MXPA97005246A - New applications of dimero de lisoz - Google Patents

Abstract

The present invention relates to pharmaceutical compositions containing a lysozyme dimer, preferably of high purity, ie with at least 10% by weight or less of unwanted by-products, and new applications thereof. Such applications include topical, oral and parenteral administration of the said compositions for non-specific immunostimulation as a measure of preventive and / or therapeutic treatment of diseases of humans and animals comprising cancer, hair growth disorders, fish diseases and diseases of bees The non-specific stimulation of the immune system is induced by a single or repeated application of the lysozyme dimer composition, preferably at concentrations of 5 to 500æg / kg body weight.

Description

NEW APPLICATIONS OF DIMERO DE LISOZIMA DESCRIPTION Background and field of the invention The present invention relates to new applications of lysozyme diiod and to compositions containing such a dimer. The new applications are based on a common principle of non-specific stimulation of the immune system and are particularly useful for the prevention and / or treatment of symptoms or diseases in relation to an impaired function of the natural defensive and regenerative systems in the human and animal body. . At the end of the eighties it was discovered that the dimer forms of certain enzymes, while substantially retaining the beneficial properties of the corresponding monomers, turned out to be much less toxic than the monomers and in some cases did not even have negative side effects at all when used in therapeutic doses. The antiviral and antibacterial compositions comprising as the active ingredient the dimer of lysozyme or other dimeric enzymes have been described in WO 89/11294. It is reported there that the lysozyme dimer is able to inhibit the proliferation of a number of bacterial strains grown in samples taken from patients, when applied at concentrations of 1.25-20 mg / ml of the culture. It is also reported that dimer is effective in treating canine parvovirus (CPV) infections when orally administered twice a day at a dose of 1-2 mg / kg body weight. Later, additional attractive features of the lysozyme dimers were found and additional therapeutic applications of the drug were developed, especially for the treatment of bacterial and viral infections as described, for example, in WO 94/01127. In WO 94/01127 a model theory is presented that can help to understand the different effects observed with the lysozyme dimer. Although the complete mode of action of the lysozyme dimer has not yet been fully understood, it appears that there is an additional curative ability that can not be explained by the bacteriolitic activity of the corresponding monomer. The inventors observed certain immunostimulatory effects of lysozyme dimer, particularly with respect to the modulation of cytokine levels. In addition, from their experiments they concluded that the lysozyme dimer appears to prevent the penetration of bacterial cells by viruses, presumably by blocking certain regions of the cell's outer surface and probably comprising viral receptor proteins. The prior art describes additional results obtained in vi tro with lysozyme dimer. Particularly, Bartholeyns and Zenebergh (Europ. J. Cancer, Vol. 15, 1979, 85-91) tested the lysozyme dimera for cytostatic activities against liver cancer cells (HCT) in vitro. They observed an inhibition of 73% ± 15% of the multiplication of the cancer cell in the cell culture (ibid., P.89, Table 2). Surprisingly, except for WO 94/01127, no experiment has yet been reported with lysozyme dimer 5 in vi vo. It is very strange and shocking and until now awaits an explanation why neither Bartholeyns and Zenebergh nor any other researcher resumes this issue to promote the further development of a promising discovery to fight cancer. A comparative sample (Fig. 1) of the purity of , Or lysozyme dimer produced according to the procedure of Sorrentino et al., Eur. J. Biochem. 124, 183-189 (1982) and the lysozyme dimer preferably used in the present invention revealed at least one possible reason: high concentrations of by-products such as monomer are found, trimer and tetramer of lysozyme in the preparation produced according to Sorrentino et al., While the product used in the present invention is highly purified, that is, it contains the desired lysozyme dimer in amounts of up to 90% by weight of the total lysozyme fraction of the preparation. A process for making such a highly purified lysozyme dimer has been described in WO 91/10731. This strongly supports the hypothesis that the purity of the lysozyme dimer of the prior art simply does not was good enough for experiments and applications in vivo because it was already known in the art 15 years ago that the monomeric form of lysozyme, in spite of its beneficial antibacterial activity, is rather toxic and can cause acute inflammations and allergies and even symptoms of toxic shock. In light of such circumstances it seems more understandable why no competent researcher including Bartholeyns and Zenebergh - although they recommend the dimer of .Lisozyme as a promising candidate for further research - have conducted additional experiments over the past 10 to 15 years to develop applications. of lysozyme dimer in vivo. Despite such a deficiency of investigative activities of the scientific world possibly due to a prejudice of the technique against the use of lysozyme dimer in vivo, the present researchers have carried out a work of development and additional research, to improve the research procedure and purification of the dimeric lysozyme and to find in vivo applications in humans and animals for the product that led, for example, to the antiviral and antibacterial applications described in WO 94/01127. On the other hand, based on their knowledge of the low toxicity of the dimeric lysozyme compared to that of the monomer and on the availability of a novel, highly purified lysozyme preparation, the present inventors tested and started cancer experiments with preparations of lysozyme dimer in vivo, although the prior art did not suggest its use to treat diseases other than bacterial or viral infections. One of the major advantages of the present invention is to prepare for the use of a highly purified lysozyme dimer for the manufacture of a pharmaceutical composition that complements or replaces the extremely toxic anti-cancer drugs usually applied in conventional chemotherapy.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 presents a comparative sample of the purity of lysozyme dimer preparations made according to two different techniques of the prior art.

Detailed description of the invention The highly purified lysozyme dimer referred to in the present invention can be used for the preparation of pharmaceutical compositions directly applicable for the treatment of animals and humans due to its low amount of toxic raonomer. As indicated above, such a purified lysozyme dimer contains about 10% by weight or less of unwanted byproducts, and can be obtained by dimerization of lysozyme monomers of any origin, for example of lysozyme monomers derived from humans, animals, eggs, plants, microorganisms, the monomers being either naturally isolated in pure form or made by chemical or genetic engineering procedures to produce lysozyme monomers thereof or essentially of the same chemical and biological nature from which they originate naturally. 5 In tests on cell cultures in vi tro y. In vivo test models, preferably test models of mice and rats, the inventors of the present invention were able to demonstrate successfully that the applied lysozyme dimer compositions present an extraordinary potential / • or in the inhibition and even in the total prevention of the proliferation of cancer tissue cells in vi tro and in vi vo. Such curative action could be confirmed by various types of cancer, including but not limited to those that are known in the art to be induced or affected by viruses. These viruses include, in particular, the human papillomavirus, Epstein-Barr virus, hepatitis B virus and retroviruses such as HTLV-I (adult T cell leukemia virus), HTLV-II (lymphoma virus) and HIV-I (human immunodeficiency virus) including subtypes. The types of cancer induced or affected by the action of these viruses comprise, ie, carcinoma of the cervix, carcinoma of the urogenital tract, Kaposi's sarcoma, primary carcinoma of the liver cell, leukemia, T-cell lymphoma, AIDS, or any subtype of them. A The object of the present invention is to prepare a pharmaceutical composition and a method for preventing and / or treating cancer in animals and humans.

Another beneficial application of the highly purified lysozyme dimer was surprisingly discovered in clinical trials when the lysozyme dimer compositions were administered to patients with more or less infected postoperative wounds, for example after amputation of a lower limb. It turned out that the local application of the dimer not only defeated the infection of the wound but also stimulated the growth and renewal of the hair within the treated area. This surprising discovery led to further investigations and finally to the elaboration of applicable pharmaceutical compositions in cases of hair growth diseases, particularly hair growth diseases based on malfunctioning or immunological dysfunctions such as, for example, in the case of alopecia areata. . Such compositions can at least partially meet the criteria of a long-desired composition by acting as a hair growth stimulant. The fundamental principle of this completely unexpected effect is believed to include among others the stimulation and improvement of immunodefensive mechanisms, improvement of blood circulation in the layers of superficial tissue and skin, general non-specific improvement of immunological functions and, probably, some effects not yet identified. Another surprising and very commercially interesting application has been found for a pharmaceutical composition containing lysozyme dimer in fish farming and beekeeping when in a mass examination experiment conventional antibiotics have been replaced with different non-specific immunostimulants also including the lysozyme dimer. The use of immunostimulants in fish culture for the prevention and / or therapy of fish diseases is a promising new development (Siwicki and Anderson 1990, Siwicki et al 1994). In the present the substances to treat fish diseases include antibiotics, drugs and chemicals used to sterilize ponds for the conservation of fish (Stoskopf, 1993). While each therapy is at least partially effective in the treatment of a particular disease, problems arise with the accumulation of these substances in the environment and in the food of the fish as well as with the appearance of pathogenic strains of microorganisms resistant to various antibiotics. In the case of beekeeping it is a widespread practice to kill bee colonies if there is evidence that the bees are affected by microbes, for example bacterial and / or viral infections. Such practice prevents the spread of infections among other colonies and due to this minimizes losses. There is therefore a need to provide an alternative and reliable method for the prophylactic and / or therapeutic treatment of honey bees that combines both the advantage of an antibiotic that further strengthens the immune system of bees and an antibiotic that will not result in the development of antibiotic-resistant microbial strains. The present invention prepares a method to achieve such a need. Furthermore, even if the lysozyme dimer were present in the final honeydew product in obnoxious concentrations this could be considered as an improvement in quality rather than a change for the worse, due to the benefit of the non-specific immunostimulatory effect of the lysozyme dimer. In general, immunostimulants comprise a group of biological and synthetic compounds that increase non-specific cellular and humoral defense mechanisms in animals and humans. Immunostimulants such as betaglucan, chitosan, levamisole, oligominerals and mineral combinations, and various derivative products Many plant and animal sources are effective in preventing diseases. Several types of beta-glucans appear to be especially promising for the stimulation of the fish's non-specific cellular and humoral immune response. Non-specific defense mechanisms that include phagocytosis and the production of oxidant radicals are rapidly stimulated by immunostimulants and are rapidly prepared to protect the fish against pathogenic microorganisms such as, for example,, viruses, bacteria, mycoplasmas and fungi, and / or against parasites or other pathogens. Thus, these mechanisms are superior to the specific immune response that requires a longer period of time for the development of a specifically adapted immune response including antibody formation and specific cell activation. The very encouraging results obtained in the first experiments in vi, which indicate a significant improvement of the immunological defense mechanisms, have led to launch a detailed research program on the effect of the lysozyme dimer on the specific cellular and humoral defense mechanisms. and not specific to fish, particularly trout and salmon, and honey bees. Therefore, it is an object of the present invention to provide new applications of lysozyme dimer comprising its use for the manufacture of a pharmaceutical composition for the stimulation of a cellular and humoral immune response in fish and honey bees. It is another object of the present invention to provide a method for the use of pharmaceutical compositions containing said lysozyme dimer to induce a non-specific stimulation of the immune system by a single or repeated application of the composition, for the prevention and / or therapy of diseases of natural origin of fish and bees. Experiments in vi have shown that in some cases, for example, where fish are easily harmed by certain chemicals or undesirable bioactive substances such as byproducts of drugs that have been administered to them, it may be preferable to treat the fish with the dimer of highly purified lysozyme reported in W091 / 10731 containing about 10% by weight or less of unwanted by-products and which is essentially free of the monomeric form of lysozyme. In order to reduce reproduction costs in fish farming and / or beekeeping, it could, however, be It is acceptable to administer a low purity lysozyme dimer preparation as long as it is applied at a dose that does not cause adverse effects due to the presence of toxic byproducts, especially of lysozyme monomer. The lysozyme dimer referred to herein can be applied either directly to patients who need it as it can be used for the manufacture of pharmaceutical compositions to be applied in customary galenic forms. Gels, ointments, or liquid compositions comprise the lysozyme dimer preferably in a concentration of about 0.1-10 mg / ml and often at a concentration of about 0.1-1.0 mg / ml. They are usually prepared as sterile and pyrogen-free compositions and optionally also comprise at least one physiologically acceptable solvent or vehicle and / or at least a suitable preservative. Pharmaceutical compositions containing lysozyme dimer are useful and are intended primarily for topical and / or parenteral application comprising a local injection, for example, in the vicinity of a solid tumor, or external applications on the surface of the body including subcutaneous injection. Intravenous injections may replace or further support topical applications during the course of therapy. However, it has also proved very effective to administer compositions of lysozyme dimer to mucous membranes, preferably by inhalation (nasal, buccal, pharyngeal mucosa) of liquid compositions or application topical (eg, vaginal, cervical mucosa) of liquid or cream compositions or buffers impregnated with lysozyme dimer material. In some cases it is preferred to apply the lysozyme dimer orally preferably in galenic forms I, * customary such as for example tablets, capsules or -gragees or in the form of agglomerates, granules, flocculated or as a powder. These solid compositions frequently contain the active drug in an amount of about 0.1-10 mg, preferably about 0.1-1.0 mg per g of the total composition. It is also preferable that they comprise, in addition, at least one suitable vehicle and / or preservative and / or other customary additives such as, for example, a flavor or a dye. In case of solid compositions to treat fish can be added to the nutrients. Alternative or Additionally, the lysozyme dimer can be dissolved in the pond water for fish conservation because it is believed that the active drug can also be reabsorbed through the fish's gills. In the case of compositions of lysozyme dimer in beekeeping can be applied by dissolving the solid compositions in drinking water, tea, sugar solutions or other usual liquids prepared for bees, for example as a substitute for feeding. However, it can also be prepared as an aqueous solution - preferably concentrated - and mixed with honey, in order to ensure the ingestion of the drug by bees. However, a composition for oral use may also be in the form of an osmotic system. In certain cases such as, for example, in order to prevent and / or treat cancer of the urogenital tract, local application of antiseptic dressings or tampons impregnated with an effective dose of the highly purified lysozyme dimer can be useful and beneficial. The various types of the lysozyme dimers compositions mentioned above are administered in a single or repeatable dose of about 0.005 to 0.5 mg / kg of body weight, especially at a dose of about 0.01 to 0.1 mg / kg of body weight. It is valid without mentioning that the required concentration of active lysozyme dimer in the final pharmaceutical composition depends mainly on the size of the human or animal patient and the type of therapy or prophylactic treatment programmed for the patient in question. NeverthelessIn many cases, the aforementioned concentration ranges are sufficient for an adequate treatment. However, it may be necessary, particularly in veterinary medicine, to increase the actual concentrations of lysozyme dimer in the composition to a value beyond 10 mg / ml and below the solubility product of the dimer in the respective solvent, i.e. to about 20 mg / ml of liquid or ointment. By doing so, the volume of the composition to be administered can maintain a reasonable minimum for ease of handling. If possible, a combined administration protocol of the aforementioned compositions is preferred to a single application therapy whether oral, parenteral or topical. Due to the essentially non-toxic nature of the highly purified lysozyme dimer compositions containing such a dimer can be administered over a prolonged period of time, i.e. months and even years without causing harmful side effects. The time intervals for prophylactic or therapeutic administration of the drug can typically range from daily doses of 1 or more times to weekly and monthly and may also comprise even larger intervals, depending on the respective patient and the urgency of the treatment as well as the efficacy of immunostimulation by the lysozyme dimer. As indicated above, the present invention was made at least partially possible through the availability of the high-grade lysozyme dimer. The striking difference in the quality of the product and, in particular, the participation of the desired lysozyme monomer, is demonstrated by Fig. 1: Line 1 represents standards of LMW (low molecular weight) proteins previously stained: Phosphorylase B 142,000 dalton , BSA (bovine serum albumin) 97,000, ovalbumin 50,000, carbonic anhydrase 35,100, trypsin inhibitor of soybean 29,700, lysozyme 21,900 (Biorad, USA); lines 2 and 3 show purified lysozyme dimer LYDIUM KLP® 602 (KLP-602), batch 506449, laboratory control; line 2 loaded with 6.6 μg and line 3 with 19.8 μg; lines 4 and 5 show another batch of KLP-602, line 4 loaded with 6.6 μg and line 5 with 19.8 μg; lines 6 and 7 show a lysozyme dimer preparation (KIW-607) prepared according to Sorrentino and J * O col., Eur. J. Bioch. 124, 183-189 (1982); line 6 loaded with 6.6 μg and line 7 with 19.8 μg. The purified lysozyme dimer preparation KLP-602 contains four times more dimer than the compared product KIW-607, while the compared product KIW607 contains six times more monomer than KLP-602. In order that the invention described herein be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and should not be interpreted as limiting this invention in no way.

Example 1: Lysozyme dimer (KLP-602) in the treatment of lymphatic leukemia in AKR mice.

Materials and procedures Seven-month-old mice of both sexes of the AKR leukemic strain, inoculated with Graffi virus from the Animal Inbreeding Center, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, were used in the study. In AKR mice the early activation of endogenous leukemic virus takes place, leading to clinical manifestations of lymphatic leukemia at the age of seven to nine months. The mice were divided into three groups of 30 animals each: Group I (experimental) - animals KLP-602 were administered subcutaneously once at a dose of 20 μg / kg in 0.3 cm3 of PBS; Group II (experimental) - the animals received KLP-602 twice, at the same dose as before; Group III (control) - animals received 0.3 cm3 of PBS twice. The mice were sacrificed by bleeding three and six weeks after the last injection of KLP-602 (10 mice from each group). Ten mice from each group were left alive. The following examinations were performed: 1. Measurement of total body weight and mass of internal organs such as spleen, thymus and external cervical lymph nodes, with estimation of the ratio of organ mass to total body weight. The relationship of the therapeutic activity was evaluated using the formula L / K x 100%, where L is the average survival time of the mice treated with KLP-602 and K is the average survival time of the controls. 2. Histopathological examinations of biopsies of the liver, spleen, thymus and lymph nodes. The sections were stained with H + E of the Gomori procedure for reticular fibers. 3. The level of free radicals in the serum was estimated using the chemiluminescence (Chl) procedure that allows the detection of mu / weak photonic luminescence of the serum. 4. Phagocytic activity of polymorphonuclear (PMN) was estimated using NBT and chemiluminescence (Chl) tests.

Results Single subcutaneous (Group I) and double (Group II) administration of KLP-602 to the highly leukemic AKR mouse strain, at a dose of 20 μg / kg, caused a statistically insignificant increase in body weight, and a decrease in the mass of internal organs such as: spleen, thymus, lymph nodes and liver (the latter was statistically insignificant) compared to control animals (Group III) measured after three weeks of study (Table I). The increase of the total body weight in both treated groups, the decrease in the mass of the spleen, thymus and lymph nodes in Group I, and the increase of the liver and thymus in Group II, when compared with the controls (Group III) occurred after six weeks of the study (Table I).

Table 2 shows the relationship of the weight of the internal organs to the total body weight. After three weeks the mass increase of the thymus was observed in Group III (control) compared to both experimental groups (I and II). After six weeks, there was an increase in the mass of the spleen, thymus and lymph nodes in Groups II and III when compared with Group I. Histopathological examinations performed after three and six weeks revealed a moderate colonization (in the experimental groups) and excessive colonization (in the control group) of the cervical lymph nodes, thymus and spleen with lymphoid cells. However, the leukemic infiltrates in the liver were only found in the control. The lymphoid cells had oval or circular hyperchromic nuclei with numerous nucleoli and atypical mitotic figures. These cells were accompanied by scarcely differentiated centroblastic cells, reticulum cells and islands of proliferating megakaryocytes in the spleen. The stroma of the lymphatic organs such as the liver, lymph nodes and thymus contained thin, scarce reticulum fibers.

Chemiluminescence Exams: The activity of free radicals in the sera of the control mice was K = 18 x 10-3 after three weeks of the study. In Group II the activity of free radicals was lower (K = 10 x 10"3) After six weeks the level of free radicals was K = 10 x 10" 3 in the control animals, lower in Group II (K = 16 x 10"3) and slightly higher in Group I (K = l, 5 x 10-3) The marked increase in phagocytic activity of PMN was observed in both experimental groups as compared to the group of control after three weeks 5 and was: 4688 Chl (Group I), 3485 Chl (Group II), and only 2052 Chl for the Control Group After six weeks the phagocytic activity in Group II was equally high (3264 Chl ) and was lower in Group I (2477 Chl) .A significant increase in measured phagocytic activity was observed / * > by a NBT test after three weeks in both treated groups and was 0.27 and 0.26 in Groups I and II, respectively. After six weeks the increase in phagocytic activity was lower and took place only in Group II. The phagocytic activity in the control group was of 0.22. The therapeutic efficacy index of KLP-602 was 128% and 150% in Groups I and II respectively, being above the minimum 125%. This indicates the marked immunostimulatory properties of KLP-602 and qualifies it for studies additional. The leukemic infiltrates were considerably smaller in both experimental groups than in the control group and were even absent in some mice. Leukemic infiltrates were present in the thymus, spleen and lymph nodes. In addition, they were found in the liver of the control animals. The results of the study indicated that KLP-602 can partially inhibit or delay the progression of leukemia in AKR mice. In contrast, the rapid growth of lymphatic organs with morphological changes, typical for well-differentiated lymphoid leukemia, occurred in animals treated with placebo. On the other hand, KLP-602 inhibits the increase of free radicals in the serum of the mice. It inhibits free radical procedures similar to the action of antioxidants such as vitamin E, C, or selenium. It is possible that the inhibition of the proliferation of leukemic cells in AKR mice treated with KLP-602 depends on the inhibition of induced free radical procedures. In the control mice (without KLP-602) the level of free radicals was higher and correlated with the intensity of the disease. KLP-602 can stimulate the phagocytic activity of MPS measured by two independent procedures, NBT and Chl, and in this way stimulates the immunity communicated to the cell. It can probably modulate the synthesis of TNF. In addition, the "tumor rejection antigens" present on the surface of leukemic cells can be recognized by T cells cooperating with MPS. Tumor anti-infiltrating lymphocytes (TIL), 50-100% more effective than activated lymphokine destructors (LAK), are among those T cells. They have CD4 or CD8 or mixed phenotype and manifest the IL-2 receptor on their surface. It is still unknown why they are paralyzed by neoplastic cells.

The reticulo-endothelial system with MPS cell stimulation is an essential immunological and antiviral mechanism. The stimulation of these cells results in the production of interferon and indirectly activates specific and non-specific immunological procedures, dependent on B cell, T cell or NK. MPS cells participate in immunological procedures through their receptors such as Fe receptors, C3 receptors or HLA class II receptors and also / '} lecithin receptors, transferrins, urokinase, insulin and other fifty lesser known receptors. Any of the changes in its expression and affinity indicates activation of the cells. MPS cells can phagocytose NO sensitive cells because they can synthesize this substance. Activated phagocytes are called "angry macrophages" or "blood thirsty" cells. MPS cells can destroy viruses through their cytotoxic and cytolytic properties, defective virion production and interferon synthesis. The promising results of the preliminary study 20 on AKR mice indicate that KLP-602 can be used successfully in the treatment of leukemia in AKR mice.

Table 1. Total body mass (g) and internal organ mass (mg) in experimental and control mice (± S) x - statistically significant difference (P 0.01) for a: b, a: c and b: c Table 2. Mass ratio of internal organs to total body mass in control and experimental mice (%) X - statistically significant difference (p 0.01) for a: b, a: c and b: c Example 2: Effect of a highly purified lysozyme dimer preparation (KLP-602) on hair growth in alopecia areata. The study was conducted to determine if a balm containing KLP-602 acts on hair growth in alopecia areata. In addition, the tolerability of this balm was estimated when applied to the skin.

Materials and Procedures The study was a comparative, controlled clinical study. Twelve individuals (two male patients and ten female patients) with alopecia areata in the "telogen" stage in good general health were included in the study. They were between 19 and 50 years old. Twelve individuals (three male patients and nine female patients) with alopecia areata treated with MINOXIDIL® were considered as a control group. Their ages ranged from 19 to 49 years. The distribution of individuals by sex and age within two treatment groups was similar for the two groups.

Criteria for patient selection Inclusion criteria 1. Alopecia areata in the "telogen" stage; 2. Age 18-50 years.

Exclusion criteria 1. Alopecia areata in the "dystrophic" stage; 2. Another type of alopecia; 3. Any other skin disease.

Concomitant medication Concomitant medication was not permitted unless it was needed to treat general health conditions.

Assignment of treatment numbers During the study, the people who met the selection criteria were numbered in test groups in successive order. The researcher entered the corresponding number in the appropriate place in each Printed Case Report (CRF). Test medication: The hair balm manufactured by NIKA was provided according to the original formula in identical 100 ml plastic bottles with a calibrated small tipped dropper.

A 0.05% KLP-602 lotion was applied to the scalp three times a day according to the instructions of the attending dermatologist. The treatment began immediately after recognizing alopecia areata by the dermatologist. A 2% MINOXIDIL® solution was applied twice daily to the affected skin areas.

The duration of treatment was 16 weeks. After treatment, patients were observed during 2 months of follow-up period.

Assessment of safety Individuals from both groups tested were examined through a complete history and medical examination at the beginning and end of the study. Blood and urine samples were taken for routine laboratory tests before and after treatment. Laboratory studies included the following tests: Hematology Hemoglobin, hematocrit, total and differential white cell count.

Serum biochemistry ALT (GPT), AST (GOT), alkaline phosphatase, bilirubin.

Urine Urinalysis in cellulose strip for protein, blood and glucose.

Patients were visited monthly during-months of therapy and 2 months of follow-up period. At each visit, individuals were asked about the presence of general or local adverse events such as irritation, burning, stinging; Treatment areas were examined for erythema or other local side effects.

Efficacy evaluation Efficacy parameters 1. The main efficacy parameter was hair growth. 2 . The secondary efficacy parameter was the healing time of the altered scalp. ' 3. Photographic documentation was obtained at the following points in the study: before the application of the hair balm with KLP-602 at the time of hair growth at the end of the application of the hair balm with KLP-602. A microscopic evaluation (trichogram) was performed. The standardized hair trichograms obtained from the limit of the alopecia areata lesions reveal useful information on the course and severity of the disease and also the effectiveness of the therapeutic measures. At each visit, the scalp was evaluated for signs of hair growth that was categorized as: - none a slightly visible regrowth (hair or a few solitary terminal hairs disseminated) - moderate - incomplete (partial) - complete Hair growth "cosmetically acceptable "was defined as sufficient to cover the scalp and hide areas of residual hair loss.

Results All the individuals registered in the study (12 treated with 0.05% KLP-602 and 12 of the control group treated with 2% Minoxidil solution) completed a therapy period of 4 months and 2 months of follow-up.

Efficacy The most useful measure of effectiveness was the researchers' evaluations at the end of the study. Usually eight of the twelve individuals (66%) in the group of KLP-602 and six of twelve individuals (50%) in the group of Minoxidil showed increased hair growth during the study. Of these, four individuals in the KLP-602 group (33%) and three in the Minoxidil group (25%) were considered to have cosmetically acceptable, complete hair growth. Partial hair growth was observed in three of twelve individuals evaluable in the KLP-602 group (25%) and three in the Minoxidil group respectively (25%). In one individual in the KLP-602 group (9%), minimal hair growth was observed. There was no hair growth in four of twelve (33%) in the KLP-602 group and in six of twelve (50%) in the Minoxidil group.

In the group of KLP-602 with alopecia in less than 25% of the scalp, hair growth was obtained in eight out of eight individuals and six out of seven in the Minoxidil group, respectively. No regrowth was observed in individuals with alopecia in an extension of more than 25% percent of the scalp in both treated groups. The mean time of the hair growth response in correlation with the duration of the treatment ranged from one to three months in both groups. Three individuals in the KLP-602 group responded after one month of treatment. In a female patient hair growth after one month was with terminal hair of around 5 mm in length and the bald scalp areas were covered with thick hair regrowths. At the follow-up visit after the second month this woman appeared with total alopecia (emotional stress divorce) and did not respond to subsequent treatment, completed the study with negative result. In one individual in the Minoxidil group, regrowth of hair was observed after one month of therapy. In four patients in the KLP-602 group, a regrowth of hair was obtained after two months of treatment. After three months of study, a regrowth of hair was observed in two individuals in the KLP-602 group and in three in the Minoxidil group, respectively. Safety No general side effects were observed during the therapy and the follow-up period. No abnormalities were observed in the laboratory blood and urine values at the beginning and end of the study in both treated groups. In local adverse cases observed during the study: A female patient in the KLP-602 group complained of burning and stinging of the scalp in the second month of treatment. Related these symptoms with wearing a new wig. The sensations disappeared within two weeks without additional treatment, did not stop the therapy and - * or completed the study with good results. An individual treated with 2% Minoxidil solution complained of oily hair, no significant clinical signs were observed on the scalp examination. The patient correlated this incident with the trial but was willing to continue the treatment. 15 No significant findings were found in correlation with hair regrowth and the age of the patients, location of bald areas and the duration of the episode in progress, which was short and similar for both groups and evaluated from one week to a maximum year. All those who do not respondents in both treated groups had alopecia areata anamnesis that lasted at least five years (5 - 15 years).

Discussion The study was carried out to evaluate the safety and efficiency of hair growth with the 0.05% KLP-602 lotion applied topically in patients with alopecia areata and to compare it with the effectiveness of a 2% Minoxidil solution during a treatment. four month therapy The hair balm containing KLP-602 was well tolerated by patients. 66% of individuals treated with KLP-602 lotion at 0, 05% showed increased hair growth during the study compared to 50% of individuals in the Minoxidil group. Cosmetically acceptable hair growth was observed in 33% of patients in the KLP-602 group and in 25% in the Minoxidil group respectively. Better treatment results were observed in patients with an extension of alopecia less than 25% of the altered scalp. The 0.005% KLP-602 lotion was easy to use topically and did not cause any adverse local or general incidents. Considering the short time of the study (16 weeks) the results obtained were very favorable.

Conclusions 1. The hair balm containing KLP-602 was well tolerated and did not cause any side effects. 2. The topical application of KLP-602 at 0.05% was easy and safe. 3. No side effects or general or side effects were observed in the laboratory values during the study. 4. Due to the short time of the study (4 months) and 2 months of follow-up, the complete evaluation of the results and treatment behavior was impossible, although this period was long enough to observe a favorable efficacy of the treatment. 5. The results obtained in the experiment showed that the 0.05% KLP-602 lotion is comparable to a 2% Minoxidil solution and even more effective in the treatment of alopecia areata. 6. The preclinical investigations and the results obtained in the study showed that KLP-602 applied topically induces hair growth in patients suffering from alopecia areata.

In summary, the study was conducted to estimate the efficacy and tolerability of a hair balm containing KLP-602 in hair growth in alopecia areata. Applications of the KLP-602 lotion at 0.05% three times a day were generally well tolerated in the 4 month treatment period. No local or systemic side effects were observed. A better hair growth response was observed in the KLP-602 group than in the Minoxidil group. The response of the hair regrowth was observed in 66% of the individuals with the KLP602 lotion at 0.05%. The percentage of patients who responded with regrowth of moderate or dense hair was greater among patients whose bald area comprised less than 25% of the scalp. The 0.05% KLP-602 seems to be a remarkable agent in the treatment of alopecia areata.

Example 3: Prophylactic application of lysozyme dimer to induce a non-specific stimulation of the fish's immune system.

Test design Three hundred healthy fish of the rainbow trout species (oncorhynchus mykiss, family of salmonids) weighing 90 - 100 g each were subjected to the experiment. The lysozyme dimer made according to W091 / 10731 (designated herein as "KLP-602") was dissolved containing about 10% by weight or less of unwanted byproducts and is essentially free of the monomeric form of lysozyme in PBS for prepare injection solutions for the administration of lysozyme dimer of 10 and 100 μg per kg of body weight, suitable for intraperitoneal injection in fish. The drug was administered once a day. The therapeutic protocol comprised different groups of fish subjected to the application of the drug on either day 1 only, day 1 and day 3, or days 1, 3 and 5, respectively. The fish of the control group was not subjected to the administration of lysozyme dimer. After 1, 2, 3 and 4 weeks after the administration of the last injection, 10 individuals from each of the treated groups were sampled and the control was sampled. The fish were immobilized and about 2 ml of blood was collected from the caudal vein by means of a heparinized blood collection tube (Greiner). The phagocytic capacity of PMN and MN cells, the activity of abrupt increase in oxygen consumption and the potential destructive activity of PMN and MN cells, MPO activity in PMN phagocytes, and serum lysozyme and gammaglobulin levels were determined. Also, 20 individuals from each group were subjected to a disease stimulation test (furunculosis). Fish were given intraperitoneal injections unique to a bacterial suspension of Aeromonas salmonidae that was grown in a nutrient broth for 48 hours. The mortality (proportion of death) of the animals was determined and their interrelation with the bacterial infection applied by the isolation of pathogens from the kidneys of the dead animals was confirmed.

Results In the groups treated with KLP-602, all immunological parameters mentioned above were significantly increased (P <; 0, 05) on the corresponding values of the control group. This surprising result was observed in all groups, that is, after a single, double or triple application of KLP-602 and lasted throughout the observation period up to 4 weeks after the last injection. Also, the mortality of the fish in the groups treated with KLP-602 was significantly lower than in the control group. Even the activity of the abrupt increase in oxygen consumption was considerably greater, as were the phagocytic indices and neutrophilic myeloperoxidase (MPO) levels of the groups treated by the double and triple application of KLP-602. 5 The known susceptibility of rainbow trout to virulent bacterial stimulation by A. salmonicida and the results observed with groups treated prophylactically with the lysozyme dimer composition suggest that the drug was indeed able to induce a & In the short term, it was recognized that the protection was greater in the groups that received two or three doses of KLP602 than in the groups treated by a single application only.

Conclusion The application of highly purified lysozyme dimer to fish increases non-specific defense mechanisms and significantly improves protection against microbial stimulation, for example, by A. salmonicida (which causes furunculosis). This observation is of great importance for a prophylactic and therapeutic treatment of the fish in the fish farming industry in order to minimize fish losses due to fish diseases that occur frequently, in particular during the fishing season. spring.

Example 4: Therapeutic application of lysozyme dimer to induce non-specific stimulation of the fish's immune system.

Test design Two hundred rainbow trout were included in this comparative study of non-specific defense mechanisms in the course of a natural incidence infection with IPNV (Infectious Pancreatic Necrosis Virus) and in the application "!"> Of a composition comprising dimeric lysozyme (KLP-602) After identification of the IPNV infection, part of the fish were treated with KLP-602, while a control group remained untreated. main characteristics that characterize the cellular and humoral defense mechanisms specimens were analyzed by immunological and serum procedures. These procedures included the weekly determination of the number of total leukocytes, relative count of leukocytes, phagocytic capacity of PMN and MN cells, activity of the increase in oxygen consumption and activity potential shredder, MPO activity in neutrophils, lysozyme and ceruloplasmin activity in the plasma, and total protein and gammaglobulin levels, over a 2-month observation period. Repeated groups of rainbow trout were fed diets containing the non-specific immunostimulant KLP-602 at a dose of 20 μg per kg of body weight. Lyophilized samples of KLP-602 were applied together with nutrients either orally in solid form (flocculated, agglomerated) or dissolved in the water of the fish conservation pond, that is, as an aqueous solution, for a period of seven days. It is assumed that drug 5 can also be reabsorbed via a pathway through the fish gills. They were then tested and tested for changes in non-specific cellular and humoral defense mechanisms at weekly intervals. Each time, 10 * fish were randomly selected for immunological assays and IPNV identification. In addition, all fish were observed daily in search of unusual behavior, morphological changes and mortality.

Results An immunosuppressive effect could be observed in fish naturally infected by IPNV. Throughout the observation time, the parameters indicating the cellular and humoral defense mechanisms were significantly reduced. specific, while a week after feeding the diet containing the lysozyme dimer some parameters, for example, the level of ceruloplasmin, activity of the abrupt increase in oxygen consumption, immunoglobulin level, and activity of lysozyme were significantly increased (for example, approximately 31% increase in ceruloplasmin level, approximately 40% increase in lysozyme activity, approximately 33% increase in activity from abrupt increase in oxygen consumption) when compared to the control group. After the application of KLP-602, immunostimulatory effects of non-specific defense mechanisms were unambiguously identified. Also, 5 cumulative mortality was the lowest in the groups fed the diet containing KLP-602.

Conclusion Fish treated with a diet containing dimer - ** lysozyme (KLP-602) showed advantageous changes of non-specific immunological defense mechanisms. These changes correlated with reduced mortality, that is, indicating protection against naturally occurring infection by IPNV, whose finding strongly recommends further develop and optimize the strategy of prevention and / or therapy of diseases of fish by administration of the lysozyme dimer as referred to herein.

Claims (20)

1. CLAIMS 1. The use of a lysozyme dimer for the preparation of a pharmaceutical composition for the stimulation of cellular and humoral defense mechanisms not specific to the immune system of a human or animal individual, in which the stimulation comprises increasing the phagocytic activity and increasing the less a parameter selected from the - * or group consisting of ceruloplasmin level, activity of abrupt increase in oxygen consumption, immunoglobulin level and lysozyme activity and / or wherein the stimulation comprises regulating serum levels of oxygen free radicals.
2. 2. Use according to claim 1, to prevent or treat a disease selected from the group consisting of leukemia, hair growth disorder, fish disease and bee disease.
3. 3. The use according to claim 1, for preventing or treating an immunosuppressive effect.
4. 4. The use according to claim 1, wherein the lysozyme dimer contains about 10% by weight or less of unwanted by-products and is practically free of a monomeric form of the lysozyme.
5. 5. The use according to claim 2, in 25 where the disease is leukemia induced by the action of a virus.
6. 6. The use according to claim 5, wherein the virus is a retrovirus.
7. 7. The use according to claim 2, wherein the disease of hair growth disorder is 5 alopecia areata.
8. 8. Use according to claim 2, wherein the fish disease is selected from the group consisting of furunculosis and IPNV infection.
9. 9. The use according to claim 8, in, *? where the fish disease is in a fish of the salmonid family.
10. 10. The use according to claim 3, wherein the immunosuppressive effect is caused by a microbial infection.
11. 11. The use according to claim 1, for the manufacture of a pharmaceutical composition for administering a single or repeatable dose of about 0.005 to 0.5 mg / kg of body weight for an animal or human individual.
12. 12. The use according to claim 11, wherein the dose is in a range of from about 0.01 to about 0.1 mg / kg of body weight.
13. 13. The use according to claim 1, for the preparation of a pharmaceutical composition in the form 25 of a gel, an ointment or a liquid composition, for topical, oral or parenteral administration and which, in addition, comprises at least one additive selected from the group consisting of a physiologically acceptable solvent, a vehicle and a suitable preservative.
14. 14. The use according to claim 13, for the manufacture of a pharmaceutical composition which comprises the lysozyme dimer in an amount of from 0.01 to 10 mg / ml.
15. 15. The use according to claim 13, wherein the pharmaceutical composition comprises the dimer of .Lisozyme in an amount of from 0.01 to 1 mg / ml. The use according to claim 1, for the manufacture of a pharmaceutical composition in solid form suitable for oral administration and wherein the pharmaceutical composition further comprises at least one pharmaceutically acceptable additive selected from the group consisting of a carrier and a preservative . 17. The use according to claim 16, wherein the pharmaceutical composition comprises the dimer of .Lisozyme in an amount of from about 0.01 to about 10 mg / g. , 18. The use according to claim 10, for administering at least a single dose of the composition to the individual prior to the microbial infection. 19. The use according to claim 10, for administering at least a single dose of the composition to the individual after identification of the microbial infection. 20. The use according to claim 10, for administering at least three doses of the composition at substantially equal intervals to the individual before the microbial infection.