RU2108804C1 - Preparation "locferon" for treatment and prophylaxis of viral diseases and a method of its preparing - Google Patents

Abstract

FIELD: biotechnology, medicine. SUBSTANCE: invention relates to curative-prophylaxis preparation based on human leukocyte interferon. Preparation "Locferon" has interferon synthesized by donor blood leukocytes induced by virus strain-inducer of virus parainfluenza-1 Sendai GKV N 2339. Preparation is concentrated and purified by ultrafiltration. Preparation has 8000 IU antiviral activity in one ampoule - interferon, cellular mediators of molecular mass 10-100 kDa, ovalbumin - less 1 ng/ml, sodium chloride and stabilizing agent. "Locferon" inhibits multiplication of broad spectrum viruses - herpes viruses, adenoviruses, enteroviruses and others. Preparation blocks intracellular replication of viral particles and activates protective cellular response reactions. EFFECT: enhanced effectiveness of preparation. 3 cl

Description

 The invention relates to biotechnology and medicine, in particular to the production of therapeutic drugs based on leukocyte interferon.

 As you know, leukocyte interferon or alpha-interferon, secreted by leukocytes in response to the inducer, is one of the most important factors in protecting the body from viral infections and several other diseases.

 It was established that leukocytes in combination with alpha-interferon protein also produce other biological active proteins with mediator activity, such as lymphokines and monokines, and most importantly, this whole complex activates the body's immune system. In this regard, the modern concept of “interferon” covers a complex of biologically active proteins secreted by leukocytes on the effects of inducers of various nature, and the degree of activity of interferon depends on the presence of cellular mediators in its composition, preserved during the purification of the drug.

 Known drug "Leukinferon" - purified human leukocyte interferon (CHLI) with the preservation of biologically active proteins, containing in one ampoule 10,000 IU of the antiviral activity of alpha-interferon, interleukin 1, tumor necrosis factor, macrophage-inhibiting factor and leukocytining factor [1].

 Leukinferon is a highly active drug, but is applicable only in the form of injections, which limits its use in widespread practice.

 A known method of producing Leukinferon preparation, which provides for the synthesis of interferon in a culture of donor blood leukocytes under the action of an inducer - Newcastle disease virus and subsequent inactivation of the virus and purification of the PLI from allantoic proteins by negative immunosorption, and then gel filtration on a column with Sephadex G 25 to remove low molecular weight fraction mass (m.m.) up to 10 kD.

 Phosphate buffer is added to the final purified fraction to a final concentration of 0.01 M, sodium chloride to a final concentration of 0.9%, and the stabilizer is mannitol [2].

 However, the stage of purification of the target product from impurities is time-consuming, lengthy and inefficient in large-scale production.

 Also known is the drug "Interlock" for the treatment and prevention of viral diseases, containing alpha-interferon and biologically active proteins formed during the activation of leukocytes and having m.m. in the range of 10-30 kD. The filler and stabilizer of the drug "Interlock" is lactose. One ampoule of the drug contains at least 5000 IU of human alpha interferon [3].

 The disadvantage of the Interlock preparation is its low efficiency due to the loss in purification of high molecular weight biologically active proteins synthesized by leukocytes at the stage of interferon biosynthesis.

 A known method for producing the Interlock drug, including incubating the culture of donor blood leukocytes with an inducer - Newcastle disease virus, separating the culture fluid containing interferon from the blood cells, purifying the PLI by precipitating it with ammonium sulfate, followed by dissolving the precipitate in 2-8 M urea and then gel filtration on a column with Akrilex P-60 gel to remove a low molecular weight fraction of up to 10 kD. Phosphate buffer is added to the resulting fraction to a final concentration of 0.01 M and a lactose stabilizer. The resulting dosage form is poured into ampoules of 2 ml and freeze-dried [3].

 However, the known method for producing CHLI does not allow a preparation with a wide range of biologically active proteins.

 In addition, toxic substances, including urea, are used in the cleaning process, which affects the reactogenicity of the final form of the drug.

 The drug "Interlock" and the method of its preparation is adopted as the closest analogue.

 An object of the invention is to obtain a drug with high antiviral activity, purified from ballast proteins and allergenic impurities, and the development of a technologically advanced method for its production, acceptable for industrial production.

 The technical result of the invention is to create a highly active antiviral drug "Lockferon" containing alpha interferon in combination with cell mediators, free of allergenic components, non-reactive and good tolerance of the final form, as well as the development of a high-performance and simple technology for its preparation.

 The invention consists in the following.

 The drug for the treatment and prevention of viral diseases Lokferon contains human leukocyte interferon synthesized by blood leukocytes under the influence of an inducer - strain of the parainfluenza virus 1 Sendai STB N 2339, concentrated and purified by micro and ultrafiltration; containing at least 8000 IU in one ampoule of the antiviral activity of alpha interferon and cell mediators with m.m. 10-100 kDa, ovalbumin - less than 1 kg / ml, free of antibiotics and heparin and containing sodium chloride - to a final concentration of 0.9 wt.%, And a stabilizer, for example, lactose or polyvinylpyrrolidone or mannitol.

 The method of obtaining the drug "Lockferon" for the treatment and prevention of viral diseases based on PLI, characterized in that for the induction of interferon use the parainfluenza virus strain 1 Sendai STB N 2339 and after acid inactivation of the virus-inducer of the intermediate product of interferon without changing pH (pH 2.0- 2.4) is subjected to three-stage cleaning: first by microfiltration on membranes with a cutoff threshold of 0.2 μm, then by ultrafiltration and diafiltration on membranes with a cutoff limit of 5 kD, and then by ultrafiltration on membranes with a cutoff limit of 100 kD. In the second stage, the filtrate is subjected to purification after microfiltration, and in the third stage, the concentrate obtained from the previous stage.

 After the purification process is completed, sodium chloride is added to the resulting filtrate, which is purified CHLI, to a final concentration of 0.9 wt. %, and a stabilizer, for example lactose, polyvinylpyrrolidone, mannitol. The solution of the drug is sterile filtered, pH is adjusted to 7.0-7.2, poured into vials and freeze-dried.

 According to the invention, the parainfluenza virus strain 1 Sendai GKV N 2339 is used to induce interferonogenesis, which leads to a significant decrease in the amount of allantoic proteins, in particular, ovalbumin in the target product, and it does not require additional operations to purify the drug from ovalbumin.

 The strain of the parainfluenza virus 1 Sendai STB N 2339 was selected in the interferon department of the Biomed company named after Mechnikov, typed with the strain of the parainfluenza virus 1 Sendai STB N 326 and differs from it by the infectious activity titer. The strain is deposited in the State virus collection of the Institute of Virology. Ivanovsky and has a registration number 2339.

 The Lockeron interferon preparation synthesized and purified using membrane technologies retains a wide range of immunomodulators with m.m. 10-100 kDa, produced by human leukocytes during biosynthesis, has high antiviral activity and, unlike other known drugs, has buffering properties, therefore, to maintain stability it does not require the addition of buffered solutions, which reduces the toxicity of the finished form.

 In addition, the production of Lockeron is carried out without the use of antibiotics, heparin, urea, which also leads to a decrease in toxicity and the absence of side effects and good tolerance of the final form.

Example 1. Leukocytes isolated from donated blood are suspended in medium 199 with appropriate additives and an inducer, a strain of the parainfluenza virus 1 Sendai GKB N 2339, is added in an amount of 8000 GAU / ml. Incubated at 37 ^o C for 18 hours. Next, the leukocytes are removed by centrifugation, 20% hydrochloric acid solution is added to the supernatant to pH 2.0-2.4 and incubated for at least 7 days.

 After acid inactivation, the semi-finished interferon is subjected to a three-stage purification with final sterilizing filtration. The entire purification of interferon, including sterilizing filtration, is carried out in the pH zone from 2.0 to 2.4.

 In the first stage of purification, the interferon prefabricated is clarified by microfiltration on membranes with a cutoff threshold of 0.2 μm.

 In the second stage, the clarified semi-finished product is concentrated 5-10 times on ultrafiltration membranes with a cut-off limit of 5 kD and then subjected to diafiltration at a constant volume with the introduction of purified distilled water in a 10-fold volume of the concentrate.

 In the third stage of purification, partially purified, free from low molecular weight impurities and concentrated semi-finished product (concentrate) of interferon is subjected to diafiltration at a constant volume on ultrafiltration membranes with a cutoff limit of 100 kD with the introduction of purified distilled water. Interferon (filtrate), purified from high molecular weight impurities, is diluted 2-4 times.

 After the purification process is completed, sodium chloride is added to the filtrate, which is purified interferon, to a concentration of 0.9% and lactose to a concentration of 1 mg / ml and sterilely filtered.

 The resulting dosage form is poured into 2 ml vials and freeze-dried.

 Example 2. Obtained in example 1, the drug "Lockferon" in the form of eye drops is used to treat ophthalmic infectious diseases, such as herpetic keratitis caused by the herpes simplex virus (vesicular, star-shaped, pinpoint, tree-like, carbohydrate); herpetic keratitis stromal with and without cornea; herpetic corneal ulcer; herpetic keratouveitis with ulceration and without ulceration of the cornea; herpetic uveitis; herpetic conjunctivitis; adenoviral conjunctivitis; epidemic keratoconjunctivitis; hemorrhagic keratoconjunctivitis.

 The effectiveness of the drug is highest in the initial stage of the disease.

 In the acute stage of the disease, Lockferon is used as an instillation in the eye, 1-2 drops 8 times a day in combination with symptomatic therapy. As the inflammatory process is stopped, the number of instillations is reduced to 2-3 times a day. The course of treatment is continued until the symptoms of the disease disappear.

 The drug has no age restrictions, has no contraindications.

 With the introduction of the drug Lockferon, no noticeable reactions of both local and general nature were noted. Sensitization with prolonged repeated administration was not detected.

 The frequency of achieving a pronounced therapeutic effect in patients with severe forms of herpetic eye lesions with the use of Lockeron was 1.5-2 times higher than with Interlock treatment.

 Example 3. Obtained in example 1, the drug "Lockferon" in the form of lotions is used to treat simple recurrent herpes in combination with vitamins B, E and C. The course of treatment was 5 days.

 Clinical symptoms and subjective sensations disappeared on the 4th day of treatment. After the treatment, persistent remission was observed for 5 months.

 Example 4. Obtained in example 1, the drug "Lockferon" in the form of drops is used for the treatment and prevention of acute respiratory diseases and flu.

 For the prevention of respiratory diseases, the drug is administered into the nasal passages once a day during the epidemic period, and for treatment - four times a day for 2-5 days.

SOURCES OF INFORMATION
1. RU, patent N 2033180, class. A 61 K 38/21, 1995.

 2. RU, patent N 297296, class. A 61 K 38/21, 1977.

 3. And 122.015-84. "Instructions on the manufacturing technology and control of purified leukocyte interferon" from 12.29.84 (based on the order of the Ministry of Health from 08.31.83).

Claims (3)

 1. The drug for the treatment and prevention of viral diseases based on human leukocyte interferon, characterized in that it contains interferon synthesized by leukocytes of donated blood under the influence of an inducer - a strain of parainfluenza virus 1 Sendai STB N 2339, concentrated and purified by micro- and ultrafiltration, containing less than 8000 IU in one ampoule of antiviral activity of α-interferon and cell mediators with m. m of 10 - 100 kD, ovalbumin - less than 1 ng / mg, free of antibiotics and heparin, and containing sodium chloride I - to a final concentration of 0.9 wt.% and a stabilizer.  2. The drug according to claim 1, characterized in that the stabilizer contains lactose, or polyvinylpyrrolidone, or mannitol.  3. A method of obtaining a drug for the treatment and prevention of viral diseases, including incubating a culture of leukocytes of donor blood with a virus-inducer, separating interferon-containing culture fluid, acid inactivation of the virus-inducer, purification of the resulting interferon, followed by the addition of a stabilizer and sodium chloride and freeze drying, characterized in that the Sendai strain GKB N 2339 strain of parainfluenza virus 1 is used as an inducer, purification is carried out at pH 2.0 - 2.4, first by microfiltration on membranes with cutoff 0.2 μg, then the filtrate is subjected to ultrafiltration and diafiltration on membranes with a cutoff limit of 5 kD, then the concentrate after diafiltration is again diafiltered on ultrafiltration membranes with a cutoff limit of 100 kD, and sodium chloride is added to the obtained filtrate, which is purified interferon to a final concentration of 0.9 wt.% and a stabilizer.