Hydrogen sulfide detoxifying agent

Abstract

The purpose of the present invention is to provide a hydrogen sulfide detoxifying agent.　The present invention provides a hydrogen sulfide detoxifying agent containing, as an active ingredient, an inclusion complex formed by the inclusion of a water-soluble metal porphyrin in a cyclodextrin dimer represented by chemical formula (1).

Description

Hydrogen sulfide antidote

[0001] The present invention relates to a hydrogen sulfide antidote.

[0002] Hydrogen sulfide poisoning frequently occurs in Japan at construction sites such as factories and underground facilities. Treatment methods include hyperbaric oxygen therapy and the use of nitrite preparations to generate oxidized hemoglobin, which then captures hydrogen sulfide. The former has limitations in terms of equipment and the inability to perform other medical procedures during treatment, and the effectiveness of the latter has been called into question in recent years, so an effective treatment has not yet been established.

[0003] The present inventors have previously studied inclusion complexes formed by cyclodextrin dimers encapsulating water-soluble metalloporphyrins and discovered that these complexes have high affinity for oxygen (O2) and carbon monoxide (CO), with an affinity for CO more than 100 times greater than that of hemoglobin (see Patent Document 1 and Non-Patent Documents 1 and 2). Patent Document 2 discloses a carbon monoxide scavenger containing as an active ingredient an inclusion complex formed by cyclodextrin dimers encapsulating water-soluble metalloporphyrins represented by a specific formula. Cyclodextrin derivatives are also described in Patent Documents 3 to 6 and Non-Patent Documents 3 and 4. Non-Patent Document 7 also demonstrates the binding of hemoCD3 to hydrogen sulfide. However, hemoCD3 has a problem in that its binding constant with hydrogen sulfide is not sufficiently large and its hydrogen sulfide decomposition efficiency, as described below, is not sufficiently high, making it unsuitable as an antidote for hydrogen sulfide poisoning.

[0004] Japanese Patent Publication No. 2006-2077 Japanese Patent Publication No. 2010-194475 Japanese Patent Publication No. 6180241 International Publication No. 2023 / 068193 International Publication No. 2021 / 153197 Japanese Patent Publication No. 2015-044961 Japanese Patent No. 7208624 Japanese Patent Publication No. 2020-143014 Japanese Patent Publication No. 2013-231111

[0005] Angew. Chem. Int. Ed., 44, 435-438 (2005)Inorg. Chem. 45, 4448-4460 (2006)ACS Med. Chem. Lett. 2011, 2, 943-947Angew. Commun (Camb). 2021 Jan 7; 57(2): 148-173.J Am Chem Soc. 2002 Aug 21; 124(33): 9937-44.Chem Commun (Camb). 2015 Mar 7;51(19):4059-61.

[0006] Therefore, an object of the present invention is to provide an antidote for hydrogen sulfide.

[0007] The present inventors have discovered that compounds such as hemoCD-I, which they have independently developed, exhibit remarkable binding ability to hydrogen sulfide (higher binding constant than oxyhemoglobin). When compounds such as hemoCD-I were administered to a model animal (mouse) of hydrogen sulfide poisoning, a significant improvement in survival rate was observed.

[0008] That is, the present invention provides the following: [Embodiment 1] A hydrogen sulfide detoxifying agent containing, as an active ingredient, an inclusion complex formed by inclusion of a water-soluble metalloporphyrin in a cyclodextrin dimer represented by the following chemical formula (1): (In the formula, n may be the same or different and represents any of the numbers 1, 2, and 3; R may be the same or different and represents any of an -OCH group, an -OH group, and a hydroxypropyl group; L may be the same or different and represents an amide bond, a thioether bond, an amine bond, or a -(CH) k-, where k is 1, 2, 3, 4, or 5; J is pyridine or imidazole substituted with one or more linear or branched hydrocarbon groups, where the hydrocarbon group has 1, 2, 3, 4, or 5 carbon atoms; and each L bonds to one carbon atom of the one or more linear or branched hydrocarbon groups in J.) [Aspect 2] The hydrogen sulfide detoxifier according to Aspect 1, wherein the cyclodextrin dimer represented by chemical formula (1) is a cyclodextrin dimer represented by the following chemical formula (9) or chemical formula (10): (In the above general formula (9), R represents a protecting group that protects the hydroxyl group of cyclodextrin, m may be the same or different and represent an integer of 1 to 2, and n may be the same or different and represent an integer of 1 to 3.) (In the above general formula (10), R represents a protecting group that protects the hydroxyl group of cyclodextrin, p's may be the same or different and represent an integer of 1 to 2, and q's may be the same or different and represent an integer of 1 to 3.) [Aspect 3] A hydrogen sulfide detoxifying agent according to aspect 1, wherein the water-soluble metalloporphyrin is represented by the following chemical formula (2): (In the formula, A represents either an anionic substituent or a neutral or cationic functional group, and Z represents either Fe or Co.) [Aspect 4] A in chemical formula (2) is a sulfo group (-SO3 - ), a sulfonamide group, an amino group, a nitro group, a carboxy group, a hydrogen atom, a chlorine atom, or a hydroxyl group, or any of the groups represented by the following chemical formulas (3) to (8), wherein n in chemical formulas (4) and (6) to (8) may be the same or different and represent an integer of 1 to 200, and R in chemical formulas (3) and (4) may be the same or different and represent a hydrogen atom or a hydrocarbon group. [Embodiment 5] The hydrogen sulfide detoxifying agent according to embodiment 1, wherein the inclusion complex formed by the cyclodextrin dimer represented by chemical formula (1) and the water-soluble metalloporphyrin undergoes an autoxidation reaction represented by the following reaction formula (11) at pH 7.0 and 25°C with a half-life of 0.01 to 5 hours: (wherein Z represents a metal atom in the water-soluble metalloporphyrin, and L represents the valence of Z in ionization.) [Aspect 6] The hydrogen sulfide antidote according to Aspect 1, wherein the acid dissociation constant of coordinated water in the inclusion complex formed by the cyclodextrin dimer represented by chemical formula (1) and the water-soluble metalloporphyrin is 5 to 9. [Aspect 7] The hydrogen sulfide antidote according to Aspect 2, wherein the cyclodextrin dimer represented by chemical formula (1) is a cyclodextrin dimer represented by chemical formula (10). [Aspect 8] A method for producing a pharmaceutical composition for treating and / or preventing hydrogen sulfide poisoning, comprising the step of mixing the cyclodextrin dimer represented by chemical formula (1) according to Aspect 1 with the water-soluble metalloporphyrin represented by chemical formula (2) according to Aspect 3. [Aspect 9] The hydrogen sulfide detoxifying agent according to Aspect 1, wherein the inclusion complex formed by the cyclodextrin dimer represented by chemical formula (1) and the water-soluble metalloporphyrin has an acid dissociation constant of coordinated water of 5 to 8. [Aspect 10] The hydrogen sulfide detoxifying agent, comprising, as an active ingredient, an inclusion complex formed by the cyclodextrin dimer represented by the following chemical formula (1) or chemical formula (12) and the water-soluble metalloporphyrin. (In formula (1) or formula (12), n may be the same or different and represent any of the numbers 1, 2, and 3; R may be the same or different and represent any of an -OCH group, an -OH group, and a hydroxypropyl group; L may be the same or different and represent an amide bond, a thioether bond, an amine bond, or a -(CH) k-, where k is 1, 2, 3, 4, or 5; J is pyridine or imidazole substituted with one or more linear or branched hydrocarbon groups, where the hydrocarbon group has 1, 2, 3, 4, or 5 carbon atoms; and each L is bonded to one carbon atom of the one or more linear or branched hydrocarbon groups in J.

[0009] The hydrogen sulfide antidote provided by this invention has the potential to become a drug that can be administered immediately at the scene of emergency medical treatment for hydrogen sulfide poisoning. It has also been shown that hemoCD-I is effective as an antidote for cyanide poisoning in addition to hydrogen sulfide poisoning. Therefore, it has the potential to become a drug that can be used for multiple gas poisonings. Furthermore, after administration, it is excreted in the urine and does not remain in the body. Therefore, its sufficiently high safety has been proven in previous studies.

[0010] 1 is a graph showing the survival rate of mice in a state of hydrogen sulfide intoxication after administration of hemoCD-I, hemoCD-P, or saline.

[0034] FIG. 1 is a graph showing the binding constants of hydrogen sulfide for hemoCD-I and hemoCD-P and the half-life of the autoxidation reaction.

[0035] FIG. 2 is a graph showing the catalytic hydrogen sulfide decomposition reaction by hemoCD.

[0036] Scheme 1: Proposed overall reaction scheme between met-hemoCD-I and sulfide.

[0037] FIG. 3 is a graph showing the rate of hydrogen sulfide decomposition by each hemoCD.

[0038] FIG. 4 is a graph showing the change in the percentage of hydrogen sulfide remaining over time in solutions treated with hemoCD-I and hemoCD-P at 25°C.

[0039] FIG. 5 is a graph showing the half-life of the autoxidation reaction of each hemoCD and hemoglobin.

[0039] FIG. 6 is a graph showing the half-life (t 1 / 2 a) Collman, JP; Boulatov, R.; Sunderland, CJ; Fu, L., Chem. Rev., 2004, 104, 561-588. The binding rate of hydrogen sulfide (k on ) and dissociation rate (k off (a) k of hemoCD-I and hemoCD-P for different [NaSH] on and (b) koff Plot of parameters.

[0011] The hydrogen sulfide antidote of the present invention contains, as an active ingredient, an inclusion complex formed by a specific cyclodextrin dimer including a water-soluble metalloporphyrin.

[0012] The inclusion complex can be prepared by a method including the step of mixing a cyclodextrin dimer with a water-soluble metalloporphyrin, for example, by mixing the cyclodextrin dimer with the water-soluble metalloporphyrin in an aqueous solvent.

[0013] Cyclodextrin dimer: The cyclodextrin dimer is represented by the aforementioned chemical formula (1) or (12). In chemical formula (1) or (12), J represents either pyridine or imidazole substituted with one or more linear or branched hydrocarbon groups, where the plural number of linear or branched hydrocarbon groups can be, for example, 2, 3, 4, 5, or 6. This cyclodextrin dimer can be produced, for example, as described in prior art documents, by tosylating and epoxidizing cyclodextrin, methylating the hydroxyl groups of the cyclodextrin, and then bonding the methylated cyclodextrin to a linker molecule. Note that the hydroxyl groups of the cyclodextrin are methylated in advance to prevent hydrogen bonds formed by the hydroxyl groups from hardening the inner cavity of the cyclodextrin, making it difficult for the water-soluble metalloporphyrin to be included in the inner cavity of the cyclodextrin dimer. The cyclodextrin dimer is more preferably represented by the aforementioned chemical formula (9) or chemical formula (10). The cyclodextrin dimer represented by the general formula (9) can be produced, for example, as described in JP 2010-194475 A, by tosylating and epoxidizing cyclodextrin, methylating the hydroxyl groups of the cyclodextrin, and then bonding the methylated cyclodextrin to a linker molecule. Protecting the hydroxyl groups of the cyclodextrin with methyl groups or the like in advance can harden the inner cavity of the cyclodextrin due to hydrogen bonds formed by the hydroxyl groups, preventing the water-soluble metalloporphyrin from being difficult to include in the inner cavity of the cyclodextrin dimer. The cyclodextrin dimer represented by the general formula (10) can be produced by tosylating and epoxidizing a cyclodextrin, methylating the hydroxyl groups of the cyclodextrin, and then bonding the methylated cyclodextrin to a linker molecule, as described in JP 2013-231111 A. Protecting the hydroxyl groups of the cyclodextrin with methyl groups or the like in advance can prevent the inner cavity of the cyclodextrin from being hardened by hydrogen bonds formed by the hydroxyl groups, making it difficult for the water-soluble metalloporphyrin to be included in the inner cavity of the cyclodextrin dimer.In general formulae (9) and (10), examples of the protecting group R include, in addition to the methyl group, an ethyl group, an acetyl group, and a hydroxypropyl group.

[0014] Examples of cyclodextrins that can be used as raw materials for the cyclodextrin dimer include α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin. Among these, β-cyclodextrin is preferred because it can easily encapsulate water-soluble metalloporphyrins. The cyclodextrin used as raw material for the cyclodextrin dimer represented by the general formula (9) can be any of α-cyclodextrin, β-cyclodextrin (n=2), and γ-cyclodextrin. However, it is preferred to use β-cyclodextrin as the raw material because it can easily encapsulate water-soluble metalloporphyrins, and to use a linker molecule with m=1. The cyclodextrin used as the raw material for the cyclodextrin dimer represented by the general formula (10) may be any of α-cyclodextrin, β-cyclodextrin (q=2), and γ-cyclodextrin. However, it is preferable to use β-cyclodextrin as the raw material because it is easy to include the water-soluble metalloporphyrin, and to use a linker molecule with p=1.

[0015] Regarding Im3CD and Py3CD used in the examples described later, Im3CD has an amide bond and Py3CD has a thioether bond in the linker structure, but the reverse may also be true, or a simple hydrocarbon -(CH2) k A similar effect can be expected with -, ether bond (-O-), or amine bond (-NH-). Nitrogen atoms such as pyridine and imidazole are necessary to coordinate to the metal atom in the water-soluble metalloporphyrin, but there is no problem if they are swapped. As will be explained later, the amide bond and imidazole of Im3CD are not essential, and the degree of packing by the cyclodextrin and the strength of the electron pushing on the metal atom in the water-soluble metalloporphyrin determine the effectiveness as an antidote, and these are determined by the half-life (t 1 / 2 ) and the acid dissociation constant of coordinated water (pK aTherefore, the inclusion complex formed by encapsulating a water-soluble metalloporphyrin in a cyclodextrin dimer preferably has a half-life (t 1 / 2 ) of 0.01 to 5 hours, compounds with an acid dissociation constant (pKa) of coordinated water of 5 to 9, and compounds with an acid dissociation constant (pKa) of coordinated water of 5 to 8. More specifically, compounds having an acid dissociation constant (pKa) of coordinated water of 5 to 9 include compounds having an acid dissociation constant (pKa) of coordinated water of 5.0 or more, 5.1 or more, 5.2 or more, 5.3 or more, 5.4 or more, 5.5 or more, 5.6 or more, 5.7 or more, 5.8 or more, 5.9 or more, 6.0 or more, 6.1 or more, 6.2 or more, 6.3 or more, 6.4 or more, 6.5 or more, 6.6 or more, 6.7 or more, 6.8 or more, 6.9 or more, 7.1 or more, 7.2 or more, 7.3 or more, 7.4 or more, 7.5 or more, 7.6 or more, 7.7 or more, 7.8 or more, 7.9 or more, 8.0 or more, 8.1 or more, 8.2 or more, 8.3 or more, 8.4 or more, 8.5 or more, 8.6 or more, 8.7 or more, 8.8 or more or 8.9 or more, and examples thereof include compounds having an acid dissociation constant (pKa) of coordinated water of 9.0 or less, 8.9 or less, 8.8 or less, 8.7 or less, 8.6 or less, 8.5 or less, 8.4 or less, 8.3 or less, 8.2 or less, 8.1 or less, 8.0 or less, 7.9 or less, 7.8 or less, 7.7 or less, 7.6 or less, 7.5 or less, 7.4 or less, 7.3 or less, 7.2 or less, 7.1 or less, 6.9 or less, 6.8 or less, 6.7 or less, 6.6 or less, 6.5 or less, 6.4 or less, 6.3 or less, 6.2 or less, 6.1 or less, 6.0 or less, 5.9 or less, 5.8 or less, 5.7 or less, 5.6 or less, 5.5 or less, 5.4 or less, 5.3 or less, 5.2 or less, or 5.1 or less. 1 / 2 ) is measured by the following procedure. An excess amount of sodium dithionite is added to an aqueous solution of oxidized hemoCD, in which the iron atom in hemoCD is trivalent, to reduce the iron atom to divalent. Next, the excess amount of sodium dithionite is passed through a gel filtration column (HiTrap TMIn the process of removing the salt using a desalting column (5 mL, Cytiva), the reduced hemoCD solution is combined with the oxygen in the aqueous solution to prepare a solution. The autoxidation reaction from the reduced form to the oxidized form is then traced using ultraviolet-visible absorption spectroscopy, and the half-life of the autoxidation reaction (t 1 / 2 ) is calculated. where A represents the absorbance at any wavelength that changes due to the autoxidation reaction, k represents the reaction rate constant, and t represents time. a The pK is measured by the following procedure: the hemoCD aqueous solution is adjusted to an acidic condition, for example, pH 1. The pH is then gradually changed to the alkaline side until it reaches, for example, pH 14, and the UV-visible absorption spectrum is measured during this period. The absorbance at any wavelength before and after the change in the UV-visible absorption spectrum resulting from the acid dissociation of water molecules coordinated to hemoCD is plotted against the pH of the aqueous solution. The midpoint of the change, i.e., the point where the same amount of hemoCD coordinated with water molecules and hemoCD coordinated with hydroxy ions are present, is called the pK. a Furthermore, for the inclusion complex in which a cyclodextrin dimer includes a water-soluble metalloporphyrin, for example, the binding constant with hydrogen sulfide at 25°C and pH 7 [M -1 ] is 1×10 5 That's it, 2 x 10 5 That's it, 3 x 10 5 That's it, 4 x 10 5 That's it, 5 x 10 5 That's it, 6 x 10 5 That's it, 7 x 10 5 That's it, 8 x 10 5 That's it, 9 x 10 5 That's it, 1 x 10 6 or more, or 2 x 10 6 Furthermore, examples of the inclusion complexes in which a cyclodextrin dimer includes a water-soluble metalloporphyrin include compounds having a binding rate with hydrogen sulfide at pH 7.0 [M -1 s -1 ] is 0.2×10 -3 Above, 0.3 × 10 -3 More than 0.4 × 10-3 Above, 0.5 x 10 -3 That's 0.6 x 10 -3 That's 0.7 x 10 -3 That's 0.8 x 10 -3 That's 0.9 x 10 -3 That's it, 1.0 x 10 -3 That's it, 1.1 x 10 -3 That's it, 1.2 x 10 -3 That's it, 1.3 x 10 -3 That's it, 1.4 x 10 -3 That's it, 1.5 x 10 -3 That's it, 1.6 x 10 -3 That's 1.7 x 10 -3 That's it, 1.8 x 10 -3 That's it, 1.9 x 10 -3 or more, or 2.0 x 10 -3 or more, and the binding rate with hydrogen sulfide at pH 7.4 [M -1 s -1 ] is 0.1×10 -3 Above, 0.2 × 10 -3 Above, 0.3 × 10 -3 That's 0.4 x 10 -3 Above, 0.5 x 10 -3 That's 0.6 x 10 -3 That's 0.7 x 10 -3 That's 0.8 x 10 -3 That's 0.9 x 10 -3 That's it, 1.0 x 10 -3 That's it, 1.1 x 10 -3 That's it, 1.2 x 10 -3 That's it, 1.3 x 10 -3 That's it, 1.4 x 10 -3 That's it, 1.5 x 10 -3 That's it, 1.6 x 10 -3 That's 1.7 x 10 -3 or more, or 1.8 x 10 -3 The above compounds are exemplified.

[0016] The roles required of dimeric cyclodextrins such as Im3CD and Py3CD are as follows:

[0017] First, the water-soluble metalloporphyrin is encapsulated to prevent the porphyrin itself from forming aggregates, which would prevent it from interacting effectively with hydrogen sulfide.

[0018] Second, the unpaired electron on the nitrogen atom of the pyridine or imidazole bridge coordinates to the metal atom at the porphyrin center, stabilizing the coordination of hydrogen sulfide. This allows hydrogen sulfide (HS) to coordinate to hemoCD, and then the hydrogen ion is released, forming an SH-hemoCD complex. The closer the acid dissociation constant (pKa) of the water molecule coordinated to the metal atom in the water-soluble metalloporphyrin is to physiological conditions (pH 7-7.4), the higher the affinity for hydrogen sulfide. This is thought to be due to the following reasons: hemoCD, with a pKa close to physiological conditions, is dominated by coordination with water molecules that are prone to exchange reactions with hydrogen sulfide, promoting the binding of hydrogen sulfide to hemoCD. Furthermore, the presence of some hydroxo forms suppresses the reverse reaction.

[0019] To achieve a higher detoxification effect, the following mechanism is also required. Specifically, hemoCD, with hydrogen sulfide coordinated, promotes the oxidation of hydrogen sulfide, detoxifying it, and then exhibits catalytic activity, capturing additional hydrogen sulfide. After coordination with hemoCD, hydrogen sulfide is oxidized by oxygen to sulfate, sulfite, or thiosulfate. The metal atoms in hemoCD are reduced by hydrogen sulfide and become reduced. Therefore, the oxidation rate of the reduced metal atom becomes the rate-limiting step in the catalytic reaction, determining the efficiency of hydrogen sulfide decomposition. For example, when iron is used as the metal atom in water-soluble metalloporphyrins, hydrogen sulfide binds to the trivalent iron atom. Homolytic cleavage of the Fe-S bond then reduces the iron atom to a divalent iron atom, generating sulfhydryl radicals. Because the sulfhydryl radical is unstable, it is thought to rapidly convert to inorganic ions such as sulfate, sulfite, or thiosulfate. However, since the reduced iron atom is divalent, it forms oxygen adducts in the presence of oxygen. If the oxygen adduct is relatively stable, i.e., the reaction rate from the oxygen adduct to the oxidized form (iron trivalent) (autoxidation reaction rate) is slow, the subsequent capture of hydrogen sulfide will be impossible. Furthermore, the iron atom in hemoCD can also be reduced by reducing agents present in the body. In other words, hemoCD compounds with a fast autoxidation reaction rate can exist stably in the body as iron trivalents, and are thought to have high hydrogen sulfide capture capacity and hydrogen sulfide decomposition efficiency. In controlling the autoxidation reaction of this iron atom, it is thought that increasing the distance between the cross-linking cyclodextrins makes it easier for water molecules to enter the vicinity of the porphyrin, thereby speeding up the autoxidation reaction rate.

[0020] Cyclodextrin dimers can be expected to have similar effects as long as they do not impair the properties of the hydrophobic cavity formed by cyclodextrin. Therefore, with respect to R in the above-mentioned chemical formula (1) or chemical formula (12), it is considered possible to partially replace the -OCH3 groups with OH groups, hydroxypropyl groups, or the like. Because it is important that the hydrophobic cavity of cyclodextrin and the hydrophobicity of porphyrin interact to form an inclusion complex, partial modification to the extent that this interaction is not impaired is thought to have no effect on the properties. However, methylation increases the flexibility of the cyclodextrin skeleton, which was previously rigid due to hydrogen bonding, resulting in a structure more suitable for interaction with water-soluble metalloporphyrins. The methylation shown in the examples below is optimal, and excessive modification may impair the properties.

[0021] In the aforementioned chemical formula (1) or chemical formula (12), n = 1 represents α-cyclodextrin, and n = 3 represents γ-cyclodextrin, which respectively have narrower and wider internal cavities than the β-cyclodextrin skeleton of Py3CD. The driving force for inclusion is the same as in the conventional case, in that the phenyl group on the porphyrin side chain interacts hydrophobically with the internal cavity of the cyclodextrin. To control the autoxidation rate for a high detoxification effect, for example, there is room for control by increasing k in chemical formula (1) or chemical formula (12) for α where n = 1, or by decreasing k for γ where n = 3.

[0022] Regarding k in the above-mentioned chemical formula (1) or chemical formula (12), in the case of hemoCD-I, the number of carbon atoms can be estimated to be approximately 2 when the amide bond is replaced with a hydrocarbon bond, so k = 3 is considered to roughly correspond to hemoCD-I.

[0023] Water-soluble metalloporphyrin: The water-soluble metalloporphyrin is, for example, a porphyrin-based compound that is soluble in water by coordinating a metal ion at the center and that can be included in a cyclodextrin dimer represented by chemical formula (1) or chemical formula (12), and is not particularly limited.

[0024] Examples of water-soluble metalloporphyrins include compounds represented by the above-mentioned chemical formula (2). More specific examples of water-soluble metalloporphyrins include 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin iron complex (FeTPPS) and 5,15-bis(3,5-dicarboxylatophenyl)-10,20-diphenylporphyrin iron complex. These compounds can be synthesized by known methods, or commercially available products (e.g., from Frontier Scientific, Tokyo Chemical Industry Co., Ltd.) can be used as they are.

[0025] A in chemical formula (2) may represent any of the groups represented by the above chemical formulas (3) to (8), and n in chemical formulas (4) and (6) to (8) may be the same or different and represent an integer of 1 to 200, where n is, for example, an integer of 200 or less, 190 or less, 180 or less, 170 or less, 160 or less, 150 or less, 140 or less, 130 or less, 120 or less, 110 or less, 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, 40 or less, 30 or less, 20 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less. Furthermore, n is an integer of, for example, 0 or more, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 110 or more, 120 or more, 130 or more, 140 or more, 150 or more, 160 or more, 170 or more, 180 or more, 190 or more, or 200 or more. A in chemical formula (2) may represent any of the groups represented by chemical formulas (3) to (8), where R in chemical formulas (3) to (4) may be the same or different and represent a hydrogen atom or a hydrocarbon group, and the number of carbon atoms in the hydrocarbon group represented by R in chemical formulas (3) to (4) may be, for example, 1 to 10.

[0026] A in chemical formula (2) is the functional group of FeTPPS, SO3 -Anionic substituents such as these are more suitable in terms of the stability of the inclusion complex because they strengthen the inclusion with cyclodextrin. On the other hand, even if a neutral or cationic functional group is used, hydrogen sulfide will bind to the metal atom at the center of the porphyrin, and the effects of the present invention will be exerted. The phenyl group is important for the inclusion with cyclodextrin. Examples of A include substituents with a molecular weight of 1 to 10,000.

[0027] As described above, there are few restrictions on A in chemical formula (2), so A may be any of the substituents shown in the above-mentioned chemical formulas (3) to (8). In addition, there are also other examples, such as TPP where A is a hydrogen atom, TPPOH where A is an OH group, and -O-CH2-CH2-(-O-CH2-CH2) q It has been confirmed that inclusion complexes can be formed even with long molecular chains such as polyethylene glycol chains represented by -OCH3 (q is a positive integer). Previous studies have confirmed that inclusion complexes can be formed with compounds with q values ​​of 44 and 112 (compounds with molecular weights of 2000 and 5000, respectively).

[0028] This is because the phenyl group attached to the porphyrin skeleton plays a significant role in the inclusion complex with cyclodextrin. While the inclusion effect is stronger when A is anionic due to the charge imbalance within cyclodextrin, neutral or cationic groups also form inclusion complexes, which are thought to have a detoxifying effect. The binding constants for the formation of large inclusion complexes (CD + FeTPPS → CD / FeTPPS) due to the substituents on A have been studied in a previous paper (J. AM. CHEM. SOC. 2002, 124, 9937-9944). As shown in Table 2 of that paper, where the solvent is EG-H2O, an inclusion complex is formed. The paper also noted that inclusion complexes do not form with PC3Py and PC7Py, which lack phenyl groups, or with TMPyP, in which the phenyl group is replaced by a cationic pyridine skeleton.

[0029] Regarding Z in chemical formula (2), the central metal can be Co in addition to Fe, and the same effect can be achieved. It is widely known that cobalt atoms bond with hydrogen sulfide in compounds such as hydroxocobalamin and cobinamide. Therefore, Z in chemical formula (2) can also be Co.

[0030] Hydrogen sulfide binds strongly to iron(III). In vivo, hemoCD is reduced to iron(II). Therefore, for effective detoxification, it is important that the iron(III) state is stable (i.e., has a short oxidation half-life) and that the binding constant K for hydrogen sulfide is sufficiently large. In carbon monoxide antidotes, CO binds to hemoCD, and in cyanide antidotes, CN binds to hemoCD, achieving therapeutic effects by removing toxic substances from the body. In contrast, in hydrogen sulfide poisoning antidotes, after hydrogen sulfide binds to hemoCD, the iron atom in hemoCD is reduced by homolytic cleavage of the Fe-S bond, and the resulting sulfhydryl radical is oxidized to a non-toxic inorganic ion. Therefore, to achieve a high therapeutic effect, the reduced hemoCD must be re-oxidized to iron(III), which readily binds to hydrogen sulfide and then captures other hydrogen sulfide, completing the catalytic hydrogen sulfide decomposition cycle.

[0031] The hydrogen sulfide antidote of the present invention can be administered to humans or other animals as the inclusion complex alone or as a pharmaceutical composition together with a known pharmaceutical carrier. The dosage form of the pharmaceutical composition is not particularly limited and may be selected appropriately as needed. Specific examples include oral preparations such as tablets, capsules, granules, fine granules, and powders, and parenteral preparations such as injections, suppositories, and liniments. The amount of hydrogen sulfide antidote in the pharmaceutical composition and the dosage of the pharmaceutical composition to a patient can be freely selected depending on the dosage form, the patient's age, weight, and severity of the disease.

[0032] The hydrogen sulfide antidote of the present disclosure may also be a pharmaceutical composition for treating and / or preventing hydrogen sulfide poisoning. Some embodiments of the present disclosure may be a pharmaceutical composition for use in the treatment and / or prevention of hydrogen sulfide poisoning, comprising a cyclodextrin dimer represented by chemical formula (1) or chemical formula (12) and a water-soluble metalloporphyrin, wherein the water-soluble metalloporphyrin and the cyclodextrin dimer form an inclusion complex. In other words, some embodiments of the present disclosure may be a pharmaceutical composition for use in the treatment and / or prevention of hydrogen sulfide poisoning, comprising an inclusion complex comprising a cyclodextrin dimer represented by chemical formula (1) or chemical formula (12) and a water-soluble metalloporphyrin as an active ingredient. The pharmaceutical composition according to the present disclosure may further comprise additional ingredients, such as a pharmaceutically acceptable carrier.

[0033] When the hydrogen sulfide antidote of the present invention is produced as an oral preparation such as a tablet, it can be produced by a known production method together with known excipients, binders, disintegrants, surfactants, lubricants, flow enhancers, etc.

[0034] The antidote for hydrogen sulfide of the present invention can also be administered orally as a suspension, emulsion, syrup, or elixir, which may contain flavoring agents, odorants, colorants, etc.

[0035] When the hydrogen sulfide antidote of the present invention is prepared as a parenteral preparation such as an injection or infusion, it can be prepared by known methods using a diluent such as distilled water for injection, a physiological saline diluent, or an aqueous glucose solution. If necessary, a disinfectant, preservative, or stabilizer may be added. For stability reasons, this parenteral preparation can be frozen after being filled into a vial or the like, and the water removed by a conventional freeze-drying process, and the freeze-dried product can be reconstituted into a liquid just before use. If necessary, an isotonic agent, stabilizer, preservative, or soothing agent may also be added.

[0036] Other examples of parenteral preparations of the hydrogen sulfide antidote of the present invention include external liquid preparations, ointments and other topical preparations, and suppositories for rectal administration, which can also be prepared according to known methods.

[0037] The hydrogen sulfide antidote of the present invention may be administered in vivo using known DDS technology, for example, by encapsulating the hydrogen sulfide antidote in a carrier such as a liposome. In this case, the hydrogen sulfide antidote of the present invention can be efficiently delivered to the target site by using a carrier that specifically recognizes cells at the target site.

[0038] The hydrogen sulfide antidote of the present disclosure can be used in a method for treating and / or preventing hydrogen sulfide poisoning in a subject in need of treatment. Some embodiments of the present disclosure can be a method for treating and / or preventing hydrogen sulfide poisoning in a subject in need of treatment, comprising administering to the subject an inclusion complex comprising a cyclodextrin dimer represented by Formula (1) or Formula (12) and a water-soluble metalloporphyrin. The route of administration can be, for example, but is not limited to, intravenous administration or inhalation administration. Administration can be, for example, to a subject diagnosed with hydrogen sulfide poisoning or suspected of having hydrogen sulfide poisoning. Administration can also be prophylactic to a subject at risk of developing hydrogen sulfide poisoning.

[0039] Furthermore, some embodiments of the present disclosure relate to the use of an inclusion complex comprising a cyclodextrin dimer represented by formula (1) or (12) and a water-soluble metalloporphyrin in the manufacture of a medicament for treating and / or preventing hydrogen sulfide poisoning. Some embodiments of the present disclosure also relate to a method for producing a pharmaceutical composition for treating and / or preventing hydrogen sulfide poisoning, the method comprising the step of mixing a cyclodextrin dimer represented by formula (1) or (12) with a water-soluble metalloporphyrin represented by formula (2).

[0040] Example 1: All animal experiments were conducted in accordance with the animal experiment regulations established by Doshisha University. Nine-week-old female BALB / cCrSlc mice (weight approximately 20 g) were used as experimental animals. Sodium hydrogen sulfide was used as the hydrogen sulfide source. Sodium hydrogen sulfide is a solid at room temperature and atmospheric pressure, making it easy to handle. It can be dissolved in aqueous solution to generate hydrogen sulfide. Sodium hydrogen sulfide was dissolved in saline (100 μL) at a concentration of 22.5 mg / kg of mouse body weight. This sodium hydrogen sulfide aqueous solution was intraperitoneally administered to mice, resulting in hydrogen sulfide intoxication. Subsequently, 1 minute after administration of the sodium hydrogen sulfide aqueous solution, hemoCD-I (7 mM, 200 μL), hemoCD-P (7 mM, 200 μL), or saline alone (200 μL) was intraperitoneally administered. Mice were observed for 45 minutes after administration of the sodium hydrogen sulfide aqueous solution to determine whether they survived. HemoCD-I was synthesized by the method described in ACS Med. Chem. Lett. 2011, 2, 943-947, and hemoCD-P was synthesized by the method described in Angew. Chem. Int. Ed. 2005, 44, 435-438. The respective reaction schemes are shown below.

[0041] The results are shown in Figure 1. The binding constants of hydrogen sulfide and the half-lives of the oxidation reaction for hemoCD-I and hemoCD-P are shown in Figure 2. As shown in Figures 1 and 2, it became clear that hydrogen sulfide poisoning can be treated by administering hemoCD-I or hemoCD-P. Furthermore, as shown in Figure 2, hemoCD-I binds strongly to hydrogen sulfide and maintains stable trivalent iron, resulting in the strongest detoxifying effect against hydrogen sulfide poisoning.

[0042] The following three points are important for a more effective detoxification of hydrogen sulfide poisoning.

[0043] First, it has a sufficiently fast binding rate (k on) and can quickly capture hydrogen sulfide in the body. The binding of hydrogen sulfide is an exchange reaction with the water molecule coordinated to the central metal of the metalloporphyrin. The hydrogen sulfide molecule exchanges with the water molecule relatively quickly. On the other hand, protons (H + ) is eliminated from hydroxo (OH - In the case of the hydroxyl group (H2O) ligand, the charge number changes before and after the exchange, slowing the exchange reaction. In other words, to rapidly bind to hydrogen sulfide, it is preferable for water molecules to be coordinated to the central metal of the metalloporphyrin at a pH range of 7.0 to 7.4, which is close to physiological conditions. This characteristic is indicated by the pKa of the water coordinated to the metal atom, with a pKa of 5.0 or higher being preferred, and a pKa of 6.5 or higher being even more preferred. The pKa of the water coordinated to the metal atom can be controlled by the axial ligand moiety to the metal atom incorporated into the linker portion of the cyclodextrin dimer. The axial ligand moiety consists of an electron-donating ligand, such as a ligand containing a nitrogen atom of pyridine or imidazole, a phenolate ligand containing an oxygen atom, or a thiolate ligand containing a sulfur atom.

[0044] Second, it has a sufficiently large binding constant (K) for hydrogen sulfide in the pH range of 7.0 to 7.4, which is close to physiological conditions, and can strongly capture bound hydrogen sulfide. To stabilize bound hydrogen sulfide, it is important to introduce an electron-donating axial ligand to the central metal of the metalloporphyrin. Increasing the electron density of the central metal allows the formation of a stable hydrogen sulfide complex. Furthermore, the binding constant is related to the binding rate (k on ) and dissociation rate (k off ) ratio, it is important to reduce the dissociation rate. In other words, the dissociation reaction is an exchange reaction between the solvent molecules, water molecules, and hydrogen sulfide molecules. After the dissociation reaction, the central metal of the metalloporphyrin is converted to a hydroxo (OH -When a cyclodextrin dimer is used as a ligand, the charge number changes before and after the reaction, which is thought to slow the exchange reaction. Therefore, to achieve a large binding constant by slowing the dissociation rate, the pKa of the water coordinated to the metal atom is preferably less than 9.0, and even more preferably less than 8.0. The pKa of the water coordinated to the metal atom can be controlled by the axial ligand moiety to the metal atom incorporated into the linker portion of the cyclodextrin dimer. The axial ligand moiety consists of an electron-donating ligand, such as a ligand containing a nitrogen atom of pyridine or imidazole, a phenolate ligand containing an oxygen atom, or a thiolate ligand containing a sulfur atom.

[0045] Third, the autoxidation rate of the oxygen complex is sufficiently fast to complete the catalytic hydrogen sulfide decomposition cycle at pH 7.0 to 7.4, which is close to physiological conditions. After the formation of a hydrogen sulfide coordination complex on the metalloporphyrin, the metal-hydrogen sulfide bond spontaneously homolytically cleaves, resulting in a single-electron reduction of the central metal. If the metal atom is an iron atom, it is reduced from trivalent iron to divalent iron. In the body, the divalent iron porphyrin complex forms an oxygen complex with oxygen molecules. This oxygen complex then undergoes autoxidation, returning to the original trivalent iron, which can then recombine with hydrogen sulfide molecules, potentially providing catalytic detoxification. In other words, the faster the autoxidation rate, the more effective the detoxification effect. The autoxidation rate can be controlled, for example, by using a linker structure that bridges the cyclodextrin dimer. The greater the number of constituent atoms, the longer the distance between cyclodextrin dimers, which increases the contact between the central metal atom of the porphyrin and water molecules, and therefore the rate of autoxidation tends to be faster. The rate of the autoxidation reaction in water at pH 7 is preferably within 30 hours, and particularly preferably within 5 hours.

[0046] In addition to the structures described in the Examples, examples of cyclodextrin dimer / water-soluble metalloporphyrin inclusion complexes that satisfy the above requirements and are effective in detoxifying hydrogen sulfide poisoning in the body include the following structures.

[0047] The parameters for each hemoCD compound are shown in Table 1 below.

[0048] Next, the decomposition rate of hydrogen sulfide by each hemoCD was investigated. The decomposition rate of hydrogen sulfide was measured using the following procedure. To a solution containing hydrogen sulfide, 40 or more equivalents of N,N-dimethyl-p-phenylenediamine sulfate and 60 or more equivalents of iron(III) chloride relative to the amount of hydrogen sulfide were added in the presence of trifluoroacetic acid. The concentration of methylene blue produced by the reaction with hydrogen sulfide was calculated from the absorbance at 665 nm. Assuming that the concentration of methylene blue was equal to the hydrogen sulfide concentration in the solution, the decomposition rate of hydrogen sulfide was calculated from the change in hydrogen sulfide concentration over time in the presence or absence of hemoCD. The results are shown in Figure 4. Furthermore, the half-life of the autoxidation reaction of each hemoCD, examined using the method described herein, was compared with the half-life of the autoxidation reaction of hemoglobin. The results are shown in Figure 5. Furthermore, the binding rate of hydrogen sulfide to each hemoCD (k on ) and dissociation rate (k off The binding rate of hydrogen sulfide (k on ) was measured using the following procedure. At least 50 equivalents of sodium hydrogen sulfide were added to the solution containing hemoCD. The absorbance at 410 nm was measured immediately after the addition, and the rate constant k was calculated by approximating the following equation: Here, t is the time immediately after addition, [A] is the absorbance at 410 nm at a certain time t, and [A]0 is the absorbance at 410 nm before addition. k was calculated for each of different sodium hydrogen sulfide concentrations. k was plotted against the sodium hydrogen sulfide concentration, and the binding rate (k on ) was calculated. off ) was calculated from the following relational expression: The results are shown in Figure 6. Here, K is the binding constant between hemoCD and hydrogen sulfide. The binding constant, K, between hemoCD and hydrogen sulfide was measured as follows: A sodium hydrogen sulfide solution was titrated with a hemoCD solution at pH 7.0 and 25°C. The UV-visible absorption spectrum of hydrogen sulfide coordination to hemoCD was measured, and the change in absorbance at a given wavelength before and after coordination was plotted against the concentration of sodium hydrogen sulfide. The titration curve was analyzed using the software "SPANA" based on the Sequential 1:1 Complex Formation System to calculate K. As shown in Figure 4, the hydrogen sulfide consumption rate was significantly increased in the presence of hemoCD compared to that in the absence of hemoCD. Furthermore, hemoCD-I consumed hydrogen sulfide at a faster rate than hemoCD-P. This, as shown in Figure 5, indicates that the catalytic hydrogen sulfide decomposition shown in Figure 3 is more efficient due to the lower stability of the oxygen adducts in hemoCD-I. Furthermore, the binding rate of hemoCD with hydrogen sulfide decreased as the pH of the aqueous solution increased, as shown in Figure 6. This is because in the high pH range, hydrogen sulfide (HS) binds to hydrogen sulfide ions (HS - ), which reduces the effective hydrogen sulfide concentration. Among these, hemoCD-I showed a faster binding rate than hemoCD-P in the pH range of 7.0 to 7.4, which is close to physiological conditions. This is because hemoCD-I has a pKa of 7.7, which is close to physiological conditions, and in the pH range of 7.0 to 7.4, the sixth ligand of hemoCD-I is dominated by water molecules. Because water molecules readily exchange with hydrogen sulfide, hemoCD-I exhibited a faster binding rate in the pH range of 7.0 to 7.4, which is close to physiological conditions. The acid dissociation constant of the coordinated water of hemoCD-P, calculated using the method described in this paper, was 5.5.

Claims

1. A hydrogen sulfide detoxifying agent containing, as an active ingredient, an inclusion complex formed by a cyclodextrin dimer represented by the following chemical formula (1) encapsulating a water-soluble metal porphyrin. (In the formula, n may be the same or different and represents any of the numbers 1, 2, and 3; R may be the same or different and represents any of an -OCH group, an -OH group, and a hydroxypropyl group; L may be the same or different and represents an amide bond, a thioether bond, an amine bond, or a -(CH) k -, where k is 1, 2, 3, 4, or 5; J is pyridine or imidazole substituted with one or more linear or branched hydrocarbon groups, where the hydrocarbon group has 1, 2, 3, 4, or 5 carbon atoms; and each L is bonded to one carbon atom of the one or more linear or branched hydrocarbon groups in J.

2. The hydrogen sulfide detoxifying agent according to claim 1, wherein the cyclodextrin dimer represented by chemical formula (1) is a cyclodextrin dimer represented by the following chemical formula (9) or chemical formula (10): (In the above general formula (9), R represents a protecting group that protects the hydroxyl group of cyclodextrin, m may be the same or different and represent an integer of 1 to 2, and n may be the same or different and represent an integer of 1 to 3.) (In the above general formula (10), R represents a protecting group that protects the hydroxyl group of cyclodextrin, each p may be the same or different and represents an integer of 1 to 2, and each q may be the same or different and represents an integer of 1 to 3.) 3. The hydrogen sulfide detoxifying agent according to claim 1, wherein the water-soluble metal porphyrin is represented by the following chemical formula (2): (In the formula, A represents either an anionic substituent or a neutral or cationic functional group, and Z represents either Fe or Co.) 4. A in chemical formula (2) is a sulfo group (-SO3 - ), a sulfonamide group, an amino group, a nitro group, a carboxy group, a hydrogen atom, a chlorine atom, or a hydroxyl group, or any of the groups represented by the following chemical formulas (3) to (8), wherein n in chemical formulas (4) and (6) to (8) may be the same or different and represent an integer of 1 to 200, and R in chemical formulas (3) to (4) may be the same or different and represent a hydrogen atom or a hydrocarbon group.

5. The hydrogen sulfide detoxifying agent according to claim 1, wherein the half-life of the autoxidation reaction represented by the following reaction formula (11) at pH 7.0 and 25°C in the inclusion complex formed by the cyclodextrin dimer represented by chemical formula (1) and a water-soluble metal porphyrin is 0.01 to 5 hours. (In the formula, Z represents a metal atom in the water-soluble metalloporphyrin, and L represents the valence of Z in ionization.) 6. The hydrogen sulfide detoxifying agent according to claim 1, wherein the acid dissociation constant of the coordinated water in the inclusion complex formed by the cyclodextrin dimer represented by chemical formula (1) and the water-soluble metal porphyrin is 5 to 9.

7. The hydrogen sulfide detoxifying agent according to claim 2, wherein the cyclodextrin dimer represented by chemical formula (1) is a cyclodextrin dimer represented by chemical formula (10).

8. A method for producing a pharmaceutical composition for treating and / or preventing hydrogen sulfide poisoning, comprising the step of mixing a cyclodextrin dimer represented by chemical formula (1) described in claim 1 with a water-soluble metal porphyrin represented by chemical formula (2) described in claim 3.

9. The hydrogen sulfide detoxifying agent according to claim 1, wherein the acid dissociation constant of the coordinated water in the inclusion complex formed by the cyclodextrin dimer represented by chemical formula (1) and the water-soluble metal porphyrin is 5 to 8.

10. A hydrogen sulfide detoxifying agent containing, as an active ingredient, an inclusion complex formed by a cyclodextrin dimer represented by the following chemical formula (1) or chemical formula (12) inclusion of a water-soluble metal porphyrin: (In formula (1) or formula (12), n may be the same or different and represent any of the numbers 1, 2, and 3; R may be the same or different and represent any of an -OCH group, an -OH group, and a hydroxypropyl group; L may be the same or different and represent an amide bond, a thioether bond, an amine bond, or a -(CH) k -, where k is 1, 2, 3, 4, or 5; J is pyridine or imidazole substituted with one or more linear or branched hydrocarbon groups, where the hydrocarbon group has 1, 2, 3, 4, or 5 carbon atoms; and each L is bonded to one carbon atom of the one or more linear or branched hydrocarbon groups in J.

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