Medicine containing methemoglobin vesicles as an active ingredient and use thereof

Abstract

The purpose of the present invention is to provide a medicine that is for use in instances of cyanide poisoning, hydrogen sulfide poisoning, and azide poisoning, that does not cause side effects, and that has excellent quick-acting properties and a high antidote effect. Specifically, the present invention is a medicine that is for use in instances of cyanide poisoning, hydrogen sulfide poisoning, and azide poisoning, and that contains, as an active ingredient, at least one selected from the group consisting of hemeproteins capable of binding to a cyanide ion (CN-), a hydrogen sulfide ion (HS-), and an azide ion (N3 -), hemeprotein-containing substances capable of binding to CN-, HS-, and N3 -, and heme derivatives capable of binding to CN-, HS-, and N3. More specifically, the present invention is a medicine or pharmaceutical composition for treatment or prophylaxis of cyanide poisoning, hydrogen sulfide poisoning, and azide poisoning.

Description

[Technical Field]

[0001] The present invention relates to a medicine for treating or preventing cyanide poisoning, hydrogen sulfide poisoning and azide poisoning. [Background technology]

[0002] Cyanide poisoning occurs when the cyanide ion (CN - It is caused by the binding of cytochrome c oxidase (CcOX) in intracellular mitochondria, directly inhibiting the cell's oxygen metabolism. Its causes are diverse, including suicide attempts, terrorist attacks, and even unintentional inhalation during the burning of synthetic fibers in fires. Symptoms progress quickly, suffocating the entire body, so treatment must begin as soon as possible.

[0003] Hydrogen sulfide (HS) is highly water-soluble, and at physiological pH, approximately two-thirds of it is converted into hydrogen sulfide ions (HS - ), one-third of which exists as undissociated H2S. Hydrogen sulfide is always present in trace amounts in the body and is considered a physiologically active gas due to its role in regulating vascular function and providing cell protection. However, hydrogen sulfide is highly toxic, and acute poisoning can occur if excessive exposure occurs. While the statistical number of annual deaths from hydrogen sulfide poisoning is unknown, many workers in industrial sectors, including agriculture and petroleum, are exposed to hydrogen sulfide. One-third of oil industry workers experience symptoms of hydrogen sulfide exposure, some of which may lead to unconsciousness. Furthermore, because hydrogen sulfide can be easily produced from simple chemicals, like cyanide ion, it has recently been used for suicide and as a raw material for terrorist weapons of mass destruction.

[0004] Sodium azide (NaN3) is a highly water-soluble white solid that is used as a preservative and a broad-spectrum insecticide. However, azide poisoning has occurred due to the accidental ingestion of preservatives. It is also frequently used as a homicide agent when intentionally mixed into food and drink.

[0005] Existing antidote methods for cyanide poisoning mainly consist of two therapies: the combined use of nitrites (amyl nitrite and sodium nitrite, etc.) and sodium thiosulfate, or the use of hydroxocobalamin alone. However, the mortality rate remains high, and these treatments cannot be considered the best options. A major problem with the combined use of nitrites and sodium thiosulfate is that it can cause hypoxemia due to nitrite. Nitrite converts hemoglobin in red blood cells to methemoglobin (MetHb), which then binds to CcOX. - It converts CcOX to MetHb, which has a higher cyanide affinity, and restores the function of CcOX (Non-Patent Document 1). Therefore, it is highly likely that the oxygen-carrying capacity of hemoglobin will be inhibited, causing hypoxemia, making it difficult to use in the event of a fire where breathing difficulties and carbon monoxide (CO) poisoning may occur (Non-Patent Document 2). A significant problem with hydroxocobalamin monotherapy is the decreased detoxification capacity. - Since it exerts its detoxifying ability by directly coordinating with nitrites, it does not cause hypoxemia like nitrites and can be used in fires. However, due to its small molecular weight, it has been reported that its detoxifying ability is significantly reduced to 1 / 600 when it binds to plasma proteins such as haptocorrin, and it is highly unlikely that it will function as a fast-acting detoxifier in the in vivo environment (Non-Patent Document 3).

[0006] The pathogenesis of hydrogen sulfide and azide poisoning is triggered by the binding of the respective substances (hydrogen sulfide or azide) to CcOX and the inhibition of oxidative phosphorylation after entering the body via various routes, including oral, transdermal, mucosal, and inhalation routes. Hydrogen sulfide and azide poisoning are acutely toxic with a high fatality rate, but no approved antidotes for hydrogen sulfide or azide poisoning exist. Interestingly, the pathogenesis of hydrogen sulfide and azide poisoning shares some commonalities with that of cyanide poisoning. Therefore, the efficacy of sodium nitrite and hydroxocobalamin, which are approved as antidotes for cyanide poisoning, as well as their analogues, as antidotes for hydrogen sulfide and azide poisoning has been investigated (Non-Patent Documents 12–17).

[0007] As described above, the development of preventive and therapeutic methods for cyanide poisoning, hydrogen sulfide poisoning, and azide poisoning is an urgent issue, and although research is being actively conducted, there are currently no preventive or therapeutic agents that are highly effective and have a strong detoxifying effect. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Baskin et al., The Journal of Clinical Pharmacology 1992;32(4):368-375. [Non-patent document 2] Brenner et al., Toxicology and Applied Pharmacology 2010;248(3):269-276. [Non-patent document 3] Watanabe et al., ACS Medicinal Chemistry Letters 2011;2(12):943-947. [Non-patent document 4] Taguchi et al., Journal of Pharmaceutical Sciences 2011;100(2):775-783. [Non-Patent Document 5] Sakai et al., Journal of Internal Medicine 2007;263(1):4-15. [Non-patent document 6] Sakai et al., Bioconjugate Chemistry 2014;25(7):1301-1310. [Non-Patent Document 7] Kettisen et al., Bioconjugate Chemistry 2015;26(4):746-754. [Non-patent document 8] Cabrales et al., Resuscitation 2007;75(1):124-134. [Non-Patent Document 9] Taguchi et al., Drug Metabolism and Disposition 2009;37(7):1456-1463. [Non-Patent Document 10] Gu et al., Arch Toxicol. 2018;92(12):3505-3515. [Non-Patent Document 11] Cambal et al., Chemical Research in Toxicology 2013;26(5):828-836. [Non-Patent Document 12] Frawley et al., Chem. Res. Toxicol. 2020;33:594-603. [Non-Patent Document 13] Praekunatham et al., Chem. Res. Toxicol. 2020;33:333-342. [Non-Patent Document 14] Jiang et al., Sci. Rep. 2016;6:1-10. [Non-Patent Document 15] Ng et al., Clin. Toxicol. 2019;57:189-196. [Non-Patent Document 16] Haouzi et al., Toxicol. Sci. 2019;168:443-459. [Non-Patent Document 17] Truong et al., Toxicology. 2007;242:16-22. Summary of the Invention [Problem to be solved by the invention]

[0009] In view of the above circumstances, an object of the present invention is to provide an antidote for cyanide poisoning, hydrogen sulfide poisoning, and azide poisoning, which has a rapid effect and a high detoxifying effect. [Means for solving the problem]

[0010] In order to solve the above problems, the present inventors investigated the use of hemoglobin vesicles (HbV), an artificial red blood cell preparation in which a high concentration of human hemoglobin is encapsulated in liposomes (Non-Patent Documents 4 and 5), as an antidote for cyanide poisoning, hydrogen sulfide poisoning, and azide poisoning. MetHb, which is generated by the oxidation of hemoglobin (Hb), does not have the ability to bind oxygen and has lost its function of transporting oxygen in vivo. However, the present inventors have discovered that MetHb is a CN - , hydrogen sulfide ion (HS - ) and azide ion (N3 - ) has been found to have the ability to bind to methemoglobin. Specifically, by using methemoglobin vesicles (MeHbV), in which Hb in HbV is converted to MetHb, it was thought that the problems with existing cyanide poisoning antidotes described above could be overcome and a highly effective cyanide poisoning antidote could be developed. Furthermore, the inventors suspected that MetHbV might also function as an effective antidote for hydrogen sulfide poisoning and azide poisoning, which share common pathogenic mechanisms, and evaluated the efficacy of MetHbV as a therapeutic and preventive agent for hydrogen sulfide and azide poisoning. The present inventors administered MetHbV to a mouse model of cyanide poisoning and evaluated its effects on survival rate and recovery time from poisoning. The results showed that, under sodium cyanide (NaCN) poisoning, the model mice administered MetHbV had a significantly higher survival rate than the untreated group administered NaCN alone, and also had a faster recovery time from poisoning than groups administered other existing drugs. Furthermore, the present inventors found that administering NaCN to mice previously administered MetHbV suppressed death from cyanide poisoning. Furthermore, the present inventors administered MetHbV to hydrogen sulfide poisoning model mice and azide poisoning model mice before and after the onset of each poisoning, and confirmed its effectiveness as a preventive administration (pre-administration) and a therapeutic administration (post-administration) in both poisoning model mice, and found that its detoxifying effect was greater than that of sodium nitrite and hydroxocobalamin. That is, the present inventors have found for the first time that MetHbV exhibits excellent effects as a therapeutic and preventive agent for cyanide poisoning, hydrogen sulfide poisoning, and azide poisoning.

[0011] That is, the present invention provides the following (1) to (6). (1) A drug for cyanide poisoning, hydrogen sulfide poisoning, and azide poisoning that contains cyanide ion (CN - ), hydrogen sulfide ion (HS - ) and azide ion (N3 - ) can bind to hemoproteins, such as CN - , H.S. - and N3 - Contains hemoproteins capable of binding CN - , H.S. - and a heme derivative capable of binding N3, as an active ingredient. (2) The above CN - , H.S. - and N3 - The pharmaceutical according to (1) above, wherein the hemoprotein capable of binding is methemoglobin. (3) The above CN- , H.S. - and N3 - The pharmaceutical according to (1) or (2) above, wherein the substance containing a hemoprotein capable of binding to methemoglobin is a metherythrocyte or a capsule containing methemoglobin. (4) The pharmaceutical according to (3) above, wherein the capsule comprises at least one selected from the group consisting of a liposome, a polymersome, and a thin polymer film. (5) A pharmaceutical agent according to any one of (1) to (4) above for the treatment of cyanide poisoning, hydrogen sulfide poisoning, and azide poisoning. (6) A pharmaceutical composition according to any one of (1) to (4) above for preventing cyanide poisoning, hydrogen sulfide poisoning, and azide poisoning. In this specification, the symbol "to" indicates a numerical range including the values ​​on either side of it. [Effects of the Invention]

[0012] The medicines and pharmaceutical compositions (hereinafter also referred to as "medicines, etc.") of the present invention for cyanide poisoning, hydrogen sulfide poisoning, and azide poisoning have the effect of being more rapidly effective than existing medicines, and being extremely unlikely to cause side effects seen in existing medicines, such as the induction of hypoxemia.

[0013] Furthermore, the medicines etc. according to the present invention are highly effective not only as therapeutic drugs but also as preventive drugs. [Brief explanation of the drawings]

[0014] [Figure 1] A transmission electron microscope (TEM) image of the prepared MetHbV is shown. [Figure 2] The particle size distribution of the prepared MetHbV is shown. [Figure 3] This shows the change in absorption spectrum when OxyHbV (oxyhemoglobin vesicle) transforms into CN-MetbV (CN-coordinated MetHbV) via MetHbV. Each absorption spectrum is shown by a solid line (MetHbV), a dotted line (OxyHbV), and a dashed line (CN-MetHbV). [Figure 4] The cytotoxicity of MetHbV at various concentrations was evaluated using CCK-8 (Cell Counting Kit-8). PC-12 cells were used. Each value is shown as the mean ± standard error (n = 3). [Figure 5] The effects of various concentrations of antidotes (MetHbV, OxyHbV, and empty vesicles (EVs)) were evaluated using CCK-8 (Cell Counting Kit-8). PC-12 cells were used. (A) Each antidote was added at 2.5 g (Hb / dL) or an equivalent volume. (B) Each antidote was added at 5 g (Hb / dL) or an equivalent volume. (C) Each antidote was added at 10 g (Hb / dL) or an equivalent volume. (D) Each antidote was added at 15 g (Hb / dL) or an equivalent volume. (E) Each antidote was added at 20 g (Hb / dL) or an equivalent volume. [Figure 6] The effects of various antidotes (metRBCs (red blood cells (RBCs) containing methemoglobin), metHb (methemoglobin), OHCbl (hydroxocobalamin), and Na2S2O3 (sodium thiosulfate)) were compared with the effect of MetHbV. (A) Each antidote was added at 2.5 g (Hb / dL) or equivalent. (B) Each antidote was added at 5 g (Hb / dL) or equivalent. (C) Each antidote was added at 10 g (Hb / dL) or equivalent. (D) Each antidote was added at 15 g (Hb / dL) or equivalent. (E) Each antidote was added at 20 g (Hb / dL) or equivalent. [Figure 7]Survival rates after a single oral administration of NaCN (12 mg / kg) in mice pretreated with an antidote are shown. (A) Mice were intravenously administered MetHbV (500, 1000, 1500, or 2000 mg / kg), empty vesicles (equivalent to 1000 mg / kg MetHbV), or saline, followed by NaCN. (B) Mice were intravenously administered NaNO2 (17 mg / kg), Na2S2O3 (61 mg / kg), NaNO2 (17 mg / kg) and Na2S2O3 (61 mg / kg) (combined), OHCbl (330 mg / kg), empty vesicles (same as above), or saline, followed by NaCN. All mice treated in (A) and (B) were observed for 7 days (n = 10 per group). [Figure 8] The survival rate of mice receiving a single oral dose of NaCN (12 mg / kg) followed by post-administration of an antidote is shown. Five minutes after NaCN administration, mice were intravenously administered MetHbV (1000 or 2000 mg / kg), MetHbV (1000 mg / kg) and Na2S2O3 (61 mg / kg) (combined), NaNO2 (17 mg / kg), Na2S2O3 (61 mg / kg), NaNO2 (17 mg / kg) and Na2S2O3 (61 mg / kg) (combined), or OHCbl (330 mg / kg). All mice were observed for 7 days (n = 10 per group). Five mice died before antidote administration. [Figure 9] The survival rates of mice receiving a single oral dose of NaCN (12 mg / kg) followed by subsequent antidote administration are shown. Ten minutes after NaCN administration, mice were intravenously administered MetHbV (1000 or 2000 mg / kg), MetHbV (1000 mg / kg) and Na2S2O3 (61 mg / kg) (combined), NaNO2 (17 mg / kg), Na2S2O3 (61 mg / kg), NaNO2 (17 mg / kg) and Na2S2O3 (61 mg / kg) (combined), or OHCbl (330 mg / kg). All mice were observed for 7 days (n = 10 per group). Five mice died before antidote administration. [Figure 10]Mice were treated with NaCN and given an antidote, and the recovery time was measured. Five minutes (A) or 10 minutes (B) after NaCN administration, mice were intravenously administered MetHbV (1000 or 2000 mg / kg), MetHbV (1000 mg / kg) and Na2S2O3 (61 mg / kg) (combination), NaNO2 (17 mg / kg), Na2S2O3 (61 mg / kg), NaNO2 (17 mg / kg) and Na2S2O3 (61 mg / kg) (combination), or OHCbl (330 mg / kg). The recovery time was then measured. All mice were observed until they recovered from the loss of righting reflex (coma). [Figure 11] The figure shows the survival rate after a single subcutaneous administration of NaHS (35 mg / kg) in mice pretreated with an antidote. The upper figure shows the administration schedule of the antidote and NaHS. The lower figure shows the time course of mouse survival rate. Mice were intravenously administered MetHbV (500 or 1000 mg / kg), NaNO2 (17 or 34 mg / kg), OHCbl (330 mg / kg), or saline, followed by NaHS administration 5 minutes later. [Figure 12] The figure shows the survival rate of mice that received a single subcutaneous dose of NaHS (35 mg / kg) followed by subsequent administration of an antidote. The upper figure shows the administration schedule of the antidote and NaHS. The lower figure shows the time course of mouse survival rate. Five minutes after the administration of NaHS to the mice, MetHbV (1000 or 1000 × 2 mg / kg), NaNO2 (34 mg / kg), and OHCbl (330 mg / kg) were administered intravenously. [Figure 13] The figure shows the survival rate after a single oral administration of NaN3 (40 mg / kg) in mice pre-administered with an antidote. The upper figure shows the administration schedule of the antidote and NaN3. The lower figure shows the time course of mouse survival rate. Mice were intravenously administered MetHbV (1000 or 1000 × 2 mg / kg), NaNO2 (17 mg / kg), OHCbl (330 mg / kg), or saline, followed by NaHS 5 minutes later. [Figure 14]The figure shows the survival rate of mice given a single oral dose of NaN3 (40 mg / kg) followed by subsequent administration of an antidote. The upper figure shows the administration schedule of the antidote and NaN3. The lower figure shows the time course of mouse survival rate. Ten minutes after NaHS administration to mice, MetHbV (500, 1000, or 1000 × 2 mg / kg), NaNO2 (17 mg / kg), OHCbl (330 mg / kg), or saline was intravenously administered. [Figure 15] The particle size distribution of stored MetHbV is shown. MetHbV suspensions at 10 g Hb / dL were stored in glass vials at 4°C, 23-28°C, and 37°C for one year. The particle size distribution of each stored MetHbV was evaluated. [Figure 16] The figure shows the survival rate of mice given a single oral dose of NaCN (12 mg / kg) followed by subsequent administration of stored MetHbV. The upper figure shows the administration schedule for stored MetHbV and NaCN. The lower figure shows the time course of mouse survival rates. Ten minutes after NaCN administration, mice were intravenously administered 1000 mg / kg of fresh MetHbV, MetHbV stored at 4°C for one year (4°C MetHbV), MetHbV stored at 23-28°C for one year (23°C MetHbV), or MetHbV stored at 37°C for one year (37°C MetHbV). [Figure 17] The figure shows the survival rate of mice that received a single subcutaneous administration of NaHS (35 mg / kg) followed by subsequent administration of stored MetHbV. The upper figure shows the administration schedule of stored MetHbV and NaHS. The lower figure shows the time course of mouse survival rate. Ten minutes after NaHS administration, mice were intravenously administered 1000 mg / kg of MetHbV (fresh MetHbV), MetHbV stored at 4°C for one year (4°C MetHbV), MetHbV stored at 23-28°C for one year (23°C MetHbV), or MetHbV stored at 37°C for one year (37°C MetHbV). [Figure 18]The figure shows the survival rate of mice given a single oral dose of NaN3 (40 mg / kg) followed by subsequent administration of stored MetHbV. The upper figure shows the administration schedule for stored MetHbV and NaN3. The lower figure shows the time course of mouse survival rates. Ten minutes after NaN3 administration, mice were intravenously administered 1000 mg / kg of fresh MetHbV, MetHbV stored at 4°C for one year (4°C MetHbV), MetHbV stored at 23-28°C for one year (23°C MetHbV), or MetHbV stored at 37°C for one year (37°C MetHbV). DETAILED DESCRIPTION OF THE INVENTION

[0015] Hereinafter, an embodiment of the present invention will be described. The first embodiment is a medicine for cyanide poisoning, hydrogen sulfide poisoning, and azide poisoning, which contains cyanide ions (CN - ), hydrogen sulfide ion (HS - ) and azide ion (N3 - ) can bind to hemoproteins, such as CN - , H.S. - and N3 - Contains hemoproteins capable of binding CN - , H.S. - and a heme derivative capable of binding N3, as an active ingredient.

[0016] Here, hemoproteins are proteins bound to porphyrin derivatives called heme, and include hemoglobin, myoglobin, and cytochrome. Hemoglobin, a typical hemoprotein, has a tetrameric structure consisting of two types of subunits (α subunits and β subunits), two of each. Each subunit of hemoglobin consists of a polypeptide called globin and heme, and heme usually contains a divalent iron ion (Fe 2+ The iron coordinated to heme is an oxidized ferric ion (Fe 3+), it is called methemoglobin (MetHb). Unlike hemoglobin, methemoglobin cannot bind to oxygen and therefore does not have the ability to transport oxygen in the body. The present inventors have found that methemoglobin is CN - , H.S. - and N3 - It has the ability to bind to CN - , H.S. - and N3 - They found that MetHb is a "heme protein capable of binding to MeHb," and investigated whether MetHb or substances containing MetHb could be used to prevent or treat cyanide poisoning, hydrogen sulfide poisoning, and azide poisoning, and demonstrated that this is indeed possible.

[0017] In this embodiment, "CN - "HS" - "Heme proteins capable of binding N3 - The term "hemoprotein capable of binding CN" also includes polymers and high molecular weight versions of the protein. - , H.S. - and N3 - When the hemoprotein that can bind to MeHb is MetHb, MetHb includes not only MetHb itself, but also intramolecularly cross-linked MetHb that prevents dissociation into its individual subunits, MetHb in which multiple MetHb molecules are intermolecularly cross-linked with glutaraldehyde or activated raffinose to increase the molecular weight of MetHb, and polymer-bound MetHb that is chemically bound to polyethylene glycol, dextran, or albumin. Hb is not limited to that purified from human blood, but also includes Hb purified from livestock (pigs, cows, etc.), other organisms (e.g., lugworms, earthworms), and genetically modified Hb. 2+ Myoglobin (Mb) and cytochromes can also be used as raw materials for hemoproteins containing heme coordinated with . Heme derivatives are chemically modified heme by covalently bonding various functional groups to create a hydrophobic environment or basic ligands, resulting in the formation of ferrous ions (Fe 2+ ) gas molecules combine to form trivalent iron ions (Fe 3+ ) when CN - For example, aqueous solutions of heme derivatives, albumin, liposomes, and micelles carrying heme derivatives can be used.

[0018] Also, "CN - , H.S. - and N3 - "Containing hemoproteins capable of binding CN" means - , H.S. - and N3 - CN is a substance containing heme proteins that can bind to - , H.S. - and N3 -When the hemoprotein capable of binding to MetHb is MetHb, examples of the MetHb-containing substance include met-erythrocytes containing methemoglobin (met-erythrocytes) and MetHb-encapsulated capsules containing MetHb. Capsule materials include polymers such as polystyrene, gum arabic, nylon, and silicone; biomaterials such as gelatin; polymers of poly-ε-caprolactam, polyethylene glycol, and biodegradable polymers such as polylactic acid and polyglycolic acid; polysaccharides; and polymer thin films prepared from materials containing copolymers of amino acid polymers. Further capsule materials include, but are not limited to, hydrogels, silica gels, polyion complexes, polymersomes, niosomes, and liposomes. MetHb-encapsulated capsules are preferably methemoglobin vesicles (MetHbV), in which MetHb is encapsulated in liposomes. In one example of a liposomal MetHb-encapsulating capsule, the structure of MetHbV consists of four components: 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol, 1,5-O-dihexadecyl-N-succinyl-L-glutamate (DHSG), and 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-PEG5000 (DSPE-PEG5000).

[0019] MetHb and MetHb-containing products can be prepared by oxidizing Hb or Hb-containing products. The following mainly describes a method for preparing MetHbV, an example of a MetHb-containing product, but MetHb can also be prepared by a similar method. MetHbV can be prepared, for example, by reacting hemoglobin vesicles prepared by known methods (e.g., Non-Patent Documents 6 and 7) with an oxidizing agent. The oxidizing agent is not particularly limited, but examples that can be used include sodium nitrite, 4-dimethylaminophenol, hydrogen peroxide, and K3[Fe(CN)6]. Specifically, a solvent (or dispersion medium, hereinafter the same) is placed in a container (glass, plastic, etc.), and an oxidizing agent is added to dissolve or disperse the oxidizing agent in the solvent. The solvent is typically, but not limited to, physiological saline. Next, hemoglobin vesicles (HbV) are added to the container containing the oxidizing agent and solvent. For example, when 1 M NaNO2 is used as the oxidizing agent, the mixing ratio of HbV to oxidizing agent (HbV:1 M NaNO2) is 10:1 to 100:1, preferably 20:1 to 80:1, and more preferably 40:1 to 60:1. The amount of solvent is sufficient to allow HbV and oxidizing agent to be thoroughly mixed. The reaction time after mixing HbV, oxidizing agent, and solvent can be adjusted by the concentration of the oxidizing agent used; the higher the oxidizing agent concentration, the shorter the reaction time. This allows HbV to react with the oxidant, converting 99% or more of HbV to MetHbV.

[0020] Thereafter, unreacted oxidizing agent is removed by ultracentrifugation, or by adding high molecular weight dextran and then by centrifugation, gel filtration (GPC), or ultrafiltration, to purify MetHbV. When ultracentrifugation is used, it may be performed once or multiple times (e.g., 2 to 3 times), depending on the specifications of the ultracentrifuge. For example, when MetHbV is adjusted at a mixing ratio (volume ratio) of HbV:1M NaNO2 = 50:1, the preferred ultracentrifugation conditions are 50,000 × g for 20 minutes. When centrifugation is performed after the addition of high-molecular-weight dextran, for example, high-molecular-weight dextran (e.g., Dextran from Leucostoc spp., Mr. 450,000-650,000) is dissolved in phosphate-buffered saline (PBS) to prepare a high-molecular-weight dextran solution. The dextran concentration of the high-molecular-weight dextran solution is 10% to 30% by weight, preferably approximately 20% by weight. Subsequently, an equal volume of PBS to the oxidized HbV dispersion and approximately 0.6 volumes of aqueous dextran solution are added to the HbV dispersion, and the mixture is centrifuged at 3000 g for approximately 20 minutes. The HbV precipitates due to the excluded volume effect of the high-molecular-weight dextran. After removing the supernatant, PBS is added to redisperse the precipitate, and excess oxidizing agent is removed. When ultrafiltration is performed after adding an oxidizing agent to HbV, an ultrafiltration membrane (e.g., Merck Millipore Biomax, cut-off Mw 1000 kDa, filtration area 0.1 m) is used. 2 The oxidized HbV is circulated at room temperature using a circulating tube. While maintaining the circulating volume, PBS is gradually added in an amount approximately six times the initial volume of HbV, and the filtrate containing the liberated oxidant is discharged. The outlet pressure of the circulating fluid is maintained at, for example, no more than 0.1 MPa. When adding an oxidizing agent to HbV and then performing gel filtration, for example, a gel filtration column packed with Sepharose CL-4B is washed with PBS and then the oxidizing agent-reacted HbV is eluted. MetHbV, which has a large particle size, flows out first, followed by the unreacted oxidizing agent, which can then be removed.

[0021] Alternatively, MetHbV can be prepared by binding NO to HbV and then contacting it with O2. Specifically, for example, an HbV dispersion is degassed with N2 and then contacted with NO to generate HbNO. Excess NO is again removed with N2, and the mixture is then contacted with O2 to prepare MetHbV.

[0022] The prepared MetHbV dispersion can be adjusted to a desired concentration and viscosity by adding an aqueous solvent whose osmotic pressure and component concentrations are physiologically acceptable.

[0023] MetHbV can also be produced by encapsulating MetHb, which is formed by oxidizing hemoglobin, into endoplasmic reticulum. The encapsulation of MetHb into endoplasmic reticulum can be carried out according to the methods described above (Non-Patent Documents 6 and 7, etc.). MetHbV has a negative zeta potential. The zeta potential of MetHbV is preferably -16.00 to -10.00, more preferably -15.00 to -11.00. The particle size of MetHbV is preferably 100 to 300 nm, more preferably 200 to 300 nm, and even more preferably 240 to 250 nm. The zeta potential and particle size can be measured by dynamic light scattering. The maximum absorption wavelength of the Soret band absorption spectrum of MetHbV is preferably 400 to 410 nm, typically around 408 nm. The absorption spectrum of MetHbV can be measured by ultraviolet-visible spectroscopy. CN - , H.S. - and N3 - By passing through the endoplasmic reticulum (lipid bilayer membrane) of MetHbV, it can bind to MetHb in the endoplasmic reticulum, thereby exerting the effect of preventing or treating various types of MetHb poisoning.

[0024] When the pharmaceutical composition according to this embodiment is administered to a subject (a person at risk of developing each of the poisonings) before the onset of cyanide poisoning, hydrogen sulfide poisoning, or azide poisoning, it can suppress the onset of each of the poisoning symptoms. Furthermore, when the pharmaceutical composition according to this embodiment is administered to a patient (a person who has developed cyanide poisoning, hydrogen sulfide poisoning, or azide poisoning) after the onset of poisoning, it can ameliorate each of the poisoning symptoms and restore the patient to a normal state. Therefore, the pharmaceutical composition according to this embodiment can be used as a therapeutic and preventive agent for cyanide poisoning, hydrogen sulfide poisoning, and azide poisoning.

[0025] The pharmaceutical composition according to this embodiment contains an active ingredient (CN - , H.S. - and N3 -Alternatively, the active ingredient may be administered as a pharmaceutical composition containing one or more common pharmaceutical additives in addition to the active ingredient. The medicament and pharmaceutical composition (hereinafter also referred to as "medicine, etc.") according to this embodiment may contain, in addition to the active ingredient, an existing active ingredient that has been confirmed to be effective in improving the symptoms of cyanide poisoning, hydrogen sulfide poisoning, and azide poisoning, such as NaSO or hydroxocobalamin. The amount of the existing active ingredient is, for example, 1% by weight or more, 90% by weight or less, or 50% by weight or less, based on the weight of the active ingredient.

[0026] The dosage form of the pharmaceutical or other drug according to this embodiment is not particularly limited, and examples thereof include infusions and injections. Liquid preparations such as infusions and injections can be prepared by dispersing, for example, MetHb or a substance containing MetHb as the active ingredient in physiological saline, and adding a buffer or preservative as necessary. The preferred viscosity range for infusions and injections is 1.0 to 5.0 cP. When dispersed in physiological saline to adjust the viscosity within this range, for example, when MetHbV is used as the active ingredient, the concentration is preferably 15 g / dL or less, more preferably 10 g / dL or less. When the concentration of MetHbV when dispersed in physiological saline is within this range, the viscosity of the MetHbV dispersion can be appropriately adjusted, making it easy to administer. Furthermore, when MetHbV is contained in the pharmaceutical composition of this embodiment, it may be MetHbV prepared just before use, or may be MetHbV that has been prepared and then stored. The storage period of MetHbV may be as long as, for example, about one year, and the temperature during storage of MetHbV may be, for example, from 0°C to 40°C, preferably from 4°C to 37°C.

[0027] The type of formulation additive used in the production of the medicament, etc. according to this embodiment, the ratio of the formulation additive to the active ingredient, and the production method of the medicament, etc. can be appropriately selected by a person skilled in the art depending on the form of the medicament, etc. As the formulation additive, an inorganic or organic substance, or a solid or liquid substance can be used, and it can be blended in an amount of 1% by weight to 90% by weight of the active ingredient.

[0028] The dosage and frequency of administration of the medicine etc. according to this embodiment are not particularly limited, and can be appropriately selected at the discretion of a physician depending on conditions such as the degree of progression of the symptoms of cyanide poisoning, hydrogen sulfide poisoning or azide poisoning in the subject of treatment or prevention, and the body weight and age of the subject of administration. When used as an injection, for example, a daily dose of 0.001 to 1000 mg (converted to hemoglobin vesicle weight) may be administered continuously or intermittently to an adult. The administration may be performed as soon as possible after the onset of cyanide poisoning, hydrogen sulfide poisoning, or azide poisoning is confirmed, or as a preventative administration before the patient is placed in a situation where cyanide poisoning, hydrogen sulfide poisoning, or azide poisoning may occur.

[0029] The pharmaceutical agent according to the present embodiment may be provided in the form of a kit together with instructions for administration, etc. In the kit, the pharmaceutical agent is supplied sealed in a container that effectively maintains the activity of the constituent components of the pharmaceutical agent for a long period of time, is not adsorbed to the inside of the container, and is made of a material that does not alter the constituent components. For example, the pharmaceutical agent may be supplied sealed in a glass or plastic container. The kit may also include instructions for use, which may be printed on paper or stored on an electromagnetically readable medium such as a CD-ROM or DVD-ROM and provided to the user.

[0030] A second embodiment is a method for treating or preventing cyanide poisoning, hydrogen sulfide poisoning, and azide poisoning, comprising administering to a subject a compound of formula (I) comprising: - , H.S. - and N3 - A hemoprotein capable of binding CN- , H.S. - and N3 - Contains hemoproteins capable of binding CN - , H.S. - and N3 - The preventive or therapeutic method comprises administering a medicine or pharmaceutical composition for cyanide poisoning, which contains as an active ingredient one or more selected from the group consisting of heme derivatives capable of binding to cyanide, to a patient suffering from cyanide poisoning or a person who may develop cyanide poisoning. Here, "treatment" means preventing or alleviating the progression and worsening of the condition in patients who have already developed cyanide poisoning, hydrogen sulfide poisoning, or azide poisoning, and refers to treatment aimed at preventing or alleviating the progression and worsening of cyanide poisoning that has already developed. Furthermore, "prevention" means preventing the onset of cyanide poisoning, hydrogen sulfide poisoning, or azide poisoning in patients who are at risk of developing the condition, and is a treatment aimed at preventing the onset of cyanide poisoning in advance.

[0031] The subject of treatment and prevention is a "mammal." Here, "mammal" means any animal classified as a mammal, including, but not limited to, humans, pet animals such as dogs and cats, and livestock animals such as cows, pigs, sheep, and horses. A particularly preferred "mammal" is a human.

[0032] The third embodiment is a CN for producing a medicine or pharmaceutical composition for cyanide poisoning, hydrogen sulfide poisoning, and azide poisoning. - , H.S. - and N3 - A hemoprotein capable of binding CN - , H.S. - and N3 - Contains hemoproteins capable of binding CN - , H.S. - and N3 - For a detailed description of this embodiment, see the description of the first embodiment.

[0033] Where this specification is translated into English and includes the singular words "a," "an," and "the," these shall include the plural as well as the singular, unless the context clearly indicates otherwise. [Example]

[0034] Examples are given below, but the scope of the present invention is not limited to these examples. In the examples, "%" means "% by weight" unless otherwise specified. 1. Materials and Experimental Methods 1-1. Experimental samples HbV and empty vesicles were prepared based on previous reports (see Non-Patent Documents 6 and 7). Commercially available special-grade reagents and solvents were used, and ultrapure water was used as the solvent.

[0035] 1-2. Preparation of MetHbV MetHbV was prepared by oxidizing HbV. Sodium nitrite (NaNO2) was dissolved in saline, and 100 μL of the resulting 1 M solution was added to 5 mL of HbV solution (10 g Hb / dL) and allowed to react for 24 hours (Non-Patent Document 8). After the reaction, the percentage of methemoglobin-containing Hb was measured (described below), and only samples with a concentration of 95% or higher were used. The prepared MetHbV solution was washed three times with saline by ultracentrifugation (50,000 g, 30 min) (Non-Patent Document 9) to remove excess NaNO2. MetHbV was dispersed in saline to adjust to the final target concentration.

[0036] 1-3. Observation using a Transmission Electron Microscope (TEM) MetHbV (45 μM or 0.3 g Hb / dL) was dropped onto a TEM grid and negatively stained with 2% samarium acetate. After removing excess liquid, the grid was observed using a JEOL JIM-1230 microscope at an accelerating voltage of 80 kV.

[0037] 1-4. Measurement of zeta potential The ζ potential of MetHbV was measured by diluting it with saline to 0.1 g Hb / dL and using dynamic light scattering (DLS) (Otsuka Electronics Co., Ltd., ELSZ-2000Z).

[0038] 1-5. Particle size measurement The particle size and polydispersity index (PDI) of MetHbV were measured by diluting the MetHbV sample with saline to 0.1 g Hb / dL using a Malvern Panalytical Zetasizer Nano Z.

[0039] 1-6. Measurement of absorption spectrum transition MetHbV (5.2 μM) was mixed with NaCN (52 μM), and the change in spectrum was measured using Infinite® M1000 PRO (manufactured by TECAN).

[0040] 1-7. Preparation of MetHbV for cell experiments The preparation procedure was the same as that for MetHbV used in the physical property evaluation, but RPMI-1640(-) was used instead of saline for dilution and washing.

[0041] 1-8. Preparation of empty endoplasmic reticulum Empty vesicles (EVs) were diluted in an excess amount of RPMI-1640(-) and stirred for 24 hours to replace the saline in the EVs. The substituted empty EV solution was ultracentrifuged (100,000 g, 1 h) (Non-Patent Document 10) and then dispersed in RPMI-1640(-) to adjust the final concentration.

[0042] 1-9. Preparation of metHb MetHb was prepared by oxidizing oxyHb. Sodium nitrite (NaNO2) was dissolved in RPMI-1640, and 100 μL of the resulting solution was added to 5 mL of Hb solution (10 g / dL) at 1 M concentration and allowed to react for 24 hours. After the reaction, the percentage of Hb converted to metHb was measured (described below), and only samples with a concentration of 95% or higher were used. The prepared metHb solution was filtered (10 kDa) to remove excess NaNO2. MetHb was dissolved in RPMI-1640 to adjust to the final target concentration.

[0043] 1-10. Preparation of OxyHbV OxyHbV was prepared by bubbling O2 gas through the HbV solution for 5 minutes. After ultracentrifugation, OxyHbV was dissolved in RPMI-1640 to the final target concentration.

[0044] 1-11. Preparation of met-erythrocytes Blood was collected from ddY mice and centrifuged (3000 rpm, 20 min) to remove plasma and leukocyte membranes. The resulting red blood cells were resuspended in an equal volume of saline. 100 μL of 1 M NaNO2 solution was added to 5 mL of the red blood cell suspension and gently stirred for 10 minutes to prepare met-erythrocytes. The percentage of methemoglobin (Hb) in the red blood cells was measured using a blood gas analyzer (Siemens Rapid Point 500). Those with a concentration of 95% or higher were used as samples. The samples were then washed with saline and centrifuged (3000 rpm, 20 min) three times to prepare washed met-erythrocytes. Hb concentration was measured using a Fuji Dry Chem (Fuji Film Wako) analyzer and adjusted to the desired concentration with saline.

[0045] 1-12.Cell culture PC-12 cells, a rat adrenal medulla pheochromocytoma, were purchased from the JCRB Cell Bank of the National Institutes of Biomedical Innovation, Health and Nutrition. They were seeded onto collagen-coated cell dishes (Iwaki, φ=100 mm) in RPMI-1640 medium (hereafter referred to as RPIMI-1640(+)) containing 5% (v / v) heat-inactivated fetal bovine serum (FBS), 10% horse serum, and antibiotics (penicillin 100 unit / mL, streptomycin 100 μg / mL). They were then cultured at 37°C under 5% CO2. After reaching semi-confluence, the cells were treated with trypsin and centrifuged (1000 rpm, 5 minutes), resuspended in RPMI-1640(+), and passaged.

[0046] 1-13. Cytotoxicity test PC-12 cells were grown in a collagen-coated 96-well culture plate (Sumitomo Bakelite) at 2 × 10 4 Cells were seeded at 100 μL per well and cultured for 24 hours to allow adhesion. After removing the medium, 50 μL of NaCN solution (0, 2.5, 5, 10, 15, 20, 30, 40, 80, and 160 mM) dissolved in RPMI-1640(-) was added to each well. Immediately after, 50 μL of each antidote was added to each well to adjust the final NaCN concentration to 0, 1.25, 2.5, 5, 7.5, 10, 15, 20, 40, and 80 mM, respectively. The plates were then cultured at 37°C under 100% humidified air for 1 hour.

[0047] 1-14. Antidote treatment group To confirm dose-dependent effects, six doses were added to all groups to achieve or correspond to Hb concentrations of 0, 2.5, 5, 10, 15, and 20 g / dL (0, 0.39, 0.78, 1.6, 2.3, and 3.1 mM). To evaluate efficacy, two groups were included: the MetHbV group, which was designed to evaluate efficacy; the metHb group and the metRBC group, which used a nitrite drug (sodium nitrite), which has already been shown to be effective, and the OHCbl group, which has a different mechanism of action. Two negative control groups were also included: the OxyHbV group and the sodium thiosulfate group. Finally, empty vesicles containing only lipid bilayers without Hb were added, each containing an amount of lipid equivalent to embedding Hb, for a total of seven groups.

[0048] 1-15. Cell viability test After culturing the cells for 1 hour, the NaCN and antidote mixture was removed, and the wells were washed three times with RPMI-1640(-). 100 μL of RPMI-1640 was added, followed by 10 μL of CCK-8 (Cell Counting Kit-8, Dojin Chemical Laboratory). After culturing for 2 hours, the absorbance at 450 nm was measured using a microplate reader (Infinite M1000, Tecan).

[0049] 1-16.EC 50 Calculation of Using the results obtained from the cell viability test, the EC (effective concentration) for each group was calculated with reference to the report by Gu et al. (Non-Patent Document 10).

[0050] 1-17. Laboratory animals 1-17-1. Mouse model for cyanide poisoning Eight-week-old ddY female mice (25-27 g) were purchased from Japan SLC Co., Ltd. and used in the experiment after one week of preliminary breeding. 1-17-2. Mice for hydrogen sulfide and azide poisoning models Eight-week-old ddY female mice (25-27 g) were purchased from Japan SLC Co., Ltd. and used in the experiment after one week of preliminary breeding.

[0051] 1-18. Study using a mouse model of cyanide poisoning 1-18-1. Creation of a mouse model of cyanide poisoning After fasting for 4 hours, ddY mice were orally administered a solution of NaCN dissolved in saline at a standardized dose of 10 mL / kg using a probe (Natsume, 23G) under unanesthetized conditions.

[0052] 1-18-2. Setting up the antidote pre-administration treatment group To confirm the dose-dependent effect, four MetHbV groups were established: 500, 1000, 1500, and 2000 mgHb / kg. A group of empty vesicles, consisting of only lipid bilayers without encapsulated Hb, was also established. The lipid content of the empty vesicles was set to be equivalent to the vesicle volume at 1000 mg / kg of MetHbV. A control group was established, receiving an equivalent volume of saline. Four control groups were established using existing drugs with confirmed efficacy: NaNO2 (17 mg / kg), NaNO2 (17 mg / kg) + Na2S2O3·5H2O (61 mg / kg), Na2S2O3·5H2O (61 mg / kg), and OHCbl (330 mg / kg), for a total of 10 groups. The dose of the existing drugs was set to be equivalent in molar ratio to the oral administration of NaCN. In other words, the LD50 of NaCN in mice was calculated. 90 :12 mg / kg, and all existing drugs were standardized to 0.245 mmol / kg.

[0053] 1-18-3. Preparation of a mouse model of cyanide poisoning by pre-administration of an antidote Each antidote was administered to mice via the tail vein at a uniform dose of 10 mL / kg under 3% isoflurane inhalation anesthesia. After administration of each antidote, the anesthesia was promptly terminated and the mice were awakened. Five minutes later, NaCN solution was administered via LD 50 (8.5 mg / kg) or LD 90 The animals were orally administered at a dose of 12 mg / kg. Animals that did not return to a fully awake state within 5 minutes due to the effects of anesthesia were not used in this study. The awake state was confirmed by the recovery of reflex responses according to the definition of awake below.

[0054] 1-18-4. Setting of antidote post-administration treatment group The MetHbV group consisted of two groups: one receiving 1000 mg / kg and the other receiving 1000 × 2 mg / kg (20 mL / kg, a double dose) (total 2000 mgHb / kg). A MetHbV 1000 mg / kg + Na2S2O3·5H2O (61 mg / kg) group was also included as a combination treatment with existing medications. Similar to the pretreatment group, four control groups were also included: the NaNO2 group, the NaNO2 + Na2S2O3·5H2O group, the Na2S2O3·5H2O group, and the OHCbl group, for a total of seven groups.

[0055] 1-18-5. Preparation of a mouse model of cyanide poisoning after antidote administration NaCN solution was administered to mice 90 After oral administration at a dose of 12 mg / kg, each antidote was administered via the tail vein under coma conditions 5 or 10 minutes later.

[0056] 1-18-6. Measurement of time from antidote administration to awakening Each antidote was administered to mice suffering from coma due to cyanide intoxication, and the time from administration of each antidote until the mice became awake was measured. Coma was defined as the loss of the righting reflex, and the mice were placed in a supine position on a board. The time until they were no longer able to return to a normal prone position was measured (Non-Patent Document 11). Awake was defined as a state in which the righting reflex had recovered and was functioning after coma. To avoid false positive reactions due to reflexes such as convulsions, the same trial was performed twice for each animal.

[0057] 1-19. Study using a mouse model of hydrogen sulfide poisoning 1-19-1. Pre-administration of antidote Each antidote was administered intravenously to 9-week-old ddY female mice under isoflurane anesthesia 5 minutes before (-5 minutes) hydrogen sulfide administration. 35 mg / kg sodium hydrosulfide (NaHS) dissolved in saline was administered subcutaneously, and the survival rate was tracked up to 60 minutes after NaHS administration. Each group had n=10 mice. 35 mg / kg NaHS was the 90% lethal dose (LD 90 The groups administered each antidote and saline (control) were set as follows: MetHbV group: 500 mg Hb / kg (5 mL / kg) 1000 mg Hb / kg (10 mL / kg) Sodium nitrite (NaNO2) administration group: 17 mg / kg (10 mL / kg), 34 mg / kg (10 mL / kg) Hydroxocobalamin (OHCbl) administration group: 330 mg / kg (10 mL / kg) Saline (physiological saline) administration group: 10 mL / kg

[0058] 1-19-2. Post-treatment administration of antidote Nine-week-old ddY female mice were subcutaneously administered 35 mg / kg of sodium hydrosulfide (NaHS) dissolved in saline. Five minutes after NaHS administration, each antidote was administered intravenously under isoflurane anesthesia, and survival rates were monitored up to 60 minutes after administration. Each group consisted of 10 mice. The antidote administration groups were as follows: MetHbV administration group: 1000 mg Hb / kg (10 mL / kg) 1000×2 mg Hb / kg (20 mL / kg) Sodium nitrite (NaNO2) administration group: 34 mg / kg (10 mL / kg) Hydroxocobalamin (OHCbl) administration group: 330 mg / kg (10 mL / kg)

[0059] 1-20. Study using a mouse model of azide poisoning 1-20-1. Pre-administration of antidote Each antidote was administered intravenously to 9-week-old ddY female mice under isoflurane anesthesia 5 minutes before (-5 minutes) administration of sodium azide (NaN3). Sodium azide (NaN3, 40 mg / kg) dissolved in saline was administered orally, and the survival rate was tracked up to 120 minutes after administration. Each group had n=10 mice. 40 mg / kg of NaN3 was approximately 90% lethal dose (LD 90 The groups administered each antidote and saline (control) were set as follows: MetHbV administration group: 1000 mg Hb / kg (10 mL / kg) 1000 x 2 mg Hb / kg (20 mL / kg) Sodium nitrite (NaNO2) administration group: 17 mg / kg (10 mL / kg) Hydroxocobalamin (OHCbl) administration group: 330 mg / kg (10 mL / kg) Saline (physiological saline) administration group: 10 mL / kg

[0060] 1-20-2. Post-treatment administration of antidote Nine-week-old female mice were orally administered 40 mg / kg of sodium azide (NaN3) dissolved in saline. 10 minutes after NaN3 administration, each antidote was administered intravenously under isoflurane anesthesia, and survival rates were assessed up to 120 minutes after NaN3 administration. Each group consisted of 10 mice. The groups administered each antidote were as follows: MetHbV group: 500 mg Hb / kg (5 mL / kg) 1000 mg Hb / kg (10 mL / kg) 1000 x 2 mg Hb / kg (20 mL / kg) Sodium nitrite (NaNO2) administration group: 17 mg / kg (10 mL / kg) Hydroxocobalamin (OHCbl) administration group: 330 mg / kg (10 mL / kg) Saline (physiological saline) administration group: 10 mL / kg

[0061] 1-21. Confirmation of the stability and detoxification effect of stored MetHbV 1-21-1. Stability of stored MetHbV Storage conditions for MetHbV MetHbV suspensions at 10 g Hb / dL were placed in glass vials and stored at 4°C (refrigerated), 23-28°C (room temperature), or 37°C (warm) for up to 1 year (12 months). Physicochemical properties of MetHbV stored for a long period (1 year (12 months)) Particle size, polydispersity index (PDI), and ζ-potential were measured using a Zetasizer Nano ZS (Malvern Panalytical Ltd. UK) and an ELSZ2KOP zeta potential analyzer (Otsuka Electronics, Japan).

[0062] 1-21-2. Confirmation of effectiveness against cyanide poisoning Nine-week-old ddY female mice were orally administered 12 mg / kg of sodium cyanide (NaCN) dissolved in saline. Ten minutes after NaCN administration, preserved MetHbV was administered intravenously, and the survival rate was assessed up to 60 minutes after administration. Each group had 10 mice. The treatment groups were as follows: Stored at 4°C. MetHbV administration group: 1000 mg Hb / kg (10 mL / kg) Store at 23-28°C. MetHbV administration group: 1000 mg Hb / kg (10 mL / kg) Stored at 37°C MetHbV administration group: 1000 mg Hb / kg (10 mL / kg)

[0063] 1-21-3. Confirmation of effectiveness against hydrogen sulfide poisoning Nine-week-old ddY female mice were subcutaneously administered 35 mg / kg of sodium hydrosulfide (NaHS) dissolved in saline. Five minutes after NaHS administration, preserved MetHbV was administered intravenously, and survival rates were assessed up to 60 minutes after administration. Each group consisted of 10 mice. The treatment groups were as follows: Stored at 4°C. MetHbV administration group: 1000 mg Hb / kg (10 mL / kg) Store at 23-28°C. MetHbV administration group: 1000 mg Hb / kg (10 mL / kg) Stored at 37°C MetHbV administration group: 1000 mg Hb / kg (10 mL / kg)

[0064] 1-21-4. Confirmation of effectiveness against azide poisoning Nine-week-old ddY female mice were subcutaneously administered 40 mg / kg of sodium azide (NaN3) dissolved in saline. Ten minutes after NaN3 administration, preserved MetHbV was administered intravenously, and the survival rate was assessed up to 120 minutes after administration. Each group consisted of 10 mice. The treatment groups were as follows: Stored at 4°C. MetHbV administration group: 1000 mg Hb / kg (10 mL / kg) Store at 23-28°C. MetHbV administration group: 1000 mg Hb / kg (10 mL / kg) Stored at 37°C MetHbV administration group: 1000 mg Hb / kg (10 mL / kg)

[0065] 1-22. Ethical Considerations All animal experiments were performed under approval numbers 18013-(0), 20046-(0), and 20067-(0) by the Keio University Institutional Animal Care and Use Committee.

[0066] 2.Results 2-1. Preparation of MetHbV and evaluation of its physicochemical properties First, MetHbV was prepared and observed by TEM. The physicochemical properties of the particles were evaluated by DLS (Digital Light Spectroscopy) to determine particle size distribution (PDI) and zeta potential. First, MetHbV was produced by converting the hemoglobin encapsulated inside HbV into metHb using the oxidizing agent sodium nitrite. TEM images showed that the particles were spherical unilamellar liposomes (Figure 1). Next, the physicochemical properties of the obtained MetHbV were evaluated, and it was found to be a monodisperse particle with an average particle diameter of 240.7 nm and a ζ potential of -13.2 mV (Figure 2 and Table 1). [Table 1]

[0067] 2-2. Evaluation of MetHbV formation and cyanide binding by spectral changes Spectral changes of MetHbV were evaluated by UV-visible spectroscopy. HbV is stored under N2-exchange conditions and exists as deoxyHbV. Upon exposure to air, it immediately binds with oxygen in the air and exhibits a peak at 415 nm, which is attributable to oxyHbV. Furthermore, the addition of sodium nitrite to the HbV solution shifted the peak wavelength to 408 nm, confirming the formation of MetHbV. Furthermore, when MetHbV was mixed with sodium cyanide solution, the MetHbV peak (408 nm) shifted to a peak at 420 nm, which is attributable to cyanide-bound MetHbV, demonstrating the cyanide-bound nature of MetHbV (Figure 3).

[0068] 2-3. Cell evaluation (in vitro) 2-3-1. Evaluation of cytotoxicity of MetHbV To evaluate the cytotoxicity of MetHbV, we added various concentrations of MetHbV to PC12 cells and compared cell viability. As a result, the group added with 20 g / dL of MetHbV showed a survival rate of approximately 95%, demonstrating almost no toxicity (Figure 4).

[0069] 2-3-2. Cytoprotective effect of MetHbV against cyanide poisoning MetHbV is CN -To investigate whether MeHbV binds to CcOX and competitively blocks its binding to CcOX in cells, we added sodium cyanide (NaCN) to cells and then compared cell viability after adding each antidote. The NaCN-only control group showed a cell viability of approximately 49% at 10 mM NaCN, while the MeHbV 2.5 g / dL group showed a significantly increased viability of approximately 87%. Furthermore, we examined the concentration-dependent increase in MeHbV relative to NaCN. At 20 mM NaCN, the MeHbV group showed a concentration-dependent increase in viability: 39% at 2.5 g / dL, 47% at 5 g / dL, and 77% at 10 g / dL. On the other hand, in comparison with MetHbV, an OxyHbV group in which hemoglobin in the liposomes was not methemoglobinized, and an empty vesicle (EV) group in which hemoglobin was not encapsulated in the liposomes were placed. These groups showed the same behavior as the control group, and CN - These results indicate that MetHbV exhibits cytoprotective (detoxifying) properties, and that the mechanism of action is due to the coordination of cyanide to methemoglobin in MetHbV (Figure 5).

[0070] 2-3-3. Comparison of the cytoprotective effects of MetHbV with existing drugs against cyanide poisoning To examine the efficacy of MetHbV against cyanide, we performed the same procedure as above with existing drugs currently in clinical use and compared cell viability. The results showed that met-erythrocytes and metHb converted with sodium nitrite had significantly higher viability than the control group treated with NaCN alone. Hydroxocobalamin (OHCbl), which detoxifies hemoglobin through a different mechanism of action and is set at an equal molar ratio to hemoglobin, also had higher viability than the control group. On the other hand, the addition of sodium thiosulfate (NaSO) showed similar behavior to the control group and was ineffective. The effective met-erythrocyte, metHb, and OHCbl groups exhibited similar behavior to the MetHbV group at all concentrations. These results demonstrate that MetHbV has comparable efficacy to existing drugs in cell-based evaluations (Figure 6).

[0071] 2-3-4. Comparison of cyanide effective concentration (EC) in the presence of each antidote To compare the toxicity of sodium cyanide to PC12 cells in the presence of each antidote, the 50% effective concentration (EC 50 As a result, when exposed to NaCN alone without the addition of an antidote, EC 50 On the other hand, the group with MetHbV 2.5 g / dL added had an EC 50 The EC value was 17.9 mM, confirming that a sufficient effect could be obtained even at a low concentration of 2.5 g / dL. In addition, met red blood cells and metHb, which have a similar mechanism of action during detoxification, also showed a significant increase in EC value in the 2.5 g / dL addition group. 50 The EC values ​​were 20.6 mM and 16.9 mM, respectively, demonstrating similar effects. OxyHbV and empty endoplasmic reticulum, which do not exhibit detoxification ability, did not show any significant effects despite the addition of a high concentration of 20 g / dL. 50 were only 10.3 mM and 10.7 mM, respectively (Table 2). [Table 2]

[0072] 2-4. Evaluation using animals (in vivo) 2-4-1. Study using a mouse model of cyanide poisoning 2-4-1-1. Evaluation of the effectiveness of MetHbV as a preventive administration (pre-administration) To verify the effectiveness of MetHbV against cyanide poisoning in vivo, we evaluated it using ddY female mice. After MetHbV administration, the median lethal dose (LD 50 When 8.5 mg / kg of NaCN, the 90% lethal dose (LD ), was orally administered, all animals (n=5 each) in the groups administered MetHbV at 500 mg / kg, 1000 mg / kg, and 2000 mg / kg, respectively, survived (data not shown). 90 When the study was performed at 12 mg / kg NaCN (the standard dose), 90% of the MetHbV 1000 mg / kg group survived. However, the higher MetHbV 1500 mg / kg and 2000 mg / kg groups only had a 70% survival rate. Furthermore, 90% of the MetHbV 500 mg / kg group died (Fig. 7a). In contrast, the combination therapy with sodium nitrite and sodium thiosulfate (NaNO2 + Na2S2O3), an existing drug currently in clinical use, and the OHCbl monotherapy group had a 90% and 50% survival rate, respectively (Fig. 7b). These results demonstrate that MetHbV is as effective as or more effective than existing drugs in the treatment of cyanide poisoning in vivo.

[0073] 2-4-1-2. Evaluation of the efficacy of MetHbV in post-administration simulating clinical conditions As described in 2-4-1-1, the effectiveness of MetHbV in pre-administration was demonstrated, so we investigated the effectiveness of MetHbV in post-administration to living organisms in a state of cyanide poisoning. 90Each antidote was administered 5 minutes after the administration of 1000 mg / kg of NaCN, and survival rates were assessed. All animals survived in the 1000 mg / kg MetHbV group and the 1000 × 2 mg / kg group (double the 1000 mg / kg MetHbV dose). All animals also survived in the group receiving 1000 mg / kg MetHbV in combination with Na2S2O3 (61 mg / kg). On the other hand, the group receiving the combination of sodium nitrite and sodium thiosulfate (NaNO2 + Na2S2O3) had an 80% survival rate (Figure 8). Furthermore, in the OHCbl group, survival was 90% at 1 hour after NaCN administration, but this rate dropped to 70% by day 7. Similarly, in the NaNO2 alone group, survival was 50% at 1 hour, but dropped to 20% by day 7 (data not shown).

[0074] Also, LD 90 We also evaluated survival rates after each antidote was administered 10 minutes after the administration of NaCN. All animals survived in the MetHbV 1000 × 2 mg / kg group and the MetHbV 1000 mg / kg + Na2S2O3 (61 mg / kg) combined group. The control OHCbl group had a survival rate of 60% (Figure 9). In contrast, all animals in the MetHbV 1000 mg / kg group survived 1 hour after administration, but the survival rate dropped to 80% by day 7. Similarly, in the NaNO2-only group, survival dropped from 30% at 1 hour to 0% by day 7 (data not shown).

[0075] Furthermore, we tracked the time to recovery from intoxication after NaCN administration in the rats used to evaluate survival rates after the antidote administration described above. Because NaCN was administered before the antidote, symptoms of intoxication appeared quickly. Therefore, the antidote was administered 5 or 10 minutes after NaCN administration, and the time from coma to recovery was measured as an indicator of symptom recovery. The shortest recovery time was approximately 1 minute for MetHbV (1000 × 2 mg / kg) at 5 minutes after NaCN administration. The MetHbV 1000 mg / kg group and the MetHbV 1000 mg / kg + Na2S2O3 (61 mg / kg) combination group were 1.1 minutes and 2 minutes, respectively. In contrast, the conventional drug groups, sodium nitrite and sodium thiosulfate (NaNO2 + Na2S2O3) combination therapy and the OHCbl monotherapy group were 2.5 minutes and 4.8 minutes, respectively (Figure 10A). Furthermore, the shortest recovery time 10 minutes after administration was approximately 2 minutes for MetHbV (1000 × 2 mg / kg), similar to that 5 minutes after administration. The recovery times were 3.6 minutes and 3 minutes for the MetHbV 1000 mg / kg group and the MetHbV 1000 mg / kg + Na2S2O3 (61 mg / kg) combination group, respectively. Meanwhile, the recovery times for the conventional drug combination therapy (NaNO2 + Na2S2O3) and the OHCbl monotherapy group were 3 minutes and 4.6 minutes, respectively (Figure 10B). These results demonstrate that MetHbV contributes to recovery from cyanide intoxication, and its effectiveness is comparable to or greater than that of conventional drugs.

[0076] 2-4-2. Study using a mouse model of hydrogen sulfide poisoning First, we evaluated the survival rate to examine the effectiveness of prophylactic (pre-administration) administration of MetHbV against hydrogen sulfide poisoning. As shown in Figure 11, the effectiveness of MetHbV as a prophylactic administration against fatal hydrogen sulfide poisoning was confirmed, and its effect was dose-dependent. Furthermore, it was more effective than sodium nitrite or hydroxocobalamin. Next, we evaluated the survival rate to examine the efficacy of MetHbV in the treatment (post-hoc) of hydrogen sulfide poisoning. As shown in Figure 12, the therapeutic effect of MetHbV on fatal hydrogen sulfide poisoning was confirmed, and the effect was dose-dependent. Furthermore, it was more effective than sodium nitrite or hydroxocobalamin.

[0077] 2-4-3. Study using azide poisoning model mice First, we evaluated the survival rate to examine the effectiveness of prophylactic (pre-administration) administration of MetHbV against azide poisoning. As shown in Figure 13, MetHbV demonstrated a preventive effect against fatal azide poisoning, and the effect was dose-dependent. Furthermore, the effect was greater than that of sodium nitrite or hydroxocobalamin. Next, we evaluated the survival rate to examine the effectiveness of MetHbV treatment (post-treatment) for azide poisoning. As shown in Figure 14, the therapeutic effect of MetHbV on fatal azide poisoning was confirmed, and the effect was dose-dependent. Furthermore, the effect was greater than that of sodium nitrite or hydroxocobalamin.

[0078] 2-5. Stability and detoxification effect of stored MetHbV 2-5-1. Stability of stored MetHbV When using MetHbV as an antidote for acute poisoning, it is desirable to be able to start treatment quickly. To achieve this, it is necessary to keep it on hand in locations where it is likely to be used, such as ambulances and chemical plants, without requiring strict storage conditions. Therefore, to investigate whether MetHbV can be used as a practical, "ready-to-use" antidote for acute poisoning, we evaluated its long-term stability under various temperature conditions. The results are shown in Table 3 and Figure 15. MetHbV was stored in suspension at three different temperatures for six months and one year: refrigerated (4°C), room temperature (23-28°C), and warm (37°C). Under all conditions, its physicochemical properties were virtually unchanged from those of fresh MetHbV. This suggests that MetHbV remains stable as liposomes even after long-term storage (Table 3 and Figure 15). [Table 3]

[0079] 2-5-2. Detoxification effect of stored MetHbV The results of examining the therapeutic effect of MetHbV after long-term storage (1 year (12 months)) on cyanide poisoning are shown in Figure 16. In a mouse model of fatal cyanide poisoning, all preserved MetHbV exhibited therapeutic effects (extension of survival time) equivalent to those of fresh MetHbV. Similarly, in hydrogen sulfide poisoning and azide poisoning model mice, all preserved MetHbVs showed therapeutic effects (extension of survival time) equivalent to those of fresh MetHbV (Figure 17: hydrogen sulfide poisoning, Figure 18: azide poisoning). [Industrial Applicability]

[0080] The medicine of this embodiment has therapeutic and preventive effects against cyanide poisoning, hydrogen sulfide poisoning, and azide poisoning, and is therefore expected to be useful in the medical field.

Claims

[Claim 1] A medicine for treating or preventing cyanide poisoning, hydrogen sulfide poisoning, and azide poisoning, comprising: CN - , H.S. - and N 3- the pharmaceutical comprises, as an active ingredient, a hemoprotein containing substance capable of binding to methemoglobin, the hemoprotein being methemoglobin, the hemoprotein containing substance being a capsule encapsulating methemoglobin, and the capsule being made of at least one selected from the group consisting of a liposome, a polymersome, and a thin polymer film.

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