CN119113089B

CN119113089B - Application of biologically active protein in preparing medicine for treating herpes virus infection and medicine composition for treating herpes virus infection

Info

Publication number: CN119113089B
Application number: CN202411128691.3A
Authority: CN
Inventors: 常天安; 邹海霞; 李锋; 尹欣然; 李炳佑
Original assignee: Jiangsu Zhengda Tianchuang Biological Engineering Co ltd
Current assignee: Jiangsu Zhengda Tianchuang Biological Engineering Co ltd
Priority date: 2024-08-16
Filing date: 2024-08-16
Publication date: 2025-04-01
Anticipated expiration: 2044-08-16
Also published as: CN119113089A

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

Application of bioactive protein in preparing medicine for treating herpesvirus infection and medicine composition for treating herpesvirus infection

Technical Field

The invention relates to application of bioactive proteins in preparation of a medicament for treating herpes virus infection and a pharmaceutical composition for treating herpes virus infection, and belongs to the technical field of medicines.

Background

Herpes viruses HSV (herpes viruses) are a class of viruses with an envelope and double stranded DNA genome. There are 8 types of herpes viruses found to infect humans, including herpes simplex virus type 1 (herpes simplex virus-1, HSV-1), herpes simplex virus type 2 (herpes simplex virus-2, HSV-2), varicella zoster virus (VARICELLA ZOSTER VIRUS, VZV), epstein-Barr virus (EBV), human cytomegalovirus (human cytomegalovirus, HCMV), human herpes virus type 6 (HHV-6), human herpes virus type 7 (HHV-7) and Kaposi's sarcoma-associated virus (Kaposi's sarcoma-associated herpesvirus, KSHV). Patients and healthy carriers are sources of infection, primarily through direct intimate and sexual contact. HSV invades the body through various routes such as oral cavity, respiratory tract, genital tract mucous membrane and damaged skin. The infection rate of the human is 80-90%, the common clinical manifestation is herpes locally accumulated on mucous membrane or skin, and serious systemic diseases can occur occasionally, and viscera are involved.

At present, no medicine capable of directly killing the herpes virus exists, and the treatment principle of the herpes virus HSV infection is oral antiviral medicines, such as acyclovir, mecobalamin for nourishing nerves to relieve pain and prevent complications such as postherpetic neuralgia. Long-term administration of the antiviral drug acyclovir may cause damage to liver or kidney function. Symptoms such as arthralgia, diarrhea, headache, nausea, vomiting, dizziness and the like appear.

Herpes simplex virus HSV belongs to the subfamily of the alpha virus of the family herpesviridae and has a viral plasmid size of about 180 nanometers. The viruses are currently classified into type 1 and type 2 in the clinical setting of venereal disease hospitals based on antigenic differences. Type 1 is mainly obtained from the labial foci, from which type 2 can be isolated. Infection is due to human-to-human contact. The number of people infected from four months to years after occurrence can reach 50-90% of the total number of people's mouths, and is one virus most vulnerable to invasion, but only a part of the diseases occur clinically. The diseases can be classified into labial herpes, herpetic keratitis, herpetic dermatitis, pudendum herpes, kaposi disease, and the like, and sometimes are causative factors of meningitis and encephalitis.

Shingles is an acute infectious skin disease caused by varicella-zoster virus. After infection of children without immunity to this virus, varicella occurs. Some patients become virally infected without symptoms. Because the virus has neurotropic property, the virus can be hidden in neurons of spinal nerve rear root ganglion for a long time after infection, when the resistance is low or tired, the virus can grow and reproduce again when the virus is infected, and the virus moves to the skin along nerve fibers, so that the affected nerves and skin generate strong inflammation.

The lovir drug is mainly used for inhibiting virus replication by interfering virus DNA polymerase, and can be combined with a growing DNA chain under the action of the DNA polymerase to cause the extension interruption of the DNA chain, thereby playing an antiviral role, but the drug is difficult to permeate capsid protein to act on DNA, so that the treatment effect is poor.

The principle of action of interferon is to induce cells to synthesize antiviral proteins, thereby exerting antiviral action, but the effect of interferon is very small when viruses are in a highly replication state.

Therefore, a medicine which can directly kill the herpes virus and has good treatment effect and small side effect needs to be searched.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide application of bioactive proteins in preparing medicaments for treating herpes virus infection and a pharmaceutical composition for treating the herpes virus infection.

The technical scheme for solving the technical problems is as follows:

in a first aspect of the invention there is provided the use of a biologically active protein selected from the group consisting of anhydrated lactoferrin, anhydrated lactoglobulin, anhydrated whey protein or anhydrated histone in the manufacture of a medicament for the treatment of herpes virus infections, said herpes virus being herpes zoster virus VZV.

Preferably, in said application, said biologically active protein is 4-hydroxy phthalic anhydride modified lactoferrin. The 4-hydroxy phthalic anhydride modified lactoferrin disclosed in the invention is 4-hydroxy phthalic anhydride modified lactoferrin disclosed in Chinese patent 202110238692.3, and the preparation method is also referred to the method in Chinese patent 202110238692.3.

The inventors have unexpectedly found, through extensive research work, that modified bioactive proteins, such as anhydrated lactoferrin, have inhibitory effects on the herpes zoster virus (VZV). The acidylated lactoferrin is derived from milk protein, and the surface of the lactoferricin obtained after acidylation modification has a large amount of negative charges, and is combined with a positive charge region of a herpesvirus capsid protein to block the process of virus invasion into host cells, thereby achieving the purpose of blocking herpesvirus infection.

In a second aspect of the invention, a pharmaceutical composition capable of preventing and treating a herpes virus infection is provided. The herpesvirus is the herpes zoster virus VZV, the pharmaceutical composition is prepared from, by weight, 0.01-25% of bioactive protein, 0.01-5% of matrix, 0.1-5% of humectant, 0.1-4% of pH regulator, 0.01-3% of preservative and 58-99.77% of solvent, wherein the bioactive protein is selected from the group consisting of anhydrated lactoferrin, anhydrated lactoglobulin, anhydrated whey protein and anhydrated histone.

The anhydrated lactoferrin, the anhydrated lactoglobulin, the anhydrated whey protein and the anhydrated histone are derived from milk protein, and the anhydrated lactoferrin, the anhydrated lactoglobulin, the anhydrated whey protein or the anhydrated histone which are obtained after modification have a large amount of negative charges and can be combined with positive charge areas of herpesvirus capsid proteins to block the process of virus invasion into host cells, thereby achieving the purpose of blocking herpesvirus infection.

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

Preferably, in the pharmaceutical composition, the matrix comprises one or more of carbomer, sodium alginate, poloxamer and hypromellose.

The matrix is a medical polymer material with stable property, no stimulation and no sensitization, can be covered on erosion damaged tissues, can isolate and prevent wound infection, repair mucous membrane injury and promote wound healing.

Preferably, in the pharmaceutical composition, the humectant is one or more selected from glycerin, propylene glycol and sodium hyaluronate.

The moisture-retaining agent has good hygroscopicity, is mild and has no irritation, a layer of protective film can be formed on the skin to isolate the invasion of air, the skin can be well moisturized, the moisture of the air can be absorbed, and the skin chapping can be effectively prevented.

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

The normal pH value range of the skin surface is 4.0-7.0, and the pH value of the medicine can be reasonably designed by adding the pH regulator, so that the skin health is facilitated, and the skin is beneficial.

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

The preservative has broad-spectrum antibacterial performance, and particularly has very strong inhibition effect on gram-positive bacteria and gram-negative bacteria. The preservative can achieve an antibacterial effect by disrupting the protein activity of the bacterial cell membrane, causing cell death and blocking its growth and reproduction.

Preferably, in the pharmaceutical composition, the solvent is purified water.

Preferably, the dosage form of the pharmaceutical composition is an external dosage form, and each 100g of the pharmaceutical composition comprises the following components in parts by mass:

Bioactive proteins	0.01g-10g
		Substrate	0.02g-2.0g
Humectant type	0.5g-3g
		PH regulator	0.2g-2g
Preservative agent	0.05g-1g
		Solvent(s)	Allowance of

The preparation process of the external preparation formulation comprises the following steps:

1) Adding the matrix with the prescription amount into part of solvent, stirring, adding the bioactive protein with the prescription amount, humectant, pH regulator and antiseptic, stirring to dissolve, adding solvent, and stirring uniformly.

2) The external ointment can be obtained by filling the above medicines into the packaging container of the ointment, the external spray can be obtained by filling the above medicines into the packaging container of the spray, and other external preparations can be obtained by filling the above medicines into the packaging containers of other external preparations.

In one preferred embodiment of the invention, the bioactive protein is 4-hydroxy phthalic anhydride modified lactoferrin, the content of the anhydride lactoferrin is 5-10%, the content of the matrix is 0.02-2%, the content of the humectant is 1.0-2.0%, the content of the pH regulator is 0.1-0.5%, the content of the preservative is 0.1-1% and the balance of the solvent.

In one preferred embodiment of the invention, the pharmaceutical composition is a bioactive protein topical ointment, and each 100g of the prescription comprises the following specific components in mass:

The preparation method of the external ointment comprises weighing each component according to the prescription of the bioactive protein external ointment, firstly taking poloxamer with prescription amount, adding purified water, stirring, then adding the anhydride lactoferrin, glycerol, naOH and nipagin esters with prescription amount, stirring to dissolve, finally adding purified water, stirring uniformly, and finally filling the medicine into a packaging container of the ointment to obtain the bioactive protein external ointment.

In one preferred embodiment of the invention, the pharmaceutical composition is a bioactive protein external spray, and each 100g of the prescription comprises the following specific components in mass:

The preparation method of the bioactive protein external spray comprises the following steps:

weighing each component according to the prescription of the bioactive protein external ointment, firstly taking carbomer with the prescription amount, adding purified water, stirring, then adding the anhydride lactoferrin, glycerol, naOH and phenoxyethanol with the prescription amount, stirring to dissolve, finally adding purified water, and stirring uniformly to obtain the bioactive protein external ointment. And filling the medicine into a packaging container of the spray to obtain the bioactive protein external spray.

The invention has the following technical effects:

1) The inventors have conducted extensive research and experimental verification work to find for the first time that modified bioactive proteins, i.e. anhydrated lactoferrin, have an inhibitory effect on herpes zoster virus (VZV), and this property is demonstrated in examples 12-13.

2) The medicine composition has reasonable auxiliary material types, dosage and main component proportion, is administered in the form of ointment, spray or other dosage forms suitable for external use, has obvious curative effect, small local toxic and side effect, low acute toxicity of skin, no irritation and sensitization, no interference to normal functions of skin, safety and effectiveness. The preparation prepared according to the prescription of the invention has good appearance, moderate viscosity and easy application and spray administration. The preparation process is feasible, the quality is controllable, the preparation process is simple, and the preparation process is suitable for industrial mass production.

Drawings

FIG. 1 is an in vitro transdermal test.

FIG. 2 is the tissue penetration rate of the antiviral drug ointment and spray of example 13.

Detailed Description

The present invention is further described in connection with the following detailed description, which is not intended to be limiting, and the detailed description is not to be taken in a limiting sense, but is defined by the appended claims, along with the full scope of equivalents to which such claims are entitled.

The anhydrides lactoferrin used in all examples of the present invention are 4-hydroxy phthalic anhydride modified lactoferrin, i.e. 4-hydroxy phthalic anhydride modified lactoferrin disclosed in chinese patent "202110238692.3", and the preparation method is also referred to the method in chinese patent "202110238692.3".

The preparation method comprises the steps of dissolving 4-hydroxy phthalic anhydride in dimethyl sulfoxide at 25 ℃ to obtain saturated 4-hydroxy phthalic anhydride solution, dissolving lactoferrin in 0.1M trisodium phosphate solution to obtain protein solution with the final concentration of 30mg/mL, adding the 4-hydroxy phthalic anhydride solution into the protein solution, stirring and mixing uniformly, keeping the final concentration of the 4-hydroxy phthalic anhydride in a reaction system to be 60mM, standing for 2 hours to obtain the anhydrated lactoferrin, carrying out ultrafiltration displacement purification by using a pH 7.2 phosphate buffer solution after carrying out suction filtration sterilization by using a 0.45uM filter membrane, carrying out suction filtration sterilization by using a 0.45uM microporous filter membrane, adding protective agent mannitol, carrying out freeze drying by using a freeze dryer, and collecting freeze-dried powder to obtain the anhydrated lactoferrin freeze-dried powder. The protein freeze-drying step comprises the following steps of a pre-freezing stage, a primary sublimation stage and a secondary sublimation stage, wherein the temperature of a baffle plate is reduced to-70 ℃ and the retention time is 6 hours, and the primary sublimation stage is kept for 12 hours and the secondary sublimation stage is kept for 7 hours.

EXAMPLE 1 determination of the bacteriostatic Activity of anhydrated lactoferrin

And inoculating the inclined plane strain with a broth peptone culture solution, performing expanded culture for 16-20 hours, counting on a nutrient agar culture medium plate, and adjusting the concentration of the bacterial suspension to 106-108 CFU/ml. Inoculating the young fungus suspension of the strain on a nutrient agar culture medium, and uniformly coating. A sterile oxford cup was placed in the center of the dish and 0.1ml of sterile microfiltered lactoferrin and the anhydrated modified lactoferrin solution of example 1 (pH 7.0) were added to the cup, with sterile water as a control. After the petri dish was placed in a 37 ℃ constant temperature incubator for 24 hours, the diameter (mm) of the inhibition zone was measured. The lowest sample concentration greater than the diameter of the control zone (8 mm) was the lowest inhibitory concentration (MIC, μg/ml).

TABLE 1 diameter of zone of inhibition (mm) produced by anhydrated lactoferrin against different microorganisms

As can be seen from Table 1, the anhydrated lactoferrin has been found to have a broad spectrum of antibacterial, antioxidant effects, and various degrees of inhibition and killing against many microorganisms, including gram-positive and gram-negative aerobic and anaerobic bacteria and some fungi.

EXAMPLE 2 detection of the inhibitory Activity of anhydrated lactoferrin against herpes zoster Virus (VZV)

1. Construction of VZV pseudoviruses

The VZV related gene (Genbank: E36506.1) is subjected to codon optimization, amplified by Polymerase Chain Reaction (PCR), cloned into eukaryotic cell expression vector, and then subjected to enzyme digestion and sequencing identification to form the expression vector of pVZV with stable expression. Sequencing was carried out to identify the correct three expression vector plasmids and the reporter plasmid pCMV-Gluc for large scale amplification. The expression vector plasmids and reporter plasmids used were purchased from the company Shanghai, inc.

293FT cells (purchased from North Narah Biotechnology Co., ltd.) are spread on a 96-well cell culture plate, after 12 hours, VZV expression plasmids are added into 0.25mol/L CaCl ₂ solution in different amounts, caCl ₂ solution of the plasmids is dripped into an equal volume of 2X HeBS (pH=6.97) solution, the mixture is uniformly mixed, after standing for 5 minutes, the mixed solution is added into pre-spread 293FT cell holes, after 12 hours, the culture medium is replaced, after transfection for 48 hours, all the well cells are collected, the same volume of lysate is used for cracking for 16 hours at 37 ℃, the supernatant is collected to obtain pseudovirus suspension, and the pseudovirus suspension is subpackaged and placed at-80 ℃ for standby.

2. Virus titer assay

The VZV pseudovirus suspension was serially diluted 10-fold from 10 ^-1 to 10 ^-6, medium was removed from 96 wells, 50 μl of pseudovirus dilution was added, 5 μl of normal medium was then added, 37 ℃ and 5% co ₂ was further cultured for 72h, 50 μl of culture supernatant was taken and detected using the merck alkaline phosphatase detection kit, and TCID50 was calculated.

3. Detection of inhibitory Activity of anhydrated lactoferrin on VZV

293FT cells were passaged into 96-well plates with a cell number of 1X105 cells per well, incubated at 37℃with 5% CO ₂ for 8h. The VZV pseudovirus (50M) of 100TCID50 was incubated with different concentrations of the anhydrified lactoferrin lyophilized powder solution, respectively, for 30min at 37 ℃. The medium in the 96-well plate was removed, the above incubated mixture was added, the culture was continued for 12 hours at 37℃with 5% CO ₂, fresh medium was changed, after 72 hours of culture, 50M culture supernatant was taken, and the IC50 (half inhibitory concentration) of the anhydrated lactoferrin was calculated using SPSS25, using the merck alkaline phosphatase assay kit. The results are shown in Table 2.

TABLE 2 detection of the inhibitory Activity of anhydrated lactoferrin against VZV

It can be seen from Table 2 that the anhydrated lactoferrin has an inhibitory effect on VZV.

EXAMPLE 3 bioactive protein topical ointment

100G of prescription, wherein the mass of each component in the prescription is as follows:

anhydrated lactoferrin	5g
		Carbomer (carbomer)	1g
Glycerol	2g
		NaOH	0.3g
Nipagin esters	0.1g
		Purified water	91.6g

Secondly, the preparation method comprises the following steps:

Weighing the components according to the prescription of the bioactive protein external ointment for standby, firstly taking carbomer with the prescription amount, adding purified water for stirring, then adding the anhydride lactoferrin, glycerol, naOH and nipagin esters with the prescription amount for stirring to dissolve, finally adding the purified water, and stirring uniformly to obtain the bioactive protein external ointment. And filling the medicines into a packaging container of the ointment to obtain the bioactive protein external ointment.

EXAMPLE 4 bioactive protein topical ointment

Secondly, the preparation method comprises the following steps:

Weighing the components according to the prescription of the bioactive protein external ointment for standby, firstly taking poloxamer with the prescription amount, adding purified water for stirring, then adding the anhydride lactoferrin, glycerol, naOH and nipagin esters with the prescription amount for stirring to dissolve, finally adding purified water, and stirring uniformly to obtain the bioactive protein external ointment. And filling the medicines into a packaging container of the ointment to obtain the bioactive protein external ointment.

EXAMPLE 5 bioactive protein topical ointment

Secondly, the preparation method comprises the following steps:

Weighing the components according to the prescription of the bioactive protein external ointment for standby, firstly taking the sodium alginate with the prescription amount, adding purified water for stirring, then adding the acid anhydride lactoferrin, glycerol, naOH and nipagin esters with the prescription amount for stirring to dissolve, finally adding the purified water, and stirring uniformly to obtain the bioactive protein external ointment. And filling the medicines into a packaging container of the ointment to obtain the bioactive protein external ointment.

EXAMPLE 6 bioactive protein topical ointment

Secondly, the preparation method comprises the following steps:

Weighing the components according to the prescription of the bioactive protein external ointment for standby, firstly taking the hydroxypropyl methylcellulose with the prescription amount, adding purified water for stirring, then adding the anhydride lactoferrin, glycerol, naOH and nipagin esters with the prescription amount for stirring to dissolve, finally adding the purified water, and stirring uniformly to obtain the bioactive protein external ointment. And filling the medicines into a packaging container of the ointment to obtain the bioactive protein external ointment.

EXAMPLE 7 bioactive protein topical spray

Secondly, the preparation method comprises the following steps:

Example 8 bioactive protein topical spray

Secondly, the preparation method comprises the following steps:

Weighing the components according to the prescription of the bioactive protein external ointment for standby, firstly taking poloxamer with the prescription amount, adding purified water for stirring, then adding the anhydride lactoferrin, glycerol, naOH and phenoxyethanol with the prescription amount for stirring to dissolve, finally adding purified water, and stirring uniformly to obtain the bioactive protein external ointment. And filling the medicine into a packaging container of the spray to obtain the bioactive protein external spray.

EXAMPLE 9 bioactive protein topical spray

Secondly, the preparation method comprises the following steps:

weighing the components according to the prescription of the bioactive protein external ointment for standby, firstly taking the sodium alginate with the prescription amount, adding purified water for stirring, then adding the lactoferritin anhydride, glycerol, naOH and phenoxyethanol with the prescription amount for stirring to dissolve, finally adding the purified water, and stirring uniformly to obtain the bioactive protein external ointment. And filling the medicine into a packaging container of the spray to obtain the bioactive protein external spray.

Example 10 bioactive protein topical spray

Secondly, the preparation method comprises the following steps:

Weighing the components according to the prescription of the bioactive protein external ointment for standby, firstly taking the hydroxypropyl methylcellulose with the prescription amount, adding purified water for stirring, then adding the anhydride lactoferrin, glycerol, naOH and phenoxyethanol with the prescription amount for stirring to dissolve, finally adding the purified water, and stirring uniformly to obtain the bioactive protein external ointment. And filling the medicine into a packaging container of the spray to obtain the bioactive protein external spray.

EXAMPLE 11 biological evaluation test of the sample of example 4

1. In vitro cytotoxicity assay (MTT method)

After leaching a sample by 0.1g/mL, adding 4 test solutions (100%, 50%, 25% and 12.5%) of the leaching solution into a 96-well culture plate (cells cultured for 24 hours in the holes), culturing for 24 hours in a CO ₂ constant-temperature incubator at 37+ -1 ℃, measuring absorbance (reference wavelength 650 nm) at 570nm wavelength of an enzyme-labeled instrument by using a tetrazolium salt (MTT) colorimetric method, and calculating relative survival rate. Cell viability (%) = test group D value/blank group D value x 100%, the results are shown in table 3.

TABLE 3 cytotoxicity test results

As can be seen from Table 3, the relative cell viability of the negative control group was 99.99% and was free from potential cytotoxicity, the relative cell viability of the positive control group was 3.06% and was potentially cytotoxic, the relative cell viability of the test group was 90.31% and was free from potential cytotoxicity, and the relative cell viability of the test group was 91.28% and was free from potential cytotoxicity.

2. Skin irritation

The samples were applied to the back skin of the experimental rabbits and the application was removed after 4h of contact. Negative controls were run in the same manner. The test sites were observed for skin tissue response and scored 1h, 24h, 48h and 72h after removal of the dressing. The skin irritation test was repeated, and after 72 hours of observation in the acute skin irritation test, the samples and the negative control were applied daily in the same way for 14 consecutive days, and skin tissue reactions at the test site were observed on time and scored. Acute skin irritation test and repeated skin irritation test the primary irritation index of the sample was calculated separately. The results are shown in tables 4-5.

TABLE 4 skin irritation response scoring results for rabbits (Single contact test)

TABLE 5 scoring results of rabbit skin irritation response (repeated contact test)

As can be seen from tables 4 to 5, the primary stimulation index of the sample for the acute skin stimulation test of the experimental rabbit was 0, and the primary stimulation index of the sample for the repeated skin stimulation test of the experimental rabbit was 0, and the skin stimulation response type was very slight stimulation response.

3. Skin sensitization reaction (closed application test)

After repeated sample contact for induction over a period of time, the test group animals were stimulated with the sample at the shaved non-tested sites. And the skin reactions at all animal sites of challenge were graded 24h, 48h after patch removal. Animals of the negative control group were operated in the same manner. The results are shown in Table 6.

TABLE 6 sensitization test observations

As can be seen from Table 6, the skin of the excitation site of the animals in the test group and the skin of the animals in the negative control group have no application test reaction, no obvious sensitization symptom is observed, the score grade is 0, the sensitization incidence rate is 0, and the sample does not cause the skin sensitization reaction of the guinea pigs in the skin sensitization test of the guinea pigs.

Example 12 comparison of the inhibitory Effect of the sample of example 4 and acyclovir cream on herpes zoster Virus

The sample of example 4 is applied to the affected part three times a day for 4-7 days. After the end of the use, the detection was carried out using a varicella-zoster IgG antibody detection kit (ELISA) (WUHF Co., ltd.).

Acyclovir cream (Fujian Pacific pharmaceutical Co., ltd.) 3% in specification, and its application method, topical application. The product is applied to the affected part for 4-6 times a day for 7 days. After the end of the use, the detection was carried out using a varicella-zoster IgG antibody detection kit (ELISA) (WUHF Co., ltd.).

TABLE 7 detection results

Note that "-" represents negative (P/N < 1.5), "+" represents positive (P/N. Gtoreq.2.1), and both P and N are values after the blank control is removed.

The detection result shows that the detection result of the sample of the example 4 is negative, and the detection result of the sample of the example 4 is positive, which indicates that the sample of the example 4 has an inhibition effect on the VZV virus, and the inhibition effect of the acyclovir emulsifiable paste on the VZV virus is weaker than that of the sample of the example 4.

EXAMPLE 13 ex vivo penetration of antiviral Agents and acyclovir cream in porcine skin tissue 1 penetration in vitro transdermal test

The pig skin tissue is prepared by taking abdomen skin, removing fat and hair tissue, soaking in physiological saline, and preserving at normal temperature.

The pig skin tissue was placed in a Franz diffusion cell with a circular portion of tissue sandwiched between two chambers of the Franz diffusion cell with an active diffusion area of 1cm ², and the impedance of the pig skin tissue was measured using a waveform generator to ensure the integrity of the tissue fragments for penetration studies. Pig skin tissue with impedance >3K omega cm ² was suitable for investigation. The receiving chamber was filled with 8ml of 5% PBS pH7.4, stirred with a 3mm magnetic stirrer bar at 600rpm, and the temperature was maintained at 37℃with a circulating water bath.

Loading of ointment in donor chamber about 200mg of antiviral drug (example 4 ointment, example 7 spray) and acyclovir cream were filled separately into tared 1mL syringes and samples were dispensed into the donor chamber. The drug and cream were spread onto the skin surface with a pre-weighed applicator. After the drug and cream were loaded and spread onto the skin surface, the weight of the 1mL syringe and applicator was noted to determine the exact amount of drug and cream loaded into the donor chamber.

Penetration and penetration studies swine vaginal penetration studies were performed over time (2 h, 4h and 8 h). After loading with the drug and cream, 500 μl of samples were withdrawn from the receiving compartments at different time intervals and the withdrawn samples were replaced each time with an equal volume of fresh receiving medium. Samples taken at each time interval were immediately stored at-20 ℃ until analysis. 2. After 4 and 8 hours, 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 wash solution (50% methanol water) or cleaned with a cotton swab.

Table 8 time course IVPT study design

Study of	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

Sample drug solutions for analysis were prepared by taking samples stored at-20 ℃ and thawing for 30min at room temperature. The samples were centrifuged at 13000rpm for 5min and an equal volume of extraction solvent was added to 200 μl of supernatant. The samples were centrifuged at 13000rpm for 5min and the supernatant transferred to a vial for analysis.

The analysis and detection method is respectively detected by referring to a fourth BCA method of four general rule 0731 protein content determination method of Chinese pharmacopoeia 2020 edition and a method under the detection item of two acyclovir of Chinese pharmacopoeia 2020 edition.

Fig. 1 is an in vitro tissue penetration test in skin tissue comparing antiviral drug ointment with 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. As the dimethyl sulfoxide transdermal agent is added in the preparation process of the anhydride lactoferrin raw material, the result shows that the antiviral drug ointment has higher permeability than acyclovir emulsifiable paste in skin tissues, has high tissue permeability and can generate high anti-VZV activity.

2. Penetration test

The experimental procedure of the penetration test was the same as above (i.e. "preparation of pig skin tissue", "loading of ointment in donor chamber", "penetration study", the specific procedure was the same as in the in vitro transdermal test of experiment 1).

Pig skin tissue (active diffusion area) was minced after IVPT. The 8mm punch that washed the active diffusion area of pig skin tissue was removed, weighed and transferred into a tube. This was followed by placing the tube in dry ice for about 15min. After a specified time, the tissue is removed and placed in a pre-cooled petri dish. Tissue was minced into smaller pieces on a petri dish with a pre-cooled scalpel. The minced tissue was transferred to a sample tube and the dish was rinsed with 1ml of 5% ph7.4 PBS solution and transferred to the same tissue sample tube. These tubes were stored at-70 ℃ until analysis.

Drug was extracted from pig skin tissue by taking out tissue samples stored in-70 ℃ shreds and thawing at room temperature for about 90min. The sample was kept at room temperature for 4h with shaking in BioShaker. After 4h, the samples were centrifuged at 13000rpm for 5min. To 100. Mu.L of the supernatant, 400. Mu.L of the extraction solvent was added and vortexed for 2min. The samples were centrifuged at 13000rpm for 5min and the supernatant transferred to a vial for analysis.

Fig. 2 is the tissue penetration rates of the antiviral drug ointment of example 4 and the spray of example 7. The spray formulation produced a similar tissue penetration rate to the ointment formulation, with an average of 58ng/mg of compound in the tissue, both with high tissue penetration rates, both producing high anti-VZV activity.

The foregoing is merely exemplary embodiments of the present invention and are not intended to limit the scope of the present invention, and all equivalent modifications made by the present invention or direct or indirect application in other related technical fields are included in the scope of the present invention.

Claims

1. Use of a biologically active protein in preparing 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, and the specific preparation method is as follows: at a temperature of 25° C., dissolving 4-hydroxyphthalic anhydride in dimethyl sulfoxide to obtain a saturated 4-hydroxyphthalic anhydride solution; dissolving lactoferrin in a 0.1M trisodium phosphate solution to obtain a protein solution with a final concentration of 30 mg/mL; adding the 4-hydroxyphthalic anhydride solution to the protein solution, stirring and mixing, wherein the final concentration of 4-hydroxyphthalic anhydride in the reaction system is 60 mM, and standing for reaction for 2 hours; obtaining anhydride-modified lactoferrin; and filtering with a 0.45 uM filter membrane for sterilization and then using 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 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.