Adenosine triphosphate scavenger and adenosine diphosphate scavenger

Abstract

To provide a component useful as an ATP scavenger and / or an ADP scavenger.SOLUTION: Transferrin is useful as an adenosine triphosphate scavenger, and butyrylcholinesterase is effective as an adenosine diphosphate scavenger.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to adenosine triphosphate sequestrants and adenosine diphosphate sequestrants. [Background technology]

[0002] In allogeneic hematopoietic stem cell transplantation, complications such as graft-versus-host disease (GVHD) and hepatic veno-occlusive disease / sinusoidal obstruction syndrome (VOD / SOS), as well as infections, greatly affect the treatment outcome. GVHD is prevented by using immunosuppressants, but it develops in approximately half of treated patients. When GVHD becomes severe and resistant to treatment, large amounts of steroid hormones or thymoglobulin, or in recent years, mesenchymal stem cells or JAK2 inhibitors, are used, and treatment outcomes have gradually improved, but there are still some fatal cases. In addition, VOD / SOS is a rare condition, but its cause is unknown, and once it develops, the prognosis is poor.

[0003] It has been reported that heparan sulfate, which is decomposed and released during tissue injury, activates dendritic cells via the Toll-like receptor TLR4, causing GVHD in mice, and that α-1 antitrypsin reduces HS and alleviates GVHD (Non-Patent Document 1). Clinical trials of α-1 antitrypsin for preventing GVHD have progressed to phase II clinical trials (Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Todd V Brennan, Liwen Lin, Xiaopei Huang, Diana M Cardona, Zhiguo Li, Keith Dredge, Nelson J Chao, Yiping Yang, Heparan sulfate, an endogenous TLR4 agonist, promotes acute GVHD after allogeneic stem cell transplantation, Blood. 2012 Oct 4;120(14):2899-908. [Non-Patent Document 2] The Safety and Efficacy of Alpha-1 Antitrypsin (AAT) for the Prevention of Graft-Versus-Host Disease (GVHD) in Patients Receiving Hematopoietic Cell Transplant (MODULAATE), [online], April 24, 2023, Good Clinical Practice Network [searched June 13, 2023], Internet<URL:https: / / ichgcp.net / clinical-trials-registry / NCT03805789> Summary of the Invention [Problem to be solved by the invention]

[0005] As mentioned above, clinical trials of alpha-1 antitrypsin are underway, but there is still insufficient information available about its effectiveness, so other options for GVHD prophylaxis are desirable.

[0006] Here, if GVHD becomes severe, it can cause thrombotic microangiopathy (TMA) throughout the body, as in VOD / SOS. In TMA, pulsated red blood cells are observed, suggesting that platelet aggregation occurs in the microvessels. ADP is released from the aggregated platelets, activating other platelets and promoting further aggregation. ATP is also released at the same time as ADP, and the released ATP competes with ADP at the receptors on the platelet surface, and may be converted to ADP, stimulating aggregation. It has also been reported that extracellular ATP affects the immune system via the P2X7 receptor in GVHD, contributing to the aggravation of the disease. Furthermore, ATP is suspected to be involved in the TMA state in VOD / SOS as well.

[0007] As such, it is believed that extracellular ATP and ADP are involved in GHVD, VOD / SOS, and TMA, and therefore it is desirable to develop an active pharmaceutical ingredient that can control the amount of extracellular ATP and ADP as an effective pharmaceutical ingredient for preventing or ameliorating these pathologies.

[0008] Therefore, an object of the present invention is to provide a component useful as an ATP scavenger and / or an ADP scavenger. [Means for solving the problem]

[0009] As a result of intensive research, the present inventors have newly found that transferrin is useful as a scavenger of adenosine triphosphate, and butyrylcholinesterase is effective as a scavenger of adenosine diphosphate. Based on these findings, the present invention has been completed through further research. That is, the present invention provides the following aspects of the invention.

[0010] Item 1. Adenosine triphosphate scavengers, including transferrin. Item 2. The scavenger according to Item 1, which is used as an agent for preventing or improving adenosine triphosphate excess. Item 3. The capture agent according to Item 2, wherein the adenosine triphosphate excess is a complication of allogeneic hematopoietic stem cell transplantation and / or a pre-transplant treatment for allogeneic hematopoietic stem cell transplantation. Item 4. The capture agent according to Item 3, wherein the complication is selected from the group consisting of graft-versus-host disease, hepatic veno-occlusive disease / sinusoidal obstruction syndrome, and thrombotic microangiopathy. Item 5. The scavenger according to Item 3 or 4, which is applied to patients with iron overload. Item 6. The capture agent according to Item 2, wherein the adenosine triphosphate excess disorder is tumor lysis syndrome. Item 7. The capture agent according to Item 1, which is supported on an adsorbent for blood purification treatment of patients with adenosine triphosphate excess. Item 8. The capture agent according to Item 1, which is used as a screening agent for compounds that bind to adenosine triphosphate. Item 9. A scavenger of adenosine diphosphate, including butyrylcholinesterase. Item 10. The capture agent according to Item 9, which is used as a preventive or therapeutic agent for a condition requiring inhibition of adenosine diphosphate. Item 11. The capturing agent according to Item 10, wherein the symptom is a complication of allogeneic hematopoietic stem cell transplantation. Item 12. The capture agent according to Item 11, wherein the complication is selected from the group consisting of graft-versus-host disease, hepatic veno-occlusive disease / sinusoidal obstruction syndrome, and thrombotic microangiopathy. Item 13. The capture agent according to Item 10, which is used as a screening agent for compounds that bind to adenosine diphosphate. Item 14. A method for testing a condition requiring inhibition of adenosine diphosphate, comprising the steps of: (i) measuring butyrylcholinesterase in a sample collected from a test animal; and (ii) detecting the condition based on the result of step (i); 4. A method for testing comprising: Item 15. The testing method according to Item 14, wherein the step (ii) is carried out by comparing the result of step (i) obtained for the test animal with the result of step (i) obtained for a normal control, and using a decrease in adenosine diphosphate as an indicator. Item 16. A diagnostic kit for a condition requiring inhibition of adenosine diphosphate, comprising a reagent capable of detecting butyrylcholinesterase. Effect of the Invention

[0011] In accordance with the present invention, there are provided moieties useful as ATP scavengers and / or ADP scavengers. [Brief description of the drawings]

[0012] [Figure 1] FIG. 2 shows a computer simulation diagram illustrating the binding mode between transferrin and adenosine triphosphate. [Diagram 2] FIG. 1 shows a computer simulation diagram illustrating the binding mode between butyrylcholinesterase and adenosine diphosphate. [Figure 3A] This result indicates that Apo-TF (transferrin) reduced the ATP concentration in a concentration-dependent manner, that is, that Apo-TF captured ATP. [Figure 3B] The results show that holo-TF (transferrin) did not capture ATP. [Figure 3C] The results show that Apo-TF (transferrin) captured ATP but not ADP. [Figure 4] These results show that BChE (butyrylcholinesterase) captures ADP. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] [1. Adenosine triphosphate scavengers] The adenosine triphosphate capture agent of the present invention is characterized in that it contains transferrin and is used to capture adenosine triphosphate.

[0014] [1-1. Transferrin] Transferrin is a protein contained in plasma that binds and transports iron ions. Transferrin is also called plasma transferrin, serotransferrin, and siderophilin.

[0015] The transferrin used in the present invention as a scavenger of adenosine triphosphate is in the apo form.

[0016] Specific examples of transferrin include transferrin derived from vertebrates, preferably transferrin derived from mammals, and more specifically, transferrin derived from primates such as humans and monkeys. These transferrins may be used alone or in combination. Among these transferrins, transferrin derived from primates is preferred, and transferrin derived from humans is more preferred.

[0017] Transferrin may be purified from the above-mentioned vertebrate transferrin source, or may be produced using genetic recombination technology. Transferrin sources include blood, kidney, intestine, liver, lung, heart, and central nervous system. Methods for producing transferrin using genetic recombination technology include a method for expressing the desired transferrin in host cells such as Escherichia coli, yeast, insect cells, plant cells, and animal cells; and a method for producing a genetically modified vertebrate and purifying the desired transferrin from the transferrin source of the genetically modified vertebrate.

[0018] FIG. 1 shows a computer simulation diagram illustrating the binding mode between transferrin and adenosine triphosphate.

[0019] [1-2. Other ingredients] In addition to the above-mentioned transferrin, the adenosine triphosphate capture agent of the present invention may contain physiologically or pharma- ceutically acceptable bases and / or additives, etc., as necessary (for example, depending on the use of the adenosine triphosphate capture agent). Examples of such bases and additives include excipients, binders, disintegrants, lubricants, isotonicity agents, plasticizers, dispersants, emulsifiers, solubilizers, wetting agents, stabilizers, suspending agents, adhesives, coating agents, glossing agents, water, oils and fats, ester oils, waxes, hydrocarbon bases, fatty acids, lower alcohols, higher alcohols, water-soluble polymers, surfactants, metal soaps, polyhydric alcohols, pH adjusters, buffers, antioxidants, UV protection agents, preservatives, flavorings, fragrances, powders, dyes, chelating agents, etc. These bases and additives may be used alone or in combination of two or more. The content of these bases and additives may be appropriately set depending on the type and / or dosage form of the added components used.

[0020] [1-3. Dosage form] The dosage form of the adenosine triphosphate capture agent of the present invention is not particularly limited and can be appropriately set according to the application. For example, the dosage form includes liquid, powder, granule, tablet, and the like, and the form supported on an insoluble carrier. The above-mentioned dosage form can be prepared using a physiologically or pharma- ceutical acceptable base and / or additive, and the type and amount of the base and additive can be appropriately set by a person skilled in the art. The above-mentioned form supported on an insoluble carrier can be prepared using a physiologically acceptable insoluble carrier (e.g., an adsorbent for blood purification treatment, etc.), and the type and method of the insoluble carrier can be appropriately set by a person skilled in the art.

[0021] [1-4.Application] The adenosine triphosphate capture agent of the present invention can be used for any application that utilizes the binding between transferrin and adenosine triphosphate.

[0022] When the adenosine triphosphate capture agent of the present invention is used, the pH (25° C.) condition is, for example, 5 or more, preferably 5.5 or more, 5.6 or more, 5.7 or more, 5.8 or more, or 5.9 or more. The upper limit of the pH condition is, for example, 8 or less, preferably 7.5 or less, more preferably 7 or less, 6.5 or less, 6.3 or less, or 6.2 or less.

[0023] Specific uses of the adenosine triphosphate capture agent of the present invention include uses as an adsorbent for blood purification treatment of patients with adenosine triphosphate excess, an agent for preventing or improving adenosine triphosphate excess, and a screening agent for compounds that bind to adenosine triphosphate.

[0024] [1-4-1. Adenosine triphosphate excess] Adenosine triphosphate excess refers to a pathological condition caused by a symptom involving excess adenosine triphosphate (e.g., inflammation, cell damage, and / or vascular endothelial damage, etc.), and specifically includes complications of allogeneic hematopoietic stem cell transplantation, pre-transplant treatment of allogeneic hematopoietic stem cell transplantation, tumor lysis syndrome, crush syndrome, traumatic shock, septic shock, myocardial infarction, muscular dystrophy, collagen disease, cancer, COVID-19 (coronavirus disease that occurred in 2019), etc. Complications of allogeneic hematopoietic stem cell transplantation include graft-versus-host disease (GVHD), hepatic veno-occlusive disease / sinusoidal obstruction syndrome (VOD / SOS), and thrombotic microangiopathy (TMA). The complication may be accompanied by iron overload. When used for adenosine triphosphate excess accompanied by iron overload, a large amount is administered so that there is more transferrin than iron in the body. It has already been reported that there are no problems with administering large amounts of transferrin (for example, 1500 mg twice a week). Pre-transplant treatment for allogeneic hematopoietic stem cell transplantation includes high-dose chemotherapy and whole-body radiation therapy.

[0025] [1-4-2. Adsorbent for blood purification treatment of patients with adenosine triphosphate excess] When used as an adsorbent for blood purification treatment of patients with adenosine triphosphate excess, the adenosine triphosphate capture agent of the present invention can be used in a state of being supported (immobilized) on an insoluble carrier. The shape of the insoluble carrier is not particularly limited, and examples thereof include fibrous and beaded forms. The form of the insoluble carrier on which the adenosine triphosphate capture agent of the present invention is supported is not particularly limited as long as it is a form that allows contact between blood and the adenosine triphosphate capture agent of the present invention, and examples thereof include a form filled in a column and a permeable membrane form.

[0026] In general, blood purification therapy refers to a method of treating a disease by removing disease-causing or disease-related substances or blood components that have accumulated in the blood using extracorporeal blood circulation or peritoneal dialysis to remove pathogenic substances from the blood, thereby maintaining the blood (body fluids) at normal levels. In the present invention, blood purification therapy refers to a method of treating a disease by removing excess adenosine triphosphate that has accumulated in the blood using extracorporeal blood circulation or peritoneal dialysis to remove excess adenosine triphosphate that has accumulated in the blood, thereby maintaining the blood (body fluids) at normal levels.

[0027] In this application, the blood of a patient with adenosine triphosphate excess is purified using an adsorbent carrying the adenosine triphosphate scavenger of the present invention, thereby lowering the level of excess adenosine triphosphate in the blood, thereby suppressing symptoms (e.g., inflammation, cell damage, and / or vascular endothelial damage) caused by excess adenosine triphosphate, and improving pathological conditions caused by these symptoms.

[0028] [1-4-3. Agents for preventing or improving adenosine triphosphate excess] When used as an agent for preventing or improving adenosine triphosphate excess, the adenosine triphosphate capture agent of the present invention can be used in the form of a pharmaceutical composition containing the capture agent as an active ingredient. This pharmaceutical composition can be administered to a subject animal in which adenosine triphosphate excess should be prevented or improved by intravenous administration, intramuscular administration, subcutaneous administration, or the like.

[0029] In the prevention or amelioration of adenosine triphosphate excess, the pharmaceutical composition is administered to an animal suffering from adenosine triphosphate excess, whereby the supplemented transferrin captures excess adenosine triphosphate and reduces its level, thereby suppressing various symptoms caused by excess adenosine triphosphate and ameliorating pathological conditions caused by these symptoms.

[0030] More specific examples of the prophylactic or ameliorating agent for adenosine triphosphate excess include, for example, an agent for preventing or ameliorating complications of allogeneic hematopoietic stem cell transplantation (GVHD (particularly severe cases), VOD / SOS (particularly severe cases), TMA (particularly severe cases), etc.); an agent for suppressing side effects of pretreatment for allogeneic hematopoietic stem cell transplantation (high-dose chemotherapy, whole-body radiation therapy, etc.) in order to suppress side effects such as cell damage that may be caused by the pretreatment; an agent for suppressing side effects of the pretreatment used in combination with the pretreatment; an agent for suppressing side effects caused by drugs (pretreatment drugs, immunosuppressants, etc.) used in organ transplantation or anticancer drugs; These include agents for suppressing side effects of the drug, which are used in combination with the drug or an anticancer drug to suppress side effects such as vascular endothelial dysfunction that may occur; blood pressure lowering therapeutic agents administered to treat hypotension caused by crush syndrome, traumatic shock, or sepsis; agents for suppressing the progression of muscular dystrophy, which are administered to improve the prognosis of muscular dystrophy; anticancer drugs administered to suppress the progression of cancers in which adenosine triphosphate has been reported to be involved; and agents for preventing or improving sepsis, collagen disease (especially severe cases), and COVID-19.

[0031] [1-4-4. Screening agent for compounds that bind to adenosine triphosphate] When used as a screening agent for a compound that binds to adenosine triphosphate, the adenosine triphosphate capture agent of the present invention can be used in the form of an analytical or research reagent. The adenosine triphosphate capture agent of the present invention in such a form can be specifically used in the following screening method. The screening method includes step 11 of contacting a sample A containing adenosine triphosphate with a candidate compound that binds to adenosine triphosphate, and step 12 of contacting the sample B obtained after the contact in step 11 with the adenosine triphosphate capture agent of the present invention. In step 12, if there is no target in sample B that is captured by the adenosine triphosphate capture agent of the present invention, or if the content of adenosine triphosphate in sample B is lower than the content of adenosine triphosphate in sample A, the candidate compound can be selected as a compound that binds to adenosine triphosphate.

[0032] [2. Adenosine diphosphate scavengers] The adenosine diphosphate scavenger of the present invention is characterized in that it contains butyrylcholinesterase and is used to capture adenosine diphosphate.

[0033] [2-1. Butyrylcholinesterase] Butyrylcholinesterase is a cholinesterase enzyme (EC 3.1.1.8) that has the activity of nonspecifically hydrolyzing many different choline-based esters. Butyrylcholinesterase is also called BChE, BuChE, pseudocholinesterase, or plasma esterase.

[0034] The specific example of butyrylcholinesterase includes the butyrylcholinesterase derived from vertebrate, preferably the butyrylcholinesterase derived from mammal, more specifically the butyrylcholinesterase derived from primates such as human and monkey.These butyrylcholinesterases can be used alone or in combination of multiple kinds.Among these butyrylcholinesterases, the butyrylcholinesterase derived from primates is preferred, and the butyrylcholinesterase derived from human is more preferred.

[0035] Butyrylcholinesterase may be purified from the butyrylcholinesterase source of the above-mentioned vertebrate, or may be produced using recombinant technology.Butyrylcholinesterase sources include blood, kidney, intestine, liver, lung, heart, and central nervous system.The method for producing butyrylcholinesterase using recombinant technology includes the method of expressing the target butyrylcholinesterase in host cells such as Escherichia coli, yeast, insect cells, plant cells, and animal cells; the method of producing recombinant vertebrate and purifying the target butyrylcholinesterase from the butyrylcholinesterase source of the recombinant vertebrate.

[0036] FIG. 2 shows a computer simulation diagram illustrating the binding mode between butyrylcholinesterase and adenosine diphosphate.

[0037] [2-2. Other ingredients] In addition to the butyrylcholinesterase, the adenosine diphosphate scavenger of the present invention may contain a physiologically or pharma- ceutically acceptable base and / or additive, etc., if necessary (for example, depending on the use of the adenosine diphosphate scavenger). Examples of such bases and additives include excipients, binders, disintegrants, lubricants, isotonicity agents, plasticizers, dispersants, emulsifiers, solubilizers, wetting agents, stabilizers, suspending agents, adhesives, coating agents, glossing agents, water, oils and fats, ester oils, waxes, hydrocarbon bases, fatty acids, lower alcohols, higher alcohols, water-soluble polymers, surfactants, metal soaps, polyhydric alcohols, pH adjusters, buffers, antioxidants, UV inhibitors, preservatives, flavorings, fragrances, powders, dyes, chelating agents, etc. These bases and additives may be used alone or in combination of two or more. The content of these bases and additives may be appropriately set depending on the type and / or dosage form of the added components used.

[0038] [2-3. Dosage form] The dosage form of the adenosine diphosphate capture agent of the present invention is not particularly limited and can be appropriately set according to the application. For example, the dosage form includes liquid, powder, granule, tablet, and the like, and the form supported on an insoluble carrier. The above-mentioned dosage form can be prepared using a physiologically or pharma- ceutical acceptable base and / or additive, and the type and amount of the base and additive can be appropriately set by a person skilled in the art. The above-mentioned form supported on an insoluble carrier can be prepared using a physiologically acceptable insoluble carrier (e.g., an adsorbent for blood purification treatment, etc.), and the type and method of supporting the insoluble carrier can be appropriately set by a person skilled in the art.

[0039] [2-4.Application] The adenosine diphosphate scavenger of the present invention can be used in any application that utilizes the binding between butyrylcholinesterase and adenosine diphosphate.

[0040] When the adenosine diphosphate capture agent of the present invention is used, the pH (25° C.) condition is, for example, 5 or more, preferably 5.5 or more, 6 or more, 6.5 or more, 7 or more, or 7.2 or more. The upper limit of the pH condition is, for example, 8 or less, preferably 7.8 or less, more preferably 7.6 or less, more preferably 7.5 or less, 7.3 or less, 7 or less, or 6.5 or less.

[0041] Specific uses of the adenosine diphosphate capture agent of the present invention include uses as an adsorbent for blood purification treatment of patients with adenosine diphosphate excess, an agent for preventing or improving adenosine diphosphate excess, and a screening agent for compounds that bind to adenosine diphosphate.

[0042] [2-4-1. Adenosine diphosphate excess] Adenosine diphosphate excess refers to a pathological condition caused in part by symptoms involving excess adenosine diphosphate (e.g., inflammation, cell damage, and / or vascular endothelial damage, etc.), and specifically includes complications of allogeneic hematopoietic stem cell transplantation, tumor lysis syndrome, crush syndrome, traumatic shock, septic shock, myocardial infarction, muscular dystrophy, collagen disease, cancer, COVID-19 (coronavirus disease that occurred in 2019), etc. Complications of allogeneic hematopoietic stem cell transplantation include graft-versus-host disease (GVHD), hepatic veno-occlusive disease / sinusoidal obstruction syndrome (VOD / SOS), and thrombotic microangiopathy (TMA).

[0043] Adenosine diphosphate excess can be diagnosed using a decrease in butyrylcholinesterase as an indicator, based on the method described below under "3. Method for testing for adenosine diphosphate excess."

[0044] [2-4-2. Adsorbent for blood purification treatment of patients with adenosine diphosphate excess] When used as an adsorbent for blood purification treatment of patients with adenosine diphosphate excess, the adenosine diphosphate capture agent of the present invention can be used in a state of being supported (immobilized) on an insoluble carrier. The shape of the insoluble carrier is not particularly limited, and examples thereof include fibrous and beaded forms. The form of the insoluble carrier on which the adenosine diphosphate capture agent of the present invention is supported is not particularly limited as long as it is a form that allows contact between blood and the adenosine diphosphate capture agent of the present invention, and examples thereof include a form filled in a column and a permeable membrane form.

[0045] In general, blood purification therapy refers to a method of treating a disease by removing disease-causing or disease-related substances or blood components that have accumulated in the blood using extracorporeal blood circulation or peritoneal dialysis to remove pathogenic substances from the blood, thereby maintaining the blood (body fluids) at normal levels. In the present invention, blood purification therapy refers to a method of treating a disease by removing excess adenosine diphosphate that has accumulated in the blood using extracorporeal blood circulation or peritoneal dialysis to remove excess adenosine diphosphate that has accumulated in the blood, thereby maintaining the blood (body fluids) at normal levels.

[0046] In this application, the blood of a patient with adenosine diphosphate excess is purified using an adsorbent carrying the adenosine diphosphate scavenger of the present invention, thereby lowering the level of excess adenosine diphosphate in the blood, thereby suppressing symptoms (e.g., inflammation, cell damage, and / or vascular endothelial damage) caused by excess adenosine diphosphate, and improving pathological conditions caused by these symptoms as one of their causes.

[0047] [2-4-3. Agents for preventing or improving adenosine diphosphate excess] When used as an agent for preventing or improving adenosine diphosphate excess, the adenosine diphosphate capture agent of the present invention can be used in the form of a pharmaceutical composition containing the capture agent as an active ingredient. This pharmaceutical composition can be administered to a subject animal in which adenosine diphosphate excess should be prevented or improved by intravenous administration, intramuscular administration, subcutaneous administration, or the like.

[0048] In adenosine diphosphate excess, butyrylcholinesterase is consumed to capture excess adenosine diphosphate, so that the level of butyrylcholinesterase (i.e., butyrylcholinesterase that is not complexed with adenosine diphosphate) is reduced.In this application, the pharmaceutical composition is administered to an animal with adenosine diphosphate excess to supplement the butyrylcholinesterase that is relatively insufficient compared to the level of excess adenosine diphosphate, and the supplemented butyrylcholinesterase captures adenosine diphosphate and reduces its level.Therefore, symptoms (e.g., inflammation, cell damage, and / or vascular endothelial damage, etc.) caused by excess adenosine diphosphate are suppressed, and pathological conditions caused by these symptoms are improved.

[0049] More specific examples of the prophylactic or ameliorating agent for adenosine diphosphate excess include an agent for preventing or ameliorating complications of allogeneic hematopoietic stem cell transplantation (GVHD (particularly severe cases), VOD / SOS (particularly severe cases), TMA (particularly severe cases), etc.); an agent for suppressing side effects of a drug used in organ transplantation (pretreatment drug, immunosuppressant, etc.) or an anticancer drug in combination with the drug or anticancer drug to suppress side effects such as vascular endothelial disorder that may be caused by the drug; a blood pressure lowering therapeutic agent administered to treat blood pressure reduction caused by crush syndrome, traumatic shock, or sepsis; an inhibitor of the progression of muscular dystrophy pathology administered to improve the prognosis of muscular dystrophy; an anticancer drug administered to suppress the progression of cancer in which adenosine diphosphate has been reported to be involved; and an agent for preventing or ameliorating sepsis, collagen disease (particularly severe cases), and COVID-19.

[0050] [2-4-4. Screening agent for compounds that bind to adenosine diphosphate] When used as a screening agent for a compound that binds to adenosine diphosphate, the adenosine diphosphate capture agent of the present invention can be used in the form of an analytical or research reagent. The adenosine diphosphate capture agent of the present invention in such a form can be specifically used in the following screening method. The screening method includes step 11 of contacting a sample A containing adenosine diphosphate with a candidate compound that binds to adenosine diphosphate, and step 12 of contacting the sample B obtained after the contact in step 11 with the adenosine diphosphate capture agent of the present invention. In step 12, if there is no target in sample B that is captured by the adenosine diphosphate capture agent of the present invention, or if the content of adenosine diphosphate in sample B is lower than the content of adenosine diphosphate in sample A, the candidate compound can be selected as a compound that binds to adenosine diphosphate.

[0051] [3. Methods for testing for adenosine diphosphate excess] Butyrylcholinesterase has a function of binding to adenosine diphosphate, and therefore has a characteristic that its level is low when the patient is in an adenosine diphosphate excess state. Therefore, butyrylcholinesterase is useful as a marker for an adenosine diphosphate excess state. Therefore, the present invention also provides a method for testing for adenosine diphosphate excess.

[0052] The method for testing for adenosine diphosphate excess of the present invention is characterized by comprising: (i) a step of measuring butyrylcholinesterase (specifically, free butyrylcholinesterase) in a sample collected from a subject animal; and (ii) a step of detecting adenosine diphosphate excess based on the results of the step (i).

[0053] [3-1. Process (i)] The subject animal is not particularly limited and may be any animal. Specific examples of the subject animal include humans and mammals other than humans. Examples of mammals other than humans include laboratory animals such as rodents, such as mice, rats, hamsters, and guinea pigs, and rabbits; livestock animals, such as pigs, cows, goats, horses, and sheep; pets, such as dogs and cats; and primates, such as humans, monkeys, orangutans, and chimpanzees. Among these subject animals, primates are preferred, and humans are preferred.

[0054] The specimen collected from the subject animal is not particularly limited as long as it contains butyrylcholinesterase, and specifically includes cell specimens, tissue specimens, and body fluid specimens.Cell specimens and tissue specimens include specimens of cancer cells and non-cancerous cells, as well as cancer tissues and non-cancerous tissues, and cancer cells and cancer tissues include those derived from primary tissues and metastatic tissues, and more specifically, those derived from organ tissues, nerve tissues, lymph nodes, etc.Specific examples of body fluid specimens include blood samples (whole blood, serum, plasma), sweat, urine, cerebrospinal fluid, lymph, saliva, sputum, alveolar lavage fluid, tears, aqueous humor, vitreous humor, etc.

[0055] When a blood sample is used as the specimen, examples of the blood sample include blood samples that have been treated with heparin or a chelating agent (e.g., ethylenediaminetetraacetic acid, sodium citrate), and from the viewpoint of further improving quantitativeness, heparin-treated blood samples are preferred.

[0056] As a method for measuring butyrylcholinesterase, a known method can be used without any particular limitation.

[0057] As an example of the method for measuring butyrylcholinesterase, butyrylcholinesterase in a specimen is reacted with a substrate (a compound designed to generate thiol by hydrolysis), a thiol compound is generated from the substrate, and the generated thiol compound is covalently bonded with a fluorescent reagent for detecting thiol (a label is designed to generate fluorescence when it is bonded with thiol, and the label is quenched when it is not bonded with thiol), and butyrylcholinesterase can be quantified based on the fluorescence intensity generated by the covalent bond.In addition to fluorescence, any signal label such as color development or radiation can be used, and butyrylcholinesterase can be quantified based on color development intensity, wavelength, or radiation intensity.

[0058] Other examples of the method for measuring butyrylcholinesterase include a method of directly or indirectly detecting the specific binding between butyrylcholinesterase in a specimen and anti-butyrylcholinesterase antibody for detection.Specific examples of such detection methods include immunoassays such as ELISA, Western blot, immunoprecipitation, radioimmunoassay (RIA), and fluorescent immunoassay.The detection of butyrylcholinesterase in a specimen by these immunoassays can be used in the form that anti-butyrylcholinesterase antibody is bound to a label such as enzyme label, fluorescent label, color label, or radioactive label.

[0059] [3-2. Process (ii)] Step (ii) can be carried out by comparing the results of step (i) obtained for the test animal with the results of step (i) obtained for a normal control, using a decrease in butyrylcholinesterase as an indicator.

[0060] Here, the normal control refers to an animal of the same species as the subject animal from which the sample subjected to step (i) was derived, which does not have adenosine diphosphate excess or is not at risk of developing adenosine diphosphate excess.

[0061] When the sample subjected to step (i) is a human sample, in step (ii), the butyrylcholinesterase activity in the blood sample can be used as an indicator that is less than 170 U / mL. The present inventor has previously confirmed that BChE is useful as an ATP scavenger, that BChE is useful as a predictive marker for TLS (ATP excess), and that low BChE (specifically, less than 170 U / mL) is a risk factor for TLS with a significant difference. It has also been confirmed that the group with low BChE (specifically, less than 170 U / mL) is highly likely to develop tumor lysis syndrome (ATP excess). Since it can be inferred that in a state in which butyrylcholinesterase shows a low value in ATP excess, ADP also shows a low value, as described above, in the case of testing for ADP excess, the butyrylcholinesterase activity in the blood sample can be used as an indicator that is less than 170 U / mL.

[0062] In step (ii), the presence or absence of adenosine diphosphate excess, the severity of adenosine diphosphate excess, the effectiveness of treatment for adenosine diphosphate excess, and the risk of developing adenosine diphosphate excess can be diagnosed.

[0063] In step (ii), for example, the presence or absence of adenosine diphosphate excess, the severity of adenosine diphosphate excess, the effectiveness of treatment for adenosine diphosphate excess, and the risk of developing adenosine diphosphate excess can be diagnosed.

[0064] Specifically, the presence or absence of adenosine diphosphate excess can be judged as adenosine diphosphate excess when the above indicators are met. The severity of adenosine diphosphate excess can be judged based on the fact that it becomes severe in inverse proportion to the level of butyrylcholinesterase. Furthermore, the therapeutic effect of adenosine diphosphate excess is judged for the subject animal during or after the treatment, and it can be judged that there is a tendency for improvement by treatment using the increase in the level of butyrylcholinesterase compared to the result of step (i) before treatment (at the time of diagnosis of adenosine diphosphate excess) as an indicator, and it can be judged as remission using the same level of butyrylcholinesterase compared to the result of step (i) obtained for normal control as an indicator.

[0065] Furthermore, the risk of developing adenosine diphosphate excess is predicted for a subject animal during a treatment that may cause adenosine diphosphate excess (for example, if the adenosine diphosphate excess is tumor lysis syndrome, this can be before the start of cancer treatment such as cytotoxic anticancer drugs and radiation therapy), and the risk of developing adenosine diphosphate excess can be predicted using a decrease in the butyrylcholinesterase level as an indicator compared to the result of step (i) obtained for a subject animal that is not at risk of developing adenosine diphosphate excess (for example, if the adenosine diphosphate excess is tumor lysis syndrome, this can be before the start of cancer treatment).

[0066] In addition, in step (ii), a correlation diagram may be prepared in advance between the butyrylcholinesterase level in the sample and the presence or absence, severity, prognosis, etc. of adenosine diphosphate excess, and the measurement results of butyrylcholinesterase in the sample of the test animal may be applied to the correlation diagram to perform evaluation.

[0067] [4. Diagnostic kit for Adenosine Diphosphate Hyperphosphatemia] As described above, butyrylcholinesterase is useful as a marker for an adenosine diphosphate excess state, and can therefore be used to test for the presence or absence of adenosine diphosphate excess. Therefore, the present invention also provides a diagnostic kit for adenosine diphosphate excess.

[0068] The diagnostic kit for adenosine diphosphate hyperphosphatemia of the present invention is characterized by comprising a reagent capable of detecting butyrylcholinesterase.

[0069] An example of a reagent capable of detecting butyrylcholinesterase is a substrate of butyrylcholinesterase that produces a compound capable of detecting a signal. An example of a substrate of butyrylcholinesterase that produces a compound capable of detecting a signal is a compound designed to produce thiol by hydrolysis by butyrylcholinesterase, more specifically, a thiocholine compound used in the Ellman method and a thioester compound such as N-alkylpiperidine methanethiol ester described in International Publication No. 2010 / 100720 (the amount of thiol compound produced by hydrolysis of the thiocholine compound or thioester compound is measured by a fluorescent reagent for detecting thiol); a choline ester compound having a radioactive label (the radioactive compound produced by hydrolysis of the choline ester compound is measured by scintillation), etc. When these substrates are included in a diagnostic kit, they can be included in the diagnostic kit in combination with an acetylcholinesterase inhibitor in order to increase the specificity for butyrylcholinesterase.

[0070] Other examples of reagents capable of detecting butyrylcholinesterase include anti-butyrylcholinesterase antibody.Anti-butyrylcholinesterase antibody can be used in the form of being bound with a label such as an enzyme label, a fluorescent label, a color label, or a radioactive label.Anti-butyrylcholinesterase antibody bound with butyrylcholinesterase can be measured based on immunoassays such as ELISA, Western blot, immunoprecipitation, radioimmunoassay (RIA), and fluorescent immunoassay.

[0071] The reagent capable of detecting butyrylcholinesterase may contain a buffer solution, a salt, a stabilizer, a preservative, etc., and may be formulated according to a conventionally known method. In addition to the above-mentioned reagent, the diagnostic kit of the present invention may contain a labeling substance, a detection agent for the labeling substance, a reaction diluent, a standard antibody, a buffer solution, a dissolving agent, a cleaning agent, a reaction stop solution, a control sample, etc., which may be required for detecting butyrylcholinesterase.

[0072] In the diagnostic kit of the present invention, the reagent capable of detecting butyrylcholinesterase may be immobilized on an insolubilized carrier.The material of the insolubilized carrier is not particularly limited as long as it does not prevent the detection of butyrylcholinesterase, and examples thereof include polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, polyvinyl chloride, fluororesin, silicone, cross-linked dextran, polysaccharide, paper, glass, silicon dioxide, metal, agarose, and mixtures or composite materials thereof.The shape of the insolubilized carrier is not particularly limited as long as it is a form that allows the immobilized reagent capable of detecting butyrylcholinesterase to come into contact with the specimen, and examples thereof include microplates, trays, spheres, fibers, rods, discs, containers, cells, test tubes, etc. EXAMPLES

[0073] The present invention will be described in detail below based on examples, but the present invention is not limited to these.

[0074] [Test Example 1] Capture of extracellular ATP by transferrin (TF) (1) Experimental procedure Apo-TF (Nakalai tesque Adenosine-5'-triphosphate disodium salt hydrate Cat. No. 34402) or iron-saturated holo-TF (Nakalai tesque Adenosine-5'-triphosphate disodium salt hydrate Cat. No. 34443) was mixed with ATP (Nakalai tesque Adenosine-5'-triphosphate disodium salt hydrate Cat. No. 01072) to prepare mixed solution a1 (final concentrations of Apo-TF and holo-TF, respectively, 200 μM, 100 μM, or 50 μM; final concentration of ATP, 200 μM). Mixture a1 (pH 6.0 for 100 μM ATP and 100 μM apo-TF, pH 7.0 for 100 μM ATP and 100 μM Holo-TF) was left for 10 minutes, and then TF (80 kDa) was removed using a 10 kDa ultrafiltration membrane to obtain permeate a2. The ATP concentration of permeate a2 was measured. As a control, the same procedure was performed except that H2O was used instead of the sample containing TF and ultrafiltration was not performed.

[0075] Separately, a mixture b1 of Apo-TF and ADP (Nakalai tesque Adenosine-5'-triphosphate disodium salt hydrate Cat. No. 01652) was prepared, and ATP was added to prepare mixture b2 (final concentration of Apo-TF: 100 μM, final concentration of ADP: 100 μM, final concentration of ATP: 100 μM). After leaving mixture b2 (pH 6.0) for 10 minutes, TF (80 kDa) was removed using a 10 kDa ultrafiltration membrane to obtain permeate b3. The ATP concentration of permeate b3 was measured. The ATP concentration was measured by the fluorometric method (excitation 535 nm / emission 587 nm) using an ATP Colometric / Fluorometric Assay kit (BioVison Cat. No. K354-100). As a control, the same procedure was performed except that H2O was used instead of the sample containing TF and ADP was not added. The same procedure was also carried out except that ADP was not added.

[0076] (2) Results As shown in FIG. 3A, the concentration of ATP in the permeate a2 was decreased in an Apo-TF concentration-dependent manner, confirming that Apo-TF binds to ATP. On the other hand, as shown in FIG. 3B, the concentration of ATP in the permeate a2 did not change regardless of the concentration of holo-TF, confirming that holo-TF does not bind to ATP. Furthermore, as shown in FIG. 3C, ADP coexisting with apo-TF did not inhibit the binding of apo-TF to ATP. In other words, apo-TF did not bind to ADP in the coexistence of ATP and ADP.

[0077] [Test Example 2] Capture of extracellular ADP by butyrylcholinesterase (BChE) (1) Experimental procedure A sample containing BChE (Cohn fraction IV-4) was mixed with ATP (Nakalai tesque Adenosine-5'-triphosphate disodium salt hydrate Cat. No. 01072) to prepare mixed solution c1 (final concentration of BChE: 10 μM, final concentration of ATP: 10 μM). After leaving mixed solution c1 (pH 7.4) for 10 minutes, BChE (83 kDa) was removed using a 10 kDa ultrafiltration membrane to obtain permeate c2. The ATP concentration of permeate c2 was measured. It is known that BChE binds to ATP.

[0078] Separately, a sample containing BChE (Cohn fraction IV-4) was mixed with ADP (Nakalai tesque Adenosine-5'-triphosphate disodium salt hydrate Cat. No. 01652) to prepare mixed solution d1, and ATP was added to prepare mixed solution d2 (BChE final concentration 10μM, ADP final concentration 50μM, ATP added amount 10μM). After leaving mixed solution d2 (pH 7.4) for 10 minutes, BChE (83kDa) was removed using a 10kDa ultrafiltration membrane to obtain permeate d3. The ATP concentration of permeate b3 was measured.

[0079] The ATP concentration was measured by the fluorescence method (excitation 535 nm / emission 587 nm) using an ATP Colometric / Fluorometric Assay kit (BioVison Cat. No. K354-100). As a control, the same procedure was performed except that PBS was used instead of the sample containing BChE.

[0080] (2) Results The results are shown in Figure 4. In the permeate d3 prepared from the mixed solution d1 to which ADP was added, the ATP concentration was higher than in the permeate c2 prepared from the mixed solution c1 to which ADP was not added. This indicates that in the process of preparing the permeate d3, the ADP added when preparing the mixed solution d1 bound to BChE first, thereby partially inhibiting the binding of the ATP added when preparing the mixed solution d2 with BChE. The fact that the binding was not completely inhibited is thought to be due to the fact that the BChE solution contained albumin, which binds to ATP but not ADP. (Computer simulations were performed using Autodock vina and NGLviewer through Galaxy Europe, and the affinity score, which indicates the strength of binding to albumin, was calculated using RDkit (version 2020.03.4).The results showed that at pH 7.4, the affinity score between albumin and ATP was -8.0 or less (specifically, -8.1 or less, meaning that it binds), and the affinity score between albumin and ADP was greater than -8.0 (specifically, -7.9 or more, meaning that it does not bind).)Based on the above, serum fraction Cohn fraction IV-4 preparations containing BChE are useful as ADP scavengers, either as is or in concentrated form.

Claims

1. Adenosine triphosphate sequestrants, including transferrin.

2. The scavenger according to claim 1, which is used as an agent for preventing or improving adenosine triphosphate excess.

3. The capture agent according to claim 2 , wherein the adenosine triphosphate excess is a complication of allogeneic hematopoietic stem cell transplantation and / or a pre-transplant treatment for allogeneic hematopoietic stem cell transplantation.

4. The capture agent of claim 3 , wherein the complication is selected from the group consisting of graft-versus-host disease, hepatic veno-occlusive / sinusoidal obstruction syndrome, and thrombotic microangiopathy.

5. The scavenger according to claim 3, which is applied to patients with iron overload.

6. The method of claim 2 , wherein the adenosine triphosphate excess is tumor lysis syndrome.

7. The capture agent according to claim 1, which is used in the form of being supported on an adsorbent for blood purification treatment of patients with adenosine triphosphate excess.

8. The capture agent according to claim 1, which is used as a screening agent for compounds that bind to adenosine triphosphate.

9. A scavengers of adenosine diphosphate, including butyrylcholinesterase.

10. The capture agent according to claim 9, which is used as a preventive or therapeutic agent for a condition requiring inhibition of adenosine diphosphate.

11. The method of claim 10, wherein the condition is a complication of allogeneic hematopoietic stem cell transplantation.

12. The capture agent of claim 11, wherein the complication is selected from the group consisting of graft-versus-host disease, hepatic veno-occlusive / sinusoidal obstruction syndrome, and thrombotic microangiopathy.

13. The capture agent according to claim 10, which is used as a screening agent for compounds that bind to adenosine diphosphate.

14. 1. A method for testing for a condition requiring inhibition of adenosine diphosphate, comprising the steps of: (i) measuring butyrylcholinesterase in a sample collected from a test animal; and (ii) detecting the condition based on the result of step (i); 4. A method for testing comprising:

15. The testing method according to claim 14, wherein the step (ii) is carried out by comparing the result of step (i) obtained for the subject animal with the result of step (i) obtained for a normal control, and using a decrease in adenosine diphosphate as an indicator.

16. A kit for diagnosing a condition requiring inhibition of adenosine diphosphate, comprising a reagent capable of detecting butyrylcholinesterase.

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