Method for treating delayed-type hypersensitivity reactions associated with changes of qualitative and/quantitative composition of blood extracellular DNA

Technical field [0001] The invention is a medical and veterinary disclosure and relates to the DNase enzyme which is used to treat diseases associated with delayed-type hypersensitivity reaction.

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 2 497 488 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 2 497 488 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 (Diabetes and the risk of infection) high efficiency and low toxicity, 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.

[0013] 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 DNAase 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 at 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.

[0014] The development of delayed-type hypersensitivity reactions 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 delayed-type hypersensitivity reactions, about the biological role of extracellular DNA in the blood. such reactions and the potential therapeutic effect of destroying extracellular DNA in the blood to treat such reactions, so considering all of the above, the invention complies with the requirements of the "novelty" (N) criteria.

[0015] As the applicant has determined, the extracellular DNA in the blood of patients with delayed-type hypersensitivity reactions 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.

[0016] It has been established that extracellular DNA in the blood, including extracellular DNA of bacteria, fungi and protozoa, promotes the development of delayed-type hypersensitivity reactions.

[0017] It has been established that the destruction of extracellular DNA in blood plasma leads to a therapeutic effect in delayed-type hypersensitivity reactions.

[0018] The above-mentioned new features of the claimed invention are based on new ideas regarding the mechanisms of the described diseases. In this way, the claimed method adapts to the requirements of the "inventive level" (IS) criterion.

Brief Description of the Drawings [0019] As described below, the invention has been explained by means of a detailed description of the embodiments, without reference to the drawings.

Preferred embodiment [0020] The claimed inventive method is carried out as follows:

Materials and methods [0021] 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).

[0022] 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, and then the phenol-chloroform mixture was separated by centrifugation (500 g for 20 minutes, room temperature).

[0023] 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. 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 gradient step into 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 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.

EP 2 497 488 B1

Example 1. Inhibition of lymphocyte activation [0024] 20 C57BI mice were immunized transdermally with a suspension of Mycobacterium Smegmatis (100 mkg antigen in 50 mkl aluminum alum) in a paw pad. Four weeks later, the mice were sacrificed and splenocytes isolated. Splenocytes of sensitized and untreated mice were cultured in petri dishes in suspension culture (2.5 x10⁶ cells / ml) in PRPMI 1640 medium with 2 mM glutamine, antibiotics and 10% fetal calf serum in the presence of Mycobacterium Smegmatis antigen (5mkg / ml) for 24 hours at 5% CO2 at 37 ° C. To determine the level of splenocyte activation in the presence of [3H] thymidine antigen 6 hours before the end of the culture was added to a concentration of 0.1 mCi / ml. After culturing, the cells were washed, dissolved in formamide and radioactivity measured.

Series 1 (5 dishes) Sensitized mouse splenocytes.

Series 2 (5 dishes) Splenocytes of non-allergic mice. Recombinant dornase alfa (Genentech) was added to the medium at a concentration of 1 mkg / ml.

Series 3 (5 dishes) Splenocytes of untreated mice.

Series 4 (5 dishes) Splenocytes of untreated mice. Extracellular blood DNA isolated from untreated mice was added to the medium 2 hours after repeated subcutaneous injection of Mycobacterium Smegmatis antigen at a dose of 200 mkg. DNA was added at a concentration of 0.05 mkg / ml.

Series 5 (5 dishes) Splenocytes of untreated mice. Extracellular blood DNA isolated from sensitized mice was added to the medium 2 hours after repeated subcutaneous injection of Mycobacterium Smegmatis antigen at a dose of 200 mkg. DNA was added at a concentration of 0.05 mkg / ml.

Series 6 (5 plates). Splenocytes of untreated mice that were cultured without the addition of antigen.

[0025] [3H] thymidine uptake by splenocytes 24 hours after culture with antigen.

[0026] The results of the experiment are shown in Table 6.

Table 6

<td colspan="2">Inhibition of lymphocyte activation</td>

<td>Series number</td><td>Lymphocyte Number (CPM)</td>

<td> 1</td><td> 115000</td>

<td> 2</td><td> 75000</td>

<td> 3</td><td> 35000</td>

<td> 4</td><td> 95000</td>

<td> 5</td><td> 40000</td>

<td> 6</td><td> 15000</td>

[0027] Thus, extracellular DNA of blood plasma increases lymphocyte-specific activation under antigenic stimulation, and the use of DNase leads to inhibition of antigenic stimulation according to the claimed method.

Example 2. Treatment of delayed-type hypersensitivity [0028] A 23-year-old man was admitted to hospital in a serious condition. 4 years ago in 1999, chronic myeloleukosis was diagnosed. Previously, hydroxyurea and interfero5 therapy was performed

EP 2 497 488 B1. Due to the acute progression of the disease, a bone marrow transplant was performed. The bone marrow was transplanted from an HLA compatible but ABO compatible donor and whole body irradiation with cyclophosphate was performed. The patient was given methotrexate to prevent graft versus host reaction. On the ninth day, the graft versus host reaction developed with general rash and diarrhea. The patient received pulse therapy with methylprednisolone and anti-lymphocytic globulin for 9 days. The patient's condition has improved. By day 30, bone marrow function returned and the patient was discharged from the hospital. A week later, the patient was repeatedly admitted to the hospital with leukopenia (leukocytes were 0.9 * 10⁹), ulceration of the oral mucosa and fever. Hypoplasia and eosinophilia were found in the aspiration biopsy. Azathioprine and leucomax were prescribed, but 6 weeks later leukopenia developed (leukocytes 0.7 * 10⁹). Azathioprine was stopped and pulse therapy with methylprednisolone and leucomax was performed. At the same time, fever and sores of the oral mucosa could not be eliminated.

[0029] Soon after the end of therapy, episodes of intravascular hemolysis with a decrease in the number of leukocytes and thrombocytes appeared. The patient was prescribed intravenous infusions of bovine pancreas deoxyribonuclease 400 mg / day (800,000 Kunitz units) 6 times a day for one hour for 2 weeks. The level of DNA hydrolysis activity in blood plasma was over 180 Kunitz units per liter of plasma in not less than 12 hours. From the 5th day of therapy, the patient's condition improved. The number of leukocytes by the 7th day increased to 1.7 * 10⁹, and by day 15 was 2.4 * 10 on average⁹. During this time, the symptoms of hemolysis disappeared, the temperature dropped, and sanitation of mouth ulcers was observed. Mouth ulcers were sanitized and the number of erythrocytes normalized. The patient was discharged from the hospital in a satisfactory condition. A month later, during a follow-up visit to the hospital, a normal blood pattern was found. [0030] Thus, the use of DNase according to the claimed method has a therapeutic effect in a delayed type hypersensitivity reaction.

Industrial use [0031] To implement the methods, well-known materials and devices manufactured under industrial conditions were used and, according to the above, the invention fulfills the requirements of the "industrial applicability" (IA) criterion.