PL176407B1 - Lysozyme dimer as well as composition containing it. novel application of lysozyme dimer and compositions containing it - Google Patents

Abstract

1. Pharmaceutical production \ veraj2ąa dimerlizOzzroujćO <for active and pharmaceutically active substances an acceptable carrier which comprises a dimer as the active ingredient and isozyme in the amount of 33 ng-10 mg / ml, preferably 0.001-10 mg / ml, in particular 0.01-10 mg / ml, optionally in admixture with an effective amount of an antibiotic, in the case of content dimerisozyme within 0.001-10 mg / ml, and preferably 0.01-10 mg / ml, this composition it always contains iisozyme dimer in a mixture with an antibiotic as active substance.

Description

The present invention relates to a new pharmaceutical composition containing a lysozyme dimer.

Enzymes in monomeric form have been known for a long time for their therapeutic effectiveness in the treatment of various diseases.

Lysozyme was discovered by Fleming in 1922, but its enzymatic properties were revealed only around 1950. Since then, the compound has been the subject of numerous studies, which resulted in reports of its multiple therapeutic effects. Among many others, antiviral, antibacterial, anti-inflammatory and antihistamine properties have been found. However, the therapeutic use of lysozyme has been limited due to the negative side effects of the monomeric form.

The above limitation of the practical use of lysozyme and other therapeutically active enzymes was overcome in the late 1980s, when it was found that the isolated dimeric forms of the enzymes, while retaining all the advantageous features of the known monomeric forms, did not show any side effects in therapeutic applications. Antiviral and antibacterial compositions containing lysozyme dimer or other dimerized enzymes as active ingredient are described in International Publication No. WO / 11294. In this application it was reported that in in vitro tests, lysozyme dimer inhibited the proliferation of a series of bacterial colonies grown on patient samples at a concentration of 5-20 mg / ml of culture. Dimer has also been reported to be effective in the treatment of canine parvovirosis (CPV) when administered orally twice daily at a dose of 1-2 mg / kg body weight.

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Based on this disclosure, Poland has granted transitional protection for a pharmaceutical agent, granting the exclusive right to manufacture and sell product No. 1. The composition protected by this law contains a lysozyme dimer in a concentration of 0.001 to 20 mg / ml of a pharmaceutically acceptable carrier adapted for intravenous or oral administration. , parenteral topical, and systemic, intrauterine, vaginal, intramammary and external and is in the form of a solution, ointment, tablets, capsules, suppositories, drops or oil.

Research works on lysozyme dimer were continued by the creator of the above solution. Further attractive features of lysozyme dimer have been discovered and new therapeutic uses for this agent have been developed, in particular for the treatment of certain dysfunctions of natural defense mechanisms.

In clinical trials conducted to confirm the efficacy of the antibacterial and antiviral activity of the lysozyme dimer, it was surprisingly found that the dimer is surprisingly effective in the treatment of acute diseases of the gastrointestinal and respiratory systems. New research has been carried out to determine the action of lysozyme dimer at failure stages of the natural defenses in various disease stages. Studies have also been carried out on the possibility of combining classical therapy based on antibiotics with the use of a lysozyme dimer.

It is well known that bacterial toxins are one group of numerous aggressive agents by which bacteria cause disease. Recent research into bacterial toxins has focused on their effects on the host's immune system. This interaction is primarily based on immunomodulation followed by the release of cytokines and other mediators, which is the cause of many toxin-induced physiological disorders. The latter effect has been studied primarily for the effects of endotoxins, which play an important role in the pathogenesis of gram-negative sepsis (see DF Bayston, J. Cohen: "Bacterial endotoxins and current concepts in the diagnosis and retreatment of endotoxaemia" by J. Med. Microbiol. 1990, 31: 73-83.). Although the role of exotoxins in infections caused by Staphylococcus aureus and Streptococcus pyogenes purulent streptococcus has long been known, it was only the isolation of the staphylococcal toxic shock syndrome that led to increased interest in the exotoxins produced by these organisms.

Toxic shock is a severe disease characterized by high fever, low blood pressure, intravascular coagulation, extensive erythroderma, mucosal erythema, renal failure, hypocalcaemia, hypoalbuminemia, and transient rashes (flaking reddening of the skin). Many cases of toxic shock have been associated with the use of vaginal tampons during the menstrual period, but there are now increasing reports of this syndrome in cases not related to the menstrual cycle, in patients of both sexes, often after surgery, where the tamponade is left large (for example, when tamponing the nose). after nose plastic surgery or acute haemorrhage). Staphylococcal strains isolated from the vaginas of patients with toxic shock syndrome (TSS) have been shown to produce Toxic Shock Syndrome (TSST-1) toxin 1, but an undisclosed infection may also be the source of the TSST-1 producing microorganism. The initial bacteremia may be latent but may lead to localized infections after several weeks or months. The appearance of such infections may be accompanied by symptoms of sepsis or septic shock. Less frequent but more dramatic bacteremia may occur when there is no gateway to infection or accompanying localized infections, and in such cases shock, endocarditis, disseminated intravascular coagulopathy, and multiple organ failure are more rapid (see DL Stevens et al: Gram-positive shock; Curret Opinions in Infectious Diseases 1992,: 355-363).

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Similar observations are known for gram-positive bacteria. For example, infection caused by purulent streptococcus - Streptococcus pyogenes, is accompanied by shock and the mortality rate is 30%. Streptococcus pneumoniae is the cause of pneumonia that has been documented to be highly resistant to penicillin and tends to develop shock syndrome. Moreover, AIDS patients have a higher incidence of pneumococcal infection than the general population.

Gram-negative infections can also lead to septic shock. Gram-negative bacilli and sticks as well as commas are the source of the most important enterotoxins. Enterotoxin is a lipopolysaccharide (LPS) component of the outer membrane of the cell walls of gram-negative bacteria. Enterotoxins primarily affect the intestinal system and usually cause diarrhea. The most common infections caused by gram-negative bacteria in humans and animals are those caused by Escherichia coli. The severe dehydration associated with such infections can kill the infected individual. According to data from the World Health Organization (WHO), acute diarrhea kills approximately 3.2 million children in developing countries each year. About 30% of all sepsis cases are caused by gram-negative bacteria.

Sepsis due to bacterial infections caused by gram-positive and gram-negative bacteria is always a serious disease, occurring in all countries. In the United States, there are approximately 400,000 cases per year and the mortality rate is approximately 50%.

Sepsis and septic shock have been the subject of numerous publications in recent years. It has been observed that in the pathophysiology of septic shock, endotoxemia and other diseases, mediators of bacterial intoxication play an important role. These include tumor necrosis factor (TNF), interleukin-1 (IL-1), interferon (IFN), platelet activity factor (PAF) and eicosandoids (arachidonic acid derivatives), with TNF being the most important metabolism as well as on the immune system and phagocytes (compare FE Berkowitz: Bacterial toxins in pathogenesis of infections; Current Opinions in Infectious Diseases, 1991 4: 332-337). It was shown that individuals who did not survive septic shock had higher concentrations of TNF and interleukin-1. Many authors have reported that septic shock patients had elevated levels of TNFot in plasma and blood. At the same time, it was noted that the toxic effects of TNF-α do not depend as clearly on the concentration of TNF as on the constant presence (excess) in the body. Many authors have investigated the modulation of the cytokine cascade in sepsis and septic shock. Effective solutions include the use of TNF monoclonal antibodies and the neutralization of lipopolysaccharides with anti-lipopolysaccharides. The antibodies, however, do not enhance the removal of bacterial debris. Partially beneficial effects have also been seen with the use of agents such as dexamethasone and pentoxifylline that block macrophage TNF production.

It is also known that other cytokines also contribute to septic shock. In this situation, treatments that modulate the cytokine cascade in septic shock have the potential to influence the development of infection because host defense is dependent on the same inflammatory cytokines. Finding an agent to prevent septic shock is a major task because of the potential benefit that this could have for a large number of patients. In the solution sought, controlling the level of TNF seems to be of paramount importance.

Similarly, TNF plays a critical role in another defense mechanism of fever, which is the physiological response to infections common to all higher species of animals and humans. To date, five pyrogenic cytokines (interleukin-1, TNF interferon, interleukin-2 and interleukin-6) have been identified as the main endogenous mediators of the fever reaction, inhibiting pre-optic heat-sensitive neurons that normally allow heat loss and reduction of heat production in the human body. Fever and its mediators have a capacity

Harming both the invasive organism and the host organism. A large body of data has accumulated in recent years to suggest that interleukin-1, TNF and interleukin-6 are mediators of pathophysiological abnormalities in the course of infection. Since endogenous pyrogens contribute to the pathological processes in numerous infections, both the mediators and the fever reaction are potentially harmful to the host organism. The most compelling evidence for this has come from research into gram-negative sepsis. There is evidence that endogenous pyrogens mediate systemic and local manifestations of sepsis caused by gram-positive bacteria, AIDS, spirochete infections, meningitis, adult respiratory failure, purulent arthritis, and mycobacteriosis. Although the data quoted contradict the observed phenomenon that a fever reaction increases the resistance of experimental animals to infection, nevertheless the preservation of a species more than keeping individuals alive is at the heart of the evolutionary process. It is considered possible that the same mechanism may cause deleterious systemic effects of pyrogenic cytokines on the outcome of general infection (e.g. gram-negative sepsis) and manifest itself in less fulminant infections as beneficial local fever effects. Thus, by hastening the death of hopelessly infected individuals, nature kills individuals who pose a threat to the species. In this way, the species as a whole can be protected against epidemic diseases (compare P.A. Maćkowiak: Mechanism of fever; Current Opinions in Infectious Diseases, 1992, 5: 348-354).

According to the basic concept of fever pathogens, exogenous pyrogens, regardless of their origin or structure, cause fever by inducing host cells (primarily macrophages) to produce endogenous pyrogens. Thus, therapeutic methods based on the use of anti-endogenous pyrogen antibodies and antagonists of endogenous pyrogen receptors may be effective in such cases. One possibility is to block TNF synthesis. Animal studies show that TNF can be synthesized upstream of IL-1 and other cytokines in the body's cascading response to infection. Thus, for many scientists, inhibition of TNF biosynthesis also means inhibition of IL-1 synthesis. But at the same time, inhibiting TNF biosynthesis also means stopping the harmful effects of some electrifying and hopeless infections.

TNF is also recognized as one of the mediators of inflammatory processes. Inflammation is in many cases the first stage of a disease that develops septic shock in its natural course. In situations where the continuity of the tissues is broken, such as wounds at risk of infection, war injuries, especially abdominal wounds (peritonitis), gastrointestinal diseases such as acute inflammation accompanying spilling appendicitis, acute bacterial and viral infections such as those occurring in post-influenza pneumonia, in neoplastic diseases, especially in the lysis phase of neoplastic tumors and the like, inflammation is the first symptom of an increase in TNF production. Controlling TNF levels would therefore be a desirable model for treating such infections.

Even more significant is the role of TNF in AIDS as such. AIDS is characterized by a significant reduction in immune immunity. A significant feature of the AIDS syndrome is the decreased number of CD4 + lymphocytes. The number of cells infected with HIV, the causative agent of AIDS, is relatively small (<1 in 100-1000) even among peripheral blood mononuclear cells (PBMCs) in AIDS patients. While CD4 + lymphocytes are preferentially infected, these cells are not the sole target of attack in HIV infection. Recent studies have shown that the spectrum of HIV attack targets is relatively broad. Clear differences were observed in the effects of HIV infection in monocytes / macrophages compared to T lymphocytes. While T lymphocytes tend to be destroyed, monocytes / macrophages allow the infection to continue. The HIV virus can therefore grow and aggregate in monocytes / macrophages as well as in other cells of the body. The kind of HIV response that is inherent in mono6

176,407 citations / macrophages may be responsible for the latent carrier of the virus by the host organism; this reaction may also cause pathogenic sequelae caused by soluble factors produced by infected cells (compare Toshifumi Matsuyama et al: Cytokines and HIV infections: Is AIDS a Tumor Necrosis factor disease ?; AIDS 1991.5: 1405-1417). It has been reported by numerous scientists that HTLV-1 infected human T cell lines are highly susceptible to HIV infection, resulting in a dramatic cytopathic effect coupled with enhanced HIV replication. In addition, HIV-infected cells are susceptible to damage due to cell supernatants. In determining the viral load after treatment with such a supernatant, it was found that the factor produced by T cells (MT-2) enhances HIV replication. This factor has been identified as TNF-β and this finding is consistent with reports that T cells (MT-2) produce TNF-β. The same effect is seen when TNF-α is used. Both TNF-α and TNF-β selectively kill HIV infected cells and enhance HIV replication. It has also been reported that HIV-infected T-cell lines and PBMCs freshly isolated from HIV-infected individuals respond to TNF by increasing TNF levels. This suggests that the same upregulation of HIV virus expression may also occur in vivo. Of course, the potentiating effect of TNF can be neutralized by anti-TNF antibodies. The increase in HIV replication after TNF-α and TNF-β treatment is more than tenfold (compare A. Yakamam et al: Tumor necrosis factor (α, β) induced by HIV -1 in peripheral blood mononuclear cells potentate virus replication; AIDS 1990, 421-427).

It has also been confirmed that a variety of cytokines can influence HIV production. Using purified mononuclear phagocytes from normal peripheral blood, induction of IL-6 and TNF-α was observed several hours after exposure to HIV. The same cytokine induction was observed with heat-inactivated HIV virus. Based on these and a number of similar observations and the report by Toshifumi Matsuyama et al. (Op. Cit.), It can be assumed that AIDS is a cytokine or TNF dependent disease. In the AIDS cytokine network, both TNF-α and TNF-β appear as nodal molecules enhancing HIV replication and inducing their own expression and the expression of other cytokines. TNF-α has been shown to stimulate the release of other cytokines in cells of various types and thus is a key cytokine of the cytokine cascade in the primary defense mechanism.

It has been suggested that many of the symptoms associated with AIDS may be explained by the release of cytokines with different biological functions. The increased production of IL-1 and TNF-α from two known pyrogens may explain the fever observed in AIDS patients. TNF-α may be associated with AIDS-related cachexia. Both TNF-α and TNF-β function as immunomodulators and effector molecules in cytotoxicity with monocytes as mediators. Furthermore, TNF is responsible for activating the immune response and can directly kill HIV infected cells contributing to HIV replication. The immune mechanism has also been proposed to explain the phenomenon of CD4-T cell depletion in AIDS patients (Matsuyama et al., Loc. Cit.). It has also been reported that Kaposi's sarcoma is induced by TNF-α: TNF-α can be produced from keratinocytes under the influence of a physiological stimulus such as ultraviolet light, which may contribute to the induction of IL-6 in the skin and the development of Kaposi's sarcoma in the the course of AIDS. In in vitro tests, TNF-α can damage myelin and oligodendrocytes; also some glioblastoma-derived cell lines have been shown to be susceptible to the anti-proliferative effects of TNF-α. These observations may lead to the conclusion that the dysfunction of the central nervous system in AIDS patients is due to the action of TNF-α. Many reports have confirmed that serum TNF-α and IL-1 levels increase significantly with the development of AIDS and ARC (AIDS-related complex), while in asymptomatic HIV carriers it remains at the same level as in the control group. from healthy people. According to Matsuyam et al. (Op. Cit.), AIDS is as much a disease associated with TNF as it is with HIV. This demonstrates that achieving control over induction of iUF can lead to effective therapy for AIDS patients.

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The object of the invention is to develop a composition useful in the prophylaxis and therapy of the above-described disease states.

This aim was achieved according to the invention thanks to the unexpected finding that:

1.Lysozyme dimer inhibits TNF synthesis,

2.Lysozyme dimer stimulates the synthesis of IFN-α,

3. Lysozyme dimer stimulates phagocytic activity, and thus has an activity that allows its use in connection with the above-described pathological conditions, including treatment, alleviation of the course, and even to some extent prophylaxis of diseases associated with excessively high levels of TNF, described above.

The pharmaceutical composition containing lysozyme dimer as active substance and a pharmaceutically acceptable carrier according to the invention is characterized in that it contains as active substance the dimerized form of lysozyme in an amount of 33 ng -10 mg / ml, preferably 0.001 mg / ml, in particular 0.01 - 10 mg / ml, optionally in a mixture with an effective amount of an antibiotic, where in the case of a lysozyme dimer content in the range 0.001-10 mg / ml, preferably 0.01-10 mg / ml, this composition always contains lysozyme dimer in mixture with an antibiotic.

The composition according to the invention, containing the lysozyme dimer and the antibiotic, is characterized in that in the in vitro test in the presence of bovine serum the antibiotic activity is up to 100% higher than in the same test without the addition of lysozyme dimer.

Preferably, the composition of the invention comprises a lysozyme dimer substantially free of monomeric, trimeric and oligomeric forms of lysozyme.

Preferably, the composition according to the invention comprises lysozyme dimer in an amount of 33 ng - 0.001 mg / ml.

When the composition according to the invention contains an antibiotic as one of the active ingredients, it comprises an antibiotic selected from the group consisting of penicillin, neomycin, erythromycin, cephalosporin.

Preferably, the composition according to the invention containing the lysozyme dimer and the antibiotic is in the form of a solution and contains the lysozyme dimer in an amount that provides an interaction of the lysozyme dimer on the cell of the microorganism at a concentration of 5 µg / ml.

Preferably, the composition according to the invention is adapted to be administered in one or more doses of 0.02 mg / kg body weight.

Preferably, the composition of the invention is in the form of an injection solution suitable for intravenous administration.

The lysozyme dimer, which is the active ingredient of the composition according to the invention, has the ability to modulate natural defense mechanisms by at least one action selected from the group consisting of: increasing phagocytic activity, increasing interferon synthesis, decreasing the level of tumor necrosis factor (TNF), suppressing tumor necrosis factor (TNF) secretion. and inhibiting tumor necrosis factor (TNF) biosynthesis in animals and humans.

Lysozyme dimer has a particularly beneficial effect on diseases associated with increasing or excessively high levels of tumor necrosis factor (TNF), including diseases that are not viral or bacterial infections. One of these conditions is HIV infection with an asymptomatic stage and the AIDS-related ARC complex. Controlling the release of the tumor necrosis factor at these stages can delay the onset of the full symptoms of AIDS.

Inflammation and associated fever as well as sepsis, septic shock and cachexia are other such conditions.

According to the invention, the lysozyme dimer is used together with an antibiotic. Lysozyme dimer therapy can also accompany conventional AZT therapy.

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The antibiotic administered together with the lysozyme dimer is more effective than the one administered alone. The effective dose of the antibiotic is reduced by about 33% to about 50% relative to the effective dose of the same antibiotic in known compositions containing the antibiotic alone. This applies in particular to penicillin, neomycin, erythromycin and cephalosporin (SEFRIL).

The composition of the present invention comprises a pharmaceutically acceptable carrier suitable for intravenous, oral, parenteral, topical, and systemic, intrauterine, vaginal, intramammary and external administration and is in the form of a solution, ointment, tablets, capsules, suppositories, drops or oil.

Preferably, the carrier is an apyrogenic sterile composition comprising a physiologically acceptable solvent and a pharmaceutically acceptable preservative. Preferably, the apyrogenic, sterile composition containing at least one physiologically acceptable solvent and / or at least one pharmaceutically acceptable preservative is apyrogenic sterilized water or an aqueous PBS solution as solvent and thiomersal as a preservative acceptable for use in pharmaceutical proteinaceous agents. Preferably the composition of the invention is in the form of an injection solution suitable for intravenous administration.

The injectable composition according to the invention can also be administered intramuscularly and subcutaneously. In some applications, it may be appropriate to administer the same liquid composition either intra-uterinally or intramammaryly - or locally or optionally in conjunction with other external formulations simultaneously or independently.

Another preferred form of using lysozyme is the use of a tampon or an aseptic dressing as a solid carrier for a therapeutically effective dose of this active ingredient. A particular example is vaginal tampons for use during menstruation.

The isolated, purified lysozyme dimer is preferably used as the dimerized form of lysozyme. In some applications it is possible to use compositions which contain small fractions of trimers and higher oligomers of that enzyme in addition to the lysozyme dimer.

The dimerized form of lysozyme is obtained by a controlled polymerization of the enzyme monomer followed by careful purification, in particular removal of the monomeric form with known toxic side effects and the trimer fractions and higher oligomers from the reaction mixture. Any known method of carrying out the polymerization may be suitable for obtaining the dimerized form. One possible method, also including purification steps, is described in International Publication No. WO 91/10731.

Previous preclinical studies of lysozyme dimer revealed no mutagenic or teratogenic properties, and showed no or only little tolerance. Toxicity determined for a single dose (acute toxicity) by oral or _{topical application - LD 50} could not be determined (> 2000 mg / kg) and for intravenous administration - LD ₅₀ was> 1000 mg / kg.

The new applications of lysozyme dimer now disclosed have a solid foundation, confirmed by in vitro tests and in vivo clinical applications backing them up. Some comparative studies have also been carried out.

The inhibitory effect on TNF release is believed to be so effective due to the broader spectrum of activity of the lysozyme dimer, in particular its ability to induce IFN release and its potentiating effect on phagocytosis. Both these additional properties are important factors in natural defense mechanisms. Thus, the therapeutic and prophylactic effects in the above-described new applications of lysozyme dimer are assisted by natural defense mechanisms potentiated by the dimer of this enzyme.

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The present invention will be further elucidated in the following Examples, in which reference will be made to the accompanying drawings graphically illustrating test results of particular interest. In the drawings:

Figure 1 illustrates the suppression of TNF release in an in vitro assay in lymphocyte culture stimulated with a suboptimal dose of ConA in the presence of various dilutions of lysozyme dimer.

Figure 2 illustrates the blood levels of TNF in calves (experimentally determined) a

Figure 3 illustrates the blood INF of calves (experimentally determined) in a comparative study of the therapeutic efficacy of the medicament of the invention.

Example I.

In order to determine the immunoactivity of the lysozyme dimer, tests on human peripheral blood PBL lymphocytes were used, the results of which were assessed using the FACS analytical method.

Mitogenic stimulation of human peripheral blood lymphocytes is a commonly used method of testing the reactivity of most cells of the immune system. To investigate the effect of drug substances on the activation and proliferation of lymphocytes from healthy blood donors, mitogen is administered at suboptimal doses and finally lymphocyte response is quantified by evaluating appropriate immunologically relevant parameters. The obtained results are evaluated by comparing them with the results obtained for the control cultures without the addition of medicinal substances.

ConA was used to stimulate lymphocytes at a concentration of 20 µg / ml of medium. The initial cell concentration was 10 ⁶ cells / ml. Short-term cultures incubated in a CO ₂ atmosphere were grown for one (IL-2 receptors on lymphocytes and HLA-Dr) or two days (all other tests). The following parameters were measured to assess cellular activity:

- neopterin (immune activation marker)

- β-2-microglobulin (also an activation marker)

- interleukin-2 (autocrine and paracrine substance derived from the T-helper lymphocyte)

- interleukin-6 (cellular differentiation hormone)

- tumor necrosis factor - TNF (vasoactive multidirectional interleukin)

- interferon-α (a differentiating factor, especially for B lymphocytes)

- thymidine kinase (enzyme, stimulated in the process of cell proliferation)

- leukocyte interleukin-2 receptor (acceptor molecule for autocrine and paracrine

IL-2)

- Ki-67 lymphocyte (antigen expressed in activated proliferated cells)

- HLA-Dr lymphocyte (class II histocompatibility antigen, stimulated during an immune response).

The following observations were made in the culture supernatants:

Neopterin - produced by T lymphocytes during the immune response and in stimulated cultures - in the present experiments was not clearly elevated under the influence of lysozyme dimer as compared to the control value observed in CoA stimulated cells without addition of the tested dimer. The neopterin values were slightly increased with the increase in the concentration of the investigated dimer.

Similarly, the values of β-2-microglobulin at all concentrations of lysozyme dimer fluctuated around the control values.

IL-2 receptors - eliminated from the lymphocyte surface during culture and providing information on the total amount of converted IL-2 receptors - were at a level comparable to

176,407 IL-2 receptors on the lymphocyte surface (see below) and showed marked suppression at higher concentrations of the tested dimer.

Interleukin-6 showed a distinct dose-dependent trend towards higher concentrations at higher concentrations. This particle is very important in haemopoiesis, cell differentiation, and the immune response. In this test, the first three values had to be extrapolated because the highest standard corresponded to 2000 pg / ml.

The results for other particles obtained in the studies are presented below in Table 1. Unlike Interleukin-6, TNF-α was contained in the culture supernatants at dramatically reduced concentrations, except for the last two dilution steps, which proved ineffective in suppressing TNF concentration. .

Table 1

Effect of lysozyme dimer on human peripheral blood lymphocytes

No A sample HLA-Dr / CD3 EL-2 Rec Ki-67 / CD8 Ki-67 / CD4 control 3.3 8.9 6.0 2.7 1. 1 mg / ml 2.2 2.6 4.8 5.3 2. 0.3 mg / ml 1.9 6.3 4.1 3.0 3. 0.2 mg / ml 3.0 6.1 6.5 (6.9) 4. 33 pg / ml 3.0 5.9 6.3 3.0 5. 10 pg / ml 3.4 6.0 7.7 3.7 6. 3.3 pg / ml 2.4 5.0 7.5 3.7 7. 1 pg / ml 2.5 4.9 7.0 4.1 8. 0.3 pg / ml 3.3 5.8 7.5 3.9 9. 0.1 pg / ml 2.8 4.0 7.9 3.8 10. 33 ng / ml 3.1 4.6 8.0 4.1

Effect of lysozyme dimer on human peripheral blood lymphocytes

No A sample TNF Kinase thymidine IFN-α control 205.11 9242 4.97 1. 1 mg / ml 38.07 923 8.81 2. 0.3 mg / ml 17.19 10914 9.44 3. 0.1 mg / ml 13.75 7254 17.96 4. 33 pg / ml 36.11 9525 3.67 5. 10 pg / ml 22.22 5492 7.54 6. 3.3 pg / ml 54.91 5198 6.26 7. 1 pg / ml 18.47 5840 33.91 8. 0.3 pg / ml 14.47 6839 10.07 9. 0.1 pg / ml 94.16 3672 4.97 10. 33 ng / ml 172.46 7312 10.07

Thymidine kinase is measured in the lymphocytic cytoplasm after freezing and thawing the cell mass. Thymidine kinase is stimulated to increase synthesis in dividing cells, which makes it a good marker of cell proliferation. The data in Table 1 show that there is a suppression at the highest lysozyme dimer concentrations tested, and no clear trend can be found at the remaining dilutions. On the other hand, interferon-α, under the same conditions, showed values higher than the control value achieved with concavalin A (ConA) alone, at the higher concentrations tested. A clear increase is seen when going from the second value to the third dilution.

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Lymphocytic markers:

HLA-Dr / CD3, which is a marker of histocompatibility, is expressed on activated T cells during the immune response. The results obtained indicate a certain percentage of activated T cells in the control culture; at different values of lysozyme dimer concentrations in the cultures, the observed values fluctuate but oscillate around the value observed in the control culture.

IL-2 receptors on lymphocytes: Interleukin-2 is a cytokine produced by T-helper lymphocytes when activated by IL-2. There is autocrine and paracrine IL-2. T-helper lymphocytes not only produce IL-2 but are also stimulated to proliferate by this particle. IL-2 receptors on the surface of T-helper lymphocytes are stimulated to increased synthesis upon activation. The data from Table 1 show that at the highest concentrations of lysozyme dimer there is a clear suppression of IL-2 receptors on lymphocytes, while the remaining values do not differ more than within the biological bandwidth. Table 1 also includes data for Ki-67 / CD8 and Ki-67CD4. Ki-67 is a proliferative molecule that appears in cells undergoing mitosis. Ki 67 is an important parameter for the evaluation of stimulated cells and in the diagnosis of neoplasms. From the presented results it can be seen that a slight inhibition of cell proliferation based on Ki-67 + suppression cells (CD8) with the highest two doses of lysozyme dimer. In helper lymphocytes (CD4), a significant percentage increase in positive cells is observed at the highest concentration. At lower doses, the percentage of cells expressing Ki-67 / CD4 is slightly higher than that of the control culture.

The clear suppression of TNF is illustrated in Figure 1.

The immunological parameters listed above and selected for their potential importance in the immune response were analyzed by a method based on testing the effect of the test substance on human peripheral blood lymphocytes stimulated suboptimally with concavalin A (ConA), well known, with high sensitivity and allowing the evaluation of many different parameters. At certain concentrations of lysozyme dimer, there were marked differences in the tests compared to the values observed with ConA-only stimulated lymphocytes, while with, for example, TNF and INF-α, clear effects were observed at all concentrations tested.

Example II. Laboratory studies have been performed to determine the effect of lysozyme dimer on phagocytamic activity of milk and blood cells in vitro. It was previously established that in standard in vitro tests, lysozyme dimer does not inhibit the proliferation of microorganisms isolated from infected cow's mammary glands. Since it has been found in clinical trials that lysozyme dimer eliminates infection of the mammary glands of cows when administered intramammary and intravenously at the same time, it has become clear that the antibacterial mechanism in the cow's mammary glands is phagocytosis. Thus, the blood and milk of both healthy and infected cows were used in in vitro tests to investigate the effect of lysozyme dimer on phagocytosis. For comparison, studies were carried out with the same amount of test substance and the same incubation time using cells isolated from healthy and infected cows, and even from infected and healthy mammary gland quarters of the same cow in order to eliminate individual differences in the observed reactions.

Both the purified form of dimerized lysozyme and a mixture of dimer and small fractions of trimers and higher lysozyme oligomers were used for the research. The test substances were added to the blood or milk of healthy and infected cows at concentrations of 25 - 0.25 pg / ml and the mixtures were incubated at 37 ° C for 0.5-24 hours. The percentage of phagocytic cells (phagocytic index according to the method of Wisniewski et al.) And the percentage of NTB-positive granulocytes (according to Park) were determined for each sample.

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Lysozyme dimer enhances the phagocytic activity of leukocytes in in vitro assays. The observed results are dose dependent and incubation time dependent. Higher concentrations of lysozyme dimer are necessary for the activation of leukocytes in the milk than for the activation of leukocytes in the blood. Excessively high concentrations of lysozyme dimer reduce phagocytic activity in vitro. Selected experimental results - showing that essentially as long as the post-reaction mixture does not contain the cytotoxic monomeric form of lysozyme, the results are comparable for highly purified dimer and for the less thoroughly purified form of dimer - are presented in Tables 2 and 3 below, in which the term "lysozyme + dimer" is used to denote a mixture containing the small fractions of trimer and higher lysozyme oligomers mentioned above.

Table 2

Effect of lysozyme dimer on the phagocytic activity of leukocytes from milk from healthy cows (dimer concentration 20 pg / ml, incubation time 3 hours)

Leukocytes from the milk of healthy cows Parameter preparation Cow No. 477 Cow number 463 control 77.8 80.0 % phagocytosis lysozyme + dim.er 100 100 lysozyme dimer 92.6 100 control 2.7 4.1 index lysozyme dimer + 4.4 6.4 phagocytes lysozyme dimer 4.8 8.4 control 3.4 2.5 % NBT reduction lysozyme dimer + 5.7 3.8 lysozyme dimer 3.4 6.8

Table 3

Effect of lysozyme dimer on the phagocytic activity of leukocytes from milk from infected cows (dimer concentration 20 pg / ml, incubation time 30 minutes)

Leukocytes from the milk of an infected cow (No. 490) Parameter preparation infected quarter * / quarter healthy control 98 58 % phagocytosis lysozyme dimer + 98 90 lysozyme dimer 100 100 index control 10.9 2.7 phagocytal lysozyme dimer + 8.7 14.9 lysozyme dimer 10.9 8.9 control 4.2 4.2 % NBT reduction lysozyme dimer + 5.9 8.4 lysozyme dimer 5.5 7.0

* / measurements at the time of infection, untreated cow

It is clear from the above that the degree of purification of the lysozyme dimer does not have a significant effect on the observed phagocytic activity of leukocytes from milk. However, it may turn out in further studies that the effector cells may be granulocytes.

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The following examples are provided in in vivo test reports. In kinetic studies, a composition containing 2 mg of dimer and isozyme in 10 ml of PBS solution was used. This composition was designated KLP-602.

Example III. It has been shown that when administered intravenously, KLP-602 dimer and isozyme stimulates the aagoccyar activity of granuiocytes in the blood in healthy and sick calves and healthy foals, as well as in sword cows after intramammary administration. This effect is manifested by an increased neutrophil titer and an increased ability to absorb staphylococci and reduce NBT. This phenomenon occurs primarily during the first 12-24 hours after injection of the agent. The effect of KLP-602 on the phagocytic activity of the udder on the dose and formulation as well as on the individual response of the animal.

Iizozyme dimer has been used to treat infectious diseases in cattle, pigs, horses and dogs. The test agent was administered in various doses and at various time intervals intravenously, intramuscularly, subcutaneously, intramammaryly and intra-uterinally. The agent was administered to 346 cows, 274 cuts, 110 sows and boars, 294 piglets, 709 unweaned piglets, 35 foals and 107 dogs. Control groups were infiltrated by alternative methods, no animal was left unprotected due to the risk of falling. Nevertheless, on the basis of the conducted research, comparative conclusions can be made, taking as a reference point the kinetic picture of new diseases known from the veterinary literature.

Example IV. The studies carried out on the cuts measured the levels of IFN and TNF in the blood of healthy and infected animals. The diseased animals were left untreated for the first 12 hours of infection, then they were treated with antibiotics and high purification iisozyme dimer. The results obtained are graphically illustrated in Figures 1 and 2 showing the results obtained for TNF and IFN, respectively. Both figures show the real resuits obtained in the four identified individuals. In both figures line 1 corresponds to the results obtained for a healthy cut. Line 2 - for cutting sick and untreated (12 hour IFN value equal to 45 U / m and not shown, and on the graph the slope of line 2 corresponds to the direction indicated by this value), after 12 hours the animal was subjected to a known method; Line 3 - for the cut with antibiotics and line 4 - for the cut with KLP-602.

In another experiment, more than 90% of chicks suffering from gastroenteritis and more than 85% of bronchial pulmonary inflammation were dissected in the group of chops treated with iisozyme dimer - one or two injections of KLP-602. It was typical for symptoms such as fever and diarrhea to resolve quickly. Treatment with the test agent did not require body fluid replacement. The time and speed of recovery were slower than in the antibiotic controlled group.

Example 5 KLP-602 was most effective in treating swine diseases. 100% or not 100% of the animals recovered from the following diseases: postpartum bloat (MMA syndrome), dysentery, purulent uterine inflammation, influenza and colonic infection. The administration of the test agent in cases of edema disease with bronchial pneumonia had a less spectacular effect, although the obtained results were still much lower than the results obtained with the known method. Rapid resolution of diarrhea (within the first 24 hours) and fever was observed, as well as recovery of lactation - especially important in cases of postpartum udder and uterine inflammation (saving piglets' life). The efficacy of KLP-602 in comparison with the results of the anthernative treatment is illustrated below with the data in Table 4.

Example VI. When using KLP-602, 100% of foals suffering from jeiit catarrh and 83% of foals with bronchial pneumonia recovered faster than the animals in the control group, conventionally bred.

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Example VII. Attempts have been made to use KLP-602 to treat certain diseases in dogs, such as folliculitis (100% cure), upper and lower respiratory tract infections, and gastrointestinal infections manifested by diarrhea. This group was dominated by parvovirosis, which is known to be a practically incurable disease. Nevertheless, recovery was achieved in approximately 75% of parvovirosis cases.

Several important observations have been made in the tests carried out on animals affected by the diseases caused by natural infections, as described above:

1. The therapy turned out to be effective and very simple in the treatment of diseases of multifactorial etiology and pathogenesis (diseases of this type dominate in populations of farm animals and are difficult to control, especially in farms where the epidemic spread of diseases is very easy). These results demonstrate the modulating effect of lysozyme dimer on natural defense mechanisms.

2. The therapy turned out to be effective in diseases whose natural development may run through the stage of endotoxic shock, or disorders such as high and prolonged fever, affecting the condition of the animal for a long time after recovery, as is the case in horses (foals) bred with intended for participation in races and other sports and other animals bred for the food industry, where the cost of rearing is important. Rapid recovery will significantly reduce the cost of treating such diseases.

3. The results observed in vivo confirm the decreased level of TNF in various naturally occurring infections treated with lysozyme dimer, thus confirming the claimed features of the invention.

Example VIII. The effect of lysozyme dimer on the activity of antibiotics against various microorganisms was investigated; in order to select bacteria for further tests, to determine the doses of antibiotics most effective against the selected microorganisms and to determine the range of lysozyme dimer concentrations in which the expected effects can be expected. In these studies, two series of freeze-dried purified lysozyme dimer were used: one produced in 1991 and the other produced in 1992. The studies used antibiotics available on the Polish market, produced by POLFY, a Polish pharmaceutical manufacturer, such as penicillin, neomycin, erythromycin, cephalosporin (SEFRIL).

The antibiotics taken for the trials were suspended in a buffered NaCl solution (PBS - Biomed) or in bovine serum. Lysozyme dimer was then added to the suspension in such a way that its concentration in the test samples was always 5 pg / ml in the test samples. The concentrations of the tested antibiotics were different in each trial due to the different sensitivity of the microorganisms used. The effect of an antibiotic alone or an antibiotic in combination with a lysozyme dimer on Escherichia coli, Salmonella enteritidis, Staphylococcus aureus and Streptococcus uberis isolated from diseased animals was investigated in vitro using suspensions of the microorganism in PBS or bovine serum. Laboratory strains of Sarcina lutea 9341 ATCC and Staphylococcus aureus 209 P were also used in the experiments.

Table 4

Animals treated with KLP Animals treated by other means number cases Time (days) Healed Unhealed number cases The preparation used AT % Healings 1 number % number % I. Colibacillosis in piglets 434 1-2 421 97 13 3 400 * / antibiotics glucose, vitamin B 3-5 75-80 * /

176 407 cont. table 4

II. Dysentery in piglets 29 1-2 29 100 - - 219 antibiotics STOLMED RIDOWET 5-9 75 III. Postpartum agrotoxicity thirty 1-2 thirty 100 - - 40 * / antibiotics cortizone, sulfa 3-5 80 * / IV. Piglet flu 38 1-5 38 100 - - 200 * / antibiotics (preparations with prolonged action) 3-9 60-80 * / V. Edema disease - 82 1-3 78 95 4 5 120 * / antibiotics penicillin, tetracycline, vitamin B, fluids + glucose 3-6 6 - * /

* / approximate data

Due to the preliminary nature of the trials, each antibiotic was tested against 1,2 or 3 different types of bacteria as follows:

1. Preparation of the plates

0.05 ml aliquots of the 18 hour culture broth of the tested bacterial strain were added to a 14 ml sample of spiked agar (Biomed). The mixture was mixed and poured onto 10 mm diameter dishes. After cooling and solidifying the agar, sterile cylinders were placed on it and filled with the solution of the tested antibiotic.

2. Preparation of Antibiotic Solutions mg a sample of the antibiotic to be tested was first dissolved in PBS and then the necessary amount of this solution was added to a predetermined volume of PBS or bovine serum to obtain a concentration of the antibiotic in the solution as close as possible to the MIC (minimum inhibitory concentration); always solutions of three different concentrations were prepared.

3. Preparation of the lysozyme dimer solution mg of the lyophilized lysozyme dimer was initially dissolved in 10 ml of PBS. Further dilutions were made using either PBS or bovine serum. The diluted solutions were added to the antibiotic solution prepared as described above. The following combinations were tested:

- antibiotic / PBS

- antibiotic / PBS + lysozyme dimer

- antibiotic / serum

- antibiotic / serum + lysozyme dimer

The concentration of the antibiotic was the same in each cylinder; the concentration of lysozyme dimer was 5 pg / ml. After filling the cylinders with the solutions, the plates were left at room temperature for two hours and then the cultures were incubated at 37 ° C.

4. Reading the results and evaluation of the activity:

The plates were removed from the oven after 18 hours of incubation and the diameter of the zone of inhibition of bacterial growth (no colonies) around the cylinders was measured.

No differences were observed in the size of the zones of inhibition of bacterial growth around the cylinders with suspensions of antibiotics in PBS without addition and with the addition of lysozyme dimer at a concentration of 5 pg / ml. However, the size of the zones of inhibition was reduced around the cylinders filled with the suspension of the antibiotic in bovine serum and enlarged around the cylinders filled with

176 407 suspension of antibiotic with the addition of lysozyme dimer in bovine serum. The size of these zones was larger than the zones around the cylinders filled with PBS suspensions and larger than around the cylinders filled with the serum suspension of antibiotic alone without the addition of lysozyme dimer.

The described phenomenon occurred in tests with penicillin used against Sarcina lutea. Ampicillin showed synergism in combination with lysozyme dimer in inhibiting the in vitro growth of Escherichia coli, Salmonella enteritidis and Staphylococcus epidermidis. Increased activity of erythromycin was observed in the presence of lysozyme dimer against Staphylococcus aureus 209 P and Streptococcus uberis, as well as SEFRIL against Staphylococcus aureus 209 P. Escherichia coli and Salmonella enteritidis were resistant to this antibiotic. The increase in the antibacterial activity of the tested antibiotics was observed only when bovine serum was used as a solvent. In general, the increase in activity averaged 50%, but in some cases the presence of lysozyme dimer resulted in a 100% increase in the activity of the antibiotic.

Conclusions:

Lysozyme dimer (without preservative) at 5 pg / ml shows synergism in vitro with some antibiotics used at MIC (minimum inhibitory concentration) in the presence of bovine serum to inhibit bacterial growth. The results obtained so far show that it will be important to continue the research.

Example IX. AZT synergy in the treatment of AIDS patients

In a recent report by Ito et al that TNF-α can antagonize the antiviral activity of AZT against HIV (M. Ito et al. "Tumor necrosis factor antagonizes inhibitors effect of azidothymidine on human immunodeficiency virus (HIV) replication in vitro" Biochem. Biophys. Res. Commun, 1990, 166; 1095-1101). Advanced AIDS patients succumb to numerous opportunistic infections. Certain pathogens can induce elevations in TNF-α, IL-6 and other cytokines that may have an immunosuppressive effect or may enhance HIV replication.

For these reasons, treatment with AZT alone is not sufficiently effective. In order to increase the antiviral activity of AZT on HIV, it is now proposed to combine AZT-based therapy with administration of a lysozyme dimer to inhibit TNF synthesis of an agent that antagonizes AZT's HIV antiviral activity.

Claims (7)

Patent claims A pharmaceutical composition containing a lysozyme dimer as active substance, a pharmaceutically acceptable carrier, characterized in that it contains as active substance lysozyme dimer in an amount of 33 ng-10 mg / ml, preferably 0.001-10 mg / ml, in particular 0.01-10 mg / ml , optionally in a mixture with an effective amount of an antibiotic, wherein, in the case of a lysozyme dimer content in the range 0.001-10 mg / ml, and preferably 0.01-10 mg / ml, the composition always contains lysozyme dimer in a mixture with the antibiotic as active substance. 2. A composition according to claim 1 The composition of claim 1, wherein the lysozyme dimer is substantially free of monomeric, trimeric and oligomeric forms of lysozyme. 3. A composition according to claim 1 The composition of claim 1 or 2, characterized in that the lysozyme dimer is present in an amount of 33 ng - 0.001 mg / ml. 4. A composition according to p. The method of claim 1, wherein the antibiotic is an antibiotic selected from the group consisting of penicillin, neomycin, erythromycin, cephalosporin. 5. A composition according to p. A composition according to claim 1 or 2, characterized in that it is in the form of a solution and contains the lysozyme dimer in an amount that ensures the effect of the lysozyme dimer on the microbial cell at a concentration of 5 pg / ml. 6. A composition according to p. The formulation according to claim 1, which is adapted to be administered in one or more doses of 0.02 mg / kg body weight. 7. The composition according to p. A pharmaceutical composition according to claim 1 which is in the form of an injection solution suitable for intravenous administration.