RU2308968C2 - Method for treating human diseases accompanied with increased dna content in extracellular spaces of tissues and medicinal preparation for its realization - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: the present innovation deals with treating wide variety of diseases accompanied with increased DNA content in extracellular spaces of human tissues and organs such as: proliferative, for example, tumoral and hyperplastic processes; diseases of infectious origin; diseases being the result of atrophic, degenerative and inflammatory alterations in organs and tissues, for example, systemic lupus erythematosus. The method for therapy includes enteral introduction of DNAase enzyme at the dosages ranged 2000 up to 500000 Kunz U/kg body weight daily. Medicinal preparation for realization of this method contains DNAase enzyme the content of which in a single dosage corresponds to 50000 up to 5000000 Kunz U. It has been first established that enteral introduction of such high dosages of DNAase provides absorption of catalytically valuable quantities of this enzyme into the blood at significant increase of DNA-hydrolytic activity of the enzyme.

EFFECT: higher efficiency of therapy.

13 cl, 12 ex, 6 tbl

Description

The invention relates to medicine and can be used for the treatment and prevention of a wide range of diseases accompanied by a high content of deoxyribonucleic acid in the extracellular spaces of human tissues and organs. The range of such diseases is wide and includes the following main groups of diseases:

- diseases of a proliferative nature, accompanied by accelerated reproduction and excessive death of the body’s own cells, for example, tumor and hyperplastic processes; a high content of extracellular DNA is associated with a poor prognosis of the disease (Lecomte, T., et al., Detection of free-circulating tumor-associated DNA in plasma of colorectal cancer patients and its association with prognosis. Int J. Cancer 2002 Aug. 10; 100 ( 5): 542-8;

- diseases of an infectious nature associated with the multiplication of an infectious agent, for example, bacterial, viral, fungal, protozoal infections, dysbacterioses; extracellular DNA in such diseases is either an independent pathogenic factor, for example, in the case of infections caused by DNA-containing viruses, or contributes to the development of microorganisms, being the basis of the extracellular matrix of their colonies (Extracellular DNA required for bacterial biofilm formation, Cynthia B Whitchurch et al., Science. 2002 Feb. 22; 295 (5559): 1487), participating in the genetic transformation of microorganisms (Transformation of malaria parasites by the spontaneous uptake and expression of DNA from human erythrocytes. Deitsch K., Driskill C., Wellems T.: Nucleic Acids Res. 2001 Feb. 1; 29 (3): 850-3), or complicates their course, forming the basis of purulent-non crustal masses (Zaman S., et al. Direct amplification of Entamoeba histolytica DNA from amoebic liver abscess pus using polymerase chain reaction, Parasitol Res. 2000 Sep; 86 (9): 724-8.); With S. Cherry and L. R. Christensen. Presence and significance of desoxyribose nucleoprotein in the purulent pleural exudates of patients. Proc. Soc. Exp. Biol. Med., 1948, 68: 179-84;

- Diseases resulting from atrophic, degenerative and inflammatory changes in organs and tissues, for example, systemic lupus erythematosus; extracellular DNA is one of the key factors in the pathogenesis of the disease (Pisetsky D.S., Immune response to DNA in systemic lupus erythematosus. Isr Med Assoc J., 2001 Nov .; 3 (11): 850-3); DNA generated from dying cells can accelerate the processes of aging and atrophy of human tissues, US 6524578.

Known methods of treating a number of these diseases by enteral use of certain enzymes; in particular, CA 2394856 describes the enteral administration of enzymes that destroy cell surface proteins, lipids and hydrocarbons for the treatment of infectious diseases. This method does not ensure the destruction of deoxyribonucleic acids, which are one of the main components of both the intercellular matrix of growing microorganisms and purulent detrital masses and, as a result, this method is not effective enough in the treatment of infectious diseases.

A known method for the treatment of diseases accompanied by inflammation by oral administration of a complex of proteolytic and lipolytic enzymes Bromelain, GB 984464. The compositions used according to the described method also do not contain enzymes that destroy deoxyribonucleic acids. Due to the low therapeutic efficacy, the described method has not found independent use in clinical pharmacology and is used only as an adjunct (Bromelain: biochemistry, pharmacology and medical use, Maurer H.R., Cell Mol Life Sci 2001 Aug 58: pp.1234-45).

A known method of systemic enzyme therapy (SET), based on the use of compositions of proteolytic enzymes administered orally or in the form of enemas in high doses (Wrba, N. & Pecher, O. Enzymes: A Drug of the Future. Ecomed Verlagsgesellschaft AG & Co., 1993 )

Also known are methods of treating human diseases accompanied by inflammation, based on the enteral administration of a complex of enzymes containing, in addition to glycolytic, proteolytic and lipolytic enzymes, also enzymes that destroy deoxyribonucleic acid (deoxyribonucleases, DNases), GB 1005985.

GB 1005985 describes a method for treating inflammatory diseases by oral administration of a combination of enzymes, including steptodornase (streptococcal DNase) and chemical anti-inflammatory substances; the patent indicates the preferred use of proteolytic enzymes derived from the pancreas; it is also concluded that there is no effect of the dose of enzymes used on the effectiveness of treatment.

Among the known methods of treatment based on the enteric use of DNase enzymes, the closest to the claimed method is a method of treating a wide range of diseases accompanied by inflammation, based on the enteral administration of the DNase enzyme by taking Varidase tablets containing a complex of stereptokinase and streptodornase enzymes (DNase). The treatment method is based on the enteral administration of 4 to 8 Varidase tablets per day for the treatment of certain diseases accompanied by inflammation (ROTE LISTE Buch 2004; ISBN 3-87193-286-8, Rote Liste Service GmbH), and is most widely used: Continued marketing of a useless drug ('Varidase') in Panama. Lee D., Lancet 1990 Mar, pp. 335: 667.

The main disadvantage of this method is its low therapeutic and prophylactic efficacy both for the treatment of diseases of the gastrointestinal tract and for the treatment of diseases of other organs, which is associated with the lack of absorption of the enzyme in the systemic circulation. This is noted, in particular, in: Orally and rectally administered streptokinase. Investigation of its absorption and activity; Oliven A., Gidron E .; Pharmacology, 1981 vol. 22: pp. 135-8.

Known drug for oral administration (Varidase), containing streptococcal enzymes streptokinase (proteolytic enzyme) and streptodornase - streptococcal deoxyribonuclease. The preparation contains 10,000 units of streptokinase and 2,500 units of streptodornase in one tablet (ROTE LISTE Buch 2004; ISBN 3-87193-286-8, Rote Liste Service GmbH).

This drug is adopted as a prototype of the claimed drug.

The low effectiveness of the drug has become one of the main reasons that Varidase tablets were discontinued and their registration was canceled as a medicine in many countries of the world and, in particular, in the USA. One of the main arguments was their pharmacological inefficiency (Department of Health and Human Services Food and Drug Administration; Federal Register, Vol.50, N240; Dec.13, 1985).

The basis of the present invention according to claims 1 to 7 is the solution to the problem of creating an effective method for the treatment of human diseases, accompanied by a high content of deoxyribonucleic acid in the extracellular spaces of tissues and organs.

According to the invention (claims 1 to 7), this problem is solved due to the fact that in the method of treating human diseases accompanied by an increased content of deoxyribonucleic acid in the extracellular spaces of tissues and organs, by enteral administration of the DNase enzyme, the DNase enzyme is administered in doses of 2000 up to 500,000 units Kunza per 1 kg of body weight per day; however, in the method, the DNase enzyme can be administered orally; however, in the method, a dosage form can be used to ensure the release of the enzyme in the oral cavity; however, in the method, a dosage form can be used to ensure the release of the enzyme in the stomach; however, in the method, a dosage form can be used to ensure the release of the enzyme in the small intestine; however, in the method, a dosage form can be used to ensure the release of the enzyme in the large intestine; however, in the method, the DNase enzyme can be introduced rectally.

The basis of the invention according to claims 8 to 13 is the solution to the problem of creating a drug for the implementation of the proposed method.

According to the invention (pp. 8-13), this problem is solved due to the fact that in a medicinal product for the treatment of human diseases, accompanied by an increased content of deoxyribonucleic acid in the extracellular spaces of human tissues and organs, containing a biologically active substance - DNase enzyme, enzyme content DNase in a single dose of the drug is from 50,000 to 5,000,000 units. Kunza; wherein the drug may be in the form of a tablet for oral administration; while the drug can be made in the form of a capsule for oral administration; while the drug can be made in the form of a suppository for rectal administration; wherein the drug may be in the form of chewing gum or an oral buccal film, or a sublingual tablet; while the drug can be made with the possibility of dosing in the form of toothpaste or tooth gel, or powder, or mouthwash, chewing gum, oral buccal film or sublingual tablet.

The applicant for the first time found that with enteral administration of only high doses of the enzyme in excess of 2000 KU / kg / day, there is a significant increase in DNA hydrolytic activity in the urine and the content of the immunoreactive enzyme DNase I in the urine increases (table 1). These changes have a dose-dependent character in the dose range from 2000 to 500,000 KU / kg / day. In general, this indicates that, when enteric doses of high DNAse are absorbed, catalytically significant amounts of the enzyme are absorbed into the blood. Such a discovery makes it possible to create effective enteric dosage forms of DNase for the treatment of diseases accompanied by an increased content of deoxyribonucleic acid in the extracellular spaces of human tissues and organs, outside the digestive tract.

Thanks to the implementation of the distinguishing features of the invention, an important new result is achieved: the effectiveness and safety of treating a wide range of diseases that are accompanied by an increased content of deoxyribonucleic acid in the extracellular spaces of tissues and organs are ensured; in addition, an important property of the method is the technical simplicity of the invention (both the method and the preparation).

In addition, as was found by the applicants, in the implementation of the proposed method for the treatment of infectious diseases, the inhibition of the formation of biofilms of pathogenic microorganisms occurs, as well as the suppression of the occurrence and selection of drug-resistant clones of microorganisms. This circumstance allows you to use the inventive method for monitoring the development and spread of drug resistance among both normal and pathogenic microflora of the human body.

The estimated amount of bovine pancreatic DNase I activity of 3500 KU / mg (manufactured by Seravac; South Africa) was dissolved in water and administered once per entosally to 24 healthy volunteers (3 volunteers per dose). After that, total urine was collected for 12 hours; determined by the viscometric method the DNA hydrolytic activity of urine, KU per 1 ml of total urine; the presence of bovine serum DNase I in the total fraction of DNase I was determined by a change in the pattern of electrophoregram during gel electrophoresis with isoelectric focusing. The results of the study are shown in table 1.

Table 1 Dose of DNase (KU / kg) once 0 250 1000 2000 20000 35,000 50,000 100,000 500,000 Urinary DNA hydrolytic activity, KU / ml 0.11 0.13 0.09 0.5 0.9 5.2 10.3 17.0 37.9 The presence of bovine pancreatic DNKase I in the total fraction of blood DNase - - - + + + ++ ++ +++

The implementation of the invention according to claims 8 to 13 is illustrated by the following examples:

Example 1

Solid dosage form of DNase for oral administration of the following composition:

Dry bovine pancreatic DNase 100,000 units Kunza Magnesium stearate 2.5 mg Microcrystalline cellulose 50 mg Lactose before tablet formation weighing 200 mg

The dosage form is prepared as follows: dry bovine pancreatic DNase with an activity of 3500 KU / mg (manufactured by Seravac; South Africa) (based on 100,000 units of Kunz (about 28 mg per 1 tablet) is mixed with magnesium stearate, moistened, granulated, mixed with lactose and microcrystalline cellulose in the amount necessary to obtain tablets weighing 200 mg, the mixture is pressed into tablets.

Example 2:

Capsule dosage form of DNase for oral administration of the following composition:

Dry bovine pancreatic DNase 1,000,000 units Kunza Magnesium stearate 2.5 mg Microcrystalline cellulose 40 mg

The dosage form is prepared as follows: dry crystalline bovine pancreatic DNAse activity of 3500 KU / mg (manufactured by Seravac; South Africa) at the rate of 1,000,000 units. Kunza (about 280 mg per 1 capsule) was mixed with magnesium stearate and microcrystalline cellulose, moistened, pressed and granulated. The granulate was poured into cellulose capsules.

Example 3

A soft dosage form for rectal administration of the following composition (for 1 suppository weighing 3 g).

Bull Pancreatic DNase 5,000,000 units Kunza Solid fat base 1600 mg

Example 4

Dosage form in the form of plates for oral use (chewing gum).

Bull Pancreatic DNase 1,000,000 units Kunza Rubber base 2500 mg Sugar, dextrose, tapioca, wax up to 3000 mg

The implementation of the claimed method is illustrated by the following examples.

Example 5

Cancer treatment.

The study included 9 patients admitted to a surgery clinic with a diagnosis of breast cancer recurrence. All patients previously underwent surgical and chemo-radiation treatment of the disease. For hospitalization, contraindications to further surgical and chemo-radiation treatment were established during hospitalization. All patients had measurable metastases in the liver and / or lungs. All patients agreed to the treatment. At the time of the start of the study, the life expectancy of patients was at least 3 months.

Group 1 patients (3 people) received placebo capsules for three months.

2 group of patients (3 people) received the capsules of example 2 in a daily dose of 5,000,000 units. Kunza for three months.

Group 3 patients (3 people) received Varidase tablets (manufactured by Wyeth) at a maximum recommended dose of 10 tablets per day (25000IU streptodornase per day) for 3 months.

One patient of group 3 was hospitalized 9 weeks after the start of treatment due to a sharp deterioration in well-being and the progression of metastasis and died in the clinic 3 days after hospitalization.

3 months after the start of treatment, the patients were re-hospitalized. All patients underwent control computed tomography, performed clinical and biochemical blood tests, and assessed the general condition according to the Karnowski scale.

All patients of groups 1 and 3 showed an increase in the size of metastatic nodes in the liver and lungs, the appearance of new metastatic foci, a decrease by an average of 30% of the Karnovsky index was revealed. In three out of five patients, blood biochemical parameters markedly worsened (a decrease in serum albumin, an increase in hepatocytolytic syndrome, anemia and biochemical signs of inflammation).

In patients of group 2 during repeated tomographic examination there were no signs of an increase in the size of previously available metastatic nodes and no new ones were noted. In one case, an increase of the Karnovsky index by 40% was noted. In two other patients, the Karnovsky index did not change. An increase in serum albumin and hemoglobin levels was noted in all three patients.

Example 6

Treatment of infectious mononucleosis caused by DNA-containing EBV virus.

The study included 20 patients aged 15 to 28 years with an immunologically confirmed diagnosis of infectious mononucleosis. Patients were divided into 3 groups:

Group 1 patients (8 people) received placebo tablets for 5 days and standard symptomatic therapy (glucocorticoids and antibiotics).

2 group of patients (6 people) received the tablets of example 1 in a daily dose of 25,000 units. Kuna per kg body weight for 5 days.

Group 3 patients (3 people) received Varidase tablets (manufactured by Wyeth) at a maximum recommended dose of 10 tablets per day (25000IU streptodornase per day) for 5 days.

The treatment results are shown in table 2.

table 2 Syndrome Terms of the disappearance of the syndrome after the onset of the disease (days) 1 group 2 group 3 group Fever 12 7 eleven Peripheral lymphadenopathy 16 9 17 Tonsillitis fifteen eleven fourteen Hepatosplenomegaly 17 12 eighteen

Example 7

Maxillofacial Phlegmon Treatment

The study included 15 patients with moderate severity who were admitted to the clinic of maxillofacial surgery with a diagnosis of phlegmon of the maxillofacial region. Patients were divided into 3 groups:

Group 1 patients (5 people) received placebo tablets for three days and standard antibiotic therapy for five days (cefotaxime 3 g per day intramuscularly).

2 group of patients (5 people) received the tablets of example 1 in a daily dose of 1,500,000 units. Kunza (15 tablets) for 3 days and standard antibiotic therapy for 5 days (cefotaxime 3 g per day intramuscularly).

Group 3 patients (5 people) received Varidase tablets (manufactured by Wyeth) at a maximum recommended dose of 10 tablets per day (25000IU streptodornase per day) for 3 days and standard antibacterial therapy for 5 days (cefotaxime 3 g per day intramuscularly).

The treatment results are shown in table 3.

Table 3 Group 1 Group 2 Group 3 Dates of cessation of fever after starting treatment 5 day 2 day 5 day Duration of wound cleansing after the start of treatment 7 day 3 day 6 day Terms of closure of the wound after the start of treatment 12 day 7 day 11 day Presence of microorganisms resistant to the antibiotic used 2 out of 5 0 1 out of 5

Example 8

Periodontitis treatment.

The study included 30 patients with moderate to severe periodontitis. At the beginning of the study, dental plaque was removed for all patients and the standard cleaning method using dental floss and interdental brushes was taught. Patients were divided into 3 groups:

Group 1 - 10 patients subsequently used only Colgate toothpaste;

Group 2 - 10 patients subsequently used Colgate toothpaste and used chewing gum with DNase as in example 4 (1/4 of the plate was chewed for 30 minutes 4 times a day) for 4 weeks.

Group 3 - 10 patients subsequently used Colgate toothpaste and used Varidase Buccal Tablets tablets for 4 weeks;

The Silens and Loe index (oral hygiene index) and the Müllermann index (gingival groove bleeding index) were determined before the study and its end.

The results of the study are shown in table 4.

Table 4 Groups Indices Before treatment After treatment 1 Group Silens and Loe 1.77 ± 0.27 1.31 ± 0.35 Müllerman 1.4 ± 0.55 0.65 ± 0.31 2 Group Silens and Loe 1.78 ± 0.37 0.71 ± 0.38 Muellerman 1.35 ± 0, b7 0.29 ± 0.21 3 Group Silens and Loe 1.74 ± 0.35 1.23 ± 0.45 Muellerman 1.48 ± 0.41 0.8 ± 0.32

Example 9

Treatment of systemic lupus erythematosus.

The study included 16 patients with an established diagnosis of systemic lupus erythematosus and laboratory symptoms of glomerulonephritis (proteinuria, microhematuria). All patients received standard therapy (non-steroidal anti-inflammatory drugs, chloroquine). Patients of the experimental group (8 people) received additional suppositories according to example 3 at a dose of 250,000 units. Kunza per kg body weight per day for 15 days. The concentration of DNA in the blood plasma was studied before and after treatment. The plasma DNA content in control patients did not significantly change. In patients of the experimental group revealed a double decrease in DNA in blood plasma at the end of a 15-day course of treatment.

Example 10

Treatment of age-related sperm motility disorder.

The study included 18 healthy male volunteers aged 50 to 55 years. Patients were divided into 3 groups:

1 group of patients (7 people) received placebo capsules for 30 days;

2 group of patients (6 people) received the capsules of example 2 in a daily dose of 35,000 units. Kunza per kg body weight for 30 days;

Group 3 patients (5 people) received Varidase tablets (manufactured by Wyeth) at a maximum recommended dose of 10 tablets per day (25000IU streptodornase per day) for 5 days.

We studied sperm motility in semen samples in patients in three groups before and after treatment. Before treatment, the percentage of motile spermatozoa in semen samples in patients of all three groups was in the range of 46-54%. Indicators of the content of motile sperm in patients of groups 1 and 3 after treatment did not change. The content of motile sperm in the semen samples of patients of the 2nd group after treatment was 58-62%.

Example 11

The influence of the proposed method on the number detected in patients with mutant bacteria resistant to rifampicin. The nasopharynx microflora was inoculated in a patient receiving rifampicin treatment (1) and a patient receiving rifampicin treatment in parallel with the treatment according to the claimed method (2). Crops were plated on agar medium containing rifampicin 50 μg / ml. The number of CFU was estimated by serial dilutions with seeding on an agar medium without antibiotic (see table 5).

Table 5 Sick CFU number The number of clones resistant to rifampicin one (3.7 ± 0.6) × 10 ⁹ 110 2 (2.0 ± 0.1) × 10 ⁸ 2-3

From table 5 it is seen that in a patient receiving an antibiotic together with treatment by the present method, an order of magnitude fewer microorganisms were sown, and among them only a few rifampicin-resistant clones.

Example 12

The influence of the proposed method on the number of sown in patients with mutants resistant to gentamicin.

Sowing feces of patients who received a course of treatment with gentamicin for acute intestinal infection. Sowing was carried out the day after the end of the course of treatment. Mixed cultures were grown in test tubes as planktonic growth for 24 hours. The number of viable cells was estimated by the number of CFU (colony forming units) after 24 hours of growth by plating on an agarized medium containing an antibiotic (Km, 50 μg / ml). Table 6 presents the averaged results for the group of control patients (Group A; 3 people) and the group of patients who received parallel treatment by the present method (Group B; 3 people) (see table 6).

Table 6 CFU number Group A 1.8 × 10 ¹⁰ Group B 2.8 × 10 ⁹

From table 6 it is seen that in patients receiving an antibiotic together with treatment by the present method, an order of magnitude smaller number of microorganisms resistant to gentamicin was sown.

Claims (13)

1. A method for the treatment of human diseases, accompanied by a high content of deoxyribonucleic acid in the extracellular spaces of tissues and organs, by enteral administration of a DNase enzyme, characterized in that the DNase enzyme is administered in doses from 2000 to 500000 units. Kunza per 1 kg of body weight per day. 2. The method according to claim 1, characterized in that the DNase enzyme is administered orally. 3. The method according to claim 2, characterized in that they use a dosage form that ensures the release of the enzyme in the oral cavity. 4. The method according to claim 2, characterized in that they use a dosage form that ensures the release of the enzyme in the stomach. 5. The method according to claim 2, characterized in that they use a dosage form that ensures the release of the enzyme in the small intestine. 6. The method according to claim 2, characterized in that they use a dosage form that ensures the release of the enzyme in the large intestine. 7. The method according to claim 1, characterized in that the DNase enzyme is administered rectally. 8. A drug for the treatment of human diseases, accompanied by a high content of deoxyribonucleic acid in the extracellular spaces of human tissues and organs, containing a biologically active substance - a DNase enzyme, characterized in that the content of DNase enzyme in a single dose of the drug is from 50,000 to 5,000,000 units. Kunza. 9. The drug of claim 8, characterized in that it is made in the form of a tablet for oral administration. 10. The drug of claim 8, characterized in that it is made in the form of a capsule for oral administration. 11. The drug of claim 8, characterized in that it is made in the form of a suppository for rectal administration. 12. The drug of claim 8, characterized in that it is made in the form of chewing gum or an oral buccal film, or a sublingual tablet. 13. The drug of claim 8, characterized in that it is made with the possibility of dispensing in the form of toothpaste or tooth gel, or powder, or mouth rinse, chewing gum, oral buccal film or sublingual tablet.