RU2557959C1 - Agent for treating body iron overload, or haemochromatosis - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves chelation of ferric (III) ions both in vivo, and in vitro with using 2-ethyl-6-methyl-3-hydroxypyridine succinate (Mexidol) to produce a stable stained complex having a high molecular weight and a complex (organic-inorganic) composition not identified among the known compounds.

EFFECT: high effectiveness of the chelation process and low toxicity of Mexidol that makes it promising for biomedical application.

4 dwg, 3 tbl, 3 ex

Description

The claimed invention belongs to the field of medicine, namely to new means of chelation of metal ions, mainly iron, which can be used in the treatment of iron overload or hemochromatosis.

Iron is a vital element that makes up hemoglobin, myoglobin, and hemin enzymes, but at the same time it can increase the production of highly reactive and toxic free oxygen radicals, especially hydroxyl radicals, thereby stimulating oxidative damage [Iron and Heme Metabolism [Electronic resource]. - Search regimen http: /themedicfbiochemistrypage.org/heme-porphyrin.pgp]. Excess iron is associated with a number of diseases, disorders and conditions, because a person does not have physiological mechanisms aimed at removing an excess of this element [Brittenham GM Disorders of Iron Metabolism: Iron Deficiency and Overload / GM Brittenham // Hematology: Basic Principles and Practice / R. Hoffman, EJ Benz, SJ Shattil [et al.]. - [3 ^rd ed.]. - New York: Churchill Livingstone, 2000 .-- P. 397-428; Fleming RE Iron overload in human disease / RE Fleming, P. Ponka // N. Engl. J. Med. - 2012. - Vol. 366, No. 4. - P. 348-359]

Hereditary hemochromatosis, a condition in which excess iron is accumulated in the body, is one of the most common genetic diseases in humans. In the United States, about one million people have signs of hemochromatosis, and about one out of every ten people can carry the gene for this metabolic disorder [Franchini M. Recent advances in hereditary hemochromatosis / M. Franchini, D. Veneri // Ann. Hematol. - 2005. - Vol. 84, No. 6. - P. 347-352; Fowler C. Hereditary hemochromatosis: pathophysiology, diagnosis, and management / C. Fowler // Crit. Care Nurs. Clin. North Am. - 2008. - Vol. 20, No. 2. - P. 191-201]. Hemochromatosis is characterized by excessive absorption of iron from food, its accumulation in the organs of the body and their damage, including such serious and even fatal health consequences as liver cirrhosis, liver cancer, heart failure, diabetes, impotence, and arthritis.

Clinical thalassemia (large and small) is a hereditary disease characterized by impaired hemoglobin synthesis, which leads to a decrease in the formation and increase in the destruction of red blood cells. [Ip H.W. Diagnosis and prevention of thalassemia / H.W. Ip, C.C. So // Crit. Rev. Clin. Lab. Sci. - 2013 .-- Vol. 50, No. 6. - P. 125-141.]. In patients with β-thalassemia or resistant anemia (for example, myelodysplastic syndrome) who receive frequent or regular red blood cell transfusions combined with increased iron absorption due to ineffective erythropoiesis, iron overload quickly develops [Survival and complications in thalassemia / C. Borgna-Pignatti, MD Cappellini, P. De Stefano [et al.] // Ann. N.Y. Acad. Sci. - 2005. - Vol. 1054. - P. 40-47; Inati A. Recent advances in improving the management of sickle cell disease / A. Inati // Blood Rev. - 2009. - Vol. 23, suppl. 1. - S. 9-13].

Iron toxicity begins to appear when its load exceeds the binding ability of blood and tissues and unbound iron accelerates the formation of oxygen free radicals, leading to peroxide damage to cells. In the absence of iron chelation, patients with constant blood transfusions inevitably experience a progressive deterioration in the functions of the pancreas, liver and heart as a result of iron overload, which usually occurs in the second decade of life [Thuret I. Post-transfusional iron overload in the haemoglobinopathies / I. Thuret // C.R. Biol. - 2013 .-- Vol. 336, No. 3. - P. 164-172].

Iron overload remains the leading cause of death among thalassemic patients in industrialized countries. Despite progress in treating such diseases, mortality among patients with β-thalassemia and sickle cell disease in the United States remains 3 times higher than among the general population [Morbidity and mortality in chronically transfused subjects with thalassemia and sickle cell disease: A report from the multi-center study of iron overload / EB Fung, P. Harmatz, M. Milet [et al.] // Am. J. Hematol. - 2007. - Vol. 82, No. 4. - P. 255-265]. For these patients, the only way to reduce the risk of serious complications is continuous therapy with iron chelators [Monitoring long-term efficacy of iron chelation therapy by deferiprone and desferrioxamine in patients with beta-thalassaemia major: application of SQUID biomagnetic liver susceptometry / R. Fischer, F. Longo , P. Nielsen [et al.] // Br. J. Haematol. - 2003. - Vol. 121, No. 6. - P. 938-948].

Although transfusion-dependent anemia is the main cause of iron overload, there are other clinical situations with a similar complication, in particular hematopoietic cell transplantation [Majhail NS Iron overload in hematopoietic cell transplantation / NS Majhail, HM Lazarus, LJ Burns // Bone Marrow Transplant. - 2008. - Vol. 41, No. 12. - P. 997-1003]. It is also known that a decrease in the iron content in individual organs and tissues, even under conditions of its normal level in the body, can be useful in neurodegenerative diseases [The role of metals in neurodegenerative diseases / LM Sayre, G. Perry, CS Atwood [et al. ] // Cell. Mol. Biol. - 2000. - Vol. 46. - P. 731-741; The transition metals copper and iron in neurodegenerative diseases / S. Rivera-Mancia, I. Perez-Neri, C. Rios [et al.] // Chem. Biol. Interact - 2010 .-- Vol. 186, No. 2. - P. 184-199; Metal chelation as a potential therapy for Alzheimers disease / MP Cuajungco, KY Faget, X. Huang [et al.] // Ann. NY Acad. Sci. - 2000. - Vol. 920. - P. 292-304], aging [Polla AS Iron as the malignant spirit in successful aging / AS Polla, LL Polla, BS Polla // Ageing Res. Rev. - 2003. - Vol. 2, No. 1. - P. 25-37], oncological diseases [Buss JL The role of iron chelation in cancer therapy / JL Buss, FM Torti, SV Torti // Curr. Med. Chem. - 2003. - Vol. 10, No. 12. - P. 1021-1034] and other conditions and diseases associated with oxidative stress, in the initiation of which iron ions play an important role [Free Radicals and Antioxidant Protocols / by ed. M. Rao Uppu, N. Subramanyam Murthy, A. William Pryor, L. Narasimham Parinandi // Series: Methods in Molecular Biology. - [2 ^nd ed.]. - 2010 .-- Vol. 610. - 480 p .; Siddique A. Review article: the iron overload syndromes / A. Siddique, KV Kowdley // Aliment. Pharmacol Ther. - 2012. - Vol. 35, No. 8. - P. 876-893].

Desferal® is considered the standard for the treatment of iron overload, as well as an antidote for acute poisoning with iron compounds. It is also known as Deferoxamine, Deferoxamine methanesulfonate, Desferan, Desferex, Desferin, DFOM, DFO. By chemical structure it is N- [5- {3 - [(5-aminopentyl) oxycarbamoyl] propionamido} pentyl] -3 - {[5- (Noxy-acetamido) pentyl] carbamoyl} propionic hydroxamic acid. Desferal belongs to the detoxifying agents, including antidotes, pharmacological group, code CAS 138-14-7, ATX: V03AC01 (brand owner - Novartis Pharma AG, Switzerland) [Desferal: [instruction] ./ [Electronic resource]. - Access mode: http://www.likar.info/lekarstva/Desferal/#1]. It has a short half-life from plasma (about 15 min) due to a combination of weak distribution and fast clearance, therefore, distribution is limited to plasma and it is impossible to maintain the reservoir in other cellular compartments. The drug is not effective when taken by mouth, since only 15% of the administered dose is absorbed.

It forms complexes with trivalent ions of iron and aluminum; to a lesser extent binds divalent ions. It is a natural siderophore chelating iron from the labile fraction of the intracellular pool, which is rapidly updated from the deposition pool. Desferal can bind iron, which is in free form or is part of ferritin and hemosiderin. The resulting compounds are excreted in the urine, which leads to a decrease in pathological deposits of iron in the tissues [Mechanism of action of iron chelators / I.V. Smirnov, G.N. Koltsova, S.P. Shcherbinina [et al.] // Hematology and transfusiology. - 2006. - No. 1. - S. 40-44].

Currently, the use of Desferal (Deferoxamine) is common in the treatment of patients with iron overload as a result of repeated blood transfusions. [Ware N.M. Evaluation and treatment of transfusional iron overload in children / H.M. Ware, J.L. Kwiatkowski // Pediatr. Clin. North Am. - 2013 .-- Vol. 60, No. 6. - P. 1393-1406]. The minimum absorption of this drug from the gastrointestinal tract and its short half-life in the blood require slow, long parenteral administration of the drug to achieve a negative iron balance as the main goal of effective chelation. Desferal is prescribed in the form of a subcutaneous infusion lasting 8-10 hours 3-5 times a week, using a special battery-powered injector (pump) [Intensification of chelating-therapy in patients with thalassemia major / H.J. Laws, U. Göbel, A. Christaras [et al.] // Klin. Padiatr. - 2005. - Vol. 217, No. 3. - P. 120-125]. Long-term (almost lifetime) use of Desferal (Deferoxamine) by patients with thalassemia is associated with long-term survival without complications associated with iron overload [Effectiveness and safety of ICL670 in iron-loaded patients with thalassaemia: a randomised, double-blind, placebo-controlled, dose -escalation trial / E. Nisbet-Brown, NF Olivieri, P.J. Giardina [et al.] // Lancet. - 2003. - Vol. 361, No. 9369. - P. 1597-1602]. Unfortunately, only a small number of patients with iron overload are prescribed such therapy all over the world because of poor susceptibility to treatment and the high cost of treatment [Yagudina RI Pharmacoeconomic analysis of chelation therapy in patients with chronic post-transfusion iron overload / R.I. Yagudina, A.Yu. Kulikov, I.S. Krysanov // Pharmacoeconomics. - 2009. - No. 3. - S. 34-38]. At the same time, in patients with β-thalassemia who poorly follow the instructions for using the drug, the probability of 25-year survival is only 32% [Survival and complications in patients with thalassemia major treated with transfusion and deferoxamine / C. Borgna-Pignatti, S. Rugolotto, P. De Stefano [et al.] // Haematologica. - 2004. - Vol. 89, No. 10. - P. 1187-1193].

The costs and inconveniences of using Desferal led to the search for an orally active iron chelator, and Deferipron has been used in recent years for the oral treatment of patients with thalassemia [Monitoring long-term efficacy of iron chelation therapy by deferiprone and desferoxamine in patients with beta-thalassaemia major: application of SQUID biomagnetic liver susceptometry / R. Fischer, F. Longo, P. Nielsen [et al.] // Br. J. Haematol. - 2003. - Vol. 121, No. 6. - P. 938-948 Chelation therapy with oral solution of deferiprone in transfusional iron-overloaded children with hemoglobinopathies / A. Makis, N. Chaliasos, S. Alfantaki [et al.] // Anemia. - 2013 .-- Vol. 2013. - P. 121762]. Deferipron (Ferriprox) was first approved for use with thalassemia in 1994 and is registered in Europe and Asia [Savulescu J. Thalassaemia major: the murky story of deferiprone / J. Savulescu // BMJ. - 2004. - Vol.328, No. 7436. - P.358-359], much later in the USA and Canada. By its chemical structure, it is 3-hydroxy-1,2-dimethylpyridin-4 (1Н) -one, code CAS 30652-11-0, АТС V03AC02 (brand owner is ApoPharma Inc. (Toronto, Canada) [Deferiprone. - Wikipedia Free Encyclopedia [Electronic resource]. - Regimen of access: http://en.wikipedia.org/wiki/Deferiprone].

The drug is used for iron overload, including with myocardial damage [Pat. IL153733 A, IPC A61K 31/44; A61K 31/4412; A61P 9/00. Use of deferiprone for the preparation of iron chelating agents / M. Spino (CA); A. Piga (CA); applicant APOTEX Inc. (CA). - No. IL20020153733; CA20002313270; applic. 12/29/2002; public. 04/29/2010]. It is more convenient for prolonged use, but the risk of developing agranulocytosis, thrombocytopathy and arthropathy limits the use of this drug [Use of deferiprone for iron chelation in patients with transfusion-dependent thalassaemia / S.S. Jamuar, A.H. Lai, A.M. Tan [et al.] // J. Paediatr. Child Health - 2011. - Vol. 47, No. 11. - P.812-817; Distal ulnar changes in children with thalassemia and deferiprone related arthropathy / R. Sharma, R. Anand, J. Chandra [et al.] // Pediatr. Blood Cancer - 2013. - Vol.60, No. 12. - P.1957-1962].

Another drug for treating iron overload with an oral route of administration is Deferazirox (Exjade, ICL670, 4- [3,5-bis- (2-hydroxyphenyl) - [1,2,4] -triazol-1-yl] benzoic acid) - a complexing drug, which is a triple ligand with a high affinity for iron (III) and binding it in a ratio of 2: 1. Long-term daily use of Deferasirox in adults and children with β-thalassemia or sickle cell anemia due to ongoing blood transfusions led to a decrease in total iron stores in the body, and the decrease in iron in the liver was similar to that for Deferrasamine [Deferasirox for managing transfusional iron overload in people with sickle cell disease / JJ Meerpohl, G. Antes, G. Rücker [et al.] // Cochrane Database Syst. Rev. - 2010 .-- Vol.8. - CD007477; Long-term safety and efficacy of deferasirox (Exjade) for up to 5 years in transfusional iron-overloaded patients with sickle cell disease / E. Vichinsky, F. Bernaudin, G.L. Forni [et al.] // Br. J. Haematol. - 2011. - Vol. 154, No. 3. - P.387-397].

Deferasirox, like other iron chelators, can cause side effects (impaired gastrointestinal function, skin rash, increased activity of liver transaminases and creatinine, high frequency of hearing loss, maculopathy, and clouding of the lens of the eye (early cataract) [A maculopapular-type eruption] associated with deferasirox administration / A. Ohshita, N. Nakai, N. Katoh [et al.] // J. Am. Acad. Dermatol. - 2013. - Vol.69, No. 5. - e265-7; Chaudhary P. Deferasirox: appraisal of safety and efficacy in long-term therapy / P. Chaudhary, V. Pullarkat // J. Blood Med. - 2013. - Vol. 4. - P. 101-110].

Deferipron and Deferazirox are chelators of non-biological origin, which are used orally in some patients who cannot receive Desferal, however, these compounds are less effective in binding iron (III) and less toxic. [Comparative efficacy and safety of deferoxamine, deferiprone and deferasirox on severe thalassemia: a meta-analysis of 16 randomized controlled trials / S. Xia, W. Zhang, L. Huang [et al.] // PLoS One. - 2013 .-- Vol. 8, No. 12. - e82662].

As you can see, the existing iron chelators have certain drawbacks, which requires the search for new methods of chelation of iron and their corresponding pharmacological agents, convenient in use, low toxic and fairly cheap, since in most cases we are talking about long-term therapy.

The process of producing a chelated iron compound with the participation of amino-formyl-glycine dipeptide is described, but the complex iron compound obtained in this way is intended for use in animal husbandry as a feed additive to fill the mineral deficiency, and cannot be used in medicine, and the method for its production is carried out outside the body [Pat. CN 103224546 A, IPC A23K 1/16; C07K 1/113; C07K 5/062. Hydration amino formyl glycine dipeptide chelating iron and preparation method thereof / Fan Mingzhi (CN); Wu Chunli (CN); Shu Xugang (CN); Wu Xin (CN); Yin Yulong (CN); Gao Junyong (CN); applicant Guangzhou Tanke Bio Technology Co Ltd; Guangzhou Jiuyi Biolog Technology Co Ltd; Zhongkai University of Agriculture and Engineering; Inst, of Subtropical Agriculture Chinese Academy of Sciences (CN). - No. CN 20131142525; applic. 05/06/2013; public. 07/31/2013].

Chelating iron compounds and their preparation are known, in particular iron bisglycinate chelate [US Pat. 2480222 RF, IPC A61K 33/26; A61K 31/194; A61K 47/20; A61K 47/26; A61P 7/06. Pharmaceutical preparations containing iron bisglycinate chelate / M. Baldachchi (IT); application and patent holder M. Baldachchi (IT). - No. 2009104689/15; declared 02/11/2009; publ. 04/27/2013]. In this case, the preparation of the chelate compound is aimed at using iron as a source of iron when it is deficient in the body and cannot be used in case of acute and chronic iron overload.

There is a complexation method for producing metal compounds, specifically iron chelate in the form of its concentrated solution, which is carried out by the interaction of an iron salt in an aqueous medium with a complexing agent, which is used N, N, N ′, N′-ethylenediaminetetraacetic and citric acid, [Pat . 2458930 RF, IPC C07F 15/02. A method of obtaining a humic iron chelate / A.V. Brykalov (RU), E.M. Golovkina (RU), E.V. Great (RU); application and patent holder of Kuban State Agrarian University (RU). - No.2010150895 / 04; declared 12/13/2010; publ. 08/20/2012]. The specified method of chelation of iron is simple, but is carried out outside the body and is aimed at obtaining a solution for feeding agricultural plants. It cannot be used for chelation of iron as a therapeutic measure in pathological conditions associated with overloading the body with this element.

It is proposed to bind iron ions with a water-insoluble polymer chelating agent having a main polymer chain and an aromatic ring attached to this chain via an —NH — CH ₂ bond, where the aromatic ring has functional groups in the form of —OH, —COOH or other radicals [Pat. EP2664333 A1, IPC A61K 31/722; A61P 13/12; A61P 3/12; A61P 39/04. Polymeric iron chelating agent / Nishida Yuzo (JP); Kohgo Yutaka (JP); Ikuta Katsuya (JP); Sasaki Katsunori (JP); applicant Disease Adsorption System Technologies Co Ltd (JP); Nat. University Corp. Asahikawa Medical University (JP). - No. EP 20120734667; JP 20110006043; applic. 01/12/2012; public. 11/20/2013]. Insolubility in water suggests the possibility of selectively chelating biologically unstable iron without being included in metabolic processes in vivo, however, such a chelator is not very suitable for medical use, especially for creating sterile dosage forms necessary for parenteral administration in case of acute poisoning with iron salts.

Known iron chelator, which is used together with pyrithione [Pat. MX2013004557 A, IPC A61K 31/555. Personal care compositions composed a pyrithione and an iron chelator / Saunders C.W .; Youngquist R.S .; Jun X .; Kelly C.P .; Domsic J.K .; Lucas R.L .; Schwartz J.R. (US); applicant Procter & Gamble (US). - No. MX 20130004557; US20100407754P; applic. 04/23/2013; public. 06/07/2013], but this composition is intended for hygienic purposes and is not suitable for the treatment of acute or chronic iron overload of the body, as well as for the elimination of iron ions in case of free-radical pathology.

Exochelins are proposed as iron chelators, but the emphasis in this invention is on the antioxidant properties of these substances and their administration in case of myocardial infarction before or simultaneously with myocardial reperfusion to prevent damage to the heart muscle as a result of formation of free radicals mediated by iron ions [Pat. MX9604499 A, IPC A01N 1/02; A61K 31/55. Novel iron chelator and inhibitor of iron-mediated oxidation / Horwitz Lawrence; Horwitz Marcus A .; Gibson Bradford W .; Reeve Joseph (US); applicant Univ. California (US). - No. MX 19960004499; US 19950383180; applic. 10/01/1996; public. 11/29/1997].

Chelation of iron is proposed to be carried out using phytic acid or its salt and lactoferin, which have complexing properties and, at the same time, as biogenic substances, have low toxicity, however, the composition is aimed only at improving the condition of the skin and the use of cosmetic or dermatological preparations [Pat . DE102005019202 A1, IPC A61K 8/18; A61K 8/55; A61K 8/67; A61K 8/98; A61Q 17/00; A61Q 19/00. Cosmetic / dermatological preparation, useful e.g. to improve the skin appearance, comprises iron chelating agent such as phytin and / or its slats or lactoferrin / Sauermann K. (DE); Sauermann C. (DE); Sauermann G. (DE); Jaspers S. (DE); Koop U. (DE); Filbry A. (DE); applicant Beiersdorf AG (DE). - No. DE 20051019202; applic. 04/20/2005; public. 10/26/2006].

Complexing agents are proposed with the general formula, which may include a carboxyl group or its derivative, hydroxyl, hydroxylated alkoxy or alkoxyalkyl group; metal group, as well as nitrogen and sulfur atoms, used to obtain diagnostic and medicines for nuclear magnetic resonance imaging, scintigraphy, ultrasound, radiotherapy, and detoxification in case of poisoning by compounds of heavy brooms [Pat. 2073005 RF, IPC C07F 11/00, C07F 15/02. Metal chelating compound / A. Berg (NO), T. Almen (SE), J. Claveness (SE), P. Rongved (SE), T. Thomassen (NO); application and patent holder Nycomed AC (NO). - No. 4743079/04; declared 07/15/1988; publ. 02/10/1997]. However, the proposed chelators are not specifically recommended for the treatment of iron overload or acute intoxication with iron salts. In addition, based on the chemical composition of the proposed complexones, it can be assumed that they have significant toxicity, in particular hepatotoxicity.

The synthesis and description of the derivatives of pyridoxal isonicotinoyl chloride hydrazone, suitable for oral dosage forms, soluble in water and capable of chelation under conditions of overload with iron and other metals [Pat. US5834492 A, IPC C07D 213/86. Water soluble orally effective iron chelator / Lewis N. (US); Patel V. (US); Terpinski J. (US); Bliss R. (US); applicant Jacobus Pharmaceutical Co (US). - No. US19970765369; applic. 04/07/1997; public. 10.11.1998], however, we are talking about substances that are at the stage of preclinical testing and can have a significant number of side effects due to structural analogy with vitamins and antagonism with them.

There is an invention that relates to chelating agents with high stability and high chelating ability with respect to iron, superior to the activity of ethylenediaminetetraacetic acid [Pat. CN 103304435 A, IPC С07С 227/10; C07C 229/36. Chelating agent with high stability and high iron chelating ability and preparation method thereof / Xue Qunxiang (CN); Gong Feixiang (CN); applicant Shaanxi Res. Design Inst, of Petroleum and Chemical Industry (CN). - No. CN 20131277722; applic. 07/03/2013; public. 09/18/2013]. A chelating agent is a compound that is derived from phenol derivatives, aliphatic diamine and an aqueous solution of glyoxalic acid in a single-step synthesis, however, the clinical prospects of the described chelator remain unclear.

The invention relates to a chelating agent that can selectively chelate iron ions and is represented by a compound or its salt containing an aromatic hydrocarbon ring or an aromatic heterocyclic ring with radicals in the form of an alkylene group, hydrogen atoms, a hydrocarbon group or a group having a chelating ability [Pat. US 2011189779 A1, IPC C07C 229/14; C07C 229/26; C07D 233/64; G01N 33/20. Iron chelating agent, method for producing same, method for determining amount of iron ions and method for trapping iron ions / Kohg Yutaka (JP); Ikuta Katsuya (JP); Sasaki Katsunori (JP); Nishida Yuzo (JP); applicant Univ. Yamagata (JP); Nat. Univ. Corp. Asahikawa Medical College (JP). - No. US 200913120126; JP 20080243095; applic. 09/18/2009; public. 08/04/2011], but, as in the previous case, the possibility of the clinical use of such a chelating agent has not been studied.

There is an invention related to the cycloalkyl derivative of 3-hydroxy-4-pyridinone, which is useful for complexation with metal ions, including iron, in chemical and biological systems [Pat. NZ529657 A, IPC A61K 31/4422; C07D 213/81. Iron chelating cycloalkyl derivatives of 3-hydroxy-4-pyridinones / Tam T .; Spino M .; Li W .; Wang Y .; Hao Y .; Shah B.H .; applicant APOTEX Inc. (CA). - No. NZ20030529657; applic. 11/20/2003; public. 07/30/2004], however, it can be assumed that such a derivative, like other known substances of this series, will have side effects that limit its possible use in patients with acute and chronic iron overload.

Chelation of iron can be carried out using a pyrozolone derivative containing aromatic radicals and halogen atoms, which, obviously, along with the elimination of excess iron in animals and humans, will lead to detoxification of the liver [Pat. JP 2004203820 A, IPC A61K 31/4152; A61P 3/10; A61P 43/00; A61P 9/10; C07D 231/26. Iron chelating agent / Tamaoki Toshiaki (JP); Tsuchiya Koichiro (JP); applicant Mitsubishi Pharma Corp. (JP). - No. JP20020376668; applic. 12/26/2002; public. 07/22/2004].

New crystalline forms of 4- [3,5-bis (2-hydroxyphenyl) - [1,2,4] triazol-1-yl] benzoic acid are proposed; methods for producing these crystalline forms, compositions containing such forms, and their use in diagnostic methods or treatment of warm-blooded organisms, especially humans [Pat. US 2012203007 A1, IPC C07D 249/08. Polymorphic forms of deferasirox (ICL670A) / M. Mutz (DE); applicant Novartis AG (CH). - No. US 201213405428; EP 20060125002; WO 2007EP62903; applic. 02/27/2012; public. 08/09/2012]. However, this invention refers to the already used Deferazirox agent and a new form or pharmaceutical composition based on it, as well as the method of their use for chelation of iron, can not radically reduce the side effects of this drug.

For iron chelation under conditions of iron overload, desferritioscin analogues are proposed and their use is described [Pat. US 2013210870 A1, IPC C07D 277/12. Desferrithiocin Polyether Analogues / Bergeron Jr. Raymond J. (US); applicant Univ. Florida (US). - No. US 201213683301; WO 2008 US 03433; applic. 11/21/2012; public. 08/15/2013]. However, the substances claimed in this invention are characterized by nephrotoxicity in animal experiments. In addition, the proposed compounds are at the stage of preclinical studies and it is impossible to say exactly what the results of clinical trials will be, since significant species specificity is traced in the disclosed data.

Known invention relates to amphiphilic metal chelators that have specificity for iron and demonstrate neuroprotective and good transport properties in lipophilic environments [Pat. US2012058945 A1, IPC A61K 31/4412; A61K 31/47; A61K 31/497; A61K 38/00; A61K 38/09; A61K 38/22; A61K 45/06; A61P 25/00; A61P 25/16; A61P 25/28; C07D 213/69; C07D 215/26; C07D 295/15; C07D 295/205; C07K 14/575; C07K 14/70; C07K 5/10; C07K 7/06; C07K 7/22; C07K 7/23. Neuroprotective iron chelators and pharmaceutical compositions composition them / M. Youdim (IL); M. Fridkin (IL); H. Zheng (IL); A. Warshawsky (IL); R. Warshawsky (IL); applicant Yeda RES & DEV (IL); Technion RES & DEV Foundation (IL). - No. US 201113296061; WO 2003IL00932; applic. 11/14/2011; public. 03/08/2012]. The function of the iron chelator is preferably provided by residues of 8-hydroxyquinoline, hydroxamate or pyridinone. The compounds of this invention are useful for the treatment and prevention of diseases, disorders and conditions associated with iron overload and oxidative stress, namely neurodegenerative and cerebrovascular diseases, tumor diseases, hemochromatosis, thalassemia, cardiovascular diseases, diabetes and a number of other diseases. The proposed N-hydroxypyridin-2-ones and 3-hydroxypyridin-4-ones are promising for oral use (hydroxypyridinone CP20, or Deferiprone is used in the clinic); some hydroxypyridinones (for example, CP20, CP24, CP94) are able to penetrate the blood-brain barrier and have structural similarities between the catechol group and the iron chelator in the DOPA structure. In another embodiment of this method, the chelation function is provided by hydroxamate, since hydroxamates are known as iron enterosorbents, and desferrioxamine B (Desferal) has long been the drug of choice for treating iron overload. As you can see, the main emphasis in this invention is on the use of previously known iron chelators according to new indications, and methods for using new agents for chelation of iron are not proposed.

Closest to the claimed tool (prototype) is a tool to reduce the content of metals, especially iron, in mammals, which is conjugates of Deferoxamine, its derivatives and analogues [Pat. US 2010273847 A1, IPC A61K 31/16; A61K 31/4188; A61P 1/16; A61P 25/00; A61P 31/10; A61P 33/06; A61P 39/04; C07C 259/06; C07D 495/04. Desferrioxamine conjugates, derivatives and analogues / R. Codd (AU); L.G. Schipanski (AU); applicant R. Codd (AU); L.G. Schipanski (AU). - US20080740957; AU 20070905998; WO 2008AU01617; applic. 10/31/2008; public. 10/28/2010.]. The compounds mentioned in this method (biotin-deferoxamine, adamantane-deferoxamine, benzo-15-crown-5-4-carboxyl-deferoxamine or 4-methyl-phenoxyacetate-deferoxamine), like Deferoxamine itself, are iron chelators and can be used for overloading the body with iron as a result of dysmetabolic disorders and constant blood transfusions. They can also be used to treat cancer, malaria, fungal infections, in the development of which the role of iron and (or) free radical oxidation has been established. Such compounds have greater hydrophobicity compared to deferoxamine and the possibility of oral administration, they are more resistant to enzymatic degradation and have a longer half-life. It is believed that the presence of functional groups in the N-terminal sequence of the new compounds will reduce the side effects associated with this section of the Deferoxamine molecule. In addition, conjugation with substances like Deferiprone or Deferazirox makes it possible for both ends of the molecule to participate in the chelation of iron. At the same time, the proposed analogues have not been adequately studied in animal experiments, much less have been studied and not used in the clinic, which does not allow us to sufficiently assess their effectiveness and safety.

Thus, the disadvantages of the known means of chelation of iron is that complexing agents are substances that have a large number of side effects, or pharmacokinetic properties that impede their clinical use (for example, short half-life, limited distribution, or the need to use only one route of administration), and also at the stage of preclinical trials with an unclear prospect of use in the clinic.

The objective of the invention is to expand the arsenal of means of chelation of iron ions, which provide a decrease in its content in the body during enteral and parenteral administration with minimal toxicity, proven in both toxicological and clinical studies.

The problem is solved by using 2-ethyl-6-methyl-3-hydroxypyridine succinate (Mexidol) as a chelating agent to treat iron overload or hyperchromatosis.

The technical result consists in the fact that when using this tool for acute and chronic iron loading, the chelation process is sufficiently high with the low toxicity of Mexidol, which ensures the prospects of its biomedical use.

Mexidol belongs to the well-known class of compounds, which includes a number of substances exhibiting biological properties [K. Dumayev The structure and reactivity of derivatives of 3-hydroxypyridine with electrophilic substitution / K.M. Dumayev, L.D. Smirnov // Successes in chemistry. - 1975. - T. 44, No. 10. - S. 1788-1804]. This class of compounds exhibits antioxidant properties and is widely used in medicine [Pharmacology of antioxidants based on 3-hydroxypyridine / V.E. Novikov, L.A. Kovaleva, S.O. Losenkova [et al.] // Access mode to the article:].

Mexidol is a substance with the empirical formula C ₁₂ H ₁₇ NO ₅ and a molar mass of 255.26 g / mol, highly soluble in water and ethanol, sparingly soluble in ether and insoluble in chloroform, with a maximum absorption at a wavelength of 297 ± 2 nm for identification UV spectroscopy [Ethylmethylhydroxypyridine succinate. Powder substance. Producer: LLC Bion. Per. number: LSR-001704 / 07-260707].

It belongs to the ATX N07XX group, CAS 2364-75-2; available in dosage forms such as capsules, solution for intravenous and intramuscular administration, film-coated tablets, film-coated tablets under the trade names Mexidol®, Medomexi, Mexibel, Mexidant, Mexicoor, Mexiprim, Mexipine [Ethyl methylhydroxypyridine succinate. Wikipedia, the free encyclopedia [Electronic resource]. - Access mode:

The owner of the Mexidol® brand is NPK Pharmasoft LLC (RF).

2-ethyl-6-methyl-3-hydroxypyridine succinate is used in the clinic and exhibits antioxidant, antihypoxic, anxiolytic, nootropic effects, improves myocardial and liver functions, has radioprotective properties [Voronina T.A. Antioxidant Mexidol. The main neuropsychotropic effects and mechanism of action / T.A. Voronin // Psychopharmacology and Biological Addiction. - 2001. - No. 1. - S. 2-12; Mexidol and combined vascular pathology of the brain and heart / R.S. Mirzoyan, T.S. Ganynina, M.A. Lebedeva [et al.] // Experiment. and wedge, pharmacology. - 2011. - T. 74, No. 6. - S. 20-23; The effect of Mexidol on the post-radiation restoration of the hematopoiesis system / V.V. Frost, Yu.B. Cheap, G.V. Sukoyan [et al.] // Radiats. biol. radioecologist. - 2009. - T. 49, No. 1. - S. 91-96].

The lethal dose of 2-ethyl-6-methyl-3-hydroxypyridine succinate, which causes the death of 50% of the animals (LD50), is 820 mg / kg for rats and 475 mg / kg for mice, and more than 3000 mg / kg for oral administration in rats and 2010 mg / kg in mice. A comparison of effective therapeutic doses (ED) of Mexidol (10-300 mg / kg) with toxic and lethal doses shows a significant therapeutic breadth and safety of the drug: the therapeutic index calculated as the ratio of LD50 / ED50 is 16.4 [T. Voronina MEXIDOL® main effects, mechanism of action, application [Electronic resource] / T.A. Voronin. - Access mode:

Said 3-hydroxypyridine derivative has beneficial pharmacokinetic properties. It is rapidly absorbed when taken orally (half-absorption period - 0.08-1 hour). When administered intramuscularly, it is determined in blood plasma within 4 hours after administration, with a maximum concentration achieved with intramuscular administration after 0.3-0.58 hours, when taken orally - 0.46-0.5 hours. It is quickly distributed in organs and tissues. The average retention time of the drug in the body with intramuscular injection is 0.7-1.3 hours, with oral administration - 4.9-5.2 hours. It is metabolized in the liver by glucuronidation. It is rapidly excreted in the urine mainly in the form of metabolites (50% in 12 hours). [Pharmacokinetic, behavioral and neurophysiological aspects of the action of 2-ethyl-6-methyl-3-hydroxypyridine in rats / A.K. Sariev, S.V. Krapivin, T.A. Voronin [et al.] // Bull. an experiment. biology and medicine. - 1998. - T. 106, No. 8. - S. 165-167; The relationship of glucuronoconjugation of mexidol and the features of its therapeutic effect in patients with organic lesions of the central nervous system / A.K. Sariev, I.A. Davydova, G.G. Neznamov [et al.] // Experiment. and wedge, pharmacology - 2001. - T. 64, No. 3. - S. 17-21].

To obtain the in vivo chelation effect, a solution of 2-ethyl-6-methyl-3-hydroxypyridine succinate containing an effective dose of the drug (100-50 mg / kg body weight in animal experiments) is administered both orally and parenterally under acute iron loading or repeatedly one of these ways to achieve a negative iron balance in chronic situations.

As a criterion for assessing the formation of a compound between 2-ethyl-6-methyl-3-hydroxypyridine succinate and iron ions, electron scanning microscopy and X-ray analysis are chosen in the model system to determine the elemental composition of the resulting compound and its correspondence to previously known substances, and the body - determining the concentration of iron in blood serum and iron-depositing organs (liver, spleen).

The invention is illustrated by the following examples.

Example 1. The interaction of Mexidol with iron ions (III) outside the body

Materials and methods. The crystalline hydrate of iron (III) chloride (FeCl ₃ ) was dissolved in distilled water in the amount necessary to prepare a 1% solution (mass / volume). The concentration of Fe ³⁺ ions before and after interaction with 2-ethyl-6-methyl-3-hydroxypyridine succinate was controlled photocolorimetrically using salicylic acid or potassium thiocyanate according to a previously prepared calibration schedule. The substance of 2-ethyl-6-methyl-3-hydroxypyridine succinate was dissolved in a 1% solution of iron (III) chloride in such an amount that their molar ratio was 2: 1. The resulting reaction product was investigated for solubility in polar and non-polar solvents and melting point. The isolated crystals were washed on a Schott filter with hot solvents in the following sequence: water-DMF-N-methylpyrrolidone-toluene-ethanol-hexane, and then dried at 90-100 ° C. The purified crystals were subjected to elemental analysis. For the quantitative determination of carbon and hydrogen, the Pregl method was used; for the quantitative determination of nitrogen, the Dumas method [Guben-Weil. Organic Chemistry Methods: Analysis Methods. T.2. - M .: Goskhimizdat, 1963. - S. 106-109, 180-188]. In addition, the resulting complex was investigated by scanning electron microscopy with spectrometry using a scanning scanning electron microscope Cam Scan 4D (CamScan Electron Optics Ltd, UK) [Gorelik S. S. X-ray and electron-optical analysis: Textbook. manual for universities: 3rd ed. add. and reslave. / S.S. Gorelik, L.N. Rastorguev, Yu.A. Skakov. - M .: MISIS, 1994. - 328 s]. On the basis of elemental analysis, the empirical formula of the resulting complex was calculated.

Carried out x-ray phase analysis of the sample [Gorelik S.S. X-ray and electron-optical analysis: Textbook. manual for universities: 3rd ed. additional and revised / S.S. Gorelik, L.N. Rastorguev, Yu.A. Skakov. - M .: MISIS, 1994. - 328 p.]. The diffraction patterns were recorded on a DRON-UM1 diffractometer (NPP Burevestnik OJSC, RF) in Co Ka emission from the anode line (1.79021 Å) with a graphite monochromator in the reflected beam with the Bragg-Brentano survey geometry (in the angular range of 4-80 degrees with a step of 0.1 hail and exposure time of 1 s. To study the thermal degradation of the reaction product, it was successively heated in air to temperatures of 120, 200, 300, and 400 ° C and diffraction patterns of the cooled samples were recorded. nnyh PDF-2 [PDF-2 PCPDFWIN Versio129619n 2.1, -. 2000, JCPDS-ICDD].

Results and discussion. In the interaction of 2-ethyl-6-methyl-3-hydroxypyridine succinate with a 1% solution of FeCl ₃ in a 2: 1 ratio, a gel-like substance of a dark red color is formed. In the supernatant, a qualitative reaction to Fe ⁺³ ions with potassium thiocyanate (KSCN) showed their absence, which indicates that the iron has completely reacted.

At low concentrations of FeCl _{3 the} gel does not form. With increasing concentration of Fe ^{+3, the} substance acquires a dark red color and a higher density. When the gel dries, white crystals and an amorphous substance of red color are formed.

White crystals are soluble in water, but most fully soluble in ethanol. When the solvent is evaporated, they have the same melting point as 2-ethyl-6-methyl-3-hydroxypyridine succinate. The mixing test does not detect melting temperature depression, which indicates that it is 2-ethyl-6-methyl-3-hydroxypyridine succinate, which has not reacted.

Since the unreacted substance is well soluble in ethanol, the complex was purified using this solvent until 2-ethyl-6-methyl-3-hydroxypyridine succinate (white needle crystals identified by melting point) ceased to be detected in the supernatant subsequent washing with hot organic solvents on a Schott filter.

Investigating the resulting reaction product by comparing the melting points, it was shown that this substance is not 2-ethyl-6-methyl-3-hydroxypyridine succinate, which has a melting point of 112 ° C. For the compound obtained, the melting point is 128-130 ° C, which indicates that it not only differs from Mexidol, but also a more complex molecular structure.

It was also established that when the gel formed in the reaction between FeCl ₃ and the 3-hydroxypyridine derivative reacts with chloride acid (1: 1), it dissolves; staining disappears. The proton of perchloric acid can attach to the nitrogen atom of the pyridine ring, which contains an undivided pair of electrons. This leads to disruption of the gel-like structure, which indirectly confirms the participation of the nitrogen atom in its formation.

The results of the study of the complex by elemental analysis showed that it contains: C - 25.63%; 25.74%; H - 4.05%; 4.22%; 3.95%; N - 1.01%; 1.04%.

When studying the reaction product between Fe ⁺³ and a 3-hydroxypyridine derivative by scanning electron microscopy with mass spectrometry, its crystal structure was shown (Fig. 1), and the content of elements in mass% was found in four stochastically selected regions: C 33.52- 35.29%; O 46.16-60.95%; Fe 4.02-18.85%. The content of elements in atom% is: C 41.98-46.10% o; O 47.62-56.52%; Fe 1.07-5.58%. The discrepancy in the results of the analysis, especially in terms of carbon content, may be due to the peculiarities of the methods used, in particular, the use of hardware carbon-containing adhesive tape during electron microscopy.

The diffraction pattern of the initial sample of the substance formed in the reaction between Fe ⁺³ and 2-ethyl-6-methyl-3-hydroxypyridine succinate is shown in Fig. 2. It is characterized by the presence of several broadened and partially overlapping diffraction peaks, which indicates a weak (low) crystallinity of the studied material. Peak characteristics are presented in Table 1.

In the framework of the x-ray diffraction database PDF-2, the substance of the reaction product is not identified. The presence of iron in the composition of the sample is confirmed by the formation of iron oxide (II, III) Fe ₃ O ₄ when the sample is heated in air to 300 ° C, which manifests itself in a change in the diffraction pattern to that characteristic of this oxide (Fig. 3, curve 4).

Comparison of the diffraction pattern of the obtained complex with the diffraction pattern of 2-ethyl-6-methyl-3-hydroxypyridine succinate [US Pat. 2453538 C1 of the Russian Federation, IPC C07D 213/65. Stable crystalline form of 2-ethyl-6-methyl-3-hydroxypyridine succinate and method for its preparation / Germanov SB. (RU), Gomzhin A.M. (RU), Germanova O.L. (RU), E. Filippova (RU), Missul A.B. (RU); application and patent holder of Pharmamed LLC (RU). - No. 20111114873/04; declared 04/06/2011; publ. 06/20/2012.] Reveals a significant difference in the position and intensity of their peaks, which may indicate that the analyte is different from the starting substances that have reacted.

Heating the sample to 120 and 200 ° C leads to a directional change in the intensity of diffraction peaks — a decrease in the intensity of peaks No. 1, 3, 5 and an increase in the intensity of peak No. 2 and, thus, to a partial destruction of its structure (see Fig. 3). A further increase in temperature causes the disappearance of the peaks characteristic of the initial sample, and the appearance of peaks of iron oxide (II, III), which indicates the destruction of the organic component of the studied material. The data on thermal transformations also support the formation of a new organo-inorganic compound based on the starting components.

The formation of a colored complex in the reaction between iron and 3-hydroxypyridine can be explained by the fact that 2-ethyl-6-methyl-3-hydroxypyridine succinate has a phenolic core, which is characterized by a reaction with FeCl ₃ , which leads to the formation of the complex base shown in fig. four.

The simplest formula of the product (or its crystalline hydrate) of the reaction between 2-ethyl-6-methyl-3-hydroxypyridine succinate and a soluble salt of iron (III) calculated by elemental analysis is FeC _11-15 H ₂₁ N ₃ O _18. , which allows to say that 3 mol of the 3-hydroxypyridine derivative is involved in the binding of 1 mol of iron ions with partial degradation of its molecule. At the same time, a comparison of this empirical formula with the formula of 2-ethyl-6-methyl-3-hydroxypyridine succinate suggests that the real atomic ratio in the molecule is a multiple of the simplest, and coordination bonds are present in the molecule. Since the substance formed in the reaction between 2-ethyl-6-methyl-3-hydroxypyridine succinate and Fe ⁺³ has the properties of a gel, the existence of hydrogen bonds in its structure is also probable.

Thus, as a result of the chemical interaction of 2-ethyl-6-methyl-3-hydroxypyridine succinate with Fe ³⁺ ions, a stable colored complex is formed with a large molecular weight and complex component (organo-inorganic) composition, not identifiable among previously known compounds.

Example 2. The effect of Mexidol in acute load of the body with iron

Materials and methods. The experiment used white male rats of the Wistar line weighing 180-200 g (PE Biomodelservice, Ukraine). During the experiment, they were kept in standard plastic cages, received granulated feed for laboratory rodents (PE Biomodelservice, Ukraine) and water as needed. To conduct the experiments, permission was obtained from the bioethics commission of the Voronezh State Medical University “Ukrainian Medical Dental Academy”.

The grouping of experiments was as follows with 6-7 animals in each group:

1. Intact rats;

2. Intact rats with the introduction of a solvent (control);

3. Acute iron load (control pathology);

4. Acute iron load + Mexidol;

5. Acute iron load + Desferal.

As a loading substance, iron (III) chloride FeCl _{3 was used} at a toxic dose of 2000 mg / kg body weight, which was administered intragastrically once using a probe in the form of a 20% FeCl ₃ solution prepared from 6-aqueous crystalline hydrate. When choosing a dose, we were guided by literature data that the introduction of anhydrous FeCl ₃ LD50 = 450 mg / kg per rat os per os [Ferrous chloride, 4-hydrate. - MSDS Number: F1678. Effective date 11/10/05. Supercedes 02/18/03 [Electronic resource]. - Regimen of access: http://2.imimg.com/data2/MM/RV/MY-893306/ferrous-chloride-tetrahydrates.pdf].

Mexidol was administered to animals 10 minutes after the administration of a solution of iron chloride at the same time intragastrally and intraperitoneally. Its solution was prepared ex tempore using the substance 2-ethyl-6-methyl-3-hydroxypyridine succinate (LLC Bion, RF). At the same time, 1 ml of a 25% solution of Mexidol (1250 mg / kg body weight) was injected into the stomach using a probe, intramuscularly - 0.5 ml of a 4% solution of Mexidol (100 mg / kg body weight). The dose was selected based on the range of effective and toxic doses of this drug [T. Voronina MEXIDOL® main effects, mechanism of action, application [Electronic resource] / T.A. Voronin. - Access mode: http://medi.ru/doc/a 070196.htm], and also based on the analogy with the use of the well-known iron chelator - Desferal in this situation. Desferal itself (Novartis Pharma, Switzerland) was used as a reference drug. Its solution was prepared from the lyophilisate in ex tempore vials and was administered intragastrically (250 mg / kg) and simultaneously intraperitoneally at a dose of 80 mg / kg. The dose and route of administration of Desferal was chosen in accordance with the recommendations for its use in acute poisoning with iron salts [Desferal: [instructions]. / [Electronic resource]. - Access mode: http://www.vidal.ru/poisk_preparatov/desferal_232.htm]. The rats of the control group were similarly injected with a solvent, water for injection. After 5 days, the animals were killed under ether anesthesia by taking blood from the heart until it stopped. In the blood serum, liver and spleen, the iron content was determined by inductively coupled plasma atomic emission spectrometry using an Optima 2100 DV instrument (Perkin Elmer, USA) [Determination of chemical elements in biological media and preparations by inductively coupled plasma atomic emission spectrometry and inductively coupled plasma mass spectrometry: 4.1.1482-03. - Officer. ed. - M.: Ministry of Health of Russia, 2003 .-- 16 p. - (Normative document of the Ministry of Health of the Russian Federation. Guidelines)]. The data obtained were statistically processed using computer programs Statistica for Windows v. 8.0.

Results and discussion. Within 5 days after the introduction of the iron salt, no changes in the behavior and appearance of animals were observed, except for staining of urine in brown-red in the groups treated with Mexidol and Desferal. When examining the internal organs of animals after their euthanasia, macroscopic changes characteristic of toxic damage were not detected.

The iron content in the serum, liver and spleen of animals is presented in Table 2.

The indicators of animal iron metabolism with the introduction of a solvent did not differ from the parameters of intact white rats (see Table 2).

The iron load was characterized by an increase in the iron content in the blood serum of animals by 3.4 times (p <0.001) relative to the control. The iron content in the liver increased by 1.4 times (p <0.05), and in the spleen increased by 1.5 times (p <0.05) in comparison with the control values.

The use of Mexidol against the background of an iron load caused a decrease in the concentration of this element in blood serum by 3 times (p <0.002) compared with the control pathology (see Table 2). In animals of this group, the iron content in the spleen decreased by 1.5 times (p <0.05) compared with the introduction of an iron salt without pharmacological correction. In the liver under the influence of Mexidol, no significant changes in the iron content were observed, but there was a tendency to a decrease in the concentration of this element (p <0.1).

When using Desferal, there was a decrease in the concentration of iron in the blood serum by 2.9 times (p <0.05) compared with the pathological background (see Table 2). The iron content in the liver of animals decreased by 1.7 times (p <0.01) and was significantly lower not only than with iron loading without pharmacological correction, but also in control groups. At the same time, the content of this element in the spleen did not change significantly and was significantly higher than with the therapeutic introduction of Mexidol.

Thus, with a single load of iron (2000 mg / kg, intragastrally), Mexidol, administered intragastrally (1250 mg / kg) and intraperitoneally (100 mg / kg), reduces the content of this element in the blood serum and “load” organs, which in degree severity in the blood is not inferior, and in the spleen exceeds the effect of the standard iron chelator - Desferal.

Example 3. The effect of Mexidol in chronic iron load

Materials and methods. The experiment used white male rats of the Wistar line weighing 180-200 g (PE Biomodelservice, Ukraine). Before and during the experiment, they were kept in standard plastic cages, received granular feed for laboratory rodents (PE Biomodelservice, Ukraine) and water as needed. To conduct the experiments, permission was obtained from the bioethics commission of the Voronezh State Medical University “Ukrainian Medical Dental Academy”.

The grouping of experiments was as follows with 8 animals in each group:

1. Intact rats;

2. Intact rats with the introduction of a solvent (control);

3. Intact rats with the introduction of a solvent + Mexidol;

4. Chronic iron load (control pathology);

5. Chronic iron load + Mexidol.

Chronic iron load was reproduced by repeated parenteral administration of an iron preparation to animals [Study of the eye and lacrimal glands in experimental iron overload in rats in vivo / M. Repanti, S.P. Gartaganis, N.M. Nikolakopoulou [et al.] // Anat. Sci. Int. - 2008. - Vol. 83. - P. 11-18]. As such a preparation, iron (III) hydroxide polyisomaltosate was used in the form of the preparation Ferrum Lek (Lek, Slovenia) [FERRUM LEC® for intramuscular administration (FERRUM LEK). Vidal Handbook “Medications in Russia” [Electronic resource]. - Access mode: ~]. It was administered to rats intraperitoneally at a dose of 7.5 mg Fe / 1 animal every 3-4 days for 4 weeks, which amounted to 10 injections. Mexidol was administered intraperitoneally to one of the animal groups at a dose of 50 mg / kg body weight. A solution of Mexidol was prepared ex tempore from its substance obtained from the manufacturer (Bion LLC, RF) using water for injection as a solvent. The animals of the control groups were injected with a solvent or Mexidol solution (50 mg / kg), following the indicated scheme. 3 days after the last injection, the animals were euthanized. During an autopsy, an examination of the internal organs was performed. The content of elemental iron was examined in blood serum, liver and spleen, as described in example 2. The data obtained were statistically processed using standard computer programs.

Results and discussion. The content of Fe in the serum, liver, and spleen of animals treated with solvent injections (control) was the same as in intact animals (Table 3).

The course administration of Mexidol at a dose of 50 mg / kg to intact animals caused a decrease in the content of iron in the blood serum and load organs compared to intact animals (p <0.1), and in the spleen, compared to the control group (p < 0.2) (see Table 3).

Chronic iron load was characterized by an increase in serum iron by 1.4 times (p <0.02) compared with intact animals and 1.6 times (p <0.005) compared to control (see Table 3). At the same time, the content of this element in the liver increased by 1.6 times compared with the intact group (p <0.05) and 1.9 times (p <0.001) compared with animals of the control group treated with solvent injections. In the spleen of rats with iron loading, an increase in iron concentration by 1.3-1.4 times (p <0.05) was observed compared with intact animals and control. Such changes may indicate an increase in both the transport pool of iron and its deposition against the background of the additional administration of this element for 4 weeks at a total dose of 375 MrFe / kg body weight.

Mexidol contributed to a 1.3-fold decrease in serum iron levels (p <0.05) compared with iron loading without pharmacological correction (see Table 3). Moreover, in the liver and spleen, the iron content decreased equally by 1.4 times (p <0.005) compared with the control pathology.

As you can see, the use of Mexidol (50 mg / kg) during chronic iron loading reduces the accumulation of this element in the body and normalizes its content in the blood serum and depot organs.

Thus, as a result of experiments, it was shown that it is possible to remove excess iron from the body in case of acute or chronic loading of this element by using derivatives of 3-hydroxypyridine, in particular 2-ethyl-6-methyl-3-hydroxypyridine succinate, which is based on chemical interaction between this derivative and iron (III) ions with the formation of a stable colored complex with a large molecular weight and complex component (organo-inorganic) composition, unidentifiable among wounds its famous compounds. The inventive method can have biomedical application, which is ensured both by its direct result in the form of elimination of excess iron from the body, and the pharmacological and toxicological properties of 2-ethyl-6-methyl-3-hydroxypyridine succinate (Mexidol), proposed as a chelating agent. An effective dose (100-50 mg / kg body weight in animal experiments) is administered both orally and parenterally during an acute load of iron or repeatedly in one of these ways to achieve a negative iron balance in chronic situations.

Claims (1)

The use of 2-ethyl-6-methyl-3-hydroxypyridine succinate as a chelating agent for the treatment of iron overload or hemochromatosis.

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