penetration and permeation studies: A time course (2h, 4h and 8h) porcine vaginal penetration study was performed. After loading with drug and cream, 500 μL samples were drawn from the receiving compartment at different time intervals and replaced with an equal volume of fresh receiving medium each time. The samples drawn at each time interval were immediately stored at -20°C until analysis. After 2, 4 and 8h, the formulation was removed from the donor compartment with the aid of a syringe and cleaned with a cotton swab. The tissue was removed and gently washed 5 times with a washing solution (50% methanol in water) or cleaned with a cotton swab.

[0134] Table 8 Time course IVPT study design

[0135] Research Acceptor fluid sampling time point Study period 2hIVPT 0 and 2h 2h 4hIVPT 0, 2 and 4 hours 4h 8hIVPT 0, 2, 4, 6 and 8 hours 8h

[0136] Preparation of sample drug solutions for analysis: Remove samples stored at -20°C and thaw at room temperature for 30 min. Centrifuge samples at 13000 rpm for 5 min, and add an equal volume of extraction solvent to 200 μL of supernatant. Centrifuge these samples at 13000 rpm for 5 min and transfer the supernatant to a vial for analysis.

[0137] The analytical and detection methods were respectively carried out in accordance with the BCA method, method 4, protein content determination method, general chapter 0731, part IV of the 2020 edition of the "Chinese Pharmacopoeia" and the [Content Determination] method under the "Acyclovir" detection item in part II of the 2020 edition of the "Chinese Pharmacopoeia".

[0138] Figure 1 This is an in vitro tissue penetration test in skin tissue, which compares the antiviral ointment and acyclovir cream. Bar A represents the tissue penetration rate of the antiviral ointment in skin tissue. Bar B represents the tissue penetration rate of the acyclovir cream in skin tissue. Since dimethyl sulfoxide transdermal agent is added during the preparation of the anhydride lactoferrin raw material, the results show that the antiviral ointment has a higher penetration rate in skin tissue than acyclovir cream, presents a high tissue penetration rate, and can produce high anti-VZV activity.

[0139] 2. Penetration test

[0140] The experimental steps of the permeation test were the same as above (i.e., the specific operations of “preparation of porcine skin tissue”, “loading of ointment in the donor chamber”, and “penetration and permeation study” were the same as those in the in vitro permeation test of Experiment 1).

[0141] Mincing porcine skin tissue (active diffusion zone) after IVPT: The 8 mm punch of the active diffusion zone of washed porcine skin tissue was removed, weighed and transferred to a tube. This tube was immediately placed in dry ice for approximately 15 min. After the designated time, the tissue was removed and placed in a pre-cooled culture dish. The tissue was minced into smaller pieces using a pre-cooled scalpel on a culture dish. The minced tissue was transferred to a sample tube, and the culture dish was rinsed with 1 ml of 5% pH 7.4 PBS solution and transferred to the same tissue sample tube. These tubes were stored at -70°C until analysis.

[0142] Extraction of drugs from porcine skin tissue: Take out the minced tissue samples stored at -70°C and thaw at room temperature for about 90 min. Keep the samples in BioShaker at room temperature for 4 h. After 4 h, centrifuge the samples at 13000 rpm for 5 min. Add 400 μL of extraction solvent to 100 μL of supernatant and vortex for 2 min. Centrifuge these samples at 13000 rpm for 5 min and transfer the supernatant to a vial for analysis.

[0143] The analytical and detection methods were respectively carried out in accordance with the BCA method, method 4, protein content determination method, general chapter 0731, part IV of the 2020 edition of the "Chinese Pharmacopoeia" and the [Content Determination] method under the "Acyclovir" detection item in part II of the 2020 edition of the "Chinese Pharmacopoeia".

[0144] Figure 2 The tissue penetration rates of the antiviral ointment of Example 4 and the spray of Example 7. The spray formulation produced a similar tissue penetration rate as the ointment formulation, with an average of 58 ng / mg of compound in the tissue, both having high tissue penetration rates and both producing high anti-VZV activity.

[0145] The above are only embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. The use of a biologically active protein in the preparation of a drug for treating herpes virus infection, characterized in that: The herpes virus is herpes zoster virus VZV, and the biologically active protein is 4-hydroxyphthalic anhydride modified lactoferrin. The specific preparation method is as follows: at a temperature of 25°C, 4-hydroxyphthalic anhydride is dissolved in dimethyl sulfoxide to obtain a saturated 4-hydroxyphthalic anhydride solution; lactoferrin is dissolved in a 0.1M trisodium phosphate solution to obtain a protein solution with a final concentration of 30 mg / mL; the 4-hydroxyphthalic anhydride solution is added to the protein solution, stirred and mixed, the final concentration of 4-hydroxyphthalic anhydride in the reaction system is 60 mM, and the reaction is allowed to stand for 2 hours; anhydride lactoferrin is obtained; after being filtered and sterilized with a 0.45uM filter membrane, a pH 7.2 Phosphate buffer solution was ultrafiltration exchange purified, and the ultrafiltration membrane used had a molecular weight cutoff of 10KD; then 0.45uM microporous filter membrane was used for filtration and sterilization, and a protective agent mannitol was added, and freeze drying was performed using a freeze dryer, and the freeze-dried powder was collected to obtain anhydride lactoferrin freeze-dried powder; the protein freeze-drying step included the following stages: pre-freezing stage: the partition temperature dropped to -70°C and maintained for 6 hours; primary sublimation stage: maintained for 12 hours; secondary sublimation stage: maintained for 7 hours.

Images