Method for treating diabetes and atherosclerosis associated with changes of qualitative and/quantitative composition of blood extracellular DNA

Technical field [0001] The invention is a disclosure in the field of medicine and veterinary medicine and relates to the enzyme DNase which is used for the treatment of atherosclerosis and diabetes.

Background of the invention [0002] The main treatment for diseases caused by bacteria, fungi and protozoa is antibiotics and chemotherapy (see Merck Manual of Diagnosis and Therapy, 16th edition). The main treatment method for drug therapy for atherosclerosis is treatment with compounds from the group of statins that inhibit cholesterol synthesis (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.) [0003] Diabetes therapy consists of three main approaches - insulin therapy, drugs that increase insulin secretion by the pancreas, drugs that increase insulin sensitivity or increase glucose utilization by tissues (Pharmacological Management of Diabetes: Recent Progress and Future Perspective in Daily Drug Treatment, Gerard Emilien et al., Pharmacol Ther. Volume 81, No. 1, p. 37-51, 1999). Type IV hypersensitivity therapy is based on immunosuppressive and immunomodulating therapy (see

Therapeutic Immunosupression, ed. AWThomson, Ser. Immunology and Medicine, volume 29, Kluwer

Acad.Publishers, Dordrecht, 2001).

[0004] For diseases caused by mutations in somatic genes and which are accompanied by the development of somatic mosaicism, there is 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 transfer 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 reasons for the ineffectiveness of the treatment (The use and resistance to antibiotics in the community. Cizman M, Int J Antimicrob Agents, 2003, Apr 21, pp. 297-307).

[0006] At present, it is widely recognized that the problem of antibiotic resistance is global threat (Mechanisms of antimicrobial resistance: their clinical relevance in the new millennium. Sefton AM, Drugs, 2002, volume 62: 557-66) and development of new, original antibiotics and a new method with a non-antibiotic mechanism affecting the infection process. For example, vancomycin is used to treat infections caused by gram positive penicillin and cephalosporin resistant granulomas. The main disadvantages of vancomycin are an increase in the number of existing vancomycin resistant strains; 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 methods of low toxicity, showing a wide spectrum of activity against all bacterial species, including antibiotic resistant strains, is still a very important task. The problems of antibiotic therapy and chemotherapy for infections caused by fungi and protozoa are similar to those for bacterial infections, for example

EP 2 497 487 B1, when the recognized drug amphotericin is used (Antifungal drug resistance to azoles and polyenes, Mar

Masiά Canuto et al., The Lancet Infectious Diseases, Volume 2, Edition 9, September 1, 2002, pages 550-563; A systematic review of the antifungal effectiveness and tolerability of amphotericin B formulations, Jane P. Barrett et al., Clinical Therapeutics, volume 25, edition 5, May 2003, pages 1295/20).

[0008] Atherosclerosis 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 different clinical manifestations (angina pectoris, stroke and so on). Symptoms particularly associated with organ dysfunction due to systemic atherosclerosis are treated with drug therapy or surgery. There is no treatment for atherosclerosis by drug therapy methods, as with any systemic disease. Established prevention that delays disease progression is treatment with 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors (Lovastatin, Pravastatin, etc.) leading to inhibition of endogenous cholesterol synthesis and increased plasma clearance of low density lipoproteins blood and weakening atherosclerosis (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). The disadvantages of this 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 effectiveness (Statins: balancing benefits, efficacy and safety., Clearfield MB, Expert Opin Pharmacother, 2002, 3 May: pp. 469-77). [0009] The main causes of disability and death among patients with type 1 and type 2 diabetes are complications associated with the development of microangiopathy and macroangiopathy. It is believed that effective metabolic control of glucose levels (maintaining glucose levels 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] But even if high-dose insulin therapy is used, the risk of complications, including fatal ones, 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 treatments for Type I and Type II diabetes, including methods for preventing complications, is still valid and widely recognized.

[0011] One recognized clinical treatment for late-type hypersensitivity is peptide, cyclosporin A (Therapeutic Immunosupression, AWThomson, Ser. Immunology and Medicine vol. 29, Kluwer Acad. Publishers, Dordrecht, 2001). Well-known disadvantages of this method are severe adverse effects, namely nephrotoxicity, hypertension and high risk of developing infections (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 is manifested by an increasing risk of transplant rejection (Renal transplantation, past, present and future, Ponticelli C, et al., J Nephrol, 1999, July-August 12 Add. 2: S105-10). Hence, for treatment that is accompanied by qualitative and / or quantitative changes in extracellular DNA in the blood, a wide range of other methods are used that have similar disadvantages: toxicity, side effects, low effectiveness of therapy. At the same time, in actual clinical practice of the disease, these defects often accompany each other. For example, treatment of late-type hypersensitivity reactions with immunosuppressants increases the risk of infectious diseases (Recent advances in the diagnosis and management of infection in the organ transplant recipient. Tolkoff2

EP 2 497 487 B1

Ruby NE, Ruby 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: 141720); several types of diabetes develop as a result of late-type hypersensitivity reactions (Evidence of islet cell autoimmunity in elderly patients with type 2 diabetes., Pietropaolo M, Barinas-Mitchell E, Pietropaolo SL, Kuller LH, Trucco M, Diabetes 2000 January 49: 32- 8) or during an infectious process (Systemic diseases caused by oral infection. Li X, Kolltveit KM, Tronstad L, Olsen I, Clin Microbiol Rev 2000 October 13: 54758) and leads to high risk of infection development (Diabetes and the risk of infection-related mortality in the US, Bertoni AG, Saydah S, Brancati FL , Diabetes Care 2001 June 24: 6 1044-9).

[0012] US 6,391607 B1 refers to variants of the amino acid sequence of human DNase I, which has increased DNA hydrolytic activity. It provides nucleic acid sequences encoding such overactive variants, thus enabling the generation of these variants in quantities sufficient for clinical use. In addition, it relates to pharmaceutical compositions and therapeutic uses for hyperactive variants of human DNase I.

[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 DNase 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), pages 66-70, XP008086562 ISSN: 0007-0920 that deoxyribonuclease treatment prevents transmitted transmission hepatic metastases to skin 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), pages 68-76, XP009124143 ISSN: 0961-2033 the use of recombinant human DNase I (rhDNase ) in patients with lupus nephritis.

Disclosure of the Invention [0016] The solution to develop a method of treatment with high efficiency and low toxicity of diabetes and atherosclerosis diseases, which is accompanied by a quantitative and / or qualitative change in the composition of extracellular DNA in blood plasma, is the basis of the present invention.

[0017] According to the invention, this task is solved by introducing an agent that destroys extracellular DNA in the blood into the systemic blood circulation to treat atherosclerosis and diabetes. As an agent that destroys extracellular DNA in the blood, the enzyme DNase can be introduced into the systemic blood circulation: the DNase enzyme can be introduced into the systemic circulation at doses that provide a change in the electrophoretic profile of extracellular DNA in the blood that can be detected by pulsed gel electrophoresis, whereby the DNase enzyme can be administered in doses and regimens that can ensure the level of hydrolytic activity against DNA measured in plasma and exceed 150 Kunitz units per liter of plasma and this level can be maintained for more than 12 hours for a total of 24 hours.

[0018] The development of diabetes and atherosclerosis of the blood vessels is accompanied by quantitative and / or qualitative changes in extracellular DNA in the blood, but the source of available data does not know about the genetic repertoire of extracellular DNA in the blood of patients with the above-mentioned diseases, about the biological role extracellular DNA in the blood in such diseases, and the potential therapeutic effect of destroying extracellular DNA in the blood to treat such diseases, so, considering all of the above, the invention complies with the requirements of the "novelty" (N) criteria.

[0019] As the applicant has determined, the extracellular DNA in the blood of patients with the above diseases contains a unique quantitative and qualitative repertoire of genes and regulatory genetic elements that is significantly different from the DNA repertoire that is described in the human genome. Unlike intracellular DNA, these patients' extracellular DNA mainly contains unique human genes. Extracellular bacterial and fungal DNA were found in the biofilm matrix and blood plasma of an infected human.

[0020] It has been established that extracellular DNA in the blood, including extracellular DNA of bacteria, fungi and protozoa, promotes the development of the diseases mentioned above.

[0021] It has been established that the destruction of extracellular DNA in blood plasma leads to a therapeutic effect in the diseases mentioned above.

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

Brief Description of the Drawings [0023] As described below, the invention has been explained by means of a detailed description of the embodiments, without reference to the drawings.

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

Materials and methods [0025] 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. 666-667).

[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 . ½ plasma was detached without affecting the rest of the cells on the Ficoll pad and further centrifuged at 10,000 g for 30 minutes to separate cell fragments and debris. The supernatant was separated without affecting the pellet and transferred to 1% sarkosil, 50MM tris-HCl, pH 7.6, 20 MM EDTA, 400 MM NaCl, and then mixed with an equal volume of phenol-chloroform mixture (1: 1). The resulting emulsion was incubated for 2 hours at t = 65 ° C, a

The phenol-chloroform mixture was then separated by centrifugation (500 g for 20 minutes, room temperature).

[0027] The deproteinisation 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. The same volume of isopropanol was then added to the resulting 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 the CsCl gradient (1M, 2.5M, 5.7M) in test tubes into the SW60Ti rotor. The volume of 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. DNA was collected from the surface in each step of the gradient into fractions. Fractions were dialyzed for 12 hours (t = 4 ° C). The presence of DNA in the fractions was determined by agar gel electrophoresis, and DNA was visualized by ethidium bromide staining. The amount of DNA was determined by spectrophotometer (Beckman DU70) in a cuvette (100 mcl) at a wavelength of 220-230 nm.

Example 1 Effect of DNase treatment on viability of pancreatic beta cells and aortic endothelium [0028] Human recombinant DNase I (Gentech) was used. Human embryonic pancreatic β cells and human aortic endothelial cells were used to create the primary cell culture. DNA isolated from the plasma of patients with severe type 2 diabetes that was complicated by atherosclerosis (0.0025 mkg per 1 ml of culture medium), was added to one of the experimental series in cell culture 24 hours after passage, and DNA extracted from blood from the same patient but DNase treated (1 mkg / ml; 37C, 30 minutes), was added to the second cell culture series. The number of viable cells was counted using trypan blue uptake techniques for 24 hours.

[0029] The results of the experiment are shown in table 4:

Table 4

<td colspan="4">Percentage of viable cells 48 hours after their growth (in percent).</td>

<td>cells</td><td>Control</td><td>Patient's DNA</td><td>DNA from a patient treated with DNase</td>

<td>Β cells</td><td> 73%</td><td> 43%</td><td> 61%</td>

<td>endothelium</td><td> 62%</td><td> 35%</td><td> 55%</td>

[0030] Thus, the extracellular DNA of the blood plasma of a patient with severe type 2 diabetes mellitus and atherosclerosis negatively affects both normal pancreatic β cells and normal endothelial cells. Destruction of the extracellular DNA of the patient's blood prevents the development of negative effects in accordance with the claimed method.

Example 2. Treatment of atherosclerosis [0031] A 54-year-old man was admitted to hospital in serious condition complaining of intense abdominal pain, diarrhea, intense leg pain that occurs when walking, weight loss. 12 years ago, type 2 diabetes was diagnosed and glibencamide prescribed. Epigastric pain after a meal appeared 15 months

EP 2 497 487 B1 earlier. Antacids were prescribed, but the pain continued to increase, and fat diarrhea appeared in the last 3 months. Due to intense pain, a few days before hospitalization, anorexia syndrome developed. During the study significant exhaustion was noted (body weight was 44 kg; weight loss was 28 kg in the last 5 months) and no aortic pulsation in the legs.

[0032] No organic changes were observed during gastroduodenoscopy and colonoscopy. ECG data were unchanged. A moderate increase in cholesterol and low density lipoprotein fraction was observed in the blood analysis. The level of glycated hemoglobin was 11%. On aortography, partial aortic occlusion below the renal artery (70%), partial iliac occlusion (90%), complete closure of the upper and lower mesenteric arteries were observed. Conservative treatment was chosen because of the inability to use surgical treatments. Intensive parenteral nutrition was started. Insulin therapy prescribed. Antiaggregant therapy was carried out. Daily intravenous infusions of bovine pancreatic DNase at a dose of 800 mg / day (1,600,000 Kunitz units) divided into 4 two-hour administrations began with the patient's consent on the 7th day after the start of therapy, the patient was allowed to take dietary food. Pain syndromes are gone. The patient received full enteral nutrition up to the 20th day of treatment. The general condition improved, body weight increased. On the 45th day of therapy, angiography was performed as part of the preoperative examination. A reduction of 20% and 30% aortic and iliac artery occlusion was observed, respectively, and blood circulation was found in the upper and lower mesenteric arteries (80% obstruction).

[0033] The extracellular DNA samples of this patient were cloned by a method that allows the construction of an unamplified library of such plasma DNA with representativeness up to one million clones with an average size of 300-500 bases.

[0034] DNA isolated by the method mentioned above was deproteinized using Proteinase K (Sigma) at 65 ° C to eliminate tightly bound proteins. DNA after deproteinization was treated with phenol chloroform at 65 ° C and sedimented with 2.5 volumes of ethanol overnight. The DNA was then processed with EcoRI restriction enzyme during 3 hours or Pfu polymerase (Stratagene) in the presence of

300 mkM of all 3'-phosphate deoxynucleotide to eliminate "sticky" ends. The finished DNA was phosphorylated with T4 polynucleotide kinase (30U, 2 hours). The resulting samples / preparations were ligated into Bluescript (Stratagene), the plasmid was digested with EcoR1 or PvuII, respectively, and dephosphorylated with CIP alkaline phosphatase (Fermentas) for 1 hour. Usually 1 mkg of vector and 0.1-0.5 mkg of serum DNA was used for ligation. Ligation was performed using a Rapid Ligation Kit (Roche) for 10 hours at 16 ° C. The volume of the ligase mixture was 50 mkl. The ligated library was transformed into DH12S cells (Life Technologies) using an electroporator (Bio-Rad). 12-20 cuvettes for electroporation were used to transform one library. Library dilutions at concentrations 10^-4, 10^-5 and 10^-6 plated for controls on 1.5% agar and LB medium plates. In both cases, the representativeness of the library was about 2-3x10⁶ clones.

[0035] Analysis of randomly selected clones with a length of 300 to 1000 units of base pairs from a library that was obtained from the extracellular DNA of a patient's blood plasma prior to treatment showed that 56 of 75 clones are unique fragments of human DNA. The function or product of the corresponding gene was identified for 11 genes by HumanGeneBank.

EP 2 497 487 B1

<td>Gene or corresponding protein product</td><td>Reported role in atherosclerosis of blood vessels and diabetes</td>

<td>Neutral endopeptidase</td><td>In atherosclerosis of blood vessels, its activity increases in endothelial cells, smooth muscle cells, endometrial stromal cells. Reduce her activity can reduce lipid buildup in the wall dishes.</td>

<td>Muskelin 1</td><td>Is a mediator of cellular response to thrombospondin 1. Thrombospondin 1 mediated processes are pathophysiological components of atherosclerotic disease artery walls.</td>

<td>Nf-kappa B.</td><td>In hyperglycemia and atherosclerosis, its activity is increased in artery wall cells.</td>

<td>Momentary receptor cationic channel potential</td><td></td>

<td>Phospholipase C, epsilon</td><td>Induces the expression of low density lipoprotein receptors.</td>

<td>CRTL1: cartilage fusion protein 1</td><td></td>

<td>17kD fetal brain protein</td><td></td>

<td>Nicotinamide nucleotide transhydrogenase</td><td></td>

<td>BAI3: specific angiogenesis inhibitor for the brain</td><td></td>

<td>GAD2: glutamate decarboxylase 2</td><td>One of the main autoantigens in type 1 diabetes.</td>

<td>E-selectin</td><td>High expression is a risk factor for the development of angiopathy in type 2 diabetes.</td>

[0036] Analysis of 50 randomly selected clones from the library obtained from the extracellular DNA of a patient's plasma 21 days after the start of treatment showed that more than 90% of the disclosed clone results are short fragments of repetitive human DNA in the main alpha-satellite DNA. At the same time, changes in the electrophoretic profile of extracellular blood DNA were recorded, which were assessed by pulse gel electrophoresis.

[0037] Thus, the use of DNase according to the claimed method has a therapeutic effect in atherosclerosis.

Example 3. Diabetes treatment [0038] Poor metabolic control of diabetes, which manifests itself in high blood glycosylated hemoglobin and low insulin sensitivity, necessitates the use of high doses of insulin and these are the main predisposing factors for the development of complications.

[0039] A 46-year-old patient has been suffering from type 2 diabetes for 3 years. Due to the unsuccessful results of oral antidiabetic compounds that were not able to lower blood glucose levels, human recombinant, short-acting insulin was prescribed at 0. , 3 U / kg (21U / day). The level of glycated hemoglobin in the blood was still high (over 10%), symptoms of diabetic angiopathy and polyneuropathy appeared, and a decrease in visual acuity. Daily insulin requirements increased to 1.2 U / kg (84 U / day). The patient was prescribed intramuscular injections of bovine pancreatic DNase at a dose of 200 mg / day (twice a day) for 4 months. Until the end of therapy, the patient's condition was better, the level of glycated hemoglobin in the blood decreased, and the daily insulin dose was also reduced twice. The results are shown in Table 5.

Table 5

<td colspan="4">Impact of DNase treatment on the patient's metabolic indexes</td>

<td></td><td>Before treatment</td><td>Three months after starting treatment</td><td>Four months after starting treatment</td>

<td>Glycated hemoglobin (%)</td><td> 13,2</td><td> 10,1</td><td> 7,2</td>

<td>Need insulin U / kg body weight</td><td> 1,2</td><td> 0,9</td><td> 0,6</td>

<td>The amount of extracellular DNA of blood plasma (%).</td><td> 100</td><td> 85</td><td> 70</td>

[0040] The amount of extracellular blood DNA was assessed by electrophoretic band densitometry. Quantity

DNA before treatment was taken as 100%.

[0041] Thus, the use of DNase in diabetes has a therapeutic effect according to the claimed method.

Industrial use [0042] To implement the methods, well-known materials and devices manufactured under industrial conditions were used, and in accordance with the above, the invention fulfills the requirements of the "industrial applicability" (IA) criterion.