New pharmaceutical uses of krill enzymes

The present invention relates to a novel therapeutic sub-application of non-immunogenic, proteinaceous materials and compositions which have enzymatic activity and have surprisingly been found to cure an incredible number of diseases in numerous clinical trials. According to the present invention, these substances are believed to function phagocytosis, i.e., they are capable of discriminating between different particles and substances according to whether they are considered normal or abnormal in a particular environment, or are capable of recognizing, targeting and killing divergent cells. However, it should be emphasized that the present invention is not intended to be limited to any of the putative modes of action.

One of the traditional methods of therapeutic use of proteolytic enzymes for therapeutic use is to treat non-healing wounds covered with proteinaceous structures instead of surgical cleaning, especially in elderly patients with ulceration due to circulatory failure. See, for example, U.S. Patent Nos. 4,801,451 and 4,963,491, which disclose a mixture of exo and endo-peptidase enzymes isolated from antarctic krill (Euphasia superba) and their use for purification purposes, which may be pharmaceutical purification (U.S. Pat. No. 4,801,451). and non-medical purification (U.S. Patent 4,963,491). The only therapeutic purification is enzymatic sebum purifying • ·

-3treatment, ie enzymatic cleaning of dead wounds. Non-medical applications include, but are not limited to, laundry applications, restoration of old paintings, and the like. International patent application WO / 85/04809 describes the use of enzymes isolated from antarctic krill as a digestive enhancer. EP-A1-0170115 discloses the use of krill as a thrombus solubilizer.

Enzymes and mixtures of enzymes have been isolated from Antarctic krill and their biological and biochemical properties have been extensively studied. Various isolation techniques have been developed to isolate these enzymes. See, for example, Anheller JE., Hellgren L., Karlstam B. and Vincent J. (1989): Biochemical and biological properties of a novel enzyme preparation extracted from Antarctic krill, which makes it suitable for treating gouty wounds. Dermatol. Gap. 281. 105-110; Axelsen NH, Kroll J. and Weeke B. (1973): A Manual of Quantitative Immuno-Electrophoresis: Methods and Applications, Scan. J. Immunol. 2, Suppl .; Bucht A. and Karlstam B. (1986): Immunological characterization of three highly purified trypsin-like enzymes from Antarctic krill (Euphausia superba) Bioi. Chem. Hoppe-Seyler 367, 366 (Suppl.); Bucht A., Johansson B. and Karlstam B. (1986): Separation and identification of proteases from Antarctic krill (Euphausia superba), 6th Int. Symp. HPLC Proteins,

-4Peptides and Polynucleotides, Baden-Baden pp. 34; Chen CS, Yan TR and Chen HY (1978): Purification and properties of trypsin-like enzymes and carboxypeptidase A from Euphausia superba, J. Food Biochem. 2, 349-366; Hellgren L., Karlstam B., Mohr V. and Vincent J. (1991): Krill Enzymes - a New Concept for Effective Debridement of Necrotic Ulcers, Int. J. Dermatol. 30, 102-103; Karlstam B. : Crossed immunoelectrophoretic krill analysis of proteins from Antarctic krill (Euphausia superba) with special reference to serine proteinases ('submitted for publication)' '; Kimoto KK, Kusama S. and Murakami K. (1983) Purification and characterization of serine proteinases from Euphausia Superba, Agric. Biol. Chem. 47, 529-534;

Lowry OH, Rosebrough NJ, Farr AL and Randal RJ (1951) Protein measurement with the Folin phenol reagent, J. Bioi. Chem. 193. 265-275; Moore S. and Stein WH (1963):

Chromatographic determination of amino acids by the use of automatic recording equipment, Methods Enzymol. 6, 819-831; Osnes KK and Mohr V. (1985): On Purification and Characterization of Three Anionic Serine-Type Peptide Hydrolases from Antarctic Krill, Euphausia superba, Comp. Biochem. Physiol. 82B, 607-619; and Osnes KK and Mohr V. (1986), On purification and characterization of exopeptidases from Antarctic krill, Euphausia superba, Comp. Biochem. Physiol.83B. 445-448.

BACKGROUND OF THE INVENTION

Our immune system is constantly actively guarded, its main functions are to protect and preserve «· ··

-5 the integrity of the organization by providing for the removal of intruders, old and used cells, divergent cells, and solutes that are considered abnormal in the environment.

Phagocytes, granulocytes, macrophages, and killer cells are the more ancient elements of our immune system, and their function is phagocytosis, engulfing pathogens and lysosomal enzymes that kill and digest them.

Lymphocytes, T-cells and B-cells are our newer immune cells that carry out guarding and defense in different ways. Some T cells release toxins with which they kill microbes, other types transmit news and control the intensity of response and B cells produce antibodies. Ancient and new cells work together in a complex way for optimal protection of our bodies.

When a piece of tissue is injured, the granulocytes gather on the spot within minutes and begin to attack alien invaders, damaged cells, and so on. against. Within a few hours, macrophages, killer cells, and T cells also respond to the chemical signaling of working granulocytes and enter the damaged area. A well-composed interplay ranges from cleaning the damaged tissue to final healing.

One of the signs of activated immune defense is inflammation caused by toxins leaking from various immune cells and microbes, so mild inflammation can be considered a healthy process. Sometimes immune defenses are overactive and acute inflammation occurs, which is more harmful than useful, and when chronic inflammation becomes chronic, an autoimmune disease

-6 can start.

The main cause of inflammation is microbial infection because, despite the versatility of immune defense, microbes have developed their own defenses, which can lead to a very dangerous condition in the long run.

Microbes can camouflage themselves so that immune cells cannot recognize them or invade cells, even immune cells, to live, reproduce and alter the host cell's essential characteristics. Some of the cancers, AIDS and opportunistic infections, which cause many complications, are examples of (incorrect) microbial behavior.

Thus, the pathogenesis of a disease depends on how well the immune system functions and normally performs its function properly. In addition to the effects of microbes, drugs are the main cause of the transient or long-term suppression of the immune response, a problem that has received considerable attention in medical research.

Summary of the Invention

An optimal drug would be an active ingredient that works in tune with immune protection, mutually helping each other, targeting the cause and symptoms of the disease and having no harmful side effects. The present invention provides a novel pharmaceutical application and a novel pharmaceutical composition which appears to possess said properties. The method by which this is achieved involves the use of one or more proteolytic enzymes from the Antarctic krill.

-7 (Euphasia superba) were isolated. As mentioned above, such enzymes are already known and, in some cases, have been proposed for therapeutic use. The results obtained using these enzymes for the purposes described herein were unexpected and indeed very surprising. The manner in which these results are achieved is illustrated in the following examples and the appended claims.

Production Example

As mentioned above, there are several ways of isolating proteolytic enzymes from krill, so the following method is only one of the possible methods for preparing the enzyme preparation of the present invention.

Frozen white krill (Euphasia superba) was thawed and homogenized. The mixture was diluted 1: 1 (v / v) with distilled water and 0.02% sodium azide was added and allowed to stand at + 4 ° C for 6 hours. The aqueous phase was collected together with the supernatant of the shells and body pieces centrifuged at 9000 rpm for 40 minutes. The aqueous phase was degreased with ethyl acetate overnight at + 4 ° C. The lower aqueous layer was collected and evaporated for several hours.

Saturated ammonium sulfate solution was then added to the residue to 60% saturation. The precipitate was centrifuged at 9000 rpm for 40 minutes, dissolved in 0.05 M phosphate buffer (pH 7.4, 0.05 M sodium chloride (PBS)) and dialyzed against PBS to give a crude extract.

The crude extracts of Sephacryl 200 (Pharmacia, · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

-8Sweden) column and the protein fractions were collected at 280 nm, and the proteolytic activity of the fractions was assayed. The active fractions were combined and lyophilized. For mixed-activity fractions, the following equivalents are hereinafter referred to as "Multi-Protein", "Multi-Enzyme", "PHIM" or "PHIM 106".

The molecular weight range of the isolated enzymes was determined (SDS-PAGE PAA4 / 30 (Pharmacia, Sweden)). At least 6 bands are separated in the range of 18,000 to 40,000, especially between 24,000 and 34,000.

One of the fractions obtained by the gel chromatography separation described above also showed endo- and exopeptidase activity, which was subjected to further separation processes. We have shown that this fraction probably contains a single enzyme of molecular weight of 26,000-32,000. Hereinafter, this enzyme is referred to as Single-Protein or Single-Enzyme.

Neither the crude extract, nor Multi-Protein nor Single-Protein showed any bactericidal activity in vitro, but presumably bacteriostatic.

In living cancers, these enzymes generally operate at temperatures between -5 and -2'C. Surprisingly, the temperature optimum of these enzymes was found to be + 55'C, and some enzymes were stable for one hour at 100'C.

Various features of PHIM

Figure 28 shows the thermal stability of PHIM prepared by the method described above, i.e. the relative activity of PHIM

Expressed as a percentage of time (activity versus amount of bovine casein digested; BioRad Protease Substrate Tablets; 30 C for 20 h) at 10, 20, 30, 40, 50, 60 and 70 C. At 70'C, activity fell below 25% after 2 hours, whereas at 60'C, this decline occurred more than 12 hours later. At 40 ° C, activity decreased below 25% after 11 days, while at 30 ° C, PHIM activity was still about 100% after 11 days.

Figure 29 shows the temperature optimum, i.e., the total proteolytic activity of PHIM (digested bovine casein area, expressed in mnr; BioRad Protease Substrate Tablets; pH 7.0; 30 ° C for 24 hours). depending on. It is interesting to note that the optimum activity was obtained at 55 ° C.

Figure 30 shows the pH stability of PHIM, i.e., the percentage of relative activity (activity expressed as amount of digested bovine casein; BioRAd Protease Substrate Tablets; 30 ° C for 20 hours) versus time, values. The reconstituted PHIM was kept at room temperature for 2-18 hours. At pH 3.5 and pH 11, activity declines rapidly over a few hours, and at pH 7 to pH 9.5, activity is greater than 70% after 18 hours.

Figure 31 shows the pH optimum of PHIM, i.e., the total proteolytic activity of PHIM (digested casein area, expressed in mnr; BioRad Protease Substrate Tablets; at 30'C for 16 hours). · · · · · ···· ·

-10függvényében. Proteolytic activity has an optimum around pH 8.

Figure 32 shows the in vitro dose-response curve of PHIM, i.e. the total proteolytic activity of PHIM (digested casein area in casein equivalents, mm)²expressed in; BioRad Protease Substrate Gel Tablets at 30'C, pH 7.0, over 24 hours). Measurement of Protein Concentration Bradford, M., Anal. Biochem. 7.2, 248 (1976). Activity increased rapidly with increasing dose of PHIM, and increased to ca. It reached its maximum at 1.5 mg PHIM and remained at this value at 15 mg PHIM.

Clinical case reports

To illustrate the therapeutic effect of the present invention, the limitations and difficulties encountered in clinical practice, the enzymes and enzyme mixtures prepared in accordance with the invention have been clinically tested for, inter alia, the following indications:

- infections

- inflammations

dead and abnormal cells

- opportunistic infections

- eye diseases

- pain

- cancer.

The trials were designed experimentally for a small number of patients for each indication. Surface wounds and infected parts were selected mainly for the study, where the clinical symptoms change.

- · · · · ·· « ··«· «

-11 we could follow it visually.

In most cases, when selecting a patient, the keyword was focused on the otherwise healthy patient vocabulary, i.e., the primary clinical problem, and the effect of other circumstances was excluded to allow a good interpretation of the results. This patient material includes some variability in overall health and basic body characteristics as the participating patients ranged in age from 20 to 85 and the indication ranged from inflammation requiring ambulatory treatment to hospital bed rest. Each test group is very homogeneous in terms of indication of general health and basic characteristics.

A brief introduction to ra.izok

1-4. Fig. 3A shows the average exudate formation rate, the average redness rate, the average swelling / edema rate and the mean pain in postoperative wounds when treated with the Single-Enzyme and MultiEnzimm of the present invention.

Figures 5 and 6 show the average reduction in pain and inflammation in the 7-day treatment with Multi-Enzyme according to the present invention for painful infections.

Figures 7 and 8 show the development of redness / swelling and pain sensation in the treatment of an upper respiratory viral infection with the Multi-Enzyme of the invention for 9 days.

Graphs 9 and 10 show mean intervals between urine excretions and pain reduction «V

-12• ·

<img file="HUT69989A_D0001.tif" />

During the treatment of bladder and urethra infection with MultiEnzimm according to the invention for four days.

Figure 11 shows the average reduction in pain when treated with Multi-Enzyme according to the invention for more than seven days for acute arthritis in racing horses.

Figure 12 shows the Single-Enzyme Disrupting Effect on Gill Surgical Wounds.

Figure 13 shows the dose response curve for Multi-Enzyme (PHIM) in the treatment of burns.

Figure 14 shows dose ranges for Multi-Enzyme (PHIM) for various indications.

Figure 15 shows the maximum accumulated amounts of Multi-Enzyme (PHIM) per kg body weight needed to cure various indications.

Figure 16 shows the minimum accumulated amounts of Multi-Enzyme (PHIM) per kg of body weight required to cure various indications.

Figure 17 is a bar graph depicting the anti-tumor effect of a single injection of PHIM 106 (IT, IP, and SC) compared to the control group.

FIG. 18 is similar to FIG. 17, but FIG

shows the effect of repeated injection.

Figure 19 is a bar graph showing tumor volume at different dosages of PHIM 106 compared to control.

Figures 20 and 21 show hi-histopathological sections of control rats in two different magnifications.

Figures 22 and 23 correspond to Figures 20 and 21

13 sections, but these are derived from rats treated according to the invention.

Figures 24 and 25 are photographs of tumor in an untreated control rat, and Figures 26 and 27 are corresponding photographs of a single subcutaneous injection rat according to the invention.

Figure 28 shows the thermal stability of PHIM, i.e., the percentage of relative activity of PHIM at various temperatures as a function of time.

Figure 29 shows the temperature optimum of PHIM, i.e., the total proteolytic activity as a function of temperature.

Figure 30 shows the pH stability, i.e. the relative activity, of PHIM as a function of time at various pH values.

Figure 31 shows the optimum pH of PHIM, i.e. total proteolytic activity as a function of pH.

Figure 32 shows the dose-response curve of PHIM, i.e., total proteolytic activity versus dose of PHIM.

1-10. Clinical example

infections

Infections and inflammations are most common when they occur together and show similar clinical signs, redness, swelling, elevated temperature, edema, exudation, pus, pain, gummy tissue, and sometimes an unpleasant odor. These clinical symptoms were followed and noted in the present invention

-14 to evaluate the course and efficacy of treatment with the Multi-Protein and Single-Protein formulations described above.

First example - Surgical wounds

In this experiment, 40 patients were examined in two groups of 20 with a total of 41 surgical wounds, of which 34 were abdominal and 7 were thoracic wounds. Each group was tested with 3 casein units / ml SingleEnzim or 5 casein units / ml Multi-Enzyme in Krill. The lyophilized white powder, free of preservative and antimicrobial additive, was dissolved in 5 ml of physiological saline.

The mean age of the patient population, 28 males and 12 females was 52 ± 16 years. The antimicrobial activity of the formulation ranged from very good to excellent in terms of clinical efficacy. Within 5 days, the infections had resolved to a non-clinical level and the clear signs of clinical infection had disappeared. There was no significant difference between the two formulations. No adverse reactions or adverse effects caused by the product were observed.

The results are summarized in Figures 1-4.

For this indication, the effective dose is from 0.01 to 100, preferably from 1 to 25 mg, per 100 cm² volt.

Second Example 1 - Small burns

In this experiment, we examined 11 patients with minor burns with S. aureus and P. aeruginosa

-15 fully infected, resistant to antibiotics and silver diazine cream. Five patients were treated with 3 units / ml of Single Enzyme Hydrocolloid Cream and 6 patients were treated with 5 units / ml Multi-Enzyme Solution. Lyophilized white powder without preservative and antibacterial additive was used. Multi-Enzyme Preparation: 1 ampoule dissolved in 5 ml of saline solution gives a concentration of 5 units of casein / ml. Single-Enzyme Preparation: 1 ampoule mixed with 5 ml hydrocolloid gel gives a final concentration of 3 units of casein per ml.

For this indication, the effective dose is from 0.01 to 100, preferably from 1 to 25 mg, per 100 cm².

after a day's treatment, the wounds were asymptomatic, which was confirmed by the result of MO cultivation. The gums, pus, fibrin fibers and bruised tissues were effectively degraded by both formulations and there was no perceptible difference between the two formulations. The preparations did not cause any adverse effects.

The results are summarized in Table 1.

• · ·

-16Summary burn injury data summary

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• · ·

Example 3 - Painful Gingivitis

The study investigated the acute or chronic infection / inflammation of 22 patients. Lyophilized white powder containing no preservative or antimicrobial additive was used. Multi-Enzyme Preparation: 1 ampoule dissolved in 5 ml of physiological saline results in a solution of 5 units of casein / ml.

Three times a day, morning, noon, and evening, one ampoule was mixed with ml of tap water and the patient rinsed the oral cavity for 5 minutes. During the two hours after treatment, patients could not eat or drink. The treatment was continued for 7 days irrespective of the result.

Patients reported a reduction in pain between 20 minutes and 12 hours after treatment. The infection and inflammation disappeared within 4 days and did not recur within 3 weeks of treatment. No adverse side effects were observed.

The results are summarized in Figures 5 and 6.

For this indication, the effective dose is 0.1 to 100, preferably 1 to 35 mg, per dose.

4th Example 1 - Upper respiratory viral infections A patient with viral influenza and secondary upper respiratory (sinusitis) bacterial infection was enrolled.

The viral infection attacked the ciliary epithelium in the respiratory tract and caused inflammatory symptoms that included redness, swelling and increased mucus production. Because damaged ciliated epithelium is no longer able to remove inhaled bacteria, the

<img file="HUT69989A_D0002.tif" />

bacterial infections are common (secondary) congenital viral infections.

Other signs of viral infection include general conditions, depression, fever, and pain.

Viruses survive and proliferate in host cells, making it difficult to kill them while leaving the host cell intact.

Multi-Enzyme preparation: lyophilized white powder without preservative and antimicrobial additive. 1 ampoule dissolved in 5 ml of physiological saline results in a solution of 5 units of casein / ml.

Approximately 0.25 mL of solution was sprayed into the nasal cavity and the oral cavity was rinsed with 4.5 mL of solution for 5 minutes. The procedure was repeated three times a day and the symptoms, redness, swelling, mucus formation, pain and adverse side effects were recorded daily.

Treatment was continued for up to 10 days or until symptoms resolved.

The patient was asymptomatic after 6 days and the other three after 9 days.

Reduction of pain in all patients within an hour to 2 days. The sputum became clear after 3 days in patients who had previously had purulent discharge. The redness, swelling disappeared within 4 days.

The results are shown in Figures 7 and 8. No adverse side effects were observed.

The effective dose is 0.1 to 100 mg, preferably 1 to 35 mg per treatment.

···

5th Example 1 - Infection of the Cavity Herpes Sinrplex virus was treated by mouth rinsing twice daily with recurrent oral Herpes Simplex infection. Lyophilized white powder without preservative or antimicrobial agent was used. Multi-Enzyme Preparation: 1 ampoule dissolved in 5 ml of physiological saline results in a solution of 5 units of casein / ml.

The ampoule was freshly prepared prior to treatment and the patient rinsed the oral cavity with the solution for 5 minutes. For 2 hours after treatment, eating and drinking were not allowed. The procedure was repeated twice a day and the symptoms, redness, swelling, pain and adverse side effects were recorded once a day.

Treatment was continued for up to 10 days or until symptoms resolved.

The reduction in pain was evident as early as 2 hours after the first treatment, although in some patients the pain returned during the first two days between treatments, but no longer thereafter.

days later, the patients were asymptomatic and herpes healed. No adverse side effects were observed.

1-5 above. Example 4 shows that bacterial, viral and fungal infections were similar in nature, all signs of infection disappeared within 3-6 days. Even drug-resistant bacterial strains such as S. aureus and P. aeruginosa responded to burns, and cell-associated viruses such as Herpes Simplex responded the same. gums ··· • · ·• ♦ · «· · ··· ·

-20 inflammations and upper respiratory viral infections were also effectively treated with 5-7 days mouthwash for only 15 minutes daily. The short application time and the presumed saliva rinse effect support the idea that the formulation is capable of adhering to cells or surfaces and acting for much longer than the actual treatment time.

Similar observations have been reported in patients with severe tearing eye infection.

The effective dose for this indication is 0.1 and 100, preferably 1 and 35 mg per treatment.

Example 6 - Decubitus ulcera infections In elderly patients, a total of 18 bedsores, which developed in the corners and in the lower body cavities, were studied. Lyophilized white powder without preservative and antimicrobial agent was used. Multi-Enzyme Preparation: 1 ampoule dissolved in 5 ml of physiological saline results in a solution of 5 units of casein / ml.

The ulcers were rinsed thoroughly with physiological saline and cleaned as far as possible, then covered with a semi-occlusive dressing after instillation of 5 ml of Multi-Enzyme preparation. The procedure was repeated twice daily for 7 days and examined for inflammation, redness, temperature, swelling, goiter, suppuration, pain and adverse side effects of the ulcers.

The infection disappeared after 4 days of treatment. 6 wounds completely healed by day 7, and a total of 11 wounds on 14 days • · · ·

-21belül. 7 wounds did not heal due to the patient's general health, but showed improvement. No adverse side effects were observed.

The effective dose is 0.1 to 100, preferably 1 to 25 mg / 100 cm⁶ volt.

Example 7 - Fistula infections

The aim of the experiment was to investigate the antimicrobial and degradative efficacy of the Krill extracted Single-Protein and Multi-Protein formulations and their utility in anal fistulae infections. Lyophilized white powder without preservative and antimicrobial agent was used.

Single-Protein preparation has endo-and exopeptidase activity: 1 ampoule mixed with 5 ml hydrogel gives a concentration of 3 casein units / ml. Multi-Protein preparation is an enzyme mixture with endo-exopeptidase activity: 1 ampoule mixed with 5 ml hydrogel gives a concentration of 5 units / ml casein.

The funnels were rinsed with sterile solution and cleaned as far as possible and treated with Single-Protein Gel or Multi-Protein Gel. The procedure was performed once a day and the patients were examined for redness, temperature, swelling, pus, pain and adverse side effects.

Treatment was continued for up to 10 days or until symptoms of infection and inflammation had disappeared.

Two patients with non-rectal anal fistulae infection, either Single-Protein or Multi • f

-22Protein preparation.

The pain had completely disappeared within 48 hours and all signs of infection and inflammation had disappeared after 4 days.

The ulcers healed within 6 to 9 days and did not resolve within 6 months of treatment. No adverse side effects were observed.

The above examples show that complicated infections which are generally very difficult to treat in the art, such as analis fistulae and decubitus ulcera, can be successfully treated with the compositions of the invention and, in most cases, a definitive cure has occurred.

The effective dose was between 0.01 and 100, preferably between 1 and 25 mg / 100 cm 2.

8th EXAMPLE I - Eye Infections A purulent eye infection of a patient was treated with an eye drop containing Multi-Enzyme Prepared twice daily from krill. (Lyophilized white powder prepared without addition of preservative and antimicrobial agent. 1 ampoule of Multi-Enzyme preparation dissolved in 25 ml water for injections resulted in 1 casein unit / ml preparation.) 2 drops, approximately 0.4 ml, in the morning and evening test liquid was added dropwise. At each instillation, the eye was examined for redness, swelling, tear formation and possible adverse side effects.

Treatment was continued for up to 10 days or until symptoms of infection and inflammation had disappeared.

·» · * &gt; 4* ···· ® · · · · ··· « « • · · ·· · *··♦

Complaints from -23Α patients resolved within 3 days. The redness and swelling of the eye area resolved within 2 days, and the excessive tearing also resolved within 2 days. Immediately after the first treatment, all patients felt that the inflamed eye had calmed down, and that the irritation and pressure sensitivity of the eye area had disappeared within minutes. No adverse side effects were observed.

The above example shows that the fastest results were obtained in the treatment of eye infections, although a very low dose of 0.4 units of casein was used per treatment.

The effective dose was 0.01 to 50, preferably 0.1 to 5 mg per treatment.

Example 9 - Preventative treatment of surgical wounds

Krill's Single-Enzyme and Multi-Enzyme formulations were tested as a preventive antimicrobial lotion for surgical wounds in a total of 60 patients, and compared to 0.9% saline.

Non-bleeding surgical wounds were removed after surgery and this was repeated twice. The wounds were rinsed and covered with sterile gauze bandage. The wound was examined for infection, inflammation, temperature, dead tissue, pain and possible damage.

salt treatment 6-12 hours thereafter, the treatment was thoroughly covered daily with the formulation solution and semi-occluded with each re-dressing for redness, swelling, bleeding, pus, bleeding, and side effects

-24A treatment was terminated when the wound was healed, with more than 90% epithelialization, or when test or control treatment failed.

Neither post-operative infection nor acute inflammation or reddening were observed in the two groups using Single-Enzyme or Multi-Enzyme solutions. 18 wounds healed during the 10 day treatment. No adverse side effects occurred.

In the control group, 4 patients were severely infected with the wound and two other patients developed acute inflammation. Redness, swelling, and pain were common in this group. 14 wounds healed within 10 days.

For this indication, the effective dose is 0.01 to 100, preferably 1 to 25 mg / 100 cm⁶ was between.

10th Example I - Bladder and urinary tract infections

In this experiment, 12 women with painful urinary tract infections were studied. Lyophilized white powder without preservative and antimicrobial agent was used. Multi-Enzyme Preparation: 6 ampoules in 50 ml physiological saline resulted in a solution of 3 units / ml casein.

The first urine was very cloudy in all patients, the second was clear or slightly cloudy.

The pain disappeared immediately and the retention of urine was also noticeably improved after two days of treatment. MO samples confirmed that no bacteria were present in the urine after 4 days, and treatment was terminated after 4 days in all patients. No adverse side effects occurred.

-25The results are summarized in Figures 9 and 10. The average MO values ​​are shown in Table 2.

Second spreadsheet

Mean MO after 4 days of treatment with Krill Multi-Enzyme

<td>MO value</td><td>MO value</td><td>MO value</td>

<td>before treatment</td><td>the 2 . on the sun</td><td>a. NAPOLI</td>

<td>1.4 x 10⁴</td><td>4.3 x 10³</td><td>&lt;1.0 x 10^:</td>

<td> 100%</td><td> 31%</td><td> &lt;1%</td>

The above examples show that treatment of bladder and urethra infections twice daily with enema has a very beneficial effect on the disease. After two days of treatment, the signs of acute inflammation and infection disappeared, and after 4 days of treatment, all symptoms disappeared.

The effective dose is from 0.01 to 200, preferably from 1 to 50 mg, per treatment.

Clinical Examples - Inflammation

The presence of inflammation was a common feature in most experiments, and the reduction in inflammation was similar for all indications. Within three to four days, the degree of inflammation was so reduced that healthy wound healing could begin. In the preventive study of postoperative wounds (Example 9), inflammation did not develop.

-26csoportokban. Also compare with Example 3 (acute and chronic yeast infections / inflammations).

11th Example 1 - Inflammated horse joints lumbar trot horse forelimb was investigated in this experiment. Lyophilized white powder without preservative and antimicrobial agent was used. Multi-Enzyme Preparation: 1 ampoule dissolved in 10 ml water for injections resulted in a solution of 2.5 casein units / ml.

ml of test solution was injected into the aching hips on days 1, 2, 4 and 7. Following injections, the horses were monitored for immediate adverse effects or signs of deterioration of their condition. Symptoms were recorded once daily.

In the latter case, pain was reduced rapidly after 30 minutes to 2 hours after the first treatment, 4 more cases within 6 hours and 2 more hours after the second injection. In 1 case, the pain ceased only after the 3rd injection, and in this case the knee was very swollen.

Heat and swelling were rapidly reduced, and in 15 cases no evidence of inflammation was observed on the second day after treatment. No adverse side effects were observed.

The results are summarized in Figure 11.

From the above example, acute arthritis in racing horses can be treated with a series of 4 intra-articular injections administered over 7 days. Symptoms of Inflammation • ·

-27 decreased rapidly, and after the second day the swelling and warmth ceased. After a week of post-treatment, the horses were back to training.

The effective dose is 0.1 to 200, preferably 1 to 50 mg per treatment.

12-16. Clinical example - Dead and degenerate cells (cf. also 3.4.5 and 10 examples)

Example 12 - Patient Wound Surgery Wounds A single Enzyme preparation made from krill was treated. Lyophilized white powder without preservative and antimicrobial agent was used. It was dissolved in 5 ml of physiological salt. SingleEnzim preparation: 3 units of casein / ml.

After normal rinsing and wound cleaning, Single-Enzyme was used. Dissolve 1 ampoule twice daily in 5 ml of physiological saline, pour the solution onto a gauze pad and completely cover the wound. The dried gauze was fixed to the wound with self-adhesive semi-occlusive material. When the bandage was replaced, we examined the redness of the wound, edema, bleeding, swelling, temperature, secretion, pus, goiter, pain, odor, possible adverse side effects, and the general health of the patient. The treatment lasted up to 7 days or until dead cells, bleeding, fibrin and pus were absorbed.

The patient group was heterogeneous with respect to the type of wounds, with surgical wounds infected, traumas, burns, gunshot wounds.

-28 wounds and diabetic wounds occurred. Patients received twice-daily polyclinic treatment. No adverse side effects were observed.

The results are summarized in Figure 12.

From the above, we can see that the gut, fibrin, pus, clot and plaques were effectively exfoliated within 1 week and some wounds healed within a week. Initially, treatment of burns and surgical wounds in diabetic patients appeared to be extremely ineffective, but in fact, at the end of treatment, there was no change in treatment. day, the gingiva was completely absorbed in the interior, remaining what was the surface nail covering the wound, and the wound was partially healed within a week.

The effective dose is from 0.01 to 100, preferably from 1 to 25 mg / 100 cm -1.

13th Example 1 - Treatment of scarring and keloids

The purpose of these studies was to investigate the utility and efficacy of the MultiProtein preparation made from krill as a possible alternative to postoperative correction. Lyophilized white powder without preservative or antimicrobial agent was used. It was dissolved in 5 ml of physiological saline. MultiProtein preparation with endo-exopeptidase activity: 1 ampoule dissolved in 5 ml water for injections yielded a solution of 5 units / ml casein.

0.2 cm Multi-Protein per cm of scar / keloid

-29 solutions were injected, up to 1 ml per wound and daily. The treatment was repeated once a day and examined for scar / keloid redness, swelling, scar temperature, bleeding, gangrene and adverse side effects.

Treatment was continued for up to 7 days or until the scar / keloid was 80% absorbed.

Multi-Protein was injected once daily into 5 facial scars, 3 hand and forearm cheloids.

Bruising scars were reduced to 25% of their original size and connective tissue scar tumors to 70% of their size after 7 days of treatment. No adverse side effects were observed during the experiment.

It can be seen that 75% and 30% of the intradermal and subcutaneous scars and keloids described above were absorbed within 7 days by injection into the tissue. For plastic correction of scars and keloids, the scar and keloids should not be absorbed too quickly, and it is rarely necessary to remove more than 60-80% of the scar to achieve a good result. The skin and underlying tissues need to slowly adjust to the new situation, otherwise there may be other problems such as wrinkling.

The effective dose is 0.1 to 50, preferably 1 to 10 mg per treatment.

14th Example I - Psoriasis and dry eczema

Psoriatic plaques and dry eczema areas were easily absorbed by the hydrogel formulation (Single-Enzyme) under the semi-occlusive layer. Within 24 hours of plaques • · ·

-30 completely disappeared, and the sensitive skin, especially in psoriatic patients, showed no other inflammatory symptoms, imitation or pain. In contrast, the affected skin areas developed less inflammation after treatment, but after the disappearance of plaques, the effectiveness of the steroid-containing creams increased and a much better effect could be achieved with these formulations.

The effective dose is 0.01 to 100, preferably 1 to 25 mg / 100 cm² quantity.

15th example - Dogs Toothpick.ia

The purpose of the experiment was to investigate the utility and effectiveness of a Multi-Enzyme preparation made from krill in canine plaque for dogs. Lyophilized white powder without preservative and antimicrobial agent was used. Multi-enzyme preparation with endo- and exopeptidase activity: 1 ampoule dissolved in 5 ml of physiological saline resulted in a solution of 5 units of casein / ml.

Carefully apply the contents of the freshly prepared ampoule to the gums and teeth. The tongue was fixed for a minimum of 2 minutes and, for 2 hours after treatment, was prevented from eating and drinking. The treatment was repeated twice daily until plaques were completely absorbed. In dogs, plaque status, saliva secretion, and adverse side effects were examined once daily.

a hunting hound was included in the study which produced abnormal plaque during special feeding and feeding. 4 days after treatment, plaques disappeared and • · ·

Experiment 31 was completed. No adverse side effects were observed.

The effective dose is 0.1 to 100, preferably 1 to 35 mg per treatment.

16th example - Human tooth plaque

The purpose of the study was to investigate the utility and effectiveness of the Krill-produced Múlti-Protein preparation for dental plaque. Lyophilized white powder without preservative or antimicrobial agent was used. Multi-Protein Preparation with Endo- and Exopeptidase Activity: 1 ampoule dissolved in 5 ml physiological saline resulted in a solution of 5 units / ml casein.

Before each treatment, 1 ampoule of Multi-Protein solution was prepared and the oral cavity was rinsed for 5 minutes. For 2 hours after treatment, eating and drinking were not allowed. The treatment was repeated twice a day and patients were tested for plaque status, salivation, dry mouth and adverse side effects once daily. Patients were not allowed to brush their teeth during the experiment.

Treatment was continued for up to seven days or until all signs of plaque had disappeared.

hours after treatment, patients felt that their teeth were smoother and softer, and that the plaque had completely decayed. At the time of the visual examination, plaque remains were visible. 2 hours after the third treatment, the plaque disappeared completely and treatment was completed. Harmful side effect

<img file="HUT69989A_D0003.tif" />

not experienced.

The effective dose is 0.1 to 100, preferably 1 to 35 mg per treatment.

17th example - Opportunistic infections

These types of infections develop in patients with cancer, AIDS, drugs that suppress the immune system, radiation, and so on. inhibit.

Pathogenic microbial strains, and normally non-pathogenic strains, can become pathogenic, causing various types of infections that eventually lead to blood poisoning and will be a direct cause of death in patients suffering from such infections.

ill, two IV. stage one uterine cancer and one had radiation infection, 7 non-healing, chronically infected surgical wounds were treated twice daily with dry wrap. After days 12, 14, and 17, the wounds healed.

The effective dose is from 0.01 to 100, preferably from 1 to 25 mg / 100 cm².

18-19. Clinical Examples - Eye Diseases

Example 18 - Dog Cataract

The purpose of this study was to investigate the utility and effectiveness of krill-derived Single-Protein and Múlti-Prote in in the treatment of canine cataracts. Lyophilized white powder without preservative and antimicrobial additive was used. Single-Protein with exo and endopeptidase activity: 1 ampoule in 5 ml water for injection ·· · ·

-33 · · · ♦ ·· · when reconstituted resulted in a solution of 3 units of casein / ml. Multi-Protein preparation, a mixture of enzymes with endo- and exopeptidase activity: 1 ampoule dissolved in 5 ml water for injections resulted in a solution of 5 units / ml casein.

Right eye 2 drops, approx. It was treated with 0.4 ml Multi-Protein in solution. The procedure was repeated once a day and the dog was examined for ophthalmic opacity and adverse side effects.

The procedure was completed when the eyes looked clear at first glance. The left eye was treated with the Single Protein solution in the same way as the Last Protein treatment,

No adverse side effects were observed.

The procedure was completed when the eyes looked clear at first glance.

days later, the opacity of the right eye was noticeably reduced, and after 10 days the eye was completely clear, at which point treatment was completed.

Similar observations were made for the left eye and treatment was completed there after 10 days. No adverse side effects were observed.

The effective dose is from 0.01 to 50, preferably from 1 to 5 mg, per treatment.

19th example - Eye Cataracts

The purpose of the experiment was to investigate the efficacy and utility of a Multi-Protein preparation made from krill in cataracts.

Multi-Protein preparation that is endo and exopeptidase

A mixture of -34 activity enzymes, a solution of 2.5 casein units / ml, was instilled into the right eye every three days.

An 80-year-old woman, who had only one light / dark eye due to cataracts, was treated with 0.4 ml of 1 casein Mu1ti-Protein preparation every three days. After 5 occasions the treatment had to be terminated for other reasons, but the opacity was visibly reduced and the lady reported that her eyesight had improved. No irritation, pain, or other discomfort occurred during or within 1 month of treatment.

The effective dose is 0.01 to 50, preferably 0.1 to 5 mg per treatment.

Clinical Examples - Pain

The pain is primarily caused by mechanical damage to the tissues and the acute sensation of pain disappears shortly after the injury. However, some sensitivity remains in the initial stages of wound healing. Infections and acute inflammation can cause various pains due to swelling of tissues, chemical reactions, biochemical activities, formation of gummy tissues, adhesion and scarring. The pain persists for a long time after wound healing, and may even persist.

Pain is a very subjective parameter and very difficult to resolve clinically. By definition, pain is defined as everything from mild itching to severe pain causing disability. Patients • · · ·

-35 people have severe pain, are getting used to pain, and the threshold is constantly increasing.

The onset of pain relief was the fastest and most obvious effect of the test compositions of the invention. Pain relief was reported 20 minutes to 2 hours after the first application, and in most cases, mild pain or tenderness remained after the second day. Patients reported similar reductions in pain, regardless of indication, for acute or chronic pain. The treated parts retained tactile sensation.

Patients who did not have pain before treatment reported that the infected area had calmed down and relaxed after the first treatment, especially when the eyes and mouth were treated. (cf. the examples above, pain is a test parameter in most experiments.)

Pain during bladder infection and gingivitis has led to physical and social discomfort in patients. As a result of the test preparation, patients rapidly regain their ability to concentrate and behave like a normal human being after 2 days of treatment. (See Examples 3 and 10)

The most objective result for the development of pain perception was obtained in the inflamed joints of horses, where the pain sensation was related to the time it took for the injured leg to become chimeric. The result is similar to that of studies in humans. (see Example 11).

An effective dose of -36A is from 0.01 to 220, preferably from 1 to 25 mg, per treatment.

20-40. Clinical Example - Mixed Cases

20th example - Gastric ulcer

A man of about 40 years with recurrent mild gastric ulcer complaints was treated with a 5 mg / capsule PHIM formulation in acid-resistant gelatin capsules. You took one capsule daily with a glass of water for 2 weeks. After the fourth day, the stomach complaints disappeared, the stomach function returned, the faeces were normal, the pain was gone, and so on.

A 55-year-old man who has had persistent gastric ulcer complaints for 20 years was treated as described above. After a few days of treatment, the complaints disappeared and the stomach functioned normally.

Gastric ulcer is an inflammatory process likely to start with a bacterial infection. First, the gastric mucosa is infected by microbes, then inflammation and then ulcer develop. This means that the cells of the gastric mucosa become abnormal at some point. This situation is quite similar to that of skin infections and wounds; the difference between the skin and the gastric mucosa is that there is a cellular system called Peyer plaque in the mucosa, which is very active in B, T cells and so on. available. This system is believed to play an important role in preventing the development of autoimmune diseases. From the fact that PHIM works effectively in this • «· · · ·

-37 system and having the ability to remove abnormal cells from the sensitive mucosal layer, it logically follows that other autoimmune diseases such as Crohn's syndrome, ulcerative colitis, rheumatoid arthritis, etc. can be effective. treatment.

The effective dose is 0.5 to 300, preferably 1 to 50 mg per treatment.

21st Example 1 - Treating Wrinkled Skin

Free radicals that form cross-links in collagen contribute most to skin wrinkling. The formation of free radicals is caused by burned or dead cells by providing an excellent medium for bacteria to accelerate the formation of free radicals. By allowing the PHIM composition to eat dead cells and bacteria, the wrinkles formed disappear.

A 33-year-old woman was treated for 60 days with a gauze dressing dipped in PHIM 106 solution every night. The amount of PHIM 106 used overnight was approximately 0.15 mg. The dressing was in contact with the skin for 30 minutes.

Treatment was performed only on one eye, and the other side was used as a control. There was a difference already after 15 days of treatment, but after 60 days there was a marked difference in frustration. The treated surface was smooth and elastic, and the number and depth of wrinkles was significantly reduced compared to the untreated area.

The effective dose is 0.01 to 100 mg, preferably 1 to 25 mg

Amount between -38η / cm.

22nd example - Octopus

We treated the rectum of a 62-year-old male. The man suffered from polyps for 3 years and received internal medical treatment. She was taking various medications, but there was no improvement.

treated with 5 g of PHIM in a solution of 5 ml of physiological saline. The treatment was repeated five times in total. All complaints were gone, and the next visit to the internist showed that the octopus had disappeared. 8 months after treatment, the man is still free of complaints.

The effective dose for this indication is 0.5 to 250, preferably 1 to 50 mg per treatment.

23rd example - Warts

A wolf on the neck of a 35-year-old male was treated with patch (Hansaplast) for 1 week dipped in PHIM 106 solution (about 0.1 mg PHIM / patch). The treatment was repeated once a day for one week. After one week of treatment, the wart completely disappeared.

The effective dose for this indication is 0.5 to 250, preferably 1 to 50 mg per treatment.

24th example - Cold

Five patients aged between 30 and 63 years were treated with PHIM 106 for several hours to develop the symptoms of colds. ··· »« · ·· «·

<img file="HUT69989A_D0004.tif" />

it consisted of mouthwash. The nasal spray dose was about 0.1 mg per nostril each time and the mouth rinse dose was 1.5 mg each time. When rinsing the mouth, the liquid was held in the oral cavity for 2-4 minutes and then swallowed. After 12 hours, the symptoms of the common cold disappeared and the patients were free of complaints.

The effective dose is 0.1 to 100, preferably 1 to 25 mg per treatment.

25th example - Heamophilus influenza

A 34-year-old woman was treated for recurrent infection with the Haemophilus influenza virus. Within a few hours of developing tension and pain in the nasal cavity, the patient was flushed with 2 mg PHIM 106 for 2 minutes with approximately 0.1 mg PHIM 106 in each nasal cavity. Spraying was repeated every three hours for 3 days. The feeling of tightness due to nasal sinus infection disappeared a few hours after the first treatment and the mucus secretion increased significantly. 3 after a day of treatment, the woman was free of complaints.

The effective dose for this indication is 0.1 to 100, preferably 1 to 55 mg per treatment.

26th example - Herpes Zoster

The face of a 70-year-old man was painful for 10 months

Herpes Zoster infection was surface treated with 1-2 mg PHIM

containing gauze bandage every three days.

Itching and Pain Even After the First Treatment «·· • ·· · ·

-40 decreased and then completely disappeared after 12 days. Because of the infection, the man had problems with chewing because his palate was sore, but after 12 days he was able to chew without problems.

The effective dose is from 0.01 to 100, preferably from 1 to 25 mg / cm.

27th example - Herpes genitalis

A 62-year-old man was treated for a 10-year history of herpes genital infection. The complaints returned regularly every 4 months and the man refrained from having sexual contact in the presence of acute complaints.

PHIM 106 was treated with a solution soaked in ca. 3 mg / dressing composition. The treatment was repeated twice a day for two days. Already after the second treatment, his complaints disappeared. The man had no complaints for 10 months after stopping treatment.

A suitable dosage range for this indication is 0.1 to 100, preferably 1 to 25 mg per treatment.

28th example - Hemorrhoid

Two subjects were treated for hemorrhoidal infection, one was a 55-year-old male and the other a 30-year-old female. The woman's problems started 3 years ago, and she had pains and minor bleeding. It was treated with dry PHIM 106 powder which was packaged in a gauze dressing and applied to the affected surface. One dose is approx. It was 5 mg. A total of 5 treatments were needed to completely resolve the complaints. She hasn't had a complaint for a year.

The man's complaints had recurred and disappeared in the past

-41években. 6 months ago, the complaints became acute, with bleeding and very severe pain. The man mg is PHIM

106 solution of gauze soaked once with pain once minutes ceased after a single treatment, all complaints disappeared and have not occurred since.

The appropriate dosage range for this indication

29th example - Diarrhea

A 41 year old man developed acute food poisoning with diarrhea (probably caused by Staphylococcus) and vomiting. One hour after the outbreak, the man is given 5 mg

She was treated with PHIM 106, which was held in her mouth for 3 minutes and then slowly swallowed. This procedure was repeated three times every 2 hours. After the fourth treatment, stomach pain resolved, vomiting and severe diarrhea were completed.

An effective dosage range for this indication is 0.5-300, preferably 1-50 mg per treatment.

30th example - baldness

A 55 and a 62 year old man were treated. Over the past 10 years they have been severely balding. The treatment consisted of impregnating the whole scalp with PHIM 106 solution for a total of ca. 5 mg

PHIM was used for a treatment. To keep moisture in the scalp, a swim cap was applied to the head for 30 minutes.

treatment was repeated once a week for approx. for 3 months.

after treatment, new hair began to grow. The reason for this success is probably that the PHIM 106 effectively removes the

Dead and abnormal cells that serve as a medium for -42 bacteria. These bacteria produce toxins that locally cause cells in the skin layer to produce, among other things, TNF, a tumor necrosis factor which, when present in large amounts, inhibits hair growth. By removing the dead and abnormal cells and bacteria mentioned above, microcirculation is also improved, which is beneficial for hair renewal.

A suitable dosage range for this indication is 0.01 to 100, preferably 1 to 15 mg / 100 cm².

31st example - Acne

One of the 29 and 30-year-old women was treated for sebaceous inflammation. The 29-year-old had severe complaints, while the 30-year-old had moderate problems, especially on the forehead.

They were treated with 0.1 mg PHIM 106 several times daily for 4-6 days. The effect was noticeable even after the first treatment and after a few days the infections disappeared and the recovery was almost complete. One week after treatment, only pigment spots were visible.

The effective dose for this indication is 0.01 to 50, preferably 1 to 10 mg per treatment.

32nd example - Bronchitis

A 55-year-old man had relatively severe bronchitis. Disease caused breathing difficulties, longer than 100 meters, without interruption, difficulty, fatigue and dry cough.

The cause of bronchitis, according to the doctor, is 3 years earlier

-43 Mycoplasma infection has occurred which has become resistant to antibiotic treatment. The infection produced pulmonary edema, which was confirmed by the Rontgen test.

<td>THE</td><td>patient was rinsed with 4 mg PHIM 106 solution</td>

<td>course.</td><td>The PHIM solution was held in his mouth for 4 minutes, then</td>

<td>slowly</td><td>He swallowed. Treatment was performed every other day</td>

first two weeks. During this time, a small amount of PHIM

<td>106 was</td><td>also inhaled.</td>

There was no improvement in the first two weeks, but after two weeks of treatment, the cervical lymph node on the left side became swollen and sore. The treatment was continued for approx. three times. After a week and a half, the problem with the lymph node had disappeared and the symptoms of bronchitis had subsided. After an additional 3 weeks of rinsing every four days, the bronchitis problems completely disappeared.

After 6.5 weeks of treatment, the patient recovered completely and was able to walk for 5 kilometers without any problems.

For this indication, an effective dose is 0.1 to 100, preferably 1 to 25 mg per treatment.

33rd example - Prostatitis

A 52-year-old man suffered from prostate inflammation every winter from the age of 20. In the last 4 years, the problems became acute and caused severe lower abdominal pain. He received various antibiotic treatments during the acute phases, but the symptoms reappeared within a few weeks after the end of the treatment. The patient had PHIM 106

-44 containing acid-resistant capsules at a dose of 5 mg / capsule upon recurrence of such moderate to severe prostatitis. He took two capsules daily for one week. The symptoms have completely disappeared and the disease has not reoccurred in the last 12 months.

An effective dosage range for this indication is 0.5-300, preferably 1-50 mg per treatment.

34th Example 1 - Infection by a resistant mycoplasma strain

A 55-year-old male was treated for resistant Mycoplasma infection. The man was treated with various antibiotics 3 years earlier when he felt sick, but the low dose developed resistance. A few weeks after the infection, he had a high fever, coughed heavily, and an X-ray showed lung edema.

Disease caused by breathing difficulties for more than 100 meters without interruption caused difficulty, fatigue and dry cough.

The patient is exposed for approx. Rinse with 4 mg PHIM 106 solution during treatment. The PHIM solution was held in his mouth for 4 minutes and then slowly swallowed. Treatment was given every other day for the first two weeks. During this time, a small amount of PHIM 106 was inhaled.

There was no improvement in the first two weeks, but after two weeks of treatment, the cervical lymph node on the left side became swollen and sore. The treatment was continued for approx. three times. After a week and a half, the problem with the lymph node had disappeared and the symptoms of bronchitis had subsided.

• ·

-45After an additional 3 weeks of rinsing every 4 days, the bronchitis problems have completely disappeared.

After 6.5 weeks of treatment, the patient recovered completely and was able to walk for 5 kilometers without any problems.

For this indication, an effective dose is 0.1 to 100, preferably 1 to 25 mg per treatment.

Example 35 - Mastitis in humans

A 28-year-old woman had milk secretion problems three days after birth, the milk secreting glands had turned to stone, and she had severe pain. The problems started half an hour after breastfeeding. In Uppsala, Sweden, they were treated at the Academic Hospital using the various methods available to them, but nothing was used.

A solution containing about 0.1 mg of PHIM was instilled into the nipple when severe pain occurred. The pain subsided after 15 minutes and the hard milk secreting glands became soft after 45 minutes.

The treatment was repeated for more than 3 months after each lactation. To find out if treatment was still needed, she sometimes waited until after the breastfeeding treatment until the pain was present. During such checks, severe pain returned and the milk secreting glands became hard.

For this indication, an effective dosage range is 0.0125, preferably 0.1-5 mg per treatment.

Example 36 - Allergic Itching

A 28-year-old woman had allergic complaints, which included itching and hives over the knee and in her sleep or under the chin. The rash on the knee looked like white, rather large protrusions, while the animal was completely covered with dots of the same color as the skin.

Gauze dressing was moistened with PHIM 106 solution and applied to the acne on the knee. After 10 minutes, the gauze dressing was removed. After 45 minutes the pimples started to disappear and after 1.5 hours they completely disappeared. The white protrusions and hives have disappeared.

an hour later we treated the chin area. This area was very itchy and even more sensitive due to scratching.

When using gauze dressing dipped in PHIM solution, itching increased and gauze dressing had to be removed after 7 minutes due to severe itching. Within 1.5 hours the itching subsided and 1.45 hours later the tel jeseir 'disappeared.

No problem appeared on either knee or face after 48 or 24 hours.

A suitable dosage range for this indication is 0.01,100, preferably 1-25 mg / 100 cm 2.

37th Example 1 - Problem of adhering to the vagina

The purpose of the experiment was to investigate the anti-adhesive properties of Krill Multi-Protein in rabbit Achilles in tear. Two rabbits left Achilles tendon separated and immediately stitched to split

-47 polishing technique, so the leg was not immobilized after surgery.

days after surgery, adhesion of the tendons to the vagina was evident and 1 mg (0.25 ml) of Multi-Protein was injected interstitially into the vagina on day 5 and treatment was repeated on days 7 and 9. Twenty-four hours after the first injection, the adhesion of the in and the vagina ceased and the in was moving freely in the vagina. Adherence did not re-occur 6 weeks after surgery.

months later, the animals were dissected and the stomach was examined macroscopically for adhesion, fibrin and collagen proliferation. The in and the sleeve were boiled separately, no sign of adhesion was found. Comparison of operative left insertion tendency with unoperated right insertion no difference in tensile strength was found.

The Multi-Enzyme preparation specifically degrades excess fibrin without affecting the fibrin needed for the perfect healing of the in and vagina.

An effective dosage range for this indication is 0.1 to 100, preferably 1 to 10 mg per treatment.

38th Example 2 - Recovering rigid wrist agility

The purpose of the experiment was to investigate the effectiveness of krill-derived Multi-Protein in the degradation of limited movable wrist fibrin tissue.

The mobility of the wrist of a woman over the age of 40 was reduced to 25% of normal mobility due to a plaster cast worn for a long time after a complicated hand fracture.

• ··

In spite of regular exercise, the mobility of the injured joint was poorly improved, and the stiffness was diagnosed to be caused by the fibrin covering the joint.

Multi-Protein preparation was injected intraarticularly at 3 days intervals for a total of four times. After day 14, mobility increased to 50-60%. After 4 months of exercise, the woman regained 70-80% of her wrist mobility

No adverse side effects were observed.

An effective dosage range for this indication is 0.1 to 100, preferably 1 to 10 mg per treatment.

39th Example 1 - Antithrombolytic / Antiembolic Activity of Single-Protein

The purpose of the experiment was to investigate the efficacy of Single-Protein produced from krill in the treatment of thromboses and emboli.

For thrombosis, an artificial stasis in the ear vein was induced until a blood clot formed. SingleProtein preparation, 0.5 mg / 2ml, was injected intravenously in front of the thrombus, 2 cm from the ischemic site. Within 30 minutes, the blood clot dissolved completely and the blood flowed freely. The treated part started to weakly gangulate but after 7 days the gland was absorbed.

In control animals, the ischemic part was completely disappeared within 4-5 days.

An effective dosage range for this indication is 0.1200, preferably 1-10 mg per treatment.

Example 40 - Treatment of Glaucoma

The aim of the experiment was to investigate the efficacy of a Multi-Protein preparation made from krill in the treatment of increased intraocular pressure.

In a 74-year-old man who reported mild pain / discomfort in his right eye, the study showed that increased intraocular pressure was the cause of the symptoms. Tests have shown that the pressure is constantly increasing and is not due to an acute problem.

daily, 0.1 mg of Múlti-Protein preparation was instilled into the eye, for a total of 3 times. After 2 weeks of post-treatment, intraocular pressure was normal.

The man reported immediate relief of pain 20 minutes after the first treatment.

No adverse side effects were observed.

An effective dosage range is 0.01 to 50, preferably 0.1 to 5 mg per treatment.

41st Example IV - Antiviral activity on HIV infected cell lines in vitro

This experiment was performed at the Swedish National Laboratory of Bacteriology (SBL). The effect of Multi-Protein preparation was compared with that of AZT and foscarnet. The formulations were tested at various concentrations ranging from undiluted to 10 µM (the highest concentration of Multi-Protein tested was 5 mg / ml). The formulations were added to 40-60% HIV-I infected cell lines under the same and standardized conditions.

At -50 dilution, AZT and foscarnet attacked and killed all cells in cell culture. Only dead and disrupted cells remained in these cultures.

Concentrated Mu1ti-Protein solution inhibited virus growth. In cultures, the number of infected cells was 40-60%, the same as at baseline, and these cells detached from the surface. The shape of these attacked cells changed and the virus in them was unable to reproduce. Healthy cells in the MultiEnzimes group were not attacked by virus released from infected cells, nor by Multi-Protein, the cells adhered to the surface and remained morphologically unchanged.

This shows that MultiProtein produced from krill is able to distinguish between healthy and divergent cells, such as those carrying the virus, and in some special way recognizes, attacks and destroys abnormal cells without harming the healthy.

A suitable dosage range for this indication is 1500, preferably 10-300 mg per treatment.

Inhibitory Effectiveness - Study 1

Virus (IC ^ q) Cell Vitality (Tox.) (HIV-1 strain HTLV IHb moi 0.4)

Multi-Protein 200 100 (deformed cell)

AZT 100 100

foscarnet

200

100 • · · ·

-51Control: 50% infected cells. Cell toxicity and percentage of infected cells were determined by immunofluorescence measurement.

Inhibitory Effectiveness - Study 2

Virus (ICtjg) Cell Vitality (Tox) (HIV-1 strain HTLV Illb moi 0.2)

Multi-Protein 200-400 100-200 (deformed cell)

AZT 100 100

Foscarnet 100 100

Control: 50% infected cells. Cell toxicity and percentage of infected cells were determined by immunofluorescence measurement.

42nd example - Epidermophytosis - swelling of the feet

The purpose of the experiment was to investigate the efficacy and utility of a Multi-Protein preparation from krill in the treatment of foot epidermophytosis.

Fungal infections of the patient were studied using 5 minutes of Multi-Protein Solution / Case in Multiple Protein Solution for 3 days and 30 minutes in a foot bath and overnight hydrogel (5 Casein / mL) for a maximum of 7 days.

The pain disappeared immediately in the majority of patients and within 2 days in the remainder. Plaques formed in the cracks, under the nail, on the surface of the skin, were easily removed, with plaques, foul odor and ···· • ···

-52 the infection disappeared after three days.

An effective dosage range for this indication is 0.01 to 100, preferably 1 to 25 mg / 100 cm².

43rd Example 1 - Eczema infections

The purpose of the experiment was to investigate the efficacy and utility of the Krill Krulti-Protein preparation for eczema seborrheic and psoriatic infections.

Forty patients were treated once or twice daily with Multi-Protein Hydrogel (2.5 units of casein / ml).

Dry eczema / plaque 2-4 treatments, 1-2 days, showed no signs of infection. Fatty type sebum plaques disappeared after 6-9 days, although the infection / inflammation disappeared within the first 2-4 days.

Patients with psoriatic plaques have experienced an increase in the effectiveness of their commonly used steroid cream, probably due to the fact that Multi-Enzyme effectively removed the plaque, making it easier for the steroids to be absorbed into the skin.

The effective dosage range for this indication is 0.01 to 100, preferably 1 to 25 mg / 100 cm².

44th Example 4 - Foreskin infections

The purpose of the experiment was to investigate the efficacy and utility of a Krill-derived Mu1ti-Protein preparation in infant infestation.

One infant at 4 and 6 weeks of age was treated with Multi-Protein at a concentration of 1 casein unit / ml twice daily

-53oldattal. The forearm was washed in the morning and evening with approximately 10 mL of solution using a standard syringe fitted with a soft catheter. After 3 days, both infants were free of symptoms and the infection did not recur within the next 2 months.

An effective dosage range for the above indication is 0.1 to 100, preferably 1 to 25 mg per treatment.

45th Example 5 - Foreskin infections in dogs

The purpose of the experiment was to investigate the efficacy and utility of the Krulti-Protein in preparation made from krill in canine infections in dogs.

The area under the foreskin of six dogs was washed once daily with Multi-Protein Solution at a concentration of 1 Casein Unit / ml. 10 ml of Multi-Protein solution was drawn into a standard disposable syringe. A soft silicone catheter was attached to the syringe, the catheter was inserted under the forearm, and the area under the forearm was slowly rinsed. Approximately 1 mL of solution was held for a minimum of 2 minutes under the foreskin and the dogs were prevented from licking this area for 30 minutes.

The purulent discharge ceased in all cases within two days and the signs of inflammation and infection disappeared within 4 days.

. The effective dosage range for this indication is 0.1 to 100, preferably 1 to 25 mg per treatment.

46th Example 1 - Eye drops for general use

The purpose of the experiment was to examine the Multi ···; . ··. ···; *:. ·. ·. · * * · · «·· ·

-54Protein preparation efficacy and usefulness in the treatment of tired and irritated eyes where there is no evidence of clinical infection.

we treated ad hoc tired and irritated eyes of a patient who had no eye disease, who had no allergic reaction to airborne dust particles, and whose eyes were not inflamed or infected. 2 drops

Multiple Protein Preparation Solution at a concentration of 0.1 Units / ml casein was instilled into the eye as needed.

within minutes, everyone was relieved of pressure or mild pain. No one reported a decrease in photosensitivity, or a change in focus or pupil dilation. Occasionally, temporary drought was experienced.

No irritation, increased tear formation or other adverse side effects have occurred.

A suitable dosage range for this indication is 0.01 to 50, preferably 0.1 to 5 mg per treatment.

47th example - Abscesses on calves

The aim of the experiment was to test the efficacy and usefulness of MultiProtein preparation on calves abscesses using Multi-Protein solution at 5 units / ml.

Two calves, with approximately 25-40 ml of abscess on the neck resulting from calcium injection, were treated once daily. 10 ml of the Multi-Enzyme solution was instilled through a drain tube and held in the abscess for 4 hours. The tube was then opened and left open until the next treatment.

··

After the third treatment, the drainage fluid was clear and after the sixth treatment the tubes were removed. The pockets were completely healed in 9 and 12 days, respectively.

The effective dosage range in this case is 0.1-200 mg, preferably 1-20 mg per treatment.

48th example - Females in dogs

The purpose of the experiment was to investigate the efficacy and utility of MultiProtein in the treatment of canine paw disorders.

Sore, infected and purulent / bleeding gums between Boxer fingers were treated twice daily with Multi-Protein Solution at 5 units / ml.

The gauze, previously dipped in 2 ml of solution and then dried, was placed on the germs. The leg was bandaged to secure the gauze and a rubber slipper was pulled on to protect it.

The pain was obviously reduced after 1 day and the purulent discharge and bleeding disappeared within 2 days. After 5-7 days, there were only faint signs of inflammation and after 11-15 days the hatching was completely gone and the dogs were able to move freely.

An effective dosage range for this indication is 0.1 to 100, preferably 1 to 25 mg per treatment.

Dosages for various indications

Figure 13 shows a dose-response curve for a specific case, burn injury, expressed as a percentage of efficacy against the antimicrobial activity of PHIM

-56 shows as a function of the PHIM concentration tested. The amount of PHIM is mg / 100 cm² wound surface area is given in units. 2 mg PHIM / 100 cm² for wound area, the efficiency is almost 100%. The dose-response curve shown may also be representative of other cases.

Figure 14 shows the ranges of PHIM dosages used for 100% efficacy, i.e. recovery, for the various indications. PHIM mg / 100 cm was used² efficiency is plotted against its function. PHIM / 100 cm² patient area) eye infections inflammation viral infections fungal infections bacterial infections pain relief

The dose range (mg mg) for the affected area is:

0,5 - 0,8

0,1 - 15

0,1 - 1

- 15

0,5 - 25

0,1 - 25

Figure 15 shows the maximum accumulated amount of PHIM per kg body weight used until recovery, that is, completion of clinical treatment, for various indications. The approximate maximum values ​​(mg PHIM / kg body weight) in each case are as follows:

eye infections 1 surface wounds 5 urinary tract infections 9 fungal infections 5

Herpes simplex infection 5 acne oily skin 18 burns 20 opportunistic infection 28

-57A Figure 16 shows the total amount used until recovery

Minimum amount of PHIM per kg body weight. The approximate minimum values ​​(mg PHIM / kg body weight) for the various cases are as follows:

eye infections &lt;1 surface wounds 3 urinary tract infections 7 fungal infections 3

Herpes simplex infection 4 acne oily skin 13 burns 13 opportunistic infection 15

1-51. conclusions from clinical examples

The very wide range of efficacy of the Single-Protein and Multi-Protein formulations of the present invention is reminiscent of the mode of action of phagocytic cells.

No clinical limitations or drawbacks could be identified for the indications studied. In contrast, the composition was effective even in cases where the immune defense was not activated, such as stagnant wounds or where the immune response was suppressed, such as opportunistic infections.

Adverse side effects

No adverse side effects were observed in any of the patients examined in the cases described above. Patients who had a history of polyallergy, food allergy, sensitive skin, or

-58 eyes were prone to dehydration, or in patients with known immunosuppressive or hyperactive disorders, and who were enrolled or studied in a separate group.

It has been shown that the Multi-Protein and Single-Protein formulations to be protected are non-toxic.

Based on the results of the above complete and preliminary clinical trials, it can also be concluded that the agents and compositions of the invention would be effective in treating a variety of other diseases and conditions in which the immune system is involved such as glaucoma, trachoma, cancer, AIDS / HIV; autoimmune diseases such as rheumatoid arthritis, Crohn's syndrome, multiple sclerosis, ulcerative colitis; diseases such as cholera, leprosy, malaria, hepatitis, typhus, sepsis; organ and tissue transplants (prevention) and plastic surgery.

49th example - Cancer

This experiment was conducted by Professor Takashi Makita at Yamaguchi University, Japan.

History

Cancers are complex and are made up of different types of diseases. Complexity is increased when, as the tumor grows, it forms primary tumor metastases in other organs, including the lymph gland. In addition to the problems caused by tumors, opportunistic infections also occur due to the inhibition of immune defense, which is caused both by the development of tumors and by chemotherapy, the most commonly used conventional therapy for cancer today.

Conventional chemotherapy treatment has many other problems. The biggest problem is that anticancer agents are unable to distinguish between a healthy cell and a cancer cell. This is the biggest problem, because chemotherapy also destroys the white blood cells needed for immune protection. As a result, cancer patients cannot be treated optimally.

Inhibited immune protection results in the tumor becoming larger and uncontrolled opportunistic infections. The limited availability of optimal treatment and opportunistic infections combine to reduce the quality of life of a cancer patient. Very few well-controlled studies have been conducted in humans, and the overall efficacy was 20-25% depending on the type of cancer.

One in Japan, well controlled II. In a study of patients with phase I primary and secondary cancers, the response rates (CR and PR) were 9.5% for adriamycin and 20% for mitomycin C [see T. Taguchi et al., Cancer Treatment (1992) 4 : 161-165].

In animal studies, efficacy is improved because higher doses are generally used than in humans, since the study of adverse side effects in animals, with the exception of objectively measurable parameters, is extremely difficult.

Cisplatin was used to treat Yoshida Sarcoma implanted in rats and cultured from cancer cells.

-60 made for studying surviving cells. The result showed that 53% of the cells survived the cisplatin treatment. (KR Harp et al., Antitumor, Toxic And Biochemical Properties of Cisplantin).

A similar experiment was performed with mitomycin C up to 0.5 mg / kg. In this case, the treatment efficiency of non-resistant YoshidaSarcoma was almost 100% in rats based on 30-day survival. The 0.5 mg / kg dose is impossible to use in humans due to severe side effects, including bone marrow suppression, which results in death of the patient. Yoshida sarcoma can easily become resistant to mitomycin C, in which case doses of 0.5 mg / kg or more are completely ineffective.

Weight loss, hair loss, decreased leukocyte count and chronic diarrhea are side effects at the above and below doses. (See Mitomycin C, Kyowo Hakko Ltd, Tokyo, Japan). Anticancer agents such as adriamycin, cisplatin, mitomycin C and the like. they are highly toxic and can only be administered intravenously or intra-arterially.

Cancer cells often develop resistance to the anticancer agent used, just as bacteria do to antibacterial agents. The development of resistance is the most serious barrier to curative treatment.

Summary of method and results

The aim of this study was to investigate the effect and efficacy of PHIM 106 in the treatment of Yoshida sarcoma in rats.

• ·

-61PHIM106 solution was injected into tumor (it), subcutaneous, (sc) and intraperitoneally (ip) in three different ways into white Wistar rats implanted with Yoshida sarcoma. Single PHIM 106 injections were given at 5 mg / kg ip, it and sc or repeatedly at 1.25 mg / kg SC twice daily for 7 days at 5 mg / kg SC every two days for a total of four times and 12.5 mg / kg in quantities of sc every other day for a total of four times.

Treatment was started when the size of the implanted tumor was 10 mm x 10 mm.

The rats were autopsied 7 days after the last injection. Tumor size was measured and compared with control animals.

The reduction in tumor size was 46% for rats given a single IT injection, 56% for those given a single SC injection, and 49% for those given a single ip injection.

In the group receiving the animals at doses of 5 mg / kg and 12.5 mg / kg sc every 2 days, for a total of 4 times, the tumor response was 53% and 69%, respectively.

One rat in the group receiving a dose of 12.5 mg / kg reduced tumor size. The rate of metastasis formation in the treated group was much lower than in the untreated group.

The treated animals, compared to the control animals, behaved normally with respect to eating and drinking. No adverse side effects were observed during the experiment.

-62 Description of the PHIM 106

As explained above, PHIM 106 is an acronym for enzymatic proteins derived from marine sources, the Antarctic krill used in the following tests. In accordance with the present invention, substantially novel properties of these proteins have been discovered. In our hypothesis, proteins first recognize diseased and abnormal cells as well as microbes, and then target cells are killed by their enzymatic properties. Healthy tissue cells remain intact.

The mechanism for perfect selectivity is still under investigation, but the working hypothesis is that the proteins are capable of responding to the same signal as our immune defense system and are able to attack any foreign intruder or abnormal cell.

Cancer cells are abnormal due to the appearance of tumor protein fragments on the cell surface, so that immune defenses recognize them as abnormal cells other than healthy tissue cells.

PHIM 106 has been tested in more than 400 patients in open-label human trials and no adverse effects were reported.

No toxicity was observed in animals. Rats were injected up to 5 g / kg body weight intravenously with PHIM 106 without showing signs of toxicity.

Because PHIM 106 consists of 24-34,000 Dalton proteins and is derived from marine organisms,

Formation of -63 could have been expected but antibody formation could not be detected when rabbits and guinea pigs were immunized.

Materials and methods used

The aim of the study.ia

The aim was to investigate, in a small number of comparative experiments, the modes of administration, the different timing of injection series and the dose response curve, as well as the overall efficacy of PHIM 106 in the treatment of implanted Yoshida sarcoma in white Wistar rats.

Our other plan was to study the targeted mode of action of PHIM 106 in the treatment of venous and lymphatic tumors.

Design of the Study Comparative experiments were conducted with PHIM 106 administered as a single ip, it, sc injection, repeated dose every two days, and a total of four sc injections. The control group remained untreated. Treatment was started when the size of the implanted tumor was approximately 10 mm x 10 mm.

The rats were autopsied 7 days after treatment and tumor size was measured. Histopathological examination was performed.

-64PHIM 106 preparation

Lyophilized white powder without preservative and antimicrobial agent was used.

The white powder was dissolved in saline for injection at a final concentration of 5 mg / ml.

Procedure x 10⁴ Yoshida sarcoma cells were implanted subcutaneously on the backs of white Wistar rats. Treatment of rats began when tumor size was approximately 10 mm x 10 mm.

Three different methods were injected intraperitoneally (ip), intratumoral (it), and subcutaneously (sc) into healthy skin at a distance of about 3-5 cm from the tumor.

The animals were divided into the following 7 groups:

First group: Control group, 11 rats, untreated

Second group: 5 rats, single injection of 5 mg PHIM 106 / kg in the middle of the tumor (s).

Third Group: Single treatment, 5 mg PHIM 106 / kg injection subcutaneous (sc)

4th group: 1 rat, intraperitoneal (ip) treatment with a single injection of 5 mg PHIM 106 / kg

5th group: 3 rats, subcutaneous (sc) treatment, 1.25 mg 'PHIM 106 / kg twice daily for 7 days.

6th group: 5 rats by subcutaneous (sc) treatment, 5 mg

PHIM 106 / kg every two days for a total of four times.

7th group: 5 rats, 12.5 mg subcutaneously (sc)

PHIM 106 / kg every two days for a total of four times.

At -657 days after completion of the experiment, rats were dissected and tumor size was measured. Control rats were autopsied on the same day as the treated rats.

Histopathological examination of the tumor was performed in each group.

Blood was collected and stored for later testing.

Results

In group 2, which received a single injection of 5 mg PHIM 106 / kg it, the tumor showed a 45% reduction compared to the control.

Group 3, which also received a single injection of 5 mg PHIM 106 / kg but sc, showed a 56% reduction in tumor, and Group 4, which received the same amount as Groups 2 and 3 but ip the tumor reduction was 49%.

Groups 6 and 7 received sc treatment with repeated injections of 5 mg PHIM 106 / kg and 12.5 mg PHIM 106 / kg, respectively, with a 53% and 69% reduction in size compared to controls.

The best efficacy was observed in group 5, which received repeated treatment for 7 days. Size reduction was 72% relative to control. See Figures 17, 18 and 19.

In all treated groups the tumors were covered by more than 50% heifers, which is a very high proportion compared to the tumors in the control group. See Figures 1 and 2.

Tumors 24 hours after PHIM 106 injection • ·

-66 they looked like jerks.

No adverse side effects were observed during or after treatment.

Twenty-four hours after injection of PHIM 106, tumors appeared to be gouty. The size of the scrotum increased, the size of the tumor decreased, and it became more distinct compared to the control rats. See Figures 3 and 4.

There was a difference in body weight between the treated and control rats. The control rats had a greater increase in body weight than the treated animals.

No adverse side effects were observed during or after treatment.

An effective dosage range for this indication is 0.1500, preferably 300 mg per treatment.

Discussion

Due to the limited number of rats, we were unable to perform statistical evaluation. The aim of this study is to investigate the relationship between the number of doses, the amount of PHIM 106 per kg body weight and the antitumor effect, and to collect data on whether the various injected PHIM 106 attacks the tumor.

Of course, the overall impression of efficiency was also a very important objective.

Immuniologically, cancer cells can be considered abnormal, so PHIM 106 is hypothesized to have properties that enable it to detect and target abnormal cells, attack and kill tumor cells,

Regardless of its mode. The results show that the antitumor effect is potent and the efficacy is similar for ip, it or sc injections.

Repeated doses of PHIM 106 are more effective than a single treatment, as expected, since Yoshida sarcoma is a rapidly growing tumor.

A greater tumor reduction was achieved in the group receiving 12.5 mg PHIM 106 / kg than in the group receiving only 5 mg PHIM 106 / kg.

On the other hand, rats treated with a total dose of 17.5 mg PHIM 106 / kg / day for 7 days had a slightly better efficacy than rats treated with a total of 50 mg PHIM 106 / kg every two days.

Yoshida sarcoma is a very malignant and rapidly growing tumor. Therefore, it was expected that the daily treatment would produce better results. The treated rats are in good condition and have a body weight of approx. It increased by 10 grams, whereas in control animals it increased by approx. 24 grams. This can only be explained by rapid tumor growth. In the treated groups, the tumor was well separated from the control animals, with metastases such as in the stomach and the like. This also means that reaching the tumor size in the control group is not easy as it covers a very large area. It is interesting to note that in the 12.5 mg PHIM 106 / kg group, one rat had no tumor growth at all. Tumor size is approx. Decreased by 15% compared to baseline.

Histopathological examination of the tumor in control and

In the PHIM 106-treated group, it was clearly shown that the tumor had a much higher goiter than the control group.

The fact that PHIM 106 is active in three different ways of administration indicates that it is capable of targeting tumor cells when entering the artery or vein. Subcutaneous administration is of particular interest because PHIM 106 is first introduced into the lymphatic vessels and a portion reaches the tumor via the lymphatic system. The PHIM 106 introduced into the lymphatic system also plays an important role in the destruction of metastases that are partially spread by this pathway. We also found that there was less transmission in the treated group compared to the control group. A large number of metastases were found in the lung, intestine and liver in the control group. In the treated group, more than 95% of the rats were free of recognizable metastases. In the remaining 5%, only small metastases developed, especially in the intestine.

Although the number of animals tested was very small in the different groups, it should be noted that all animals responded to the treatment. The possibility of using an anticancer agent without limiting the route of administration, with no apparent adverse side effects, places PHIM 106 in a new class of anticancer agents where efficacy is likely to be similar to that of the natural depends on its characteristics.

The antimicrobial activity of PHIM 106, which was observed in the orientation experiments, is another important result • «

-69 for the treatment of cancer patients who also suffer from opportunistic infection. Yoshida sarcoma is a much more malignant tumor than known tumors in humans.

Example 50 - Treatment of Belly

A four-day-old boy was treated with PHIM 106 to remove gingival tissue and prevent the spread of infection.

For treatment, gauze dipped in PHIM 106 solution was used; PHIM 106 was 2 mg per gauze. The gauze was wrapped around the umbilical cord so that it covered the healthy, infected and already gummy parts. The dressing was changed four times in 12 hours. After 12 hours, the PHIM 106 removed the gummy and infected parts, while the healthy portion remained intact. The skin was not damaged in the healthy area, no signs of infection, signs of skin irritation or inflammation were observed. The healthy navel was treated 4 more times.

Six days after birth, the baby's condition was checked in the hospital and, to their surprise, doctors found that the umbilical cord was healthy and showed no signs of infection compared to other babies examined for the same reason.

For this indication, a suitable dosage range is 0.01 to 100, preferably 1 to 25 mg per treatment.

51st example - Infected wound

A 3-month-old boy undergoing surgery for water and testicular hernia 10 days after surgery severely wounds • · ·

-70 infected and degenerated. The surgical wound opened in one place after the suture was removed.

The boy was treated with PHIM 106 solution for 3 days, equivalent to 4 mg PHIM 106 per treatment. After 3 days of treatment, the entire infection was gone and the wound healed.

The total surgical wound was 15 cm long.

The effective dosage range for this indication is 0.0111, preferably 1-25 mg per treatment.

Toxicity in mice

No toxicity could be detected in humans or animals, despite the use of very high doses up to 100 times the effective dose. The toxicity of PHIM was also tested in subcutaneously implanted P388 murine leukemic (a chemical-induced cancer) mice, and doxorubicin (DOX), a well-known anticancer agent, was used as a comparator. The results are summarized in the table below. It can be seen that none of the mice in the PHIM-treated or control group died or died, whereas in the doxorubicin-treated group, except the lowest dose group, all mice died and died. It is worth noting that the highest dose of PHIM (20 mg / kg) is much higher than the doses used in the clinical examples described herein earlier.

• · ·

-71·«

Intravenously, once daily for 9 consecutive days

<td>submitted</td><td>PHIM and</td><td>Toxicity of DOX in mice</td><td>P388 murine leukemia</td>

<td>agent</td><td>Dose (Mg / kg)</td><td>mortality</td><td>Weight change (G)</td>

<td>control</td><td> 0</td><td> 0/6</td><td> + 2,3</td>

<td>PHIM</td><td> 20</td><td> 0/6</td><td> + 3,3</td>

<td></td><td> 10</td><td> 0/6</td><td> + 3,3</td>

<td></td><td> 5</td><td> 0/6</td><td> + 3,3</td>

<td>DOX</td><td> 5</td><td> 6/6</td><td> -2,3</td>

<td></td><td> 2,5</td><td> 6/6</td><td> -0,6</td>

<td></td><td> 1,25</td><td> 0/6</td><td> + 0,5</td>

<td></td><td colspan="2">The dosage is general</td><td>Conditions</td>

Concerning the toxicity test described above, it can be concluded that no toxic effect was observed despite the severe overdose. This is true not only for murine leukemia but also for all clinical examples. Overdose did not lead to a reduction in efficacy in clinical trials. In other words, the upper dose range is much higher than the effective dose, so virtually any (reasonable) dose can be safely used.

Minimum effective dose from condition to be treated • · ·

-72 varies slightly and the presently preferred dosage regimen for the various indications is described below.

Methods of administration and preparations

Due to their negligible toxicity, the substances of the invention can be safely used in the treatment of humans and animals.

The mode of therapy for the various clinical syndromes should be tailored, as always, to the pathological condition as well as the route of administration, the form in which the composition is to be administered, the age, weight and the general health of the subject being treated.

The substances of the invention may be administered or used in the form of solutions. The solution may be applied topically, i.e., applied to the surface (e.g., surface wounds, intact skin); enema (urinary system, foreskin); in the form of eye drops; as a rinsing solution, by inhalation, injection (intraarticular, intraarterial, intravenous, intraperitoneal, subcutaneous, intramuscular); and delivered into the nasal cavity (nasally).

The substances of the invention may also be applied in the form of a gel, for example, topically and dripping (for fistula, for ulcerative wounds).

The dry powder material of the invention may be administered topically, in acid-resistant capsules, either intestinal or by inhalation.

The substances of the invention are contained in dry powder ampoules, with 15 casein in the case of Single-Protein.

-73 units, 25 casein units for Multi-Protein. For immediate use, the contents of the ampoule are dissolved in sterile sodium chloride solution or sterile water to give a solution containing 0.1-25 Units of Casein Multi-Protein or Single-Protein.

When the substance of the present invention is used in gel form, an immediate use hydrogel composition is prepared by adding the powdered Multi-Protein or Single-Protein to the hydrogel required to achieve the desired concentration. The hydrogel itself consists of low molecular weight hydrolyzed starch and has a water content of&gt; 90%.

From our experiments, it can be established that, using the above dosage regimens, the substances of the invention find their target via the circulatory system via arteries, veins and lymphatic vessels. In surface treatment, the enzyme is in direct contact with its substrate.

Medicinal preparations

Powder - Ampoule Casein Unit Single-Protein or 25 Casein Units Multi-Protein is filled into a standard vial in a known manner.

Use immediately

The contents of the ampoule (see above) are dissolved in sterile saline (saline) or sterile water to give a concentration of 0.1 to 25 Units / ml of Casein in Multi or Single Protein.

• ·

-74Hidrogél

A low molecular weight hydrolyzed starch containing&gt; 90% water is prepared in a manner known per se. The gel is packaged in batches and sterilized (autoclave) in each batch.

Immediately use hydrogel

Mixture of powdered Mu1ti-Protein or Sing1e-Protein with a hydrogel (see above) in the desired ratio gives, for example, 2.5 or 5 units of casein / ml.

Detailed description of the drawings

First Figure: Average secretion from krill Single-Enzyme (-_—-) and Multi-Enzyme (· □ ·) for 5 days.

Single-Enzyme: A total of 20 patients from Day 2 to Day 2, 18 patients from Day 2 to 3.5. days and data of 9 patients in the 3.5. days until the end of the experiment.

Multi-Enzyme: 20 patients with 21 wounds from the start of treatment to 2.5. days, 18 patients with 19 wounds at 2.5. from day 4 to day 4 and 12 patients with 12 wounds from day 4 to the end of the experiment.

Everyone reached the end of treatment by day 5 at the latest, with no clinical sign of infection.

Second Figure 3: Mean redness of skin after 5 days of treatment with SingleEnzim (--Jj--) and Multi-Enzyme (·· □ ·) isolated from krill.

• * • · » » ·· · ···· ·

-75More intense redness is probably due to decreased edema and swelling in the surrounding tissues.

Third Mean swelling / edema isolated from krill

During 5 days of treatment with Single-Enzyme (-—) and Multi-Enzyme (· · · ΓΊ - ·).

4th Mean pain during 5 days of treatment with Single-Enzyme (--®r-) and Multi-Enzyme (··· □ ') isolated from krill. The patient's self-rated pain sensation on an analogue scale from 0 to 5, No pain Unbearable pain.

5th The mean pain reduction is isolated from krill

During 7 days of treatment with Multi-Enzyme (- ·· □ ··).

6th Mean inflammation is isolated from krill

During 7 days of treatment with Multi-Enzyme (--- 0-) preparations.

7th Redness and swelling as a characteristic of inflammation after 9 days of treatment with Multi-Enzyme (••• ')' preparations isolated from krill.

8th The degree of pain perception is isolated from krill

Multi-Enzim {--- 0^-- ) for 7 days with preparations.

9th Figure 5-1: Mean time between urine excretions with 4-day treatment with Multi-Enzyme (- · □) preparations isolated from krill.

10th Mean depression of pain during 4 days of treatment with Multi-Enzyme (- · Q ·) preparations isolated from krill.

• ·

11th The mean pain reduction is isolated from krill

Multi-Enzim {--- 0^-- ) for 7 days with preparations. Definitions: severe pain: the horse does not use the sore foot; moderate pain: the horse sometimes uses the sore foot for more than 30 seconds; mild pain: the horse uses the aching leg continuously for more than 2 minutes each time.

12th Degradation effect of Single-Enzyme preparation isolated from krill on 7-day treatment of gouty purulent wounds, fibrin, clot.

--B--: black gummy fabric; --Y--: yellow purulent and fibrinous tissues; · -R- · -: Red crushed area and epithelial tissue.

13th Dose-effect curve - Effectiveness of PHIM in terms of antimicrobial activity in burns.

14th Dose ranges for PHIM - 100% efficacy, that is, the doses used to achieve complete recovery of patients.

A = eye infection; B = inflammation; C = viral infection; D = fungal infection; E = bacterial infection; F = analgesia.

15th The accumulated dose of PHIM administered to patients is the maximum amount of PHIM / kg body weight until recovery.

A = eye infection; G = superficial wound infection; H = urinary tract infection; I = fungal infection; J = Herpes simplex infection; K = oily skin patches; L = burns and M = opportunistic infections.

16th The accumulated dose of PHIM administered to patients is a minimum amount of PHIM / kg body weight until recovery.

Α = eye infection; G = superficial wound infection; H = urinary tract infection; I = fungal infection; J = Herpes simplex infection; K = oily skin patches; L = burns and M = opportunistic infections.

<td>Figure 17:</td><td>The antitumor effect of a single injection of PHIM 106</td>

<td>for control</td><td>compared to.</td>

<td>Figure 18:</td><td>Antitumor effect of repeated injection of PHIM 106</td>

<td>for control</td><td>compared to.</td>

<td>Column (n = 4)</td><td>sc 1.25 mg / kg twice daily. Q1D 1-7;</td>

Column (n = 5) sc 5 mg / kg. Q4D D 1, 3, 5, 7; Column (n = 5)

sc 12.5 mg / kg. Q4D D 1, 3, 5, 7.

19th Tumor volume in control and treated groups.

20th Control (low magnification)

21st Control (high magnification)

22nd Subcutaneous (low magnification)

23rd Subcutaneous (high magnification)

24th Control without treatment.

25th Control without treatment.

26th Figure 1: Single subcutaneous injection.

27th Figure 1: Single subcutaneous injection.

28th Figure 5A: Temperature stability assay of PHIM at pH 7.0 at 30 ° C for 20 hours.

29th Temperature optimum of PHIM. Total proteolytic activity is expressed as the digested area of ​​bovine casein (Bio Rad Protease Substrate Tablets); pH 7.0; 30 ° C for 24 hours.

30th Figure 3B: pH stability of PHIM. Solve PHIM

Keep for 2-18 hours at room temperature. The entire ··· ·· ···" ·

-78proteolytic activity is expressed as the digested area of ​​bovine casein (Bio Rad Protease Substrate Tablets), 30 C, 20 h.

31st Figure: The pH optimum of PHIM. Total proteolytic activity is expressed as the digested area of ​​bovine casein (Bio Rad Protease Substrate Tablets); 30 ° C, 16 hours, pH 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0.

32nd Dose activity curve of PHIM. Protein concentration according to Bradford [Bradford, M. Anal. Biochem., 7, 248 (1976)]. Total proteolytic activity is expressed as the digested area of ​​bovine casein (Bio Rad Protease Substrate Gel Tablets, pH 7.0, temperature: 30'C for 24 hours. Doses: 0.1, 0.3, 0.5, 0.9, 2.5, 5.0 and 15 mg.