Quinone for use in the treatment of erythrocyte enzyme disorders

Abstract

A class of quinones for use in the treatment of erythrocyte enzyme disorders. This treatment increases ATP in non-mitochondrial cells. Therefore, the present invention relates to a class of quinones for use in the treatment of diseases or conditions associated with or caused by deficiencies in the erythrocyte glycolysis pathway.

Description

Detailed description of the invention

[0001] [Field of Invention] This invention relates to the discovery that a class of quinones has the ability to restore, normalize, and / or improve ATP in mitochondria-deficient cells, such as red blood cells (RBCs). Red blood cells rely solely on the anaerobic conversion of glucose via the Emden-Meyerhof pathway (glycolysis) for the production and storage of high-energy phosphates, such as ATP, which are necessary for several biological functions. Therefore, this invention relates to a class of quinones for use in the treatment of diseases or conditions associated with or caused by deficiencies in erythrocyte glycolysis.

[0002] [background] Human red blood cells are unique in that they become anucleated upon maturation. Immature red blood cells have a nucleus, but during early erythrogenesis, before becoming circulating reticular red blood cells, they remove the nucleus and other organelles, such as mitochondria, the endoplasmic reticulum, and the Golgi apparatus, to make space for hemoglobin, which carries oxygen. As a result of lacking mitochondria, mature red blood cells do not utilize the oxygen they carry at all and economically synthesize adenosine triphosphate (ATP), like other normally differentiated cells. Instead, red blood cells rely entirely on anaerobic glycolysis, and to maintain the integrity and flexibility of their cell membranes as they travel through blood vessels, they also produce nicotinamide adenine dinucleotide (NAD). + ) circulates and ATP, ATPase-dependent K + / Na + and Ca 2+ It generates the essential energy source primarily used to drive the pump.

[0003] During their intravascular lifespan, red blood cells require energy to maintain many bodily functions, including glycolysis. Lacking a nucleus and mitochondria, mature red blood cells cannot produce energy via the oxidative Krebs cycle. Instead, red blood cells rely on the anaerobic conversion of glucose via the Emden-Mayerhof pathway for ATP production and storage.

[0004] Erythrocyte enzyme abnormality can be in any of the steps in the erythrocyte glycolysis system and can cause various diseases. It is necessary to identify substances that can treat erythrocyte enzyme abnormality and diseases caused by or related to enzyme abnormality.

[0005] The present invention is based on the unique discovery that a class of quinones has been found that increases the ATP level in erythrocytes and increases the ATP level in erythrocytes in which the enzyme pyruvate kinase (PK) is inhibited.

[0006] [Summary] In its broadest aspect, the present invention relates to quinone compounds that can be used for the recovery, normalization and / or improvement of ATP in cells lacking mitochondria, such as erythrocytes. The quinone compounds can be used in medical conditions / diseases that require the recovery, normalization and / or improvement of ATP in cells lacking mitochondria, such as erythrocytes. Specific examples are the use of quinones in erythrocyte enzyme abnormality and diseases caused by or related to enzyme abnormality. Examples include abnormalities in erythrocyte metabolism, such as in erythrocyte metabolism, for example, deficiencies in glucose-6-phosphate dehydrogenase and pyruvate kinase or other enzymes that are part of erythrocyte metabolism.

[0007] It is contemplated that the quinone compounds may also be effective in the treatment of hemolytic anemia, such as anemia caused by hemolytic anemia and ineffective erythropoiesis. Hemolytic anemia is · Defects in erythrocyte membrane production (such as in hereditary spherocytosis and hereditary elliptocytosis), · Defects in hemoglobin production (such as in thalassemia, sickle cell disease and congenital dyserythropoietic anemia), · Abnormalities in erythrocyte metabolism (such as in the erythrocyte glycolysis system and pentose phosphate pathway with deficiencies in glucose-6-phosphate dehydrogenase and pyruvate kinase or other enzymes that are part of erythrocyte metabolism), and can be broadly classified based on this.

[0008] Hemolytic anemia is characterized by a reduced lifespan of circulating red blood cells, and it can be argued that boosting ATP increases red blood cell lifespan by enhancing glycolysis and ATP production. Furthermore, some of these conditions are accompanied by ineffective erythropoiesis, and it can be expected that enhancing glycolysis and ATP production will similarly improve erythropoiesis. The latter reason is the underlying cause of anemia caused by chronic diseases, myelodysplastic syndromes, and sideroblastic anemia.

[0009] In specific embodiments, the present invention is a) Restoration, normalization and / or improvement of ATP in cells lacking mitochondria, such as red blood cells, and / or b) Treatment of erythrocyte enzyme disorders, and / or c) Treatment of deficiencies in red blood cell metabolism, such as deficiencies in the red blood cell glycolysis pathway, and / or d) Erythrocyte enzyme disorders accompanied by pyruvate kinase (PK) deficiency in red blood cells, and / or e) Erythrocyte enzyme disorders involving pyruvate kinase (PK) deficiency in red blood cells, wherein the PK deficiency is accompanied by a lack or reduction of R-type PK (PKR) activity in red blood cells, and / or f) Treatment of hemolytic anemia caused by erythrocyte metabolic disorders (such as erythrocyte glycolysis and the pentose phosphate pathway with deficiencies in glucose-6-phosphate dehydrogenase and pyruvate kinase). This relates to quinone compounds that can be used in the field.

[0010] As described above, the quinone of the present invention can generally be used to treat hemolytic anemia, specifically, hemolytic anemia caused by a deficiency of ATP in cells lacking mitochondria, such as red blood cells. More specifically, the quinone of the present invention can be used to treat hemolytic anemia caused by a deficiency in the erythrocyte glycolysis pathway.

[0011] Such treatments include, a) Treatment of hemolytic anemia, non-spherocytic hemolytic anemia, hereditary spherocytosis, hereditary elliptocytosis, acquired hemolytic anemia (e.g., congenital anemia (e.g., enzyme abnormality)), or anemia caused by chronic diseases, b) i) Hemolytic anemia diseases characterized by a decrease in the survival period of circulating red blood cells, ii) Anemia associated with abnormal red blood cell metabolism or enzyme abnormalities including non-spherocytic hemolytic anemia, glucose-6-phosphate dehydrogenase deficiency, hexokinase deficiency, and pyruvate kinase deficiency, iii) Anemia associated with red blood cell deformation including hereditary spherocytosis and hereditary elliptocytosis, iv) Treatment of diseases associated with ineffective erythropoiesis including anemia caused by chronic diseases and myelodysplastic syndrome, or c) Treatment of diseases characterized by a decrease in the survival period of circulating red blood cells or ineffective erythropoiesis including hemolytic anemia, non-spherocytic hemolytic anemia, glucose-6-phosphate dehydrogenase deficiency, hemolytic anemia caused by hereditary spherocytosis, hereditary elliptocytosis, anemia caused by chronic diseases, and myelodysplastic syndrome may be mentioned.

[0012] More specifically, the present invention relates to a compound of formula (I) for use in restoring, normalizing and / or improving ATP in cells lacking mitochondria, such as red blood cells [Chemical formula] (wherein, R 16 and R 17 are the same and are alkoxy or alkyl, or are linked together to form an aryl ring, R 18 is methyl or is linked to R 19 as a heterocyclic ring, R 19 is long-chain alkyl or forms a heterocyclic ring with R 18 through O-alkyl, alkyl is each C 1~10 linear or branched alkyl or C 3~7 cyclic alkyl, The long-chain alkyl group is optionally substituted and optionally unsaturated C 10~20 (It is a linear alkyl group.) This relates to compounds or their reduced forms or pharmaceutically acceptable salts, hydrates, solvates, or tautomers thereof. A specific example is their use in the treatment of erythrocyte enzyme disorders, particularly in the treatment of deficiencies in erythrocyte glycolysis.

[0013] In relation to the present invention, the reduced form of the quinone of formula (I) is a semiquinone or its hydroquinone, that is, a quinone in which one or both oxygen atoms in the quinone are reduced to -OH.

[0014] Specific examples of compounds include: [ka] Or its reduced form, its pharmaceutically acceptable salt, hydrate, solvate, or tautomer, [ka] or its reduced form or its pharmaceutically acceptable salt, hydrate, solvate, or tautomer, [ka] Alternatively, examples include its reduced form or pharmaceutically acceptable salts, hydrates, solvates, diastereomers, or tautomers.

[0015] As demonstrated in the examples herein, quinone compounds have been shown to increase ATP levels in red blood cells. Furthermore, the NAD(P)+ content in red blood cells increases, and the reactive oxygen species decrease. Generally, this leads to a favorable metabolic and redox state in red blood cells.

[0016] [Detailed explanation] As described above, the present invention relates to formula (I) for use in restoring, normalizing and / or improving ATP in cells lacking mitochondria, such as red blood cells. [ka] (In the formula, R 16 and R 17 They are the same and are alkoxy or alkyl, or linked together to form an aryl ring. R 18 It is either methyl or as a heterocycle R 19 It is connected to, R 19 It is either a long-chain alkyl or R via O-alkyl. 18 It forms a complex ring, Alkyls are each C 1~10 Linear or branched alkyl or C 3~7 It is a cyclic alkyl, The long-chain alkyl group is optionally substituted and optionally unsaturated C 10~20 (It is a linear alkyl group.) This relates to the compound or its reduced form or its pharmaceutically acceptable salt, hydrate, solvate, or tautomer.

[0017] Specifically, red blood cells are cells that lack mitochondria.

[0018] A specific example is the use of quinone (I) or its reduced form in the treatment of erythrocyte enzyme disorders. Typically, erythrocyte enzyme disorders are deficiencies in erythrocyte metabolism, such as deficiencies in erythrocyte glycolysis. In particular, erythrocyte enzyme disorders are associated with pyruvate kinase (PK) deficiency in erythrocytes.

[0019] Typically, PK deficiency is associated with a lack or reduction in R-type PK (PKR) activity in red blood cells.

[0020] Furthermore, as described above, the quinones of the present invention can generally be used to treat hemolytic anemia, specifically, hemolytic anemia caused by a deficiency of ATP in cells lacking mitochondria, such as red blood cells. More specifically, the quinones of the present invention can be used to treat hemolytic anemia caused by a deficiency in the erythrocyte glycolysis pathway. Specific types of hemolytic anemia are described herein, and it is intended that such anemia can be treated using the quinones described herein.

[0021] In this embodiment, the compound of formula (I) is R 16 and R 17 Both are OMe, R 18 It is methyl, R 19 is a long-chain alkyl, and The compound is one in which the long-chain alkyl group is as defined by formula (I).

[0022] Specifically, such compounds are [ka] Alternatively, it may be its reduced form, or a pharmaceutically acceptable salt, hydrate, solvate, or tautomer thereof.

[0023] Other compounds of formula (I) are: R 16 and R 17 They are linked together to form an aryl ring, R 18 R is a complex algebra. 19 It is connected to, R 19 R via O-alkyl 18 It forms a complex ring, Alkyl is C 1~10 Linear or branched alkyl or C 3~7 It may be a compound that is cyclic alkyl.

[0024] Specifically, such compounds are [ka] Alternatively, it may be its reduced form, or a pharmaceutically acceptable salt, hydrate, solvate, or tautomer thereof.

[0025] Other compounds of formula (I) are: R 16 and R 17 It is methyl, R 18 It is methyl, R 19 However, it is a long-chain alkyl group that is substituted with one -OH group and contains three double bonds in the alkyl chain. Long-chain alkyl is C 10~20 It may be a linear alkyl compound.

[0026] Specifically, such compounds are [ka] Alternatively, it may be its reduced form or a pharmaceutically acceptable salt, hydrate, solvate, diastereomer, or tautomer thereof.

[0027] Use in restoring, normalizing, and / or improving ATP in cells lacking mitochondria, such as red blood cells. Use of quinones in the treatment of hemolytic anemia caused in particular by ATP deficiency in red blood cells and / or by deficiencies in red blood cell metabolism, especially the red blood cell glycolysis pathway. Its use also includes the treatment of red blood enzyme disorders and other red blood cell diseases.

[0028] A specific example is its use in the treatment of erythrocyte enzyme disorders, particularly deficiencies in erythrocyte metabolism, such as glycolysis.

[0029] As described herein, the present invention relates to quinone compounds that can be used to restore, normalize, and / or improve ATP in cells lacking mitochondria, such as erythrocytes. A specific example is its use in the treatment of erythrocyte enzyme disorders, particularly in the treatment of deficiencies in erythrocyte metabolism, such as glycolysis. As described herein, erythrocytes rely solely on the anaerobic conversion of glucose via the Emden-Meyerhof pathway (glycolysis) for the production and storage of high-energy phosphates, such as ATP, which are necessary for several biological functions. Enzyme disorders are deficiencies in erythrocyte glycolysis. Therefore, erythrocyte enzyme disorders are enzyme deficiencies in erythrocyte glycolysis.

[0030] Quinone compounds are generally intended to be useful in the treatment of hemolytic anemia. Specifically, anemia is, • Red blood cell metabolic disorders (such as erythrocyte glycolysis and the pentose phosphate pathway with deficiencies in glucose-6-phosphate dehydrogenase and pyruvate kinase), • Deficiency in red blood cell membrane production (such as hereditary spherocytosis and hereditary ellipticosis), • Deficiency in hemoglobin production (such as congenital hematopoietic anemia) This can be caused by...

[0031] Hemolytic anemia is associated with a reduced survival time of circulating red blood cells, and it can be argued that ATP boosting extends red blood cell survival time by enhancing glycolysis and ATP production. Furthermore, some of these conditions are accompanied by ineffective erythropoiesis, and it can be expected that enhancing glycolysis and ATP production will similarly improve erythropoiesis. The latter reason is the underlying cause of anemia caused by chronic diseases and myelodysplastic syndromes.

[0032] However, the present invention focuses on the use of quinones in the restoration, normalization, and / or improvement of ATP in cells lacking mitochondria, such as red blood cells, as described herein.

[0033] In specific embodiments, the present invention is a) Treatment of red blood cell enzyme disorders, b) Treatment of erythrocyte enzyme disorders involving enzyme deficiencies in erythrocyte glycolysis, c) Treatment of erythrocyte enzyme disorders accompanied by pyruvate kinase (PK) deficiency in erythrocytes, d) Restoration, normalization, and / or increase of ATP levels in red blood cells, e) Treatment of PK deficiencies associated with the absence or reduction of R-type PK (PKR) activity in the blood. This relates to quinone compounds that can be used in the field.

[0034] As described above, the compounds are a) Treatment of hemolytic anemia or ineffective red blood cell production, b) Treatment of hemolytic anemia, nonspherocytic hemolytic anemia, hereditary spherocytosis, hereditary ellipticosis, acquired hemolytic anemia (e.g., congenital anemia (e.g., enzyme deficiency)) or anemia caused by chronic disease, c)i) hemolytic anemia characterized by a reduced survival time of circulating red blood cells, ii) anemia associated with erythrocytic metabolic disorders or enzyme disorders, including nonspherocytic hemolytic anemia, glucose-6-phosphate dehydrogenase deficiency, hexokinase deficiency and pyruvate kinase deficiency, iii) anemia associated with erythrocytosis, including hereditary spherocytosis and hereditary ellipticosis, iv) treatment of disorders associated with ineffective erythropoiesis, including anemia caused by chronic diseases and myelodysplastic syndromes, or d) Treatment of diseases characterized by reduced survival time of circulating red blood cells or ineffective erythropoiesis, including hemolytic anemia, nonspherocytic hemolytic anemia, glucose-6-phosphate dehydrogenase deficiency, hemolytic anemia resulting from hereditary spherocytosis, hereditary ellipticosis, anemia caused by chronic disease, and myelodysplastic syndromes. It can be used for this purpose.

[0035] Anemia is a blood disorder characterized by a reduced ability of the blood to carry oxygen, due to a lower-than-normal red blood cell count or a decrease in hemoglobin levels. When anemia progresses slowly, symptoms are often vague, including fatigue, shortness of breath, headaches, and decreased physical activity. When anemia is acute, symptoms may include confusion, a feeling of losing consciousness, loss of consciousness, and increased thirst. Anemia inevitably becomes severe before a person becomes noticeably pale. The symptoms of anemia vary depending on how quickly hemoglobin decreases. Other symptoms may occur depending on the underlying cause.

[0036] In connection with the present invention, the term "hemolytic anemia" is used as a general term for anemia caused by hemolysis in blood vessels (intravascular hemolysis) or elsewhere in the human body (extravascular), abnormal destruction of red blood cells. This most commonly occurs in the spleen, but can also occur in the reticuloendothelial system or mechanically (artificial valve damage). Hemolytic anemia accounts for 5% of all existing anemias. It has many possible prognoses, from systemic symptoms to life-threatening systemic effects. The general classification of hemolytic anemia is either endogenous or exogenous. Treatment varies depending on the type and cause of hemolytic anemia. However, as stated herein, treatment of hemolytic anemia is generally contemplated insofar as hemolytic anemia is caused by a deficiency in the amount of ATP produced by red blood cells, and that deficiency is caused by an erythrocyte enzyme disorder such as a deficiency in erythrocyte glycolysis.

[0037] The symptoms of hemolytic anemia are similar to those of other forms of anemia (fatigue and shortness of breath), but the destruction of red blood cells also leads to jaundice, and increases the risk of certain long-term complications such as gallstones and pulmonary hypertension.

[0038] The symptoms of hemolytic anemia are similar to the general signs of anemia. Common signs and symptoms include fatigue, pallor, shortness of breath, and tachycardia. In children, growth retardation may occur with any form of anemia. In addition, symptoms associated with hemolysis may be present, such as chills, jaundice, dark urine, and splenomegaly. Certain aspects of the medical history may suggest the cause of hemolysis, such as medications, side effects of medications, autoimmune diseases, transfusion reactions, the presence of artificial heart valves, or other medical conditions.

[0039] Chronic hemolysis can increase the excretion of bilirubin into the bile ducts, potentially leading to cholelithiasis. Continuous release of free hemoglobin is associated with the development of pulmonary hypertension (increased blood pressure in the pulmonary arteries), which then leads to episodes of syncope, chest pain, and progressive shortness of breath. Pulmonary hypertension ultimately leads to right ventricular heart failure, characterized by peripheral edema (accumulation of fluid in the skin of the legs) and ascites (accumulation of fluid in the abdominal cavity).

[0040] As stated above, the term "hemolytic anemia" is a general term. However, in relation to the present invention, it is preferable that it be limited to anemia caused by a deficiency in red blood cell metabolism, i.e., a deficiency in the red blood cell glycolysis system. As discussed herein, red blood cell metabolism is improved by the quinones discussed herein, and glycolysis and ATP production are enhanced.

[0041] Furthermore, the quinones of the present invention can be used as discussed below: The quinones discussed herein lead to improved ineffective erythropoiesis by enhancing glycolysis and ATP, and therefore, quinones may increase the survival time of circulating erythrocytes as needed. The quinones discussed herein reduce the risk of hemolytic crisis by enhancing glycolysis and ATP in the presence of increased stress from infections, etc.

[0042] In relation to the present invention, the term “associated with” in phrases such as “anemia associated with erythrocyte metabolic disorders or enzyme disorders” is intended to mean diseases caused by or in which erythrocyte metabolic disorders or enzyme disorders are observed, and diseases that cause hemolytic anemia. Similarly, the term “anemia associated with erythrocyte deformation” is intended to mean diseases in which erythrocyte deformation occurs or is observed, and the term “anemia associated with ineffective erythropoiesis” is intended to mean diseases in which ineffective erythropoiesis occurs or is observed. As described above herein, erythrocyte metabolic disorders or erythrocyte enzyme disorders are preferably caused by enzyme deficiencies in the erythrocyte glycolysis pathway.

[0043] In relation to the present invention, the expressions "decreased compared to normal" or "increased compared to normal" are intended to mean a decrease or increase of 5% or more compared to the normal value, for example, 7.5% or more, 10% or more, or 5% or more, 7.5% or more, 10% or more, 15% or more, or 20% or more. Accordingly, the expression "decreased survival time of circulating red blood cells" is intended to mean a red blood cell count that is 20% or less compared to the normal value, for example, 15% or less, 10% or less, 7.5% or less, or 5% or less, for example, about 20% or about 15% or about 10% or about 7.5% or about 5% lower than the normal value.

[0044] [Deficient in erythrocyte glycolysis and related deficiencies] The glycolysis pathway is as follows: Step 1. Glucose is phosphorylated by hexokinase to form glucose-6-phosphate. This step requires 1 mole of ATP. Step 2. Glucose-6-phosphate is isomerized by glucose phosphate isomerase to form fructose-6-phosphate. Step 3. Fructose-6-phosphate is phosphorylated by phosphofructokinase to form fructose-1,6-bisphosphate. This step requires 1 mole of ATP. Step 4. Fructose-1,6-bisphosphate is split by aldolase into two separate sugar molecules, dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. Step 5. The dihydroxyacetone phosphate molecule is isomerized by triose phosphate isomerase to form a second glyceraldehyde-3-phosphate molecule. Step 6. Glyceraldehyde-3-phosphate is phosphorylated by glyceraldehyde-3-phosphate dehydrogenase to form 1,3-bisphosphoglycerate. This step requires NAD+ as a cofactor. Step 7.1,3-bisphosphoglycerate is converted to 3-phosphoglycerate (3-PG) by phosphoglycerate kinase. This step involves the transfer of a phosphate molecule to ADP, forming 1 mole of ATP. In mature red blood cells, 2,3-BPG is also formed via the Rubeling-Rapoport pathway, in which bisphosphoglycerate mutase catalyzes the transfer of phosphoryl groups from C1 to C2 of 1,3-BPG, yielding 2,3-BPG. 2,3-bisphosphoglycerate, the most concentrated organophosphate in red blood cells, forms 3-PG under the action of bisphosphoglycerate phosphatase. Step 8.3-Phosphoglyceric acid is rearranged by the enzyme phosphoglyceric acid mutase to form 2-phosphoglyceric acid. Step 9.2 - Phosphoglyceric acid is dehydrated by the enzyme enolase to produce phosphoenolpyruvic acid. Step 10. Phosphoenolpyruvate is converted to pyruvate by pyruvate kinase. This step involves the transfer of a phosphate molecule to ADP, forming 1 mole of ATP. Step 11. Pyruvate is converted to lactate by the enzyme lactate dehydrogenase. [1] This step involves the oxidation of NADH to NAD+, which allows glycolysis to continue through the glyceraldehyde-3-phosphate dehydrogenase reaction (see Step 6, above).

[0045] In red blood cells, the pentose phosphate pathway, a metabolic pathway parallel to glycolysis, is also important because it produces NADPH. A deficiency in any one of these processes can lead to disease. Red blood cells have no other means of producing ATP and NADPH other than through these glycolytic pathways. The pentose phosphate pathway requires the use of glucose-6-phosphate dehydrogenase. Deficiencies in this enzyme are also included in the scope of this invention. A deficiency in glucose-6-phosphate dehydrogenase is known as broad bean poisoning.

[0046] The overall rate of glycolysis in erythrocytes is regulated by three rate-limiting enzymes: i) hexokinase (HK), ii) phosphofructokinase (PFK), and iii) pyruvate kinase (PK).

[0047] [Hexokinase (HK) deficiency] Hexokinase catalyzes the phosphorylation of glucose to glucose-6-phosphate (G6P) using ATP as a phosphoryl donor. As an initial step in glycolysis, HK is one of the rate-limiting enzymes in the pathway. Hexokinase activity is significantly higher in reticulocytes compared to mature red blood cells, where it is very low. In fact, of all glycolytic enzymes, HK has the lowest enzymatic activity in vitro.

[0048] Hexokinase deficiency (OMIM235700) is a rare autosomal recessive genetic disorder characterized primarily by congenital nonspherocytic hemolytic anemia (CNSHA). As with most glycolytic erythrocyte enzyme deficiencies, the severity of hemolysis varies from severe neonatal hemolysis and death to fully compensated chronic hemolytic anemia. Splenectomy is generally beneficial.

[0049] [Glucose-6-phosphate isomerase (GPI) deficiency] Glucose 6-phosphate isomerase (GPI) catalyzes the interconversion of G6P to fructose-6-phosphate (F6P) in the second step of the Emden-Meyerhof pathway / glycolysis. As a result of this reversible reaction, the product of the hexose monophosphate shunt can be recycled back into G6P. Unlike HK and other age-related enzymes, GPI activity in reticulocytes is only slightly higher than in mature erythrocytes. Beyond its role in glycolysis, GPI exerts cytokine properties extracellularly and is involved in several extracellular processes. GPI is considered a critically important enzyme because GPI knockout mice die in developmental states.

[0050] GPI deficiency (OMIM172400) is an autosomal recessive disorder, and among glycolytic enzyme disorders, it is the second most common after PK deficiency. Homozygous or compound heterozygous individuals have chronic hemolytic anemia of varying severity and exhibit enzyme activity less than 25% of normal. Hemolytic crises can be caused by viral or bacterial infections. Fetal hydrops appears to be more common in GPI deficiency than in other enzyme deficiencies. 32 In rare cases, GPI deficiency can affect non-erythrocyte tissues, leading to neurological symptoms and granulocyte dysfunction. Normally, GPI is very stable, but a striking characteristic of almost all GPI variants is their increased heat retention, while their kinetic properties remain largely unaffected.

[0051] [Phosphofructokinase (PFK) deficiency] Phosphofructokinase catalyzes the rate-limiting ATP-mediated phosphorylation of fructose-1,6-bisphosphate (FBP) via F6P.

[0052] Three distinct subunits—PFK-M (muscle), PFK-L (liver), and PFK-P (platelets)—have been identified in humans. These subunits are expressed in a tissue-specific manner, and five isozymes with varying subunit compositions (M4, M3L1, M2L2, ML3, and L4) can be identified in erythrocytes.

[0053] PFK deficiency (OMIM171850) is a rare autosomal recessive genetic disorder. Since red blood cells contain both the M and L subunits, mutations affecting either gene affect enzyme activity. Therefore, mutations in the L subunit result in red blood cells containing only M4, which are partially PFK deficient. In such cases, patients exhibit mild hemolytic disease without myopathy. Similarly, deficiency of the M subunit results in the absence of muscle PFK and further PFK deficiency in red blood cells. Thus, deficiency of the M subunit causes myopathy and mild hemolytic disease. Red blood cells expressing only L4PFK also exhibit metabolic inhibition in the PFK step of glycolysis and decreased 2,3-DPG levels.

[0054] [Aldolase deficiency] Aldolase catalyzes the reversible conversion of FBP to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate (DHAP).

[0055] Aldolase deficiency (OMIM103850) is an extremely rare disease. All patients presented with moderate chronic hemolytic anemia. Severe hemolytic anemia, as well as hemolytic myopathy, can occur. Enzyme stability may be reduced.

[0056] [Triose phosphate isomerase (TPI) deficiency] Triose phosphate isomerase (TPI) is the most active glycolytic enzyme in the body. TPI catalyzes the interconversion of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate (DHAP).

[0057] TPI deficiency (OMIM190450) is a rare autosomal recessive disorder characterized by hemolytic anemia at onset and often accompanied by neonatal hyperbilirubinemia requiring exchange transfusion. Furthermore, patients exhibit progressive neurological dysfunction, increased susceptibility to infection, and cardiomyopathy. Patients show a 20- to 60-fold increase in DHAP concentration in red blood cells, consistent with metabolic inhibition in the TPI process. The majority of affected individuals die in childhood before the age of six, although there are notable exceptions.

[0058] [Glyceraldehyde-3-phosphate dehydrogenase, monophosphoglycerate mutase, enolase, and lactase dehydrogenase] Red blood cell deficiencies of glyceraldehyde-3-phosphate dehydrogenase and enolase have been described in relation to hemolytic anemia.

[0059] [Phosphoglycerate kinase (PGK) deficiency] Phosphoglycerate kinase produces 1 mole of ATP by catalyzing the reversible conversion of 1,3-bisphosphoglycerate to 3-phosphoglycerate. This reaction can be bypassed by the Rapoport-Rubeling shunt, thereby preventing the formation of a second ATP molecule.

[0060] PGK deficiency (OMIM311800) is characterized by chronic hemolytic anemia, central nervous system dysfunction, and myopathy.

[0061] [Pyruvate kinase (PK) deficiency] In pyruvate kinase deficiency (PKD), two major characteristic metabolic biochemical abnormalities are ATP depletion and an associated increase in 2,3-diphosphoglycerate, which coincides with the accumulation of upstream glycolytic intermediates. Furthermore, one of the consequences of the decreased ATP and pyruvate levels is reduced lactate levels, which are then converted into NAD by lactate dehydrogenase for further use in glycolysis. +The cells become unable to regenerate. ATP deficiency disrupts the cation gradient across the red blood cell membrane, leading to loss of potassium and water, which in turn causes dehydration, shrinkage, and crenate formation of cells, as well as premature destruction and reduced lifespan of red blood cells (RBCs). Such deficient RBCs are destroyed in the spleen, and the excessive hemolysis in the spleen leads to the development of hemolytic anemia. The exact mechanism by which PKD isolates newly matured RBCs in the spleen and effectively shortens the overall half-life of circulating RBCs remains unclear, but recent studies suggest that metabolic dysregulation affects not only cell survival but also the maturation process, resulting in ineffective erythropoiesis.

[0062] Pyruvate kinase catalyzes the transfer of the phosphoryl group from phosphoenolpyruvate (PEP) to ADP, producing 1 mole of pyruvate and 1 mole of ATP. The enzyme requires Mg to facilitate the catalyst. 2+ and K +It possesses the absolute requirement of a cation. PK functions as the final crucial step in glycolysis, as it is an essentially irreversible reaction under physiological conditions. In addition to its role in synthesizing one of the two ATP molecules from the metabolism of glucose to pyruvate, pyruvate kinase is also an important cellular metabolic regulator. It regulates carbon flux at lower glycolysis levels to provide essential metabolite intermediates for supply biosynthetic processes, particularly the pentose phosphate pathway, in maintaining healthy cellular metabolism. Due to these critical functions, pyruvate kinase is tightly regulated both at the gene expression and enzymatic allosteric levels. In mammals, fully activated pyruvate kinase exists as a tetrameric enzyme. Four different isozymes (M1, M2, L, and R) are expressed from two separate genes. The erythrocyte-specific isozyme PKR is expressed from the PKLR gene ("L gene") located on chromosome 1q21. This same gene also encodes the PKL isozyme, which is primarily expressed in the liver. PKR consists of 12 exons, with exon 1 being erythrocyte-specific and exon 2 being liver-specific. The other two mammalian isozymes, PKM1 and PKM2, are produced from the PKM gene ("M gene") by alternative splicing events regulated by the hnRNP protein. The PKM2 isozyme is expressed in fetal tissues and adult proliferating cells, such as cancer cells. Both PKR and PKM2 are actually expressed in proerythroblasts. However, as erythrocytes differentiate and mature, PKM2 expression gradually decreases and is progressively replaced by PKR in mature erythrocytes.

[0063] Clinically, hereditary PKR deficiency manifests as nonspherocytic hemolytic anemia. The clinical severity of this disease ranges from asymptomatic anemia with fully compensated hemolysis to potentially fatal severe anemia requiring chronic transfusion and / or splenectomy at the onset of the disease or during physiological stress or severe infection. Paradoxically, the majority of asymptomatic affected individuals do not require treatment due to enhanced oxygen transfer capacity. This enhanced oxygen transfer capacity may be due to increased 2,3-DPG levels. However, in a subset of the majority of severe cases, there are no disease-modifying treatments available to these patients beyond symptomatic treatment, given the extremely rare population with an estimated prevalence of 51 per million. These patients with hereditary nonspherocytic hemolytic anemia (HNSHA) clearly demonstrate unmet medical needs.

[0064] The exact mechanism leading to the shortened lifespan of mature PK-deficient red blood cells remains unknown. Clinical presentations range from severe hemolysis resulting in neonatal death to well-compensated hemolytic anemia. Some PK-deficient patients present with fetal hydrops. Reticulocytosis is almost always observed. Splenectomy often improves hemolysis and further increases reticulocyte count, especially in severe cases.

[0065] Heterogeneous mutations in PKR lead to dysregulation of its catalytic activity. Currently, nearly 200 different mutations have been reported worldwide that are associated with this disorder. These mutations represent a wide range of genetic disorders, including deletions and biochemical abnormalities of transcription or translation, but by far the most common type is missense mutations in the coding region that affect conserved residues within the domain, which are structurally important for optimal catalytic function of PKR. The prevalence patterns appear to be heterogeneously distributed across specific ethnic backgrounds.

[0066] PK deficiency is recognized in dogs and mice. In both species, the deficiency causes severe anemia and marked reticulocyte hypercytosis, closely resembling human PK deficiency.

[0067] [Pharmaceutical composition] The quinone used in accordance with the present invention may be provided in the form of a pharmaceutical composition comprising the quinone together with one or more pharmaceutically acceptable diluents or carriers.

[0068] Quinone or its compositions may be administered by any conventional method, for example, parenterally, orally, topically (including buccal, sublingual, or transdermal administration), by inhalation or infusion (subcutaneous or intramuscular) using a medical device (e.g., a stent). Treatment may consist of a single dose or multiple doses over a period of time.

[0069] Treatment may involve administration once, twice, three times, or four times a day. Treatment may also involve continuous administration, such as intravenous administration via infusion.

[0070] While quinones can be administered alone, it is preferable to provide them as a pharmaceutical formulation with one or more acceptable carriers. The carriers must be "acceptable" in the sense that they are compatible with the compounds of the present invention and are not harmful to their recipient. Examples of suitable carriers are described in more detail below.

[0071] The formulations may, for convenience, be given in unit dosage forms and may be prepared by any method well known in the field of pharmacy. Such methods include the step of associating a quinone with a carrier constituting one or more auxiliary components. Generally, formulations are prepared by uniformly and closely associating a quinone with a liquid carrier, a fine powder solid carrier, or both, and then, if necessary, forming the product.

[0072] Quinones are typically administered intravenously, orally, or via either parenteral route, in the form of quinone-containing pharmaceutical formulations, optionally in the form of non-toxic organic or inorganic acids or bases, or addition salts, in pharmaceutically acceptable dosage forms. Depending on the disease being treated, the patient, and the route of administration, the composition may be administered in various doses.

[0073] The pharmaceutical composition must be stable under manufacturing and storage conditions and, therefore preferably, should be protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof, or, in the case of a solid or semi-solid composition, it may be a solid or semi-solid carrier.

[0074] For example, quinone may also be administered orally, orally, or sublingually, in the form of tablets, capsules, ovules, elixirs, solutions, or suspensions for immediate-release, delayed-release, or controlled-release applications, which may contain flavoring agents or coloring agents.

[0075] Preparations suitable for oral administration may be provided as separate units such as capsules, cachets, or tablets containing a predetermined amount of quinone, as well as in powder or granule form, as a solution or suspension in an aqueous or non-aqueous solution, or as an oil-in-water or water-in-oil liquid emulsion. Quinone may also be provided as pills, licks, or pastes.

[0076] A quinone solution or suspension suitable for oral administration may also contain excipients, such as N,N-dimethylacetamide, dispersants, such as polysorbate 80, surfactants and solubilizers, such as polyethylene glycol, and Phosal 50PG (consisting of phosphatidylcholine, soybean fatty acid, ethanol, mono / diglycerides, propylene glycol, and ascorbic acid palmitate). The formulation according to the present invention may also take the form of an emulsion, and the compound according to formula (I) may exist as a water-oil emulsion. The oil may be an oil-like substance, such as soybean oil or safflower oil, or a medium-chain triglyceride (MCT oil) such as coconut oil or palm oil, or a combination thereof.

[0077] The tablets may contain microcrystalline cellulose, lactose (e.g., lactose monohydrate or lactose anhydrous), sodium citrate, calcium carbonate, dicalcium phosphate and glycine, butylated hydroxytoluene (E321), crospovidone, hypromellose, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycolate, sodium croscarmellose, and certain complex silicates and granulation binders such as polyvinylpyrrolidone, hydroxypropyl methylcellulose (HPMC), hydroxypropylcellulose (HPC), macrogol 8000, sucrose, gelatin and acacia, as well as excipients. Furthermore, lubricants such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

[0078] Tablets may be manufactured by compression or molding with one or more auxiliary components as optional. Compressed tablets may be manufactured by compressing a quinone in a readily flowable form, such as a powder or granules, which is optionally mixed with a binder (e.g., povidone, gelatin, hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate, cross-linked povidone, cross-linked carboxymethylcellulose sodium), a surfactant, or a dispersant in a suitable machine. Molded tablets may be manufactured by molding a mixture of powder compounds moistened with an inert liquid diluent in a suitable machine. Tablets may be optionally coated or notched and may be formulated to provide sustained release or controlled release of the quinone component therein, for example, using hydroxypropyl methylcellulose in various ratios to provide a desired release profile.

[0079] Similar types of solid compositions can also be used as excipients in gelatin capsules. In this regard, preferred excipients include lactose, starch, cellulose, lactose, or high molecular weight polyethylene glycol. For aqueous suspensions and / or elixirs, the compounds of the present invention can be combined with various sweeteners or flavorings, colorants or pigments, emulsifiers and / or suspending agents, and diluents such as water, ethanol, propylene glycol, and glycerin, and combinations thereof.

[0080] Formulations suitable for topical administration in the oral cavity include lozenges containing quinone in a flavored base, usually sucrose and acacia or tragacanth gum, oral tablets containing quinone in an inert base such as gelatin and glycerin or sucrose and acacia, and mouthwashes containing quinone in a suitable liquid carrier.

[0081] Pharmaceutical compositions adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, impregnating bandages, sprays, aerosols or oils, transdermal devices, powders, etc. These compositions containing quinones can be prepared by conventional methods. Therefore, they may also include suitable conventional carriers and additives, such as preservatives, solvents to aid drug penetration, emollients in creams or ointments, and ethanol or oleyl alcohol for lotions. Such carriers may be present in about 1% to about 98% of the composition. Furthermore, they usually form up to about 80% of the composition. For illustrative purposes only, a cream or ointment containing about 5-10% by weight of the compound is prepared by mixing a hydrophilic material and water in an amount sufficient to produce a cream or ointment with the desired consistency.

[0082] Pharmaceutical compositions adapted for transdermal administration may be provided as separate patches intended to maintain close contact with the recipient's epidermis for an extended period. For example, quinones may be delivered from the patch by iontophoresis.

[0083] With regard to application to external tissues, such as the mouth and skin, the composition is preferably applied as a topical ointment or cream. When formulated as an ointment, the quinone may be used with either a paraffinic or water-miscible ointment base.

[0084] Alternatively, quinone can be formulated as a cream using an oil-in-water cream base or a water-in-oil base.

[0085] For parenteral administration, liquid unit dosage forms are prepared using quinone and sterile excipients, such as, but not limited to, water, alcohol, polyol, glycerin, and vegetable oil, with water being preferred. Depending on the excipient and concentration used, the quinone may be in a colloidal, suspended, or dissolved state within the excipient. In solution preparation, the quinone is dissolved in sterile water for injection, filled into appropriate vials or ampoules, and filtered and sterilized before sealing.

[0086] Advantageously, activating agents such as preservatives and buffers can be dissolved in the excipients. To enhance stability, the composition can be frozen after filling the vial, and water is removed under vacuum. The lyophilized powder is then sealed in the vial, and an accompanying vial of water for injection may be supplied to reconstitute the liquid before use.

[0087] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the composition may take the form of a sterile powder for the immediate preparation of such injectable sterile solutions or dispersions. In all cases, the final injectable form must be sterile and effectively fluid for easy syringe compatibility.

[0088] Parenteral suspensions are prepared in substantially the same manner as solutions, except that the quinone is suspended in an excipient instead of being dissolved, and sterilization cannot be performed by filtration. The quinone can be sterilized by exposure to ethylene before being suspended in a sterile excipient. Advantageously, surfactants or wetting agents are included in the composition to promote the uniform distribution of the quinone.

[0089] In particular, in addition to the components mentioned above, the formulations of the present invention may include other conventional agents in the art, depending on the type of formulation. For example, flavoring agents are examples of agents suitable for oral administration. How to select and prepare an appropriate formulation will be known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences 18th Ed. or later literature). How to select an appropriate route of administration and dosage will also be known to those skilled in the art.

[0090] Those skilled in the art will recognize that the optimal dosage and interval of individual doses of the compounds of the present invention are determined by the nature and severity of the condition being treated, the form of administration, the route and site of administration, and the age and condition of the specific subject being treated, and that the appropriate dosage to be used is ultimately determined by a physician. This dosage is often repeated as needed. If side effects occur, the dosage and / or frequency of administration may be changed or reduced in accordance with the usual medical examination.

[0091] [Method for preparing quinones used according to the present invention] Orthoquinone compounds used in the treatment of erythrocyte enzyme disorders are prepared by standard methods known to those skilled in the art. Multiple routes exist to the compounds used. Typically, the approach begins with an available bicyclic compound, to which a third ring (heterocyclic) is incorporated by attaching the appropriate functional group and then performing ring closure. This includes standard strategies for heterocyclic synthesis [e.g., Katritzky et al, Handbook of Heterocyclic Chemistry, Elsevier 2010; Eicher and Hauptmann, The Chemistry of Heterocycles, 2003, Wiley]. Generally, by synthesis, the central ring is aromatic with substituents (protected or unprotected groups), and a quinone is introduced by oxidation of the ring to regioselectively introduce a second oxygen as a hydroxyquinone, which is then converted to an orthoquinone.

[0092] Appropriate methods for synthesizing orthoquinone compounds used in the treatment of enzyme disorders are described in International Publication No. 2016159577A2, International Publication No. 2022039460, and International Publication No. 2020175851.

[0093] In embodiments where the orthoquinone compound used to treat enzyme disorders is β-lapachone, it can then be synthesized as described above. Alternatively, it can be extracted from a plant or part of a plant that naturally produces the compound. Such a method is described by Karthikeyan et al [Karthikeyan R, Sai Koushik O, Kumar PV (2016) Isolation, Characterization and Antifugal Activity of β-Lapachone from Tecomaricapensis (Thunb.) Spach Leaves, Med. Aromat. Plants 5:239].

[0094] Paraquinone compounds such as idebenone and napabucasin, used to treat erythrocyte enzyme disorders, are commercially available from various suppliers.

[0095] [General Overview] It should be understood that any of the features and / or embodiments discussed above in relation to the compounds according to the present invention are applicable by analogy to the methods described herein.

[0096] The following drawings and embodiments are provided below to illustrate the present invention. They are intended to be illustrative and should not be construed as limiting in any way.

[0097] [Specific implementation details] Specific embodiments include: 1. Formula (IV) for use in the treatment of hemolytic anemia such as erythrocyte enzyme disorders. [ka] (In the formula, R 16 and R 17 They are the same and are alkoxy or alkyl, or linked together to form an aryl ring. R 18 It is either methyl or as a heterocycle R 19 It is connected to, R 19 It is either a long-chain alkyl or R via O-alkyl. 18 It forms a complex ring, Alkyls are each C 1~10 Linear or branched alkyl or C 3~7 It is a cyclic alkyl, The long-chain alkyl group is optionally substituted and optionally unsaturated C 10~20 (It is a linear alkyl group.) Compounds thereof or their pharmaceutically acceptable salts, hydrates, solvates, enantiomers, diastereomers, tautomers, or prodrugs.

[0098] 2.R 16 and R 17 Both are OMe, R 18 It is methyl, R 19 It is a long-chain alkyl, and The long-chain alkyl compound is as defined in claim 13, for use according to item 1.

[0099] 3. [ka] or compounds for use as described in item 2, which are pharmaceutically acceptable salts, hydrates, solvates, enantiomers, diastereomers, tautomers, or prodrugs thereof.

[0100] 4.R 16 and R 17 They are linked together to form an aryl ring, R 18 R is a complex algebra.19 It is connected to, R 19 R is via O-alkyl 18 It forms a complex ring, Alkyl is C 1~10 Linear or branched alkyl or C 3~7 A compound for use as described in item 1, which is a cyclic alkyl compound.

[0101] 5. [ka] The compounds for use as described in item 4, which are pharmaceutically acceptable salts, hydrates, solvates, enantiomers, diastereomers, tautomers, or prodrugs thereof.

[0102] 6.R 16 and R 17 It is methyl, R 18 It is methyl, R 19 It is a long-chain alkyl group that is substituted with one -OH group and contains three double bonds in the alkyl chain. Long-chain alkyls are C 10~20 A linear alkyl compound for use as described in item 1.

[0103] 7. [ka] or a pharmaceutically acceptable salt, hydrate, solvate, enantiomer, diastereomer, tautomer or prodrug thereof, the compound for use as described in item 6.

[0104] 8. A compound for use described in any one of items 1-7 for erythrocyte enzyme disorders involving enzyme deficiencies in erythrocyte glycolysis.

[0105] 9. A compound for use described in any one of items 1-8, for erythrocyte enzyme disorders involving pyruvate kinase (PK) deficiency in erythrocytes.

[0106] 10. The treatment involves a compound for use described in any one of items 1-9, which restores, normalizes, and / or increases ATP levels in red blood cells.

[0107] 11. PK deficiency is associated with the lack or reduction of R-type PK (PKR) activity in red blood cells, and the compounds for use described in item 9.

[0108] 12. Compounds for use in any one of items 1-11, for use in the treatment of hemolytic anemia or ineffective red blood cell production.

[0109] 13. Compounds for use as described in any one of items 1 to 12, for the treatment of anemia caused by hemolytic anemia, nonspherocytic hemolytic anemia, sickle cell anemia, thalassemia (e.g., β-thalassemia), hereditary spherocytosis, hereditary ellipticosis, acquired hemolytic anemia (e.g., congenital anemia (e.g., enzyme deficiency)), or chronic disease.

[0110] 14. Compounds for use in any one of items 1-11 for the treatment of: i) hemolytic anemia characterized by a decrease in the survival time of circulating red blood cells; ii) anemia associated with erythrocytic abnormalities or enzyme disorders, including nonspherocytic hemolytic anemia, glucose-6-phosphate dehydrogenase deficiency, hexokinase deficiency and pyruvate kinase deficiency; iii) anemia associated with erythrocytosis, including hereditary spherocytosis and hereditary ellipticosis; iv) abnormal hemoglobin disorders, including thalassemia and sickle cell disease; v) anemia associated with ineffective erythropoiesis, including anemia caused by chronic diseases, myelodysplastic syndrome and sideroblastic anemia.

[0111] 15. Compounds for use as described in any one of items 1 to 11, for the treatment of diseases characterized by reduced survival time of circulating red blood cells or ineffective erythropoiesis, including hemolytic anemia, nonspherocytic hemolytic anemia, hemolytic anemia resulting from glucose-6-phosphate dehydrogenase deficiency, hereditary spherocytosis, hereditary ellipticosis, thalassemia, sickle cell disease, anemia caused by chronic disease, myelodysplastic syndrome or sideroblastic anemia. [Brief explanation of the drawing]

[0112] [Figure 1A] This shows changes in ATP levels, redox state, and cell size of human red cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. Shikonin dose-dependently reduced ATP levels. [Figure 1B] This graph shows changes in ATP levels, redox state, and cell size of human red cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. Shikonin dose-dependently reduced ATP levels. The bar graph shows the levels at the final measurement point. [Figure 1C] This shows changes in ATP levels, redox state, and cell size of human red cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. Shikonin increased NAD(P)H autofluorescence in a dose-dependent manner. [Figure 1D] This graph shows changes in ATP levels, redox state, and cell size of human red cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. Shikonin increased NAD(P)H autofluorescence in a dose-dependent manner. The bar graph shows the levels at the final measurement point. [Figure 1E] This shows changes in ATP levels, redox state, and cell size of human red cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. Shikonin increased cell size in a dose-dependent manner. [Figure 1F]This graph shows changes in ATP levels, redox state, and cell size of human red cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. Shikonin increased cell size in a dose-dependent manner. The bar graph shows the levels at the final measurement point. [Figure 2A] This shows changes in ATP levels, redox state, and cell size of human red cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. Mitapibat, KL1333, and idebenone all counteracted RK inhibition at selected concentrations - ATP levels. [Figure 2B] This graph shows changes in ATP levels, redox state, and cell size of human red cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. Mitapivat, KL1333, and idebenone all counteracted RK inhibition at selected concentrations - ATP levels. The bar graph shows levels at the final measurement. [Figure 2C] This shows changes in ATP levels, redox state, and cell size of human RBCs in an in vitro model of pyruvate kinase (PK) deficiency. Mitapivat, KL1333, and idebenone all counteracted RK inhibition at selected concentrations of NADH. [Figure 2D] This graph shows changes in ATP levels, redox state, and cell size of human red cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. Mitapivat, KL1333, and idebenone all counteracted RK inhibition at selected concentrations of NADH. The bar graph shows the levels at the final measurement. [Figure 2E] This graph shows changes in ATP levels, redox state, and cell size of human red cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. Mitapivat, KL1333, and idebenone all counteracted RK inhibition at selected concentrations - FSC. The bar graph shows levels at the final measurement point. [Figure 3A] This shows changes in ATP levels, redox state, and cell size of human red cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. KL1333 dose-dependently counteracted RK inhibition. ATP levels. [Figure 3B]This graph shows changes in ATP levels, redox state, and cell size of human red cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. KL1333 dose-dependently counteracted RK inhibition. ATP levels: The bar graph shows the levels at the final measurement. [Figure 3C] This shows changes in ATP levels, redox state, and cell size of human red cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. KL1333 dose-dependently counteracted RK inhibition. NAD(P)H. [Figure 3D] This graph shows changes in ATP levels, redox state, and cell size of human red cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. KL1333 dose-dependently counteracted RK inhibition. NAD(P)H. The bar graph shows the levels at the final measurement. [Figure 3E] This graph shows changes in ATP levels, redox state, and cell size of human red cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. KL1333 dose-dependently counteracted RK inhibition. FSC. The bar graph shows levels at the final measurement. [Figure 4A] This shows changes in ATP levels, redox state, and cell size of human red cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. The combination of KL1333 and mitapibat counteracted the RK inhibitory effect to a greater extent than mitapibat alone. (ATP levels.) [Figure 4B] This shows changes in ATP levels, redox state, and cell size of human red cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. The combination of KL1333 and mitapibat counteracted the RK inhibitory effect to a greater extent than mitapibat alone. ATP levels are shown; the bar graph indicates levels at the final measurement. [Figure 4C] This shows changes in ATP levels, redox state, and cell size of human RBCs in an in vitro model of pyruvate kinase (PK) deficiency. The combination of KL1333 and mitapibat counteracted the RK inhibitory effect to a greater extent than mitapibat alone. NAD(P)H. [Figure 4D]This graph shows changes in ATP levels, redox state, and cell size of human red cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. The combination of KL1333 and mitapibat counteracted the RK inhibitory effect to a greater extent than mitapibat alone. NAD(P)H. The bar graph shows the levels at the final measurement. [Figure 4E] This shows changes in ATP levels, redox state, and cell size of human red cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. The combination of KL1333 and mitapibat counteracted the RK inhibitory effect to a greater extent than mitapibat alone. FSC. Bar graphs show levels at the final measurement point. [Figure 5A] This shows changes in ATP levels, redox state, and cell size of human red cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. Mitapibat, KL1333, and idebenone all counteracted the PK inhibitory effect induced by phenylalanine methyl ester at selected concentrations. ATP levels. [Figure 5B] This shows changes in ATP levels, redox state, and cell size of human red cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. Mitapivat, KL1333, and idebenone all counteracted phenylalanine methyl ester-induced PK inhibition at selected concentrations. ATP levels are shown. The bar graph indicates levels at the final measurement. [Figure 5C] This shows changes in ATP levels, redox state, and cell size of human red cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. Mitapivat, KL1333, and idebenone all counteracted phenylalanine methyl ester-induced PK inhibition at selected concentrations. FSC(C). Bar graphs show levels at the final measurement point. [Figure 6A]This shows the changes in ATP levels of human red blood cell cytoplasm (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. In this experiment, the PK inhibitory effect was partially counteracted by β-rapacon at a concentration of 1 μM. KL1333 was more effective. No significant effect was observed at β-rapacon at 10 μM. ATP levels. [Figure 6B] This graph shows the changes in ATP levels in human red blood cell blasts (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. In this experiment, the PK inhibitory effect was partially counteracted by β-rapacon at a concentration of 1 μM. KL1333 was more effective. No significant effect was observed at β-rapacon at 10 μM. ATP levels. The bar graph shows the levels at the final measurement. [Figure 7A] This shows the changes in ATP levels of human red blood cell cytoplasm (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. In this experiment, the effect of PK inhibition was discretely affected by baciquinone at a concentration of 1 μM. KL1333 was more effective. No effect was observed at a concentration of 10 μM. ATP levels. [Figure 7B] This graph shows the changes in ATP levels in human red blood cell cells (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. In this experiment, the PK inhibitory effect was discretely affected by baciquinone at a concentration of 1 μM. KL1333 was more effective. No effect was observed at a concentration of 10 μM. The bar graph shows the ATP levels at the final measurement. [Figure 8] This shows the changes in reactive oxygen species (ROS) levels in uninhibited healthy human RBCs after treatment with 1 μM KL1333. Administration of KL1333 resulted in a significant decrease in intracellular ROS levels, as determined by tracking the fluorescence of 2-hydroethidium by flow cytometry. [Figure 9] This shows the changes in ATP levels of human red blood cell cytoplasm (RBCs) in an in vitro model of pyruvate kinase (PK) deficiency. In this experiment, the PK inhibitory effect was counteracted by napabucasin at a concentration of 1 μM. KL1333 was more effective. The effect of β-rapacon at 1 μM was similar to that of napabucasin at 1 μM.

[0113] The results from the examples are described herein.

[0114] Example 1 presents an established flow cytometry-based in vitro model of pyruvate kinase (PK) deficiency in human erythrocytes (RBCs). The PK inhibitor shikonin dose-dependently increased magnesium green fluorescence, NAD(P)H autofluorescence, and FSC (forward light scattering).

[0115] Magnesium green fluorescence is inversely proportional to intracellular changes in ATP levels. It is a dye that measures Mg2+. Because ATP binds to Mg2+ with a higher affinity than ADP, the magnesium concentration [Mg2+] in the cytosol increases with ATP hydrolysis (and therefore magnesium green fluorescence also increases).

[0116] NAD(P)H autofluorescence is related to the redox state of cells. High autofluorescence indicates high intracellular levels of NADPH and NADH.

[0117] Forward light scattering (FSC) is related to cell diameter. Much of the ATP produced by RBCs is used to drive membrane ion pumps to maintain cellular integrity. It is known that ATP deficiency in RBCs transiently increases size, and over the long term, cells take up calcium, release potassium, and become "dehydrated," i.e., smaller. In our 2-hour experiment following PK inhibition, cell size increased.

[0118] Thus, inhibiting pyruvate kinase with shikonin reduces red blood cell ATP levels, increases NAD(P)H levels, and increases cell size. All of these effects are dose-dependent.

[0119] Except for Example 5, which uses another PK inhibitor, phenylalanine methyl ester, to demonstrate the findings with shikonin, the rest of the examples use this model of pyruvate kinase (PK) deficiency. In Example 8, PK is not inhibited.

[0120] In Example 2, the effects of mitapibat, KL1333, and idebenone are compared. The results show that administration of mitapibat, KL1333, or idebenone at selective concentrations to shikonin-treated RBCs counteracts the PK inhibitory effect, as indicated by an increase in ATP levels, a decrease in NAD(P)H levels, and a decrease in cell size (compared to shikonin-treated RBCs without administration of mitapibat, KL1333, or idebenone).

[0121] In Example 3, the dose-response relationship of KL1333 was investigated. The results showed that KL1333 dose-dependently counteracted PK inhibition, as indicated by a dose-dependent increase in ATP levels, a decrease in NAD(P)H levels, and a decrease in cell size in shikonin-treated RBCs.

[0122] In Example 4, the effect of simultaneous treatment with mitapibat and KL1333 was investigated. From the results, it can be seen that the combination of KL1333 and mitapibat counteracted the PK inhibitory effect to a greater extent than mitapibat alone.

[0123] In Example 5, the effects of treating human RBCs with KL1333, idebenone, or mitapibat in an in vitro model of pyruvate kinase deficiency induced by another PK inhibitor, phenylalanine methyl ester, were investigated. Similar to the findings with the PK inhibitor shikonin, the results showed an increase in ATP and a decrease in cell size after treatment with KL1333, idebenone, or mitapibat (comparing phenylalanine methyl ester-treated RBCs with no administration of mitapibat, KL1333, or idebenone).

[0124] In Example 6, the effects of two different concentrations of β-rapacone (compared to 1 μM of KL1333) on shikonin-induced pyruvate kinase-deficient human RBCs were investigated. The results showed a partial increase in ATP levels compared to 1 μM of β-rapacone (and KL1333).

[0125] In Example 7, the effect of bathyquinone on pyruvate kinase-deficient human RBCs induced by shikonin was investigated. The results showed a slight increase in ATP levels in response to 1 μM of bathyquinone.

[0126] In Example 8, the effect of KL1333 on intracellular reactive oxygen species was investigated. The results showed that ROS decreased when KL1333 was administered.

[0127] In Example 9, the effect of napabucasin 1 μM on shikonin-induced pyruvate kinase-deficient human RBCs (compared to KL1333 1 μM and β-rapacone 1 μM) was investigated. The results showed a clear increase in ATP levels for the three quinones at the 1 μM concentrations compared (KL1333 was the most effective).

[0128] In Example 10, the enzymes and cofactors that reduce the quinone compound KL1333 and idebenone were investigated. The results showed that, in addition to the NQO1 activation described above, KL1333 and idebenone also activate NQO2 activity, and that KL1333 activates CYB5R3, thereby changing the redox states of the nicotinamide adenine dinucleotide-related cofactor, NRH, and NADH. Unlike NQO1, which is not expressed, both NQO2 and CYB5R3 are expressed in erythrocytes.

[0129] [Methods and Materials] [In vitro assays in human erythrocytes] [Flow cytometry - Measurement of intracellular ATP, cell diameter (cell size), and NAD(P)H autofluorescence] Flow cytometry based on magnesium green, NAD(P)H, and forward scatter (FSC) was used to detect mitochondrial ATP production, NAD(P)H levels, and cell size in human erythrocytes (RBCs), respectively. RBCs were gated from human blood based on forward and side scattering characteristics. Free Mg 2+ When bound to ATP, magnesium green fluorescence exhibits a higher affinity for ATP than for ADP. Therefore, as the ATP level increases, the magnesium green fluorescence decreases (1). The NAD(P)H level was measured using its intrinsic autofluorescence properties, which are excited by 360 nm ultraviolet light and emit light at 470 nm.

[0130] RBCs (50 million / ml) were stained in MiR05 buffer containing 5 mM glucose (MiR05: 0.5 mM EGTA, 3 mM MgCl2, 60 mM K-lactobionic acid, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 110 mM sucrose, free fatty acids, 1 g / L bovine serum albumin, pH 7.1) using the intracellular and cell-penetrating fluorescent dye magnesium green AM (M3735, molecular probe, 1 mM) at 37°C for 60 minutes. Next, RBCs were treated for 20 minutes with either the pyruvate kinase (PK) inhibitor phenylalanine methyl ester (1 mM), shikonin (1, 7, 15, or 30 μM), or DMSO, before adding selective concentrations of idebenone, β-lapacone, napabucasin, batiquinone, KL1333, mitapibat, or excipient solution (DMSO). BD LSR Fortessa and BD LSR Fortessa X20 enabled fluorescence excitation measurements using UV laser (355 nm), blue laser (488 nm), and violet laser (405 nm). Magnesium green fluorescence and NAD(P)H autofluorescence were detected using 530 / 30 LP filters and 450 / 50 filters, respectively.

[0131] [Flow Cytometry - Intracellular Radical Oxygen Species (ROS)] Dihydroethidium (DHE)-based flow cytometry was used to visualize cytoplasmic reactive oxygen species (ROS) levels in human erythrocytes (RBCs). RBCs were gated from blood based on forward and lateral scattering characteristics. DHE is oxidized to its fluorescent form, 2-dihydroethidium, by superoxide or hydrogen peroxide present throughout the cell. 5 billion RBCs / mL were stained with 50 μM DHE for 30 minutes at 37°C in MiR05 buffer containing 5 mM glucose. RBCs were then treated with KL1333 or 1 μM DMSO and followed for 2 hours. After treatment, DHE fluorescence was read every 15 minutes. Fluorescence excitation using a blue laser (488 nm) was measured using a BD LSR Fortessa. DHE was detected using a 610 / 20 band-filter (PI channel).

[0132] [Enzyme kinetics.] A 96-well plate was filled with pH 7.5 buffer (140 μL of 50 mM Tris hydrochloride (Apollo Scientific, BIT1513, catalog no. 1185-53-1), 0.14% BSA (Sigma-Aldrich, A9418-5G, catalog no. 9048-46-8)). 20 μL of substrate solution (KL1333 (ISC01-878-s8) or idebenone (BLD, BD134310, catalog no. 58186-27-9)) was added, followed by 20 μL of enzyme solution. Finally, 20 μL of cofactor solution was added, the resulting solution was mixed, foam was removed, and the plate reader was immediately started. The plate reader devices used were BioTek Epoch (Agilent) and BioTek Synergy. The plates used were H1 (Agilent), 96-well roundwell, flat-bottom microplates (Starlab, E2996-1600). Using readings over 2–12 minutes, the absorbance at 340 nm was measured at room temperature to record the average rate (au / min). The enzymes used were recombinant human CYB5R3 (antibodies.com, A59277), recombinant human NQO2 (Abcam, AB93933-1001 and antigenies.com, enz-515), recombinant human NQO1 (Sigma-Aldrich, D1315), and cofactors NADH (Glentham Life Sciences, GK2960-1G, catalog no. 606-68-8), NADPH (Apollo Scientific, BIB3014, catalog no. 2646-71-1), and NRH (BLD, BD221061, catalog no. 19132-12-8).

[0133] [Data Analysis] Mean fluorescence intensity (MFI) values ​​were compared using FlowJo software v.10 (BD Biosciences). Changes in magnesium green fluorescence were reversed and shown as changes in ATP levels. Statistical analysis (one-way ANOVA with Dunnett's multiple comparison post-hoc test) was performed using Graph Pad PRISM version 9 (La Jolla, California, USA).

[0134] [material] All reagents used were obtained from commercial suppliers.

[0135] KL1333 is formula: [ka] It holds.

[0136] [Examples] [Example 1 - Simulation experiment of pyruvate kinase (PK) deficiency in human red blood cells (RBCs) using shikonin (pyruvate kinase inhibitor)] Example 1 presents an established flow cytometry-based in vitro model of pyruvate kinase (PK) deficiency in human erythrocytes (RBCs). The PK inhibitor shikonin dose-dependently increased magnesium green fluorescence, NAD(P)H autofluorescence, and FSC (forward light scattering).

[0137] To allow monitoring of intracellular ATP changes, human RBCs were first stained with magnesium green AM (cell-permeable) (magnesium green fluorescence increases with decreasing cellular ATP). Subsequently, PK was inhibited by administering excipients or the pyruvate kinase inhibitor shikonin at progressively increasing concentrations. Changes in ATP levels, NAD(P)H, autofluorescence (redox state), and FSC (forward scattered light, cell diameter / size) were repeatedly evaluated over the experimental period using flow cytometry.

[0138] Magnesium green fluorescence is inversely proportional to intracellular changes in ATP levels. It is a dye that measures Mg2+. Because ATP binds to Mg2+ with a higher affinity than ADP, cytosolic magnesium concentration [Mg2+] increases with ATP hydrolysis (and therefore magnesium green fluorescence also increases).

[0139] NAD(P)H autofluorescence is associated with the redox state of a cell. High autofluorescence is associated with high intracellular levels of NADPH and NADH.

[0140] Forward light scattering (FSC) is related to cell diameter. Much of the ATP produced by RBCs is used to drive membrane ion pumps to maintain cellular integrity. ATP deficiency in RBCs has been shown to transiently increase size, and over the long term, cells become "dehydrated," i.e., smaller, by taking up calcium and releasing potassium. Our 2-hour experiment showed an increase in cell size following PK inhibition.

[0141] Thus, inhibiting pyruvate kinase with shikonin reduces red blood cell ATP levels, increases NAD(P)H levels, and increases cell size. All of these effects are dose-dependent.

[0142] These results are shown in Figure 1.

[0143] Except for Example 5, which uses another PK inhibitor, phenylalanine methyl ester, to demonstrate the findings with shikonin, the remaining examples use this model of pyruvate kinase (PK) deficiency. In Example 8, PK is not inhibited.

[0144] [Example 2 - Treatment of an in vitro human model of pyruvate kinase (PK) deficiency (induced by shikonin) - Effects of mitapibat, KL1333, and idebenone] In Example 2, the effects of mitapibat, KL1333, and idebenone are compared.

[0145] To allow monitoring of intracellular ATP changes, human RBCs were first stained with magnesium green AM (cytopermeable). Subsequently, PK was inhibited by administering an excipient or 7.5 μM shikonin to the cells. Following this (20 minutes later), the excipient, 5 μM mitapibat, 1 μM KL1333, and 10 μM idebenone were administered. Then, changes in ATP levels, NAD(P)H, autofluorescence (redox state), and FSC (forward scattered light, cell diameter / size) were repeatedly evaluated throughout the experimental period using flow cytometry.

[0146] The results show that administration of mitapibat, KL1333, or idebenone at selective concentrations in shikonin-treated RBCs counteracts the PK inhibitory effect, as indicated by increased ATP levels, decreased NAD(P)H levels, and reduced cell size (compared to shikonin-treated RBCs not treated with mitapibat, KL1333, or idebenone).

[0147] The results are shown in Figure 2.

[0148] [Example 3 - Treatment of an in vitro human model of pyruvate kinase (PK) deficiency (induced by shikonin). KL1333 - Evaluation of dose-response relationship] In Example 3, the dose-response relationship of KL1333 is investigated.

[0149] To allow monitoring of intracellular ATP changes, human RBCs were first stained with magnesium green AM (cytopermeable). Next, 7.5 μM of an excipient or shikonin was administered to inhibit PK, followed (20 minutes later) by administration of the excipient or KL1333 at progressively increasing concentrations. Flow cytometry was then used to repeatedly assess ATP levels, NAD(P)H, autofluorescence (redox state), and FSC (forward scattered light, cell diameter / size) throughout the experiment.

[0150] These results indicate that KL1333 dose-dependently counteracted PK inhibition, as demonstrated by a dose-dependent increase in ATP levels, a decrease in NAD(P)H levels, and a decrease in cell size in shikonin-treated RBCs.

[0151] The results are shown in Figure 3.

[0152] [Example 4 - Treatment of an in vitro human model of pyruvate kinase (PK) deficiency (induced by shikonin) - Effect of simultaneous treatment with mitapibat and KL1333] In Example 4, the effect of simultaneous treatment with Mitapibat and KL1333 will be investigated.

[0153] Human RBCs were first stained with magnesium green AM (cytopermeable) to allow monitoring of intracellular ATP changes. Then, excipients, mitapibat 500 nM, and mitapibat 500 nM + KL1333 500 nM were administered. Subsequently, ATP levels, NAD(P)H, autofluorescence (redox state), and FSC (forward scattered light, cell diameter / size) were repeatedly evaluated using flow cytometry throughout the experimental period.

[0154] The results indicate that the combination of KL1333 and mitapibat counteracted the PK inhibitory effect to a greater extent than mitapibat alone.

[0155] The results are shown in Figure 4.

[0156] [Example 5 - Treatment of an in vitro human model of pyruvate kinase (PK) deficiency (induced by phenylalanine methyl ester) - Effects of mitapibat, KL1333, and idebenone] In Example 5, the effects of treatment with KL1333, idebenone, or mitapibat on human PBC in an in vitro model of pyruvate kinase deficiency induced by another PK inhibitor, phenylalanine methyl ester, are investigated.

[0157] To allow monitoring of intracellular ATP changes, human RBCs were first stained with magnesium green AM (cytopermeable). Next, 1 mM of an excipient or phenylalanine methyl ester was administered to inhibit PK, followed (20 minutes later) by administration of the excipient, 5 μM of mitapibat, or 1 μM of KL1333. ATP levels and FSC (forward scattered light, cell diameter / size) were then repeatedly assessed using flow cytometry throughout the experimental period.

[0158] Similar to the findings with the PK inhibitor shikonin, the results indicate an increase in ATP and a decrease in cell size (compared to phenylalanine methyl ester-treated RBCs that were not treated with mitapibat, KL1333, or idebenone) following treatment with KL1333, idebenone.

[0159] The results are shown in Figure 5.

[0160] [Example 6 - Treatment of an in vitro human model of pyruvate kinase (PK) deficiency (induced by shikonin) - Effect of β-rapacone] In Example 6, the effects of two different concentrations of β-rapacone (compared to 1 μM KL1333) on pyruvate kinase-deficient human RBCs induced by shikonin are investigated.

[0161] To allow monitoring of intracellular ATP changes, human RBCs were first stained with magnesium green AM (cytopermeable). Next, excipients or shikonin 7.5 μM were administered to the cells to inhibit PK. Following this (20 minutes later), excipients, β-rapacon 1 μM, β-rapacon 10 μM, and KL1333 1 μM were administered. ATP levels were then repeatedly assessed throughout the experiment using flow cytometry.

[0162] The results show a partial increase in ATP levels with β-rapacon (and KL1333) 1 μM.

[0163] The results are shown in Figure 6.

[0164] [Example 7 - Treatment of an in vitro human model of pyruvate kinase (PK) deficiency (induced by shikonin) - Limited effects of baciquinone] In Example 7, the effect of bathiquinone on pyruvate kinase-deficient human RBCs induced by shikonin was investigated. Human RBCs were first stained with magnesium green AM (cell-permeable) to allow tracking of intracellular ATP changes. Subsequently, 7.5 μM of the excipient or shikonin was administered to the cells to inhibit PK. Following this (20 minutes later), the excipient, 1 μM of bathiquinone, 10 μM of bathiquinone, and 1 μM of KL1333 were administered. Then, ATP levels were repeatedly assessed throughout the experimental period using flow cytometry.

[0165] The results show a small increase in ATP levels in response to 1 μM of bathyquinone.

[0166] The results are shown in Figure 7.

[0167] [Example 8 - Effect of KL1333 on intracellular reactive oxygen species, determined using RBCs stained with dihydroethidium (DHE)] In Example 8, the effect of KL1333 on intracellular reactive oxygen species is investigated.

[0168] Human RBCs were first stained with DHE to allow monitoring of changes in intracellular reactive oxygen species (ROS). The RBCs were then treated with KL1333 or DMSO 1 μM, followed by flow cytometry for 2 hours. After additional treatment, DHE fluorescence was read every 15 minutes.

[0169] The results show that administering KL1333 reduces ROS.

[0170] The results are shown in Figure 8.

[0171] [Example 9 - Treatment of an in vitro human model of pyruvate kinase (PK) deficiency (induced by shikonin) - Effect of napabucasin compared to β-rapacon and KL1333] In Example 9, the effect of napabucasin 1 μM (compared to KL1333 1 μM and β-rapacone 1 μM) on pyruvate kinase-deficient human RBCs induced by shikonin is investigated.

[0172] To allow monitoring of intracellular ATP changes, human RBCs were first stained with magnesium green AM (cytopermeable). Subsequently, PK was inhibited by administering an excipient or shikonin 7.5 μM to the cells. Following this (20 minutes later), the excipient, napabucasin 1 μM, β-rapacon 1 μM, or KL1333 1 μM were administered. Then, ATP levels were repeatedly assessed throughout the experiment using flow cytometry.

[0173] The results show a clear increase in ATP levels for the three quinones at a comparative concentration of 1 μM (KL1333 was the most effective).

[0174] The results are shown in Figure 9.

[0175] [- Reductive metabolism of compound KL1333 and idebenone, and activation of enzymes NQO1, NQO2 and CYB5R3] In Example 10, the enzymes and cofactors that reduce the quinone compound KL1333 and idebenone were investigated.

[0176] Enzyme kinetics were established using a 96-well plate reader to measure the absorbance of substrate, enzyme, and cofactor solutions at 340 nm.

[0177] The results are shown in Table 1.

[0178] [Table 1]

[0179] The results show that, in addition to the NQO1 activation described above, KL1333 and idebenone also activate NQO2 activity, and KL1333 activates CYB5R3, thereby altering the redox states of nicotinamide adenine dinucleotide-related cofactors, NRH and NADH. Unlike NQO1, which is not expressed, both NQO2 and CYB5R3 are expressed in erythrocytes.

Claims

1. Formula (I) for use in restoring, normalizing, and / or improving ATP in cells lacking mitochondria, such as red blood cells. 【Chemistry 1】 (In the formula, R １６ and R １７ They are the same and are alkoxy or alkyl, or linked together to form an aryl ring. R １８ It is either methyl or as a heterocycle R １９ It is connected to, R １９ It is either a long-chain alkyl group or R via an O-alkyl group. １８ It forms a complex ring, Alkyls are each C １～１０ Linear or branched alkyl or C ３～７ It is a cyclic alkyl, The long-chain alkyl is optionally substituted and optionally unsaturated C １０～２０ is a linear alkyl) The compound or its reduced form, or a pharmaceutically acceptable salt, hydrate, solvate or tautomer of formula (I), or its reduced form.

2. The compound for use according to claim 1, wherein the cell lacking mitochondria is a red blood cell.

3. The compound for use according to claim 1 or 2, wherein the use is in the treatment of erythrocyte enzyme disorders.

4. The compound for use according to any one of claims 1 to 3, wherein the use is in the treatment of a deficiency in red blood cell metabolism, such as a deficiency in red blood cell glycolysis.

5. R １６ and R １７ Both are OMe, R １８ It is methyl, R １９ It is a long-chain alkyl, and The compound for use according to any one of claims 1 to 4, wherein the long-chain alkyl is as defined in claim 1. 【Request Item 6】 【Chemistry 2】 A compound for use according to any one of claims 1 to 5, which is a reduced form thereof or a pharmaceutically acceptable salt, hydrate, solvate, or tautomer thereof.

7. R １６ and R １７ They are linked together to form an aryl ring, R １８ R is a complex algebra. １９ It is connected to, R １９ R is formed via O-alkyl １８ It forms a complex ring, Alkyl is C １～１０ Linear or branched alkyl or C ３～７ A compound for use according to any one of claims 1 to 4, wherein the compound is a cyclic alkyl group. 【Request Item 8】 【Chemistry 3】 The compound for use according to claim 7, or the reduced form thereof or a pharmaceutically acceptable salt, hydrate, solvate, or tautomer thereof.

9. R １６ and R １７ It is methyl, R １８ It is methyl, R １９ It is a long-chain alkyl group that is substituted with one -OH group and contains three double bonds in the alkyl chain. Long-chain alkyls are C １０～２０ A compound for use according to any one of claims 1 to 4, wherein the compound is a linear alkyl group. 【Request Item 10】 【Chemistry 4】 The compound for use according to claim 9, or the reduced form thereof or a pharmaceutically acceptable salt, hydrate, solvate, diastereomer, or tautomer thereof.

11. A compound for use according to any one of claims 1 to 4, selected from idebenone, napabucasin, and baciquinone.

12. The compound for use according to any one of claims 1 to 11, wherein the erythrocyte enzyme disorder is accompanied by an enzyme deficiency in the erythrocyte glycolysis pathway.

13. The compound for use according to claim 12, wherein the erythrocyte enzyme disorder is accompanied by pyruvate kinase (PK) deficiency in erythrocytes.

14. The compound for use according to any one of claims 1 to 13, wherein the treatment restores, normalizes and / or increases ATP levels in red blood cells.

15. The compound for use according to claim 13, wherein the PK deficiency is associated with a lack or reduction of R-type PK (PKR) activity in red blood cells.

16. Formula (I) for use in the treatment of hemolytic anemia or ineffective red blood cell production. 【Transformation 5】 (In the formula, R １６ and R １７ They are the same and are alkoxy or alkyl, or linked together to form an aryl ring. R １８ It is either methyl or as a heterocycle R １９ It is connected to, R １９ It is either a long-chain alkyl group or R via an O-alkyl group. １８ It forms a complex ring, Alkyls are each C １～１０ Linear or branched alkyl or C ３～７ It is a cyclic alkyl, The long-chain alkyl group is optionally substituted and optionally unsaturated C １０～２０ (It is a linear alkyl group.) The compound or its reduced form, or a pharmaceutically acceptable salt, hydrate, solvate or tautomer of formula (I), or its reduced form.

17. The compound for use according to claim 16, wherein the hemolytic anemia or ineffective red blood cell production is caused by a deficiency in red blood cell metabolism.

18. The compound for use according to claim 16 or 17, wherein the hemolytic anemia or ineffective red blood cell production is caused by a deficiency in the red blood cell glycolysis pathway.

19. R １６ and R １７ Both are OMe, R １８ It is methyl, R １９ It is a long-chain alkyl, and The compound for use according to any one of claims 16 to 18, wherein the long-chain alkyl is as defined in claim 1. 【Request Item 20】 【Chemistry 6】 A compound for use according to any one of claims 16 to 19, which is the reduced form thereof or a pharmaceutically acceptable salt, hydrate, solvate, or tautomer thereof.

21. R １６ and R １７ They are linked together to form an aryl ring, R １８ R is a complex algebra. １９ It is connected to, R １９ R is formed via O-alkyl １８ It forms a complex ring, Alkyl is C １～１０ Linear or branched alkyl or C ３～７ A compound for use according to any one of claims 16 to 18, wherein the compound is a cyclic alkyl. 【Request Item 22】 【Chemistry 7】 The compound for use according to claim 21, or the reduced form thereof or a pharmaceutically acceptable salt, hydrate, solvate, or tautomer thereof.

23. R １６ and R １７ It is methyl, R １８ It is methyl, R １９ It is a long-chain alkyl group that is substituted with one -OH group and contains three double bonds in the alkyl chain. Long-chain alkyls are C １０～２０ A compound for use according to any one of claims 16 to 18, wherein the compound is a linear alkyl group. 【Request Item 24】 【Chemistry 8】 The compound for use according to claim 23, or the reduced form thereof or a pharmaceutically acceptable salt, hydrate, solvate, diastereomer, or tautomer thereof.

25. A compound for use according to any one of claims 16 to 18, selected from idebenone, napabucasin, and baciquinone.

26. A compound for use according to any one of claims 16 to 25, for treating hemolytic anemia, nonspherocytic hemolytic anemia, hereditary spherocytosis, hereditary ellipticosis, acquired hemolytic anemia (e.g., congenital anemia (e.g., enzyme deficiency)), or anemia caused by a chronic disease.

27. Compounds for use according to any one of claims 16 to 25 for treating anemia associated with ineffective red blood cell production, including i) hemolytic anemia characterized by a reduced survival time of circulating red blood cells; ii) anemia associated with red blood cell metabolic disorders or enzyme disorders, including nonspherocytic hemolytic anemia, glucose-6-phosphate dehydrogenase deficiency, hexokinase deficiency and pyruvate kinase deficiency; iii) anemia associated with red blood cell deformation, including hereditary spherocytosis and hereditary ellipticosis; iv) abnormal hemoglobinopathy; and iv) anemia caused by chronic diseases and myelodysplastic syndromes.

28. Compounds for use according to any one of claims 16 to 25, for treating diseases characterized by reduced survival time of circulating red blood cells or ineffective red blood cell production, including hemolytic anemia, nonspherocytic hemolytic anemia, hemolytic anemia due to glucose-6-phosphate dehydrogenase deficiency, hereditary spherocytosis, hereditary ellipticosis, anemia caused by chronic disease, and myelodysplastic syndromes.

Images