UA69457C2 - Aloferons immunomodulating peptides - Google Patents

Abstract

Disclosed are biologically active peptides specifically stimulating antiviral, antimicrobic and antitumor activity of immune system of humans and animals.

Description

part of the alloferon molecule. Compounds 5-20 in Table 1 represent examples of modifications of the original structure obtained by replacing amino acids in the variable regions of the alloferon molecule. In addition, the authors of the invention conducted a search for structural analogues of alloferon using databases of known proteins and peptides. Analysis of the BUMLIZORAOT database, performed using a computer system

SepotemMei did not find structural similarity between alloferon and known immunomodulating peptides. At the same time, the structural similarity of alloferon and the hemagglutinin fragment of the envelope of the influenza B virus (positions 374-384) was established. Hemagglutinin is a key structure of the virus, responsible for the fusion of the viral envelope and the host cell membrane; features of the hemagglutinin structure affect the formation of an antiviral immune response (A.I. Korotyaev, S.A. Babichev, Medical Microbiology, Immunology and Virology, St. Petersburg, "Special Literature", 1998, p. 265).

In the process of creating a valid invention, alloferon 1 was used as the basic structure. Alloferon 1 is a linear peptide with a molecular weight of 1265Da, consisting of 13 amino acids (Table 1). Comparison with analogs shown in Table 1 allows to identify functionally important elements of the alloferon molecule and to predict possible modifications of the basic structure that do not have a significant impact on its biological activity. Thus, a comparison with the structure of alloferons 2-4 shows that the region Zeg-Cs1u-Niv-Stu-c1Ip-Nivz-Siu-Ma is necessary for the manifestation of the biological activity characteristic of alloferon (the ability to stimulate the cytotoxic activity of lymphocytes in relation to target cells)! , which corresponds to the structure of alloferon-4. Positions 1-3 in the alloferon molecule may be absent or be represented by sequences of at least 1 amino acid. Comparison with the sequence of the homologous fragment of hemagglutinin of virus B also suggests that positions 4 and 5, represented in the alloferon molecule by the amino acids serine (5e) and glycine (Schu), can also be replaced by other amino acids selected from the group of aliphatic, aromatic or heterocyclic. For example, serine can be replaced by threonine (TpHg), and glycine by serine. Thus, the totality of the available data allows us to consider, for example, the first five amino acids in the alloferon molecule as a variable region, that is, they may be absent or contain at least 1 amino acid. Taking into account this circumstance, it is proposed to designate the indicated section in the structural formula of alloferon as

Hee. Similarly, positions 14-15 in the alloferon-1 molecule may be absent or be represented by sequences with at least 1 amino acid. Therefore, section 14-15 of the structural formula of alloferon is proposed to be designated as Khz. In addition, a comparison of the structure of alloferon and hemagglutinin suggests that position 8, occupied by glutamine in the alloferon molecule, is also variable, in particular, glutamine can be replaced by alanine.

Accordingly, position 8 of the structural formula of alloferon is proposed to be designated as X?, which can simply be a peptide bond connecting Su and Ni5 or contain at least 1 amino acid.

By analogy with the structure of hemagglutinin of the influenza virus, it is also possible to add the amino acid sequence of alloferon to the composition of a larger molecule, for example, a carrier protein, without significantly changing the nature of its biological activity.

Comprehensive immunological, pharmacological and toxicological studies, the results of which are reflected in the examples below, revealed a number of potentially useful properties of alloferon. The obtained experimental data show that alloferon is a cytokine-like peptide whose mechanism of action includes stimulation of recognition and lysis of defective cells by cytotoxic lymphocytes, as well as induction of endogenous interferon synthesis. Alloferon is intended for the correction of the deficiency of the interferon system and the system of natural killers, the treatment of viral, fungal and oncological diseases associated with this deficiency.

The compound is non-toxic, does not have a teratogenic, embryotoxic or mutagenic effect. Alloferon is biodegraded quickly and without residue by extracellular and intracellular proteases.

The experimentally determined properties of alloferon using the example of alloferon 1 are summarized in Table 2.

The spectrum of revealed pharmacological activity of alloferon as a whole corresponds to the known properties of alpha-interferon in the part related to the effect on the system of natural killers and resistance to viral infection. On this basis, the drug can be attributed to the group of interferonomimetics. At the same time, it is assumed that the drug does not have the full spectrum of interferon effects.

The mechanism of action of alloferon on the target cell is implemented in extremely low concentrations - less than 0.001 nanogram (109 grams) per ml. In this respect, alloferon is not inferior to interferons and interleukins. In addition, alloferon stimulates the formation of interferon in the body. This allows alloferon to be classified as an interferon inducer.

The combination of interferonomimetic and interferon-inducing action in combination with the almost complete non-toxicity of the drug favorably distinguishes alloferon from currently used cytokines and interferon inducers. Data on the biological activity of alloferon allow us to count on its use as a medicine for the therapy and prevention of various infectious and tumor diseases, including:

Viral influenza A and B

Acute and chronic viral hepatitis A, B and C.

HIV infection and secondary viral and fungal infections in AIDS

Kaloshi's sarcoma

Acute and chronic leukemia, multiple myeloma and other oncological diseases in which interferon treatment is indicated.

Candidiasis, cryptococcosis and other deep mycoses, in which interferon treatment is indicated.

Despite the significant similarity of the achieved result, alloferon is fundamentally different from interferons in a number of key features. Yes, interferons are glycoproteins with a molecular weight of 17,000 - 80,000 Da. Glycosylation of the amino acid chain is a necessary condition for the functional activity of interferons, as well as their tissue and species specificity. Alloferon is a non-glycosylated oligopeptide with a molecular weight of 1265 Da, which is 13-60 times less than the weight of interferons. Computer analysis of the database

ZUMIZ5RAOT performed using the SepoteMei system did not reveal any structural similarity between alloferon and any fragment of the amino acid sequence or interferons of other known immunomodulators. There are also significant differences in the functional relationship between alloferon and interferons. Thus, alloferon increases the formation of endogenous interferons and thus ensures the development of a cascade of protective reactions mediated by these cytokines. The introduction of exogenous interferon leads rather to the suppression of the formation of the patient's own interferons according to the principle of negative feedback. Interferons are also characterized by a number of disadvantages noted in the section "Level of technology".

According to the present invention, there is provided a pharmaceutical composition containing a peptide or mixture of peptides, according to the present invention, taken in an effective amount in combination with a pharmaceutically acceptable carrier, such as, for example, albumin. The broader concept of a pharmaceutically acceptable carrier refers to any inert, non-toxic, solid or liquid filler or diluent for the capsule material that does not adversely interact with the active ingredient or the patient's body.

Particularly suitable peptides for use in a pharmaceutical composition are alloferons 1-4. The conducted experiments showed that the pharmaceutical composition has immunomodulatory activity. Suitable methods of administration include intramuscular injection, subcutaneous injection, intravenous injection, and oral or intranasal administration.

A dose of the proposed pharmaceutical composition may contain the amount of alloferon required for a single dose. The optimal and acceptable therapeutic doses for a specific patient depend on many factors, such as the specific activity of the substance under investigation, the patient's age, weight, gender, general state of health, nature of nutrition, duration and method of administration of the substance, speed of its elimination, as well as goals its application (treatment or prevention) and the nature of the disease to which the treatment is directed. The daily dose can be, as a rule, from 0.01 to 10000 μg of peptide per kg of the patient's weight.

Data confirming the possibility of implementing the invention. Synthesis of alloferon.

The peptide, consisting of 13 amino acids, which correspond to the structure of alloferon 1, was synthesized by the method of solid-phase synthesis (Meiytak 9. apa 9.-R. Vpyap YuOemeyurtepi oi a shu ashotaisei tiyisNappei reriide zupineiiigeg m/p ipiedgaseid TEA sieamade sarabiiu, Reriide Nezeagsi, 1993 , mo!.6, p.219) using

Ethos- (M9-fluorenyl | methoxycarbonyl)- substituted amino acids. Purification of the synthesized peptide involved two stages. At the first stage, purification was carried out on Ber-Rak Mas columns with C18 sorbent (Uaiegv) by elution of the column with 4095 acetonitrile acidified with 0.059565 trifluoroacetic acid. After lyophilization, the peptide was purified to a homogeneous state by the HPLC method on a Vesktap cSoii buziet device with an Adoaroge 005 Rger 10 C18 column (100x10tt, Vgozhmpieye) using a linear gradient of 0.0590 trifluoroacetic acid and acetonitrile, acidified with 0.059565 trifluoroacetic acid (0-20954 during acetonitrile flow rate 2.5mol/min and detector wavelength 225nm). The purity of the peptide was confirmed by the MAGOI-TOEB mass spectrometry method. The correctness of the synthesis was confirmed by the method of microsequencing according to Zdman on an automatic sequencer by Arriya Viozuvietv. About 100 peptides were obtained. Peptides can be isolated in the form of an acid, its ester, a pharmaceutically acceptable salt, and also in the form of an amide.

Example 71. The effect of alloferon 71 on the cytotoxic activity of mouse spleen lymphocytes. The method of testing the cytotoxicity of mouse spleen lymphocytes corresponded to that described in the literature (Filatova et al.,

Cytology, 1990, vol. 32, Mob, pp. 652-658). Males of the SZNA line served as the source of splenocytes. Splenocytes were obtained by mechanically crushing the spleen in ARMI1640 medium and passing fragments of the organ through needles of decreasing diameter using a syringe. Intact cell aggregates were removed by filtration, then erythrocytes were lysed by short-term hypotonic shock. Splenocytes were precipitated by centrifugation at 10 rpm. at 200025 and resuspended in ARMI1640 medium supplemented with glutamine, gentamicin and RNAse. The titer of splenocytes was adjusted to the value of 2x109U cells/mol and immediately used for setting up the test.

Cells of human erythromyeloid leukemia line K5O62 served as targets for the cytotoxicity test. Cell culture was contained in plastic culture dishes in ARMI1640 medium supplemented with glutamine, gentamicin and fetal calf serum.

To determine the cytotoxicity of splenocytes, a standard method of assessing the effect of drugs on the functional activity of natural killers was used, based on the analysis of the release of NH-uridine-labeled

RNA from lysed K5b62 cells (Khaiytov et al., Vidomosti Pharm. Komitemu, 1999, 1, p. 31-36). For this purpose, NH-uridine was injected into the medium with K562 cells at a concentration of 2.5 μKCure/mol and incubated for 2 hours. Then the cells were washed by centrifugation (1Okhv., 20025) from the non-included label, precipitated by centrifugation, resuspended in the incubation medium, the number of cells was counted in a hemacytometer and diluted with the same medium to 10 cells/mol. 96-well round-bottom immunological tablets were used to perform the test. 0.1 mol of a suspension of NH-uridine-labeled K562 cells (10,000 cells per well) and 0.1 mol of a splenocyte suspension in a ratio of 5:1 or 20:1 to the number of target cells were introduced into the well of the tablet. In the control version, instead of the splenocyte suspension, 0.1 mol incubation medium. Then the drug was introduced into the wells in 0.02 mol of medium (in the control - 0.02 mol of medium). Tablets were incubated for 18 hours in

СО"- thermostat at 377"С and 695 СО". Then the contents of the wells were transferred to Whatman paper filters, the filters were washed three times with 5 mol of 596 THU, dried with 9690 ethanol and placed in vials with scintillation liquid. The radioactivity of the filters was determined on a WesKtap counter. Index cytotoxicity (IC) of splenocytes was calculated according to the formula:

IC(1-ro/Ps)x100, where Ro is radioactivity in wells containing target cells and splenocytes, Re is radioactivity in wells containing target cells without splenocytes.

The results of the analysis of the effect of alloferon on the cytotoxicity of mouse splenocytes are presented in Table 3. In each variant, the data of 13 independent determinations of cytotoxicity are summarized.

The introduction of alloferon in the incubation medium at a concentration of 0.05-50 nanograms/mol caused a highly reliable increase in the lytic activity of natural killers in relation to tumor target cells.

The lower threshold of effective concentrations of the drug was less than 0.05 nanograms/mol, and the maximum stimulating effect was achieved at 0.5 ng/mol. A 1000-fold excess of the optimal concentration eliminated the stimulation effect, however, even in this case, splenocytes maintained normal functional activity (at the control level), which indicates that the drug does not have pronounced immunosuppressive properties.

Conclusion

Thus, alloferon exhibits a pronounced stimulating effect on the activity of mouse natural killers in extremely low concentrations, which allows to characterize it as a specific cytokine-like inducer of cell-mediated cytotoxicity.

Example 2. Effect of alloferon 1 on the cytotoxic activity of human peripheral blood lymphocytes

Lymphocytes were isolated from fresh donor blood. For this purpose, a solution of 2.5906 gelatin in a ratio of 1:10 was added to 50 ml of whole heparinized blood. The mixture was allowed to stand at room temperature for 30-40 minutes. The plasma was applied to a solution of Piziorak 1077 in plastic conical tubes with a volume of 1 bmol in a ratio of 2:11 and centrifuged at ЗОхв. at 70025 for sedimentation of erythrocytes. The contents of the interphase rings formed during centrifugation were transferred to plastic conical tubes, diluted with 10 mol of isotonic phosphate buffer without Ca and Mod ions and precipitated by centrifugation at 20025 for 10 minutes. The sediment was resuspended in a fresh portion of the same buffer, the cells were again sedimented by centrifugation and resuspended in ARMI1640 medium supplemented with glutamine, gentamicin and RNAse. The number of lymphocytes was counted using a hemocytometer, diluted with the same medium to a cell concentration of 2x10b/mol and immediately used for cytotoxicity analysis (see section 1.1 for analysis methodology). Each option included 6 repetitions.

Data on the effect of aloferon 1 on the lysis of tumor cells by cytotoxic lymphocytes of human peripheral blood are shown in Table 4. The introduction of alloferon into the incubation medium caused a sharp increase in the cytotoxicity of lymphocytes in relation to tumor cells. The maximum effect was caused by a concentration of 0.O5 nanograms/mol (increasing the activity of natural killers by approximately 3 times compared to the control).

Interferon-alphagr, which was used as a positive control, showed a similar stimulating effect. However, the effect of alloferon in a concentration corresponding to the molarity of interferon (0.5ng/mol versus 5ng/mol of interferon) was probably higher. Alloferon had a similar effect on the activity of natural killers in relation to tumor cells of the A431 line (a test line for determining the activity of the MS subpopulation of natural killers).

Conclusion

The obtained data indicate a pronounced potentiating effect of alloferon on the lysis of tumor cells by human peripheral blood lymphocytes. In the conditions of this experiment, alloferon did not yield or even exceeded the effectiveness of the natural inducer of killer activity - alpha-interferon.

Example 3. Comparative analysis of the effect of alloferon 1 and alpha-interferon 2pb on the cytotoxic activity of lymphocytes from healthy donors.

The task of the actual study included the analysis of the variability of the response to alloferon and interferon-alpha in the population of healthy donors and obtaining data on the comparative effectiveness of the two drugs on this basis.

The killer activity of freshly collected samples of donor blood was studied according to the protocol outlined in example 2. The purpose of the study was to assess the variability of the response to alloferon in a population of clinically healthy people and to obtain comparative characteristics of the effectiveness of alloferon and alpha-interferon.

In total, blood samples from 17 donors were examined using two types of target cells: leukemic cells of the K562 line, sensitive to the lysis of MK (pashgai! KiiTeg) lymphocytes, and cells of the solid tumor line A431, sensitive to the lysis of MC (pashgai! suyuuhis) lymphocytes. The effector:target ratio in the experiment was 20:1. In each option, the results of cytotoxicity determinations are summarized. A statistically significant (P<0.05) increase in the cytotoxicity of lymphocytes served as a criterion of effectiveness.

The obtained experimental data are summarized in Table 5. The data in the table shows significant variability in the background activity of donor lymphocytes, in particular, the different ability to recognize and lyse tumor cell lines

K562 and A431. Significant differences in the reaction of lymphocytes from different donors to the administration of drugs were also revealed. However, in most donors, there is a clear correlation of the reaction to alloferon and alpha-interferon: donors who respond positively to interferon, in most cases, also respond positively to alloferon, and vice versa.

The total efficiency indicators of the two drugs, calculated on the basis of the data in Table 5, are given in Table b.

The majority of donors responded to the introduction of interferon (10 donors out of 17) or alloferon (also 10 donors) by increasing the cytotoxicity of lymphocytes in relation to one or another target. At the same time, a clear correlation of the reaction to one and another drug was observed: 9 donors responded equally to both drugs, and one donor was selectively sensitive only to interferon or only alloferon.

Conclusion

The study of the variability of the response of cytotoxic lymphocytes to alloferon and alpha-interferon in a sample of healthy donors showed that the effectiveness of the two drugs in the conditions of this study turned out to be identical. The revealed correlation of donors' reactions to interferon and alloferon testifies to the interchangeability of the two drugs.

Example 4. Comparative analysis of the effect of alloferon 1 and alpha-interferons on the cytotoxic activity of lymphocytes of cancer patients.

Blood samples from patients with various oncological diseases were examined according to the protocol outlined in examples 2 and 3. The purpose of the study was to analyze the characteristics of the reaction to alloferon in a randomized sample of cancer patients and to obtain comparative characteristics of the effectiveness of alloferon and alpha-interferon. In total, blood samples from 18 patients were examined using two types of target cells: leukemic cells of the K562 line (a specific target for determining the activity of MK lymphocytes) and cells of a solid tumor of the A431 line (a target for determining the activity of MC lymphocytes). The effector:target ratio in the experiment was 20:11. In each variant, the results of 6 determinations are summarized. A statistically significant (P<0.05) increase in the cytotoxicity of lymphocytes served as a criterion of effectiveness. Data on the initial activity and reaction to alloferon and interferon of cytotoxic lymphocytes of each examined patient are shown in Table 7.

The results of the research are summarized in the table. 8 and 9. The data in Table 8 show that the natural killers of approximately half of cancer patients respond positively to the administration of drugs. The frequency of positive responses in general does not differ significantly from the indicators of healthy donors. The exception is the reaction of lymphocytes treated with alloferon to K562 cells. In this case, the frequency of positive responses was significantly lower than when treated with interferon. Although the probability of differences between the response to alloferon and interferon in cancer patients does not reach a statistically significant value (Р-0.071), this circumstance may indicate a lower effectiveness of alloferon in the case of impact on the population of MK lymphocytes of cancer patients. At the same time, during the lysis of cells of the A431 line, the reactivity of lymphocytes in this group when treated with alloferon and interferon turns out to be the same.

The special nature of the immune status of cancer patients in comparison with healthy donors is also confirmed by the data in Table 9. If the background activity (during the absence of treatment with the drug) in relation to the Ab31 target is almost the same in both, the cytotoxicity in relation to the K562 target in cancer patients is clearly reduced. This defect of lymphocytes of cancer patients probably affects the essential mechanism of stimulating effect of alloferon and, to a lesser extent, interferon. Another important aspect of the effect of drugs on the lymphocytes of cancer patients is demonstrated by comparing groups that respond and do not respond to cytokine stimulation. As it turned out, patients with a low or zero background level of lymphocyte activity show a positive response. At the same time, the lack of reaction is associated with high background activity, which is much higher than the average level in the population. Therefore, the use of alloferon (as well as interferon) is most appropriate in patients with a deficiency of the link of natural killers. Selection of suitable patients using laboratory tests of cytotoxicity of lymphocytes will allow, as can be hoped, to achieve a much higher therapeutic effect of the drug.

Conclusion

Alloferon has a stimulating effect on the lysis of tumor cells by cytotoxic lymphocytes of cancer patients. At the same time, there are significant differences in the effectiveness of stimulation depending on the type of tumor cells. In this contingent of patients, the stimulation of the subpopulation of MS lymphocytes is more effective.

The positive effect of alloferon was noted in patients with acute and chronic leukemia (5 out of 9 cases). The frequency of positive responses is lower in lymphomas (2 out of 6 cases). A positive effect was also observed in a patient with lung cancer. In most of the studied cases, the stimulating effect of alloferon, like alpha-interferon, correlates with low or zero initial cytotoxicity of lymphocytes. This fact can be used to predict the effectiveness of alloferon treatment.

Example 5. Effect of structural analogues of alloferon 1 on the cytotoxic activity of human lymphocytes.

The activity of analogues of alloferon 1, alloferons C and 4 was studied according to the protocol outlined in examples 2 and 3. For this, the fraction of mononuclear cells isolated from the blood of healthy donors and tumor cells of the line

A431 was incubated in the presence of the studied drugs: alpha-interferon (positive control), alloferon 1, alloferon C or alloferon 4. The initial concentration of all drugs was 5ng/mol. The activity of the drugs was judged by the change in the lymphocyte cytotoxicity index compared to the control. As in the experiments described in the previous examples, alloferon and interferon stimulated the cytotoxic activity of lymphocytes in some donors. In such donors, the reaction to alloferon C and 4 was similar to the reaction to alloferon 1 and alpha-interferon (Table 10). Quantitatively, the stimulating effect of alloferons C and 4 was not inferior or even slightly superior to the effect of interferon and alloferon.

Conclusion.

A comparative analysis of the stimulating effect of structural analogs of alloferon 1 on the cytotoxic activity of human lymphocytes allows us to establish possible modifications of the amino acid sequence of alloferon that do not affect its biological activity. Such modifications include the removal or replacement of amino acids 1-4 and/or 14-15 in the alloferon 1 molecule.

Example 6. The effect of alloferon on the resistance of mice to the influenza A virus.

The antiviral effect of alloferon was studied on a model of lethal viral infection of mice with the influenza A virus.

A suspension of a strain of the virus pathogenic for mice was administered to the experimental animal intranasally in a dose corresponding to 10 LD»o. Alloferon 1 in 0.5 mol of 0.995 sodium chloride solution was administered intraperitoneally one day before virus inoculation, then 1, 2, 4, 6, and 8 days after inoculation. The drug was tested in doses of 25 and 2.5 micrograms. In the control, mice were injected with an equal volume of solvent. The criterion of effectiveness was the survival of animals 10 days after infection with the virus.

For the experiment, sexually mature white mice, males, weighing 20.0-22 Og were selected.

60 white mice were used in the experiment.

Administration of alloferon to infected animals caused a dose-dependent protective effect (Table 11).

The drug in a dose of 25 micrograms caused a significant increase in the number of animals that survived during their observation.

The effect of this dose is highly reliable. The lower dose did not significantly increase survival.

Conclusion

Alloferon in a dose of 25 micrograms shows a pronounced antiviral effect on mice infected with the influenza A virus.

Example 7. The effect of alloferon 1 on the resistance of mice to the influenza B virus.

The study of the effect of alloferon on the resistance of mice to the influenza B virus was carried out according to the protocol outlined in example 6. The mice were infected with the GEE 1/40 strain of the influenza B virus, taken in infectious doses corresponding to 3 and 30 LDvo. The specific antiviral drug Virazol (ribavirin) was used as a positive control. Alloferon was administered intraperitoneally in a dose of 25 μg 1 day before infection with the virus, then after 1,2,4,61 8 days.

In the control, intranasal introduction of the virus caused severe pneumonia with high mortality, regardless of the administered dose of the pathogen (Table 12). Against this background, virazol had an effective protective effect at a low dose of the virus (3 LDvo), at a higher infectious load (30 LD»5o) virazol was ineffective under the conditions of this experiment. At the same time, alloferon showed a pronounced protective effect both at high and low infectious load.

Conclusion

Alloferon in a dose of 25 μg exhibits a pronounced antiviral effect on mice infected with the influenza B virus.

The protective effect of alloferon in the conditions of this experiment exceeded the protective effect of virazol, a well-known antiviral agent.

Example 8. Effect of alloferon 1 on the synthesis of interferon by mice.

The quantitative content of interferon in the blood serum of mice was determined by titration of the analyzed samples over a monolayer of cells of the I-929 line. The cells were incubated in the presence of the samples for 24 hours at 37°C in an atmosphere of 595°C.

Then the samples were replaced with a solution of the test virus in the supporting medium (OMEM.29o blood serum KROS medium). The vesicular stomatitis virus (Indiana strain) with 100 activity was used as a test virus

TCDZ5BO. Incubation of cells with the test virus was carried out for 2 days until 90-9595 destruction of the cell monolayer was achieved in the samples of the negative control (only the test virus). A unit of serum interferon activity was taken to be the value inverse of the final serum dilution, at which protection of 50 95 cells from the cytotoxic effect of the test virus was observed. The degree of cell monolayer destruction was assessed after fixation/staining with 0.195 crystal violet in 3095 ethanol. In each variant, serum aliquots of 4 animals were studied. The well-known interferon inducer cycloferon was used as a positive control.

The interferon-inducing activity of alloferon was investigated in two series of experiments. In the first series of experiments (Table 13), alloferon was used in doses of 2.5 and 25 μg. The comparison drug (cycloferon) was used in a dose of 500 μg per mouse. The dynamics of interferon concentration in response to administration of different doses of alloferon is illustrated in Figure 1.

In the next series of experiments, the effect of alloferon and cycloferon was monitored within 48 hours after the administration of the drugs (Table 14). With a longer period of observation, the two-peak nature of the response of interferon to a single injection is clearly revealed (Fig. 2).

The data of two series of experiments to determine the interferon-inducing activity of alloferon are summarized in

Table 15.

Conclusion

The results of a comparative analysis of data on the effect of alloferon and cycloferon (comparison drug) on the dynamics of interferon titer in mice show that alloferon has a pronounced interferon-inducing effect. Unlike cycloferon, the maximum effect of which appears 2-4 hours after administration, alloferon causes a maximum increase in interferon titer 24 hours after administration. According to this parameter, alloferon can be classified as long-acting interferon inducers. An important advantage of alloferon is its effectiveness in much lower doses - 20-200 times lower than the effective dose of cycloferon.

Example 9. Assessment of toxicity and other side effects of alloferon.

The study of acute toxicity of alloferon 1 was carried out according to the method of Litchfield and Wilcoxon (Bilenky

M.L., 1963). 15 white mice were used in the experiment - 5 animals in each group. The study of acute toxicity of the drug was carried out with a single subcutaneous injection. The observation period was 14 days. The 1st group of animals was injected with the tested drug in a 10-fold dose - 0.25 mg/gol; 2nd group - in a 70-fold dose - 1.75 mg/head. The other 5 mice (group 3) served as controls.

The drug was diluted with a sterile 0.995 physiological solution and injected into the animals subcutaneously in the amount of

Oh, 5 ml. The control group of animals was injected with 0.995 saline solution. The data are presented in Table 16.

No mice died after 14 days of observation. Motor activity, behavioral reactions, physiological responses, food and water intake in the experimental and control groups did not differ, the animals did not lose weight (Table 17). The dynamics of body weight indicators in the animals of the experimental and control groups are practically the same, the differences are unreliable (Р»0.05).

After euthanasia on the 14th day, macroscopic examination of internal organs (liver, spleen, brain) was performed. According to the results of observations, no visible changes in internal organs were detected. Similar results were obtained with intraperitoneal administration of alloferone.

Thus, the conducted studies showed that alloferon 1 does not have acute toxicity upon single administration to mice, including at the maximum tested dose of 87.5 mg/kg, which is 700 times higher than the therapeutic dose of 0.125 mg/kg, sufficient to induce interferon synthesis in mice mice On this basis, the drug Alloferon can be classified as a substance that is almost completely devoid of acute toxicity.

Chronic toxicity of alloferon was also not detected when a dose of 12.5 mg/kg of alloferon, which was 100 times higher than the therapeutic dose, was administered to mice daily for 5 days. In animals, no changes were noted in the behavior, growth, anatomical structure of internal organs, biochemical and hematological indicators, with the exception of indicators of immune status associated with the specific immunomodulatory effect of the drug.

In studies on rabbits, alloferon showed no signs of acute and chronic toxicity, immunotoxicity, local sensitizing resorptive action, or pyrogenicity.

A study on guinea pigs showed that alloferon does not have an allergenic effect. In studies on rats, alloferon did not cause teratogenic and embryotoxic effects. Studies on the drosophila Ogozorniiia teiapodavzieg and the yeast Zasspagotusez segemigea did not reveal the mutagenic effect of alloferon.

Conclusion.

According to currently available data, alloferon can be classified as a drug that is almost completely devoid of toxicity and other negative side effects.

Table 1

The structure of alloferon and its analogues

- Cleanliness for life |1)28|4)5 6 7 in 9

Alloferoni | Niv |Siu|Mai| Zeg/ Siu |Niv|Yi Siu| - |Stsh| - (NivISsu| Ma /nNivIssu!| - | 95796

Alloferon2 | - (su Ma! |Zegi stu |Niv|siu| - |St!| - |niv|su|Ma|Niv| stu! - | 85495

Alloferon3 | - / - |MaZegistu|Niv|siu| - |St!| - |niv|su|Ma|Niv| - | - | 92195

Alloferoni | - / - | - |Zeg|Su|Niv|Siu| - (Sp) - |nivIsu|Ma! | |/-1 920956 oAloferon5 |Rgo ZegGesh|TigiIstu|Niv|Siu| - |(Rpe| - |niv|Su|Ma Tu Ar! -| -

Alloferonb |Rpe|Ne|Ma|Zeg Aia|Niv!iu| - (Avr| - /NivISSu Ma - 1 -1-Yi -

Alloferon7 | - / -|- | - |Tig|Niv|su| - |(St| - |nvisu|Ma/-/-,1-7/ -

Alloferon 8th | - / -|- | - | - |niv|smu| -| - | - |niv|su Ma nve|s,/)-| -

Alloferon | - Ptesh|Aa|Zegiyesh|Niv|Siu| - |(Sp| - |ni|Isu|Ma!/ -/-71-7/ - o Aloferon 10 |Suz Ma Ma Tig|stu|Niv|siu| - |Zeg| - |niv|Su|MaRne|Ma /|-|-

Alloferoni!i | - | - |Ne|Zeg Ssiu|Niv|Ysiu| - |Xiu| - /nNivISSu Ma Ryu/ -|-| -

Alloferon1!2 | - | - | - |SuviStuu|Niv|sSiu| - |(Avp| - |niv|su|Ma|nNev| -/|-| - oAloferon 13 | No Ma Aa|Agi No |Niz|Siu| - |Sip|AvpI no (Su eat/ - | - 1- 7 -

Alloferon!4 | - / - | - | - | - |niv|Siu|ZegAvriSu|niv|Siu Ma |Sipi ni|YisSu| --

Alloferoni!5 | - | - | - |Rpe suu|NivYisu| -| | - (nv/isu|ma/! - 1 -1-7 -

Alloferon!b | - / - | - | - | - |niv|siu| - |(Avp| - |niv|su|Ma Petz ila| -| - oAloferon 17 )|Niv Siu|Avr|Zegistu|Niv|Siu| - |St!| - |niv|su|MaTSAvrI| -/|-| -

Alloferon!8 | - / - | - | - | - |niv|Ssu| - | - | - |niv|Su Ma Ro eat|-| - oAloferon19 | - / - | - |Zeg|Ssu|Niv|Siu| - (Aa/Ma | niv|Ssu|Ma Me! -/1-| -

Alloferon20 |Tug|Aa|MetsZeg Siu|NivYisiu| - | - | - /NivISSu Ma Rneine!Й -| Sh -

PEV, effect thorn era, | 017

Consensus-. . spas 11 | |hineyem! 1 |not|ivory!

Table 2

Specific pharmacological activity of alloferon-1 1. Stimulation of cytotoxic activity Therapy of infectious and oncological influenza A virus and influenza B virus

And oncological diseases 5. Stimulation of cytotoxic activity Therapy and prevention of infectious and natural killer lymphocytes of cancer patients with cytotoxic lymphocytes

Table C

The effect of alloferon 1 on the cytotoxic activity of mouse splenocytes in relation to K562 human erythromyeloid leukemia cells

Concentration, o Control// | 70777771 Y7771717171717121330 | 7777777777771711l1oyu 0.5 39.33.97 185 34.324.577 161 3723457 175 500 20.353.6 95 yarebd.o1; ed. 001

Table 4

The effect of alloferon 1 on the cytotoxic activity of natural killers of human peripheral blood in relation to K5b2 tumor cells

Control.///7777777711111111111Ї11111711011111111111111111111273573 | forehead

Alloferon 0.0005 62.056.4 weight 0.005 73.821.7 td 0.05 1719.855.0 292 0.5 179.832.8 292 6b.8-7.2 245 50 68.055.3 49 500 68.854.4 Bal yah reb.00

Table 5

Variability of the response to alloferon (5 ng/ml) and alpha-interferon (5 ng/ml) in a population of healthy male donors 1

Drug "" stimulation (Р«0.05) 1 Control 5.3544.4 53.826.5

Interferon 50.21.67 55.718.6 sh -

Alloferon 30.25.57 83.72.9777 I I 2 Control 14.25 3.3 -64.5-40.6

Interferon 50.22.57 -88.3240.4 sh

Alloferon 39.81 -86.7122.8 -

With Control 29.87 4 -53.6419.9

Interferon 21.857.5 27.0-6.577 sh

Alloferon 30.826.8 39.713.37 - 4 Control 30.0zh2.9 83.853.6

Interferon 42.85 24 89.5522.3 sh

Alloferon 45.42. 91.220.9 - 5 Control 43.353.5 -14.858.3

Interferon 642557 35.45.90 sh sh

Alloferon 51.051.7 24.44 - -

Control 41.7-10.5 50.055.7

Interferon 30.55 8.6 43.755.0

Alloferon 37.65.1002 53.754.А 7 Control 40.353.2 17.355.5

Interferon 58.2532.57 11.828.3 sh

Alloferon 59.25 5.27 -10.258.0 -

Control 23.053.5 -20.06.3

Interferon 19.252.3 74bjo lyh sh

Alloferon 29.246.3 80.71. -

Control 6.2211.3 7.8515.6

Interferon 25.054.6 38.553.A4

Alloferon 25.0-12.3 24.55215.1 | Control 34.321.3 74.0-4.5

Interferon 51.8242.977 82.7-31.9 sh -

Alloferon 44.83.57 88.41.27 - - 11 | Control 57.054.А 37.216.7

Interferon 52.012.2 50.819.2

Alloferon 42.855.9 46.57 .6 12 | Control 61.853.4 55.6:4.9

Interferon 71.822.97 51.453.6 sh

Alloferon 47.84 1 56.453.1 - 13 | Control 44.0213.1 52.816.5

Interferon 62.757.5 58.216.1

Alloferon 49.753.9 54.854.0 14 | Control 30.26.11 -6.028.1

Interferon 14.758.7 48.74.57 sh

Alloferon 2.0-44.2 49.8412.977 - ish 1 oi-e | what is HER 110111

Interferon 2.213.8 25.055.7

0 IAloferon./// | 19534687 | - 277437 | -: (| 7777 16 | Control -3.557 A 61.47 1

Interferon 5.024.7 69.212.1 -

Alloferon 18.51.77 68.72.0 - 17 | Control 23.355.6 57.8141

Interferon 29.053.3 62.5:1.7

Alloferon 24.7-4.0 64.354 1

Table 6

Comparative characteristics of the effectiveness of alloferon and alpha-interferon in the effect on cytotoxic lymphocytes of the peripheral blood of healthy donors (based on the materials of Table 5)

In positive answers":

K562 7117-5196 6/17-3590

A431 4/17-2496 6/17-3590

K562 or A531 10/17-59965 10/17-5995

Number of positive answers:

On interferon and alloferon 9/17-5390

Only on interferon 1/17-695

Only for alloferon 1/17-695 x Positive response - a statistically significant increase in the index of cytotoxicity of lymphocytes under the influence of the drug.

Table 7

Reactions to alloferon (5ng/ml) and alpha-interferon2go (Zng/ml) of peripheral blood lymphocytes of oncological patients Yan Ten | indestructible DECO

Diagnosis Drug stimulation (Р« 0.05) 1 Chronic leukemia Control 11.2210.6 -4.258.7

Interferon -5.527.9 11.72412.5

Alloferon -35.355.4 -9.0512.7 2 |Chronic leukemia Control 4.855.4 37,055.6

Interferon -10.754.9 33.329.0

Alloferon 1.3322.3 8.2518.3

With Chronic leukemia Control -8.322.3 -16.2210.6

Interferon -3.5-4.0 33.5:57.47 sh

Alloferon -12.8:43.7 26.23.87 - 4 |Chronic leukemia Control -11.053.6 28.215.9

Interferon -9.257.0 18.854.3

Alloferon -1.5525.1 19.216.3 |Chronic leukemia Control -23.055.3 15.258.3

Interferon -4.6:29.6 46.04.00 sh

Alloferon -8.0-418.8 42.53.97 -

Acute leukemia Control 42.312.7 6.2116.5

Interferon 25.24.07 43.56.47 sh

Alloferon 20.26.87 48.5513.57 - 7 | Acute leukemia Control 29.215.6 -18.7-414.6

Interferon 50.22.3777 24.8210.17 sh sh

Alloferon 28.212.6 20.0-6.97 - -

Acute leukemia Control 15.2529.4 25.216.5

Interferon 40.053.4 17.8258.3 sh

Alloferon 23.555.5 40.4-6.2 -

Acute leukemia Control 23.518.2 -4.8:29.0

Interferon 25.154.6 51.55.5657 sh

Alloferon 13.854.4 51.84. - Lung cancer Control 11.5523.7 -1.2510.2

Interferon 27.244.27 59.5:153.9х sh sh

Alloferon -0.3326.1 55.52.77 - - 11 | Lymphoma Control 11.526.4 82.8:31.1

Interferon 13.83.00 80.2512.0 -

Alloferon 17.72.68 91.5:550,875 -

that's en

Interferon 39.3524.9 95.20.7977 -

Alloferon 27.1-4.8 90.20.47 -

WE ARE ON EE AND NOT HONORED

Interferon 50.84.97 93.5:20.9 sh

Alloferon 24.04 4 94.350.4 - shen lymphoma Interferon 31.259.2 26.08.9777

Alloferon 36.759.1 15.34.87 - lymphoma debit Interferon 47.55126.67 62.255.2

Alloferon 37,354.7 64.055.0 - 16 | Nekhodzhkinskaya Control 35.858.3 4725104

Mn SHE SHE SHE SHE SHE

Alloferon 37.357.0 56.354.4 o she to n 1:11 lymphoma Interferon 48.823.5 67.053.3 -

Alloferon 68.34.67 64.355.1 - 18 | Nekhodzhkinskaya Control 6.3214.9 29.854.6

Msssn ZSH ONLY: NIS

Alloferon -10.7-414.1 32.859.2

Table 8

Peculiarities of background activity and response to alloferon and interferon cytotoxicity of lymphocytes of cancer patients

In positive answers":

K562 6/18-3395 1/18-695

A431 8/18-44965 8/18-4495

K562 or A531 10/18-5695 9/18-5090 and 0

On interferon and alloferon 7/18-3890

Only for interferon 4/18-2296

Only for alloferon 2/18-11965

Table 9

The background activity of natural killers in cancer patients and healthy donors is as high as kyu ryu

Healthy donors (according to Table 6) 17 29.354.4 27.3210.6

Be iii nina NOYA PNYA NO SSV interferon prnsnvnnennnennia || ("| pits:. -359. And alloferon and interferon

Table 10

Comparative assessment of the stimulating effect of interferon-alpha, alloferons 1, C and 4 on the cytotoxic activity of human peripheral blood lymphocytes in relation to target cells of the A431 line. Drug concentration - Zng/ml 111 Drug (Bone studies, Indexcytotoxicity, Meta. b6/////| P control 17111761 svbv11Ї1111111111111111 in interferon. |Ї771717611117 17717171 48574467 | 77710001

Alloferon 3rd 17171176 60227771 "00

Table 11

The effect of alloferon 1 on the resistance of mice to the influenza virus

Kontvol/-/:/// | 777-117 1717117120 | 771717171717114 HER

Table 12

The effect of alloferon on the resistance of mice to the influenza B virus

Virus dose (LDzo) Mortality 10 days after the introduction of the virus is low

Control 0) 13 10 77

From 10 8 80

Virazol 0) 10 bo

From 10 (sh

Alloferon 0) 10 2 h

With 10 (0) (sh yared.o1 and p«0.001

Table 13

The influence of alloferon on the synthesis of interferon: the effect of the dose of the drug

Drug Dose, μg

Control./-:-/:// | 07777777 Y7777777171717171111111111111111111116о1111111111

Cycloferon. | /7/50077. | ....ЮЮ7И-.77171717117177171717171711401 |177711115255 shD

Table 14

The effect of alloferon on the synthesis of interferon within 48 hours after the administration of the drug

Drug | Dozaumk | Time after drug administration, hours

Drug Dose, mk

Control./-/:/ | - ГЇ777777777777777777717171711111111111201111111111111111

Cycloferon. | 500 | (y | 85 | 50 | 20 | «45 | 65 (

Alloferon.//// | 25 | | 45 | 25 | with 35 | 55 | with35 /

Table 15

Summary data on the interferon-inducing activity of alloferon (25mcg) and cycloferon (500mcg)

I ad Y Iitr of interferon

Drug Time after injection, h. Number of animals (Mam), u.e.

Kontvol/:/ ОЙ 7777777777771717171717171711Й7171717108611111111111111 15351711

Cycloferon. | 4 | 8 | 95345

Cycloferon. | -:( 24..:// | 8 2 live | 493.87

Alloferon..//// | : 2 uBB...Y | 8 | 315138

Alloferon..//// | 4 24uB...( | (8 | 2153.7

Alloferon./// | (:(AI 24...:-/ | 8 / T1516.2y xR-0.05

Table 16

Alloferon 1 doses used for acute toxicity analysis 18.90 19.350.6

C (control) 19.8:0.4 0.990 physiological solution

Table 17

Dynamics of body weight of experimental animals (p-5)

"TRU" groups After 7 days After 14 days 19.0520.3 18.920.1 18.9:20.2 19.6520.2 19.8:0.2 19.720.3 18.8:50.3 18.9 :20.3 19.0201 19.920 1 19.80 2 19.950.3 100 --25 МКГ 80 s I zn 25 МКГ (3rd

VO r z i i v ! . nn 0

I

20 o'clock 2 4 4

Hours after the injection at 9 p.m.

Ge

In and to "ex

IS

Zh zhk o and o 2 4 6 u

Hours after injection