RU2222345C2 - Pharmaceutical composition with cytokine and immunomodulating effects - Google Patents

Abstract

FIELD: medicine, chemical- -pharmaceutical industry, pharmacy. SUBSTANCE: invention relates to pharmaceutical composition eliciting cytokine and immunomodulating effect used for prophylaxis and treatment of diseases caused by disorders of immunologic functions. Addition of copolymer of 1,4-ethylenepiperazine N-oxide and (N-carboxyethyl)-1,4-ethylenepiperazinium bromide to an active substance in pharmaceutical composition consisting of at least one cytokine or conjugation with the latter allows creating the novel immunotherapeutic preparation with the enhanced effectiveness as compared with the known analogs. Advantage of invention involves the creation of preparation eliciting ability to modulate cytokine effect and it comprises new additional biological properties also. EFFECT: enhanced effectiveness of composition, valuable medicinal properties. 9 tbl, 8 ex

Description

 The invention relates to medicine and pharmacology, and relates to biomedical drugs and their compositions with cytokine and immunomodulatory effects, used for the prevention and treatment of diseases caused by impaired immunological functions. Since any pathology is based on or associated with a change in the reactions of the immune system, the drug according to the invention can be used in all areas of clinical medicine, the most important of which are: dentistry, ENT, dermatology, allergology, immunology, ophthalmology, etc.

 Advances in modern immunology and molecular biology have revealed a number of biologically active substances that can be successfully used to correct pathological conditions caused by impaired functions of the immune system.

 These substances are called cytokines, i.e. produced by cells (cytos-cell). Cytokines are low molecular weight proteins produced primarily by activated cells of the immune system. They are mediators of intercellular communications in the immune response, hematopoiesis, inflammation, as well as intersystem interactions. Currently known cytokines are divided into four main types: immunomodulatory, chemotactyl, fibrogenesis regulators, peptide growth factors. Having a wide spectrum of biological activity, they determine not only an adequate level of immunoreactivity, but also regulate the interactions of the main biological integrative systems of the body: nervous, immune and endocrine. The structure and mechanism of action of most cytokines are quite fully characterized. Using the methods of genetic engineering and modern biotechnology, many cytokines are currently obtained in the form of recombinant (bioengineered) drugs that are completely identical to endogenous molecules, and the amount of released cytokines is sufficient for their clinical use.

 Currently, immunotherapy with recombinant cytokines and drugs based on them is one of the most promising and constantly expanding areas of immunopharmacology (1. Royt A. Fundamentals of immunology (translated from English). - M .: Mir, 1991. 328 p .; Ketlinsky S.A., Simbirtsev A.S., Vorobev A.A. Endogenous immunomodulators .-- St. Petersburg: Hippocrates, 1992.256 s .; Freidlin I.S. System of mononuclear phagocytes. - M .: Medicine, 1984. 272 p. )

 When using recombinant cytokines and drugs based on them, the effectiveness of immunotherapy increases significantly, providing an adequate and targeted medical correction of immune dysfunctions. Cytokines introduced into the body, filling the deficit of endogenous regulatory molecules, fully reproduce their effects. This is especially important in conditions of severe or chronic pathology, when the use of traditional immunomodulators or inducers of cytokine synthesis is useless due to the depletion of the compensatory capabilities of the immune system.

 However, drugs based on cytokines do not satisfy clinicians, since they have many shortcomings: rapid destruction in the body, which forces a significant increase in the dose and frequency of administration; poor passage in tissue; adverse, including allergic, reactions, etc. This necessitates the search for new dosage forms for the use of cytokines in order to achieve a better biological effect. One of the results of this search was the creation of pegylates, that is, compounds of cytokines, primarily interferons, with “polyethylene glycol” (hereinafter referred to as PEG) (Clin Pharmacokinet 2001; 40 (7): 539-51. Pegylation: a novel process for modifying pharmacokinetics Harris JM, Martin NE, Modi M.). This process is called pegylation.

 PEG - in this case, the collective name for many isomers of polyethylene glycol, as well as its chemical modifications (PEG oxides - polyethylene oxide, acetates, etc.). PEG is a chemical substance with properties that allow it to be used as a carrier for bioorganic molecules or compounds. For example, on it (in it) molecules are agglomerated, which allows you to create a depot of a drug substance. In addition, due to local increased concentration of the active substance, its conduction into the tissues is enhanced. PEG and compounds based on it are widely used in cosmetology (A. N. Decina. Theory of soft cosmetic effects. Modern cosmetology. - Novosibirsk, 2001, 507 pp.). Pegylation allows prolonging the action of cytokines: to obtain a long-term therapeutic effect with a single use of the drug; avoid fluctuations in the concentration of drugs in the blood, causing undesirable side effects; reduce the toxicity of the drug substance (O. N. Minushkin, L. V. Maslovsky. Etiotropic therapy of chronic viral hepatitis. - “Kremlin medicine. Clinical Bulletin”, 2000, 1).

The following types of pegylates are known, for example:
1) A pharmaceutical composition of cytokine and immunomodulating action, the active substance of which consists of erythropoietin (cytokine), and PEG is used as a carrier (an additional chemical substance that performs the function of improving the dosage form) (BrJ Haematol 1999 Jan; 104 (1): 119- 26. The in vitro effect of pegylated recombinant human megakaryocyte growth and development factor (PEGrHuMGDF) on megakaryopoiesis in patients with aplastic anaemia. Brereton ML, Adams JA, Briggs M, Liu Yin JA .; (Clin Pharmacokinet 2001; 40 (7): 539-51. Pegylation: a novel process for modifying pharmacokinetics. Harris JM, Martin NE, Modi M.).

 2) A pharmaceutical composition of cytokine and immunomodulating action, the active substance of which consists of a megakaryocytic growth factor (cytokine), and PEG is used as a carrier (BrJ Haematol 1999 Jan; 104 (1): 119-26. The in vitro effect of pegylated recombinant human megakaryocyte growth and development factor (PEGrHuMGDF) on megakaryopoiesis in patients with aplastic anaemia. Brereton ML, Adams JA, Briggs M, Liu Yin JA.).

 3) A pharmaceutical composition of cytokine and immunomodulating action, the active substance of which consists of interferon alpha (cytokine), and PEG is used as a carrier (Blood 2001 Sep 15; 98 (6): 1708-13. Phase 1 study of polyethylene glycol formulation of interferon alpha-2B (Schering 54031) in Philadelphia chromosome-positive chronic myelogenous leukemia. Talpaz M, O'Brien S, Rose E, Gupta S, Shan J, Cortes J, Giles FJ, FaderI S, Kantarjian HM .; Rev Clin Esp 2001 Apr; 201 (4): 205-12. [Pegylated interferons: preliminary review of their pharmacokinetic characteristics.] Azanza Perea JR.; BioDrugs 2001; 15 (7): 419-29. Kozlowski A, Charles SA, Harris JM. Development of pegylated interferons for the treatment of chronic hepatitis C. BioDrugs. 2001; 15 (7): 419-29. Review. PMID: 11520253 [PubMed - indexed for MEDLINE].

 In the examples of the references cited, illustrating both particular examples of conjugates and the general principles of the cytokine-PEG compound, PEG is attached to the cytokine by a covalent bond (conjugation), as a result of which a chemical substance, the PEG-cytokine conjugate, is obtained. PEG is polyethylene glycol or its derivatives (products based on it, also serving as inert carriers).

 The effect of pegylates in relation to all cytokines has not been studied, mainly interferons. But it has been proved that due to pegylation, there is no sufficient improvement in the biological effect, there is no modulation of the cytokine effect, and no new additional biological properties of the pharmaceutical composition that are not inherent in its cytokines, their compounds and PEG are manifested.

 It has been fully proven that the nature and frequency of effects and deviations of laboratory parameters are similar when using PEG-interferon and conventional interferon, which indicates that pegylation does not improve or expand the immunomodulating effect of cytokines (Ready KR, Weight TL, Pockros PS et al. Efficacy and safety of pegylated (40-kd) interferon alpha-2a compared with interferon alpha-2a in noncirrhotic patients with chronic hepatitis C (Efficacy and safety of PEG- (40-kd) interferon alpha-2a compared to interferon alpha -2a in patients with chronic hepatitis C without cirrhosis). Hepat ology 2001 Feb; 33 (2): 433-438).

 The closest medicinal substance of the same purpose to the claimed invention according to the totality of signs is PegIntron (international non-proprietary name - Peginterferon-2b): a pharmaceutical composition of cytokine and immunomodulating action, the active substance of which consists of recombinant interferon alpha-2b (cytokine) conjugated with monometholoxol monomethoxol PEG) used as a carrier.

 Pharmachologic effect. It has immunostimulating and immunomodulating properties (PegIntron has an immunomodulating effect as interferon alpha-2b (antiviral effect)). The biological activity of pegylated isomers is qualitatively similar to that of free interferon alpha-2b, but weaker. By binding to the cell membrane, interferon initiates a sequence of intracellular reactions, which include the induction of certain enzymes, which leads to suppression of virus replication in infected cells, inhibition of cell proliferation, increased macrophage phagocytic activity and specific lymphocyte cytotoxicity against target cells.

 Indications. Histologically confirmed chronic hepatitis C (as monotherapy for intolerance to ribavirin or the presence of contraindications to its use).

 Contraindications Hypersensitivity (including to other interferons), autoimmune hepatitis, a history of autoimmune disease; thyroid dysfunction, not amenable to medical correction; severe impairment of renal and / or liver function, severe mental illness, severe mental disorders in history, epilepsy and other disorders of the central nervous system, pregnancy, lactation, childhood and adolescence up to 18 years (safety and efficacy have not been studied). With caution: congestive heart failure, myocardial infarction, arrhythmias, chronic obstructive pulmonary disease, bleeding disorders, including thrombophlebitis, pulmonary embolism, severe myelodepression, psoriasis, diabetes mellitus, prone to the development of ketoacidosis.

Side effects:
On the part of the nervous system: dizziness, hyperesthesia, paresthesia, emotional lability, nervousness, drowsiness, depression; rarely - suicidal thoughts and suicide attempts, agitation, confusion.

 From the digestive system: dry mouth, flatulence, vomiting, diarrhea or constipation and other dyspeptic symptoms; rarely - pain in the right hypochondrium, hepatopathy. From the cardiovascular system: decrease or increase in blood pressure, arrhythmia.

 From the respiratory system: nasal congestion, sinusitis; rarely - cough, shortness of breath, lung infiltrates of unknown etiology.

 On the part of the sensory organs: conjunctivitis, in rare cases - pain in the eyes, decreased visual acuity or restriction of visual fields, retinal hemorrhages, focal changes in the retina, obstruction of the artery or vein of the retina, hearing loss.

 From the endocrine system: impaired thyroid function, diabetes mellitus, menstrual irregularities (including menorrhagia).

 Allergic reactions: itching, dry skin, rash, urticaria, erythema, angioedema, bronchospasm, anaphylactic shock.

 Laboratory indicators: neutropenia, granulocytopenia, thrombocytopenia, the appearance of autoantibodies.

 Other: malaise, sweating, fever, chest pain, flu-like syndrome, viral infections, hot flashes, decreased libido.

 Interaction. Pharmaceutically incompatible with other drugs. A single injection of the drug does not affect the activity of cytochromes CYP1A2, CYP2C8 / C9, CYP2D6 and hepatic CYP3A4 and N-acetyltransferase. However, it must be borne in mind that other forms of interferon alpha caused a 50% decrease in theophylline clearance (which is a CYP1A2 substrate) and a 2-fold increase in its plasma concentration. With the simultaneous use of ribavirin, there were no signs of pharmacokinetic interaction.

 In 2000, the EORTC (European Organization for Research and Treatment of Cancer) team for the study of melanoma began a study of the effectiveness of PegIntron as an adjuvant treatment for patients with stage IIB-III melanoma.

 It is known that the drug was not well tolerated. More than 1/3 of the patients had to reduce the dose and / or take breaks due to toxic effects characteristic of interferon therapy; all in all, 77% of patients showed 3-4 grade toxicity during therapy (American Joint Committee on Cancer. Manual for Staging of Cancer, 4th edn. JB Lipincott Co., Philadelphia, 1992, pp. 143-148. Kirkwood JM, Strawderman MH, Ernstoff MS et al. Interferon alfa-2b adjuvant therapy of high-risk resected cutaneous melanoma: the Eastern Cooperative Oncology Group trial. J Clin Oncol 1996; 14: 7-17. Grob JJ, Dreno B, de la Salmoniere P et al. Randomized trial of interferon a2a as adjuvant therapy in resected primary melanoma thiker than 1.5 mm without clinically detectable node metastases. Lancet 1998, 351: 1905-1910. Pehamberger H, Soyer HP, Steiner A et al. Adjuvant interferon a2a treatment in resected primary stage II cutaneous melanoma. J Clin Oncol 1998, 16: 1425-1429).

 The reasons that impede the achievement of the technical result indicated below when using the well-known pharmaceutical composition adopted as a prototype include the fact that, despite some advantages compared to non-pegylated cytokines, this drug is not active enough, there is no modulation of the cytokine effect and inhibitory mechanisms that contribute to it, and there are undesirable side effects effects. This is explained, in particular, by the fact that PEG is inert as an immunomodulator (does not have its own positive biological effect). PEG simply serves as a structural lattice and allows one to obtain conglomerates of molecules, but is not an immunomodulator itself (in addition, PEG itself can be toxic).

 The present invention solves the problem of expanding the arsenal of effective biologically active agents.

 The problem is solved by the creation of new, more effective immunotherapeutic drugs based on cytokines with modulation of the cytokine effect, as well as new additional biological properties.

 The specified technical result during the implementation of the invention is achieved by the fact that the active substance of the known pharmaceutical composition of the cytokine and immunomodulating action, consisting of at least one cytokine, further comprises a copolymer of 1,4-ethylene piperazine N-oxide and (N-carboxyethyl) -1, 4-ethylene piperazinium bromide.

 A copolymer of 1,4-ethylene piperazine N-oxide and (N-carboxyethyl) -1,4-ethylene piperazinium bromide (hereinafter - COPOLYMER), a chemically synthesized substance used for immunocorrection (trade name POLYOXIDONIUM, approved since 1996, registration number 96 / 302/9, Pharmacopoeia article FS 42-3906-00).

 The copolymer itself has numerous immunomodulatory effects (the mechanism of action and clinical use of the domestic immunomodulator polyoxidonium. - Moscow, State Scientific Center - Institute of Immunology of the Ministry of Health of the Russian Federation, 2001, 110 pp.), But since it is not similar to any of the known cytokine molecules , does not possess a cytokine effect. Attention should be paid to the pronounced positive effect of the COPOLYMER as an adjuvant, an agent that enhances the immune response to a specific antigen added, and / or attached to it.

 So, COPOLYMER is an active substance that enhances (improves the pharmacodynamics and pharmacokinetics of the cytokine) and modulates (regulates and modifies) the cytokine effect, and also has active properties in relation to the regulation of immunity. Due to this, in the interaction of the active substances (cytokine - COPOLYMER) in the pharmaceutical composition according to the invention, new additional biological properties appear that are not inherent in the active substances (cytokine and COPOLYMER) of the pharmaceutical composition, but are the result of a new biologically active complex (mixture and / or compound of biologically active molecules). Modulation is regulation: leading to the optimal ratio of the number and functions of cytokine (s) in relation to a given state of the body.

 Obtaining when using the invention a technical result, which consists in modulating the cytokine effect, as well as the manifestation of the pharmaceutical composition of new additional biological properties, is demonstrated by the following examples.

Example 1
The kinetics of hepatitis C virus (HCV) and clinical data for therapy with interferon alpha-2b (realdiron), PEG-interferon alpha-2b (PegIntron) and a mixture of interferon alpha-2b with a copolymer were evaluated.

 38 patients with chronic hepatitis C were examined. All patients were divided into 4 groups. Group 1 (12 people) received Realdiron (interferon alfa-2b), 3 million ME three times a week. Group 2 (10 people) received PegIntron 1 μg / kg of weight 1 time per week. Group 3 (10 people) received a mixture of Realdiron (5 million ME) and Copolymer (0.012 g) once a week. Group 4 (6 people) received a copolymer (0.006 g) 3 times a week.

 The effectiveness of the treatment was evaluated by determining the PCR reaction (polymerase chain reaction) of HCV RNA for 12 weeks after the start of therapy.

 The data obtained were compared with data from a general clinical examination.

 After 2, 6, 12 weeks, a study was made of the content of HCV RNA in the blood serum of patients.

 A decrease in the concentration of HCV RNA (virus content in the blood) after 2 weeks from the start of treatment was observed in 13 percent of patients of the 1st group, 18 percent of the 2nd group, 29 percent of the 3rd group. In group 4, HCV RNA reduction was not noted, and therefore patients were transferred to standard treatment.

 It was established that in the group that received interferon according to the standard regimen (group 1), the time to achieve complete negativity in viremia (complete disappearance of the virus from the blood) exceeded 12 weeks, PegIntron (group 2) - from 42 to more than 84 days, interferon + copolymer ( 3 group) - from 25 to 45 days.

 The biochemical response in the 1st and 2nd groups was almost the same. In the 3rd group, the level of transaminases at the end of the 2nd week was significantly higher than in the first two groups, but subsequently was significantly lower (P <0.05).

 Severe side effects requiring medical intervention developed in 8 of 12 (66%) patients of the 1st group, in 5 of 10 (50%) patients of the 2nd group and in 3 of 10 (33%) patients of the 3rd group .

 In addition, 6 patients of the 1st group (50%), 5 patients of the 2nd group (50%) and 1 patient of the 3rd group (10%) showed malabsorption in the intestine, accompanied by steatorrhea. The nature and frequency of side effects and deviations of laboratory parameters were similar in all groups.

 The copolymer in this study alone did not have an antiviral effect. The antiviral effect to varying degrees was possessed by preparations of Interferon alpha-2c. The combination of Interferon (Realdiron) + Copolymer had the greatest effect. Interferon (Realdiron) alone had a less pronounced effect.

 Thus, if we simply summarize the effect of Copolymer and Interferon in groups 1 and 4, then the effect will be equal to the isolated use of Interferon. Whereas the mixture of the Copolymer with Interferon had a much more pronounced effect. Therefore, in this case we are talking about obtaining a synergistic ultramount antiviral effect. It should be noted that the connection of Interferon with another carrier (PEG - polyethylene glycol) (group 2) did not give such an effect.

 Based on the data of the example, we can conclude that the Copolymer enhances the antiviral effect of IFN and due to this there is a decrease in the level of transaminases. A brief increase indicates the massive destruction of the virus and cells affected by the virus, which proves the synergy of the action.

 Decreased absorption and steatorrhea are the consequences of damage to the liver and pancreas, in this case, the virus. A decrease in the severity of these symptoms is a consequence of successful treatment, primarily, elimination of the virus.

 As with transaminases, a decrease in the severity of these symptoms is primarily associated with a decrease in the concentration of the virus and, as a consequence, the level of liver damage under the influence of Interferon. According to the example (group 3), the greatest improvement was achieved when using the Interferon-Copolymer composition, which is not connected with the direct effect of the Copolymer on the virus, but is achieved by enhancing the effect of Interferon.

 Side effects - a new quality, reduced toxicity. A copolymer is an antioxidant, but it does not have direct antiviral activity; interferon is toxic.

 Thus, when using the copolymer-interferon alpha-2b mixture, a more pronounced clinical and biochemical effect is observed compared to other methods of interferon therapy, as well as new additional biological properties of the pharmaceutical composition are manifested: elimination of the side effect of interferon alpha-2, a significant increase in antiviral effect, reduction tissue destruction.

Example 2
The activity of interferon alfa-2c (Realdiron) was studied in vitro according to the generally accepted method of suppressing the cytopathogenic effect (CPD) of vesicular stomatitis virus (BBC) in a culture of L41 cells (human lymphoid cells) and expressed in international units of IU / ml. The results are presented in table 1.

 As can be seen from the table, the copolymer did not have an interferon-like effect and did not exert a depressing effect on the cytopathogenic effect of the virus. Interferon alfa-2b had this effect. But the Interferon-Copolymer mixture gave a pronounced effect, which was more than 2 times higher than the Interferon effect.

 Thus, the use of a mixture of Cytokine (Interferon alpha-2B) - Copolymer gave a pronounced total effect.

Example 3
The study of the antiviral activity of a mixture of Interferon alpha-2b and Copolymer
The study of the Preparation of a mixture of Interferon alpha-2b and Copolymer was carried out in order to detect both its direct antiviral effect and mediated by the induction of interferon by cells.

 For this, a Tr cell culture (transplantable cattle trachea cells) with the VVS test virus (vesicular stomatitis virus) in a working dose was used in the experiment. A preparation of a mixture of Interferon alpha-2b and Copolymer, Controls: 1. Copolymer (1 μg / ml), 2. Interferon alpha-2b (Realdiron - 300 IU / ml) in an amount of 0.2 ml was added to the tubes 30 minutes before and after making a test virus. The experiment was accompanied by controls of cell culture - CPD without the introduction of Drugs and virus (3) and virus - CPD without the introduction of drugs (4) and drugs - CPD without the introduction of the virus (5, 6, 7). Accounting was made after the virus was triggered in the control. The results are presented in table 2.

 As can be seen from the table, none of the drugs had its own cytopathogenic effect (controls 5, 6, 7). When the virus was introduced without drugs, the cytopathogenic effect (destruction of target cells) was most pronounced. As can be seen from the table, the introduction of the Copolymer into the cell culture before infection resulted in a decrease in the cytopathogenic effect of the virus. The introduction after infection did not give such an effect. The use of Interferon gave an equal effect both before infection of the cells and after the introduction of the virus. The mixture of Copolymer and Interferon was most active when the use of the drug made it possible to remove the cytopathogenic effect of the virus when the drug was added before infection and significantly reduced it when applied after infection.

 Thus, the mixture of Interferon and the Copolymer had a significant over-total effect.

Example 4
Determination of the biological activity of erythropoietin preparations using normocytemic mice
The study is based on measuring the stimulation of the formation of reticulocytes in normocytemic mice and was performed in accordance with the requirements of Pharmacological article FS 42-3658-98 of 02/11/1999.

 2 days before the start of the study, F1 (CDF * C57BL) 7-9 week old mice were seeded with 6 animals per cell.

 Solutions were prepared: recombinant human erythropoietin + Polyethylene glycol (m.v. 2000) (hereinafter EPO-PEG), recombinant human erythropoietin + Copolymer (hereinafter EPO-POL) and a standard, industrial sample of human recombinant erythropoietin (Erythrostim, hereinafter ESR) phosphate-albumin buffer (pH 7.2) with concentrations: 80, 40 and 20 IU / ml.

 Each group of mice was injected subcutaneously with 0.5 ml of a solution of one of the concentrations.

 A control group of 1 mice was injected with 0.5 ml of phosphate-albumin buffer pH 7.2.

 The control group of 2 mice was injected with 6 μg of the drug Copolymer.

 4 days after injection, blood samples were taken from animals and the number of reticulocytes was determined.

 For this, 1 μl of whole blood was added to 1 ml of a thiazole orange solution in a concentration convenient for the determination of reticulocytes. After staining for 3-10 minutes, reticulocytes were counted microluorometrically in a flow cytometer using a red fluorescence histogram analysis. The calculation of the actual value of the activity of erythropoietin (Ad, IU / ml) was performed using the number of reticulocytes per 1 million cells.

The method of regression analysis calculated the relationship between the logarithm of the concentration of erythropoietin (EPO) in each group and ESR and the number of reticulocytes. Comparing the regression dependence for erythropoietin with the same dependence for ESR, we calculated the coefficients of the ratio of nominal concentrations of erythropoietin and the corresponding concentrations of ESR
Ki = Yi / Xi,
where Ki is the coefficient of the ratio of the concentrations of ESR / EPO at dilution i;
Xi - nominal value of EPO concentration at dilution i (Xi = 80; 40; 20 IU / ml);
Yi is the ESR concentration value corresponding to the efficiency (i.e., the number of reticulocytes) of EPO concentration in dilution i, IU / ml.

где Кср - средний коэффициент соотношения концентраций СОЭ/ЭПО;
К1, К2, К3 - коэффициенты соотношения концентраций СОЭ при определенном разведении ЭПО;
3 - количество взятых для сравнения с СОЭ концентраций ЭПО.Based on at least three calculated coefficients, its average value was determined

where Ksr - the average ratio of the concentration of ESR / EPO;
K1, K2, K3 - coefficients of the ratio of ESR concentrations at a certain dilution of EPO;
3 - the number of EPO concentrations taken for comparison with ESR.

Multiplying the nominal value of EPO activity (Anom) by the average coefficient (Ksr), we obtained the actual value of EPO activity (Ad) in IU / ml
Hell = Ksr • Anom.

 It was established that the actual value of EPO activity for the EPO + PEG group was 2238 ME; for the EPO + POL group - 2648 ME, the EPO activity in the standard sample (ESR) (Anom) was 2000 ME.

 Thus, the addition of a copolymer to a cytokine (erythropoietin) significantly increased its biological activity in relation to the stimulation of erythroopoiesis.

 Table 3 below shows the level of reticulocytes in mice of the studied groups, including control ones. The number of reticulocytes per 1000 erythroid cells (see table 3).

 As can be seen from the table, the introduction of phosphate-albumin buffer, as well as the Copolymer did not cause an increase in the number of reticulocytes and, thus, did not stimulate erythropoiesis.

 Thus, the copolymer does not have the ability to stimulate the production of reticulocytes and erythropoiesis. When studying the activity of EPO + Copolymer, it was found that this activity is significantly increased in comparison with the standard EPO sample, as well as in comparison with the EPO - Polyethylene glycol compound.

 Considering the absence of the Copolymer effect, the lower activity of isolated EPO can be considered that the composition of EPO-Copolymer has a synergistic super-total effect.

 To confirm this effect, an activity study was performed in the immunology laboratory of the Institute of Biotechnology of the Republic of Lithuania in the international standard test for studying the activity of human recombinant erythropoietin (International Standard for Human Recombinant EPO, NIBSC code 87/684) in vitro using TF-1 cell line). The results are presented in table 4.

 As can be seen from the table, the copolymer does not affect erythropoiesis and does not have erythropoietin activity. The use of the EPO-Copolymer conjugate yielded significantly greater activity than the use of standard EPO. This indicates that the connection of the Copolymer with EPO significantly improves the properties of the latter.

 In the studies described in Example 3, the level of cytokines was determined in the collected blood samples and, using the method of radioactive chromium release, the natural killer activity (MK activity) of mouse splenocytes was determined against the Yac-1 tumor cell line.

 The data obtained are presented in tables 5 and 6 below.

 Plasma cytokines are shown in table 5.

 As can be seen from the table, under the influence of the Copolymer there is a significant stimulation of the synthesis of Interleukin 6 (IL-6) and a decrease in the synthesis of Interleukin 12 (IL-12). The level of tumor necrosis factor alpha (TNFa) has not changed.

 The effect of erythropoietins (standard erythropoietin and pegylated erythropoietin) did not practically differ from each other and was expressed in a decrease in the concentration of TNF alpha (TNFa).

 Under the action of EPO-POL (erythropoietin copolymer), the amount of IL-6, IL-12 and TNF alpha increased uniformly. (Modulation is the restoration of the normal quantitative and functional correlation of parts of the immune system. In this case, it was the EPO-POL mixture that had the greatest immunomodulating effect, since since the mice were normal (healthy), it is not necessary to selectively stimulate any one of the links of the immune system and it is important to maintain a normal ratio between them.The introduction of other drugs in this case gave a stimulating (POL) or depressive (EPO, EPO-PEG) effect).

 Interleukin 6, depending on the initial state of the immune system, can act both as a pro and anti-inflammatory cytokine. In addition, he is involved in the stimulation of thrombocytopoiesis and antitumor immunity.

 Interleukin 12 has the ability to stimulate the formation of T-lymphocytes (T-helpers - type 1 helpers), stimulate antitumor cytotoxic reactions, potentiate the effect of vaccines and antitumor immunity.

 Tumor necrosis factor is involved in the processes of tumor cell lysis, is a pro-inflammatory cytokine, and is involved in the processes of stimulation of atherosclerotic lesions.

 From the above data it follows that the Copolymer had an immunomodulating effect, which manifested itself mainly in the form of activation of pro-inflammatory reactions. Erythropoietins, on the contrary, had a mild anti-inflammatory effect. The most harmonious effect in this case was noted for the EPO-POL complex (COPOLYMER). This was expressed in stimulating the level of all studied cytokines while maintaining their normal ratio.

 In addition, it should be noted separately the stimulation under the influence of the EPO-POL complex of IL-12 production. Since IL-12 is a very promising immunotherapeutic agent, but its use in its pure form is impossible due to its high toxicity, the fact of stimulation of the production of this cytokine by the EPO-POL complex opens up new prospects for immunotherapy.

 "NK" activity (natural cytotoxicity) are shown in table 6.

 As can be seen from the table, stimulation of the antitumor cytotoxic effect was noted only in the LP and EPO-LP groups, but was not noted in other groups.

 It should be noted that the stimulation of erythropoiesis in combination with the stimulation of antitumor immunity is a very important positive quality, since most tumors occur against the background of anemia, which is aggravated by chemo- and radiation therapy. This fact emphasizes the novelty and practical value of the proposed composition.

 Thus, the copolymer does not have the ability to stimulate the production of reticulocytes and erythropoiesis. When studying the activity of EPO + Copolymer, it was found that this activity is significantly increased in comparison with the standard EPO sample, as well as in comparison with the EPO - Polyethylene glycol compound.

 Considering the absence of the Copolymer effect, the lower activity of isolated EPO can be considered that the composition of EPO-Copolymer has a synergistic super-total effect.

 To confirm this effect, an activity study was performed in the immunology laboratory of the Institute of Biotechnology of the Republic of Lithuania in the international standard test for studying the activity of human recombinant erythropoietin (International Standard for Human Recombinant EPO, NIBSC code 87/684) in vitro using a TF-1 cell line. The results are presented in table 7.

 As can be seen from the table, the copolymer does not affect erythropoiesis and does not have erythropoietin activity. The use of the EPO-Copolymer conjugate yielded significantly greater activity than the use of standard EPO. This indicates that the connection of the Copolymer with EPO significantly improves the properties of the latter.

 In the above examples, the effect of the EPO-POL complex was significantly different from both Erythropoietin (EPO) in its pure form, Copolymer in its pure form, and its analogue, EPO-PEG. Moreover, the differences extended to both erythropoiesis and immunomodulation, which emphasizes the novelty of the biological effect of this pharmaceutical composition.

 In the above example 4, in addition to modulating the cytokine effect, the following new additional biological properties of the pharmaceutical composition are manifested: significant stimulation of erythropoiesis and antitumor effect, stimulation of IL-12 production, uniform harmonious stimulation of the production of interleukins.

 For a broader understanding of the interaction of Cytokines and the Copolymer, an additional study was carried out: the effect of the connection of the Copolymer and one of the most important cytokines, the Tumor Necrosis Factor alpha, was studied. The data obtained are presented below.

Study of the influence of tumor necrosis factor alpha (TNF-a), copolymer and their conjugate on phagocytosis
Used the following drugs:
1. TNF alpha 10 and 50 PG / ml
2. Copolymer 1 and 0.1 μg / ml
3. The conjugate corresponding to a mixture of 10 PG / ml TNF-α + 0.1 μg / ml Copolymer
4. The conjugate corresponding to a mixture of 50 pg / ml TNF-α + 1 μg / ml Copolymer
5. A mixture of 10 PG / ml TNF-α + 0.1 μg / ml Copolymer
6. A mixture of 50 PG / ml TNF-A + 1 μg / ml Copolymer
7. Saline solution (control)
The study of phagocytosis using the blood of healthy donors was carried out when setting the method with latex (0.05 ml latex, the same amount of blood and the drug), keeping the samples in the thermostat for 30 minutes, centrifuging for 5 minutes at 1500 rpm and then making smears. The study of the NBT test was carried out in a standard way with nitro-blue tetrazolium [1]. The experimental data are presented in table 8.

 As can be seen from table 7, the addition of all the studied drugs led (with the exception of the Copolymer at a dose of 0.1 μg / ml) to a significant stimulation of phagocytic activity compared with the control.

 When analyzing the differences between the individual groups, it was found that the use of the Conjugate of 10 pg / ml TNF-α + 0.1 μg / ml of the Copolymer led to a significant increase in the phagocytic number and, most importantly, the phagocytic index (P <0.05) as compared to the copolymer in any used dose, and with TNFa in any used dose. Similar differences (P <0.05) were also found for the Conjugate 50 pg / ml TNF-a + 1 μg / ml Copolymer, Mixtures 10 pg / ml TNF-a + 0.1 μg / ml Copolymer, Mixtures 50 pg / ml TNF-a + 1 μg / ml of copolymer.

 It should be noted that this effect cannot be considered simply the addition of individual effects. First, the use of a mixture and / or conjugates of Copolymer and cytokine-TNF-A alone gave a greater effect both in terms of the phagocytic number and, especially, the phagocytic index, than each of the drugs used separately. The copolymer at a dose of 0.1 μg / ml generally did not give a reliable effect. In addition, the more pronounced effect of the Mixture (conjugate) of TNF-α and Copolymer with respect to stimulation of the phagocytic index characterizing the digesting ability of phagocytes is noteworthy. This effect is more pronounced and more clinically important, since it indicates not only the ability of the phagocyte to capture the microorganism (phagocytic number), but also to digest it.

 These data were confirmed by evaluating the function of phagocytic enzyme systems using the HCT test.

 As can be seen from table 9, neither TNF-α nor Copolymer in small doses of 10 pg / ml and 0.1 μg / ml, respectively, had an effect in the HCT test. At the same time, both the TNF-copolymer mixture and their conjugates in similar doses had a significant effect. And in large doses, this effect was significantly superior to the results obtained with the separate use of drugs.

 Thus, the mixture or conjugate of Cytokine - TNF-α and Copolymer had an extra-cumulative effect, as well as new properties, expressed in a more selective stimulation of phagocytosis.

 So, from the above studies it follows that we are talking about a new phenomenon, when the addition of the Copolymer leads not only to a super-total effect, but also to the appearance of fundamentally new properties in cytokines.

 It should be noted that the combination of cytokines with the Copolymer forms new substances that possess, along with the well-known, new physicochemical and biological properties that still need to be studied.

Literature
1. S. A. Ketlinsky, N.M. Kalinin. Immunology for the doctor. - St. Petersburg, 1998, 156 p.

 2. Voitkevich K.L. Determination of the total redox activity of neutrophils using the histochemical dye of nitro blue tetrazolium // Lab. a business. - 1977. - N3. - S. 147-148.

 The following are examples of specific pharmaceutical compositions and methods for their preparation, confirming the possibility of carrying out the invention to obtain the above technical result.

Example 5
The pharmaceutical composition, which is a mixture with EPO and with COPOLYMER, as active substances containing the following ingredients in the following ratio, wt.%:
EPO - 0.001-5.5
Copolymer - 0.001-7.0
Mannitol - 0.001-30
Polyvinylpyrrolidone low molecular weight medical - 10-20
Beta Carotene - 0.01
Distilled Water - Up to 100
Cooking method
Erythropoietin - a substance is obtained in a biotechnological way by introducing a cloned human erythropoietin gene as a part of a plasmid into cells of the transplanted line of the ovary of a Chinese hamster. Prepare a solution of EPO in phosphate buffer (pH 7.2-7.4) with an activity of at least 2000 ME in 1 ml. To the resulting EPO solution, a Copolymer solution is added under sterile conditions. The copolymer is obtained by chemical synthesis. Then produce filtration and packaging.

Example 6
A pharmaceutical composition consisting of suppositories (rectal, vaginal), urethral sticks, with Interferon alpha-2 and a copolymer as active substances and with a fat-based excipient, containing the following ingredients in the following ratio, wt.%:
Interferon alfa-2 - 0.000001-0.01
COPOLYMER - 0.0005-0.07
Solid Fat Base - Up to 100
The tool is prepared as follows. Active substances are obtained as described above (Interferon alpha-2 - by biotechnological, and Copolymer - by chemical synthesis). Then, a copolymer is attached to a portion of IFN by covalent binding (conjugation). The other part is mixed: a Copolymer solution is added to the IFN solution. Then the components are mixed and fed through dispensers to the machine for the manufacture and packaging of suppositories. The homogeneous mass is stirred at room temperature. Then filtering and packaging.

Example 7
The pharmaceutical composition, which is a solution for inhalation, with erythropoietin, interferon alfa-2, interleukin 4, copolymer as active substances and with a water-based excipient, containing the following ingredients in the following ratio, wt.%:
EPO - 0.000005-0.018
IFN alpha-2 - 0.000001-0.01
Interleukin 4 - 0.000001-0.01
Copolymer - 0.0005-0.07
Sodium Chloride - 2-5
Water - Up to 100
The tool is prepared as follows. Cytokines (erythropoietin, interferon alpha-2, interleukin) are obtained by a biotechnological method. The copolymer is a chemical synthesis method. Then each individually cytokine is attached by conjugation to the copolymer. After that, the IFN-Copolymer solution and the IL-4 - Copolymer solution are added to the EPO-Copolymer solution. Mixed. Sterilized by filtration. Then add the Copolymer solution. Stir, filter. A method of obtaining a solution for inhalation includes the following sequence of operations: obtaining a total solution of biologically active substances, dissolving sodium chloride, mixing the solutions, sterilizing the filtration of the resulting solution, filling the solution into a vial and sealing it.

Example 8
A pharmaceutical composition comprising eye drops (nasal drops), with Interferon alfa-2b and Copolymer as active substances and with a water-based excipient, containing the following ingredients in the following ratio, wt.%:
Interferon alfa-2v - 0.000001-0.01
Copolymer - 0.0005-0.07
Sodium Chloride - 0.9-1
Water - Up to 100
The tool is prepared as follows. Active substances are prepared as described above. The method of obtaining the claimed dosage form includes the following sequence of operations: attachment (by covalent binding - conjugation) of Interferon to the Copolymer, dissolution of the active substance and sodium chloride in water for injection with heating the solution to t 25 ^o C, sterilizing the filtration of the resulting solution, pouring the solution into a vial and its sealing.

Thus, the above information indicates the fulfillment of the following set of conditions when using the claimed invention:
- a pharmaceutical composition embodying the claimed invention in its implementation, is intended for use in medicine, namely for the prevention and treatment of diseases caused by impaired immunological functions;
- for the claimed pharmaceutical composition as described in the independent clause of the claims, the possibility of its implementation using the means and methods described in the application and known prior to the priority date is confirmed;
- a tool embodying the invention in its implementation, is able to achieve the specified technical result: the creation of a new, more effective than the known analogues of an immunotherapeutic drug based on cytokines, with the ability to modulate the cytokine effect, as well as new additional biological properties.

Claims (1)

A cytokine-based pharmaceutical composition, characterized in that it is a mixture or conjugate of a cytokine with a copolymer of 1,4-ethylene piperazine N-oxide and (N-carboxyethyl) -1,4-ethylene piperazine bromide.

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