EA014669B1 - Interferon-based formulation - Google Patents

Abstract

The invention relates to the field of medicine, veterinary medicine, chemical and pharmaceutical industry, biotechnology and can be used at production of medical and preventive immunobiological drugs, having durable immunomodulatory and antiviral action. In particular, the invention relates to the bioactive formulations on the basis of recombinant interferons. It is suggested an interferon based formulation, comprising α-interferon, stabilizer and water, which contains at least one fat-soluble vitamin in the form of colloidal particles as the stabilizer and additionally contains sodium benzoate as a preservative and sodium chloride as an agent, providing osmoticity of solution at the following components content per 1 liter of solution:A formulation can preferably contain as a stabilizer one fat-soluble vitamin in the form of colloidal particles, chosen from the group of vitamins A, D, E, B or a mixture of fat-soluble vitamins A, D, E in the form of colloidal particles, if necessary, with the water-soluble vitamins C and B12. The preparation presents a stable solution, having improved therapeutic action and providing preservation of a high level of immunomodulatory and antiviral activity in the body for a long period of time, as well as a faster and more effective action of interferon at early stages after administration into the body.

Description

The invention relates to the field of medicine, veterinary medicine, the pharmaceutical industry, biotechnology and can be used to obtain therapeutic and prophylactic immunobiological preparations with a prolonged immunomodulating and antiviral effect. In particular, the invention relates to biologically active preparations based on recombinant interferons.

Currently, for the treatment of infectious diseases of humans and animals, the most promising are drugs based on immunomodulators, including interferons. Interferon is a protein cytokine that regulates the immune response of infected organisms and thus inhibits the multiplication of viruses. Alpha-interferon is highly effective in the treatment of various viral diseases of animals and humans. The main requirements for interferon preparations are storage stability, maximum activity and duration of action.

In veterinary medicine, the injection drug based on recombinant α-interferons, Mixoferon [1], which has a direct antiviral effect, stimulates immune processes, and also increases the non-specific resistance of animals, is widely used.

Most proteins easily undergo denaturation and lose their biological activity during storage in the form of aqueous solutions. Mixoferon is also unstable in aqueous solutions. Another disadvantage is the short half-life of the active substance in the tissues and blood flow, which prevents the achievement of the maximum therapeutic effect.

For this reason, many pharmaceutical companies have developed lyophilized forms of interferon preparations. However, these forms have several disadvantages. In dry form, protein cytokines are stored much better, but lose some of the activity directly in the process of lyophilization. In addition, the need to dilute the drug before use creates inconvenience in its use.

It is known to use an aqueous interferon composition in which human serum albumin is a stabilizing factor [2]. However, human serum albumin can contain infectious pathogens and can cause immune reactions, which is why it has not been recommended to be used recently.

To stabilize interferon in various liquid dosage forms, disaccharides [3], nonionic detergents [4, 5], vitamins C and E (α-tocopherol acetate) [6], and heavy water [7] were used. To prolong the action of interferon, synthetic and natural polymers (polyvinylpyrrolidone, carboxymethyl cellulose, methyl cellulose) and their combinations were used [8, 9]. Polyethylene oxide was used to obtain the interferon preparation as an ointment [10].

It is known that vitamins, in particular vitamin E, can enhance the action of protein cytokines. To enhance the therapeutic effect, vitamin B12 was also added to interferon preparations [11, 12]. There are interferon preparations in the form of suppositories containing supplements of vitamins A, E, C [13].

Known prolonged solution of α-2-interferon [8] for medical use. The difference between this drug and other known dosage forms [14-18] consists in the use of antioxidants (citric acid and unitiol), antimicrobial agents (boric acid and sodium tetraborate) along with α-2-interferon, and also as an excipient-stabilizer of esters cellulose in a concentration of 0.5-5.0%. The drug is a clear, viscous solution that can be used in medicine for topical application.

The disadvantages of the known prolonged solution of α-2-interferon are the use of boric acid and sodium tetraborate as antimicrobial substances, which are surface antiseptics, are characterized by a mild therapeutic effect compared to antibiotics, belong to the class of low toxic substances and are allowed in veterinary medicine and only external use [19].

It is also known that to obtain a therapeutic effect, interferon and vitamin E are usually administered separately, since vitamin E is insoluble in water.

The closest technical solution to the claimed is an interferon preparation containing interferon α-2, buffer-forming salts, a stabilizing mixture of α-tocopherol acetate and organic acids [6].

The disadvantage of this complex preparation of interferon is the low content of α-tocopherol acetate (0.1 mg per 1-100 million IU of interferon), the result is only its stabilizing effect. In addition, the disadvantage of the prototype is that the vitamins in its composition are contained in the form of a lyophilized or dried alcohol solution, which leads to the loss of part of the activity of the cytokine and requires careful monitoring of the residual alcohol content. The authors do not report how the solution is restored after adding water to the lyophilisate, since vitamin E is insoluble in water, and its presence in the form of an oil is undesirable, as it can cause inflammation at the injection site.

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The objective of the invention is to obtain a preparation based on interferon in the form of a stable solution having an improved therapeutic effect and ensuring the maintenance of a high level of immunomodulating and antiviral activity in the body for a long time. An additional positive quality of the drug is the faster and more effective effect of interferon in the early stages after administration to the body.

This object is achieved in that the preparation based on interferon, including α-interferon, a stabilizer and water, as a stabilizer contains at least one fat-soluble vitamin in the form of colloidal particles and additionally contains sodium benzoate as a preservative and sodium chloride as an agent providing osmolarity of the solution, with the following ingredients per 1 liter of solution: a-interferon sodium chloride sodium benzoate, at least one fat-soluble vitamin based on the content of 1 · 10 ² -1 · 10 ¹⁰ IU

8.8- 9.1 g

1.8- 2.2 g

1.0-100 g of water is the rest.

The inventive preparation in various preferred forms of implementation as a stabilizer may contain fat-soluble vitamin A in the form of colloidal particles in an amount based on the content of 1-10 ⁷ -5-10 ⁸ IU;

fat-soluble vitamin I) in the form of colloidal particles in an amount based on the content of 5-10 ⁶ -1-10 ⁸ IU;

fat-soluble vitamin E in the form of colloidal particles in an amount of 1-100 g;

a mixture of fat-soluble vitamins A, I), E in the form of colloidal particles in the above amounts, as well as water-soluble vitamins C in an amount of 10-100 g / l and B12 in an amount of 0.01-1.0 g / l.

The quantitative content of α-interferon and fat-soluble vitamins A and I in units of mass (mg, μg, etc.) can be determined by a specialist in this field in each case from the calculation of the above content in IU taking into account the particular preparation used (in particular , depending on the degree of its purification, etc.).

The use of identical or similar ingredients or quantitative composition for the problem being solved is not known from the prior art.

The results of the claimed invention are to improve the retention of the activity of the cytokine (α-interferon) in liquid dosage form and increase the effectiveness of its therapeutic effect due to the vitamins contained in the solution, in particular due to the presence of fat-soluble vitamins in the form of colloidal particles, which simultaneously serve as a stabilizer, which makes it possible prolonging the action of the drug.

The technical result of the claimed invention is achieved due to the fact that the composition of the immunomodulating antiviral agent includes enhancing the action of interferon fat-soluble vitamins in the form of colloidal particles stabilized by a nonionic surfactant and containing polyethylene oxide residues on the surface that have a stabilizing effect on interferon and enhancing its immunomodulatory and activity with a prolonged mode of action;

sodium benzoate as a preservative with antimicrobial activity and sodium chloride as an agent for the osmosis of the solution.

The preparation of the inventive preparation having an antiviral and immunomodulatory effect and containing vitamins will be discussed further on some preferred, but not limiting examples.

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Examples

Example 1

9.0 g of sodium chloride (accurately weighed), 2.0 g of sodium benzoate (C ₆ H ₅ COO # 1) are placed in 500 ml of water for injection and mixed until completely dissolved. Without stopping mixing, add 250 ml of a concentrate of colloidal solution of vitamin A containing retinol acetate or retinol palmitate 1 -10 ⁵ IU / ml. Stirred for 5 min and add a substance containing recombinant α-interferon (human, avian, pork, bovine or a mixture thereof) with an activity of 1-10 ⁸ IU. After dissolving the substance, the volume of the resulting solution with stirring is adjusted with water for injection to 1000 ml, sterilization filtration is carried out. The solution is poured under aseptic conditions into sterile vials of 5-100 ml; sealed with rubber stoppers with run-in metal caps.

Example 2

The preparation of the claimed drug with antiviral effect and containing vitamin E is carried out analogously to example 1 with a vitamin E content of 50.0 g per 1 liter of solution.

Example 3

The preparation of the claimed drug with antiviral effect and containing vitamin осуществляют is carried out analogously to example 1 with a vitamin I content of 5-10 ⁷ IU.

Example 4

The preparation of the claimed drug with antiviral effect and containing vitamins Α, Ό and E is carried out analogously to example 1 using a multivitamin solution in the above concentrations.

Example 5

The preparation of the claimed drug with antiviral effect and containing vitamins A, B12, C, I and E is as follows.

In 500 ml of water for injection, 9.0 g of sodium chloride (accurately weighed), 2.0 g of sodium benzoate (CH ₅ COO # 1) are placed. 100 g of sodium ascorbate, 0.5 g of vitamin B12 and mix until completely dissolved. Without stopping mixing, add 250 ml of a concentrate of a colloidal solution of fat-soluble vitamins Α, Ό, E when the content of vitamins in quantities according to examples 1-3. Stirred for 5 min and add a substance containing recombinant α-interferon (human, avian, pork, bovine or a mixture thereof) with an activity of 1-10 ⁸ IU. After dissolving the substance, the volume of the resulting solution with stirring is adjusted with water for injection to 1000 ml, sterilization filtration is carried out. The solution is poured under aseptic conditions into sterile vials of 5-100 ml; sealed with rubber stoppers with run-in metal caps.

In the claimed preparation, the activity of α-interferon and the content of vitamins are determined by high performance liquid chromatography (HPLC, HPLC). In addition, they verify compliance with all requirements for a biologically active drug for injection: the absence of mechanical impurities, transparency, color, pH, rheological properties of the solution, sterility, etc.

Example 6. The study of the immunomodulatory activity of the drug containing α-interferon and vitamin E.

The drug is a colorless slightly opalescent solution. The active substance is α-interferon (5.0-10 ⁵ IU / ml) and a 5% colloidal solution of vitamin E.

The invention is illustrated in the drawing, which presents the results of a study of the pharmacokinetics of the immunomodulating activity of commercial lyophilized and the claimed drugs. Immunomodulating activity was determined by measuring the excess bactericidal activity of blood serum (BASK) of white mice after injection of the appropriate doses of drugs.

From the data shown in the drawing, it follows that the presence of colloidal particles of vitamin E in the interferon solution (curve marked ♦) contributes to an earlier manifestation of the immunomodulatory activity of the drug and to maintain its sufficiently high level for a longer time in comparison with the model aqueous solution of lyophilized interferon ( curve marked).

In the lyophilized preparation of interferon, immunomodulatory activity begins to appear 12 hours after administration to white mice and is observed over the next 18 hours, that is, after 30 hours it is no longer detected (Table 1). The inventive preparation exhibits up to 19% of the total immunomodulatory effect already 8 hours after administration of interferon in equivalent doses, and the activity persists for 54 hours of observation (Table 1).

Thus, the above combination in the composition of the inventive preparation of α-interferon and fat-soluble vitamins in the form of a stabilized colloidal solution leads to an earlier manifestation of the activity of the drug and prolongation of its action

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Table 1

Dynamics of immunomodulatory activity of drugs

A drug Immunomodulating activity, (%) Sampling time after drug administration, (hour) 4 8 12 24 thirty 48 54 The inventive drug 0 18.8 16.6 16.6 15,2 17.3 15,5 Lyophilized preparation 0 0 44.59 55,4 0 0 0

Example 7. The study of the stability of the preservation of the properties of the drug during storage.

The drug was stored in glass penicillin vials sealed with a rubber stopper with an aluminum cap. During storage, samples were taken and a study was conducted on sterility, immunomodulating and antiviral activity.

Sterility persisted for 18 months of observation.

Immunomodulating activity was determined by the excess bacteriocidal activity of blood serum (BASK) of white mice weighing 20-25 g, measured for the claimed drug, after 8 hours, and for mixoferon - 24 hours after injection. The preparations completely retained immunomodulatory activity during storage during 18 months of observation (Table 2).

Table 2 The stability of the immunomodulatory activity of drugs during storage

A drug Immunomodulating activity, (c.u.) Sampling time, (months) 0 6 12 eighteen The inventive drug 1.22 1.24 1.22 1.20 Lyophilized preparation 1.24 1.26 1.25 1.24

The antiviral activity of the preparations was determined by neutralizing the human vesicular stomatitis virus using an A-549 transplantable cell culture. The results show that the antiviral activity of the proposed drug stably persists for 18 months of storage (table. 3).

Table 3

Stability of antiviral activity of drugs during storage

A drug Antiviral activity of drugs, (x 10 ^& IU) Sampling time, (months) 0 6 12 eighteen The inventive drug 5.9 5,6 5,4 5.5 Lyophilized preparation 5,4 5.5 5,4 5.5

Thus, the proposed drug in liquid form stably maintains antiviral and immunomodulatory activity during storage for 12-18 months.

Information sources

1. Big Veterinary Encyclopedia: Mixoferon.

2. Patent FROM 4847079.

3. Patent No. 2008017.

4. Patent No. 2113845.

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11. Patent FROM 20030086901.

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13. Patent No. 2,148,988.

14. Ershov F.I. Interferon and their inducers (from molecules to drugs). - M .: GEOTAR - Media, 2005, 356 p.

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15. Patent No. 2121364.

16. Patent EC2726470.

17. Patent No. 2236866.

18. The methodological basis for the use of reaferon - human recombinant interferon a-2B protein. // Collection of materials of a scientific conference for medical practitioners, Koltsovo, January 25-29, 1993, p. 15-19.

19. Mashkovsky M.D. Medicines - Vilnius: ZAO Gamta, 2004, v. 2, p. 348.

Claims (4)

CLAIM 1. A preparation based on interferon, including α-interferon, a stabilizer and water, characterized in that as a stabilizer it contains at least one fat-soluble vitamin in the form of colloidal particles and additionally contains sodium benzoate as a preservative and sodium chloride as an agent that provides osmoticity of the solution, with the following content of ingredients per 1 liter of solution: OS-interferon based on the content of 1 · 10 ² - 1 · 10 ¹⁰ IU sodium chloride 8.8 - 9.1 g sodium benzoate 1.8 - 2.2 g at least one fat-soluble vitamin 1.0 - 100 g water rest. 2. The drug according to claim 1, characterized in that the stabilizer contains fat-soluble vitamin in the form of colloidal particles, selected from the group of vitamins A in an amount based on the content of 1 · 10 ⁷ -5 · 10 ⁸ IU, and in an amount from the calculation of content 5 · 10 ⁶ -1 · 10 ⁸ IU, E in the amount of 1-100 g 3. The drug under item 1, characterized in that as a stabilizer contains a mixture of fat-soluble vitamins A, I, E in the form of colloidal particles. 4. The preparation according to claim 1, characterized in that it contains a mixture of fat-soluble vitamins A, I, E in the form of colloidal particles as a stabilizer and additionally contains water-soluble vitamins C in an amount of 10-100 g / l and B12 in an amount of 0.01- 1.0 g / l.