Methods for determining decline in mitochondrial function

Abstract

To provide a novel bio-marker for determining neurodegenerative disease such as a Parkinson disease or Altzheimer disease and the like, or a long COVID disease state, or to provide a method for pre-predicting or post-determining an effect of an ATP enhancing therapy with respect to these patients.SOLUTION: A method is provided that determines a decline in a mitochondria function, including a step of measuring inosine in a sample derived from a patient, in which the sample is a sample derived from a cerebrospinal fluid of the patient. A method is provided that predicts or determines an effect of an ATP enhancing therapy with respect to the patient, including the step of measuring the inosine in the sample derived from the patient, in which the ATP enhancing therapy is a therapy that includes a step of administering to the patient an ATP enhancing agent in which following (A) and (B) are an active ingredient, and the sample derived from the patient is the sample from the cerebrospinal fluid of the patient. (A) a xanthine oxidoreductase (XOR) inhibitor, and (B) hypoxanthine or a substance producing the hypoxanthine in a body.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for determining a decline in mitochondrial function in a patient, and more particularly to a method for determining a decline in mitochondrial function in a patient based on the concentration of inosine or hypoxanthine in a patient-derived sample. [Background technology]

[0002] There is ample evidence that ATP deficiency due to mitochondrial dysfunction is the cause of neurodegenerative diseases and long-term COVID-19. Multiple extensive epidemiological studies have demonstrated that febuxostat, a xanthine oxidoreductase (XOR) inhibitor, can suppress Alzheimer's disease. Its mechanism of action is revealed to be increasing hypoxanthine in the blood, which then transports to the central nervous system and enhances ATP. Combining XOR inhibitors with inosine, a precursor of hypoxanthine (ATP augmentation therapy), further increases hypoxanthine, and clinical trials have demonstrated its effectiveness in improving mitochondrial disease and Parkinson's disease. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 2020-201068 [Patent Document 2] Patent Publication No. 2021-161090 [Non-patent literature]

[0004] [Non-Patent Document 1] Fox IH, Palella TD, Kelley WN. Hyperuricemia: a marker for cell energy crisis. N Engl J Med 317, 111-112, 1987 [Non-patent document 2] Atkinson DE, Walton GM. Adenosine triphosphate conservation in metabolic regulation. Rat liver citrate cleavage enzyme. J. Biol. Chem. 242: 3239-3241, 1967. [Non-patent document 3] Johnson TA, Jinnah HA and Kamatani N. Shortage of cellular ATP as a cause of diseases and strategies to enhance ATP. Front Pharmacol. 10:98, 2019. [Non-patent document 4] Short KR, Bigelow ML, Kahl J, et al. Decline in skeletal muscle mitochondrial function with aging in humans. Proc Natl Acad Sci US A. 102:5618-5623, 2005. [Non-Patent Document 5] Schwarzschild MA, Schwid SR, Marek K, et al. Serum urate as a predictor of clinical and radiographic progression in Parkinson disease. Arch Neurol. 65:716-723, 2008. [Non-patent document 6] Kobylecki CJ, Nordestgaard BG, Afzal S. Plasma urate and risk of Parkinson's disease: A mendelian randomization study. Ann Neurol. 84:178-190, 2018. [Non-Patent Document 7] Yuan H, Yang W. Genetically Determined serum uric acid and Alzheimer's disease risk. J Alzheimers Dis. 65:1259-1265, 2018. [Non-patent document 8] Jucker M, Walker LC. Self-propagation of pathogenic protein aggregates in neurodegenerative diseases. Nature. 501:45-51, 2013. [Non-Patent Document 9] https: / / doi.org / 10.14283 / jpad.2022.30 [Non-Patent Document 10] Dam T, Boxer AL, Golbe LI, et al. Safety and efficacy of anti-tau monoclonal antibody gosuranemab in progressive supranuclear palsy: a phase 2, randomized, placebo-controlled trial. Nat Med. 27:1451-1457, 2021. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention aims to provide a new biomarker for assessing the pathology of neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, or long COVID, and also to provide a method for predicting or assessing the effectiveness of ATP-augmenting therapy in these patients. [Means for solving the problem]

[0006] Previous studies have shown that hypouricemia occurs in Parkinson's disease and Alzheimer's disease, which is thought to reflect reduced ATP consumption due to impaired mitochondrial function. However, because uric acid is a waste product that cannot be reused, we conducted intensive clinical research based on the hypothesis that inosine and hypoxanthine, which are further upstream and can be reused as ATP, also decrease. As a result, we have demonstrated reduced hypoxanthine in serum and cerebrospinal fluid, and reduced inosine in cerebrospinal fluid, in patients with Parkinson's disease and Alzheimer's disease. We discovered that the concentration of inosine in cerebrospinal fluid is approximately 1 / 10 of that in serum, and that there is little transfer of inosine from the blood, making inosine in cerebrospinal fluid a biomarker for mitochondrial dysfunction in the central nervous system. Hypoxanthine in CSF has a similar concentration to that in serum and is a biomarker of systemic mitochondrial dysfunction. Since the lower the serum hypoxanthine level, the more effective ATP augmentation therapy is. Therefore, CSF inosine and hypoxanthine in CSF or serum are good indicators for predicting the efficacy of ATP augmentation therapy. Furthermore, since the primary goal of ATP augmentation therapy is to increase hypoxanthine in the blood, measuring serum hypoxanthine is a good biomarker for confirming the effectiveness of ATP augmentation therapy. Since inosine in the cerebrospinal fluid is an indicator of central nervous system mitochondrial function, it is a good indicator of the improvement of central mitochondrial function by ATP enhancers. Since long COVID is also strongly suspected to be caused by mitochondrial dysfunction, measuring hypoxanthine in serum and cerebrospinal fluid (CSF) is a good biomarker of whole-body mitochondrial dysfunction in long COVID patients, and measuring inosine in CSF is an indicator of central nervous system mitochondrial function. These can be used to predict the effectiveness of ATP-augmentation therapy. Furthermore, these are thought to be good indicators of mitochondrial function improvement in long COVID patients with ATP-augmentation therapy. That is, the present invention has the following configuration. <1> measuring inosine in a patient-derived sample; 1. A method for determining mitochondrial dysfunction in a patient, comprising: The method, wherein the sample is derived from the patient's cerebrospinal fluid. <2> The patient is a patient with a neurodegenerative disease or long COVID. <1> The method described below. <3> The patient with a neurodegenerative disease is a patient with Parkinson's disease or Alzheimer's disease. <2> The method described below. <4> Mitochondrial dysfunction is a mitochondrial dysfunction in the central nervous system. <1> ~ <3> A method according to any one of the preceding claims. <5> measuring inosine in a patient-derived sample A method for predicting or determining the effect of ATP augmentation therapy on a patient, comprising: The method is characterized in that the ATP enhancement therapy comprises a step of administering to a patient an ATP enhancer having the following active ingredients (A) and (B), and the patient-derived sample is a sample derived from the patient's cerebrospinal fluid. (A) Xanthine oxidoreductase (XOR) inhibitors (B) Hypoxanthine or a substance that produces hypoxanthine in the body <6> The patient is a patient with a neurodegenerative disease or long COVID. <5> The method described below. <7> The patient with a neurodegenerative disease is a patient with Parkinson's disease or Alzheimer's disease. <6> The method described below. <8> measuring hypoxanthine in a patient-derived sample 1. A method for determining mitochondrial dysfunction in a patient, comprising: The above method, wherein the patient-derived sample is a serum or plasma-derived sample from the patient. <9> The patient is a patient with a neurodegenerative disease or long COVID. <8> The method described below. <10> The patient with a neurodegenerative disease is a patient with Parkinson's disease or Alzheimer's disease. <9> The method described below. <11> measuring hypoxanthine in a patient-derived sample A method for predicting or determining the effect of ATP augmentation therapy on a patient, comprising: The ATP enhancement therapy is a therapy comprising a step of administering to a patient an ATP enhancer having the following active ingredients (A) and (B), and the patient-derived sample is a serum or plasma-derived sample from the patient. (A) Xanthine oxidoreductase (XOR) inhibitors (B) Hypoxanthine or a substance that produces hypoxanthine in the body <12> The patient is a patient with a neurodegenerative disease or long COVID. <11> The method described below. <13> The neurodegenerative disease patient is a Parkinson's disease patient, an Alzheimer's disease patient, or a long COVID patient. <12> The method described below. <14> measuring hypoxanthine in a patient-derived sample 1. A method for determining mitochondrial dysfunction in a patient, comprising: The above method, wherein the patient-derived sample is a cerebrospinal fluid-derived sample from the patient. <15> The patient is a patient with a neurodegenerative disease or long COVID. <14> The method described below. <16> The neurodegenerative disease patient is a Parkinson's disease patient, an Alzheimer's disease patient, or a long COVID patient. <15> The method described below. <17> measuring hypoxanthine in a patient-derived sample A method for predicting or determining the effect of ATP augmentation therapy on a patient, comprising: The method is characterized in that the ATP enhancement therapy comprises a step of administering to a patient an ATP enhancer having the following active ingredients (A) and (B), and the patient-derived sample is a sample derived from the patient's cerebrospinal fluid. (A) Xanthine oxidoreductase (XOR) inhibitors (B) Hypoxanthine or a substance that produces hypoxanthine in the body <18> The patient is a patient with a neurodegenerative disease or long COVID. <17> The method described below. <19> The neurodegenerative disease patient is a Parkinson's disease patient, an Alzheimer's disease patient, or a long COVID patient. <18> The method described below. [Effects of the Invention]

[0007] Measuring inosine in cerebrospinal fluid samples from patients can determine mitochondrial dysfunction in the central nervous system, providing a new biomarker for determining mitochondrial dysfunction in patients with neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, as well as long COVID-19 patients. Measuring inosine in the cerebrospinal fluid or hypoxanthine in the cerebrospinal fluid or serum of patient samples can determine mitochondrial dysfunction in patients, providing a new biomarker for determining mitochondrial dysfunction in patients with neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease, as well as in patients with long COVID-19. Furthermore, these measurements can be used to predict the therapeutic effect of ATP augmentation therapy beforehand or to assess the effect afterward, allowing for more effective treatment. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows the pathways for ATP production, consumption, and degradation. [Figure 2] FIG. 1 shows the mechanism of brain ATP enhancement by XOR inhibitors and inosine. [Figure 3] FIG. 1 shows the sensitivity, specificity, and AUC for distinguishing between healthy subjects and Parkinson's disease patients using serum hypoxanthine. [Figure 4] FIG. 1 shows the results of comparing the concentrations of inosine and hypoxanthine in the cerebrospinal fluid of healthy subjects and patients with Parkinson's disease. [Figure 5] FIG. 1 shows the sensitivity, specificity, and AUC for distinguishing between healthy subjects and Parkinson's disease patients using inosine in cerebrospinal fluid. [Figure 6] FIG. 1 shows the sensitivity, specificity, and AUC for distinguishing between healthy subjects and Parkinson's disease patients using hypoxanthine in cerebrospinal fluid. DETAILED DESCRIPTION OF THE INVENTION

[0009] (Method for measuring hypoxanthine) Any method can be used to measure hypoxanthine (sometimes called hypoxanthine), as long as it accurately reflects the value in each sample. When measuring blood or blood components, it is important to note that hypoxanthine levels tend to rise rapidly after blood collection. This is because adenine nucleotides (especially ATP) in red blood cells break down to produce hypoxanthine. To avoid this, it is desirable to separate serum quickly. Since separating serum requires waiting for blood clotting to occur in a test tube, it is also possible to measure hypoxanthine in plasma, which can be collected without waiting for blood clotting. Furthermore, since an enzymatic reaction is required to produce hypoxanthine from adenine nucleotides, a method of deproteinization using perchloric acid or the like as soon as possible after blood collection is also preferably used. In this case, whole blood is used as the target, rather than serum or plasma. In the case of whole blood, the hypoxanthine concentration may differ between red blood cells and plasma, so it is preferable to express it in terms of amount rather than concentration. In the measurement of hypoxanthine in the present invention, any of cerebrospinal fluid, serum, plasma, and whole blood can be used as samples.

[0010] An example of a method for measuring hypoxanthine levels in whole blood is described below. Peripheral blood was collected in a test tube containing EDTA-2Na. Immediately after collection, the blood was thoroughly mixed, and 500 μL of blood was mixed with 500 μL of ice-cold 8% PCA. The mixture was then vortexed immediately. The mixture was then centrifuged at 12,000 × g for 5 seconds at 4°C, and the supernatant was collected. 40 μL of 2M K2CO3 in 6M KOH was added to 650 μL of the supernatant to simultaneously precipitate the PCA and neutralize the solution. The mixture was centrifuged at 12,000 × g for 10 minutes at 4°C, and 40 μL of the supernatant was added to 160 μL of mobile phase and subjected to HPLC. The above is an example of a method for measuring hypoxanthine levels in whole blood; other methods that accurately measure hypoxanthine levels may also be used.

[0011] An example of a method for measuring hypoxanthine concentrations in plasma is described below. Peripheral blood is collected in a test tube containing EDTA-2Na and immediately centrifuged at 1,200 × g for 3 minutes at room temperature. 500 μL of the supernatant plasma is mixed with 500 μL of ice-cold 8% PCA, and the mixture is immediately vortexed. Subsequent procedures are the same as those described above for measuring hypoxanthine levels in whole blood. The above is an example of a method for measuring hypoxanthine concentrations in plasma; other methods that accurately measure hypoxanthine concentrations are also acceptable.

[0012] Here is an example of a method for measuring hypoxanthine concentrations in serum. Blood is collected in a blood collection tube, and after clotting, it is centrifuged at 1,200 × g for 3 minutes at room temperature. 500 μL of the supernatant serum is mixed with 500 μL of ice-cold 8% PCA, and the mixture is immediately vortexed. Subsequent procedures are the same as those described above for measuring hypoxanthine levels in whole blood. The above is an example of a method for measuring hypoxanthine concentrations in serum; other methods that accurately measure hypoxanthine concentrations are also acceptable.

[0013] This section describes an example of a method for measuring hypoxanthine concentrations in cerebrospinal fluid. Cerebrospinal fluid is obtained by standard lumbar puncture in a fasting state, transported on ice, and centrifuged at 1500 g for 10 minutes in a centrifuge cooled to 4°C. After 10 minutes of centrifugation at 1500 g, the fluid is aliquoted and stored in a freezer. At the time of measurement, the fluid is thawed, impurities are removed using a column, and then measured using tandem mass spectrometry (MS / MS analysis). The above is an example of a method for measuring hypoxanthine concentrations in cerebrospinal fluid; other methods that accurately measure hypoxanthine concentrations are also acceptable.

[0014] (Method for measuring inosine) This section describes an example of a method for measuring inosine concentrations in cerebrospinal fluid (CSF). CSF samples are collected by standard lumbar puncture in a fasting state, placed on ice, and then centrifuged at 1500 g for 10 minutes in a centrifuge cooled to 4°C. After 10 minutes of centrifugation at 1500 g, the samples are aliquoted and stored in a freezer. The samples are thawed before measurement, and impurities are removed using a column. Measurements are then performed using tandem mass spectrometry (MS / MS). The above is an example of a method for measuring inosine concentrations in CSF; other methods that accurately measure inosine concentrations are also acceptable.

[0015] (patient-derived samples) The sample to be measured for inosine may be a sample derived from a patient's cerebrospinal fluid, and the sample to be measured for hypoxanthine may be a sample derived from a patient's cerebrospinal fluid, plasma, or serum. These samples may be collected from the patient and then diluted with a diluent or pretreated before use.

[0016] (neurodegenerative disease, long COVID) The diseases for which the present invention determines that mitochondrial function has decreased are diseases caused by decreased mitochondrial function, such as neurodegenerative diseases and long COVID. Neurodegenerative diseases are a term used to describe diseases that primarily damage neurons in the human brain. Neurons are cells that are responsible for transmitting information throughout the nervous system, including the brain and spinal cord. In adults, neurons do not regenerate, and even if damaged, it is difficult to restore function. Neurodegenerative diseases are incurable, progressive disorders that result in the progressive degeneration and death of neurons. Examples of neurodegenerative diseases include Parkinson's disease, Parkinsonism, dementia with Lewy bodies, frontotemporal dementia, Alzheimer's disease, amyotrophic lateral sclerosis, multiple sclerosis, and Huntington's disease. Alzheimer's disease is synonymous with Alzheimer's disease, Lewy body dementia is synonymous with diffuse Lewy body disease, and frontotemporal dementia is synonymous with frontotemporal lobar degeneration. Frontotemporal dementia is synonymous with Pick's disease. Parkinson's syndrome includes conditions that exhibit similar symptoms to Parkinson's disease (parkinsonism) but have different causes. In a broader sense, Parkinson's syndrome is a general term for a group of diseases that share the symptoms known as parkinsonism, which includes tremors, muscle rigidity, bradykinesia, and impaired postural reflexes. Furthermore, the neurodegenerative diseases targeted by the present invention are included within the scope of the present invention as long as they involve a pathology of decreased intraneuronal ATP similar to Parkinson's disease.

[0017] Like Parkinson's disease and Alzheimer's disease, long COVID is a disease strongly suspected of being caused by mitochondrial dysfunction. Long COVID is also known as post-COVID-19 condition, post-acute COVID-19, or long-haul COVID. Its Japanese translation includes "post-COVID-19 symptoms," but we will refer to it as "long COVID" throughout this specification. Long COVID refers to a condition in which symptoms persist after the acute phase of COVID-19 caused by the novel coronavirus (SARS-CoV-2) has ended. Reported symptoms of long COVID are diverse, including fatigue, shortness of breath, taste disturbances, joint pain, psychiatric symptoms, sleep disorders, exercise intolerance, persistent low-grade fever, swollen lymph nodes, joint pain, headache, brain fog (a state of impaired thinking and concentration, as if experiencing a "fog" in the head), and a variety of autonomic nervous system symptoms (Non-Patent Document 36). Furthermore, a study using large amounts of insurance data has reported an increase in neurological disorders after the acute phase of COVID-19 (Non-Patent Document 37). Specifically, a massive search of US Medicare data revealed an increase in cases of intracerebral hemorrhage, cerebral ischemia, Parkinson's disease, dementia, anxiety syndromes, and other psychiatric disorders (Non-Patent Document 37). Furthermore, recently, using data from a longitudinal study in the UK Biobank, a comparison of brain images before and after COVID-19 infection reported morphological changes, including a reduction in overall brain size (Non-Patent Document 38). Therefore, there are concerns that the incidence of Parkinson's disease and Alzheimer's disease may increase later among COVID-19 patients (Non-Patent Documents 37, 38).

[0018] [Serum uric acid level as a biomarker of ATP consumption] ATP, which is important for bioenergy, can be produced from glucose in the cytoplasm through glycolysis, but the overwhelming majority of it is produced in mitochondria through oxidative phosphorylation using oxygen. On the other hand, ATP is consumed in many reactions that require energy, such as muscle contraction, and as a result, ATP is converted mainly into ADP and partly into AMP. AMP reacts with ATP via adenylate kinase (AK) to produce two molecules of ADP, which is then converted into ATP and reused in the glycolytic pathway and mitochondria (Figure 1). When ATP is urgently needed, cells can also synthesize ATP and AMP from two ADP molecules using AK (Figure 1). Most of the AMP produced by this reaction is recycled as ATP, but some is converted to IMP by AMP deaminase (AMPD). Most of the IMP is converted back to AMP and recycled, but some is converted to inosine and then to hypoxanthine (Figure 1). Most of the hypoxanthine is converted to IMP by hypoxanthine-guanine phosphoribosyltransferase (HGPRT), and then to AMP and recycled. Some of the hypoxanthine is converted to xanthine and then to uric acid by xanthine oxidoreductase (XOR) (Figure 1). Most of this is excreted from the body by the kidneys, and some by the intestinal tract. Intense muscle exercise, fructose administration, alcohol consumption, and increased brain activity are known to cause rapid ATP consumption, resulting in hyperuricemia. This is called hyperuricemia associated with energy crisis (Non-Patent Document 1). The mechanism is thought to be that when AMP accumulates due to the large consumption of ATP, energy charge decreases, and AMP is broken down by AMPD to prevent this. Energy charge is the amount expressed by the following formula, and indicates the cell's energy supply capacity (Non-Patent Document 2). TIFF0007811359000001.tif1549In other words, when ATP is consumed rapidly, uric acid increases, which is thought to be because the reaction of IMP → inosine → hypoxanthine increases, and some of the hypoxanthine converts to xanthine to produce uric acid (Figure 1). Therefore, when ATP is consumed rapidly, inosine and hypoxanthine increase along with uric acid. Serum uric acid levels are frequently measured in clinical tests, and many reports have been published on the subject. Uric acid has been reported as a biomarker of ATP consumption. Uric acid levels increase with increased ATP consumption and decrease with decreased ATP consumption. Examples of decreased ATP consumption include aging, Alzheimer's disease, Parkinson's disease, and malnutrition, and hypouricemia is observed in these diseases and conditions (Non-Patent Document 3). This is thought to be due to a decline in the number and function of mitochondria with aging (Non-Patent Document 4), which leads to a decrease in both ATP production and consumption. Malnutrition is thought to result in a decrease in ATP production and consumption due to a lack of energy sources such as glucose and fatty acids. Alzheimer's disease and Parkinson's disease occur in elderly patients with particularly severe mitochondrial dysfunction, which is thought to lead to hypouricemia (Non-Patent Documents 5-7). Thus, hypouricemia is thought to be an indicator of mitochondrial dysfunction (Non-Patent Document 3).

[0019] [Potential of inosine and hypoxanthine as biomarkers of ATP consumption] Thus, uric acid is often measured as a biomarker of ATP consumption, and a decrease in uric acid levels is thought to be an indicator of mitochondrial dysfunction. However, its precursors, inosine and hypoxanthine, are rarely measured in clinical settings. However, metabolic maps suggest that uric acid cannot be reused for ATP production, whereas inosine and hypoxanthine can be reused, making them more important for ATP production (Figure 1). Therefore, the inventors focused on measuring the concentrations of inosine and hypoxanthine. Specifically, we investigated the possibility that the concentrations of inosine and hypoxanthine in serum and cerebrospinal fluid could be indicators of mitochondrial dysfunction in Alzheimer's disease and Parkinson's disease.

[0020] [Mitochondrial disorders in Parkinson's disease and Alzheimer's disease] Parkinson's disease and Alzheimer's disease share common characteristics, including the accumulation of pathological proteins (α-synuclein in Parkinson's disease, tau and amyloid-β in Alzheimer's disease) and the associated breakdown of micro- and macro-level circuits (Non-Patent Document 8). However, the underlying causes of why pathological proteins accumulate in specific regions and then spread throughout the brain remain unknown. Furthermore, the accumulation of α-synuclein and amyloid-β does not correlate with clinical symptoms, and the removal of amyloid-β (Non-Patent Document 9) or tau (Non-Patent Document 10) by antibody therapy does not lead to improvement of clinical symptoms, which is problematic. On the other hand, both diseases share common features of widespread mitochondrial functional and morphological abnormalities, as well as impaired glucose and fatty acid metabolism, which are essential for ATP production. Purine nucleotides, which constitute ATP, are produced through two pathways: 1) the de novo synthesis pathway derived from the pentose monophosphate shunt, and 2) the salvage pathway, in which ATP is produced from hypoxanthine via IMP via HGPRT (Figure 1). The de novo pathway is inefficient because it consumes seven ATP molecules to produce one nucleotide molecule. Therefore, the salvage pathway is considered more efficient from an energy-saving perspective (Non-Patent Document 3). Hypoxanthine, which plays a central role in purine nucleotide production in the salvage pathway, crosses the cerebrospinal fluid barrier (Non-Patent Documents 11, 12). Therefore, increasing blood hypoxanthine levels is expected to enhance ATP production in the central nervous system (Figures 1 and 2).

[0021] [ATP augmentation therapy and hypoxanthine as a marker] The present inventors have previously conceived of an ATP-enhancing therapy that involves co-administering an XOR inhibitor, such as febuxostat, which inhibits the breakdown of hypoxanthine into xanthine and uric acid, with inosine, a precursor of hypoxanthine (Figure 1). Administration of this therapy to humans demonstrated an average 7.7-fold increase in blood hypoxanthine levels and an increase in erythrocyte ATP (Non-Patent Document 13). Administration of febuxostat alone also resulted in a slight increase in hypoxanthine (average 1.2-fold) (Non-Patent Document 13). Based on these results, the present inventors conducted a clinical study of two patients with mitochondrial disease and observed significant improvements in biomarkers (Non-Patent Document 14). Specifically, patients with mitochondrial cardiomyopathy showed a significant increase in BNP, an indicator of heart failure, due to mitochondrial dysfunction in the myocardium, but this improved with treatment. In patients with mitochondrial diabetes, mitochondrial dysfunction in the beta cells of the pancreatic islets of Langerhans caused insulin secretion dysfunction, but treatment increased insulin secretion and reduced blood glucose levels, resulting in a significant improvement in the insulin index. Furthermore, Watanabe et al. conducted a clinical trial in which 30 patients with Parkinson's disease were administered ATP augmentation therapy for two months, and confirmed a significant increase in plasma hypoxanthine and clinically significant improvement in motor symptoms in 26 evaluable cases (Non-Patent Document 15). Furthermore, they found that the treatment effect was more pronounced in patients with lower hypoxanthine levels before treatment (Non-Patent Document 16). Febuxostat does not cross the blood-brain barrier, and the target enzyme of febuxostat, XOR, is not expressed in the brain (Non-Patent Document 3). Therefore, febuxostat does not act on the brain, but rather acts on XOR in the liver to increase hypoxanthine, which then migrates to the blood and crosses the blood-brain barrier, increasing brain hypoxanthine and further increasing brain ATP (Figure 2) (Non-Patent Document 3). When febuxostat is administered in combination with inosine, blood hypoxanthine increases more than febuxostat, further increasing brain ATP (Figure 2) (Non-Patent Document 13). In other words, the essential mechanism of ATP-potentiating therapy is to increase blood hypoxanthine, and blood hypoxanthine concentration is an important marker (Non-Patent Document 3). Recently, multiple extensive epidemiological studies have revealed that febuxostat suppresses Alzheimer's disease. Specifically, extensive health insurance data from the United States (Non-Patent Document 17) and South Korea (Non-Patent Document 18) revealed that patients taking febuxostat had a lower incidence of dementia. Furthermore, AI research in the United States identified febuxostat as a drug for improving Alzheimer's disease, and extensive epidemiological studies have proven that patients taking febuxostat are less likely to develop Alzheimer's disease (Non-Patent Document 19). These studies support the view that inhibiting XOR and increasing hypoxanthine suppress the onset of Alzheimer's disease. From a series of studies, we came up with the idea that hypoxanthine and its precursor, inosine, may be reduced in the central nervous system in Parkinson's disease and Alzheimer's disease, which show mitochondrial dysfunction and reduced ATP production from the early stages of the disease. To prove this hypothesis, we first measured inosine concentrations in cerebrospinal fluid and blood in Parkinson's disease patients, and hypoxanthine concentrations in cerebrospinal fluid and blood. We also performed similar measurements in Alzheimer's disease patients.

[0022] (ATP augmentation therapy) The ATP enhancement therapy of the present invention may be any therapy that can enhance ATP in the cells of a patient, and ATP enhancement refers to a therapy that increases or suppresses the decrease of ATP in the cells present in the patient's body. ATP enhancement therapy includes a treatment method comprising the step of administering to a patient an ATP enhancer containing the following (A) and / or (B) as an active ingredient: (A) Xanthine oxidoreductase inhibitor (B) Hypoxanthine or a compound that can be converted to hypoxanthine in the body Examples of xanthine oxidoreductase inhibitors (A), which are active ingredients of ATP enhancers, include febuxostat (trade name Feburic (Teijin Pharma)), topiroxostat (trade name Uriadec (Sanwa Chemical Research Institute) and Topirolic (Fuji Yakuhin)), and allopurinol (trade name Zyloric (GlaxoSmithKline)). Pharmaceutically acceptable salts of these compounds are also included in the active ingredient (A) of the present invention. The other active ingredient (B) of the ATP enhancer may be one or more compounds selected from inosine, inosinic acid, hypoxanthine, adenosine, AMP, ADP, ATP, succinyladenosine, S-adenosylhomocysteine, S-adenosylmethionine, and pharmaceutically acceptable salts thereof, with inosine being preferred. Of these, the ATP enhancer of the present invention is preferably an ATP enhancer comprising a combination of the above (A) and (B).

[0023] (Combined use) In the ATP augmentation therapy of the present invention, "combined use of (A) and (B)" means that both component (A) and component (B) are administered, and the ATP augmenting agent "comprising a combination of (A) and (B)" is used to mean all drugs that are administered in combination with (A) and (B). Therefore, it includes both a combination drug (combined drug) in which component (A) and component (B) are mixed to form a composition, and drugs that are physically separate without being mixed but are administered together so that they can be administered at the same time. Examples of combination drugs (compound drugs) include those that have been mixed and formulated, such as granules, powders, solids, and liquids for oral administration, inhalants, etc. Examples of drugs that are physically separate but are packaged together so that they can be administered at the same time include so-called kits and drugs packaged together in a single bag. "Simultaneous administration" does not necessarily mean "at the same time" in the strict sense, and also includes cases where there is an interval between administrations as long as the effect is maintained. For example, taking one before a meal and the other after a meal corresponds to "administration at the same time" in the present invention.

[0024] (Dosage and administration method) The dosage of the ATP enhancer used in the ATP potentiation therapy of the present invention may be an effective amount, and the following dosages are desirable for each: (A) febuxostat is desirable at 10 to 80 mg / day, topiroxostat at 40 to 160 mg / day, and allopurinol at about 50 to about 800 mg / day. (B) Inosine is desirable at 0.5 to 4.0 g / day. The above-mentioned dosages can be administered once a day or in divided doses of two or more times a day. Among these, febuxostat is preferably administered twice a day, rather than once a day as in conventional febuxostat administration. In addition, inosine is preferably administered twice a day rather than once a day. Therefore, it is more desirable to administer both inosine and febuxostat twice a day. When combined, the daily dose and administration method can be adjusted accordingly, and febuxostat and inosine are preferably combined in a dosage of 20 mg or 40 mg of febuxostat with 0.5 g, 1 g, 1.5 g, or 2 g of inosine. 20 mg of febuxostat and 0.5 g of inosine per tablet are even more preferable. [Example]

[0025] [Example 1] 1. Test Method 1-1. Target (1) Parkinson's disease (PD) patients The study included 45 PD patients (29 men, 16 women, age at time of examination 68.6 ± 7.4 years, mean disease duration 88.5 ± 54.0 months) who met the criteria for probable Parkinson's disease according to the Movement Disorder Society Criteria 2015 (Mov Disord. 2015;30:1591). Serum samples were taken from 30 age- and sex-matched healthy individuals (16 men, 14 women, age at time of examination: 68.9 ± 9.1 years), and cerebrospinal fluid samples were taken from 20 age-matched individuals with no history of neuromuscular disease (16 men, 4 women, age at time of examination: 68.15 ± 8.9 years). (2) Alzheimer's disease (AD) patients A similar study was performed on the serum and cerebrospinal fluid of one patient who complained of memory impairment, had a CDR of 1.0, and objective decline in logical memory, fulfilled the diagnostic criteria of the Neurologic and Communicative Disorders and Stroke and the Alzheimer Disease Related Disorders Association (NINCDS-ADRDA) (Alzheimers Dement. 2011;7(3):263), and was diagnosed with Alzheimer's disease based on MRI and cerebral blood flow tests.

[0026] (3) Sample preparation Blood samples were collected early in the morning on an empty stomach (at least 6 hours after the last meal). Serum was collected into a tube containing serum separator and coagulation promoter that had been chilled on ice, mixed by inverting 8-10 times, then placed on ice and removed. Serum was centrifuged at 1500g for 10 minutes in a centrifuge cooled to 4°C, then dispensed into aliquots, and stored in a -80°C freezer. Plasma was collected into a tube containing EDTA-2Na that had been chilled on ice, mixed by inverting 8 to 10 times, then placed on ice and removed. Plasma was centrifuged at 1500g for 10 minutes in a centrifuge cooled to 4°C, then dispensed into aliquots, and stored in a -80°C freezer. CSF was collected by standard lumbar puncture under fasting conditions. If there were signs of bloody CSF at the time of collection, the first 1-2 ml was discarded and the subsequent collected sample was used for analysis. The first approximately 2 ml was collected in a sterile syringe and checked for protein, glucose, and cell count. The subsequent 6-7 ml was collected directly into a 15 ml Asian Instruments PP screw syringe. The syringe was placed on ice and transferred without inversion. After centrifugation at 1500 g for 10 minutes in a 4°C centrifuge, the collected fluid was aliquoted and stored in a -80°C freezer.

[0027] (4) Measurement method for inosine and hypoxanthine Inosine and hypoxanthine, used as standards, were obtained from Fujifilm Wako (Osaka, Japan). After separation using a Nexera UHPLC system (Shimadzu, Kyoto, Japan) and an Intrada Organic Acid column (150 x 2 mm, Imtakt, Kyoto, Japan) at 60 °C, tandem mass spectrometry (MS / MS) was performed using an LCMS-8060 or LCMS-8060NX triple quadrupole mass spectrometer (Shimadzu) in positive ion MRM mode. The mass transitions were hypoxanthine m / z 137 > 119 and inosine m / z 269 > 137. Because hypoxanthine levels are expected to increase with increasing time between collection and storage due to the breakdown of ATP in red blood cells, we first compared hypoxanthine levels in whole blood samples from three healthy volunteers collected and stored for 1 hour and two hours, respectively. Results showed increases of 1.28-fold, 1.31-fold, and 2.06-fold, respectively. Therefore, for all subjects, blood was stored in a freezer controlled at -80°C for 60-70 minutes. Furthermore, the correlation between serum and plasma hypoxanthine levels was examined in eight subjects, and the correlation coefficient was found to be extremely high at 0.973 (p < 0.0001). Therefore, we performed the analysis using serum samples. Statistical analysis was performed using JMP 16.

[0028] 2. Test Results 2-1. Parkinson's disease patients (1) Serum inosine Serum inosine was 4.63±5.62 μmol / L in Parkinson's disease patients and 6.22±10.09 μmol / L in healthy subjects, with no significant difference between the two groups (Table 1). (2) Serum hypoxanthine Serum hypoxanthine levels were significantly lower in Parkinson's disease patients (2.28±1.19 μmol / L) and healthy controls (6.72±6.73 μmol / L) (p<0.0001) (Table 1). The receiver operating characteristic curve (ROC) showed an AUC (area under the curve) of 0.78, and a cutoff of 2.612 μmol / L enabled differentiation of Parkinson's disease patients from healthy controls with a sensitivity of 75.56% and a specificity of 73.33% (Figure 3). (3) Inosine in cerebrospinal fluid CSF inosine levels were significantly lower in Parkinson's disease patients (0.50 ± 0.09 μmol / L) than in healthy controls (0.64 ± 0.14 μmol / L) (p < 0.0001) (Figure 4; the horizontal line indicates the overall mean for both healthy controls and Parkinson's disease patients). The receiver operating characteristic curve (ROC) showed an area under the curve (AUC) of 0.835, and a cutoff of 0.579 μmol / L resulted in a sensitivity of 80.0% and a specificity of 80.0%, enabling differentiation of Parkinson's disease patients from healthy controls (Figure 5). The inosine concentration in CSF was approximately 1 / 10 that of serum (Table 1: inosine in serum, 4.63 ± 5.62 μmol / L; Figure 4: inosine in CSF, 0.50 ± 0.09 μmol / L). No correlation was observed between CSF and serum levels (not shown). (4) Hypoxanthine in cerebrospinal fluid Hypoxanthine levels in cerebrospinal fluid were significantly lower in Parkinson's disease patients (5.04±1.70 μmol / L) than in healthy controls (5.93±1.61 μmol / L) (p = 0.0299) (Figure 4). The receiver operating characteristic curve (ROC) showed an AUC of 0.67, and a cutoff of 4.972 μmol / L enabled differentiation of Parkinson's disease patients from healthy controls with a sensitivity of 60.0% and a specificity of 80.0% (Figure 6). (5) Relationship with clinical scores In examining the relationship with clinical scores in Parkinson's disease, no items showed significant correlations with serum inosine and serum hypoxanthine (Table 2). (6) Xanthine, uric acid Serum uric acid was significantly decreased in Parkinson's disease patients (273.10±75.49 μmol / L) compared with healthy controls (399.31±114.52 μmol / L) (p<0.0001) (Table 1). Serum xanthine was also decreased in Parkinson's disease patients (1.88±1.05 μmol / L) compared with healthy controls (3.62±4.79 μmol / L), but the difference was not significant (Table 1).

[0029] 2-2. Alzheimer's disease patients In patients with Alzheimer's disease, inosine in the cerebrospinal fluid was 0.55 μmol / L, and hypoxanthine in the serum was 0.64 μmol / L, both of which were lower than those in the healthy controls. Therefore, it was found that measuring inosine in the cerebrospinal fluid or hypoxanthine in the serum of patients with Alzheimer's disease can distinguish between patients with Alzheimer's disease and healthy controls.

[0030] [Table 1]

[0031] [Table 2]

[0032] 3. Discussion (1) In patients with Parkinson's disease, inosine levels in cerebrospinal fluid were significantly lower than in healthy controls, but serum inosine levels were not. There have been no reports comparing the dynamics of inosine in cerebrospinal fluid and blood. The present invention revealed, for the first time, that inosine concentrations in cerebrospinal fluid (CSF) were extremely low, approximately one-tenth of those in serum, in both Parkinson's disease patients and healthy controls, and that there was no correlation between the concentrations of inosine in cerebrospinal fluid and serum. This finding indicates that inosine in CSF is not affected by inosine in blood and is primarily derived from nucleotides in the central nervous system. Parkinson's disease is generally known to be associated with mitochondrial dysfunction, glucose metabolism disorders, and fatty acid metabolism disorders, and PET (Positron Emission Tomography) studies also reveal decreased brain glucose metabolism (Non-Patent Document 3). The present invention confirms that decreased inosine levels in CSF in patients with Parkinson's disease reflect decreased central mitochondrial function and decreased energy metabolism. Therefore, inosine in the cerebrospinal fluid of Parkinson's disease patients can be a biomarker for decreased mitochondrial function and decreased energy metabolism in the central nervous system of Parkinson's disease patients, and measuring these concentrations makes it possible to diagnose and determine decreased mitochondrial function and decreased energy metabolism in the patient.

[0033] (2) Mitochondrial dysfunction is a common cause of energy metabolism disorders in neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. However, there are currently no biomarkers that reflect mitochondrial dysfunction in the central nervous system. 18 The only other finding was a decrease in glucose uptake by F-FDG-PET. Since cerebrospinal fluid inosine levels are also decreased in Alzheimer's disease, they are thought to be a good indicator of mitochondrial dysfunction in the central nervous system and the resulting decrease in energy (ATP). Interestingly, cerebrospinal fluid inosine levels did not show a strong correlation with clinical indicators of Parkinson's disease, and because they were decreased even in early and mild cases, they were thought to be a consistent feature from the early stages of the disease. Therefore, by measuring the inosine concentration in cerebrospinal fluid in the early stages of Parkinson's disease or Alzheimer's disease, it is possible to determine the decline in mitochondrial function, leading to early treatment.

[0034] (3) Serum and cerebrospinal fluid hypoxanthine levels were significantly lower in Parkinson's disease patients than in healthy individuals, with the decrease being more pronounced in serum. Furthermore, there was no significant difference in serum and cerebrospinal fluid hypoxanthine concentrations in healthy individuals. This suggests that hypoxanthine easily crosses the blood-brain barrier (Non-Patent Documents 11 and 12). Cybrid studies have also shown that patients with Parkinson's disease and Alzheimer's disease exhibit reduced mitochondrial function in platelets (Non-Patent Documents 20 and 21). Therefore, decreased serum hypoxanthine may reflect decreased peripheral mitochondrial function, and because hypoxanthine crosses the blood-brain barrier, hypoxanthine in cerebrospinal fluid may also reflect decreased peripheral mitochondrial function. On the other hand, according to the present invention, the level of inosine in cerebrospinal fluid was approximately 1 / 10 of that in serum, suggesting that inosine does not cross the blood-brain barrier and that inosine in cerebrospinal fluid reflects decreased mitochondrial function in the central nervous system. From the above, it was found that measuring inosine in cerebrospinal fluid can determine mitochondrial dysfunction, particularly mitochondrial dysfunction in the central nervous system, and can provide a very useful biomarker for Parkinson's disease patients and Alzheimer's disease patients suffering from mitochondrial dysfunction in the central nervous system. Furthermore, it was found that the measurement of hypoxanthine in serum or cerebrospinal fluid can be used to assess mitochondrial dysfunction, providing a highly useful biomarker for Parkinson's disease and Alzheimer's disease patients suffering from mitochondrial dysfunction.

[0035] 2. Methods for predicting or assessing the effectiveness of ATP augmentation therapy In recent years, evidence has been accumulating that abnormal energy metabolism plays an important role in the pathogenesis and progression of Parkinson's disease and Alzheimer's disease. iPSC-derived cells established from Parkinson's disease and Alzheimer's disease patients commonly exhibit widespread mitochondrial dysfunction (Non-Patent Document 26). Furthermore, PET scans of both diseases reveal impaired glucose metabolism prior to the onset of brain atrophy (Non-Patent Documents 27, 28). A decrease in ATP can explain not only the pathological conditions such as the accumulation of pathological proteins associated with abnormalities in the proteasome system, and the increased production of reactive oxygen species and neuroinflammation, but also the site-specific lesions observed in both diseases. Neuronal axons in the substantia nigra compacta, one of the earliest lesions in Parkinson's disease, are highly branched (Non-Patent Document 29) and exhibit rhythmic firing (Non-Patent Document 30), which are closely related to vulnerability to mitochondrial dysfunction (Non-Patent Document 31). On the other hand, in Alzheimer's disease, the earliest lesions occur in the entorhinal cortex, which is composed of a complex network of excitatory-inhibitory-excitatory neurons (Non-Patent Document 32) and exhibits rhythmic firing (Non-Patent Document 33). In particular, layer 2 neurons, where Alzheimer's disease lesions are most likely to occur, are Reelin-positive and require high levels of energy (Non-Patent Document 34), making them vulnerable to mitochondrial dysfunction and ATP depletion. Regarding the spread of lesions at the macro level, Watanabe used MRI to analyze large-scale brain networks and showed that brain hub regions, which integrate multiple brain regions, function as a compensatory mechanism in healthy aging, but become a common site of lesions in Parkinson's disease and Alzheimer's disease (Non-Patent Document 35). These findings suggest that when an insufficient supply of ATP is achieved due to age-related decline in mitochondrial function, abnormal glucose metabolism, or abnormal lipid metabolism, increased reactive oxygen species, neuroinflammation, and accumulation of pathological proteins associated with proteasome dysfunction may occur in areas with high demand for ATP, such as the substantia nigra and entorhinal cortex, which are rich in synapses and consume 64% of the brain's energy, and in brain hubs that show high activity in glucose metabolism PET. These may lead to neurodegeneration. Therefore, our findings, particularly the decrease in inosine in CSF, can be a simple indicator of central nervous system energy decline (mitochondrial dysfunction) and a biomarker for evaluating the effectiveness of ATP-enhancing therapy. In other words, by measuring inosine in patients' CSF samples, we can predict the effectiveness of ATP-enhancing therapy in patients suffering from central nervous system mitochondrial dysfunction and determine the effectiveness of ATP-enhancing therapy. In particular, it is possible to predict in advance the effectiveness of ATP augmentation therapy for Parkinson's disease patients and Alzheimer's disease patients who suffer from decreased mitochondrial function in the central nervous system, and also to determine the effectiveness of ATP augmentation therapy. Similarly, by measuring hypoxanthine in a patient's serum or cerebrospinal fluid sample, it is possible to predict the effectiveness of ATP-augmenting therapy in patients suffering from mitochondrial dysfunction and to determine the effectiveness of ATP-augmenting therapy. In particular, it is possible to predict in advance the effectiveness of ATP augmentation therapy for Parkinson's disease patients and Alzheimer's disease patients suffering from mitochondrial dysfunction, and also to determine the effectiveness of ATP augmentation therapy.

[0036] [Example 2] Long COVID and mitochondrial disorders 1. Determining mitochondrial dysfunction One recent study that has attracted attention regarding the mechanism of damage in COVID-19 is mitochondrial damage caused by SARS-CoV-2. It has been revealed that during the acute phase of COVID-19, the virus invades mitochondria and replicates its own RNA there (Non-Patent Documents 44, 45). Furthermore, it has been reported that the virus hijacks or kidnaps mitochondria, using them for its own maintenance and proliferation, causing mitochondrial dysfunction such as reduced energy supply (Non-Patent Documents 46-51). It has also been suggested that such mitochondrial dysfunction occurring during the acute phase may be delayed, leading to long COVID (Non-Patent Documents 52, 53). Indeed, the inflammation, immune abnormalities, cytokine storm, and even hypercoagulation and release of reactive oxygen species proposed as mechanisms of long COVID have all been reported to occur as a result of mitochondrial damage, which is completely consistent with this theory (Non-Patent Documents 46, 51, 54-59). Further supporting the involvement of mitochondrial dysfunction in Long COVID, a PET study showed that mitochondrial dysfunction in the frontal-parietal lobes of 29 COVID-19 patients who had passed the acute phase was associated with 18 Decreased F-FDG (fluorodeoxyglucose) uptake was found in the brain (Non-Patent Document 60). Another PET study showed decreased glucose metabolism in the bilateral orbitorectal gyrus and the right medial temporal lobe (Non-Patent Document 61). Because glucose is primarily used by mitochondria to produce ATP, the decreased glucose uptake likely reflects decreased ATP production due to mitochondrial dysfunction, similar to that observed in the brains of patients with Alzheimer's disease (Non-Patent Document 62). Based on the above, it is highly likely that mitochondrial dysfunction is present in long COVID patients, and measurement of hypoxanthine and inosine is considered useful as biomarkers, similar to Parkinson's disease and Alzheimer's disease shown in Example 1. In particular, measurement of inosine in cerebrospinal fluid is considered useful as an indicator of mitochondrial dysfunction in the central nervous system in long COVID patients.

[0037] 2. Method for predicting or assessing the effect of ATP augmentation therapy As mentioned above, there is ample evidence that ATP-enhancing therapy using the co-administration of febuxostat and inosine improves central nervous system energy decline and compensates for mitochondrial dysfunction. Furthermore, Mitani et al. of Tokyo Women's Medical University discovered that adding febuxostat to nematodes exerts mitochondrial protective effects and extends lifespan. They have filed a patent application for a mitochondrial protector containing the co-administration or combination of febuxostat and inosine as active ingredients (Patent Document 2). COVID-19 (coronavirus) infection is listed as a target disease for this mitochondrial protector. As mentioned above, ATP augmentation therapy for Parkinson's disease patients was more effective the lower their pretreatment serum hypoxanthine concentrations. This is thought to be because ATP augmentation therapy is more effective in Parkinson's disease patients, who are more susceptible to mitochondrial dysfunction. Therefore, hypoxanthine in serum and cerebrospinal fluid (CSF) may also be useful for predicting the effectiveness of ATP augmentation therapy in long COVID patients. Furthermore, because inosine in CSF reflects mitochondrial function in the central nervous system, it may also be useful for predicting the effectiveness of ATP augmentation therapy in advance. Furthermore, the primary goal of ATP augmentation therapy was to increase hypoxanthine in the blood, followed by increasing hypoxanthine in the cerebrospinal fluid (Figure 2). Therefore, measuring hypoxanthine in serum and CSF may be effective for post-mortem confirmation of the effectiveness of ATP-augmented therapy for long COVID. Furthermore, because inosine in CSF reflects mitochondrial function in the central nervous system, measuring inosine in CSF may be effective for post-mortem confirmation of the effects of ATP-augmented therapy on the central nervous system.

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[0039] According to the present invention, by measuring inosine in the cerebrospinal fluid of patients with neurodegenerative diseases or long COVID-19, it is possible to determine the decline in mitochondrial function in the central nervous system. This can be useful for early treatment. Furthermore, the effectiveness of ATP augmentation therapy can be predicted in advance or assessed after the fact, allowing for more effective treatment of patients.

Claims

1. measuring hypoxanthine in a patient-derived sample A method for providing an index for determining mitochondrial dysfunction in a patient based on the measurement of hypoxanthine, comprising: The above method, wherein the patient-derived sample is a serum or plasma-derived sample from the patient.

2. The method of claim 1, wherein the patient is a patient with a neurodegenerative disease.

3. The method according to claim 2, wherein the patient with a neurodegenerative disease is a patient with Parkinson's disease or Alzheimer's disease.

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