Alloferons—immunomodulatory peptides

Abstract

The invention belongs to the field of biologically active peptides specifically stimulating antiviral, antimicrobial and antitumor activity of the human and animal immune system.

Description

The present invention is concerned with immunomodulatory materials of natural origin. In particular, the present invention is concerned with peptides of invertebrate origin and pharmaceutical preparations comprising such peptides which are useful in the treatment of immune deficient conditions, infections and oncological diseases.

In the state of the art various pharmaceutical preparations of natural origin containing materials of animal, including insect, and plant tissues able to stimulate the immune system's efficacy are known.

A process for obtaining cellular protein having anti-HIV activity from CD4-positive T cells or myeloid cells is disclosed in U.S. Pat. No. 5,480,782.

A topic formulation comprising a Ginkgo biloba extract exhibiting antibacterial and antiviral properties is disclosed in DE 43 34 600 A1.

WO 96/04005 discloses a pharmaceutical composition for stimulation of the immune response of an organism comprising as the active ingredient major histocompatibility complex antigens extracted from animal tissues, serum or cells. The tissues, cells or sera are chosen from goat, veal or pig liver and bovine red blood cells.

A pharmaceutical composition containing an extract of the plant Nigella sativa is disclosed in U.S. Pat. No. 5,482,711 for treating cancer, preventing the side effects of anticancer chemotherapy, and for increasing the immune functions in humans.

WO 81/03124 discloses a polypeptide fraction isolated from the mussel Mytilus edulis and used as antibiotic composition effective against various viruses, bacteria and protozoa.

Antibacterial peptides from honey bees and a process for their isolation, production and applications have been disclosed in EP 0 299 828 A1.

Antibacterial peptides isolated from the Coleopteran insects, Tenebdo molitor and Leptinotarsa decemlineata are disclosed in WO 90/14098.

Antibacterial protein isolated from the Lepidopteran insect, Hyalophora gloveri is disclosed in EP 0 856 519 A2.

Antimicrobial peptides structurally similar with arginine-containing fragments of lentivirus transmembrane proteins are disclosed in U.S. Pat. No. 5,714,577.

Antiviral and antimicrobial peptides isolated from porcine leucocytes are disclosed in U.S. Pat. No. 5,804,558.

Immunomodulatory peptides specifically binding major histocompatibility complex class II antigens and decreasing in that way a possibility of autoimmune disease are disclosed in U.S. Pat. No. 5,827,516.

EP 0 320 528 A1 discloses the use of hemocyanins and arylphorins isolated from various molluscs and arthropods including the insect Calliphora erythrocephala as stimulants the production of specific antibodies and the antitumor activity of antibody-dependent T-lymphocytes.

The preparations mentioned above and analogous natural pharmaceutical preparations enhance the recent arsenal of medicines suitable for treatment of immune deficient conditions, infections and oncological diseases. However, the pharmaceuticals which are available up to now do not cover existing demands in immunomodulatory medicines.

Therefore, it is an object of the present invention to provide a pharmaceutical preparation having immunomodulatory activity and in particular being useful for the treatment of immune deficient conditions, infections and oncological diseases.

It has now surprisingly been found that specific peptides exhibit the desired immunomodulatory activity.

Thus, the present invention relates to a peptide consisting of up to 30 amino acid residues and having the following general structural formula (1) (SEQ ID NO: 31):

X 1 -His-Gly-X 2 -His-Gly-Val-X 3   (1)

wherein

X 1 is absent or represents at least one amino acid residue,

X 2 is a peptide bound or represents at least one amino acid residue, and

X 3 is absent or represents at least one amino acid residue,

or a pharmaceutically acceptable salt or ether thereof,

the peptide exhibiting immunomodulatory activity.

The present invention provides a new class of immunomodulatory peptides, designated “alloferons” herein, representative members of which were isolated from the blood of bacteria challenged larvae of an insect, blow fly Calliphora vicina R.-D. (Diptera, Calliphoridae).

The alloferons of the invention have been found to stimulate cytotoxic anticancer activity of animal (mouse) and human natural killer cells. Experimental data on the alloferons' immunomodulatory activity show that they are able to stimulate the cytotoxic anticancer activity of human and mouse lymphocytes at extreme low concentrations. The minimum effective concentration was determined to be about 0.0005 nanogram/ml. The optimum concentration was found to be 0.05-0.5 nanogram/ml. Assuming the important role of natural cytotoxicity as effector mechanism of innate immunity (Trinchieri G., Advances in Immunology, 1989, vol. 47, 187-375; Brittenden J., Heys S. D., Ross J. and Eremin O., 1996, vol. 77, 1126-1243), alloferons may be useful as antiviral, antimicrobial and anticancer medicines of immunomodulatory mode of action.

Moreover, with regard to the stimulation of the anticancer activity of the cytotoxic lymphocytes, alloferons were found to induce intensive and prolonged interferon synthesis in experimental animals. Interferons are a group of key antiviral (alpha- and beta-interferons) and immunomodulatory (gamma-interferon) cytokins produced in the organism in response to viral infection and some other external stimuli. Elevation of interferons concentration in the blood helps to cure or mitigate a broad range of viral, oncological and autoimmune disorders. Injections of recombinant or natural interferons are successfully used in the immunotherapy of hepatitis C (Bekkering et al., J. Hepathology, 1998, 28, 6, p. 960-964), herpes (Cardamakis et al., Gynecol. Obstet. Invest., 1998, 46, 1, p.54-57), 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 H. S., Cancer, 1998, 83, 6, p.1205-13), multiple sclerosis (Durelli et al., Mult. Scler, 1995, 1, suppl. 1, p. 32-37), atopic dermatitis (Schneider et al., Ann. Allergy Asthma Immunol., 1998, 80, 3, p. 263-268), fungal infections (Kullberg, Eur. J. Clin. Microbiol. Infect. Dis., 1997, 16, p. 51-55) etc. Moreover exogenic interferons, inducers of endogenic interferon synthesis such as bropirimine, a phenylpyrimidinone analog, might be used to achieve similar therapeutic results (Akaza et al., Eur. Urol., 1998, 34, p. 107-110).

Experimental data show that alloferons effectively induce interferon synthesis and stimulate some immunological reactions (natural killers activity) in a manner similar to interferons. Therefore alloferons are believed to have similar therapeutic use compared to interferons and interferon inducers including but not limited to treatment of interferon-sensitive viral and cancer diseases. Direct confirmation of this hypothesis is obtained in experiments with virus infected mice. It is shown that alloferon administration significantly increase the survival rate in mice intrapulmonary infected with a lethal dose of human influenza virus A and B.

The chemical structure of alloferons has no similarity with interferons, other known cytokines and interferon inducers as well as any other materials of medical importance. The chemical structure of alloferons and the mode of biological activity are also quite different of those of arylphorin isolated from Calliphora and demonstrating immunologic and antitumor activity as it is disclosed in EP 0 320 528 A1. Alloferons preferably have a molecular mass close to 1200 Da and belong to the unique peptide family which has not been described so far. Calliphora arylphorin has a molecular mass of about 500 000 Da (Naumann U. and Scheller K. Biochem. Biophys. Res. Communications, 1991, 177, p. 963-971) and is proposed to be used as an adjuvant in the course of specific vaccination and specific stimulation of antibody-dependent T-lymphocytes antitumor activity as it is disclosed in EP 0 320 528 A1. No data concerning a possible effect of arylphorin on the natural killer cell activity and interferon synthesis are available up to now.

Alloferons are linear peptides having a unique amino acid sequence represented by the general formula as follows (SEQ ID NO: 31):

X 1 -His-Gly-X 2 -His-Gly-Val-X 3

where:

X 1 is absent or represents at least one amino acid residue

X 2 is a peptide bond or represents at least one amino acid residue, and

X 3 is absent or represents at least one amino acid residue.

The alloferons of the present invention have up to 30, preferably up to 20 and most preferable 5-13 amino acid residues.

Examples of alloferons of the present invention are summarized in Table 1.

TABLE 1

Amino acid sequences of alloferons, (SEQ ID NOS 1-21), the homologous to alloferon

fragment of influenza B virus precursor protein and general formula (1).

Position

Peptide

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

Alloferon 1

His

Gly

Val

Ser

Gly

His

Gly

—

Gln

—

His

Gly

Val

His

Gly

—

Alloferon 2

—

Gly

Val

Ser

Gly

His

Gly

—

Gln

—

His

Gly

Val

His

Gly

—

Alloferon 3

—

—

Val

Ser

Gly

His

Gly

—

Gln

—

His

Gly

Val

His

—

—

Alloferon 4

—

—

—

Ser

Gly

His

Gly

—

Gln

—

His

Gly

Val

—

Alloferon 5

Pro

Ser

Leu

Thr

Gly

His

Gly

—

Phe

—

His

Gly

Val

Tyr

Asp

—

Alloferon 6

Phe

Ile

Val

Ser

Ala

His

Gly

—

Asp

—

His

Gly

Val

—

—

—

Alloferon 7

—

—

—

—

Thr

His

Gly

—

Gln

—

His

Gly

Val

—

—

—

Alloferon 8

—

—

—

—

—

His

Gly

—

—

—

His

Gly

Val

His

Gly

—

Alloferon 9

—

Leu

Ala

Ser

Leu

His

Gly

—

Gln

—

His

Gly

Val

—

—

—

Alloferon 10

Cys

Val

Val

Thr

Gly

His

Gly

—

Ser

—

His

Gly

Val

Phe

Val

—

Alloferon 11

—

—

Ile

Ser

Gly

His

Gly

—

Gln

—

His

Gly

Val

Pro

—

—

Alloferon 12

—

—

—

Cys

Gly

His

Gly

—

Asn

—

His

Gly

Val

His

—

—

Alloferon 13

Ile

Val

Ala

Arg

Ile

His

Gly

—

Gln

Asn

His

Gly

Val

—

—

—

Alloferon 14

—

—

—

—

—

His

Gly

Ser

Asp

Gly

His

Gly

Val

Gln

His

Gly

Alloferon 15

—

—

—

Phe

Gly

His

Gly

—

—

—

His

Gly

Val

—

—

—

Alloferon 16

—

—

—

—

—

His

Gly

—

Asn

—

His

Gly

Val

Leu

Ala

—

Alloferon 17

His

Gly

Asp

Ser

Gly

His

Gly

—

Gln

—

His

Gly

Val

Asp

—

—

Alloferon 18

—

—

—

—

—

His

Gly

—

—

—

His

Gly

Val

Pro

Leu

—

Alloferon 19

—

—

—

Ser

Gly

His

Gly

—

Ala

Val

His

Gly

Val

Met

—

—

Alloferon 20

Tyr

Ala

Met

Ser

Gly

His

Gly

—

—

—

His

Gly

Val

Phe

Ile

—

Influenza vi-

His

Gly

Tyr

Thr

Ser

His

Gly

Ala

His

Gly

Val

rus B precur-

sor (positions

377-387)

general

X 1

His

Gly

X 2

His

Gly

Val

X 3

formula (1)

Alloferons 1 and 2 are natural peptides isolated from the blood of bacteria challenged larvae of an insect, Calliphora vicina in the course of purposeful screening of cytokine-like materials able to stimulate cytotoxic activity of mammalian natural killer cells. Alloferons 3 and 4 are truncated forms of alloferon 1, which were chemically synthesized in order to determine possible biologically active modifications of the natural prototype molecule.

Comparative study of the effect of alloferon 1-4 on the cytotoxic activity of lymphocytes demonstrated that all of them are bioactive molecules. See Example 5. This makes possible to distinguish conservative (functionally important) and variable parts of the alloferon structure. Alloferons 5-20 are examples represented to show preferred modifications of variable fragments of the basic structure of alloferon.

A data base search did not reveal peptides of natural origin or bioactive synthetic peptides having close similarity to the alloferon structure. Therefore alloferons are believed to belong to a new family of bioactive peptides. Nevertheless, alloferons have, to certain extent, structural analogy with fragments of some functionally important proteins. For example, alloferon 1 has 63% identity with fragment 377-387 of the influenza virus B hemagglutinin precursor. Hemagglutinin is known to be a key membranotropic protein of the virus envelope responsible for the integration with the cell membrane of the host.

Alloferon 1 was used as a prototype molecule in the course of the development of the invention. Alloferon 1 is a linear peptide having a molecular mass of 1265 Da consisting of 13 amino acids. See Table 1. A comparison with alloferons 2-4 allows to determine functionally important elements of the structure of alloferon, which are necessary for its efficacy as a stimulant of NK cell's cytotoxicity and, other activities, and to predict possible structural modifications, which do not change the biological activity of the peptide.

Comparison of alloferon 1 with the structure of alloferons 2-4 shows that the presence of the fragment Ser-Gly-His-Gly-Gln-His-Gly-Val (SEQ ID NO: 32) is sufficient to conserve the biological activity as since all the peptides exhibit similar activities in NK cell cytotoxicity test. Therefore, this fragment or a part of the fragment represents the core conservative structure in alloferon sequences. Positions 1-3 in the alloferon 1 molecule can be missing or can be replaced by one or more amino acids.

Furthermore, a comparison with the homologous fragment of the influenza virus hemagglutinin reveals that positions 4 and 5, represented in the alloferon 1 sequence by amino acids serin (Ser) and glycin (Gly), can be replaced by some other amino acid preferably chosen from the group of aliphatic, aromatic or heterocyclic amino acids. For instance, serin can be replaced by threonin (Thr) and glycin by serin.

Thus, the available data reveals that the first five amino acids in the alloferon 1 sequence are a variable fragment which can be absent or contain at least one amino acid. Therefore, this fragment is marked in the alloferon structural formula (1) as X 1 . Advantageously, X 1 is selected from the group consisting of nothing, His-Gly-Val-Ser-Gly-(SEQ ID NO: 22), Gly-Val-Ser-Gly-(SEQ ID NO: 23), Val-Ser-Gly-, Ser-Gly-, Pro-Ser-Leu-Thr-Gly-(SEQ ID NO: 24), Phe-Ile-Val-Ser-Ala-(SEQ ID NO: 25), Thr-, Leu-Ala-Ser-Leu-(SEQ lID NO: 26), Cys-Val-Val-Thr-Gly-(SEQ ID NO: 27), Ile-Ser-Gly-, Cys-Gly-, Ile-Val-Ala-Arg-Ile-(SEQ ID NO: 28), Phe-Gly-, His-Gly-Asp-Ser-Gly-(SEQ ID NO: 29), Ser-Gly- and Tyr-Ala-Met-Ser-Gly-(SEQ ID NO: 30).

Similarly, positions 14-15 in the alloferon 1 molecule can be missing or can be replaced by a sequence of one or more amino acids. Therefore, this fragment is marked in the alloferon structural formula (1) as X 3 . Advantageously, X 3 is selected from the group consisting of nothing , -His-Gly, -His, -Tyr-Asp, -Phe-Val, -Pro, -Gln-His-Gly, -Leu-Ala, -Asp, -Pro-Leu, -Met and -Phe-Ile.

Moreover, comparison of alloferon and the corresponding hemagglutinin fragment reveals that position 9, occupied in the alloferon molecule by glutamin, is also variable and glutamin can be replaced by some other amino acid, for example, by alanin. Consequently, position 9 of the alloferon structural formula (1) is marked as X 2 ,which can be a peptide bond linking Gly and His or contain not less then 1 amino acid, preferably 0-3 amino acids, more preferably 0-2 amino acids and most preferable 1 amino acid, in particular -Gln-.

Advantageously, X 2 is selected from the group consisting of a peptide bond, -Gln-, -Phe-, -Asp-, -Ser-, -Asn-, -Ala-, -Gln-Asn-, -Ala-Val- and -Ser-Asp-Gly-.

Incorporation of the alloferon sequence into a larger molecule such as a carrier protein without significant alteration of the biological activity of the alloferon is also possible. Thus, the present invention also relates to chemical compounds such as peptides or proteins comprising an amino acid sequence having the above general formula (1), provided that the peptide or protein is not naturally occurring, and in particular not the influenza virus B precursor.

Complex immunological, pharmacological and toxicological studies summarized in the examples below demonstrate a range of useful properties of alloferons. The obtained data show that alloferon is a new cytokine-like peptide. The mode of action of alloferon comprises stimulation of nonself or aberrant self recognition of cells and lysis by the cytotoxic lymphocytes as well as induction of interferon synthesis. Therefore, alloferon is useful as immunomodulatory medicine to correct a deficiency in the production of interferons and activity of natural killer cells, treatment of viral, oncological and other diseases dependent on the said deficiency. Alloferon is practically nontoxic, has no teratogenic, embryotoxic or mutagenic properties as it is shown in advanced preclinical studies.

The experimentally established properties of alloferon 1 are summarized in Table 2.

TABLE 2
Experimentally proved pharmacological activities of alloferon 1.
	Effective in vivo dose
	(mg/kg body mass) or
	in vitro concentration
Activity	(ng/ml)	Medical use
1. Stimulation of mouse	0.05-50	ng/ml	Therapy of infectious
spleen lymphocytes'			and oncological
cytotoxic activity			diseases
2. Stimulation of mice'	1.25	mg/kg	Influenza therapy
resistance to the
influenza virus A
infection
3. Stimulation of mice'	1.25	mg/kg	Influenza therapy
resistance to the
influenza virus B
infection
4. Interferon synthesis	0.125-1.25	mg/kg	Therapy and
induction in mice			prophylaxis of
			viral and oncological
			diseases
5. Stimulation of human	0.0005-500	ng/ml	Therapy and
peripheral blood			prophylaxis of
lymphocytes' cytotoxic			viral and oncological
activity			diseases
6. Stimulation of	5	ng/ml	Adjuvant therapy
peripheral blood			of cancer
lymphocytes' cytotoxic
activity in cancer patients

The pharmacological activity spectrum, in general, corresponds to known properties of interferon-alpha concerning the influence on the cytotoxic activity of natural killer cells and antiviral resistance. In that aspect, alloferon can be characterized as interferon-alpha functional analog. The mode of action of alloferon, regarding in vitro stimulation of NK cells cytotoxic activity, is observed at very low concentrations—about 1 picogram/ml (10 −9 g/ml). In that aspect alloferon is as active or more active as endogenous cytokines, interferons and interleukins.

Moreover, alloferon is able to induce, alone or in cooperation with interleukin 12, the synthesis of endogenic interferons, including interferon-gamma. Therefore, alloferon can be attributed to the group of interferon inducers.

Preclinical studies of the in vivo activity of alloferon show that it has potent antiviral activity when tested using as a model mice infected by human influenza virus. In this model wild type males were infected intranasally by a suspension of the human influenza virus and alloferon 1 was injected intraperitoneally one day before infection and then 1, 2, 4, 6 and 8 days after. Alloferon effectively protected mice from pulmonary lesions and death. Thus, alloferons are useful in the preparation of a pharmaceutical preparation for the treatment or prevention of viral infections.

Neither an acute nor a chronic toxicity of alloferon 1 was found in the course of in vivo and in vitro studies.

It is understood that the pharmaceutical preparations of the present invention may also comprise conventional additives like excipients or carriers. The preparations may be administered to the patient by intranasal, enteral, such as oral or rectal, and parenteral, such as intraperitoneal, intramuscular, intravenous or subcutaneous route. The preparations may be administered in dosage forms such as intranasal dropping solutions, sprays, liposomes, capsules, tablets and suppositories. For parenteral use the pharmaceutically active components are preferably in the form of an injectable solution.

Thus, alloferons are useful in the treatment or prophylaxis of various infectious or oncological diseases where improvement of innate immunity, including interferon system and natural cell mediated cytotoxicity, can have therapeutic significance. The examples of conditions under which alloferons application is prospective comprise influenza virus and other respiratory viral infections, viral hepatitis, AIDS and AIDS relevant secondary infections and oncological conditions, acute and chronic leukemia and other cancers where interferon treatment efficacy is proved, fungal systemic infections sensitive to the interferon treatment etc.

In spite of a certain similarity in biological activity (NK cell cytotoxic activity stimulation, indirect antiviral activity), alloferons differ very much of interferons in terms of structure and mode of action. Thus, interferons are glycoproteins with molecular mass ranging from 17 000 to 80 000 daltons. Glycosilation of amino acid chain is a necessary condition of interferon functional activity as well as their tissue and species specificity. Alloferon is a preferably nonglycosilated oligopeptide having molecular mass of preferably about 1265 Da, 13-60 times less then interferons molecular masses. The amino acid sequence of alloferon has no similarity with any fragment of interferon sequences. There are essential differences between alloferons and interferons in functional aspect as well. Thus, alloferon induces the production of endogenic interferons and promotes in this way a cascade of defense responses mediated by interferons. Exogenic interferon application may rather suppress endogenic interferon synthesis by means of negative feedback mechanism.

The peptides of the present invention can be isolated from natural sources or synthesized by known methods. The peptides of this invention can also be produced by recombinant DNA techniques. Thus, the invention comprises cultivating a cell host previously transformed with a suitable vector containing a DNA sequence, e.g. a cDNA encoding a peptide sequence including any of the peptides of this invention, said DNA sequence being placed under the control of a promoter and followed by termination signals recognized by the cell host machinery such as to authorize the expression said DNA sequence of said peptide sequence, and recovering the peptide sought from the expression of products of the cell culture. Advantageous host cells belong to Lactobacillus strains, E. coli , Agrobacterium or Bacillus strains. Alternatively the peptides can be easily produced by well known chemical synthesis.

The invention is further illustrated by the following examples:

EXAMPLE 1

Isolation of Alloferons from Insect Blood, Structural Characterization and Chemical Synthesis

Alloferons were initially discovered in the blood of the immunized (bacteria challenged) insect, blowfly Calliphora vicina . Postfeeding C. vicina larvae maintained in the laboratory conditions as described (Chernysh S. I., Simonenko N. P., Numata H. Appl. Entomol. Zool., 1995, Vol. 30, No. 3, p. 498-499) were bacteria challenged by the pricking off cuticle with a needle soaked in a suspension of heat-killed Escherichia coli and Micrococcus luteus cells. The hemolymph of septically injured larvae was collected, centrifuged and applied onto a Sep-Pak C18 chromatographic column (Waters Co). The column was washed with 0.05% trifluoroacetic acid. Then the target materials were eluted with 50% acetonitril acidified with 0.05% trifluoroacetic acid. The eluted composition was lyophilized and used to stepwise chromatographic purification of the active principle. The biological activity of the fractions was monitored during purification steps using mouse spleen lymphocytes as cytotoxic cells and H3-uridine labeled K562 cancer cells as a target.

As a result of the purification steps, two close oligopeptides demonstrated potent immunomodulatory activity and referred to as alloferons 1 and 2 were isolated from the primary composition and chemically characterized. The amino acid sequence of the peptides was determined using an automated Edman degradation method on a model 473A sequenator (Applied Biosystems). The structure of alloferon 1 and 2 was determined as follows:

His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly (alloferon1) (SEQ ID NO: 1)

Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly (alloferon 2) (SEQ ID NO: 2)

The peptides were further analyzed by MALDI-TOF ionozation mass spectrometry on a Bruker (Bremen) BIFLEX matrix-assisted laser desorption time-of-flight mass spectrometer and their molecular masses were experimentally determined as 1265 Da (alloferon 1) and 1126 Da (alloferon 2). The masses of alloferon 1 and 2 deduced from the amino acid sequencing data and the masses determined by mass spectrometry are in good agreement confirming that alloferon 1 and 2 are linear peptides having no posttranslational modifications.

Alloferon 1 was selected as a prototype molecule for biological and preclinical studies. In order to get sufficient amount of alloferon 1 for the further experimentation, it was chemically synthesized by means of solid phase peptide synthesis technology (Neimark J. and J.-P. Brian, Peptide Research, 1993, vol.6, p. 219) The peptide purification protocol included two major steps. First step was performed on the Sep-Pak Vac columns with C18 sorbent (Waters) by means of the column elution by 40% acetonitril acidified by 0.05% trifluoroacetic acid. Finally the peptide was purified to homogeneity using a Beckman Gold System chromatograph equipped with an Aquapore ODS Prep 10 C18 (100×10 mm, Brownlee) column in the linear gradient of 0.05% trifluoroacetic acid and acidified acetonitril (0-20% acetonitril during 40 min under flow rate 2.5 ml/min and detector wave length 225 nm). The peptide purity was confirmed by MALDI-TOF mass spectrometry. The amino acid sequence's accuracy was confirmed by microsequencing.

Truncated forms of alloferon 1, alloferons 3 and 4, were synthesized, purified and controlled in the same way as described for alloferon 1.

EXAMPLE 2

Effect of alloferon 1 on the cytotoxic activity of mouse spleen lymphocytes

To analyze the effect of alloferon on the mouse spleen lymphocytes' cytotoxic activity, the standard cytotoxicity assay was used (Hashimoto J. and Sudo E., Gann, 1971, vol. 62, 139-145; Filatova N. A., Malygin A. M., Goryunova L. B., Fel V. Ya. and Khavinson V. K., Tsitologia,m 1990, Vol. 32, No. 6,652-658). H3-uridine labeled K562 human leukemia cells were used as targets for a cytotoxic lymphocytes' attack. Fresh spleen lymphocytes and target cells were co-incubated during 18 hours in the presence or absence of the preparation. Then the proportion of killed and normal target cells and the corresponding cytotoxicity indices were determined in control and experimental groups.

A typical result of the synthetic alloferon 1 stimulatory activity is shown in Table 3.

Alloferon administration to the incubation medium in a broad concentrations range (0.05-50 ng/ml) induced statistically significant amplification of the cytotoxic activity of natural killer cells against target tumor cells. A concentration of 500 ng/ml was not stimulatory. However, in that case the cytotoxic activity was not suppressed below the control level as well.

Therefore, even 10000-times excess of the minimum effective concentration was not harmful to the cytotoxic activity of NK cells.

Thus, alloferon efficiently stimulates the cytotoxic activity of mouse spleen lymphocytes at very low concentrations characteristic to specific cytokines such as interferon-alpha or interleukin 2 responsible for NK cells activation. At the same time, alloferon did not demonstrate immunosuppressive properties even under 10000-fold excess of the effective concentration.

TABLE 3
in vitro effect of alloferon 1 on the mouse spleen
lymphocytes' cytotoxicity to K562 human leukemia cells
	Concentration	Cytotoxicity index
Treatment	ng/ml	Average, % (n = 18)	% to control
Control	0	21.3 ± 3.0	100
Alloferon	0.05	35.2 ± 4.0**	165
	0.5	39.3 ± 3.9***	185
	5	34.3 ± 4.5**	161
	50	37.2 ± 4.5**	175
	500	20.3 ± 3.6	95
**P < 0.01;
***P < 0.001

EXAMPLE 3

Effect of Alloferon 1 on the Cytotoxic Activity of Human Peripheral Blood Lymphocytes

The determination of the cytotoxicity index has been performed as described in Example 2. Human peripheral blood lymphocytes were released from fresh donor blood and purified of erythrocytes by centrifugation using the histopak 1077 solution (Sigma). After centrifugation the lymphocytes were resuspended in phosphate buffer, centrifuged and resuspended again in RPMI 1640 medium supplemented with RNAase. The lymphocytes were diluted up to 2×10 6 cells/ml and immediately used for the cytotoxicity analysis. Interferon-alpha 2b (Intron, Shering-Plough), a natural stimulant of NK cells' cytotoxic activity was used as a positive control.

The PBLs' cytotoxicity against K562 cancer cells was significantly increased when the preparation was added to the incubation medium in a concentration starting from 0.0005 nanogram/ml, however, the stimulatory activity reached a plato at a concentration of about 0.05 nanogram/ml (Table 4). Interferon-alpha 2b administered in a concentration of 5 ng/ml was less effective as compared to the alloferon administered in the optimal concentration rang of 0.05-0.5 ng/ml.

These experimental data demonstrate a strong stimulatory effect of alloferon on the in vitro cytotoxic activity of human peripheral blood lymphocytes directed to the lysis of tumor cells.

TABLE 4
Effect of alloferon and inteferon-alpha 2b on the
cytotoxicity of human periferal blood lymphocytes.
	Concentration	Cytotoxicity index
Treatment	ng/ml	%	% to control
Control	0	27.3 ± 7.3	100
Inteferon-alpha 2b	5	64.3 ± 3.8	236***
Alloferon	0.0005	62.0 ± 6.4	227***
	0.005	73.8 ± 1.7	270***
	0.05	79.8 ± 5.0	292***
	0.5	79.8 ± 2.8	292***
	5	66.8 ± 7.2	245***
	50	68.0 ± 5.3	249***
	500	68.8 ± 4.4	252***
***P < 0.001

EXAMPLE 4

Comparative Study of Alloferon 1 and Interferon-alpha 2b Efficacy Variation Regarding Stimulation of the Cytotoxic Activity of Lymphocytes in the Population of Healthy Donors

PBLs' cytotoxic activity was monitored in the random sampling of 17 healthy donors in order to evaluate diversity and correlation of the lymphocytes' responses to alloferon and interferon-alpha 2b stimulation. The cytotoxic activity was evaluated as described in Examples 2 and 3. Two kinds of target cells were used in the study simultaneously: the K562 cell line originated from erythromyeloid leukemia cells and the A431 cell line originated from the solid colorectal tumor. The proportion of lymphocytes and target cells (E/T ratio) in the trial was 20:1.

The data obtained are summarized in Table 5. Each figure in the Table comprises results of 6 cytotoxicity determinations. The data show significant variation of the cytotoxic activity of the lymphocytes in the control groups, particularly a different capacity to recognize and eliminate the target cells of different origin. Responses to the preparation's administration are also different. Nevertheless a clear correlation of the responses to the alloferon and interferon administration was found in most donors: donors positively responding to interferon in most cases also positively respond to alloferon and vice versa.

Generalized figures of the efficacy of two preparations calculated on the basis of the data in Table 5 are shown in Table 6. Most donors were responding to interferon (10 out of 17 donors) and alloferon (also 10 donors) by statistically approved increase of the cytotoxic activity against K562 or A431 targets. Responsivenesses to alloferon and interferon administration were in good correlation: 9 donors were equally responsive to both preparations and each one donor was selectively responsive to interferon or alloferon, respectively.

Thus, an analysis of the healthy donors' responsiveness shows that alloferon and interferon-alpha 2b possess similar efficacy in this model. Moreover, two preparations seem interchangeable as stimulants of the activity of cytotoxic lymphocytes in most although not all individuals.

TABLE 5
Effect of alloferon and interferon-alpha 2b (K fin = 5 ng/ml) on the
cytotoxicity of peripheral blood lymphocytes (PBL) in healthy
donors (lymphocyte : target ratio = 20:1).
	Cytotoxicity index,	Statistically significant
	Prepara-	M ± m, %	stimulation (P ≦ 0.05)
N°	tion	K562	A431	K562	A431
1	Control	5.3 ± 4.4	53.8 ± 6.5
	Interferon	50.2 ± 1.6***	55.7 ± 8.6	+	−
	Alloferon	30.2 ± 5.5**	83.7 ± 2.9**	+	+
2	Control	14.2 ± 3.3	−64.5 ± 40.6
	Interferon	50.2 ± 2.5***	−88.3 ± 40.4	+	−
	Alloferon	39.8 ± 1.5***	−86.7 ± 22.8	+	−
3	Control	29.8 ± 7.4	−53.6 ± 19.9
	Interferon	21.8 ± 7.5	27.0 ± 6.5**	−	+
	Alloferon	30.8 ± 6.8	39.7 ± 3.3***	−	+
4	Control	30.0 ± 2.9	83.8 ± 3.6
	Interferon	42.8 ± 2.4**	89.5 ± 2.3	+	−
	Alloferon	45.4 ± 2.3***	91.2 ± 0.9	+	−
5	Control	43.3 ± 3.5	−14.8 ± 8.3
	Interferon	64.2 ± 5.7***	35.4 ± 5.2***	+	+
	Alloferon	51.0 ± 1.7	24.4 ± 4.0**	−	+
6	Control	41.7 ± 10.5	50.0 ± 5.7
	Interferon	30.5 ± 8.6	43.7 ± 5.0	−	−
	Alloferon	37.6 ± 10.2	53.7 ± 4.4	−	−
7	Control	40.3 ± 3.2	17.3 ± 5.5
	Interferon	58.2 ± 2.5**	11.8 ± 8.3	+	−
	Alloferon	59.2 ± 5.2**	−10.2 ± 8.0	+	−
8	Control	23.0 ± 3.5	−20.0 ± 6.3
	Interferon	19.2 ± 2.3	74.0 ± 2.7***	−	+
	Alloferon	29.2 ± 6.3	80.7 ± 1.8***	−	+
9	Control	6.2 ± 11.3	7.8 ± 15.6
	Interferon	25.0 ± 4.6	38.5 ± 3.4	−	−
	Alloferon	25.0 ± 12.3	24.5 ± 15.1	−	−
10	Control	34.3 ± 1.3	74.0 ± 4.5
	Interferon	51.8 ± 2.9**	82.7 ± 1.9	+	−
	Alloferon	44.8 ± 3.5*	88.4 ± 1.2**	+	+
11	Control	57.0 ± 4.4	37.2 ± 6.7
	Interferon	52.0 ± 2.2	50.8 ± 9.2	−	−
	Alloferon	42.8 ± 5.9	46.5 ± 7.6	−	−
12	Control	61.8 ± 3.4	55.6 ± 4.9
	Interferon	71.8 ± 2.9*	51.4 ± 3.6	+	−
	Alloferon	47.8 ± 4.1	56.4 ± 3.1	−	−
13	Control	44.0 ± 13.1	52.8 ± 6.5
	Interferon	62.7 ± 7.5	58.2 ± 6.1	−	−
	Alloferon	49.7 ± 3.9	54.8 ± 4.0	−	−
14	Control	30.2 ± 6.1	−6.0 ± 8.1
	Interferon	14.7 ± 8.7	48.7 ± 4.5**	−	+
	Alloferon	2.0 ± 4.2	49.8 ± 12.9**	−	+
15	Control	16.8 ± 2.3	11.2 ± 6.8
	Interferon	2.2 ± 3.8	25.0 ± 5.7	−	−
	Alloferon	19.5 ± 6.8	27.7 ± 3.7	−	−
16	Control	−3.5 ± 7.4	61.4 ± 7.1
	Interferon	5.0 ± 4.7	69.2 ± 2.1	−	−
	Alloferon	18.5 ± 1.7*	68.7 ± 2.0	+	−
17	Control	23.3 ± 5.6	57.8 ± 4.1
	Interferon	29.0 ± 3.3	62.5 ± 1.7	−	−
	Alloferon	24.7 ± 4.0	64.3 ± 4.1	−	−
*P < 0.05; **P < 0.01; ***P < 0.001

TABLE 6
Comparative characteristics of the efficacy of alloferon and
interferon-alpha 2b regarding PBLs' cytotoxic activity stimulation
in the random sampling of healthy donors (from the data of Table 5).
Indicatior	Interferon	Alloferon
Number of donors	17
Positive responses* proportion
(taking into account target specificity):
K562	7/17 = 41%	6/17 = 35%
A431	4/17 = 24%	6/17 = 35%
K562 or A531	10/17 = 59%	10/17 = 59%
Positive responses* coincidence:
Interferon and alloferon sensitive donors	9/17 = 53%
Donors selectively sensitive to interferon	1/17 = 6%
Donors selectively sensitive to alloferon	1/17 = 6%
*Positive response = statistically significant increase of lymphocyte cytotoxicity index

EXAMPLE 4

Comparative Study of the Efficacy Variation of Alloferon 1 and Interferon-alpha 2b Regarding Stimulation of the Lymphocytes' Cytotoxic Activity in the Random Sampling of Cancer Patients.

Blood samples from 18 patients with different malignancies have been tested according to the same protocol as healthy donors of Example 3. Results are shown in Table 7. Positive responses to alloferon treatment were registered in various patient groups, particularly those suffered by chronic and acute leucosis (5 of 9 cases). Positive responses were also detected in patients suffered by non-Hodgkin lymphoma (2 of 6 cases) and one lung cancer patient.

The proportion of individuals positively responding to interferon or ailoferon was slightly decreased in cancer patients compared to healthy donors but also significant (56% and 50% of the total number, respectively). The correlation of responsiveness to interferon and alloferon was also weaker compared to healthy donors although evidential as well. According to the given test, alloferon seems to be an adequate replacement of injectable interferon in some part of cancer patients. However, assuming that alloferon not only mimics interferon activity but also stimulates the production of endogenic interferons, alloferon appears to be even more prospective as replacement of injectable interferon because of the expected double stimulation of the immune response via direct activation of natural cytotoxicity and induction of endogenic interferon synthesis.

TABLE 7
Effect of alloferon and interferon-alpha 2b on the cytotoxicity of
peripheral blood lymphocytes (PBL) in cancer patients
(lymphocyte:target ratio = 20:1).
			Statistically
			significant
			stimulation
	Diag-		Cytotoxicity index, %	(P ≦ 0.05)
N°	nosis	Treatment	K562	A431	K562	A431
1	Chronic	Control	11.2 ± 10.6	−4.2 ± 8.7
	leucosis	Interferon	−5.5 ± 7.9	11.7 ± 12.5	−	−
		Alloferon	−35.3 ± 5.4	−9.0 ± 12.7	−	−
2	Chronic	Control	4.8 ± 5.4	37.0 ± 5.6
	leucosis	Interferon	−10.7 ± 4.9	33.3 ± 9.0	−	−
		Alloferon	1.3 ± 2.3	8.2 ± 8.3	−	−
3	Chronic	Control	−8.3 ± 2.3	−16.2 ± 10.6
	leucosis	Interferon	−3.5 ± 4.0	33.5 ± 7.4**	−	+
		Alloferon	−12.8 ± 3.7	26.2 ± 3.8**	−	+
4	Chronic	Control	−11.0 ± 3.6	28.2 ± 5.9
	leucosis	Interferon	−9.2 ± 7.0	18.8 ± 4.3	−	−
		Alloferon	−1.5 ± 5.1	19.2 ± 6.3	−	−
5	Chronic	Control	−23.0 ± 5.3	15.2 ± 8.3
	leucosis	Interferon	−4.6 ± 9.6	46.0 ± 4.0**	−	+
		Alloferon	−8.0 ± 18.8	42.5 ± 3.9**	−	+
6	Acute	Control	42.3 ± 2.7	6.2 ± 16.5
	leucosis	Interferon	25.2 ± 4.0**	43.5 ± 6.4*	−	+
		Alloferon	20.2 ± 6.8**	48.5 ± 13.5*	−	+
7	Acute	Control	29.2 ± 5.6	−18.7 ± 14.6
	leucosis	Interferon	50.2 ± 2.3**	24.8 ± 10.1*	+	+
		Alloferon	28.2 ± 2.6	20.0 ± 6.9*	−	+
8	Acute	Control	15.2 ± 9.4	25.2 ± 6.5
	leucosis	Interferon	40.0 ± 3.4**	17.8 ± 8.3	+	−
		Alloferon	23.5 ± 5.5	40.4 ± 6.2	−	−
9	Acute	Control	23.5 ± 8.2	−4.8 ± 9.0
	leucosis	Interferon	25.1 ± 4.6	51.5 ± 6.5***	−	+
		Alloferon	13.8 ± 4.4	51.8 ± 4.8***	−	+
10	Lung	Control	11.5 ± 3.7	−7.2 ± 10.2
	cancer	Interferon	27.2 ± 4.2**	59.5 ± 3.9***	+	+
		Alloferon	−0.3 ± 6.1	55.5 ± 2.7***	−	+
11	Uterus	Control	30.5 ± 1.3	93.3 ± 1.0
	cancer	Interferon	50.8 ± 4.9***	93.5 ± 0.9	+	−
		Alloferon	24.0 ± 4.4	94.3 ± 0.4	−	−
12	Hodg-	Control	22.2 ± 8.7	90.3 ± 0.95
	kin	Interferon	39.3 ± 4.9	95.2 ± 0.79**	−	+
	lym-	Alloferon	27.7 ± 4.8	90.2 ± 0.47	−	−
	phoma
13	Non-	Control	11.5 ± 6.4	82.8 ± 1.1
	Hodg-	Interferon	13.8 ± 3.0	80.2 ± 2.0	−	−
	kin	Alloferon	17.7 ± 2.8	91.5 ± 0.8***	−	+
	lym-
	phoma
14	Non-	Control	16.3 ± 9.6	−40.0 ± 20.5
	Hodg-	Interferon	31.2 ± 9.2	26.0 ± 8.9**	−	+
	kin	Alloferon	36.7 ± 9.1	15.3 ± 4.8**	−	+
	lym-
	phoma
15	Non-	Control	19.2 ± 12.1	57.7 ± 4.4
	Hodg-	Interferon	47.5 ± 6.6*	62.2 ± 5.2	+	−
	kin	Alloferon	37.3 ± 4.7	64.0 ± 5.0	−	−
	lym-
	phoma
16	Non-	Control	35.8 ± 8.3	47.2 ± 10.4
	Hodg-	Interferon	43.2 ± 3.5	43.5 ± 4.4	−	−
	kin	Alloferon	37.3 ± 7.0	56.3 ± 4.4	−	−
	lym-
	phoma
17	Non-	Control	49.4 ± 3.2	66.7 ± 6.2
	Hodg-	Interferon	48.8 ± 3.5	67.0 ± 3.3	−	−
	kin	Alloferon	68.3 ± 4.6**	64.3 ± 5.1	+	−
	lym-
	phoma
18	Non-	Control	6.3 ± 14.9	29.8 ± 4.6
	Hodg-	Interferon	−3.0 ± 8.7	33.2 ± 7.4	−	−
	kin	Alloferon	−10.7 ± 14.1	32.8 ± 9.2	−	−
	lym-
	phoma
*P < 0.05; **P < 0.01; ***P < 0.001

TABLE 8
Comparative characteristics of the efficacy of alloferon and
interferon-alpha 2b regarding PBLs' cytotoxic activity stimulation
in the random sampling of cancer patients (from the data of Table 7).
Indicatior	Interferon	Alloferon
Number of donors	18
Positive responses* proportion
(taking into account target specificity):
K562	6/18 = 33%	1/18 = 6%
A431	8/18 = 44%	8/18 = 44%
K562 or A531	10/18 = 56%	9/18 = 50%
Positive responses* coincidence:
Interferon and alloferon sensitive donors	7/18 = 38%
Donors selectively sensitive to interferon	4/18 = 22%
Donors selectively sensitive to alloferon	2/18 = 11%
*Positive response = statistically significant increase of lymphocyte cytotoxicity index

EXAMPLE 5

Influence of Alloferon 1 Structural Analogs on the Cytotoxic Activity of Human Peripheral Blood Lymphocytes

The activity of alloferon 1 analogs, alloferons 3 and 4, was investigated according to the protocols described in Examples 2 and 3. The mononuclear fraction isolated from the blood of healthy donors and target cell of the A431 cell line were co-incubated in the presence of one of the preparations: alloferon 1, alloferon 3, alloferon 4 or interferon-alpha 2b (positive control). The preparation's concentration in all cases was 5 ng/ml. Cytotoxicity index excess over the untreated control was used as the efficacy criterion.

The data of Table 9 demonstrate similar efficacy of alloferons 3 and 4 compared to alloferon 1 and interferon-alpha 2b.

Thus, the comparative analysis of the efficacy of alloferon 1 structural analogs, alloferons 3 and 4, shows that positions 1-4 and/or 14-15 in the alloferon 1 amino acid sequence (See Table 1) are unnecessary for it's specific pharmacological activity and, therefore, represent variable parts of the alloferon 1 structure which can be changed or replaced without activity loss.

TABLE 9
Effect of alloferons 1, 3 and 4 and interferon-alpha 2b
on the cytotoxic activity of human peripheral blood
lymphocytes against A431 tumor cells
	Experiments	Average cytotoxicity index,
Preparation	number	M ± m, %	P
Control	6	−6.0 ± 8.1
Interferon	6	48.7 ± 4.6	<0.001
Alloferon 1	4	49.8 ± 12.8	<0.01
Alloferon 3	6	60.2 ± 2.7	<0.001
Alloferon 4	5	60.8 ± 2.4	<0.001

EXAMPLE 6

In vivo Antiviral Activity of Alloferon on Mice Infected by Human Influenza Virus A

The antiviral activity of alloferon was investigated using the model of lethal infection of mice by the human influenza virus A. A suspension of the pathogenic to mice virus strain A/Aichi/2/68 (cerotype H3N2) was administered intranasally in a dose equal to 10 LD 50 doses to wild type males with body mass ranging from 20 to 22 g. Alloferon 1 dissolved in 0.5 ml of 0.9% NaCl was injected intraperitoneally one day before virus inoculation, then 1, 2, 4, 6 and 8 days after inoculation. The preparation was tested in two doses: 25 and 2.5 microgram per mouse (0.5 and 0.05 microgram/kg). Control mice were injected with an equal volume of the solvent. The mortality of the mice was monitored during 10 days after infection.

The data of Table 10 show significant decrease of post infection mortality in the group treated with 25 micrograms of alloferon. The 2.5 microgram dose was not effective.

TABLE 10
Alloferon antiviral activity in mice inoculated by human influenza virus A
		Dosage,		Mortality 10 days after
		microgram		virus inoculation
	Treatment	per mouse	N	N	%
	Control	—	20	14	70
	Alloferon	2,5	20	13	65
	Alloferon	25	20	5	25*
	*P < 0.05

Similar results were obtained in the experiment where the antiviral activity of alloferon was compared with that of remantadin. See Table 11. Remantadin (amantadine derivative) is one of the most powerful antiviral agents specifically effective against influenza virus A (Ershov F. I. Antiviral preparations, Medicina, Moscow, 1998, 187 pp). Alloferon was injected subcutaneously in a dose of 25 microgram one day before virus inoculation, then one hour before inoculation, then 1 and 2 days post infection. Remantadin in the dose 1000 microgram was given per os one day before virus inoculation, then one hour before inoculation, then 1, 2 and 3 days post infection.

Both alloferon and remantadin effectively protected most of the infected animals from lethal pulmonary lesions caused by the influenza virus. Remantadin appears to be slightly more effective in the case of influenza virus A infection, however it must be used in a significantly larger dosage (about 40 times higher) compared to the alloferon. Moreover, remantadin is known to be ineffective in the case of influenza virus B and other viral infections contrary to alloferon which is equally effective to A and B strains of the influenza virus.

TABLE 11
Antiviral activity of alloferon and remantadin in mice
infected with human influenza virus A
		Dosage,		Mortality 10 days after
		microgram		virus inoculation
	Treatment	per mouse	N	N	%
	Control	—	20	13	65
	Alloferon	25	18	4	22*
	Remantadin	1000	19	1	5***
	*P < 0.05
	***p < 0.001

EXAMPLE 7

In vivo Antiviral Activity of Alloferon on Mice Infected by Human Influenza Virus B

The anti-virus B efficacy of alloferon was tested according to the protocol described in Example 6. Animals were intranasally inoculated with pathogenic to mouse Lee {fraction (1/40)} human influenza virus B strain in doses equal to 3 and 30 LD 50 . Ribavirin, 1-beta-D-ribofuranosyl-1,2,4, -triazole-3-carboxamide, an antiviral agent effective against various influenza virus strains (Liao H. J. and Stollar V. Antiviral Res., 1993, 22, 285; Ershov F. I. Antiviral preparations, Medicina, Moscow, 1998, 187 pp) was used as a positive control.

The results are shown in Table 12. The infection caused severe pneumonia with high mortality rate in both control groups, independent of the virus dosage. Ribavirin effectively protected mice inoculated with a lower virus dose (3 LD 50 ), however it was not effective against a higher virus dose (30 LD 50 ). At the same time, alloferon was equally effective in both cases. Therefore alloferon demonstrated better antiviral efficacy compared to the known antiviral agent, ribavirin. Moreover, alloferon is effective at a dose approximately 10 times less of ribavirin's therapeutic dose.

TABLE 12
Antiviral activity of alloferon and ribavirin in mice
infected with human influenza virus B
	Virus dose		Mortality 10 days after
	(LD 50	Animals	virus inoculation
Treatment	equivalents)	number	Number	%
Control	30	13	10	77
	3	10	8	80
Ribavirin,	30	10	6	60
250 microgram	3	10	0	0***
Alloferon,	30	10	2	20**
25 microgram	3	10	0	0***
**P < 0.01
***P < 0.001

EXAMPLE 8

In vivo Effects of Alloferon on the Interferon Synthesis in Mice

In order to investigate the possible mode of alloferon's antiviral activity, the in vivo effect of the preparation on the interferon synthesis in mice was studied. The interferon concentration in the blood serum of alloferon treated and control (untreated) animals was determined using as a model a monolayer of L-929 cells. Vesicular stomatitis virus (Indiana strain) has been used as a test-virus in a dose 100 times exceeding the 50% cytopathogenic doses. One unit of interferon activity is expressed as a value reciprocal to the mouse serum dilution protecting 50% L-929 cells against the cytotoxicity of the test-virus. Cycloferon was used as a positive control. Cycloferon is an interferon inducer which belongs to the chemical group of acridanons (Ershov F. I. Antiviral preparations, Medicina, Moscow, 1998, 187 pp). Alloferon or cycloferon were injected intraperitoneally in a dose of 25 microgram and 500 microgram, respectively.

The results of two series of the experiment are summarized in Table 13. In each series the experimental groups comprised 4 animals. Blood samples were collected from each animal, then individual serum aliquots were combined and used for the interferon determination. Alloferon stimulated statistically significant the growth of the interferon concentration with a maximum efficacy reached 24 h post treatment.

TABLE 13
Effect of alloferon on the interferon synthesis in mice.
		Animals	Experiments	Interferon titre,
Treatment	Hours	number	number	Arbitrary units
Control		8		15 ± 5
Cycloferon,	4	8	2	95 ± 45
500 microgram	24	8	2	49 ± 3.8*
Alloferon,	2	8	2	31 ± 13.8
25 microgram	4	8	2	21 ± 3.7
	24	8	2	71 ± 16.2*
*P < 0.05

Another example of the in vivo effect of alloferon on the interferon synthesis in mice is shown in the Table 14. The method was the same as in the previous experiment except that alloferon was administered subcutaneously and individual blood samples were analyzed separately. Both alloferon and cycloferon stimulated the interferon production in short-term prospect, 6 hours after treatment. During 24 hours the titre of interferon returned to the control level both in alloferon and cycloferon treated animals.

Thus, the data above confirm that alloferon injection has an in vivo interferon inducing activity on the level similar to those of cycloferon, a known interferon inducer. The length of the stimulatory effect can range from 6 to 24 hours or more, depending, probably, on the physiological state of the animal and way of administration. Compared to traditional chemical interferon inducers such as cycloferon alloferon has the advantage to be effective at a much lower dosage (about 20 times less).

TABLE 14
Short-term effect of alloferon on the interferon synthesis in mice.
		Time,	Animals	Interferon titer,
	Treatment	h	number	Arbitrary units
	Control		7	7.1 ± 1.8
	Cycloferon,	6	7	32.9 ± 10.7*
	500 microgram	24	7	11.4 ± 2.8
	Alloferon,	6	8	40.6 ± 11.4*
	25 microgram	24	8	14.4 ± 4.3
	*P < 0.05

EXAMPLE 9

Alloferon Toxicity Evaluation

The toxicity of alloferon was tested using a panel of in vitro and in vivo models. See Table 15. No signs of acute or chronic toxicity, allergenic activity, embriotoxicity or harmful effect on the reproductive function in animal and microbial models, or a cytotoxicity in human in vitro models were registered so far. Therefore it is concluded that alloferon has very low toxicity or is a practically nontoxic material.

TABLE 15
Summary of alloferon safety analysis
Activity	Method	Results	Conclusion
Acute	Single	Mortality was not registered in any dose including	Toxicometric
toxicity	subcutaneous	highest dose. No changes in animal growth, feeding,	data are
	and intragastric	macroscopical structure of brain, inner and endocrine	positive.
	administration,	organs as well as skin and subcutaneous cellular	Toxic/thera-
	500-6000 mg/kg	tissue around injection place were found at the any	peutic dose
	in mice and	dosage.	ratio >35700
	300-5000 mg/kg		times in rats
	in rats		and >42800
			times in mice.
Sub-	0.2, 2 and 20	None animal died during 90 day period.	Alloferon has
acute	mg/kg once a	Analysis of animal growth, feeding, rectal	no toxic
and	day during 90	temperature, ophthalmological (mucous surfaces	effects on the
chronic	days	state and eye morphometry), neuropsychological	rats under long
toxicity		(excitability threshold, spontaneous locomotors	term daily
in rats		activity) cardiovascular (systolic arterial pressure,	administration
		heart beating rate, ECG) liver (hexenal sleep),	at therapeutic
		kidney (urine composition, phenol red excretion),	dose and
		hematological (hemogram, leukocyte formula,	doses 10 and
		coagulogram) indices, biochemical indices of	100 times
		peripheral blood (proteinemia, urea, creatinin,	exceeding
		glucose, lipids, cholesterol, bilirubin, enzymatic	therapeutic.
		activity, electrolyte composition), blood forming
		indices, pathomorphological and histological (heart,
		lungs, tracheas, stomach, pancreas, epithelial
		tissues, thymus, liver, spleen, kidney, adrenal, brain,
		testicles, ovary) data did not show negative changes
		in the organism's functions.
Allerge-	Anaphylactic	No signs of anaphylaxy were found at therapeutic	Alloferon has
nic	shock	dosage. Minimal response (short term unrest, nose	no detectable
activity		scratch and quickened breath) was registered in 1	allergenic
in		male but 12 guinea pigs which obtained 10-fold	activity.
guinea		therapeutic dose.
pigs
	Immune	The reaction was not found under alloferon
	complexes	therapeutic and 10-fold administration
	reaction
	Indirect reaction	Fat cells degranulation rate was not changed under
	of fat cells	alloferon therapeutic and 10-fold administration
	degranulation
	Conjunctive	The reaction was not found under alloferon
	probe	therapeutic and 10-fold administration
	Reaction of	The reaction was not found under alloferon
	delayed	therapeutic and 10-fold administration
	hypersensitivity
Embrio-	Subcutaneous	Alloferon administration did not cause negative	Alloferon has
toxicity	injection 1.5 and	effects in the course of embryonic development and	no
and	15 mg/kg into	postnatal development in offsprings as well as	embryotoxic
influ-	pregnant rats	alterations of male and female reproductive function	activity as well
ence on			influence on
the			the
repro-			reproductive
ductive			activity in rats
function
Mutage-	Dominant lethal	Alloferon in the dose 15 mg/kg did not induce	Alloferon has
nic	mutations in the	dominant lethal mutations in the mouse germ cells	no mutagenic
activity	mouse germ		and potential
	cells		cancerogenic
			activity
	Chromosome	Alloferon in the single dose 0.5 and 15 mg/kg and
	aberrations in	fivefold dose 0.5 mg/kg did not induce chromosome
	the mouse bone	aberrations in the mouse bone marrow cells
	marrow cells
	Ames test	Alloferon in the concentration range 0.1 - 1000
		microgram/Petri dish did not induce gene mutations
		in 3 test line of Salmonella tiphimurium .
	DNA SOS-	Alloferon has no DNA-damaging activity in the E. coli
	reparation	test-line PQ37

32 1 13 PRT Calliphora vicina 1His Gly Val Ser Gly His Gly Gln His Gly Val His Gly1 5 10 2 12 PRT Calliphora vicina 2Gly Val Ser Gly His Gly Gln His Gly Val His Gly1 5 10 3 10 PRT Artificial Sequence Description of Artificial Sequence Synthetic alloferon 3 3Val Ser Gly His Gly Gln His Gly Val His1 5 10 4 8 PRT Artificial Sequence Description of Artificial Sequence Synthetic alloferon 4 4Ser Gly His Gly Gln His Gly Val1 5 5 13 PRT Artificial Sequence Description of Artificial Sequence Synthetic alloferon 5 5Pro Ser Leu Thr Gly His Gly Phe His Gly Val Tyr Asp1 5 10 6 11 PRT Artificial Sequence Description of Artificial Sequence Synthetic alloferon 6 6Phe Ile Val Ser Ala His Gly Asp His Gly Val1 5 10 7 7 PRT Artificial Sequence Description of Artificial Sequence Synthetic alloferon 7 7Thr His Gly Gln His Gly Val1 5 8 7 PRT Artificial Sequence Description of Artificial Sequence Synthetic alloferon 8 8His Gly His Gly Val His Gly1 5 9 10 PRT Artificial Sequence Description of Artificial Sequence Synthetic alloferon 9 9Leu Ala Ser Leu His Gly Gln His Gly Val1 5 10 10 13 PRT Artificial Sequence Description of Artificial Sequence Synthetic alloferon 10 10Cys Val Val Thr Gly His Gly Ser His Gly Val Phe Val1 5 10 11 10 PRT Artificial Sequence Description of Artificial Sequence Synthetic alloferon 11 11Ile Ser Gly His Gly Gln His Gly Val Pro1 5 10 12 9 PRT Artificial Sequence Description of Artificial Sequence Synthetic alloferon 12 12Cys Gly His Gly Asn His Gly Val His1 5 13 12 PRT Artificial Sequence Description of Artificial Sequence Synthetic alloferon 13 13Ile Val Ala Arg Ile His Gly Gln Asn His Gly Val1 5 10 14 11 PRT Artificial Sequence Description of Artificial Sequence Synthetic alloferon 14 14His Gly Ser Asp Gly His Gly Val Gln His Gly1 5 10 15 7 PRT Artificial Sequence Description of Artificial Sequence Synthetic alloferon 15 15Phe Gly His Gly His Gly Val1 5 16 8 PRT Artificial Sequence Description of Artificial Sequence Synthetic alloferon 16 16His Gly Asn His Gly Val Leu Ala1 5 17 12 PRT Artificial Sequence Description of Artificial Sequence Synthetic alloferon 17 17His Gly Asp Ser Gly His Gly Gln His Gly Val Asp1 5 10 18 7 PRT Artificial Sequence Description of Artificial Sequence Synthetic alloferon 18 18His Gly His Gly Val Pro Leu1 5 19 10 PRT Artificial Sequence Description of Artificial Sequence Synthetic alloferon 19 19Ser Gly His Gly Ala Val His Gly Val Met1 5 10 20 12 PRT Artificial Sequence Description of Artificial Sequence Synthetic alloferon 20 20Tyr Ala Met Ser Gly His Gly His Gly Val Phe Ile1 5 10 21 11 PRT Influenza virus B 21His Gly Tyr Thr Ser His Gly Ala His Gly Val1 5 10 22 5 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide fragment 22His Gly Val Ser Gly1 5 23 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide fragment 23Gly Val Ser Gly1 24 5 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide fragment 24Pro Ser Leu Thr Gly1 5 25 5 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide fragment 25Phe Ile Val Ser Ala1 5 26 4 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide fragment 26Leu Ala Ser Leu1 27 5 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide fragment 27Cys Val Val Thr Gly1 5 28 5 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide fragment 28Ile Val Ala Arg Ile1 5 29 5 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide fragment 29His Gly Asp Ser Gly1 5 30 5 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide fragment 30Tyr Ala Met Ser Gly1 5 31 80 PRT Artificial Sequence Description of Artificial Sequence General structural formula sequence 31Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa His Gly Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa His Gly Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 32 8 PRT Artificial Sequence Description of Artificial Sequence Synthetic peptide fragment 32Ser Gly His Gly Gln His Gly Val 1 5

Claims

1. A peptide exhibiting immunomodulatory activity consisting of up to 30 amino acid residues, wherein the peptide has the following general structural formula (1) (SEQ ID NO: 31):X1-His-Gly-X2-His-Gly-Val-X3 Wherein:X1 is absent or represents at least one amino acid residue, X2 is a peptide bond or represents at least one amino acid residue, and X3 is absent or represents at least one amino acid residue; or a pharmaceutically acceptable salt or ether thereof.

2. The peptide of claim 1 consisting of up to 20 amino acid residues.

3. The peptide of claim 1 wherein X2 represents 0-3 amino acid residues.

4. Peptide of any of the preceding claims wherein X1 is selected from the group consisting of nothing, His-Gly-Val-Ser-Gly-(SEQ ID NO: 22), Gly-Val-Ser-Gly-(SEQ ID NO: 23), Val-Ser-Gly-, Ser-Gly-, Pro-Ser-Leu-Thr-Gly-(SEQ ID NO: 24), Phe-Ile-Val-Ser-Ala-(SEQ ID NO: 25), Thr-, Leu-Ala-Ser-Leu-(SEQ ID NO: 26), Cys-Val-Val-Thr-Gly-(SEQ ID NO: 27), Ile-Ser-Gly-, Cys-Gly-, Ile-Val- a-Arg-Ile-(SEQ ID NO: 28), Phe-Gly-, His-Gly-Asp-Ser-Gly-(SEQ ID NO: 29), Ser-Gly- and Tyr-Ala-Met-Ser-Gly-(SEQ ID NO: 30).

5. The peptide of claim 1 wherein X2 is selected from the group consisting of a peptide bond, -Gln-, -Phe-, -Asp-, -Ser-, -Asn-, -Ala-, -Gln-Asn-, -Ala-Val- and -Ser-Asp-Gly-.

6. The peptide of claim 1 wherein X3 is selected from the group consisting of nothing, -His-Gly, -His, -Tyr-Asp, -Phe-Val, -Pro, -Gln-His-Gly, -Leu-Ala, -Asp, -Pro-Leu, -Met and -Phe-Ile.

7. The peptide of claim 1 which is selected from the group consisting of (SEQ ID NOS 1-20):His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly, Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly, Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His, Ser-Gly-His-Gly-Gln-His-Gly-Val, Pro-Ser-Leu-Thr-Gly-His-Gly-Phe-His-Gly-Val-Tyr-Asp, Phe-Ile-Val-Ser-Ala-His-Gly-Asp-His-Gly-Val, Thr-His-Gly-Gln-His-Gly-Val, His-Gly-His-Gly-Val-His-Gly, Leu-Ala-Ser-Leu-His-Gly-Gln-His-Gly-Val, Cys-Val-Val-Thr-Gly-His-Gly-Ser-His-Gly-Val-Phe-Val, Ile-Ser-Gly-His-Gly-Gln-His-Gly-Val-Pro, Cys-Gly-His-Gly-Asn-His-Gly-Val-His, Ile-Val-Ala-Arg-Ile-His-Gly-Gln-Asn-His-Gly-Val, His-Gly-Ser-Asp-Gly-His-Gly-Val-Gln-His-Gly, Phe-Gly-His-Gly-His-Gly-Val, His-Gly-Asn-His-Gly-Val-Leu-Ala, His-Gly-Asp-Ser-Gly-His-Gly-Gln-His-Gly-Val-Asp, His-Gly-His-Gly-Val-Pro-Leu, Ser-Gly-His-Gly-Ala-Val-His-Gly-Val-Met and Tyr-Ala-Met-Ser-Gly-His-Gly-His-Gly-Val-Phe-Ile.

8. A chemical compound exhibiting immunomodulatory activity comprising an amino acid sequence as defined in claim 1 or a pharmaceutically active salt or ether thereof, provided that the chemical compound is not a naturally occurring peptide or protein.

9. The peptide of claim 2, which consists of 5 to 13 amino acid residues.

10. The peptide of claim 3, wherein XX is one amino acid residue.