CN118078964A - Recombinant human interferon ointment and its preparation method - Google Patents

Recombinant human interferon ointment and its preparation method Download PDF

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CN118078964A
CN118078964A CN202410496819.5A CN202410496819A CN118078964A CN 118078964 A CN118078964 A CN 118078964A CN 202410496819 A CN202410496819 A CN 202410496819A CN 118078964 A CN118078964 A CN 118078964A
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recombinant human
human interferon
ointment
skin
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CN118078964B (en
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刘景会
刘琳琳
王莹
王宇
刘玉林
黄曼
郑全莉
杨红育
富瑞丽
王江林
刘晨
徐小明
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Changchun Institute of Biological Products
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Abstract

The invention discloses a recombinant human interferon ointment and a preparation method thereof, wherein the recombinant human interferon ointment comprises the following raw material components in parts by weight: 0.015-0.025 parts of recombinant human interferon alpha-2 b, 550-600 parts of stabilizing agent, 10-20 parts of thickening agent, 2-3 parts of penetration enhancer, 5-8 parts of surfactant, 1-3 parts of pH regulator, 1-2 parts of preservative and 350-450 parts of water. The recombinant human interferon ointment with excellent stability and curative effect is successfully prepared by carefully selecting and proportioning the raw material components. Compared with the prior art, the invention not only improves the permeation efficiency and bioavailability of the medicine, but also optimizes the use experience of patients, improves the compliance and safety of treatment, and provides an effective local administration new choice for clinical application of recombinant human interferon alpha-2 b.

Description

Recombinant human interferon ointment and its preparation method
Technical Field
The invention relates to the technical field of pharmaceutical preparations, in particular to a recombinant human interferon ointment and a preparation method thereof.
Background
The recombinant human interferon is a medicine with antiviral, antitumor and immunoregulation functions, and is prepared through gene recombination technology and has similar structure and function to human body naturally produced interferon. In the medical field, the medicine plays a role in enhancing the resistance of cells to viruses, inhibiting the replication of the viruses, activating immune cells, improving the immunity of human bodies and effectively treating viral diseases and tumors.
Recombinant human interferon alpha-2 b (rhIFN alpha-2 b) is one of recombinant human interferon and has multiple functions of antivirus, anti-tumor, immunoregulation and the like. rhIFN alpha-2 b activates intracellular signaling by binding to cell surface receptors, exerting therapeutic effects. It is used mainly in treating chronic viral hepatitis, such as hepatitis B and C, condyloma acuminatum, certain hematopathy, such as hairy cell leukemia and chronic granulocytic leukemia, lymphoma, melanoma, etc. In addition, it is also suitable for other viral diseases such as influenza virus pneumonia. During treatment, doctors can make personalized schemes according to the illness state of patients. The use of rhIFN alpha-2 b may be accompanied by adverse reactions, requiring patient response monitoring and treatment adjustment under the direction of a physician. Different dosage forms, such as injection, gel, ointment, etc., can influence the absorption and curative effect of the medicine. The recombinant human interferon alpha-2 b can be prepared into ointment for local application, so that the medicine can directly act on an affected part, and the local medicine concentration is improved, thereby enhancing the treatment effect. In addition, the ointment can reduce systemic side effects of the medicine and improve the medication comfort of patients. However, ointment formulations may suffer from the disadvantages of poor skin penetration of the drug, initiation of skin irritation and poor stability, affecting the therapeutic effect and patient experience, and therefore, there is a need to continually explore new techniques and methods to improve the disadvantages.
CN104043112B discloses an ointment pharmaceutical composition containing recombinant human interferon alpha-2B (pseudomonas), the medicine provided by the invention comprises the following components: recombinant human interferon alpha-2 b, human serum albumin, glycerol, sodium carboxymethyl cellulose, citric acid, sodium citrate, sodium dodecyl sulfate, chlorogenic acid and purified water. The chlorogenic acid is added into the formula to be prepared into an ointment preparation together with the recombinant human interferon alpha-2 b, so that the skin irritation is greatly reduced, the release speed of the medicine can be accelerated, and the absorption of the medicine and the use of the preparation are facilitated. However, the poor permeability of the pharmaceutical composition may lead to the fact that the drug cannot effectively penetrate to the target site, and the drug accumulates in other non-target sites, resulting in the waste of the drug and unnecessary side effects, not only reducing the therapeutic effect, but also possibly increasing the discomfort and risk of the patient.
CN110559425A discloses a stable recombinant human interferon ointment and its production method, the ointment raw material provided by the invention includes recombinant human interferon alpha-2 b and ointment base material, in which the emulsifiable base material is formed from glycerine, dextran 40, ethyl p-hydroxybenzoate, water for injection, white vaseline, glyceryl monostearate, distearate and polysorbate-80. The preparation process of the recombinant human interferon ointment provided by the invention comprises four steps of water phase preparation, oil phase preparation, stock solution dilution and matrix emulsification, and is simple and convenient to operate and convenient for practical production and application. The ointment can be stored for a long time under low temperature, and has good quality and stability. However, the ointment may irritate the skin, which causes discomfort, pain, redness, itching and other adverse reactions to the patient, and the compliance of the patient is poor, which is unfavorable for the use of the medicine.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention provides a recombinant human interferon ointment and a preparation method thereof, wherein recombinant human interferon alpha-2 b is used as a main active ingredient, and the prepared recombinant human interferon ointment has excellent storage stability and transdermal penetration effect by reasonably proportioning with other raw materials, so that the transdermal drug administration effect of the drug is optimized, skin irritation is reduced, and comfort and treatment compliance of patients are improved.
The invention aims at providing a recombinant human interferon ointment, which comprises the following raw material components in parts by weight:
0.015-0.025 parts of recombinant human interferon alpha-2 b, 550-600 parts of stabilizing agent, 10-20 parts of thickening agent, 2-3 parts of penetration enhancer, 5-8 parts of surfactant, 1-3 parts of pH regulator, 1-2 parts of preservative and 350-450 parts of water.
Preferably, the penetration enhancer is selected from at least two of β -caryophyllene, amino acid derivatives and 1, 2-propanediol.
Preferably, the mass ratio of the beta-caryophyllene, the amino acid derivative and the 1, 2-propylene glycol in the penetration enhancer is 0.5-1.5:0.5-1.5:0.75-1.5.
Preferably, the amino acid derivative is selected from one of L-leucine methyl ester hydrochloride, L-proline methyl ester hydrochloride and L-proline dodecyl ester hydrochloride.
Further preferably, the preparation method of the L-proline dodecyl ester hydrochloride comprises the following steps in parts by weight:
1.3 to 1.4 parts of L-proline, 2.5 to 2.8 parts of n-dodecanol and 2.2 to 2.4 parts of p-toluenesulfonic acid are taken and dissolved in 100 to 120 parts of toluene, reflux reaction is carried out for 4 to 5 hours at the temperature of 125 to 135 ℃, the reaction solution is extracted for 2 to 3 times by 200 to 220 parts of sodium hydroxide aqueous solution with the mass percent of 5 to 6 percent, the organic layer obtained by extraction is dried for 10 to 12 hours by anhydrous sodium sulfate, and a crude product is obtained by drying by a rotary evaporator; petroleum ether: performing column chromatography on the obtained crude product by using mixed solution with the volume ratio of isopropanol of 7-10:1 as an eluent to obtain colorless liquid, namely L-proline dodecyl ester; introducing hydrogen chloride gas into the L-proline dodecyl ester for 4-5 h according to the molar ratio of the hydrogen chloride gas to the L-proline dodecyl ester of 1.1-1.2:1, and drying by using a rotary evaporator to obtain viscous yellow liquid, namely the L-proline dodecyl ester hydrochloride.
Preferably, the content of the recombinant human interferon alpha-2 b is 1X 10 4～2×108 IU/g ointment according to the active mass ratio.
Preferably, the pH value of the recombinant human interferon alpha-2 b ointment is 5.5-6.5.
Preferably, the thickener is selected from one or more of sodium carboxymethyl cellulose, carbomer, poloxamer, xanthan gum, hydroxypropyl guar, and sodium carboxymethyl cellulose.
Preferably, the surfactant is selected from one or more of polyethylene glycol-7, stearate, polysorbate-80, stearic acid, and glyceryl monostearate.
Preferably, the pH regulator is selected from one or more of citric acid, tartaric acid, acetic acid, sodium hydroxide and triethanolamine.
Preferably, the preservative is selected from one or more of ethyl parahydroxybenzoate, phenoxyethanol, polylysine and potassium sorbate.
Preferably, the stabilizer is selected from one or both of glycerin and polyelectrolyte complex.
Further preferably, the preparation method of the polyelectrolyte complex in the stabilizer comprises the following steps in parts by weight:
dissolving 4-4.5 parts of chitosan in 100-120 parts of 1-2 mg/mL of acetic acid aqueous solution to obtain chitosan solution; 2 to 2.5 parts of chitosan solution is dropwise added into 10 to 12 parts of 1 to2 weight percent of cellulose sodium sulfate aqueous solution at the rate of 600 to 650 mu L/min by using a constant flow pump, and the mixture is stirred for 15 to 20 minutes at the temperature of 23 to 25 ℃ to obtain a polyelectrolyte compound; washing the obtained polyelectrolyte complex with water for 2-3 times, and then freeze-drying for 10-12 hours for standby;
Or, dissolving 4-4.5 parts of polyethylene glycol chitosan in 100-120 parts of 1-2 mg/mL acetic acid aqueous solution to obtain polyethylene glycol chitosan solution; 2 to 2.5 parts of the polyethylene glycol chitosan solution is dropwise added into 10 to 12 parts of 1 to 2 weight percent of cellulose sodium sulfate aqueous solution at the rate of 600 to 650 mu L/min by using a constant flow pump, and the mixture is stirred for 15 to 20min at the temperature of 23 to 25 ℃ to obtain a polyelectrolyte compound; the polyelectrolyte complex is washed for 2-3 times by water, and then freeze-dried for 10-12 hours for standby.
Further preferably, the preparation method of the polyethylene glycol chitosan comprises the following steps of, by weight:
Dissolving 0.025-0.03 part of chitosan in 50-60 parts of 2-3 mg/mL of acetic acid aqueous solution, and stirring for 22-24 hours; then adjusting the pH value to 6-6.3 by using 5-6 mol/L sodium hydroxide aqueous solution to obtain chitosan solution; dissolving 0.4-0.45 part of methoxy-polyethylene glycol-succinimidyl valerate in 8-10 parts of anhydrous dimethyl sulfoxide to obtain methoxy-polyethylene glycol-succinimidyl valerate solution; then adding methoxy-polyethylene glycol-succinimidyl valerate solution into chitosan solution, and stirring for 48-50 h to obtain mixed solution; and (3) placing the mixed solution into a dialysis bag, dialyzing with 500-600 parts of water for 4-5 d, and then freeze-drying the dialyzed mixture to obtain the polyethylene glycol chitosan.
The second object of the present invention is to provide a method for preparing the recombinant human interferon ointment, comprising the following steps:
Mixing and stirring the stabilizer and water for 0.5-1.5 h, and then carrying out pressurized heating sterilization treatment under the conditions of 0.05-0.1 MPa and the temperature of 115-135 ℃ to obtain a mixed system A; mixing and stirring the recombinant human interferon alpha-2 b with the mixed system A for 0.5-1.5 h to obtain recombinant human interferon alpha-2 b liquid; the thickening agent, the penetration enhancer, the surfactant, the pH regulator, the preservative and the water are subjected to pressurized heating sterilization treatment under the conditions of 0.05-0.1 MPa and the temperature of 115-135 ℃, then are placed in a stirring container, stirred and mixed for 1-1.5 h under the conditions of 80-85 ℃ and 50-100 rpm, and then are cooled to below 25 ℃ to obtain an emulsified matrix; mixing recombinant human interferon alpha-2 b liquid and an emulsifying matrix, stirring for 0.5-1.5 h to obtain ointment, filling the ointment into a medicinal ointment tube, and packaging the ointment into a finished product after verification, wherein the weight of the content of the finished ointment is 4.85-5.15 g/count.
Glycerol: glycerol is an excellent humectant, which not only can absorb and retain moisture, maintain the proper moisture content of the ointment, but also can soften the stratum corneum of the skin and increase the permeability of the medicine. Meanwhile, the solvent characteristic of the glycerol is favorable for uniformly dispersing the recombinant human interferon alpha-2 b in an ointment matrix, so that the stability and the curative effect of the medicine are improved.
Citric acid: the citric acid is used as a pH regulator to provide a stable acidic environment for the recombinant human interferon alpha-2 b, which is helpful for improving the stability of the medicine. In addition, the chelation of citric acid can reduce the adverse effect of metal ions and further protect the medicine from degradation.
Polysorbate-80: polysorbate-80, as a surfactant, can reduce interfacial tension between the drug and the skin and promote permeation of the drug. The emulsification of the ointment is also beneficial to the uniform dispersion of the medicine in the ointment, and the stability of the ointment and the bioavailability of the medicine are improved.
Ethyl p-hydroxybenzoate: the ethyl p-hydroxybenzoate has antibacterial and antiseptic effects, can effectively prevent microbial contamination of the ointment during storage and use, protect recombinant human interferon alpha-2 b from microbial degradation, and prolong the effective period of the ointment.
Sodium carboxymethyl cellulose: sodium carboxymethylcellulose is used as a thickener to increase the viscosity and stability of ointments, making them easier to apply and uniformly cover the skin surface. The three-dimensional network structure formed by the sodium carboxymethyl cellulose is used as a drug carrier, which is favorable for uniform dispersion and slow release of the drug, thereby promoting effective penetration and absorption of the drug.
Beta-caryophyllene: beta-caryophyllene is used as a natural permeation enhancer, and has longer carbon chains and higher fat solubility. Beta-caryophyllene can interact with skin stratum corneum lipids, interfere with the lipid alignment of the stratum corneum, and cause lipid molecules to change from ordered crystal structures to disordered liquid crystal structures, and the structural change facilitates permeation of drug molecules. And moreover, the beta-caryophyllene can slowly move in the lipid bilayer, and is beneficial to retention in the stratum corneum and improvement of drug permeability by being tightly combined with the lipid. In addition, beta-caryophyllene can temporarily reduce the skin barrier function by increasing the water molecule transmittance of the skin, the reduction is reversible, the self-recovery capability of the skin after the drug permeation is ensured while the drug permeation is promoted, and the long-term damage to the skin barrier can be minimized, so that the potential irritation of the drug to the skin is reduced. Compared with a chemically synthesized penetration enhancer, the beta-caryophyllene has higher biocompatibility, low cytotoxicity to epidermal keratinocytes, no obvious skin erythema reaction, great potential and advantages in transdermal administration, and capability of obviously improving the transdermal absorption efficiency of the medicine.
Proline dodecyl ester hydrochloride: due to the molecular size and complexity of the macromolecular drugs themselves, the delivery capacity of β -caryophyllene for macromolecular drugs is limited by the drug molecular weight, and the inventors have found that the permeation enhancing effect of β -caryophyllene is limited for recombinant human interferon α -2b of larger molecular weight. The dodecyl proline hydrochloride is used as a chemical permeation promoter, a dodecyl ester chain in the structure can interact with lipid in skin stratum corneum, so that the lipid bilayer structure can be further disturbed, the mobility between lipid molecules is increased, and the dodecyl proline hydrochloride can interact with amino acid residues of keratin, so that the conformation of the keratin is changed, and the compactness of a keratin network is reduced. The above two-point nature of proline dodecyl ester hydrochloride helps to form a channel favorable for penetration of recombinant human interferon alpha-2 b, reduces resistance of skin barrier, and enhances penetration ability of recombinant human interferon alpha-2 b in skin. The inventor discovers that the synergistic use of the proline dodecyl ester hydrochloride and the beta-caryophyllene can make up for the defect that the delivery capacity of the beta-caryophyllene for macromolecular drugs with high molecular weight is limited by the molecular weight of the drugs, reduce the resistance of skin barriers together, create a more favorable environment for drug permeation, and obviously improve the skin permeation effect of the recombinant human interferon alpha-2 b. In addition, when proline dodecyl ester hydrochloride and beta-caryophyllene are used together, the proline dodecyl ester hydrochloride and the beta-caryophyllene can act on the recombinant human interferon alpha-2 b together to form a synergistic dissolution effect, so that the solubility of the medicine on the surface of the skin is further increased, and the diffusion of medicine molecules to the deep layer of the skin is promoted.
Propylene glycol: propylene glycol, as a polyhydric alcohol, can co-act with the dodecyl ester chain in the proline dodecyl ester hydrochloride to more effectively disturb the lipid bilayer structure of the skin stratum corneum, increase the mobility of lipid molecules and provide more penetration paths for recombinant human interferon alpha-2 b. Meanwhile, the propylene glycol has excellent hydration capability, can absorb and retain moisture, so that the skin horny layer becomes softer, and the softening effect reduces the barrier function of the horny layer, so that the recombinant human interferon alpha-2 b can penetrate the skin more easily, thereby improving the transdermal absorption efficiency of the medicine. In addition, propylene glycol can improve the solubility of recombinant human interferon alpha-2 b in the preparation, and provide more drug molecules for penetrating the skin, which is important for improving the bioavailability and therapeutic effect of the drug.
The invention has the beneficial effects that:
Compared with the prior art, the recombinant human interferon ointment with excellent stability and curative effect is successfully prepared by carefully selecting and proportioning the raw material components such as the recombinant human interferon alpha-2 b, glycerol, citric acid, polysorbate-80, ethyl p-hydroxybenzoate, sodium carboxymethyl cellulose and the like. The ointment not only maintains the original advantages of each component, such as moisturizing and skin softening effects of glycerin, pH adjustment and chelation effects of citric acid, permeation promotion and emulsification effects of polysorbate-80, antibacterial and antiseptic effects of ethyl p-hydroxybenzoate, thickening and drug carrier functions of sodium carboxymethylcellulose, but also realizes synergistic effects through the synergistic effects of each component. The synergistic effect not only improves the permeation efficiency and bioavailability of the medicine, but also optimizes the use experience of patients, improves the compliance and safety of treatment, and provides an effective local administration new choice for the clinical application of the recombinant human interferon alpha-2 b.
The permeation promoter is obtained by combining three components of beta-caryophyllene, L-proline dodecyl ester hydrochloride and propylene glycol, and the permeation promoter is used in the prepared recombinant human interferon alpha-2 b ointment, and has obvious permeation promoting effect and good safety. Beta-caryophyllene, L-proline dodecyl ester hydrochloride and propylene glycol provide more penetration ways for drug molecules and increase the permeability of the drug through the adjustment of complementarity promoting mechanism, synergistic dissolution, lipid fluidity and arrangement and reduction of skin barrier resistance. The synergistic effect of the three components not only improves the transdermal absorption efficiency of the drug, but also realizes uniform and continuous drug release by adjusting the release and penetration rate, thereby improving the treatment effect and reducing the skin irritation. The formulation optimizes the transdermal drug delivery effect while ensuring high biocompatibility, and provides an effective drug delivery strategy for clinical treatment.
In addition, the polyelectrolyte complex formed by electrostatic interaction of the cellulose sodium sulfate and the polyethylene glycol chitosan provides a stable drug delivery system for the recombinant human interferon alpha-2 b, and simultaneously reduces skin irritation. The three-dimensional network structure of the polyelectrolyte compound enhances the stability of the medicine and slows down aggregation, and the introduction of polyethylene glycol chains promotes water solubility and flexibility, which is beneficial to the penetration of the medicine in biological membranes. In addition, the slow release characteristic of the polyelectrolyte complex controls the drug release rate, reduces the concentration of the drug on the surface of the skin and relieves the irritation. In addition, the biocompatibility of the pegylated chitosan reduces immune and inflammatory reactions, and hydration softens the stratum corneum of the skin, reducing friction. These mechanisms work together to not only improve the efficacy of recombinant human interferon alpha-2 b, but also optimize the use experience of the patient.
Detailed Description
Parameters of specific chemicals are used, sources.
Recombinant human interferon alpha-2 b is manufactured by Beijing Bai Albo technology Co., ltd., product number: JN0421;
Sodium carboxymethyl cellulose, purity: 99%, manufacturer is guangzhou Tianjia biotechnology limited, EINECS No.: 618-378-6;
Polysorbate-80, purity: 99%, manufacturer is guangzhou Tianjia biotechnology limited, EINECS No.: 500-019-9;
Ethyl p-hydroxybenzoate, purity: 99%, manufacturer is Tian Hengchang chemical industry Co., ltd., product number: HC0430;
beta-caryophyllene, pale yellow liquid, purity: 99%, the manufacturer is Wuhan energy kernel pharmaceutical chemical industry Co., ltd., product number: s0735;
methoxy-polyethylene glycol-succinimidyl valerate, purity: 98%, manufacturer is Shanghai Seikovia biotechnology Co., ltd., product number: SCMP-30053;
The purity of the sodium cellulose sulfate is more than or equal to 95 percent, and the manufacturer is Hubei Yangxin medical science and technology Co., ltd., CAS number: 9005-22-5;
Chitosan, white powder, purity: 95% of manufacturers are the chemical industry Co., ltd, EINECS number: 618-480-0.
Example 1
A method for preparing recombinant human interferon ointment, comprising the following steps:
575g of stabilizer and 173g of purified water are mixed and stirred for 1h, and then pressurized and heated sterilization treatment is carried out under the conditions of 0.06MPa and 125 ℃ to obtain a mixed system A; mixing and stirring 0.02g of recombinant human interferon alpha-2 b with the mixed system A for 1h to obtain recombinant human interferon alpha-2 b liquid; 15g of sodium carboxymethyl cellulose, 2.5g of penetration enhancer, 6g of polysorbate-80, 2g of citric acid, 1.5g of ethyl p-hydroxybenzoate and 225g of purified water are subjected to pressurized heating sterilization treatment under the conditions of 0.06MPa and 125 ℃, then placed in a stirring container, stirred and mixed for 1h under the conditions of 82 ℃ and 80rpm, and then cooled to below 25 ℃ to obtain an emulsified matrix; mixing recombinant human interferon alpha-2 b liquid with emulsified matrix, stirring for 1 hr to obtain ointment, filling into medicinal ointment tube, and packaging to obtain final product with ointment content weight of 5 g/count.
The content of recombinant human interferon alpha-2 b in the ointment is 2.0X10 4 IU/g ointment, and the pH value of the ointment is 6.
The stabilizer is glycerol.
The penetration enhancer is prepared by mixing 0.75g of beta-caryophyllene, 0.75g L-leucine methyl ester hydrochloride and 1g of 1, 2-propylene glycol.
Example 2
A method for preparing recombinant human interferon ointment, which is different from example 1 in that the penetration enhancer is prepared by mixing 0.75g of beta-caryophyllene, 0.75g L-proline methyl ester hydrochloride and 1g of 1, 2-propylene glycol.
Example 3
A method for preparing recombinant human interferon ointment, which is different from example 1 in that the penetration enhancer is prepared by mixing 0.75g of beta-caryophyllene, 0.75g L-proline dodecyl ester hydrochloride and 1g of 1, 2-propylene glycol.
The preparation method of the L-proline dodecyl ester hydrochloride comprises the following steps:
Dissolving 1.37g L-proline, 2.7g n-dodecanol and 2.3g p-toluenesulfonic acid in 110g toluene, carrying out reflux reaction for 4.5h at 130 ℃, extracting the reaction solution with 210g of 5% sodium hydroxide aqueous solution for 3 times, drying the organic layer obtained by extraction with anhydrous sodium sulfate for 11h, and drying with a rotary evaporator to obtain a crude product; performing column chromatography on the crude product by using a mixed solution of petroleum ether and isopropanol with a volume ratio of 8:1 as an eluent to obtain colorless liquid, namely L-proline dodecyl ester; introducing hydrogen chloride gas into the L-proline dodecyl ester for 4.5h according to the molar ratio of the hydrogen chloride gas to the L-proline dodecyl ester of 1.15:1, and drying by using a rotary evaporator to obtain viscous yellow liquid, namely the L-proline dodecyl ester hydrochloride.
Example 4
A method for preparing recombinant human interferon ointment is different from example 3 in that the penetration enhancer is prepared by mixing 1.25g of beta-caryophyllene and 1.25g L-proline dodecyl ester hydrochloride.
Example 5
A method for preparing recombinant human interferon ointment, which is different from example 3 in that the penetration enhancer is prepared by mixing 1.25g L-proline dodecyl ester hydrochloride 1.25g of 1, 2-propylene glycol.
Example 6
A method for preparing recombinant human interferon ointment, which is different from example 3 in that the penetration enhancer is prepared by mixing 1.25g of beta-caryophyllene and 1.25g of 1, 2-propanediol.
Example 7
A method of preparing a recombinant human interferon ointment, differing from example 3 in that the stabilizer is formed by mixing 437.5g of glycerol and 137.5g of polyelectrolyte complex.
The preparation method of the polyelectrolyte compound comprises the following steps:
4.3g of chitosan was dissolved in 110g of 1mg/mL of acetic acid aqueous solution to obtain a chitosan solution; 2.2g of chitosan solution is added into 11g of 2wt% cellulose sodium sulfate aqueous solution drop by drop at a rate of 640 mu L/min by using a constant flow pump, and the mixture is stirred for 18min at 25 ℃ to obtain a polyelectrolyte compound; the resulting polyelectrolyte complex was washed 3 times with water, and then freeze-dried for 12 hours for use.
Example 8
A method of preparing a recombinant human interferon ointment, differing from example 3 in that the stabilizer is formed by mixing 437.5g of glycerol and 137.5g of polyelectrolyte complex.
The preparation method of the polyelectrolyte compound in the stabilizer comprises the following steps:
Dissolving 4.3g of polyethylene glycol chitosan in 110g of 1mg/mL acetic acid aqueous solution to obtain polyethylene glycol chitosan solution; 2.2g of the polyethylene glycol chitosan solution is dropwise added into 11g of 2wt% cellulose sodium sulfate aqueous solution at a rate of 640 mu L/min by using a constant flow pump, and the mixture is stirred for 18min at 25 ℃ to obtain a polyelectrolyte compound; the resulting polyelectrolyte complex was washed 3 times with water, and then freeze-dried for 12 hours for use.
The preparation method of the polyethylene glycol chitosan comprises the following steps:
0.028g of chitosan was dissolved in 552 g/mL of acetic acid aqueous solution and stirred for 24h; then adjusting the pH to 6.1 by using a 6mol/L sodium hydroxide aqueous solution to obtain a chitosan solution; 0.42g of methoxy-polyethylene glycol-succinimidyl valerate is dissolved in 8.5g of anhydrous dimethyl sulfoxide to obtain a methoxy-polyethylene glycol-succinimidyl valerate solution; then adding methoxy-polyethylene glycol-succinimidyl valerate solution into chitosan solution, and stirring for 48 hours to obtain mixed solution; the mixed solution was placed in a dialysis bag (14 kDa), dialyzed against 550g of ultrapure water for 4.5d, and the dialyzed mixture was then lyophilized to obtain polyethylene glycol chitosan.
Comparative example 1
A method for preparing recombinant human interferon ointment is different from example 1 in that the penetration enhancer is azone.
Comparative example 2
A method for preparing recombinant human interferon ointment is different from example 1 in that the penetration enhancer is menthol.
Comparative example 3
A method for preparing recombinant human interferon ointment is different from example 3 in that citric acid is not added.
Test example 1
In vitro transdermal penetration-promoting activity evaluation experiment
Permeation rate (J) describes the amount or rate of drug passing through the skin per unit time, with higher permeation rates meaning that the transdermal enhancer is more effective in enhancing the drug's passage through the skin barrier. Thus, by measuring the permeation rates of different transdermal enhancers, it is possible to compare their efficacy and select the transdermal enhancer that is the most effective; the permeation Enhancer Ratio (ER) represents the ratio of the permeation rate of a drug after the transdermal enhancer is used to the permeation rate when not in use, and the higher the permeation enhancer ratio, the more remarkable the enhancement effect of the transdermal enhancer on the permeation of the drug. By calculating the permeation enhancement ratio, the enhancement degree of the transdermal enhancer on the drug permeation capacity can be more intuitively understood. The permeation rate (J) and the permeability increasing ratio (ER) are key indexes for evaluating the effect of the transdermal enhancer, and jointly reflect the action effect of the transdermal enhancer.
Preparation of animal skin
Healthy rabbits weighing about 3kg are adapted to be bred for 10d, then the skin is killed, the abdominal skin is taken, the skin is dehaired by 10% sodium sulfide, subcutaneous fat is removed, the rabbit is repeatedly washed by purified water until no turbidity exists, and the rabbit is soaked in normal saline and is frozen and preserved in a refrigerator at the temperature of minus 20 ℃ for standby. Before each experiment, the rabbit skin was thawed at normal temperature and the integrity of the rabbit skin was carefully checked without any breakage.
Solution configuration
Preparation of the receiving cell solution: phosphate buffer ph 7.4;
preparation of blank feed solution: recombinant human interferon alpha-2 b was accurately weighed and dissolved in physiological saline at a concentration of 2%. Then, the drug solution is treated by ultrasonic for 10min to fully dissolve the blank supply solution, so as to carry out an in-vitro percutaneous penetration promoting activity evaluation experiment;
Preparation of experimental feed solution: the permeation enhancers of examples 1 to 6 and comparative examples 1 to 2 of the present invention were added to physiological saline, respectively, to prepare permeation enhancer solutions having a concentration of 2%. Thereafter, recombinant human interferon alpha-2 b was accurately weighed and dissolved in the prepared permeation enhancer solution at a concentration of 2%. Then, the drug solution was sonicated for 10min to allow the drug to be sufficiently dissolved for the in vitro transdermal penetration-promoting activity evaluation experiment.
In vitro percutaneous permeation experiments
In vitro transdermal permeation experiments were performed in Franz diffusion cells. The diffusion area of the diffusion cell was 2.5cm 2 and the receiving cell volume was 7mL. Before the experiment, the skin of the mice is checked to be intact, after the skin is confirmed to be intact, the skin of the mice is placed in normal temperature normal saline to be naturally thawed and cleaned, and then the moisture on the skin surface is sucked by filter paper. The mouse skin was immobilized in the middle of the diffusion device with the stratum corneum facing the supply reservoir. The prepared phosphate buffer receiving solution is added into the receiving tank, bubbles are removed to enable the receiving solution to fully contact with the skin, the receiving solution and the skin are balanced for 1h at 35 ℃, 2ml of prepared sample solution is added into the supply tank, and the experiment is carried out under constant-temperature magnetic stirring with the temperature of 35+/-1 ℃ and the rotating speed of 400 rmp. At predetermined time intervals (0, 1,2, 4, 6,8, 10 and 12 h), 1mL of the receiving liquid sample was taken out of the receiving tank, and immediately an equal volume of the phosphate buffer solution of the same temperature was added to the receiving tank, so that the sedimentation condition was maintained throughout the experiment. The drug content was measured by high performance liquid chromatography after the sample of the received liquid was filtered through a 0.45 μm microporous membrane. Each sample was tested in parallel 4 times. By calculating the cumulative transdermal permeation quantity Q n of the drug, the steady state permeation rate (J) is obtained from the slope of the straight line portion of the drug cumulative transdermal permeation quantity-time curve.
The cumulative transdermal penetration Q n of the drug was calculated from the following formula:
Qn=∑CnV/S
Wherein C n is the concentration of the drug in the receiving liquid at the moment t, V is the volume of the receiving liquid in the receiving tank, and S is the effective diffusion area.
The penetration enhancer to drug penetration Enhancer Ratio (ER) is calculated by the formula:
ER=J/J0
Where J is the permeation rate of the drug after the addition of the permeation enhancer and J 0 is the inherent permeation rate of the drug.
The test results were averaged and specific test data are shown in table 1 below:
As is clear from Table 1, examples 1 to 6 and comparative examples 1 to 2 significantly promoted the penetration of the skin into the drug, compared with the blank, indicating that the recombinant human interferon ointment prepared according to the present invention can improve the penetration rate of recombinant human interferon alpha-2 b in the stratum corneum. Comparative examples 1 to 6 and comparative examples 1 to 2, wherein the permeation rate and permeation enhancement ratio of recombinant human interferon alpha-2 b of examples 1 to 6 are superior to those of comparative examples 1 to 2, respectively, wherein example 3 is optimal, and the permeation rate and permeation enhancement ratio reach 23.31 mug/cm 2/h, 2.71, respectively, and have excellent transdermal enhancement efficacy of recombinant human interferon alpha-2 b. The reason for this analysis may be that the recombinant human interferon alpha-2 b is used as a macromolecular protein drug with higher hydrophilicity, and the ability of the recombinant human interferon alpha-2 b to penetrate the skin barrier is limited during the percutaneous permeation because the cell gap of the skin horny layer is small and the skin horny layer is mainly composed of hydrophobic lipids. And recombinant human interferon alpha-2 b as a protein may have a negative charge on its surface or may exhibit a neutral charge, which may be charge-repulsive to skin having a negative charge, increasing the difficulty of penetrating the skin. Aiming at the problem of poor skin permeability of the recombinant human interferon alpha-2 b, the invention uses beta-caryophyllene, L-proline dodecyl ester hydrochloride and propylene glycol cooperatively, thereby improving the skin permeation effect of the recombinant human interferon alpha-2 b in the process of medication. Beta-caryophyllene increases the transdermal permeability of drug molecules by temporarily reducing the skin barrier function, interfering with the multiple mechanisms such as the alignment of stratum corneum lipids, while L-proline dodecyl ester hydrochloride also has the property of interacting with the stratum corneum lipids of the skin, and can change the conformation of keratin by interacting with keratin, reduce the compactness of the keratin network, form channels which contribute to the skin permeation of recombinant human interferon alpha-2 b, and increase the permeability of the skin barrier. The beta-caryophyllene and the L-proline dodecyl ester salt are cooperatively used, so that the defect that the delivery capacity of the beta-caryophyllene on macromolecular medicaments with high molecular weight is limited by the molecular weight of the medicaments can be overcome, the lipid bilayer structure is more effectively disturbed, the mobility among lipid molecules is increased, and the permeation of the macromolecular medicaments of recombinant human interferon alpha-2 b is facilitated. Propylene glycol further softens the stratum corneum by increasing its fluidity and hydration capacity, reducing the drag of the drug through the skin. Meanwhile, the propylene glycol can improve the drug solubility, and further improve the distribution uniformity and permeability of drug molecules in the skin. The three components act on the skin barrier through different mechanisms, complement each other, promote permeation of drug molecules together, carry different charges which can be neutralized with charges on the skin surface, possibly contribute to reducing repulsive interaction with the charges on the surface of the recombinant human interferon alpha-2 b, reduce the difficulty of penetrating the skin, and can obviously improve the transdermal absorption efficiency of the drug.
In conclusion, the synergistic effect of the beta-caryophyllene, the L-proline dodecyl ester hydrochloride and the propylene glycol improves the transdermal absorption efficiency of the medicine by complementing and enhancing the respective permeation promotion mechanism, and obviously increases the permeation promotion effect of the recombinant human interferon alpha-2 b.
Test example 2
Skin irritation test
The recombinant human interferon alpha-2 b ointment has remarkable curative effect in antiviral treatment, but has certain skin irritation defect when in use. Discomfort such as localized redness, itching or pain may occur after use by some patients, mainly due to interactions of the drug with skin cells or irritation of the drug itself. The reduction of skin irritation is important because the stimulatory response not only reduces the patient's medication experience, but may also affect the sustained use and therapeutic effect of the drug. The skin irritation of the recombinant human interferon ointment prepared by the invention is determined by animal experiments, and the commercial recombinant human interferon alpha-2 b cream is selected as a control to increase the experimental persuasion.
Test drug: the ointments prepared in example 3 and examples 7-8 and the commercially available recombinant human interferon alpha-2 b cream (Changchun biological product research all liability company, national drug standard S20153008, production lot number 20220414, specification: 2 ten thousand IU/g,5.0 g/count).
40 Male healthy guinea pigs weighing 250-300 g were adapted to 7d and then randomly divided into 4 groups of 10 animals each, recorded as commercial, example 3 and examples 7-8 groups. The back skin (4.0 cm 2) of each group of guinea pigs was dehaired, the dehaired area skin was equally divided into 2 pieces and recorded as skin A and skin B, and no damage was examined after 75% alcohol was wiped. Skin a was given the test drug and skin B was the blank. After being smeared evenly, the chest is bound and fixed by a rubber band with proper length, taken down after being smeared for 24 hours, and washed by warm water. And (5) visually observing whether edema and erythema exist at the smearing part 1h, 3h, 10h and 24h after the tested medicines are removed. Skin irritation response scoring criteria are shown in Table 2, and skin irritation intensity (combined score for erythema and edema) scoring criteria are shown in Table 3:
As is clear from Table 4, in example 3 and examples 7 to 8, there was no erythema or edema irritation to the skin compared with the commercial group, and no irritation was judged according to the irritation intensity scoring criteria, whereas the commercial group showed slight irritation, which indicates that the recombinant human interferon ointment prepared in the present invention can effectively reduce the irritation of recombinant human interferon alpha-2 b to the skin, and improve the safety and comfort of the recombinant human interferon ointment as a transdermal administration preparation. The reason for this is probably because, first, when β -caryophyllene, L-proline dodecyl ester hydrochloride and propylene glycol are used in combination, the natural nature and high biocompatibility of β -caryophyllene, in combination with L-proline dodecyl ester hydrochloride and propylene glycol, enhances the safety of the overall formulation, and the synergistic effect thereof can effectively reduce the concentration requirement of a single component, and reduces the irritation and allergic reaction risk to skin. In addition, the reversible disturbance of the beta-caryophyllene on the skin barrier ensures the self-recovery capability of the skin after the medicine permeates, and avoids skin injury possibly caused by long-term use. The hydration capacity of propylene glycol softens the stratum corneum and reduces the barrier function of the stratum corneum and the friction irritation of the pharmaceutical ingredients. The combination of the three can optimize the even distribution of the medicine in the skin, prevent the accumulation of the local high-concentration medicine and further reduce the local irritation.
Secondly, sulfate anions on cellulose sodium sulfate and amino cations on polyethylene glycol chitosan form a polyelectrolyte complex with a three-dimensional network through electrostatic interaction, and the use of the polyelectrolyte complex effectively reduces skin irritation while improving the stability of recombinant human interferon alpha-2 b. The polyelectrolyte complex effectively disperses the drug through its three-dimensional network structure, preventing aggregation of the drug, thereby reducing local skin irritation. The slow release property of the polyelectrolyte complex can control the drug release rate, reduce the concentration of the drug on the surface of the skin and relieve the direct stimulation. As a protective carrier, the polyelectrolyte complex can reduce the direct contact between the medicine and the skin, further reduce the irritation and improve the medication safety. The polyethylene glycol chitosan reduces skin immunity and inflammatory reaction by virtue of biocompatibility, and simultaneously, hydration of polyethylene glycol chains can soften stratum corneum, reduce friction and irritation when the medicine penetrates, and reduce medicine irritation by dual effects. The polyelectrolyte complex formed by the cellulose sodium sulfate and the polyethylene glycol chitosan loads recombinant human interferon alpha-2 b, so that the percutaneous administration effect of the medicine is optimized, the skin irritation is reduced, and the comfort level and the treatment compliance of patients are improved.
Test example 3
Stability test
Skin herpes caused by HSV-1 virus infection is a common skin disease, and is mainly characterized by erythema on mucous membrane parts such as lips, nose and the like, wherein the erythema clusters on the skin herpes, commonly called as "excessive internal heat" and clinically called as herpes simplex. This condition is susceptible to recurrent attacks, especially when ingested with spicy, irritating, fried foods or when the body's immunity is reduced. The recombinant human interferon alpha-2 b has remarkable effect on treating skin herpes caused by HSV-1. It can be used as broad-spectrum antiviral drug, and can effectively inhibit replication of HSV-1 virus, relieve herpes symptom, and accelerate healing of herpes. Meanwhile, the recombinant human interferon alpha-2 b can also enhance the immune function of the organism, enhance the resistance of the organism to HSV-1 virus and prevent the recurrence of herpes by improving the phagocytosis of macrophages and the killing effect of T cells. However, the stability of recombinant human interferon alpha-2 b has a significant impact on its therapeutic efficacy. If the stability of the drug is poor, it may lose activity during storage or use, resulting in poor therapeutic efficacy. Therefore, ensuring the stability of the drug is important for treating skin herpes caused by HSV-1. The stability of the recombinant human interferon ointment prepared by the invention is tested by establishing a guinea pig skin herpes model and carrying out a guinea pig skin infection herpesvirus treatment test, and commercially available recombinant human interferon alpha-2 b cream is selected as a control to increase the experimental persuasion.
Virus: HSV-1 virus was purchased from the university of Wuhan collection center (CCTCC).
Test drug: recombinant human interferon ointment prepared in example 3 and examples 7 to 8, comparative example 3;
Control drug: recombinant human interferon alpha-2 b cream (Changchun biological products research all liability company, national drug standard S20153008, production batch number: 20220414, specification: 2 ten thousand IU/g,5.0 g/branch) is commercially available.
And (3) molding: 150 male healthy guinea pigs with the weight of 250-300 g are taken and are adaptively fed for 7d. The guinea pig back skin (4.0 cm 2) was then dehaired and the dehaired area skin was aliquoted into 2 pieces, recorded as skin A and skin B. After skin disinfection with 75% alcohol, the center of each dehairing area was deeply pricked with a sterile 7-needle plum-blossom needle, after stopping the needle for 2 minutes, 30 μl of virus stock was dropped on the pricked skin, and the pricked skin was rubbed with a sterile glass rod to cause infection in guinea pigs. On day 4 after infection with the virus, the skin of the back of the guinea pig shows typical herpes lesions, and the lesion degree reaches 3.6-4.0 cm 2.
150 Guinea pigs infected with herpes were then randomly divided into 5 groups of 30, each, recorded as commercial, example 3 and examples 7-8, comparative example 3. The skin of each group of guinea pigs was administered with the test drug and the control drug, respectively, 4 times/d, 0.25 g/time, and the administration was continued for 7d. Wherein the medicines smeared on the skin A are test medicines and control medicines before the accelerated stability test, and the medicines smeared on the skin B are test medicines and control medicines after the accelerated stability test; wherein the accelerated stability test is performed as follows: the test drug and the control drug were placed at a temperature of 30.+ -. 2 ℃ and a relative humidity of 60.+ -. 5 ℃ for 6 months, respectively.
The symptom of each dehairing area is observed every day, the number of eruptions and the skin state of the herpes are registered, the ulcers or blisters on the affected part of the skin herpes of the mice start to scab, the surrounding red and swelling and inflammatory reaction are reduced, and no new blister is judged to be effective in treatment; after scabbing on all the affected parts of the herpes of the skin of the mice, and no new blister grows, judging that the mice are healed, and calculating the treatment effective rate and the healing rate according to the final treatment condition, wherein the calculation formula of the treatment effective rate and the healing rate is as follows:
Treatment efficacy (%) = treatment efficacy mice/number of herpesmice infected with each group
Recovery rate (%) = number of mice recovered/number of mice infected with herpes for each group
The specific test results are shown in Table 5 below:
As is clear from Table 5, the groups of example 3 and examples 7 to 8 and comparative example 3 each have a better therapeutic effect on skin herpes than the commercially available group. The treatment effective rate and the cure rate before and after the acceleration stabilization test are compared, the treatment effects of the example 3, the comparative example 3 and the commercial group after the acceleration stabilization test are obviously reduced, the stability is poor, the treatment effect of the examples 7-8 is less in the reduction trend, wherein the treatment effect change of the example 8 before and after the acceleration stabilization test is minimum, the treatment effective rate and the cure rate are better than those of the other groups before and after the acceleration stabilization test, the treatment effect is optimal, and the stability of the recombinant human interferon ointment prepared in the example 8 is better, and the drug effect is not easily influenced. The analytical reasons are probably because the polyelectrolyte complex with a three-dimensional network is formed by electrostatic interaction of sulfate anions on cellulose sodium sulfate and amino cations on pegylated chitosan in the preparation process of the recombinant human interferon ointment, so that an effective drug delivery system is provided for loading the recombinant human interferon alpha-2 b, a protective environment is provided for the recombinant human interferon alpha-2 b, the recombinant human interferon alpha-2 b is prevented from being directly contacted with degradation factors, and the stability of the recombinant human interferon alpha-2 b is improved. Meanwhile, the three-dimensional network structure of the polyelectrolyte complex may limit the movement of the recombinant human interferon alpha-2 b molecule, and reduce the aggregation phenomenon of the drug, thereby improving the stability of the drug, while the introduction of polyethylene glycol chains may increase the water solubility and flexibility of chitosan, thereby being beneficial to forming a more compact three-dimensional network structure and enhancing the stability of the complex.
From the aspect of improving the drug effect, the slow release characteristic of the polyelectrolyte complex is beneficial to controlling the release rate of the recombinant human interferon alpha-2 b, prolonging the half life of the recombinant human interferon alpha-2 b in vivo, improving the pharmacokinetic characteristic and improving the bioavailability and the curative effect of the drug. Meanwhile, the polyelectrolyte complex is used as a carrier, so that the recombinant human interferon alpha-2 b can be effectively targeted to the lesion site, and the release of the medicine in target tissues is ensured, thereby further improving the treatment effect. In addition, the introduction of polyethylene glycol chain increases the hydrophilicity and flexibility of chitosan, can enhance the penetration capability of polyelectrolyte complex on biological membrane, promote the cellular uptake of recombinant human interferon alpha-2 b, and is helpful for improving the drug effect.
In conclusion, the polyelectrolyte complex formed by the cellulose sodium sulfate and the polyethylene glycol chitosan can enable the recombinant human interferon alpha-2 b to be more stable in the storage and use processes, reduce the degradation and inactivation of the medicine, and remarkably improve the medicine effect of the recombinant human interferon alpha-2 b.

Claims (9)

1. The recombinant human interferon ointment is characterized by comprising the following raw material components in parts by weight:
0.015-0.025 parts of recombinant human interferon alpha-2 b, 550-600 parts of stabilizing agent, 10-20 parts of thickening agent, 2-3 parts of penetration enhancer, 5-8 parts of surfactant, 1-3 parts of pH regulator, 1-2 parts of preservative and 350-450 parts of water;
The penetration enhancer is selected from at least two of beta-caryophyllene, amino acid derivatives and 1, 2-propylene glycol; the mass ratio of the beta-caryophyllene, the amino acid derivative and the 1, 2-propylene glycol in the penetration enhancer is 0.5-1.5:0.5-1.5:0.75-1.5.
2. The recombinant human interferon ointment according to claim 1, wherein said amino acid derivative is selected from one of L-leucine methyl ester hydrochloride, L-proline dodecyl ester hydrochloride.
3. The recombinant human interferon ointment according to claim 2, wherein the preparation method of the L-proline dodecyl ester hydrochloride comprises the following steps in parts by weight:
1.3 to 1.4 parts of L-proline, 2.5 to 2.8 parts of n-dodecanol and 2.2 to 2.4 parts of p-toluenesulfonic acid are taken and dissolved in 100 to 120 parts of toluene, reflux reaction is carried out for 4 to 5 hours at the temperature of 125 to 135 ℃, the reaction solution is extracted for 2 to 3 times by 200 to 220 parts of sodium hydroxide aqueous solution with the mass percent of 5 to 6 percent, the organic layer obtained by extraction is dried for 10 to 12 hours by anhydrous sodium sulfate, and a crude product is obtained by drying by a rotary evaporator; petroleum ether: performing column chromatography on the obtained crude product by using mixed solution with the volume ratio of isopropanol of 7-10:1 as an eluent to obtain colorless liquid, namely L-proline dodecyl ester; introducing hydrogen chloride gas into the L-proline dodecyl ester for 4-5 h according to the molar ratio of the hydrogen chloride gas to the L-proline dodecyl ester of 1.1-1.2:1, and drying by using a rotary evaporator to obtain viscous yellow liquid, namely the L-proline dodecyl ester hydrochloride.
4. The recombinant human interferon ointment of claim 1, wherein: the content of the recombinant human interferon alpha-2 b is 1X 10 4～2×108 IU/g ointment based on the active mass ratio.
5. The recombinant human interferon ointment of claim 1, wherein: the thickener is selected from one or more of sodium carboxymethyl cellulose, carbomer, poloxamer, xanthan gum, hydroxypropyl guar gum and sodium carboxymethyl cellulose; the surfactant is one or more selected from polyethylene glycol-7, stearate, polysorbate-80, stearic acid and glyceryl monostearate.
6. The recombinant human interferon ointment of claim 1, wherein: the pH regulator is one or more selected from citric acid, tartaric acid, acetic acid, sodium hydroxide and triethanolamine; the preservative is one or more selected from ethyl p-hydroxybenzoate, phenoxyethanol, polylysine and potassium sorbate; the stabilizer is selected from one or two of glycerin and polyelectrolyte complex.
7. The recombinant human interferon ointment according to claim 6, wherein the preparation method of the polyelectrolyte complex in the stabilizer comprises the following steps in parts by weight:
dissolving 4-4.5 parts of chitosan in 100-120 parts of 1-2 mg/mL of acetic acid aqueous solution to obtain chitosan solution; 2 to 2.5 parts of chitosan solution is dropwise added into 10 to 12 parts of 1 to2 weight percent of cellulose sodium sulfate aqueous solution at the rate of 600 to 650 mu L/min by using a constant flow pump, and the mixture is stirred for 15 to 20 minutes at the temperature of 23 to 25 ℃ to obtain a polyelectrolyte compound; washing the obtained polyelectrolyte complex with water for 2-3 times, and then freeze-drying for 10-12 hours for standby;
Or, dissolving 4-4.5 parts of polyethylene glycol chitosan in 100-120 parts of 1-2 mg/mL acetic acid aqueous solution to obtain polyethylene glycol chitosan solution; 2 to 2.5 parts of the polyethylene glycol chitosan solution is dropwise added into 10 to 12 parts of 1 to 2 weight percent of cellulose sodium sulfate aqueous solution at the rate of 600 to 650 mu L/min by using a constant flow pump, and the mixture is stirred for 15 to 20min at the temperature of 23 to 25 ℃ to obtain a polyelectrolyte compound; the polyelectrolyte complex is washed for 2-3 times by water, and then freeze-dried for 10-12 hours for standby.
8. The recombinant human interferon ointment according to claim 7, wherein the preparation method of the pegylated chitosan comprises the following steps in parts by weight:
Dissolving 0.025-0.03 part of chitosan in 50-60 parts of 2-3 mg/mL of acetic acid aqueous solution, and stirring for 22-24 hours; then adjusting the pH value to 6-6.3 by using 5-6 mol/L sodium hydroxide aqueous solution to obtain chitosan solution; dissolving 0.4-0.45 part of methoxy-polyethylene glycol-succinimidyl valerate in 8-10 parts of anhydrous dimethyl sulfoxide to obtain methoxy-polyethylene glycol-succinimidyl valerate solution; then adding methoxy-polyethylene glycol-succinimidyl valerate solution into chitosan solution, and stirring for 48-50 h to obtain mixed solution; and (3) placing the mixed solution into a dialysis bag, dialyzing with 500-600 parts of water for 4-5 d, and then freeze-drying the dialyzed mixture to obtain the polyethylene glycol chitosan.
9. The method for preparing the recombinant human interferon ointment according to any one of claims 1 to 8, comprising the steps of:
Mixing and stirring the stabilizer and water for 0.5-1.5 h, and then carrying out pressurized heating sterilization treatment under the conditions of 0.05-0.1 MPa and the temperature of 115-135 ℃ to obtain a mixed system A; mixing and stirring the recombinant human interferon alpha-2 b with the mixed system A for 0.5-1.5 h to obtain recombinant human interferon alpha-2 b liquid; the thickening agent, the penetration enhancer, the surfactant, the pH regulator, the preservative and the water are subjected to pressurized heating sterilization treatment under the conditions of 0.05-0.1 MPa and the temperature of 115-135 ℃, then are placed in a stirring container, stirred and mixed for 1-1.5 h under the conditions of 80-85 ℃ and 50-100 rpm, and then are cooled to below 25 ℃ to obtain an emulsified matrix; mixing recombinant human interferon alpha-2 b liquid and an emulsifying matrix, stirring for 0.5-1.5 h to obtain ointment, filling the ointment into a medicinal ointment tube, and packaging the ointment into a finished product after verification, wherein the weight of the content of the finished ointment is 4.85-5.15 g/count.
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