Method for treating systemic bacterial infection associated with changes of qualitative and/or quantitative composition of blood extracellular dna

Technical Field The invention is a disclosure in the field of medicine and veterinary and refers to a DNAse enzyme that is used to treat a systemic bacterial infection that is accompanied by quantitative and qualitative changes in extracellular DNA in the blood.

Background of the Invention [0002] The main method of treating diseases caused by bacteria, fungi and protozoa are antibiotics and chemotherapy (see Merck Manual of Diagnosis and Therapy, 16th edition). The main method of drug therapy for atherosclerosis is therapy with statin compounds that inhibit cholesterol synthesis (see New Concepts and Paradigms in Cardiovascular Medicine: The Later Gould, The AMERICAN JOURNAL OF MEDICINE, volume 104.22 June 1998, 2s - 17s.) [0003] Diabetes therapy consists of three main approaches - insulin therapy, drugs that increase the secretion of insulin by the pancreas, drugs that increase the sensitivity of tissues to insulin or increase the utilization of glucose by tissues (Pharmacological Management of Diabetes: Recent Progress and Future Perspective in Daily Drug Treatment, Gerard Emilien et al., Pharmacol. Ther. vol. 81, No. 1, pp. 37-51, 1999). Type IV hypersensitivity therapy is based on immunosuppressive and immunomodulatory treatment (see Therapeutic Immunosupression, ed. AWhomson, Ser. Immunology and Medicine, vol. 29, Kluwer Acad. Publishers, Dordrecht, 2001).

[0004] Diseases caused by mutations in somatic genes and accompanied by the development of somatic mosaicism have no etiological therapy, see Youssoufian H, Pyeritz RE. Mechanisms and Consequences of Somatic Mosaicism in Humans ,. Nature Reviews Genetics, 2002; 3: 748-758.

[0005] Drug resistance is considered to be the main problem of antibiotic therapy for bacterial infections. The transmission of antibiotic-resistant strains in the circulation and the emergence of new ones in the treatment process (for example, as a result of biofilm formation in the patient's body) are the main cause of ineffectiveness of treatment (Cizman M, Int J Antimicrob Agents, 2003, Apr 21, pp. 277-307).

[0006] At present it is widely recognized that the problem of antibiotic resistance is a global threat (Mechanisms of antimicrobial resistance: the clinical milieu in Sefton AM, Drugs, 2002, vol. 62: 557-66) and is needed development of new, original antibiotics and a new method with a non-antibiotic mechanism influencing the infectious process. For example, vancomycin is used to treat infections caused by gram-positive granulomas resistant to penicillin and cephalosporin. The main disadvantages of vancomycin are the increase in the number of vancomycin-resistant strains existing; high toxicity; relatively narrow spectrum of activity (The threat of vancomycin resistance. PerlTM, Am J Med May 1999 106: 26 S-37S.).

[0007] The above data indicate that the development of new, effective low toxicity methods that display a broad spectrum of activity for all bacterial species, including those resistant to antibiotics, is still a very important task. Problems of antibiotic therapy and chemotherapy

Infections caused by fungi and protozoa are similar to those for the treatment of bacterial infections, for example, when the recognized drug amphotericin is used (Antifungal drug resistance to azoles and polyenes, Mar Masia Canuto et al., The Lancet Infectious Diseases, Vol. 2, 9th edition, 1st September 2002, pages 550-563, A systematic review of the antifungal effects and tolerability of amphotericin B formulations, Jane P. Barrett et al., Clinical Therapeutics, vol. 25, 5th edition, May 2003, pages 1295-1220).

[0008] Atherosclerosis of cardiovascular vessels is a systemic disease that is accompanied by the formation of specific atherosclerotic plaques in the walls of large and medium-sized arteries. Depending on the location, degree and size of atherosclerotic plaques, the disease has various clinical symptoms (angina pectoris, stroke and so on). Symptoms particularly associated with organ dysfunction caused by systemic atherosclerosis are treated by means of drug therapy or surgery. There is no treatment for atherosclerosis of the blood vessels by drug therapies, as with any systemic disease. An established prevention measure that delays the progression of the disease is treatment with 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (HMG-CoA) (Lovastatin, Pravastatin, etc. ) leading to the inhibition of endogenous cholesterol synthesis and the increase in the clearance of low-density lipoprotein in the blood plasma and the weakening of atherosclerosis (New Concepts and Paradigms in Cardiovascular Medicine: The Non-invasive Management of Coronary Artery Disease, K. Lance Gould, THE AMERICAN JOURNAL OF MEDICINE, volume 104, June 22, 1998, 2s-17s). The disadvantages of such treatment are side effects (A safety look at currently available statins, Moghadasian MH, Expert Opin Drug Saf 2002 September 1, pp. 269 -74) and limited efficacy (Statins: balancing benefits, efficacy and safety., Clearfield MB, Expert Opin Pharmacother, 2002, May 3, pp. 469-77). [0009] The main cause of disability and death in patients with type 1 and type 2 diabetes is complications associated with the development of microangiopathy and macroangiopathy. It is believed, that effective metabolic control of glucose (maintaining the glucose level and glycated hemoglobin levels within normal limits) prevents the development of complications. Insulin therapy, including intensive insulin therapy, is the method of choice when it is impossible to achieve metabolic control with other drugs (Outpatient insulin therapy in type 1 and type 2 diabetes mellitus: scientific review, DeWitt DE, Hirsch IB, JAMA, 2003, May 289: pp. 2254-64).

[0010] However, even if high-dose insulin therapy is used, the risk of complications, including deaths, is still high enough (Cause-specific mortality in a population with diabetes: South Tees Diabetes Mortality Study, Roper NA, et al., Diabetes Care, 2002 June 25: pp. 43-8). Accordingly, the task of developing new therapies for type I and type II diabetes, including methods for preventing complications, is still valid and widely recognized.

[0011] One of the recognized clinical methods for the treatment of late type hypersensitivity reactions is the administration of the peptide cyclosporin A (Therapeutic Immunosupression, published by AWThomson, Ser. Immunology and Medicine vol. 29, Kluwer Acad. Publishers, Dordrecht, 2001). Well-known disadvantages of this method are severe side effects, namely nephrotoxicity, hypertension and high risk of infection (Cyclosporine: mechanisms of action and toxicity., Graham RM, Cleve Clin J Med, 1994, July-August 61: pp. 308-13). Another problem is the loss of efficacy during long-term treatment, which manifests itself as a growing risk of transplant rejection (Renal transplantation, past, present and future, Ponticelli C, et al., J Nephrol, 1999, July-August 12, Appendix 2: S105-10) . Hence, for treatment, which are accompanied by qualitative and / or quantitative changes in extracellular DNA in the blood, a wide variety of other methods are used that have similar disadvantages: toxicity, side effects, and low efficacy of therapy. At the same time, in actual clinical practice these diseases often accompany each other. For example, the treatment of hypersensitivity reactions of the late type with immunosuppressive drugs multiplies the risk of infectious diseases (Recent

EP 1 661 579 B1 advances in the diagnosis and management of transplant recipients. Tolkoff-Rubin NE, Rubin RH; Semin Nephrol 2000 March 20: 148-63); atherosclerosis is a very common complication of diabetes (Diabetes and atherosclerosis: epidemiology, pathophysiology, and management, Beckman JA, Creager MA, Libby P; JAMA 2002 May 287: 2570-81) and is often accompanied by a systemic infectious process (Infection and atherosclerosis: potential roles of pathogen burden and molecular mimicry., Epstein SE, Zhu J, Burnett MS, Zhou YF, Vercellotti G, Hajjar D, Arterioscler Thromb Vasc Biol 2000 June 20: 1417-20); Several types of diabetes develop as a result of late-type hypersensitivity reactions (Evidence of islet cell autoimmune in elderly patients with diabetes type, Pietropaolo M, Barinas-Mitchell E, Pietropaolo SL, Kuller LH, Trucco M,

[0012] US 6391607 B1 relates to amino acid sequence variants of human DNase I, which has increased DNA hydrolytic activity. It provides nucleic acid sequences encoding such overactive variants, thereby allowing the production of these variants in amounts sufficient for clinical use. In addition, it relates to pharmaceutical compositions and therapeutic applications of overactive human DNase I variants.

[0013] According to DE 40 24 530 A1, viruses and viral diseases are controlled in humans and animals by treatment with nucleases (I) isolated from bovine pancreas. (I) also include ribonucleases and deoxyribonucleases, especially DNases I.

[0014] SUGIHARA S ET AL. describe in BRITISH JOURNAL OF CANCER, NATURE PUBLISHING GROUP, LONDON, GB, volume 67, No. 1, January 1, 1993 (1993-01-01), pp. 66-70, XP008086562 ISSN: 0007-0920, that deoxyribonuclease treatment is prevented by of hepatic metastases to the liver of dermally transplanted tumor cells in mice.

[0015] DAVIS JC JR ET AL: describe in LUPUS, BASINGSTOKE, GB, volume 8, No. 1, January 1, 1999 (199901-01), pp. 68-76, XP009124143 ISSN: 0961-2033 application of recombinant human DNase I (rhDNase ) in patients with lupus nephritis.

Disclosure of the Invention [0016] A solution for developing a method for treating a systemic bacterial infection with high efficacy and low toxicity, which is accompanied by a quantitative and / or qualitative change in the composition of extracellular DNA in the blood plasma, is the basis of the present invention.

According to the invention, this task is solved by introducing a destructive agent of the extracellular DNA in the blood into the systemic blood circulation for the treatment of a systemic bacterial infection associated with qualitative and / or quantitative changes in extracellular DNA in the blood that is observed. As a means of destroying the extracellular DNA in the blood for systemic blood circulation, the DNase enzyme can be introduced: the DNAase enzyme can be introduced into the systemic circulation at doses that change the electrophoretic profile of the extracellular DNA in the blood, which can be detected by pulse-gel electrophoresis, with the enzyme DNase can be administered in doses and regimens that can provide a level of DNA hydrolytic activity measured in the blood plasma and exceed 150 units

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Kuntza per liter of plasma and this level can be maintained for more than 12 hours over a total of 24 hours.

[0018] The development of a systemic bacterial infection is accompanied by quantitative and / or qualitative changes of extracellular DNA in the blood, but the available data does not know the genetic repertoire of extracellular DNA in blood of patients with such infection, the biological role of extracellular DNA in the blood in such a contamination, and on the potential therapeutic effect of destroying extracellular DNA in the blood for the purpose of treating such an infection, thus, taking into account all of the foregoing, the invention is in accordance with the requirements of the "novelty" criteria (N).

[0019] As established by the applicant, extracellular DNA in the blood of patients with systemic bacterial infection contains a unique quantitative and qualitative repertoire of genes and regulatory genetic elements that is significantly different from the repertoire of DNA that is described in the human genome. Unlike intracellular DNA, the extracellular DNA of these patients mainly contains unique human genes. Extracellular bacterial and fungal DNA was found in the biofilm matrix and blood plasma of the infected human.

[0020] It has been established that extracellular DNA in the blood, including the extracellular DNA of bacteria, fungi and protozoa, promotes the development of systemic bacterial infection.

[0021] It has been established that the destruction of extracellular DNA in the blood plasma leads to a curative effect on systemic bacterial infection.

[0022] The aforementioned new features of the claimed invention are based on new ideas regarding the mechanisms of the disease described. In this way, the claimed method adapts to the requirements of the "inventive step" criterion (IS).

Brief description of the drawings [0023] According to the following, the invention has been explained by means of a detailed description of embodiments without reference to the drawings.

Preferred embodiment [0024] The claimed inventive method is implemented as follows:

Materials and Methods The following agents were used to destroy extracellular DNA in the blood: bovine pancreatic DNase (Sigma and Samson-Med), recombinant human DNase I (Gentech), anti-DNA DNA hydrolyzing antibodies isolated from the blood of patients with lupus erythematosus according to from Shuster AM (Shuster AM et al., Science, vol.256, 1992, pp. 665-667).

[0026] Extracellular DNA from blood plasma was isolated as follows: fresh plasma (no more than 3-4 hours after collection) with the addition of anticoagulant (sodium citrate) was centrifuged in Ficoll-PlaquePlus (Amersham-Pharmacia) for 20 minutes at 1500 g at room temperature . Plasma was detached, without affecting the rest of the cells on Ficoll infusion and further centrifuged at 10,000g for 30 minutes to separate cell fragments and debris. The supernatant was separated, without affecting the precipitate and transferred to 1% sarco4

EP 1 661 579 B1, strength 50_mM tris-HCl, pH 7.6, 20 MM EDTA, 400 mM NaCl, and then mixed with an equal volume of phenol-chloroform (1: 1). The resulting emulsion was incubated for 2 hours at t = 65 ° C, and then the phenol-chloroform mixture was separated by centrifugation (500 g for 20 minutes, room temperature). The deproteinization procedure with the phenol-chloroform mixture was repeated 3 times, and then the aqueous phase was treated with chloroform and diethyl ether. Separation from organic solvents was carried out by centrifugation at 5000 g for 15 minutes. Then the same volume of isopropanol was added to the obtained aqueous phase and the mixture was incubated overnight at 0 ° C. After sedimentation, the nucleic acids were separated by centrifugation at 10,000 g for 30 minutes. The nucleic acid pellet was dissolved in 10 mM tris-HCl buffer, pH 7, 6 with 5 mM EDTA and added to a CsCl gradient (1M, 2.5M, 5.7M) in test tubes for the SW60Ti rotor. The volume of the DNA solution was 2 ml, the volume of each CsCl step was 1 ml. The ultracentrifugation was performed in a L80-80 centrifuge (Beckman) for 3 hours at 250,000 g. The DNA was collected from the surface at each step of the gradient to the fraction. Fractions were dialyzed for 12 hours (t = 4 ° C). The presence of DNA in the fractions was determined by agar gel electrophoresis and the DNA was visualized by staining with ethidium bromide. The amount of DNA was determined with a spectrophotometer (Beckman DU70) in a cuvette (100 mcl) at a wavelength of 220-230 nm. The DNA was collected from the surface at each step of the gradient to the fraction. Fractions were dialyzed for 12 hours (t = 4 ° C). The presence of DNA in the fractions was determined by agar gel electrophoresis and the DNA was visualized by staining with ethidium bromide. The amount of DNA was determined with a spectrophotometer (Beckman DU70) in a cuvette (100 mcl) at a wavelength of 220-230 nm. The DNA was collected from the surface at each step of the gradient to the fraction. Fractions were dialyzed for 12 hours (t = 4 ° C). The presence of DNA in the fractions was determined by agar gel electrophoresis and the DNA was visualized by staining with ethidium bromide. The amount of DNA was determined with a spectrophotometer (Beckman DU70) in a cuvette (100 mcl) at a wavelength of 220-230 nm.

Example 1

Treatment of experimental sepsis caused by Candida Albicans (not part of the invention) and St. Aureus [0028] Group 1 - 30 mice were inoculated intraorbitalia with 1x10 bacteria of the pathogenic Staphylococcus aureus VT-2003R strain. Recombinant dornase-alpha (Genentech) was given intraperitoneally at a dose of 500 mkg / kg at 2, 6, 10 and 14 hours after inoculation.

[0029] A group of 2 - 10 mice were inoculated with extrynegal irradiation of 1x10 bacteria of the pathogenic strain Staphylococcus aureus VT-2003R. The phosphate buffer was given intraperitoneally at 2, 6, 10 and 14 hours after infection. [0030] Following the last vaccination with dornase, 24 mice from group 1 were divided into two subgroups (1a and 1b).

[0031] Mice from subgroup 1a (8 mice) - 2 hours after the last administration of dornase, extracellular DNA in the blood isolated from a number of other mice that were retro-orbital inoculated with 1 x 10 bacteria of the pathogenic strain was injected intravenously (at a dose of 0.1 mkg per animal). Staphylococcus aureus VT2003R 15 hours before DNA isolation.

[0032] Mice from subgroup 1a (8 mice) - 2 hours after the last administration of dornase, intravenously injected (at a dose of 0.1 mkg per animal) extracellular DNA in the blood was isolated from a number of other mice that were intravenously infected with a dose of LD₅0 Candida albicans bacteria 3 days before DNA isolation. [0033] Animal viability was assessed 32 hours after infection. The results are shown in Table 1.

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Table 1

Mouse viability at various times after infection

<td></td><td>0 h.</td><td>2 h.</td><td>4 h.</td><td>6 h.</td><td>8 h.</td><td>12 h.</td><td>24 h.</td><td>28 h.</td><td>32 h.</td>

<td>Group 1</td><td>100%</td><td>100%</td><td>100%</td><td>90%</td><td>90%</td><td>80%</td><td>50%</td><td>40%</td><td>thirty%</td>

<td>Group 2</td><td>100%</td><td>100%</td><td>70%</td><td>60%</td><td>50%</td><td>40%</td><td>thirty%</td><td>20%</td><td>20%</td>

<td>1a</td><td></td><td></td><td></td><td></td><td></td><td></td><td>20%</td><td>10%</td><td>10%</td>

<td>1b</td><td></td><td></td><td></td><td></td><td></td><td></td><td>50%</td><td>50%</td><td>40%</td>

[0034] A group of 3 - 10 mice. Clinical isolates of Candida albicans in an LD50 dose were administered intravenously. Recombinant dornase alfa (Genentech) was given intraperitoneally at a dose of 1 mg / kg twice a day on the 2nd day, on the 3rd day and on the 4th day after the infection.

[0035] A group of 4 to 10 mice. Clinical isolates of Candida albicans in an LD50 dose were administered intravenously. Amphotericin B (Genentech) was given intraperitoneally at a dose of 20 mg / kg twice daily on the 2nd day, the 3rd day and the 4th day after infection.

[0036] A group of 5-10 mice. Clinical isolates of Candida albicans were administered intravenously at the LD50 dose. The phosphate buffer (Genentech) was administered intraperitoneally as a negative control twice daily on the 2nd day, the 3rd day and the 4th day after infection.

[0037] Mouse viability and weight were evaluated 7 days after infection. The results are shown in Table 2.

Table 2

<td colspan="5">Mouse viability at various times after infection</td>

<td></td><td>1 Day</td><td>3rd day</td><td>5 day</td><td>7 day</td>

<td>Group 3</td><td>100%</td><td>100%</td><td>100%</td><td>100%</td>

<td>Group 4</td><td>100%</td><td>100%</td><td>100%</td><td>100%</td>

<td>Group 5</td><td>100%</td><td>80%</td><td>50%</td><td>50%</td>

The weight of mice from group 4 of the 7th day of the experiment was 20% less than in group 3. This fact indicates that amphotericin B is more toxic than dornase alfa, although their protective efficacies are equal. [0039] Thus, the extracellular DNA in the blood of infected animals has a negative effect on the development of the infection process and, according to the claimed method, its destruction is effective in the treatment of bacterial and fungal infections.

Example 2. Treatment of general infection (sepsis) [0040] A 38-year-old man was admitted to the Department of Internal Medicine in severe condition. Twelve days earlier he was diagnosed with acute respiratory distress syndrome. Due to the subfebrile state, weakness and pain in the right half of the chest, 5 days before hospitalization, inflammation was diagnosed.

EP 1 661 579 B1. Cefazolin injection and roxytomycin were prescribed per os, but no improvement was observed, and two days before admission to the hospital developed a fever (39.5-40 ° C), nausea, headache. On the last day before hospitalization, hemorrhagic skin rash, muscle pain, jaundice and diarrhea appeared. Until the time of admission to the hospital the temperature was 38.3 C, blood pressure 100/60; tachycardia was found with 120 beats per minute, negative meningeal symptoms, cold and blue limbs. Laboratory data: moderate leukocytosis and left hemogram shift (18% of the young form), neutrophil appearance with toxic granulation, increase in conjugated bilirubin, AST and ALT were present. During the liver ultrasound examination numerous, small zones of heterogeneity were found. The patient was diagnosed with sepsis. Bacteriological blood control was performed. Oral gentamicin / ampicillin / metronidazole, heparin, and vasodilators were used for the treatment. Despite the antimicrobial chemotherapy, haemabsorption and transfusion of blood plasma, the patient's condition deteriorated. Vancomycin infusions were recommended because S. pneumoniae was isolated from the patient's blood. In the next 48 hours, despite the infusion with vancomycin, the patient's condition continued to deteriorate. Symptoms of multiorgan failure appeared. Following the consent of the parents, intravenous continuous infusions of bovine DNase from the pancreas at a dose of 800 mg / day (1,600,000 Kunitz units) were initiated. The symptoms of stabilization of the condition appeared twelve hours after the start of the DNase infusion. These included: improvement of peripheral blood circulation and systemic hemodynamic indices, appearance of urine. DNase infusions were continued for 5 consecutive days.

[0041] Therefore, the use of DNase according to the invention has a curative effect on systemic bacterial infection.

Industrial Applicability [0042] To implement the methods, well-known materials and devices manufactured under factory conditions have been used, and accordingly, the invention meets the requirements of the "industrial suitability" (IA) criterion.