RU2269359C2 - Method for prophylaxis of oncological diseases, or infections mordibidized by bacteria or fungi and protozoa, or arteriosclerosis, or diabetes mellitus, or diseases mediated by delayed hyperresponsiveness reaction, or diseases mediated by somatic cell gene mutations (variants) - Google Patents

Abstract

FIELD: medicine and veterinary.

SUBSTANCE: invention relates to method for prophylaxis of oncological diseases, or infections mordibidized by bacteria or fungi and protozoa, or arteriosclerosis, or diabetes mellitus, or diseases mediated by delayed hyperresponsiveness reaction, or diseases mediated by somatic cell gene mutations. In the first embodiment of invention blood extracellular DNA destroying agent, such as DNAase, is administered into blood. In the second embodiment agent, binding to blood extracellular DNA, such as anti-DNA antibody is administered into blood. According to the third embodiment enzyme altering of blood extracellular DNA chemical structure is administered into blood. According to the forth embodiment agent, stimulating synthesis and/or activity of endogenic deoxyribonuclease or agent stimulating synthesis of antibody binding to blood extracellular DNA are administered into blood.

EFFECT: effective method for treatment of abovementioned diseases without side effects when prolonged using of preparation affected on blood extracellular DNA.

7 cl, 11 tbl, 18 ex, 5 dwg

Description

The invention relates to medicine and veterinary medicine and can be used for the prevention of cancer or infections caused by bacteria, fungi and protozoa, or atherosclerosis, or diabetes mellitus, or diseases associated with a delayed-type hypersensitivity reaction, or diseases developing due to mutations in somatic cell genes .

The listed diseases are the main causes leading to disability and mortality. Currently, the generally accepted concept is the preference of preventive intervention (preventing the development of a disease or its relapse) in relation to the treatment of an already developed disease or its relapse. In accordance with this, well-known and intensively developed new methods of drug prevention of the development and recurrence of these diseases are intensively used.

It is generally recognized that vaccination is an effective and reliable way to prevent and relapse infections, but effective vaccines at the moment have been created only to prevent a very limited range of bacterial, parasitic and fungal infections, see G. Ada, A. Ramsey; Vaccines, Vaccination and the Immune Responce; Lippincott-Raven, NY, 1997. For the majority of clinically relevant infections, the only chemoprophylaxis method currently remains. Chemoprophylaxis of a widespread infection caused by the simplest malaria drugs chloroquin and mefloquin can be cited as examples, see Merck Manual of Diagnosis and Therapy; 16 ^th Edition.

A known method of chemoprophylaxis is prophylaxis with antibiotics and sulfa drugs of bacterial infections, see Merck Manual of Diagnosis and Therapy; 16 ^th Edition, as well as the prevention of fungal infections in patients with immunodeficiency with Amphotericin, see Prophylaxis and treatment of fungal infections associated with haematological malignancies. Sibel Ascioglu et.al., International Journal of Antimicrobial Agents, Volume 15, Issue 3, July 2000, Pages 159-168.

The main method of drug treatment of atherosclerosis is the therapy with statin drugs - inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase (Lovastatin, Parvastatin, etc.), which inhibit the synthesis of cholesterol in the body and lead to an acceleration of clearance of low density lipoproteins from blood plasma that slows the development of atherosclerosis, see New Concepts and Paradigms in Cardiovascular Medicine: The Noninvasive Management of Coronary Artery Disease, K. Lance Gould, THE AMERICAN JOURNAL OF MEDICINE, Volume 104, June 22, 1998, 2s-17s.

Medicinal methods for primary prevention of cancer with proven efficacy (except for methods for primary prevention of rare specific forms of cancer) have not been developed (Enzyme Induction and Dietary Chemicals as Approaches to Cancer Chemoprevention: The Seventh DeWitt S. Goodman Lecture. Allan H. Conney, Cancer Research , vol. 63, pp. 7005-7031, November 1, 2003). Methods based on antitumor chemotherapy, immunotherapy and hormone therapy are widely used to prevent recurrence of cancer, see Adjuvant treatment for colorectal cancer., Van Laethem JL, Acta Gastroenterol Beig 2001 Jul-Sep 64: 263-7; Adjuvant therapy for breast cancer patients: treatment decision tree from a French cancer network, Bachelot T, et.al., Bull Cancer, 2002, Oct 89: pp.897-903 used in the so-called "adjuvant mode".

Medicinal methods for the prevention of diabetes with proven effectiveness also do not exist. There are known attempts to prevent type I diabetes in high-risk groups using insulin therapy, see Effects of insulin in relatives of patients with type 1 diabetes mellitus., New Engi J Med 2002 May 346: pp. 1685-91, and type II diabetes high-risk groups with biguanide drugs, see Metfbrmin & lifestyle intervention prevent Type 2 diabetes: lifestyle intervention has the greater effect., Doggrell SA, Expert Opin Pharmacother, 2002, Jul 3: pp. 1011-3.

The most common way to prevent reactions associated with delayed hypersensitivity is a method based on therapeutic immunosuppression with cyclosporine, see Therapeutic Immunosupression, ed. A.W. Thomson, Ser. Immunology and Medicine vol. 29, Kluwer Acad. Publishers, Dordrecht, 2001.

There are no methods for drug prevention of diseases developing as a result of mutations in somatic cell genes. (Youssoufian H, Pyeritz RE. Mechanisms and Consequences of Somatic Mosaicism in Humans, Nature Reviews Genetics, 2002; 3: 748-758).

Analyzing the healing properties of the known methods for the prevention of the diseases under consideration, we can distinguish the following main disadvantages:

1. The considered methods exhibit toxicity or cause the development of complications (adjuvant chemotherapy of tumor diseases, therapeutic immunosuppression with cyclosporine, prophylaxis with statin drugs).

2. The considered methods are not effective enough or lose their effectiveness during prolonged use (insulin prophylaxis, biguanides, statins, anticancer drugs, chemoprophylaxis of infections, anticancer drugs, cyclosporine).

3. The considered methods are active within a narrow circle of diseases or even in relation to individual forms of the same disease.

Currently, there are no methods that would allow the prevention of the above diseases in the complex, as well as prevent their relapses. In this regard, there is no opportunity to take any well-known technical solution for the prototype of the present invention.

The basis of all variants of the present invention is the solution of the problem of creating a non-toxic and effective method for long-term prophylactic and anti-relapse use in diseases accompanied by changes in the qualitative and / or quantitative composition of extracellular blood DNA, namely, oncological diseases, infections caused by bacteria, fungi and protozoa, atherosclerosis, diabetes mellitus, allergic diseases associated with the delayed-type hypersensitivity reaction, diseases, developing arising due to mutations in somatic cell genes.

According to the first embodiment of the invention, this problem is solved due to the fact that an agent that destroys extracellular blood DNA is introduced into the blood; an agent that destroys extracellular DNA of the blood, can be introduced for life; as an agent that destroys extracellular DNA of the blood, the enzyme DNase can be used;

according to a second embodiment of the invention, this problem is achieved by introducing into the blood an agent that binds extracellular blood DNA; as an agent for binding extracellular DNA to blood, anti-DNA antibodies can be used;

according to a third embodiment of the invention, this problem is solved by introducing into the blood an enzyme that alters the chemical structure of extracellular blood DNA;

according to a fourth embodiment of the invention, this problem is solved by introducing into the blood an agent that stimulates the synthesis and / or activity of endogenous deoxyribonuclease, or an agent that stimulates the synthesis of antibodies that bind extracellular blood DNA.

The development and recurrence of the diseases under consideration is accompanied by qualitative and quantitative changes in the extracellular blood DNA, however, sources known to the applicant lack knowledge on the genetic repertoire of the extracellular blood DNA of patients with the diseases under consideration, the biological role of the extracellular blood DNA in the diseases under consideration, and the possible therapeutic effect of its destruction for the prevention of development and recurrence of these diseases.

According to the applicants, the extracellular DNA of the blood of patients with the diseases under consideration contains a unique in its qualitative and quantitative composition repertoire of genes and regulatory genetic elements, which differs sharply from the DNA repertoire described in the human genome. Unlike intracellular DNA, the extracellular DNA of the blood of patients with the diseases under consideration contains mainly unique human genes. The presence of bacterial extracellular DNA and extracellular DNA of fungi in the biofilm matrix and in the blood plasma of an infected person has been established.

It has been established that extracellular DNA of blood, including extracellular DNA of bacteria, fungi and parasites, contributes to their development and recurrence in the diseases under consideration.

It was found that the destruction, as well as the modification and binding of extracellular blood DNA in the diseases under consideration, leads to preventive and anti-relapse effects.

The above new properties of the claimed invention, based on fundamentally new ideas about the role of extracellular DNA of blood plasma in the development of the diseases under consideration and their recurrence, allow us to conclude that the claimed method meets the criterion of "inventive step".

The claimed method is implemented as follows.

Materials and methods.

In the first embodiment, bovine pancreatic DNase (Sigma), bovine pancreatic DNase (Samson-Med) and recombinant human DNase I (Dornase; Genetech) were used as a DNA destroying agent. A DNase solution for administration was prepared by dissolving a DNase matrix solution in sterile phosphate buffer immediately before administration.

In the second embodiment, antibodies against DNA isolated from the blood of patients with systemic lupus erythematosus using the method of Shuster A.M. were used as a DNA binding agent. (Shuster A.M. et.al., Science, v. 256, 1992, pp. 665-667). Such anti-DNA antibodies are capable of not only binding but also carrying out hydrolysis of DNA.

In the third embodiment, bacterial Sss I Methylase (CpG Methylase), (New England Biolabs) was used as a DNA altering agent.

In the fourth embodiment, an anti-G-f actin antibody (Calbiochem) was used as an agent that stimulates the synthesis and / or activity of endogenous biopolymers that bind, or destroy, or modify the chemical composition, and / or conformation, and / or polymerization of extracellular blood DNA without its destruction. ) G-Actin is an inhibitor of the activity of endogenous DNase I. The binding of actin to antibodies increases the activity of endogenous DNase I.

Plasma DNA was isolated as follows: fresh (no more than 3-4 hours after collection) blood plasma with an added anticoagulant (sodium citrate) was unscrewed on a pillow of Ficoll-Plaque Plus (Amersham-Pharmacia) at 1500 g for 20 minutes at room temperature. Plasma (1/2 of the total number) was carefully selected without touching the remainder of the cells on the Ficoll pillow, and unscrewed at 10,000 g for 30 minutes to get rid of cell debris and debris. The supernatant was collected without affecting the precipitate, up to 1% sarcosyl, up to 50 mM Tris-HCl, pH 7.6, up to 20 mM EDTA, up to 400 mM NaCl and an equal volume of 1: 1 phenol-chloroform mixture were added. The resulting emulsion was incubated at 65 ° C for 2 hours, then phenol-chloroform was separated by centrifugation at 5000 g for 20 minutes at room temperature. The phenol-chloroform deproteinization procedure was repeated in the same way three times, after which the aqueous phase was treated with chloroform and then diethyl ether. Separation from organic solvents was carried out by centrifugation at 5000 g for 15 minutes. An equal volume of isopropanol was added to the resulting aqueous phase and incubated overnight at 0 ° C. After precipitation, nucleic acids were separated by centrifugation at 0 ° C, 10,000 g for 30 minutes. The nucleic acid precipitate was dissolved in a buffer containing 10 mM Tris-HCl, pH 7.6, 5 mM EDTA, and applied onto a cushion of step cesium chloride (1 M, 2.5 M, 5.7 M) in a centrifuge tube for the SW60Ti rotor. The DNA volume was 2 ml, the volume of each step of CsCl - 1 ml. Ultracentrifugation was carried out in an L80-80 (Beckman) instrument for 3 hours at 250,000 g. DNA was collected from the surface of the 5.7M step in fractions. Fractions were dialyzed for 12 hours at 4 ° C. The presence of DNA in the fractions was determined by agarose electrophoresis with visualization of DNA with ethidium bromide. The amount of DNA was determined spectrophotometrically (Beckman DU70) in a 100 μl cuvette, taking a spectrum from 220 to 320 nm.

The implementation of the method is illustrated by examples.

Example 1. Prevention of recurrence of a malignant tumor.

Antibodies against DNA were isolated from the blood of patients with systemic lupus erythematosus according to the method of Shuster A.M. (Shuster A.M. et.al., Science, v. 256.1992, pp. 665-667). Such anti-DNA antibodies are capable of not only binding but also carrying out hydrolysis of DNA.

Group 1 - 10 mice vaccinated with Ehrlich carcinoma (control). On the 10th day after transplantation of the tumor, the mice received a single intraperitoneal injection of cyclophosphamide at a dose of 200 mg / kg.

Group 2 - 10 mice vaccinated with Ehrlich carcinoma. On the 10th day after transplantation of the tumor, the mice received a single intraperitoneal injection of cyclophosphamide at a dose of 200 mg / kg. On the 15th day after transplantation of the tumor, the mice received an intravenous injection of a fraction of human anti-DNA antibodies (IgG) of 200 μg per animal.

Group 3 - 10 mice vaccinated with Ehrlich carcinoma. On the 10th day after transplantation of the tumor, the mice received a single intraperitoneal injection of cyclophosphamide at a dose of 200 mg / kg. On the 15th day after transplantation of the tumor, the mice received an intravenous injection of a fraction of non-specific human immunoglobulin (IgG) of 200 μg per animal.

Group 4 - 10 mice vaccinated with Ehrlich carcinoma. On the 10th day after transplantation of the tumor, the mice received a single intraperitoneal injection of cyclophosphamide at a dose of 200 mg / kg. On the 15th day after transplantation of the tumor, the mice received another intraperitoneal injection of cyclophosphamide at a dose of 200 mg / kg.

The anti-relapse effect of binding of extracellular blood DNA was determined by the survival of animals on the 30th day after tumor inoculation. In the first and third and fourth groups, all animals died. In the second group, 50% of the animals remained alive.

Thus, the use of an agent that binds extracellular DNA of blood plasma, slows down the recurrence of a malignant tumor according to the claimed method. The efficiency of binding of extracellular blood DNA is superior to the effectiveness of the anti-relapse cyclophosphamide chemotherapy repeat course.

Example 2. Prevention of recurrence of a malignant tumor.

The patient is 57 years old. Two years ago, stage IV lung carcinoma was detected with metastases in the bone and brain. The patient received 2 courses of chemotherapy with taxol, however, due to the absence of a tumor reaction and severe toxicity, she refused to continue chemotherapy treatment. The patient underwent pneumectomy, and she was discharged under the supervision of a regional oncologist. After 3 months, the patient developed symptoms of compression of the superior vena cava. With repeated hospitalization, a relapse of the tumor in the area of the bronchus stump was revealed. The patient refused radiological and chemotherapeutic treatment. With the patient's consent, she was prescribed intramuscular injections of bovine pancreatic DNase at a dose of 400 mg per day (2 injections per day; 2000 units of Kunz per 1 mg). The patient was discharged. Over the next 2 months, the symptoms of compression of the superior vena cava gradually disappeared. The patient’s health improved. On follow-up visits after 3, 6 and 12 months - the absence of clinical and radiological signs of disease progression.

The decrease in the content of extracellular blood DNA (VDNA) (ng / ml) of the patient before the start of the anti-relapse course according to the claimed method, 2, 3, 6 and 12 months after the start of the anti-relapse course is shown in the graph (figure 1).

Thus, the use of DNase according to the claimed method has an anti-relapse effect in a malignant tumor.

Example 3. Prevention of the development of autoimmune diabetes.

Diabetes in NOD mice occurs due to the development of autoimmune damage to pancreatic b cells. Group 1 mice (10 females of the NOD line) at the age of 6 weeks received intramuscular injections of dornase-alpha (Genentech) at a dose of 500 μg / kg (4 times a day) for 4 weeks. Group 2 mice (10 females of the NOD line) received an intravenous injection of a 200 μg human anti-DNA antibody (IgG) fraction per mouse at 6, 7, 8, 9, and 10 weeks old. 10 mice in the control of the 3rd group received four-fold intramuscular injections of phosphate buffer from 6 to 10 weeks. Starting from week 15, the level of glucose in the blood of mice was measured every three days. If the glucose level exceeded 250 mg / dl in two subsequent measurements, diabetes was detected. Estimated the number of cases at 15, 18, 24 and 28 weeks of mice.

The results are shown in table 1.

Table 1
The ratio of healthy and sick mice (The first digit is the number of healthy animals in the group; the second digit is the number of sick animals in the group): 15 week 18 week 24 week 28 week Group 1 8/0 8/2 7/3 6/4 Group 2 8/0 7/3 6/4 5/5 Group 3 8/2 4/6 2/8 2/8

Thus, the destruction and binding of extracellular DNA in blood plasma has a prophylactic effect in the development of autoimmune diabetes.

Example 4. The influence of various methods of destruction of binding and modification of extracellular DNA on its pathogenic properties.

C57B1 mice were vaccinated with a high metastatic or low metastatic LLC tumor strain. On the 9th day after the inoculation, the animals were euthanized and the total blood plasma of mice was collected. The total fraction of extracellular blood DNA after isolation was stored at -20 ° C in phosphate buffer.

The experiment involved 7 groups of mice inoculated with a low metastatic LLC strain.

Group 1-6 mice with a low metastatic LLC strain inoculated.

A group of 2-6 mice with a grafted low metastatic LLC strain + double intravenous (on the seventh and eighth day after transplantation) administration of the total extracellular DNA fraction of mice with a grafted high metastatic strain (0.05 μg of DNA was dissolved in 500 μl of fresh heparinized blood before administration).

A group of 3-6 mice with a grafted low metastatic LLC strain + double intravenous (on the seventh and eighth day after transplantation) administration of the total DNA fraction of mice with a grafted high metastatic strain (0.05 μg of DNA was dissolved in 500 μl of fresh plasma before administration). Before the introduction, the DNA sample was subjected to photochemical disinfection (addition of 1 μM methylene blue, followed by red light exposure for 10 minutes (~ 60,000 Lux).

A group of 4-6 mice with a grafted low metastatic LLC strain + double intravenous (on the seventh and eighth day after grafting) administration of the total DNA fraction of mice with a grafted high metastatic strain (0.05 μg of DNA was dissolved in 500 μl of fresh plasma before administration). Before administration, the DNA sample was mixed with 10 μg of anti-DNA antibodies.

A group of 5-6 mice with a grafted low metastatic LLC strain + intravenous twofold (on the seventh and eighth day after grafting) administration of the total DNA fraction of mice with a grafted high metastatic strain (0.05 μg of DNA was dissolved in 500 μl of fresh heparinized blood before administration). Before introducing into the sample, 1 μg of Ricin plant toxin fragment A was added and incubated for 1 hour at 37 ° C. Ricin is a member of the RIP family (ribosome inactivating proteins) of toxins that are widely used to create immunotoxins. In addition to the ability to inactivate ribosomes, these proteins have the ability to deadenylate DNA. To realize the toxic effect, the catalytic unit A of RIP type II toxins must be delivered to the cell by subunit B. In the absence of subunit B, chain A is not toxic, however, the polynucleotide adeninglycosidase activity of chain A can be used to inactivate DNA circulating in plasma.

Group 6 - 6 mice with a low metastatic LLC strain vaccinated + intravenous twofold (on the seventh and eighth day after transplantation) administration of the total DNA fraction of mice with a high metastatic strain vaccine (0.05 μg of DNA before dissolution was dissolved in 500 μl of fresh heparinized blood. DNA sample before enzymatic methylation was introduced (I. Muiznieks et.al., FEBS Letters, 1994, v. 344, pp. 251-254).

Group 7 - 6 mice with a low metastatic LLC strain inoculated + double intravenous (on the seventh and eighth day after transplantation) administration of the total extracellular DNA fraction of mice inoculated with a low metastatic LLC strain.

Group 8 — 6 mice with a low metastatic LLC strain vaccinated + intravenous two-day (on the seventh and eighth day after transplantation) administration of the total DNA fraction of mice with a high metastatic strain vaccinated (0.05 μg of DNA before dissolution was dissolved in 500 μl of fresh heparinized blood. DNA sample before administration was incubated in the presence of 200 ng / ml dornase-alpha for 30 minutes at 37 ° C.

The number of metastatic nodes in the lungs was evaluated on day 15 after inoculation.

The experimental results are shown in table 2.

table 2 Group Ncp. one 12.0 2 22.5 3 14.1 four 15,5 5 15.1 6 12.3 7 13.3 8 13.5

Thus, the extracellular DNA of the blood of mice with a high-grade tumor strain enhances the metastasis of a less malignant tumor. The destruction, binding and modification of extracellular blood DNA prevent this according to the claimed method.

Example 5. Prevention of experimental atherosclerosis.

The experiment used 18 white outbred rats aged 20 months. Under general anesthesia, the left external carotid artery was cannulated by an emboloectomy catheter and mechanical trauma to the endothelium of the common carotid artery was inflicted. Over the next 6 days, 6 control rats received 4 intramuscular injections of phosphate buffer, 6 rats in group 1 received intramuscular injections of dornase-alpha, and 6 rats in group 2 received 2 intravenous injections of anti-DNA antibodies at a dose of 600 μg (in the first and third day after surgery).

On the 12th day, damaged carotid arteries were removed, fixed in formalin and paraffinized. In a serial analysis of histological sections, the intensity of subendothelial changes in the area of mechanical injury was analyzed. In mice of the 1st and 2nd groups, the total area of the formed “neointima” was on average 60% and 40% less, respectively, compared with animals of the control group.

Thus, the destruction and binding and modification of extracellular blood DNA inhibit the development of "early" atherosclerotic changes according to the claimed method.

Example 6. Prevention of bacterial and fungal infections.

In the experiment used white outbred mice weighing 23-25 grams.

Group 1 (30 mice) - the mice were injected into the retroorbital sinus with bacteria of the pathogenic strain of Staphylococcus aureus VT-2003R at a dose of 1 × 10 ¹⁰ bacterial cells per mouse. Recombinant dornase-alpha (Geneyech) was administered at a dose of 500 μg / kg intraperitoneally at 2, 6, 10 and 14 hours after infection.

Group 2 (10 mice) - mice were injected into the retroorbital sinus with bacteria of the pathogenic strain of Staphylococcus aureus VT-2003R at a dose of 1 × 10 ¹⁰ bacterial cells per mouse. Phosphate buffer was administered intraperitoneally 2, 6, 10, and 14 hours after infection.

After the last administration of dornase, subgroups 1a and 1b were isolated from 24 mice of group 1.

Subgroup 1a (8 mice) - 2 hours after the last injection of dornase, mice were injected with extracellular blood DNA (0.1 μg per mouse) obtained from mice infected with Staphylococcus aureus VT-2003R at a dose of 1 × 10 ¹⁰ bacterial cells per mouse through 15 hours after the introduction of bacteria into the retroorbital sinus.

Subgroup 1c (8 mice) - 2 hours after the last injection of dornase, mice were injected with extracellular blood DNA (0.1 μg per mouse) obtained from mice infected with Candida Albicans at a dose of LD50 per mouse, 3 days after intravenous administration of fungi.

The survival rate of animals was evaluated 32 hours after infection.

The results are shown in table 3.

Table 3
The survival of mice at different times after infection: 0 hour 2 hours 4 hours 6 hours 8 hours 12 hours 24 hours 28 hours 32 hours Group 1 one hundred% one hundred% one hundred% 90% 90% 80% fifty% 40% thirty% Group 2 00% one hundred% 70% 60% fifty% 40% thirty% twenty% twenty% 1a twenty% 10% 10% 1c fifty% fifty% 40%

Group 3 (10 mice) - mice were injected intravenously with fungi subcultured from the pathogenic clinical isolate of Candida Albicans, at a dose of LD50. Recombinant dornase-alpha (Geneyech) was administered at a dose of 1 mg / kg intraperitoneally twice a day at 2, 3 and 4 days after infection.

Group 4 (10 mice) - mice were injected intravenously with subcultured fungi of the pathogenic clinical isolate Candida Albicans at a dose of LD50. Amphotericin B was administered at a dose of 20 μg / kg intraperitoneally twice a day at 2, 3 and 4 days after infection.

Group 5 (10 mice) - mice were injected intravenously with subcultured fungi obtained from the pathogenic Coin isolate Candida Albicans at a dose of LD50. As a negative control, phosphate buffer was administered intraperitoneally twice a day at 2, 3, and 4 days after infection.

The survival and weight of the mice were evaluated on day 7 after infection.

The results are shown in table 4.

Table 4
The survival of mice at different times after infection: 1 day 3 day 5 day 7 day Group 3 one hundred% one hundred% one hundred% one hundred% Group 4 one hundred% one hundred% one hundred% one hundred% Group 5 one hundred% 80% fifty% fifty%

In group 4 on day 7, the weight of the animals averaged 20% less than in group 3. This suggests that, with equal protective efficacy, dornase-alpha and amphotericin B showed greater toxicity.

Thus, the extracellular DNA of the blood of infected animals has an increasing effect on the course of the infectious process, and its destruction according to the claimed method has a preventive effect both in bacterial and fungal infections.

Example 7. Prevention of autoimmune diabetes and malignant tumors.

In the experiment, 20 female NOD mice and 20 female C3H mice were used.

Diabetes in NOD mice occurs due to the development of autoimmune damage to pancreatic b cells. Group 1 mice (10 females of the NOD line) received an intravenous injection of a fraction of mouse anti-G-f actin antibodies (Calbiochem) at 200 μg per mouse at 6, 7, 8, 9, and 10 weeks old. 10 mice in the control of the 3rd group received four-fold intramuscular injections of phosphate buffer from 6 to 10 weeks. Starting from week 15, the level of glucose in the blood of mice was measured every three days. If the glucose level exceeded 250 mg / dl in two subsequent measurements, diabetes was detected. The number of cases was estimated at 15, 18, 24 and 28 week mice

The results are shown in table 5.

Table 5
The ratio of healthy and sick mice (The first digit is the number of healthy animals in the group; the second digit is the number of sick animals in the group): 15 week 18 week 24 week 28 week Group 1 9/1 8/2 6/4 6/4 Group 2 7/3 5/5 3/7 3/7

Tumors were induced by intradermal injection of 3-methylcholanthrene (1 mg of 3-methylcholanthrene dissolved in 50 μl of olive oil per mouse) at four weeks of age. The number of mice with developing tumors was estimated at 10 weeks after carcinogen administration.

Group 3 mice (10 females of the SZN line) received intravenous injection of a 200 μg mouse anti-G-actin antibody fraction per mouse at 5, 6, 7, 8, 9, and 10 weeks old.

Group 4 mice (10 females of the C3H line) received intravenous injections of phosphate buffer at the age of 5, 6, 7, 8, 9, and 10 weeks.

The presence of tumors in animals at the age of 14 weeks was determined.

The results are shown in table 6.

Table 6
The ratio of healthy and sick mice (The first digit is the number of healthy animals in the group; the second digit is the number of sick animals in the group): 14 week Group 3 2/8 Group 4 4/6

Thus, suppressing the activity of intrinsic inhibitors of endogenous DNase I has a prophylactic effect in the development of autoimmune diabetes and cancer.

Example 8. Prevention of the spread of a mutant gene. A number of human diseases arise due to the development of a state of somatic mosaicism - expansion of a mutant gene in a population of somatic cells (Youssounan H, Pyeritz RE. Mechanisms and Consequences of Somatic Mosaicism in Humans. Nature Reviews Genetics 2002; 3: 748-758).

As a model for the development of somatic mosaicism, the mutation frequency of the HPRT gene in blood T-lymphocytes was studied. The human HPRT gene (Chromosome Xq26) encodes a constitutively expressed but not essential enzyme involved in the metabolism of purine bases. Cloning was performed according to the method described by Bigbee W (Bigbee W. Et al., Mutation Res., 1998, v.397, pp. 119-136). The peripheral blood lymphocytes were cloned in 8 patients who received a course of three-week immunostimulating therapy with Neovir after surgical removal of the tumor. Of 8 patients, 4 patients additionally received human recombinant DNase I (Genetech) therapy (200 μg / kg intravenously, 4 times a day for 3 weeks). The frequency of occurrence of HPRT-deficient clones in the blood of patients receiving DNase I therapy was on average 3 times lower than that in the blood of patients receiving only immunostimulating therapy. The addition of extracellular DNA from the blood of patients who did not receive DNase into the culture medium during the cloning of T-lymphocytes of patients treated with DNase increases the frequency of occurrence of HPRT-deficient clones during cloning of the latter.

Thus, the destruction of the extracellular DNA of the patient’s blood with the enzyme DNase reduces the frequency of occurrence of the mutant gene in the population and prevents the development of somatic mosaicism according to the claimed method.

Example 9

Prevention of recurrence of Markiafava-Miqueli disease

Markiafava-Mikeli disease is a very rare disease associated with the presence of a somatic gene mutation at the PI gene locus (phosphatidyl glycan class A). Patient O., 17 years old, suffers from this disease for 3 years. Over the past 2 years, the disease has been able to compensate for the use of glucocorticosteroid hormones. Over the past year, the symptoms of the disease reappeared and began to intensify: episodes of hemolysis (2-3 times a month), hemoglobinuria, hepatosplenomegaly, macrocytosis, anisocytosis. 4 blood transfusions were performed. Four months ago, the patient developed an episode of acute renal failure (creatinine - 8.2 mg / dl) due to an intense episode of intravascular hemolysis against a background of purulent tonsillitis. Within a week, the patient underwent hemodialysis procedures. After compensating for renal function, with the patient's consent, she was prescribed weekly infusions of human donor abzyme DNA isolated in patients with systemic lupus erythematosus, in a single dose of 500 mg (1, 2, 3 and 4 weeks). During this month, there was only one disease paroxysm (2 weeks) of significantly lower intensity than the previous one. The crisis was easily stopped by a three-day course of prednisone at a dose of 50 mg per day. At week 5, the patient was prescribed daily single intramuscular injections of DNase I at a dose of 2,000,000 units of Kunz per day. Over the next three months of observation, the patient returned to normal blood count, jaundice and hepatosplenomegaly disappeared. Paroxysms of the disease were not repeated (see figure 2).

Example 10. Prevention of the progression of odontogenic soft tissue infection

The study included patients with odontogenic abscesses and phlegmon of soft tissues admitted to the clinic of maxillofacial surgery. Upon admission, the condition of the patients was evaluated on a scale of M.M.Soloviev. Patients with a Soloviev index of 91 to 159 points have a 50% chance of infection progressing after the start of treatment. In total, 25 patients with a Soloviev index of 91 to 159 points were selected for the study. The control group receiving standard surgical benefits and antibiotic therapy included 15 patients. In the experimental group, which received an additional intramuscular injection of DNase I in a daily dose of 1020,000 units. Kunza, 10 patients were included. On the third day after the start of treatment in the control group in 7 patients, local dissemination of infection was detected. Of 10 patients in the experimental group, none of the local dissemination of infection occurred.

Eletrophoresis of extracellular blood DNA of patients with odontogenic soft tissue infection on day 3 from the start of treatment (Fig.3).

Lane 1 - A patient from the control group with local dissemination

Lane 2 - Patient from the experimental group

Lane 3 - Patient from the experimental group

Lane 4 - Patient from the experimental group

Lane 5 - Molecular Weight Markers

Prevention of the progression of malignant tumors.

The study included 9 patients with various forms of malignant tumors (breast cancer - 2; stomach cancer - 3; lung cancer - 4), who were admitted to the clinic of thoracic surgery after the previous ineffective chemoradiation treatment. All patients at the beginning of treatment showed signs of rapid growth of metastases (according to repeated computed tomograms). All patients received a 30-day course of DNase I treatment daily intravenously in the form of six 30-minute infusions per day in a daily dose of 2,100,000 Kunza units. During the subsequent control computed tomography study, only two patients showed progression of the disease. In these patients, an increase in the amount of extracellular blood DNA was detected and high titers of antibodies to DNase I were determined. All other patients had a distinct decrease in the level of extracellular blood DNA (Fig. 4).

Table 7
Deoxyribonuclease Antibody Titers in Patients 3 and 7 Patient 3 Patient 7 1 Week 4 week Breeding 1 Week 4 week Breeding 2,089 2,127 fifty 1,621 2,163 fifty 0.989 1,149 250 1,281 2,017 250 0.320 0.401 1250 0.383 1,768 1250 0.125 0.154 6250 0,207 1,125 6250

Example 12

Prevention of diabetes and atherosclerosis progression

The ongoing study included 10 patients with an established diagnosis of type 2 diabetes. All patients were previously transferred to treatment with long-acting recombinant human insulin due to the inability to provide normoglycemia with oral antidiabetic agents. The average age of patients was 54 years. The average duration of the disease from the time of diagnosis was 4.5 years. Five patients of the experimental group were prescribed intramuscular injections of bovine pancreatic DNase at a dose of 200 mg per day (two injections per day) for 6 months. We studied the content of glycosylated hemoglobin in the blood, the index of atherogenicity of blood lipoproteins (as an indicator of atherogenesis activity; it is known that vascular complications of diabetes naturally occur due to rapidly developing atherosclerosis of large arteries). The daily requirement for insulin was calculated (as an indicator of the activity of the underlying disease).

The results of the experiment are shown in Table 8

Table 8
The effect of treatment with DNase on metabolic parameters in the group of patients receiving treatment by the present method. Before treatment After 4 months After 8 months Glycosylated hemoglobin (%) 12.7 10.3 7.9 Blood Lipoprotein Atherogenic Index 7.31 6.1 4,56 Insulin requirement U / kg body weight 1.34 0.87 0.63 Extracellular DNA DNA content; ng / ml 170 125 90

Prior to the study, two patients of the experimental group underwent perfusion myocardial scintigraphy with thallium 201 due to suspected coronary atherosclerosis and a “painless” form of coronary heart disease. In both patients, diffuse focal zones with impaired accumulation of the radiopharmaceutical were revealed, indicating the presence of coronary atherosclerosis. Patients were prescribed standard therapy (beta-blockers + nitrates). At the end of the study, the patients were referred for repeated perfusion myocardial scintigraphy. The dynamics showed an improvement in the pattern of accumulation of the radiopharmaceutical in the myocardium, indicating an improvement in blood flow in the pool of coronary arteries.

Example 13

Prevention of relapse of malignant tumors In September - October 2003, palliative surgery to remove the affected lung with excision of single lungs was performed in three patients (2 men aged 47 and 55 years and one woman aged 51 years) with thoracic surgery clinic in thoracic surgery clinic metastatic nodes in the opposite lung. All patients previously received chemotherapy and radiation treatment. After surgery, all patients, with their consent, were prescribed daily intramuscular injections of deposited prolonged DNase at a dose of 5,000,000 Kunza Units per day. To date (observation period - 10 months, follow-up examinations every 2 months), none of the observed patients showed signs of recurrence of lung cancer. At the same time, the data of retrospective control indicate that the likelihood of developing a relapse by this period and in this clinical situation is approaching 100%.

Example 14. Prevention of bacteremia.

The study included 10 patients who applied to a dental clinic for acute toothache. All patients were diagnosed with acute purulent apical periodontitis and removal of a diseased tooth was indicated. Patients were divided into 2 groups of 5 people. With the consent of the patients of the experimental group, 30 minutes before the operation, they were injected intravenously with DNase I at a dose of 2,000,000 Kunza Units. 2 hours after surgery, all patients had blood culture. In 5 patients of the experimental group, the sowing was sterile. In 3 patients of the control group Streptococcus was seeded from the blood.

Example 15. Prevention of delayed-type hypersensitivity.

30 C57B1 mice were immunized with a suspension of Mycobacterium Smegmatis (100 μg antigen in 50 μl aluminum alum) subcutaneously in the paw pad. After 4 weeks, a resolving dose of antigen (50 μg) was injected into the mice of the opposite paw.

The mice of the first group (10 mice) were injected anti-DNA antibodies intravenously at a dose of 200 μg per mouse 30 minutes before the resolution dose.

Mice of the second group (10 mice) were injected intravenously with enzymes containing bacterial Sss I Methylase (CpG Methylase, NewEngland Biolabs) 30 minutes prior to the administration of the resolving dose. In the Sss I experiments, methylase was included in the composition of small unilamellar liposomes (SUVs) in the ratio of 1u of the enzyme per 1 μg of lipids (enzymesosomes) at a dose of 750 μg per mouse.

Mice in the control group (10 mice) were injected with phosphate buffer 30 minutes before the resolution dose.

The intensity of paw edema was evaluated at 1, 2, 5, and 24 hours after the administration of the resolving dose of antigen.

The results are shown in table 9.

Table 9
The intensity of paw edema at different times after the introduction of a resolving dose of antigen. In 30 minutes 1 hour 2 hours 5 o'clock 24 hours The control 2.1 3.6 3,7 4.1 4.2 Group 1 2.1 2,8 3.0 2.9 2.9 Group 2 2.1 3.0 3,1 3.4 3.0

Example 16. Prevention of relapse of a malignant tumor in animals

As an agent stimulating the synthesis of endogenous deoxyribonuclease, human umbilical cord blood stem cells (UPCS) used were transfected with in vitro cDNA of the human Deoxyribonuclease 1 gene.

Group 1 - 7 mice vaccinated with Ehrlich carcinoma - control.

Group 2 - 8 mice vaccinated with Ehrlich carcinoma, received on the third day after the transplantation of the tumor an intravenous injection of 25 million SCPC.

A group of 3 to 8 mice vaccinated with Ehrlich carcinoma, received on the third day after tumor transplantation an intravenous injection of 25 million SCPC transfected with in vitro cDNA of the human Deoxyribonuclease 1 gene.

The effect was determined by the size of the tumor on day 7 after inoculation

Table 10
Tumor size 7 days after inoculation: Group Tumor volume mm ³ one 117 +/- 12 2 111 +/- 17 3 43 +/- 15

Example 17

Prevention of recurrence of a malignant tumor. In January 2004, in a clinic for thoracic surgery, two patients (a man aged 57 years old and a woman aged 55 years old) with a diagnosis of lung cancer in the T4N2M + stage underwent palliative surgery to remove the affected lung. All patients previously received chemotherapy and radiation treatment. After surgery, patients, with their consent, underwent a procedure for the mobilization of peripheral blood stem cells (GM-CSF of 5 μg / kg subcutaneously three times within 3 days), followed by stem cell isolation by cytapheresis using a Haemonetic apparatus. The isolated cells were transfected with cDNA of the human dornase-alpha gene (Deoxyribonuclease I) by cationic liposomes and 50,000,000 cells were injected intravenously. The procedure was repeated after 3, 6 and 9 months. To date (observation period - 14 months, control examinations every 3 months), none of the observed patients showed signs of disease progression. At the same time, the data of retrospective control indicate that the likelihood of developing a relapse by this period and in this clinical situation is approaching 100%. Figure 5 shows information about the content of extracellular DNA of the blood of patients before treatment and during treatment by the present method.

Example 18. Prevention of relapse of malaria

Patient D., 49 years old, was admitted to the hospital on the 20th day from the onset of the disease with complaints of fever up to 40-41 ° C, tremendous chills in the morning and afternoon, repeated after 48 hours, lasting 2.5-3 hours, changing to fever for 4 hours, without sweating, weakness, intense diffuse headache, poor appetite, intermittent sleep. 2 months ago I came from India, where I spent my vacation. Microscopy revealed Plasmodium Vivax (3-5 in the field of view). The patient was prescribed Delagil at a course dose of 2.5 g and Primakvin at 0.015 g per day at a course dose of 0.2 g. On the fourth day from the start of treatment, microscopy in the blood kept Plasmodium Vivax, and continued attacks of fever and hepatosplenomegaly. With the patient's consent, she received intradermal injections of DNA from a calf thymus mixed with Freund's incomplete adjuvant (50 mg / 50 mg). Only 3 injections with an interval of 48 hours. Symptomatic treatment was carried out for 2 weeks. After 2 weeks, a repeated course of Delagil was prescribed, at the end of which there was a sanation of blood from the parasite, the disappearance of fever attacks and normalization of well-being.

The table shows information on the content of extracellular DNA of the patient’s blood and the titer of anti-DNA antibodies during treatment (Day 0 - upon admission to the hospital)

Table 11 Days from the start of treatment 0 four 8 12 fourteen eighteen 22 26 thirty in blood DNA ng / ml 117 123 155 138 73 46 12 7 5 Anti-DNA antibody titer (1:50 dilution) 0.113 0.124 0.354 0.614 1,534 2,211 2,655 2,644 2,520

Claims (7)

1. A method for preventing the development of cancer, or infections caused by bacteria, fungi and protozoa, or atherosclerosis, or diabetes mellitus, or diseases associated with a delayed-type hypersensitivity reaction, or diseases developing due to mutations in somatic cell genes, characterized in that blood is injected with an agent that destroys extracellular blood DNA. 2. The method according to claim 1, characterized in that the introduction of an agent that destroys extracellular DNA of the blood is carried out for life. 3. The method according to claim 1, characterized in that the enzyme DNase is used as an agent that destroys extracellular blood DNA. 4. A method for preventing the development of cancer, or infections caused by bacteria, fungi and protozoa, or atherosclerosis, or diabetes mellitus, or diseases associated with a delayed-type hypersensitivity reaction, or diseases that develop due to mutations in somatic cell genes, characterized in that blood is injected with an extracellular blood DNA binding agent. 5. The method according to claim 4, characterized in that as an agent that binds extracellular blood DNA, use anti-DNA antibodies. 6. A method for preventing the development of cancer, or infections caused by bacteria, fungi and protozoa, or atherosclerosis, or diabetes mellitus, or diseases associated with a delayed-type hypersensitivity reaction, or diseases that develop due to mutations in somatic cell genes, characterized in that blood is injected with an enzyme that changes the chemical structure of extracellular blood DNA. 7. A method for preventing the development of cancer, or infections caused by bacteria, fungi and protozoa, or atherosclerosis, or diabetes mellitus, or diseases associated with a delayed-type hypersensitivity reaction, or diseases that develop due to mutations in somatic cell genes, characterized in that in the blood an agent stimulating the synthesis or activity of endogenous deoxyribonuclease, or an agent stimulating the synthesis of antibodies that bind extracellular blood DNA is administered.

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