Alloferon-immunomodulating peptides

 

(57) Abstract:

Describes new compounds of General formula I: X1-His-Gly-X2-His-Gly-Val-X3or their pharmaceutically acceptable salts, or esters, or amides, where X1missing or contains at least 1 amino acid, X2contains at least 1 amino acid, or represents a peptide bond; X3missing or contains at least 1 amino acid, whereby these amino acid selected from groups: aliphatic, aromatic or heterocyclic. Compounds stimulate antiviral, antimicrobial and antitumor activity of the human immune system. 2 s and 5 C.p. F.-crystals, 2 ill., 17 table.

The present invention relates to proteins or biologically active peptides, specifically stimulating antiviral, antimicrobial and antitumor activity of the human immune system, also to medicines on the basis of them.

Art

Known connections peptide, polypeptide and protein nature, used in medicine as an antiviral, antimicrobial and anticancer agents.

A known mixture of polypeptides isolated from shellfish and possessing anti-virus, Academy of Sciences of the s-peptide, isolated from the haemolymph of insects (Europatent WO 90/14098). Known peptides collection cryptodev with a broad spectrum antibiotic activity, was isolated from the cells of the intestinal epithelium (U.S. patent N 5422424). Known antimicrobial peptides structurally similar to contains arginine fragments of transmembrane proteins of lentiviruses (U.S. patent N 5714577). Known cysteine-containing antimicrobial peptides from the family protegrins (U.S. patent N 5804558). All of the above peptides and polypeptides are compounds on the basis of which received drugs direct antimicrobial action (peptide antibiotics). As analogues of the present invention for use, these compounds have a fundamentally different mechanism of action, which is characterized by several disadvantages. In particular, to achieve a direct antibiotic effect usually requires the creation of high concentrations of the antibiotic and its long-term presence in the blood and other tissues. This creates significant drug load on the patient's body. In addition, a well-known fact is the rapid development of pathogenic microorganisms resistance to antibiotics.

Known anticancer peptides is). Bleomycine have a direct cytotoxic effect on tumor cells, but the possibility of their application in the clinic is limited pronounced side effects, primarily by the lungs and kidneys.

Known immunomodulatory peptides that act on the electoral decline of the presentation of antigen receptor major histocompatibility complex (MHC II) (U.S. Pat N 5827516). However, the effect of these peptides does not include modulating the mechanisms of natural immunity, in particular, systems of interferons and natural killer cells.

It is known the use of natural (derived from donor blood) and recombinant (derived through genetic engineering) of interferons as specific activators of natural immunity (Borden EC. N Engl J Med 1992; 326: 1491-1492). Under the interferon is a group of glycoproteins produced in response to viral infection or other incentives by leukocytes (interferon-alpha), fibroblast (IFN-beta), T-lymphocytes and natural killer cells (interferon-gamma). Molecular weight interferon ranges from 17000-45000 Yes, the alpha - and beta-interferons and from 20000 to 80000 Yes gamma-interferon. It is believed that alpha - and BA. I. Korotyaev, S. A. Babichev Medical Microbiology, immunology and Virology, St. Petersburg. publishing house "Special literature", 1998, S. 161-163).

Alpha-interferon is recognized as one of the most effective means of immunotherapy in the treatment of various viral diseases and cancer. In particular, clinical trials have confirmed its efficacy in hepatitis C (Bekkering et al. , J. Hepathology, 1998, 28, 6, p. 960-964), herpes (Cardamakis et al., Gynecol. Obstet. Invest., 1998, 46, I, p.54-57), multiple sclerosis (Durelli et al., Mult. Sclerosis, 1995, 1, Suppl.l, p. S32-S37), multiple myeloma (Zee et al., J. Clin. Oncol., 1998, 16, 8, p. 2834-2839), Hodgkin's disease (Aviles et al. Leuk. Lymphoma, 1998, 30, 5-6, p. 651-656), myeloid leukemia (Gilbert, Cancer, 1998, 83, 6, R. 1205-13). Interferons are also of interest for immunotherapy of deep mycosis (Kullberg, Eur. J. Clin. Environ. Infect. Dis., 1997, 16, p. 51-55). Interferons are the closest analogues of the present invention on the achieved results in the field of application.

However, the high cost of recombinant alpha-interferon makes it unaffordable for wide clinical use. Another limitation are side effects associated with possible pyrogenalum, immunogenicity and other undesirable properties of recombinant interferon. Primanti. Chemical synthesis of interferon, due to the complexity of their structure, is currently unrealistic.

The essence of the invention.

The present invention is the search for new compounds with immunomodulatory activity, which can be used to create medicines and development of methods of treatment and prevention of viral and microbial infections and tumors. To this end developed a new family of biologically active peptides that differ from interferons and other immunomodulatory analogs their characteristic structure and mechanism of action, and more affordable.

The proposed immunomodulators are linear peptides-alloferon, the structure of which is described by the following structural formula:

X1-His-Gly-X2-His-Gly-Val-X3< / BR>
or their pharmaceutically acceptable salts, or esters, or amides,

where X1missing or contains at least 1 amino acid,

X2contains at least 1 amino acid, or represents a peptide bond,

X3missing or contains at least 1 amino acid, whereby these amino acid selected from groups: aliphatic, aromatic or GE is Intesa and characterized by HPLC and may be allocated in the form of their various derivatives, such as esters, pharmaceutically acceptable salts or amides (see tab. 1).

Alloferon structure 1 or 2 are synthetic peptides containing 11-13 amino acid residue, characterized in targeted search for regulators of the activity of cytotoxic lymphocytes of humans and animals. Alloferon structure 3 or 4 are analogous to alloferon 1 and 2 with short amino acid chain, which were synthesized in order to clarify the possible modifications of the structure alloferon preserving its characteristic biological activity. In the comparative studies of the biological activity of alloferon 1-4, it was found that they all have similar stimulating effect on the cytotoxic activity of lymphocytes of humans and animals. This helped to determine the functionally important part of the molecule alloferon.

In addition, the inventors have conducted a search of structural analogues of alloferon using databases of known proteins and peptides. Analysis of the SWISSPROT database, implemented using a computer system GenomeNet did not reveal structural similarities of alloferon and known immunomodulatory peptides. At the same time was a). Hemagglutinin is a key structure of the virus responsible for the fusion of viral envelope and the cell membrane of the host; features of the structure of hemagglutinin have a decisive influence on the formation of the antiviral immune response (A. I. Korotyaev, S. A. Babichev. Medical Microbiology, immunology and Virology, St. Petersburg, publishing house "Special literature", 1998, S. 265). Comparison with the structure of alloferon suggests that the area 377-387 molecules of hemagglutinin plays the role of a immunomodulator used by the organism-host of the virus to activate systems natural antiviral immunity.

In the creation process of the present invention, the basic structure was used alloferon 1. Alloferon 1 is a linear peptide with a molecular mass 1265 Yeah, consisting of 13 amino acids (Table. 1). Comparison with analogues shown in Table. 1 reveals functionally important elements of the molecule alloferon and to predict possible modification of the basic structure have no significant impact on its biological activity. Thus, comparing with the structure of allopurinol 2-4 shows that it is necessary for the manifestation of the characteristic alloferon biological whom she) is a plot of Ser-Gly-His-Gly-Gln-His-Gly-Val the corresponding structure alloferon-4. Positions 1-3 in the molecule alloferon-1 may not be present or to be represented by a sequence of at least 1 amino acids. Comparison with sequences homologous to a fragment of the hemagglutinin of the virus In lets also assume that the positions 4 and 5, are presented in the molecule alloferon amino acids serine (Ser) and glycine (Gly), can also be replaced by other amino acids, selected from the group of aliphatic, aromatic or heterocyclic. For example, serine can be replaced by a threonine (Thr), and glycine - serine. Thus, the totality of the available data allows us to consider, for example, the first five amino acids in the molecule alloferon variable plot, i.e., they may be missing or contain at least 1 amino acids. With this in mind, it is proposed to designate a specified area in the structural formula alloferon as X1. Similarly positions 12-13 molecule alloferon-1 may not be present or to be represented by a sequence of at least 1 amino acids. Therefore, section 12-13 of the structural formula alloferon are encouraged to identify as X3. In addition, a comparison of patterns of alloferon and hemagglutinin call the particular glutamine may be replaced by alanine. Accordingly, the position of the 8 structural formula alloferon are encouraged to identify as X2that may just be a peptide bond connecting Gly and His, or at least 1 amino acid.

By analogy with the structure of the hemagglutinin of influenza virus is also possible the accession of the amino acid sequence of alloferon part of a larger molecule, for example, of carrier protein without significant changes in the nature of its biological activity.

Complex immunological, pharmacological and Toxicological studies, the results of which are demonstrated in the following examples, identified a number of potentially useful properties alloferon. The obtained experimental data show that alloferon is cytogenetically peptide, mechanism of action which involves the stimulation of recognition and lysis of defective cells by cytotoxic lymphocytes, and induction of the synthesis of endogenous interferon. Alloferon is intended for the correction of system deficiency of interferon and natural killer cells, treatment related deficit viral, fungal diseases and cancer. The connection is not toxic, does not have teratogenic, mocnymi and intracellular proteases.

Experimentally determined properties alloferon for example alloferon 1 are summarized in Table. 2. Range of identified pharmacological activity of alloferon generally corresponds to the well-known properties of alpha-interferon in the part concerning the impact on the system's natural killer cells and resistance to viral infection. On this basis, the drug can be attributed to the group of interferometic. It is possible that the drug does not possess the full range of effects of interferon.

The mechanism of action of alloferon on target cells is implemented in extremely low concentrations - less than 0.001 nanogramme (10-9grams) per ml In this respect, alloferon not inferior to the interferons and interleukins. In addition, alloferon stimulates the formation of interferon in the body. This can be attributed to alloferon to inducers of interferon. The combination interferoninducible and interferon-inducing actions, combined with the almost complete toxicity of the drug to distinguish alloferon from currently used cytokines and interferon inductors.

Data on the biological activity of alloferon allow for its use as a medicine is - virus influenza A and B,

- acute and chronic viral hepatitis A, B and C,

- HIV infection and secondary viral and fungal infections in AIDS,

- Kaposi's sarcoma,

- acute and chronic leukemia, multiple myeloma and other cancers, which shows the treatment of interferon,

- candidiasis, cryptococcosis, and other deep mycoses, which shows treatment with interferon.

Despite the substantial similarity of the achieved result, alloferon on a number of key characteristics fundamentally different from the interferon. So, interferons are glycoproteins with a molecular weight of 17000 - 80000 Yes. Glycosylation of amino acid chains is a necessary condition for the functional activity of interferons and their tone and withspecific. Alloferon is deglycosylation the Oligopeptide with a molecular mass 1265 Yes, 13-60 times less than the mass of interferons. Computer analysis of the SWISSPROT database, performed using system GenomeNet did not reveal structural similarities of alloferon and any fragment of the amino acid sequence of interferon or other known immunomodulators. Functional relationship between allopurinol and interferon also it is thus the development of a cascade of defense responses, mediated by these cytokines. Introduction of exogenous interferon leads rather to the suppression of the formation of the patient's own interferon on the principle of negative feedback. For interferons also characterized by a number of deficiencies identified in the section "prior art".

In accordance with the present invention presents a pharmaceutical composition comprising a peptide or mixture of peptides according to the present invention taken in effective amounts in combination with a pharmaceutically acceptable carrier, such as albumin. Particularly suitable peptides for use in pharmaceutical compositions are alloferon 1-4. The experiments showed that the pharmaceutical composition has immunomodulatory activity. Suitable methods of application can serve as an intramuscular injection, subcutaneous, intravenous, and oral or intranasal use. The daily dose may be generally from 0.01 to 1000 μg of peptide per 1 kg of patient's weight.

Information confirming the possibility of carrying out the invention. Synthesis of alloferon.

A peptide consisting of 13 amino acids corresponding to the structure of alloferon the ated TFA cleavage capability, Peptide Research, 1993, vol.6, p. 219) using Fmoc-(N-[9-fluorenyl] methoxycarbonyl)-substituted amino acids. Purification of the synthesized peptide consisted of two stages. At the first stage of purification was carried out on columns (Sep-Pak Vac with sorbent C18 (Waters) by elution of the column with 40% acetonitrile, acidified with 0.05% triperoxonane acid. After lyophilization, the peptide was purified to a homogeneous state by HPLC instrument Beckman Gold System with column Aquapore ODS Prep 10 C18 (h mm, Brownlee) using a linear gradient of 0.05% triperoxonane acid and acetonitrile, acidified with 0.05% triperoxonane acid (0-20% acetonitrile over 40 min at a flow rate of 2.5 ml/min and the wavelength detector 225 nm). The purity of the peptide was confirmed massspectrometric method MALDI-TOF of massspectrometry. The correctness of the synthesis was confirmed by the method of mikroekonomia on Adminu on automatic sequencing machine Applied Biosystems. We got about 100 peptides. The peptides can be isolated in the form of acid, its ester, pharmaceutically acceptable salts, as well as amide.

Example 1. The impact of alloferon 1 on cytotoxic activity of lymphocytes in the spleen of the mouse.

The method for testing cytotoxicity of lymphocytes in the spleen of the mouse with the s line SN. Splenocytes were obtained by mechanical grinding of the spleen in the medium RPMI 1640 and transmission using a syringe fragments of the body through needles of decreasing diameter. Intact cellular aggregates were removed by filtration, then was literally erythrocytes using short hypotonic shock. Splenocytes were besieged by centrifugation 10 min at 200 G and resuspendable in medium RPMI 1640 supplemented with glutamine, gentamicin and RNA asoi. The titer of splenocytes brought to a value of 2106cells/ml and immediately used for the production test.

Targets in the formulation of the test for cytotoxicity served cells eritromicina human leukaemia of K 562 line cells. Culture cells were kept in plastic culture plates in RPMI medium 1640 supplemented with glutamine, gentamicin, and fetal calf serum. To determine the cytotoxicity of splenocytes used the standard method of assessing the impact of drugs on the functional activity of natural killer cells, based on the analysis of the output labeled H3-uridine RNA from lysed cells K562 (Khaitov R.M and other statements Pharm. Committee, 1999, 1, S. 31-36). To do this in an environment with K562 cells were injected H3-uridine at a concentration of 2.5 µci/ml and inkberrow what trifoliorum, resuspendable in the incubation medium, counted the number of cells in hemacytometer and diluted with the same medium up to 105cells/ml For setting the test used a 96-well round-bottom immunological tablets. In the hole of the tablet was made in 0.1 ml of suspension labeled H3-uridine K562 cells (10,000 cells per well) and 0.1 ml of the suspensions of splenocytes in the ratio of 5:1 or 20:1 to the number of target cells. In the control variant instead of suspensions of splenocytes were injected 0.1 ml of the incubation medium. Then in the wells made the drug in 0.02 ml of medium (control - 0.02 ml of medium). The plates were incubated for 18 hours in CO2- thermostat at 37oC and 6% CO2. Then the contents of the wells were transferred to paper filters Whatman, three times washed filters 5 ml of 5% THU, dried 96% ethanol and placed in vials with scintillation fluid. The radioactivity of the filters was determined on a Beckman counter.

Index of cytotoxicity (IC) splenocytes were calculated by the formula:

CI=(1-Pabout/Pwith)100,

where Paboutradioactivity in the wells containing target cells and splenocytes,

Pwithradioactivity of the wells containing target cells without splenocytes.

The results of the impact analysis allothermic definitions of cytotoxicity.

Introduction to the incubation environment alloferon at a concentration of 0.05-50 ng/ml caused a highly reliable increase in lytic activity of natural killer cells against tumor target cells. The lower threshold of effective concentrations of the drug were less than 0.05, nanogram/ml, and the maximum stimulatory effect was achieved at 0.5 ng/ml 1000-fold excess of the optimum concentration was eliminated the effect of the stimulation, but in this case, the splenocytes were kept normal functional activity (level of control), indicating that the absence of drug expressed immunosuppressive properties.

Conclusion

Thus, alloferon has a marked stimulatory effect on the activity of natural killer cells mouse in extremely low concentrations, which allows to characterize it as a specific cytogenetically inducer of cell-mediated cytotoxicity.

Example 2. The impact of alloferon 1 on cytotoxic activity of lymphocytes in human peripheral blood

Lymphocytes were isolated from fresh blood. For this, 50 ml of heparinised whole blood was added a solution of 2.5% gelatin in a ratio of 1: 10. The mixture otstoya test tubes of 10 ml in 2:1 and centrifuged for 30 min at 700 G for sedimentation of erythrocytes. Content formed during centrifugation interphase rings carried in a plastic conical tubes, diluted with 10 ml of isotonic phosphate buffer without Ca ions and Mg and precipitated by centrifugation at 200 G for 10 min. the Precipitate resuspendable in a fresh portion of the same buffer, the cells were again besieged by centrifugation and resuspendable in medium RPMI 1640 supplemented with glutamine, gentamicin and RNA asoi. The number of lymphocytes was calculated using hemocytometer, was diluted in the same medium to a cell concentration of 2106/ml and immediately used for analysis of cytotoxicity (method of analysis, see 1.1). Each option consisted of 6 replicates.

Data on the influence of alloferon 1 on the lysis of tumor cells by cytotoxic lymphocytes in human peripheral blood are shown in table. 4. Introduction to the incubation environment alloferon caused a dramatic increase in cytotoxicity of lymphocytes against tumor cells. The maximum effect was caused by a concentration of 0.05 nanogram/ml (increased activity of natural killer cells approximately 3-fold compared with control). Interferon-alpha 2b, used as a positive control, had a similar stimulating deml interferon), was significantly higher.

A similar effect alloferon had on the activity of natural killer cells against tumor cell line A431 (test line for determining the activity of NC subpopulation of natural killer cells).

Conclusion

The data obtained indicate pronounced potentiating effect alloferon on the lysis of tumor cells by lymphocytes in human peripheral blood. Under the conditions of this experiment alloferon was not inferior or even superior to the efficiency of the natural inducer killer activity of interferon alpha.

Example 3. Comparative analysis of the impact of alloferon 1 and alpha-2b on the cytotoxic activity of lymphocytes of healthy donors.

The objective of this study was to analyze the variability of response to alloferon and interferon-alpha in a population of healthy donors and receive on the basis of data on the comparative efficacy of the two drugs. Biocidal activity of the freshly harvested blood samples have been investigated in accordance with the Protocol set forth in example 2. The aim of the study was to assess the variability of response to alloferon in a population of clinically healthy people and comparative characteristic is whether the two types of target cells: leukemia K562 cells, sensitive to lysis by NK (natural killer) lymphocytes, and cells of the solid tumor lines A431, sensitive to lysis NC (natural cytotoxic) lymphocytes. The ratio of effector : target was in the experience of 20:1. In each case summarizes the results of 6 definitions of cytotoxicity. The criterion of effectiveness was statistically significant (P 0.05) increase in the cytotoxicity of lymphocytes. The obtained experimental data are summarized in Table. 5. From the data table is a considerable variability of the background activity of lymphocytes of donors, particularly the ability to recognize and lyse tumor cells lines C and A431. Found substantial differences in the response of lymphocytes from different donors to the medications. However, the majority of donors, there is a clear correlation response alloferon and alpha-interferon: donors respond positively to interferon, in most cases, respond positively and alloferon, and Vice versa. Summary indicators of the effectiveness of two drugs, calculated on the basis of the data Table. 5, are given in Table. 6. Most donors responded to interferon (10 donors from 17) or alloferon (10 donors) increase the cytotoxicity of lymphocytes in relation to creation responded to both drugs and one donor was selectively sensitive only to interferon or only alloferon.

Conclusion

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 terms of this test were identical. Found the correlation of the responses of donors to interferon and alloferon indicates the interchangeability of the two drugs.

Example 4. Comparative analysis of the impact of alloferon 1 and alpha-2b on the cytotoxic activity of lymphocytes of cancer patients.

The blood samples of patients with various cancer have been investigated in accordance with the Protocol outlined in examples 2 and 3. The aim of the study was the analysis of the characteristics reaction to alloferon in a randomized sample of cancer patients and the comparative performance characteristics of alloferon and alpha-interferon. All samples were studied in the blood of 18 patients using two types of target cells: leukemia K562 cells (a specific target for determining the activity of NK lymphocytes and cells of the solid tumor lines A431 (the target for determining the activity of NC cells). The ratio of effector:target was in about the key significant (P 0.05) increase in the cytotoxicity of lymphocytes.

Data source activity and response to alloferon and interferon cytotoxic lymphocytes each examined patient are given in Table. 7.

The results of the study are summarized in Table. 8 and 9. The Data Table. 8 show that natural killer about half of cancer patients will respond to the medications. The frequency of positive answers as a whole is not materially different from indicators of healthy donors. The exception is the reaction of lymphocytes treated with allopurinol, cells C. In this case, the frequency of positive responses was significantly lower than during treatment with interferon. Although the likelihood of differences between responses on alloferon and interferon in cancer patients does not reach a statistically significant value (P= 0.071), this fact may indicate lower efficiency alloferon in the case of impact on a population of NK lymphocytes of cancer patients. At the same time when the lysis of cell line A431 lymphocyte reactivity in this group handling allopurinol and interferon is the same.

The special nature of the immune status of cancer patients compared with healthy donors confirmed the other is almost the same, the cytotoxicity against target K in cancer patients is clearly reduced. This defect lymphocytes of cancer patients likely to affect the mechanism of the stimulating action of alloferon and, to a lesser extent, interferon. Another important aspect of the action of drugs on the lymphocytes of cancer patients demonstrates the comparison groups, reacting and not reacting to the stimulation by cytokines. As it turned out, the positive response detect mainly patients with low or zero level of background activity of lymphocytes. At the same time, the lack of response is associated with a high background activity, much higher than its average level in the population. Consequently, the use of alloferon (as interferon) is most appropriate in patients with a deficit level of natural killer cells. The selection of appropriate patients by means of laboratory tests of cytotoxicity of lymphocytes will, hopefully, achieve a significantly higher therapeutic effect of the drug.

Conclusion

Alloferon has a stimulating effect on the lysis of tumor cells by cytotoxic lymphocytes of cancer patients. However, there are), the Aulnay more effective stimulation subpopulations NC lymphocytes. The positive effect of alloferon noted in patients with acute and chronic leukemia (5 out of 9 cases). The frequency of positive answers below lymphomas (2 of 6 cases). A positive effect was also observed in a patient with lung cancer. In most of the cases examined, the stimulating effect of alloferon as alpha-interferon correlates with low or zero initial cytotoxicity of lymphocytes. This fact can be used to predict the effectiveness of treatment allopurinol.

Example 5. The effect of structural analogues of alloferon 1 on cytotoxic activity of human lymphocytes.

The activity of the analogs alloferon 1, allopurinol 3 and 4 investigated in accordance with the Protocol outlined in examples 2 and 3. For this fraction of mononuclear cells isolated from blood of healthy donors, and the tumor cell line A431 cells were then incubated in the presence of the studied drugs: interferon-alpha (positive control), alloferon 1, alloferon 3 or alloferon 4. The initial concentration of all drugs was 5 ng/ml On the activity of the preparations was assessed by the change in the index of cytotoxicity of lymphocytes compared to control.

As in the experiments described in the previous examples, Ariccia on alloferon 3 and 4 was similar to the reaction to alloferon 1 and alpha-interferon (PL. 10). In quantitative terms, the stimulating effect of alloferon 3 and 4 are not inferior or even slightly exceeded the effect of interferon and alloferon.

Conclusion.

A comparative analysis of the stimulating effect of structural analogues of alloferon 1 on cytotoxic activity of human lymphocytes allows you to set the possible modifications of the amino acid sequence of alloferon without affecting its biological activity. Such modifications include the removal or replacement of amino acids 1-4 and/or 11-13 in the composition of the molecules alloferon 1.

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

Antivirus action alloferon studied on the model of lethal viral infection of mice with influenza virus A. the Suspension is pathogenic for mice of the strain of virus was administered to experimental animals intranasally at a dose corresponding to 10 LD50. Alloferon 1 in 0.5 ml of 0.9% solution of sodium chloride was injected intraperitoneally one day before inoculation of the virus, then after 1, 2, 4, 6 and 8 days after inoculation. The drug was tested at doses of 25 and 2.5 micrograms. Control mice were administered an equal volume of solvent. Efficacy criterion was the survival of animals after 10 sushi, males weight 20,0-22,0,

In the experiment were used 60 white mice.

The introduction of infected animals alloferon caused a dose-dependent protective effect (Table. 11). The drug at a dose of 25 micrograms caused a significant increase in the number of survivors during the observation period the animals. The effect of this dose vysokopostavlen. The lower dose did not cause a significant increase in survival.

Conclusion

Alloferon at a dose of 25 micrograms has a strong antiviral effect on mice infected with influenza virus A.

Example 7. The impact of alloferon 1 on resistance of mice to influenza B.

The study of the impact of alloferon on the resistance of mice to influenza was conducted in accordance with the Protocol set forth in example 6. Mice infected with strain LEE 1/40 of influenza virus In samples taken in infectious doses, corresponding to 3 and 30 LD50. As a positive control used specific antiviral drug virazole (ribavirin). Alloferon was administered intraperitoneally at a dose of 25 µg per 1 day before infection with a virus, then after 1, 2, 4, 6 and 8 days.

In the control intranasal introduction of the virus caused severe pneumonia with visitee effect at a low dose of virus (3 LD50), with a high infectious load (30 LD50) mirasol under the conditions of this experiment proved ineffective. At the same time, alloferon had a marked protective action at both high and low infectious load.

Conclusion

Alloferon dose of 25 mcg has a strong antiviral effect on mice infected with influenza B. Protective effect of alloferon under the conditions of this experiment exceeded the protective effect of virazole known anti-virus tools.

Example 8. The impact of alloferon 1 on the synthesis of interferon in mice.

The quantitative content of interferon in the serum of mice was determined by titration of the analyzed samples above the monolayer cell line L-929. Cells were incubated in the presence of the samples for 24 hours at 37oC in an atmosphere of 5% CO2. Then the samples were replaced with a solution of the test virus in a supportive nutrient medium (DMEM.2% serum of cattle). As a test virus used vesicular stomatitis virus (strain Indiana) activity 100 TCD50. Incubation of the cells with the test virus was carried out for 2 days to achieve 90-95% destruction of the cell monolayer in samples negative control (only test view serum which was observed to protect 50% of the cells from the cytotoxic effect of the test virus. The degree of destruction of the monolayer of cells was evaluated after fixation/staining with 0.1% crystal violet in 30% ethanol. In each case investigated aliquots of serum 4 animals. As a positive control used is a known inducer of interferon cold.

Interferon-inducing activity of alloferon was investigated in two series of experiments. In the first series of experiments (Table. 13) alloferon was applied at doses of 2.5 and 25 mg. Comparator drug (cold) was used at a dose of 500 μg per mouse.

The dynamics of the concentration of interferon in response to the introduction of various doses of alloferon illustrated in Fig. 1.

In the next series of experiments the effect of alloferon and cycloferon was traced within 48 hours after drug injection (PL. 14). When a longer period of observation is obvious two-peak character of the interferon response to a single injection (Fig. 2). The data of the two series of experiments to determine the interferon-inducing activity of alloferon summarized in Table. 15.

Conclusion

The results of the comparative analysis of data on the impact of alovera is t expressed interferon-inducing effect. Unlike a cold, the maximum effect which manifests itself in 2-4 hours after injection, alloferon causes the maximum increase in the titer of interferon 24 hours after injection. This parameter alloferon can be classified as inducers of interferon prolonged action. An important advantage of alloferon also is effective in significantly lower doses in 20-200 times lower than the effective dose of cycloferon.

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

The study of the acute toxicity of alloferon 1 was carried out according to the method of Litchfield and Wilcoxon signed (Belenky, M. L., 1963). In the experiment were used 15 white mice - 3 groups of 5 animals each. Study of acute toxicity of the preparation was carried out in a single subcutaneous injection. The observation period was 14 days.

the 1st group of animals were administered the test drug at 10x the dose of 0.25 mg/goal; the 2nd group - 70 times the dose of 1.75 mg/goal. The remaining 5 mice (group 3) were used as control. The drug was diluted with sterile 0,9% saline and was administered to the animals subcutaneously in the amount of 0.5 ml of the Control group animals were injected with 0.9% saline. Data predtechenskii reaction, physiological excretions, eating and drinking and in the experimental and control groups did not differ, the animals weight is not lost (PL. 14). Dynamics of body weight in animals of the experimental and control groups are almost identical, the differences unreliable (P > 0,05).

After euthanasia at 14 days was performed macroscopic examination of internal organs (liver, spleen, brain). According to the results of observations of apparent changes of internal organs is not detected. Similar results were obtained by intraperitoneal injection of alloferon.

Thus, studies have shown that alloferon 1 has no acute toxicity after a single dose to mice, including the maximum investigated the dose of 87.5 mg/kg, which is 700 times greater than therapeutic dose of 0.125 mg/kg, sufficient for the induction of interferon synthesis in mice. On this basis, the drug alloferon can be attributed to substances almost completely devoid of acute toxicity. Alloferon also not revealed chronic toxicity when administered to mice daily for 5 days dose of 12.5 mg/kg alloferon 100 times greater than therapeutic. Animals are not marked changes in behavioral what ucheniem indicators of immune status, associated with specific immunomodulatory effects of the drug.

Studies in rabbits have alloferon not revealed signs of acute and chronic toxicity, immunotoxicity, sensitizing local resorptive action, progenote. In a study in Guinea pigs demonstrated the absence of alloferon allergenic action. In studies on rats alloferon did not cause teratogenic and embryotoxic effects. Studies on the fruit fly Drosophila melanogaster and Saccharomyces cerevizea showed no mutagenic effect of alloferon.

Conclusion

According to currently available data alloferon can be attributed to the drugs, almost completely devoid of toxicity and other negative side effects.

1. Alloferon - immunomodulatory peptides of General formula I

X1- His - Gly - X2- His - Gly - Val - X3< / BR>
or their pharmaceutically acceptable salts, or esters, or amides,

where X1missing or contains at least 1 amino acid;

X2contains at least 1 amino acid, or represents a peptide bond,

X3missing or contains at least 1 amino acid, and these am the th of peptides under item 1, preserving together with immunomodulating activity.

3. Peptides on PP.1 and 2, having the properties of inductors of interferon synthesis.

4. Peptides on PP.1 and 2, having the properties of stimulants cytotoxic activity of lymphocytes of humans and animals.

5. Peptides on PP.1 and 2 having antiviral activity.

6. Peptides on PP.1 and 2, with antitumor activity.

7. Pharmaceutical composition having immunomodulatory activity, containing a peptide or mixture of peptides on PP.1 and 2 or pharmaceutically acceptable salts or their esters, in combination with a pharmaceutically acceptable carrier.

 

Same patents:

The invention relates to the field of cell biology, in particular to new peptides with the ability to interact with intracellular transduction signal, thereby inhibiting the process of signal transmission, leading to proliferation and cell motility

The invention relates to biotechnology

The invention relates to new peptides with organizaitnal activity with high biological activity of the same type as the natural compound HRV, but with a shorter amino acid chain

The invention relates to peptides of formula (I): X - A1- A2- Thr - Ala - Val - Gly - His - Leu - psi - A9- Q, where X represents a hydrogen, a simple relationship linking the alpha-amino group of A1with gamma-carboxyl part 3-propionyloxy part of A2if A2is Glu[-], or a group of the formula R1CO-, where R1selected from the group comprising: hydrogen, C1- C10- alkyl, or phenyl C1- C10- alkyl, A1is a D - or L-amino acid residue selected from the group consisting of: Phe, p - Hl - Phe, pGlu, Nal, Pal, Tpi, unsubstituted Trp or Trp substituted in the benzene ring by one or more substituents from the group comprising C1- C3- alkyl, or A1represents a peptide bond linking the acyl part of R1CO with alpha aminocyclo A2if A2represents Gln, Glu/-/ Glu (Y) or His, where /-/ is a simple relationship linking the gamma-carboxyl group of A2with the alpha-amino group of A1if A2is Glu, where X represents a simple bond, Y represents - or SIG5where R5is hydrogen, C1- C3- alkyl or phenyl; Leu - psi - is a reduced form Lой adjacent A9- balance is pseudopeptides communication; A9is a TAS, Ista, or DМТас; and Q represents NH2or CQ1where Q1is hydrogen, and pharmaceutically acceptable acids or salts, and pharmaceutical compositions, which has antagonistic activity against bombezin and to a method of treating cancer in mammals on the basis of the peptides of formula (I)

The invention relates to medicine, molecular biology and Virology and represents the biologically active compound is a synthetic oligopeptides corresponding to amino acid residues (and

The invention relates to new biologically active compounds, specifically to a peptide of General formula: R-GLu-Leu-Lys-Pro-Leu-X-Glu-Val-Leu-Asn-Leu-R', where R is CH3CO-(Ac), Ac-Glu, Ac-Leu-Glu-Glu-, HCO-(Form), Form-Glu-, Form-Glu-Glu, Form-Leu-Glu-Glu-, CH(CH)nCO-CH(CH)nCO-Glu-, CH(CH)nCO-Glu-Glu-, CH(CH)nCO-Leu-Glu-Glu-, n - 8 - 18, X - L-Glu, D-Glu, R' - OMe or NH2who are the inductors and/or potentiators growth stimulating activity of macrophages in vitro, have regenerative-reparative action in vivo, and can find application in medicine for treatment of several diseases involving generalized activation of macrophages (hepatitis, toxic liver damage, cirrhosis of the liver, skin wounds, burns, polytrauma, diabetes and others)

The invention relates to novel antagonists of LH-RF, and above all, peptidomimetics and peptide with a modified side chain, of the formula V, where D-Xxx, R1- R8described in the description, and their salts with pharmaceutically acceptable acids, as well as it describes their therapeutic use as analogue releasing factor, luteinizing hormone (LH-RF) with high antagonistic activity and not detecting unwanted side effects, especially no incentive edema

New peptides // 2162855

The invention relates to biotechnology and can be used for Introduzione nucleic acids into cells

The invention relates to the field of cell biology, in particular to new peptides with the ability to interact with intracellular transduction signal, thereby inhibiting the process of signal transmission, leading to proliferation and cell motility

The invention relates to new peptide derivatives, which are suitable for use as anti-inflammatory drugs

The invention relates to the derivatives of antibiotics lipopeptides A complex 1437, method of their production and their use as medicines
Up!