PL176407B1 - Pharmaceutical composition containing lysozyme dimer - Google Patents

Abstract

1. A pharmaceutical composition comprising a dimer of an active substance and a pharmaceutically acceptable carrier, characterized in that it contains as an active substance a dimer of an isozyme in a concentration of 33 ng-10 mg/ml, preferably 0.001-10 mg/ml, and in particular 0.01-10 mg/ml, optionally in a mixture with an effective amount of an antibiotic, wherein in the case of a dimer of an isozyme content in the range of 0.001-10 mg/ml, and preferably 0.01-10 mg/ml, the composition always contains as an active substance a dimer of an isozyme in a mixture with an antibiotic.

Description

The present invention relates to a new pharmaceutical composition comprising 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 not revealed until around 1950. Since then, the compound has been the subject of numerous studies, resulting in reports of its multiple therapeutic effects. Among other properties, antiviral, antibacterial, anti-inflammatory, and antihistaminic properties have been noted. However, the therapeutic use of lysozyme has been limited due to the negative side effects of the monomeric form.

The above limitation in the practical use of lysozyme and other therapeutically active enzymes was overcome in the late 1980s when it was found that isolated dimeric forms of the enzymes, while retaining all the advantageous characteristics of the known monomeric forms, exhibit no side effects when used therapeutically. Antiviral and antibacterial compositions containing a lysozyme dimer or other dimerized enzymes as an active ingredient are described in International Publication No. WO/11294. This application reports that in in vitro tests the lysozyme dimer inhibited the proliferation of a range of bacterial colonies grown on patient samples at a concentration of 5-20 mg/ml of culture. It also reports that the dimer was effective in the treatment of canine parvovirus (CPV) infection when administered orally twice daily at a dose of 1-2 mg/kg of body weight.

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

Research on lysozyme dimer continued with the inventor of the above-mentioned solution. Further attractive properties of lysozyme dimer were discovered, and new therapeutic applications for this agent were developed, particularly for treating certain dysfunctions of natural defense mechanisms.

Clinical trials conducted to confirm the antibacterial and antiviral efficacy of lysozyme dimer have unexpectedly revealed its effectiveness in treating acute gastrointestinal and respiratory diseases. New studies have been conducted to determine the effect of lysozyme dimer in stages of various diseases where natural defense mechanisms fail. Studies have also been conducted on the possibility of combining classical antibiotic therapy with lysozyme dimer.

It is well known that bacterial toxins constitute one of numerous aggressive factors through the action of which bacteria cause disease. Recent studies of bacterial toxins have focused on their effects on the host immune system. This effect primarily involves immunomodulation and subsequent release of cytokines and other mediators, which is the cause of many toxin-induced physiological disorders. This latter effect has been studied primarily in relation to the action of endotoxins, which play an important role in the pathogenesis of sepsis caused by gram-negative bacteria (see D.F. Bayston, J. Cohen: "Bacterial endotoxins and current concepts in the diagnosis and retreatment of endotoxaemia," J. Med. Microbiol. 1990, 31:73-83). Although the role of exotoxins in infections caused by Staphylococcus aureus and Streptococcus pyogenes has long been known, it was only the identification of staphylococcal toxic shock syndrome that led to increased interest in the exotoxins produced by these organisms.

Toxic shock is a serious illness characterized by high fever, low blood pressure, intravascular coagulation, widespread erythroderma, mucosal erythema, renal failure, hypocalcemia, hypoalbuminemia, and transient rashes (skin redness that peels off). Many cases of toxic shock have been associated with the use of vaginal tampons during menstruation, but there is now an increasing number of reports of this syndrome in cases unrelated to the menstrual cycle, in patients of both sexes, often following surgical procedures where the tamponade is left in place (for example, during nasal packing after plastic surgery or acute hemorrhage). Staphylococcal strains isolated from the vaginas of patients with toxic shock syndrome (TSS) have been shown to produce toxic shock syndrome toxin 1 (TSST-1), but the source of the TSST-1-producing organism may also be an undiagnosed infection. Initial bacteremia may be latent, but after several weeks or months it can lead to localized infections. The manifestation of such infections may be accompanied by symptoms suggestive of sepsis or septic shock. Less frequent but more dramatic bacteremia may occur in the absence of a portal of entry or associated localized infections, and in such cases shock, endocarditis, disseminated intravascular coagulopathy, and simultaneous multiorgan failure have a more severe course (see D.L. Stevens et al.: Gram-positive shock; Curret Opinions in Infectious Diseases 1992, pp. 355-363).

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Similar observations are known in cases of gram-positive bacteria. For example, infection with Streptococcus pyogenes is accompanied by shock and a mortality rate of 30%. Streptococcus pneumoniae causes pneumonia, which has been documented to be highly resistant to penicillin and prone to shock syndrome. Furthermore, AIDS patients have a higher incidence of pneumococcal infections than the general population.

Infections caused by gram-negative bacteria can also lead to septic shock. Gram-negative bacilli and vibrio bacteria are the source of the most important enterotoxins. Enterotoxin is a lipopolysaccharide (LPS) component of the outer membrane of gram-negative bacterial cell walls. Enterotoxins primarily affect the intestinal tract and typically 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 cause death in the infected individual. According to the World Health Organization (WHO), acute diarrhea kills approximately 3.2 million children in developing countries each year. Approximately 30% of all sepsis cases are caused by gram-negative bacteria.

Sepsis, caused by bacterial infections caused by both gram-positive and gram-negative bacteria, is a serious disease that occurs in all countries. In the United States, approximately 400,000 cases are reported annually, with a mortality rate of approximately 50%.

In recent years, sepsis and septic shock have been the subject of numerous publications. It has been observed that mediators of bacterial intoxication play a significant role in the pathophysiology of septic shock, endotoxemia, and other diseases. These include tumor necrosis factor (TNF), interleukin-1 (IL-1), interferon (IFN), platelet activity factor (PAF), and eicosandoids (arachidonic acid derivatives), the most important of which is TNF, which affects metabolism, the immune system, and phagocytes (see F.E. Berkowitz: Bacterial toxins in the pathogenesis of infections; Current Opinions in Infectious Diseases, 1991, 4:332-337). It has been shown that individuals who did not survive septic shock had higher concentrations of TNF and interleukin-1. Many authors have reported that patients with septic shock had elevated TNFα levels in plasma and blood. It has also been noted that the toxic effects of TNF-α do not depend as clearly on the concentration of TNF as on its persistent (excessive) presence in the body. Many authors have investigated the possibility of modulating the cytokine cascade in sepsis and septic shock. Proposed effective solutions include the use of monoclonal TNF antibodies and neutralization of lipopolysaccharides with anti-lipopolysaccharides. However, these antibodies do not enhance the clearance of bacterial debris. Partially beneficial effects have also been observed with agents such as dexamethasone and pentoxifylline, which block TNF production by macrophages.

Other cytokines are also known to contribute to septic shock. In this situation, treatments that modulate the cytokine cascade in septic shock have the potential to influence the progression of infection, as host defenses rely on these same inflammatory cytokines. Finding a means of preventing septic shock is paramount due to the potential benefit it could bring to a large number of patients. Controlling TNF levels appears to be crucial in this search.

TNF plays a similarly critical role in another defense mechanism: fever, a physiological response to infections common to all higher animal species and humans. Five pyrogenic cytokines (interleukin-1, TNF interferon, interleukin-2, and interleukin-6) have been identified to date as the main endogenous mediators of the fever response, inhibiting preoptic heat-sensitive neurons that normally facilitate heat loss and reduce heat production in the human body. Fever and its mediators have the ability to

176,407 damaging both invading organisms and the host. In recent years, considerable data have accumulated suggesting that interleukin-1, TNF, and interleukin-6 mediate the pathophysiological abnormalities of infection. Because endogenous pyrogens contribute to the pathological processes in numerous infections, both the mediators and the febrile response are potentially harmful to the host. The most compelling evidence for this claim has been gathered from studies of gram-negative sepsis. There is evidence that endogenous pyrogens mediate the systemic and local manifestations of sepsis caused by gram-positive bacteria, AIDS, spirochetal infections, meningitis, adult respiratory failure, pyrosis, and mycobacteriosis. Although the cited data contradict the observed phenomenon that the febrile response increases resistance to infection in experimental animals, the preservation of the species, rather than the survival of individual individuals, nevertheless constitutes the essence of the evolutionary process. It is considered possible that the same mechanism could cause the harmful effects of systemic pyrogenic cytokines on the outcome of general infections (e.g., gram-negative sepsis) and manifest in less fulminant infections as beneficial local febrile effects. Thus, by hastening the death of hopelessly infected individuals, nature kills individuals that pose a threat to the species. In this way, the species as a whole can be protected from 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. Therefore, therapeutic methods based on the use of antibodies against endogenous pyrogens and antagonists of endogenous pyrogen receptors may be effective in such cases. One possibility is blocking TNF synthesis. Animal studies indicate that TNF can be synthesized before IL-1 and other cytokines in the body's cascade response to infection. Therefore, for many scientists, inhibiting TNF biosynthesis also means inhibiting IL-1 synthesis. However, inhibiting TNF biosynthesis also means halting the harmful effects of some fulminant and fatal infections.

TNF is also recognized as a mediator of inflammatory processes. In many cases, inflammation is the first stage of the disease, which naturally progresses to septic shock. In situations of tissue disruption, such as wounds susceptible to infection, war injuries, especially abdominal wounds (peritonitis), gastrointestinal diseases such as acute inflammation accompanying appendicitis, acute bacterial and viral infections such as those occurring in post-influenza pneumonia, and cancers, especially during the lysis phase of tumors, inflammation is the first symptom of increased TNF production. Controlling TNF levels would therefore be a desirable model for treating such infections.

The role of TNF in AIDS itself is even more significant. AIDS is characterized by a significant weakening of immune defenses. A hallmark of the AIDS syndrome is a reduced number of CD4+ lymphocytes. The number of cells infected with HIV, the etiological agent of AIDS, is relatively low (<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 exclusive target of attack in HIV infections. Recent studies have shown that the spectrum of HIV target cells is relatively broad. Distinct differences in the effects of HIV infection have been observed in monocytes/macrophages compared to T lymphocytes. While T lymphocytes tend to be destroyed, monocytes/macrophages allow the infection to persist. The HIV virus can therefore grow and aggregate in monocytes/macrophages as well as in other body cells. The type of response to HIV infection that is specific to mono6

176,407 cytoplasmic cells/macrophages may be responsible for latent viral carriage by the host; this response may also result in pathogenic sequelae induced 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). Numerous researchers have reported that human T cell lines infected with HTLV-1 are highly susceptible to HIV infection, resulting in a dramatic cytopathic effect coupled with enhanced HIV replication. Furthermore, HIV-infected cells are susceptible to damage by their supernatants. Determining viral load after challenge with such a supernatant has shown that a factor produced by T cells (MT-2) enhances HIV replication. This factor was identified as TNF-β, and this finding is consistent with reports that T cells (MT-2) produce TNF-β. The same effect is observed with TNF-α. 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 its levels. This suggests that the same upregulation of HIV expression may also occur in vivo. Of course, the potentiating effect of TNF can be neutralized by anti-TNF antibodies. The enhancement of HIV replication after treatment with TNF-α and TNF-β is more than tenfold (compare A. Yakamam et al.: Tumor necrosis factor (α,β) induced by HIV -1 in peripheral blood mononuclear cells potentiates virus replication; AIDS 1990, 421-427).

It has also been confirmed that various 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 using heat-inactivated HIV. Based on these and a number of similar observations, as well as the report by Toshifumi Matsuyama et al. (op. cit.), one may be led to believe that AIDS is a cytokine- or TNF-dependent disease. In the AIDS cytokine network, both TNF-α and TNF-β appear as key molecules that enhance HIV replication and induce their own expression and the expression of other cytokines. TNF-α has been shown to stimulate the release of other cytokines in various cell types, making it a key cytokine in the cytokine cascade in the basic defense mechanism.

It has been suggested that many symptoms associated with AIDS may be explained by the release of cytokines with various biological functions. Increased production of IL-1 and TNF-α, two known pyrogens, may explain the fever observed in AIDS patients. TNF-α may be associated with AIDS-associated 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 may directly kill HIV-infected cells, contributing to HIV replication. An immunological mechanism has also been proposed to explain the reduction of CD4-T cells in AIDS patients (Matsuyama et al., op. cit.). Kaposi's sarcoma has also been reported to be induced by TNF-α: TNF-α can be produced from keratinocytes in response to physiological stimuli such as ultraviolet light, which may contribute to the induction of IL-6 in the skin and the development of AIDS-related Kaposi's sarcoma. In vitro, TNF-α can cause damage to myelin and oligodendrocytes, and some glioma-derived cell lines have been shown to be susceptible to the antiproliferative effects of TNF-α. These observations may lead to the conclusion that central nervous system dysfunction in AIDS patients is a result of TNF-α. Several reports have confirmed that serum levels of TNF-α and IL-1 increase significantly with the development of AIDS and ARC (AIDS-related complex), whereas in asymptomatic HIV-positive individuals, they remain at the same levels as in healthy controls. According to Matsuyama et al. (op. cit.), AIDS is as much a disease associated with TNF as with HIV. This demonstrates that achieving control over iUF induction can lead to effective therapy for AIDS patients.

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The aim of the invention is to develop a composition useful in the prevention and treatment of the above-described disease states.

This object is 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 therefore exhibits activity that allows its use in connection with the above-described pathological conditions, including in the treatment, alleviation of the course and even to some extent in the prevention of diseases associated with excessively high levels of TNF, described above.

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

The composition according to the invention, comprising a lysozyme dimer and an antibiotic, is characterized in that in an in vitro test in the presence of bovine serum the activity of the antibiotic is up to 100% higher than in the same test without the addition of the 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 of the invention contains lysozyme dimer in an amount of 33 ng - 0.001 mg/ml.

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

Preferably, the composition according to the invention containing a lysozyme dimer and an antibiotic is in the form of a solution and contains a lysozyme dimer in an amount ensuring the effect of the lysozyme dimer on the microbial cell at a concentration of 5 pg/ml.

Preferably, the composition of 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 injectable solution suitable for intravenous administration.

The lysozyme dimer constituting 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 the secretion of tumor necrosis factor (TNF) and inhibiting the biosynthesis of tumor necrosis factor (TNF) in animals and humans.

Lysozyme dimer is particularly beneficial in diseases associated with elevated or excessively high levels of tumor necrosis factor (TNF), including those not caused by viral or bacterial infections. One such condition is HIV infection, which includes asymptomatic stages and the AIDS-related ARC complex. Controlling the release of tumor necrosis factor (TNF) during these stages may delay the onset of full-blown AIDS symptoms.

Other conditions of this type include inflammation and accompanying fever, as well as sepsis, septic shock and cachexia.

According to the invention, lysozyme dimer is used in conjunction with an antibiotic. Lysozyme dimer therapy can also be used alongside standard AZT therapy.

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Antibiotics administered in combination with lysozyme dimer are more effective than those administered alone. The effective dose of the antibiotic is reduced by approximately 33% to approximately 50% compared to the effective dose of the same antibiotic in known formulations containing the antibiotic alone. This is particularly true for penicillin, neomycin, erythromycin, and cephalosporin (SEFRIL).

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

Preferably, the carrier is a non-pyrogenic sterile composition comprising a physiologically acceptable solvent and a pharmaceutically acceptable preservative. Preferably, the non-pyrogenic sterile composition comprising at least one physiologically acceptable solvent and/or at least one pharmaceutically acceptable preservative is non-pyrogenic sterilized water or an aqueous PBS solution as solvent and thiomersal as a preservative acceptable for use in protein pharmaceuticals. Preferably, the composition of the invention is in the form of an injectable solution suitable for intravenous administration.

The injectable composition of the invention can also be administered intramuscularly and subcutaneously. In some applications, it may be appropriate to simultaneously or independently administer the same liquid composition intrauterinely or intramammarily – or locally, optionally in conjunction with other external preparations.

Another beneficial use of lysozyme is in a tampon or 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 dimerized form of lysozyme is preferably an isolated, purified lysozyme dimer. In some applications, it is possible to use compositions that, in addition to the lysozyme dimer, contain small fractions of trimers and higher oligomers of this enzyme.

The dimerized form of lysozyme is obtained by controlled polymerization of the enzyme monomer, followed by careful purification, in particular by removing the monomeric form with known toxic side effects and the trimer and higher oligomer fraction from the reaction mixture. Any known polymerization method can be used to obtain the dimerized form. One possible method, including purification steps, is described in International Publication No. W0 91/10731.

Previous preclinical studies of lysozyme dimer revealed no mutagenic or teratogenic properties and showed no or only low tolerability. The single-dose toxicity (acute toxicity) following oral or topical administration ( _LD50 ) could not be determined (>2000 mg/kg), and following intravenous administration, the _LD50 was >1000 mg/kg.

The new applications of lysozyme dimer now being disclosed have a solid foundation in in vitro testing and in vivo clinical applications to support them. Some comparative studies have also been conducted.

It is believed that the inhibitory effect on TNF release has been so effective due to the broader spectrum of activity of the lysozyme dimer, particularly its ability to induce IFN release and its potentiating effect on phagocytosis. Both of these additional properties are important factors in natural defense mechanisms. Therefore, the therapeutic and prophylactic effects of the novel applications of lysozyme dimer described above are supported by the natural defense mechanisms enhanced by the dimer of this enzyme.

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The present invention will be further explained 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 performed in lymphocyte cultures stimulated with a suboptimal dose of ConA in the presence of lysozyme dimer at various dilutions.

Figure 2 illustrates the level of TNF in the blood of calves (determined experimentally), and

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

Example I.

In order to determine the immunoactivity of lysozyme dimer, tests were performed on human peripheral blood lymphocytes (CLL), the results of which were evaluated using the FACS analytical method.

Mitogenic stimulation of human peripheral blood lymphocytes is a commonly used method to study the reactivity of most immune cells. To examine the effect of therapeutic substances on the activation and proliferation of lymphocytes from healthy blood donors, the mitogen is administered at suboptimal doses, and the lymphocyte response is ultimately measured quantitatively by assessing appropriate immunologically relevant parameters. The obtained results are evaluated by comparison with those obtained from control cultures without the addition of therapeutic substances.

Lymphocyte stimulation was achieved with ConA at a concentration of 20 pg/ml of medium. The initial cell concentration was ¹⁰⁶ cells/ml. Short-term cultures incubated in a _CO2 atmosphere were maintained for one (IL-2 receptors on lymphocytes and HLA-Dr) or two days (all other assays). 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 T-helper lymphocytes)

- interleukin-6 (cellular differentiation hormone)

- tumor necrosis factor - TNF (vasoactive multi-target interleukin)

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

- thymidine kinase (an enzyme activated 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 proliferating cells)

- HLA-Dr lymphocyte (class II histocompatibility antigen, activated during the immune response).

The following observations were made in the supernatants of the cultures conducted:

Neopterin—produced by T lymphocytes during the immune response and in stimulated cultures—was not significantly elevated in the present experiments by lysozyme dimer compared to the control value observed in CoA-stimulated cells without the addition of the tested dimer. Neopterin values were slightly elevated with increasing concentrations of the tested dimer.

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

IL-2 receptors - eliminated from the lymphocyte surface during culture and giving information about the total number of transformed IL-2 receptors - were at a level comparable to

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

Interleukin-6 exhibited a clear, dose-dependent trend toward higher values at higher concentrations. This molecule is crucial for hematopoiesis, cell differentiation, and the immune response. In our assay, the first three values had to be extrapolated because the highest standard corresponded to 2000 pg/ml.

The results for other molecules obtained in the conducted 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 levels, which proved ineffective in suppressing TNF concentration.

Table 1

The effect of lysozyme dimer on human peripheral blood lymphocytes

No. 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

The effect of lysozyme dimer on human peripheral blood lymphocytes

No. Sample TNF Thymidine kinase 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 lymphocyte cytoplasm after freezing and thawing of the cell mass. Thymidine kinase is stimulated to increase its synthesis in dividing cells, making it a good marker of cell proliferation. The data in Table 1 show that suppression occurs at the highest tested concentrations of lysozyme dimer, while no clear trend can be detected at the remaining dilutions. Interferon-α, on the other hand, under the same conditions exhibits values higher than the control value achieved with concavalin A (ConA) alone at the higher tested concentrations. A significant increase is visible from the second to the third dilution.

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Lymphocyte markers:

HLA-Dr/CD3, a marker of histocompatibility, is expressed on activated T lymphocytes during the immune response. The obtained results indicate a certain percentage of activated T cells in the control culture; at different lysozyme dimer concentrations in the cultures, the observed values vary 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 upon activation by IL-2. IL-2 exists in both autocrine and paracrine forms. T-helper lymphocytes not only produce IL-2 but are also stimulated to proliferate by this molecule. IL-2 receptors on the surface of T-helper lymphocytes are stimulated to increased synthesis upon activation. Table 1 shows that at the highest concentrations of lysozyme dimer, there is a marked 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 on 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 assessment of stimulated cells and in the diagnosis of cancer. The presented results show a slight inhibition of cell proliferation based on Ki-67+ suppressor cells (CD8) at the highest two doses of lysozyme dimer. A significant increase in the percentage of positive cells was observed in helper lymphocytes (CD4) at the highest concentration. At lower doses, the percentage of cells expressing Ki-67/CD4 was slightly higher than in the control culture.

The marked suppression of TNF is illustrated in Figure 1.

The immunological parameters mentioned above and selected due to their potential importance in the immune response were analyzed using a method based on testing the effect of the test substance on human peripheral blood lymphocytes stimulated suboptimally with concavalin A (ConA), a well-known, highly sensitive agent that allows for the assessment of many different parameters. At certain concentrations of lysozyme dimer, there were clear differences in the studies compared to the values observed for lymphocytes stimulated only with ConA, while in the case of, for example, TNF and INF-α, clear effects were observed at all tested concentrations.

Example II. Laboratory studies were conducted to determine the effect of lysozyme dimer on the phagocytic activity of milk and blood cells in vitro. It had previously been determined that, in standard in vitro tests, lysozyme dimer did not inhibit the proliferation of microorganisms isolated from infected mammary glands of cows. Because clinical studies demonstrated that lysozyme dimer eliminates infection in the mammary glands of cows when administered intramammarily and simultaneously intravenously, it became clear that the antibacterial mechanism in the mammary glands of cows is phagocytosis. Therefore, blood and milk from both healthy and infected cows were used in in vitro tests to examine the effect of lysozyme dimer on phagocytosis. For comparison, studies were conducted with the same amount of the test substance and the same incubation time, using cells isolated from healthy and infected cows, and even from infected and healthy mammary quarters of the same cow, to eliminate individual differences in the observed responses.

Both a purified form of dimerized lysozyme and a mixture of the dimer and small fractions of trimers and higher oligomers of lysozyme were used in the study. 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 (phagocytosis index according to the method of Wiśniewski 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- and incubation-time-dependent. Higher concentrations of lysozyme dimer are necessary to activate leukocytes in milk than to activate leukocytes in blood. Excessively high concentrations of lysozyme dimer reduce phagocytic activity in vitro. Selected experimental results—showing that, as long as the post-reaction mixture does not contain the cytotoxic monomeric form of lysozyme, comparable results are obtained for the highly purified dimer and the less thoroughly purified dimer—are presented in Tables 2 and 3 below, in which the term "lysozyme dimer +" is used to designate a mixture containing small fractions of the trimer and higher lysozyme oligomers mentioned above.

Table 2

The 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 No. 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

The 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 milk of an infected cow (No. 490) Parameter preparation quarter infected */ healthy quarter control 98 58 % phagocytosis lysozyme dimer+ 98 90 lysozyme dimer 100 100 index control 10.9 2.7 phagocytic 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

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

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The following examples include reports of in vivo studies. In the clinical studies, a composition containing 2 mg of the isozyme dimer in 10 ml of PBS solution was used. This composition was designated KLP-602.

Example III. When administered intravenously, the KLP-602 isozyme dimer has been shown to stimulate the phagocytic activity of blood granulocytes in healthy and sick calves and healthy foals, as well as in lactating cows after intramammary administration. This effect is manifested by increased neutrophil counts and an increased ability to absorb staphylococci and reduce NBT. This phenomenon occurs primarily during the first 12-24 hours after injection. The effect of KLP-602 on phagocytic activity in the udder depends on the dose and form of the agent, as well as on the individual response of the animal.

The isozyme dimer was used to treat infectious diseases in cattle, pigs, horses, and dogs. The tested agent was administered at various doses and at various time intervals intravenously, intramuscularly, subcutaneously, intramammarily, and intrauterinely. The agent was administered to 346 cows, 274 calves, 110 sows and boars, 294 piglets, 709 unweaned piglets, 35 foals, and 107 dogs. Control groups were treated using alternative methods; no animal was left untreated due to the risk of death. Nevertheless, comparative conclusions can be drawn based on the conducted studies, taking as a reference the clinical picture of the treated diseases known from the veterinary literature.

Example IV. In studies conducted on cuts, IFN and TNF levels were measured in the blood of healthy and infected animals. Diseased animals remained untreated for the first 12 hours of infection and were then treated with antibiotics and a highly purified isozyme dimer. The results are graphically illustrated in Figures 1 and 2, which present the results obtained for TNF and IFN, respectively. Both figures represent the actual results obtained in four individually identified cuts. In both figures, line 1 corresponds to the results obtained for a healthy cut. Line 2 corresponds to the results obtained for a diseased and untreated cut (the IFN value at 12 hours, equal to 45 U/m, is not shown, but the slope of line 2 on the graph corresponds to the direction of this value). After 12 hours, the animal was treated by a known method; line 3 - for the cut treated with antibiotics and line 4 - for the cut treated with KLP-602.

In another study, in a group of chicks treated with the isozyme dimer—one or two injections of KLP-602—over 90% of chicks suffering from gastroenteritis and over 85% of chicks suffering from bronchopneumonia recovered. Rapid resolution of symptoms such as fever and diarrhea was typical. Treatment with the study agent did not require fluid replacement. The time and rate of recovery were faster than in the control group treated with antibiotics.

Example V. KLP-602 was most effective in curing diseases in pigs. Full or almost 100% of animals recovered from the following diseases: postpartum mastitis (MMA syndrome), dysentery, purulent metritis, influenza, and E. coli infections. Administration of the test agent to cases of edema disease and bronchopneumonia had a less dramatic effect, but the results were nevertheless significantly better than those obtained with the established method. Rapid resolution of diarrhea (usually within the first 24 hours) and fever was observed, as well as return of lactation—particularly significant in cases of postpartum mastitis and metritis (saving the lives of piglets). The effectiveness of KLP-602 treatment compared to the results of alternative treatment is illustrated below in Table 4.

Example VI. When treated with the KLP-602 preparation, 100% of foals suffering from enteritis and 83% of foals with bronchopneumonia recovered faster than animals in the control group treated traditionally.

176 407

Example VII. KLP-602 was tested in the treatment of several canine diseases, including folliculitis (100% cure rate), upper and lower respiratory tract infections, and gastrointestinal infections manifesting as diarrhea. Parvovirus, known to be a virtually incurable disease, predominated in this group. Nevertheless, recovery was achieved in approximately 75% of parvovirus cases.

In tests carried out on animals affected by the diseases resulting from natural infections described above, a number of important observations were made:

1. The therapy proved effective and very simple in treating diseases with multifactorial etiology and pathogenesis (diseases of this type dominate farm animal populations and are difficult to eradicate, especially in farms where epidemic spread of diseases is very easy). These results demonstrate the modulatory effect of lysozyme dimer on natural defense mechanisms.

2. The therapy has proven effective in diseases whose natural progression may progress through a stage of endotoxic shock or disorders such as high and prolonged fever, which impact the animal's condition long after recovery, as is the case with horses (foals) bred for racing and other sports, and other animals bred for the food industry, where breeding costs are a significant factor. Rapid recovery will translate into a significant reduction in the cost of treating such diseases.

3. The results observed in vivo confirm the reduced 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 antibiotic activity against various microorganisms was studied to select bacteria for further testing, determine the most effective antibiotic doses against the selected microorganisms, and establish the lysozyme dimer concentration range within which the desired effects could be expected. Two batches of lyophilized purified lysozyme dimer were used in these studies: one produced in 1991, and the other in 1992. Antibiotics available on the Polish market, manufactured by POLFA, a Polish pharmaceutical manufacturer, were used in the studies, including penicillin, neomycin, erythromycin, and cephalosporin (SEFRIL).

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

Table 4

Animals treated with KLP Animals treated with other means Number of cases Time (days) Healed Unhealed Number of cases Preparation used U % 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 Table 4 continued

II. Dysentery in Piglets 29 1-2 29 100 - - 219 Antibiotics STOLMED RIDOWET 5-9 75 III. Postpartum agalactia 30 1-2 30 100 - - 40*/ antibiotics cortisone, sulfa drugs 3-5 80*/ IV. Piglet Flu 38 1-5 38 100 - - 200*/ Antibiotics (long-acting preparations) 3-9 60-80*/ V. Edematous 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. Preparing the tiles

A 0.05 ml aliquot of an 18-hour culture broth of the bacterial strain being tested was added to a 14 ml sample of enriched agar (Biomed). The mixture was mixed and poured onto 10 mm diameter dishes. After the agar cooled and solidified, sterile cylinders were placed on the agar and filled with the antibiotic solution being tested.

2. Preparation of antibiotic solutions: A sample of the antibiotic under test was first dissolved in PBS and then the necessary amount of this solution was added to a previously determined volume of PBS or bovine serum to obtain an antibiotic concentration in the solution as close as possible to the MIC (minimum inhibitory concentration) value; solutions of three different concentrations were always prepared.

3. Preparation of Lysozyme Dimer Solution: 1 mg of lyophilized lysozyme dimer was initially dissolved in 10 ml of PBS. Further dilutions were prepared 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 antibiotic concentration was the same in each cylinder; the lysozyme dimer concentration 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 assessing the activity:

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

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

176,407 suspension of the 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 the suspensions in PBS and larger than those around the cylinders filled with the antibiotic alone in serum without the addition of lysozyme dimer.

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

Conclusions:

Lysozyme dimer (preservative-free) at a concentration of 5 pg/ml exhibits in vitro synergism with certain antibiotics used at MIC (minimal inhibitory concentration) in the presence of bovine serum in inhibiting bacterial growth. The results obtained so far indicate that further research will be important.

Example IX. Synergism with AZT in the treatment of AIDS patients

Ito et al. recently reported that TNF-α can antagonize the antiviral activity of AZT against HIV (M. Ito et al. “Tumor necrosis factor antagonizes the inhibitory effect of azidothymidine on human immunodeficiency virus (HIV) replication in vitro.” Biochem. Biophys. Res. Commun, 1990, 166; 1095-1101). AIDS patients in advanced stages of the disease are subject to numerous opportunistic infections. Some pathogens can induce an increase in TNF-α, IL-6, and other cytokines, which may have an immunosuppressive effect or may enhance HIV replication.

For these reasons, treatment with AZT alone is not sufficiently effective. To enhance the antiviral activity of AZT against HIV, it is currently proposed to combine AZT-based therapy with the administration of a lysozyme dimer to inhibit the synthesis of TNF, a factor that antagonizes the antiviral activity of AZT against HIV.

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.