Hydrogen sulfide detoxification agent

Abstract

An object of the present application is to provide a hydrogen sulfide antidote. The present application provides a hydrogen sulfide antidote containing, as an effective ingredient, an inclusion compound obtained by inclusion of a water-soluble metalloporphyrin in a cyclodextrin dimer represented by the following formula (1).

Description

Technical Field

[0001] This invention relates to an antidote for hydrogen sulfide poisoning. Background Technology

[0002] Hydrogen sulfide poisoning incidents frequently occur at construction sites, such as factories and underground facilities, within Japan. Treatment methods include hyperbaric oxygen therapy and the generation of oxyhemoglobin using nitrite preparations, which then captures hydrogen sulfide. The former is limited by equipment and other medical procedures cannot be performed during treatment; the latter, due to doubts about its efficacy in recent years, currently lacks an established effective treatment method.

[0003] The inventors have long been dedicated to researching inclusion complexes formed by encapsulating water-soluble metalloporphyrins with cyclodextrin dimers, and have discovered that these inclusion complexes have a high affinity for oxygen (O2) and carbon monoxide (CO), with an affinity for CO exceeding that of hemoglobin by more than 100 times (see Patent Document 1, and Non-Patent Documents 1-2). Patent Document 2 discloses a carbon monoxide remover containing an inclusion complex, as shown in a specific chemical formula, formed by encapsulating water-soluble metalloporphyrins with cyclodextrin dimers as its active ingredient. Cyclodextrin derivatives are also described in Patent Documents 3-6 and Non-Patent Documents 3-4. Furthermore, Non-Patent Document 7 indicates that hemoCD3 can bind to hydrogen sulfide. However, due to the insufficient binding constant of hemoCD3 to hydrogen sulfide and the relatively low decomposition efficiency of hydrogen sulfide (described later), it is unsuitable as an antidote for hydrogen sulfide poisoning.

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: Japanese Patent Application Publication No. 2006-2077

[0007] Patent Document 2: Japanese Patent Application Publication No. 2010-194475

[0008] Patent Document 3: Japanese Patent No. 6180241

[0009] Patent Document 4: International Publication No. 2023 / 068193

[0010] Patent Document 5: International Publication No. 2021 / 153197

[0011] Patent Document 6: Japanese Patent Application Publication No. 2015-044961

[0012] Patent Document 7: Japanese Patent No. 7208624

[0013] Patent Document 8: Japanese Patent Application Publication No. 2020-143014

[0014] Patent Document 9: Japanese Patent Application Publication No. 2013-231111

[0015] Non-patent literature

[0016] Non-patent literature 1: Angew. Chem. Int. Ed., 44, 435-438 (2005)

[0017] Non-patent literature 2: Inorg. Chem. 45, 4448-4460 (2006)

[0018] Non-patent literature 3: ACS Med. Chem. Lett. 2011, 2, 943-947

[0019] Non-patent literature 4: Angew. Chem. Int. Ed., 52, 6894-6897 (2013)

[0020] Non-patent literature 5: Chem Commun (Camb). 2021 Jan 7; 57(2): 148-173.

[0021] Non-patent literature 6: J Am Chem Soc. 2002 Aug 21; 124(33): 9937-44.

[0022] Non-patent literature 7: Chem Commun (Camb). 2015 Mar 7;51(19):4059-61. Summary of the Invention

[0023] The problem the invention aims to solve

[0024] Therefore, the object of the present invention is to provide a hydrogen sulfide antidote.

[0025] Solution to the problem

[0026] The inventors discovered that compounds such as hemoCD-I, independently developed by the inventors, exhibit a significant binding capacity for hydrogen sulfide (its binding constant is higher than that of oxidized hemoglobin). Therefore, after administering compounds such as hemoCD-I to hydrogen sulfide poisoning model animals (mice), a significant increase in survival rate was observed.

[0027] That is, the present invention provides the following technical solution:

[0028] [Aspect 1]

[0029] A hydrogen sulfide antidote contains an inclusion complex consisting of a cyclodextrin dimer containing a water-soluble metalloporphyrin as the active ingredient.

[0030]

[0031] (In the formula, n can be the same or different, representing any number from 1, 2, and 3; R can be the same or different, representing any one of -OCH3 group, -OH group, or hydroxypropyl group; L can be the same or different, representing amide bond, thioether bond, amine bond, or -(CH2) bond.) k - represents any of the hydrocarbon groups, where k represents any number from 1, 2, 3, 4, 5; J represents any of the pyridine or imidazole groups substituted with one or more straight-chain or branched hydrocarbon groups, where the hydrocarbon group has any number of carbons from 1, 2, 3, 4, 5; L is bonded to one carbon atom of each of the one or more straight-chain or branched hydrocarbon groups in J.

[0032] [Aspect 2]

[0033] According to aspect 1, the hydrogen sulfide antidote is a cyclodextrin dimer represented by chemical formula (1) that is represented by chemical formula (9) or chemical formula (10).

[0034]

[0035] (In the above general formula (9), R represents the protecting group of the hydroxyl group of the cyclodextrin; m can be the same or different, representing integers from 1 to 2 respectively; n can be the same or different, representing integers from 1 to 3 respectively.)

[0036]

[0037] (In the above general formula (10), R represents the protecting group protecting the hydroxyl group of the cyclodextrin; p can be the same or different, representing integers from 1 to 2 respectively; q can be the same or different, representing integers from 1 to 3 respectively.)

[0038] [Aspect 3]

[0039] According to aspect 1, the hydrogen sulfide antidote is a water-soluble metal porphyrin represented by the following chemical formula (2).

[0040]

[0041] (In the formula, A represents any one of anionic substituents, neutral or cationic functional groups; Z represents any one of Fe or Co.)

[0042] [Aspect 4]

[0043] According to the hydrogen sulfide antidote described in aspect 3, A in chemical formula (2) represents a sulfonyl group (-SO3). -), sulfonamide group, amino group, nitro group, carboxyl group, hydrogen atom, chlorine atom, hydroxyl group, or, representing any one 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, representing integers from 1 to 200 respectively, and R in chemical formulas (3) to (4) may be the same or different, representing hydrogen atom or hydrocarbon group respectively.

[0044]

[0045] [Aspect 5]

[0046] According to aspect 1, the hydrogen sulfide antidote, in an inclusion complex consisting of a cyclodextrin dimer containing a water-soluble metalloporphyrin, undergoes an auto-oxidation reaction represented by the following reaction formula (11) at pH 7.0 and 25°C, with a half-life of 0.01 hours to 5 hours.

[0047]

[0048] (In the formula, Z represents the metal atom in the water-soluble metalloporphyrin; L represents the valence of Z during the ionization process.)

[0049] [Aspect 6]

[0050] According to aspect 1, the hydrogen sulfide antidote is composed of a cyclodextrin dimer containing a water-soluble metal porphyrin, and the acid dissociation constant of the coordinating water is 5 to 9.

[0051] [Aspect 7]

[0052] According to aspect 2, the hydrogen sulfide antidote is a cyclodextrin dimer represented by chemical formula (1) and is represented by chemical formula (10).

[0053] [Aspect 8]

[0054] A method for manufacturing a pharmaceutical composition for treating and / or preventing hydrogen sulfide poisoning, comprising: mixing a cyclodextrin dimer represented by chemical formula (1) according to aspect 1, and a water-soluble metalloporphyrin represented by chemical formula (2) according to aspect 3.

[0055] [Aspect 9]

[0056] According to aspect 1, the hydrogen sulfide antidote is composed of a cyclodextrin dimer containing a water-soluble metal porphyrin, and the acid dissociation constant of the coordinating water is 5 to 8.

[0057] [Aspect 10]

[0058] A hydrogen sulfide antidote contains an inclusion complex consisting of a water-soluble metalloporphyrin encapsulated by a cyclodextrin dimer represented by the following chemical formula (1) or (12) as the active ingredient.

[0059]

[0060] (In formula (1) or formula (12), n can be the same or different, representing any number among 1, 2, and 3 respectively; R can be the same or different, representing any one of -OCH3 group, -OH group, and hydroxypropyl group respectively; L can be the same or different, representing amide bond, thioether bond, amine bond, and -(CH2) bond respectively.) k - represents any of the hydrocarbon groups, where k represents any number from 1, 2, 3, 4, 5; J represents any of the pyridine or imidazole groups substituted with one or more straight-chain or branched hydrocarbon groups, where the hydrocarbon group has any number of carbons from 1, 2, 3, 4, 5; L is bonded to one carbon atom of each of the one or more straight-chain or branched hydrocarbon groups in J.

[0061] Invention Effects

[0062] The hydrogen sulfide antidote provided by this invention is expected to become a drug formulation for immediate treatment of hydrogen sulfide poisoning at the scene of an emergency. In addition to hydrogen sulfide, hemoCD-I is also known to be effective as an antidote for cyanide poisoning. Therefore, it is expected to become a therapeutic drug for various types of gas poisoning. Furthermore, it is excreted in the urine after administration, thus leaving no residue in the body. Therefore, it has extremely high safety, as demonstrated by previous studies. Attached Figure Description

[0063] Figure 1 A graph showing the survival rate of mice in a state of hydrogen sulfide poisoning after administration of hemoCD-I, hemoCD-P, or saline.

[0064] Figure 2 A graph showing the binding constants of hydrogen sulfide with hemoCD-I and hemoCD-P and the half-life of the auto-oxidation reaction is presented.

[0065] Figure 3 A diagram illustrating the catalytic hydrogen sulfide decomposition reaction by hemoCD is shown. Scheme 1: The proposed overall reaction scheme between met-hemoCD-I and sulfides.

[0066] Figure 4 A graph showing the decomposition rates of hydrogen sulfide by each hemoCD is presented. The graph also shows the change in the residual proportion of hydrogen sulfide in the solution after treatment with hemoCD-I and hemoCD-P at 25°C over time.

[0067] Figure 5 A graph showing the half-lives of various hemoCDs and hemoglobins in the auto-oxidation reaction. Half-lives (t) of O2 adducts of various sulfide acceptors at 25°C. 1 / 2 ). a) Collman, JP; Boulatov, R.;Sunderland, CJ; Fu, L., Chem. Rev., 2004, 104, 561-588.

[0068] Figure 6 The binding rates (kJ / kJ) of each hemoCD with hydrogen sulfide were shown. on ) and dissociation velocity (k off The figure shows the (a)k values ​​of hemoCD-I and hemoCD-P for different [NaSH]. on and (b)k off Plotting the parameters. Detailed Implementation

[0069] The hydrogen sulfide antidote of the present invention contains an inclusion complex formed by encapsulating a water-soluble metalloporphyrin with a specific cyclodextrin dimer as the active ingredient.

[0070] This inclusion compound can be manufactured by a process including mixing a cyclodextrin dimer and a water-soluble metalloporphyrin. For example, the inclusion compound can be manufactured by mixing a cyclodextrin dimer and a water-soluble metalloporphyrin in an aqueous solvent.

[0071] Cyclodextrin dimers:

[0072] The cyclodextrin dimer is represented by the aforementioned chemical formula (1) or chemical formula (12). In chemical formula (1) or chemical formula (12), J represents any one of pyridine or imidazole substituted with one or more straight-chain or branched hydrocarbon groups, wherein the number of the multiple straight-chain or branched hydrocarbon groups can be, for example, 2, 3, 4, 5, or 6. For example, as described in the prior art literature, the cyclodextrin dimer can be manufactured by first subjecting the cyclodextrin to toluenesulfonation and epoxidation treatment, then methylating the hydroxyl group of the cyclodextrin, and then combining the methylated cyclodextrin with a linker molecule. It should be noted that the hydroxyl group of the cyclodextrin is methylated in advance to prevent the hydrogen bonds generated by the hydroxyl group from hardening the pores of the cyclodextrin, making it difficult for water-soluble metalloporphyrins to be encapsulated in the pores of the cyclodextrin dimer. In addition, the cyclodextrin dimer is more preferably represented by the aforementioned chemical formula (9) or chemical formula (10). For example, as described in Japanese Patent Application Publication No. 2010-194475, a cyclodextrin dimer represented by the general formula (9) can be manufactured by first subjecting the cyclodextrin to toluenesulfonation and epoxidation, then methylating the hydroxyl groups of the cyclodextrin, and finally combining the methylated cyclodextrin with linker molecules. Pre-protecting the hydroxyl groups of the cyclodextrin with methyl groups or the like prevents hydrogen bonds generated by the hydroxyl groups from hardening the pores of the cyclodextrin, making it difficult for water-soluble metalloporphyrins to be encapsulated in the pores of the cyclodextrin dimer. For example, as described in Japanese Patent Application Publication No. 2013-231111, a cyclodextrin dimer represented by the general formula (10) can be manufactured by first subjecting the cyclodextrin to toluenesulfonation and epoxidation, then methylating the hydroxyl groups of the cyclodextrin, and finally combining the methylated cyclodextrin with linker molecules. Pre-protecting the hydroxyl groups of cyclodextrin with methyl groups or similar compounds can prevent the hydrogen bonds generated by the hydroxyl groups from hardening the pores of the cyclodextrin, making it difficult for water-soluble metalloporphyrins to be encapsulated in the pores of the cyclodextrin dimer. In addition to methyl groups, the protecting group R in general chemical formulas (9) and (10) can also be, for example, ethyl, acetyl, hydroxypropyl, etc.

[0073] The cyclodextrin used as a raw material for cyclodextrin dimers can be, for example, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, etc., among which β-cyclodextrin is preferred because it readily encapsulates water-soluble metalloporphyrins. The cyclodextrin used as a raw material for cyclodextrin dimers represented by the general formula (9) can be any one of α-cyclodextrin, β-cyclodextrin (n=2), and γ-cyclodextrin. Since β-cyclodextrin readily encapsulates water-soluble metalloporphyrins, β-cyclodextrin is preferred as a raw material, and the linker molecule is m=1. The cyclodextrin used as a raw material for cyclodextrin dimers represented by the general formula (10) can be any one of α-cyclodextrin, β-cyclodextrin (q=2), and γ-cyclodextrin. Since β-cyclodextrin readily encapsulates water-soluble metalloporphyrins, β-cyclodextrin is preferred as a raw material, and the linker molecule is p=1.

[0074] For the Im3CD and Py3CD used in the following embodiments, as linker structures, Im3CD has amide bonds and Py3CD has thioether bonds, even if the situation is reversed, or only hydrocarbon-(CH2). k -Ether bonds (-O-) or amine bonds (-NH-) can also achieve the same effect. Although nitrogen atoms such as pyridine and imidazole are required due to coordination with the metal atoms in water-soluble metalloporphyrins, they can be replaced with others. As will be discussed later, the amide bond or imidazole in Im3CD is not essential; the resulting cyclodextrin stacking and the strength of electron injection into the metal atoms in water-soluble metalloporphyrins determine its effectiveness as an antidote, which in turn is determined by the half-life (t) of the auto-oxidation reaction. 1 / 2 ) and the acid dissociation constant of coordinated water (pK) a The parameters such as pH and temperature determine the half-life (t) of the auto-oxidation reaction (oxidation reaction starting from the reduced state after oxygenation) carried out under conditions of pH 7.0 and 25°C. Therefore, as an inclusion complex formed by encapsulating water-soluble metalloporphyrins with cyclodextrin dimers, the half-life (t) of the auto-oxidation reaction is preferably determined by parameters such as pH and temperature. 1 / 2 The acid dissociation constant (pK) of compounds with a time range of 0.01 h to 5 h for coordinated water a Compounds with a value of 5–9, and the acid dissociation constant (pK) of coordinated water. a Compounds with a value of 5–8. The acid dissociation constant (pK) of coordinated water. a Compounds with a value of 5 to 9 can specifically be those with an acid dissociation constant (pK) of coordinated water. a Compounds with an acid dissociation constant (pK) of 5.0 or higher, 5.1 or higher, 5.2 or higher, 5.3 or higher, 5.4 or higher, 5.5 or higher, 5.6 or higher, 5.7 or higher, 5.8 or higher, 5.9 or higher, 6.0 or higher, 6.1 or higher, 6.2 or higher, 6.3 or higher, 6.4 or higher, 6.5 or higher, 6.6 or higher, 6.7 or higher, 6.8 or higher, 6.9 or higher, 7.1 or higher, 7.2 or higher, 7.3 or higher, 7.4 or higher, 7.5 or higher, 7.6 or higher, 7.7 or higher, 7.8 or higher, 7.9 or higher, 8.0 or higher, 8.1 or higher, 8.2 or higher, 8.3 or higher, 8.4 or higher, 8.5 or higher, 8.6 or higher, 8.7 or higher, 8.8 or higher, or 8.9 or higher, and the acid dissociation constant (pK) of coordinated water. aCompounds with a pH value below 9.0, 8.9, 8.8, 8.7, 8.6, 8.5, 8.4, 8.3, 8.2, 8.1, 8.0, 7.9, 7.8, 7.7, 7.6, 7.5, 7.4, 7.3, 7.2, 7.1, 6.9, 6.8, 6.7, 6.6, 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, or 5.1. The half-life (t) of the auto-oxidation reaction carried out at pH 7.0 and 25°C is also considered. 1 / 2 The determination was performed according to the following steps. Excess sodium dithionite was added to an aqueous solution of the oxidized form of hemoCD (where iron atoms are in the valence trivalent) to reduce the iron atoms to the valence divalent. Then, the solution was filtered through a gel filtration column (HiTrap). TM The desalting column (5 mL, manufactured by Cytiva) removes excess sodium dithionite. In this process, a reduced hemoCD aqueous solution is produced that combines with oxygen in the aqueous solution. Subsequently, the auto-oxidation reaction from the reduced form to the oxidized form is tracked by UV-Vis absorption spectroscopy, and the half-life (t) of the auto-oxidation reaction is calculated according to the following formula. 1 / 2 ).

[0075]

[0076]

[0077] Where A represents the absorbance at any wavelength that changes due to the auto-oxidation reaction, k represents the reaction rate constant, and t represents time.

[0078] Acid dissociation constant of coordinated water (pK) a The determination was performed according to the following steps. The hemoCD aqueous solution was adjusted to an acidic condition, for example, pH 1. The pH was then gradually changed towards alkalinity until, for example, pH 14, and the UV-Vis absorption spectrum was measured during this period. For the change in UV-Vis absorption spectrum caused by the acid dissociation of water molecules coordinated with hemoCD, the absorbance at any wavelength before and after the change was plotted against the pH of the aqueous solution. The midpoint of the change, i.e., the point where hemoCD coordinated with water molecules and hemoCD coordinated with hydroxyl ions exist in equal amounts, was taken as pK. a Please provide a solution.

[0079] In addition, inclusion complexes formed by encapsulating water-soluble metalloporphyrins with cyclodextrin dimers can, for example, have a binding constant [M] with hydrogen sulfide at 25°C and pH 7. -1 [1×10] 5 Above, 2×10 5 Above, 3×10 5 Above, 4×10 5 Above, 5×10 5 Above, 6×10 5 Above, 7×10 5 Above, 8×10 5 Above, 9×10 5 Above, 1×10 6 Above, or 2×10 6 The above compounds.

[0080] Furthermore, inclusion complexes formed by encapsulating water-soluble metalloporphyrins with cyclodextrin dimers can, for example, exhibit a binding rate [M] with hydrogen sulfide at pH 7.0. -1 s -1 [0.2×10] -3 Above, 0.3×10 -3 Above, 0.4×10 -3 Above, 0.5×10 -3 Above, 0.6×10 -3 Above, 0.7×10 -3 Above, 0.8×10 -3 Above, 0.9×10 -3 Above, 1.0×10 -3 Above, 1.1×10 -3 Above, 1.2×10 -3 Above, 1.3×10 -3 Above, 1.4×10 -3 Above, 1.5×10 -3 Above, 1.6×10 -3 Above, 1.7×10 -3 Above, 1.8×10 -3 Above, 1.9×10 -3 Above, or 2.0 × 10 -3 The above compounds, and their binding rate with hydrogen sulfide at pH 7.4 [M] -1 s -1 [0.1×10] -3 Above, 0.2×10 -3 Above, 0.3×10 -3 Above, 0.4×10 -3 Above, 0.5×10 -3 Above, 0.6×10-3 Above, 0.7×10 -3 Above, 0.8×10 -3 Above, 0.9×10 -3 Above, 1.0×10 -3 Above, 1.1×10 -3 Above, 1.2×10 -3 Above, 1.3×10 -3 Above, 1.4×10 -3 Above, 1.5×10 -3 Above, 1.6×10 -3 Above, 1.7×10 -3 Above, or 1.8 × 10 -3 The above compounds.

[0081] Divalent cyclodextrins such as Im3CD and Py3CD need to perform the following two functions.

[0082] First, it encapsulates water-soluble metalloporphyrins, preventing the porphyrins from forming polymers. Once polymerized, they cannot interact sufficiently with hydrogen sulfide.

[0083] Second, the unpaired electrons on the nitrogen atoms of the pyridine or even imidazole at the cross-linking sites coordinate with the metal atom at the porphyrin center, stabilizing the coordination of hydrogen sulfide. Thus, after hydrogen sulfide (H₂S) coordinates with hemoCD, the hydrogen ion decouples, forming an SH-hemoCD complex. The acid dissociation constant (pK) of the water molecule coordinated with the metal atom in the water-soluble metalloporphyrin is... a The closer the pH is to physiological conditions (pH 7–7.4), the higher the affinity for hydrogen sulfide. This is attributed to the following reason: having a pH close to physiological conditions... a The coordination of hemoCD is dominated by water molecules that readily undergo exchange reactions with hydrogen sulfide, thus promoting the binding of hydrogen sulfide to hemoCD. Moreover, the presence of partial hydroxyl groups can inhibit the reverse reaction.

[0084] To achieve a higher detoxification effect, the following action is required: the hemoCD coordinated with hydrogen sulfide acts as a catalyst, promoting the oxidation of hydrogen sulfide and rendering it non-toxic, before capturing more hydrogen sulfide. After coordination with hemoCD, hydrogen sulfide is oxidized by oxygen, transforming into sulfate ions, sulfite ions, and thiosulfate ions. The metal atoms in the hemoCD are reduced by the reduction of hydrogen sulfide. Therefore, the oxidation rate of the reduced metal atoms becomes the rate-limiting step of the catalytic action, determining the hydrogen sulfide decomposition efficiency, in order to combine with other hydrogen sulfides. For example, when iron atoms are used as the metal atoms in water-soluble metalloporphyrins, hydrogen sulfide combines with trivalent iron atoms. Subsequently, the Fe-S bond undergoes homolytic cleavage, causing the iron atoms to be reduced to divalent iron, generating SH radicals. Since SH radicals are unstable, they should rapidly convert into inorganic ions such as sulfate ions, sulfite ions, and thiosulfate ions. On the other hand, since the reduced iron atom is in the valent II state, it will form an oxygen adduct in an oxygen-containing environment. At this point, the oxygen adduct is relatively stable, meaning the reaction rate (autooxidation rate) from the oxygen adduct to the oxide of ferric iron (valent III) is slow, and it cannot capture subsequent hydrogen sulfide. Furthermore, the iron atom in hemoCD can also be reduced by reducing agents present in the body. In other words, a hemoCD compound with a faster autooxidation rate can exist stably in the body as ferric iron and should have a high hydrogen sulfide capture capacity and hydrogen sulfide decomposition efficiency. In controlling the autooxidation reaction of this iron atom, increasing the distance between cross-linked cyclodextrins allows water molecules to easily reach the vicinity of the porphyrin, thereby accelerating the autooxidation rate.

[0085] Cyclodextrin dimers can achieve the same effect as long as the properties of the hydrophobic cavity formed by the cyclodextrin are not compromised. Therefore, a portion of the R,-OCH3 group in formula (1) or formula (12) should be replaced by an OH group or a hydroxypropyl group, etc. This is because the hydrophobic cavity of the cyclodextrin interacts with the hydrophobicity of the porphyrin to form an inclusion complex, and therefore partial modification without impairing the interaction should not affect the properties. However, methylation increases the flexibility of the originally rigid cyclodextrin skeleton due to hydrogen bonding, thus forming a structure more suitable for interaction with water-soluble metalloporphyrins. The methylation shown in the later examples is the optimal approach; excessive modification may impair its properties.

[0086] In the aforementioned chemical formula (1) or chemical formula (12), n=1 is α-cyclodextrin and n=3 is γ-cyclodextrin, whose internal cavities are narrower and wider than those of β-cyclodextrin, which forms the Py3CD backbone, respectively. The driving force for inclusion is the same as before, in that the phenyl group of the porphyrin side chain interacts hydrophobically with the internal cavity of the cyclodextrin. To obtain a higher detoxification effect, the auto-oxidation rate can be controlled, for example, by increasing k in chemical formula (1) or chemical formula (12) for α-cyclodextrin with n=1, and decreasing k for γ-cyclodextrin with n=3, etc.

[0087] For k in the aforementioned chemical formula (1) or chemical formula (12), if it is hemoCD-I, if the amide bond is replaced with a hydrocarbon bond, its carbon number can be estimated to be about 2. Therefore, k=3 can be considered to be roughly equivalent to hemoCD-I.

[0088] Water-soluble metalloporphyrins:

[0089] Water-soluble metal porphyrins are, for example, porphyrin compounds that have a metal ion coordinated at the center and are soluble in water. There are no particular restrictions on them, as long as they are porphyrin compounds that can be encapsulated by cyclodextrin dimers represented by chemical formula (1) or chemical formula (12).

[0090] Water-soluble metalloporphyrins can be, for example, compounds represented by the aforementioned chemical formula (2). More specifically, water-soluble metalloporphyrins can be 5,10,15,20-tetra(4-sulfonylphenyl)porphyrin iron complex (FeTPPS), 5,15-bis(3,5-dicarboxylated phenyl)-10,20-diphenylporphyrin iron complex, etc. It should be noted that these compounds can be synthesized, for example, by known methods, or commercially available products can be used directly (e.g., Frontier Scientific, Tokyo Chemical Industry Co., Ltd., etc.).

[0091] In chemical formula (2), A can represent any of the groups represented by chemical formulas (3) to (8), wherein n in chemical formulas (4) and (6) to (8) can be the same or different, and can represent integers from 1 to 200, such as less than 200, less than 190, less than 180, less than 170, less than 160, less than 150, less than 140, less than 130, less than 120, less than 110, less than 100, less than 90, less than 80, less than 70, less than 60, less than 50, less than 40, less than 30, less than 20, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 20, less than 10, less than 9, less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less than 2, or less than 1. Additionally, n is, for example, an integer greater than or equal to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200. A in chemical formula (2) can represent any of the groups represented by chemical formulas (3) to (8), wherein R in chemical formulas (3) to (4) can be the same or different, representing either a hydrogen atom or a hydrocarbon group, and the number of carbon atoms in the hydrocarbon group of R in chemical formulas (3) to (4) can be, for example, 1 to 10.

[0092] For A in chemical formula (2), the functional group such as SO3 in FeTPPS - Such anionic substituents enhance inclusion with cyclodextrins, and are therefore preferred from the viewpoint of inclusion compound stability. On the other hand, since hydrogen sulfide binds to the metal atom at the porphyrin center, even neutral or cationic functional groups can exert the effects of this invention. The phenyl group is crucial for inclusion with cyclodextrins. A can, for example, be a substituent with a molecular weight of 1 to 10,000.

[0093] Thus, since there are fewer restrictions on A in chemical formula (2), A can be any of the substituents represented by the aforementioned chemical formulas (3) to (8). Furthermore, it has been confirmed that even TPP with A as a hydrogen atom, TPPOH with A as an OH group, or even A with -O-CH2-CH2-(-O-CH2-CH2) q Long molecular chains, such as polyethylene glycol chains represented by -OCH3 (q being a positive integer), can also form inclusion complexes. It should be noted that existing studies have confirmed that compounds with q values ​​of 44 and 112 (compounds with molecular weights of 2000 and 5000, respectively) can also form inclusion complexes.

[0094] This is because the phenyl group bound to the porphyrin skeleton has a significant impact on the inclusion complex with cyclodextrin. Given the uneven charge distribution within the cyclodextrin, anionic groups (A) result in a stronger inclusion complex, but even neutral or cationic groups can form inclusion complexes and thus achieve a detoxification effect. Previous papers have also investigated the binding constants of substituents in A when forming large inclusion complexes (CD + FeTPPS → CD / FeTPPS) (J. AM. CHEM. SOC. 2002, 124, 9937-9944). As shown in Table 2 of that paper, solvent belonging to the EG-H2O class forms an inclusion complex. It should be noted that this paper also discloses that PC3Py and PC7Py without phenyl groups, as well as TMPyP with the phenyl group replaced by a cationic pyridine skeleton, cannot form inclusion complexes.

[0095] For Z in chemical formula (2), the same effect is achieved when the central metal is Co, in addition to Fe. As is well known, in compounds such as hydroxycobalamin and cobalaminamide, cobalt atoms combine with hydrogen sulfide. Therefore, Z in chemical formula (2) can also be Co.

[0096] Hydrogen sulfide has a strong affinity for ferric iron (Fe3+). In vivo, hemoCD is reduced to ferrous iron (Fe2+). Therefore, for optimal detoxification, maintaining the stability of ferric iron (i.e., a short oxidation half-life) and a sufficiently large binding constant K with hydrogen sulfide are crucial. In carbon monoxide antidotes, CO binds to hemoCD, while in cyanide antidotes, CN binds, achieving a therapeutic effect by removing the toxic substance from the body. In contrast, with hydrogen sulfide antidotes, the Fe-S bond homolytically cleaves after hydrogen sulfide binds to hemoCD, reducing the iron atoms in hemoCD and oxidizing the generated SH radicals into non-toxic inorganic ions. Therefore, to achieve higher therapeutic efficacy, a catalytic hydrogen sulfide decomposition cycle needs to be driven, i.e., the reduced hemoCD is re-oxidized to form ferric iron, which readily binds to hydrogen sulfide, and then captures other hydrogen sulfide.

[0097] The hydrogen sulfide antidote of the present invention comprises a pharmaceutical composition consisting of the inclusion complex monomer or a known pharmaceutical carrier, and can be administered to humans or other animals. The dosage form of the pharmaceutical composition is not particularly limited and can be selected as needed. Specifically, it can be an oral preparation such as tablets, capsules, granules, fine granules, powders, etc., or a non-oral preparation such as injections, suppositories, or topical applications. It should be noted that the dosage of the hydrogen sulfide antidote in the pharmaceutical composition and the amount of the pharmaceutical composition administered to the patient can be freely selected based on the dosage form, the patient's age, weight, and the severity of the disease.

[0098] The hydrogen sulfide antidote disclosed herein can also be a pharmaceutical composition for treating and / or preventing hydrogen sulfide poisoning. Some embodiments of this disclosure may be a pharmaceutical composition for use in treating and / or preventing 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 forms an inclusion complex with the cyclodextrin dimer. In other words, some embodiments of this disclosure may be a pharmaceutical composition for use in treating and / or preventing hydrogen sulfide poisoning, containing an inclusion complex comprising a cyclodextrin dimer represented by chemical formula (1) or chemical formula (12) and a water-soluble metalloporphyrin as the active ingredient. The pharmaceutical compositions involved in this disclosure may further comprise pharmaceutically acceptable carriers and other additional ingredients.

[0099] When the hydrogen sulfide antidote of the present invention is formulated into oral preparations such as tablets, it can be manufactured together with known excipients, binders, disintegrants, surfactants, lubricants, flow aids, etc., according to known manufacturing methods.

[0100] Furthermore, the hydrogen sulfide antidote of the present invention can also be administered orally as a suspension, emulsion, syrup, or elixir. In this case, it may contain flavoring agents, deodorizing agents, coloring agents, etc.

[0101] When the hydrogen sulfide antidote of the present invention is formulated into non-oral preparations such as injections or infusions, it can be manufactured using known methods, together with diluents such as distilled water for injection, physiological saline, or glucose solution. It should be noted that bactericides, preservatives, and stabilizers can be added as needed. Furthermore, for stability considerations, the non-oral preparation can be frozen after being filled into vials or other containers, and the moisture can be removed through conventional freeze-drying. The freeze-dried material can then be reconstituted into a liquid before use. Additionally, isotonic agents, stabilizers, preservatives, and analgesics can be added as needed.

[0102] Other examples of non-oral formulations of the hydrogen sulfide antidote of the present invention may be topical liquids, ointments, or suppositories for rectal administration, which may also be manufactured according to known methods.

[0103] It should be noted that the hydrogen sulfide antidote of the present invention can be delivered using known DDS (drug delivery systems) technology. For example, the hydrogen sulfide antidote of the present invention can be encapsulated in a carrier such as liposomes for in vivo administration. In this case, if a carrier or similar device capable of specifically recognizing cells at the target site is used, the hydrogen sulfide antidote of the present invention can be efficiently delivered to the target site.

[0104] The hydrogen sulfide antidote disclosed herein can be used in methods for treating and / or preventing hydrogen sulfide poisoning in a subject. Partial embodiments of this disclosure can be a method for treating and / or preventing hydrogen sulfide poisoning in a subject, comprising administering to the subject an inclusion complex containing a cyclodextrin dimer represented by chemical formula (1) or (12) and a water-soluble metalloporphyrin. The route of administration may be, for example, intravenous or inhalation administration, but is not limited thereto. For example, administration may be given to a subject diagnosed with hydrogen sulfide poisoning or a subject suspected of having hydrogen sulfide poisoning. Furthermore, prophylactic administration may be given to a subject at risk of hydrogen sulfide poisoning.

[0105] Additionally, some embodiments of this disclosure also relate to the use of an inclusion complex comprising a cyclodextrin dimer represented by chemical formula (1) or chemical formula (12) and a water-soluble metalloporphyrin in the manufacture of a medicament for treating and / or preventing hydrogen sulfide poisoning. Some embodiments of this disclosure also relate to a method for manufacturing a pharmaceutical composition for treating and / or preventing hydrogen sulfide poisoning, comprising: mixing a cyclodextrin dimer represented by chemical formula (1) or chemical formula (12) and a water-soluble metalloporphyrin represented by chemical formula (2).

[0106] Example

[0107] Example 1:

[0108] All animal experiments were conducted in accordance with the animal experiment regulations established by Doshisha University. Nine-week-old female BALB / cCrSlc mice (approximately 20 g in weight) were used as experimental animals. Sodium bisulfide was used as the hydrogen sulfide source. Since sodium bisulfide is solid at room temperature and under normal pressure, it is easy to handle; dissolving it in an aqueous solution generates hydrogen sulfide. Sodium bisulfide was dissolved in physiological saline (100 μL) to achieve a sodium bisulfide concentration relative to the mouse body weight of 22.5 mg / kg. The mice were administered this sodium bisulfide aqueous solution intraperitoneally to induce hydrogen sulfide poisoning. One minute after administration of the sodium bisulfide aqueous solution, a control experiment was performed by intraperitoneal administration of hemoCD-I (7 mM, 200 μL) solution, hemoCD-P (7 mM, 200 μL) solution, or physiological saline (200 μL) alone. Mice were observed for 45 minutes after administration of the sodium bisulfide aqueous solution to confirm survival. It should be noted that hemoCD-I was synthesized according to the method shown in ACS Med. Chem. Lett. 2011, 2, 943-947, while hemoCD-P was synthesized according to the method shown in Angew. Chem. Int. Ed. 2005, 44, 435-438. Their respective reaction formulas are shown below:

[0109]

[0110] The result is Figure 1 As shown in the image. Additionally... Figure 2 The binding constants of hydrogen sulfide with hemoCD-I and hemoCD-P, as well as the half-life of the oxidation reaction, are shown. For example... Figures 1-2 As shown, hydrogen sulfide poisoning can be treated by administering hemoCD-I or hemoCD-P. Furthermore, as... Figure 2 As shown, hemoCD-I has a strong binding force with hydrogen sulfide and can stably maintain ferric iron, thus having the highest detoxification effect on hydrogen sulfide poisoning.

[0111] To achieve a more effective detoxification effect against hydrogen sulfide poisoning, the following three points are important:

[0112] First, it exhibits a sufficiently fast binding rate (kJ / mL) to hydrogen sulfide within a pH range close to physiological conditions, from 7.0 to 7.4. on The metalloporphyrin (H+) can rapidly capture hydrogen sulfide within the body. The binding of hydrogen sulfide is an exchange reaction between hydrogen sulfide and water molecules coordinated to the central metal of the metalloporphyrin. This exchange between hydrogen sulfide and water molecules occurs rapidly. On the other hand, if a proton (H+) is removed from the water molecule coordinated to the H+, the exchange can proceed rapidly. + ) hydroxyl group (OH) - If the ligand is involved, the charge number changes before and after the exchange, thus slowing down the exchange reaction. In other words, for rapid binding with hydrogen sulfide, water molecules are preferably coordinated to the central metal of the metalloporphyrin within a pH range close to physiological conditions (7.0 to 7.4). This characteristic is reflected in the pKa of the coordinated water with the metal atom, which is preferably in the range of 5.0 or higher, and more preferably in the range of 6.5 or higher. The pKa of the coordinated water with the metal atom can be controlled by the axial ligand portion coordinated to the metal atom at the linker site of the cyclodextrin dimer. The axial ligand portion consists of electron-donating ligands, such as ligands containing pyridine or imidazole nitrogen atoms, and phenolic salt ligands containing oxygen atoms or thiolate ligands containing sulfur atoms.

[0113] Second, within the physiological pH range of 7.0 to 7.4, it possesses a sufficiently large binding constant (K) for hydrogen sulfide, enabling strong capture of bound hydrogen sulfide. To stabilize the bound hydrogen sulfide, it is crucial to introduce an electron-donating axial ligand onto the central metal of the metalloporphyrin. By increasing the electron density of the central metal, a stable hydrogen sulfide complex can be formed. Furthermore, since the binding constant is proportional to the binding rate (k... on ) and dissociation velocity (k off The ratio of hydroxyl (OH) to hydrogen sulfide is crucial for reducing the dissociation rate. That is, the dissociation reaction is an exchange reaction between water molecules (as solvent molecules) and hydrogen sulfide molecules. After the dissociation reaction, if the hydroxyl (OH) -When ligands are coordinated to the central metal of a metalloporphyrin, the change in charge before and after the reaction slows down the exchange reaction. Therefore, to obtain a larger binding constant by reducing the dissociation rate, the pKa of the coordinated water of the metal atom is preferably less than 9.0, and more preferably less than 8.0. The pKa of the coordinated water of the metal atom can be controlled by the axial ligand portion of the metal atom embedded in the linker site of the cyclodextrin dimer. The axial ligand portion consists of electron-donating ligands, such as ligands containing pyridine or imidazole nitrogen atoms, phenolic salt ligands containing oxygen atoms, or thiolate ligands containing sulfur atoms.

[0114] Third, within the physiological pH range of 7.0 to 7.4, the auto-oxidation rate of the oxygen complex is fast enough to drive the catalytic decomposition cycle of hydrogen sulfide. After the formation of a hydrogen sulfide coordination complex on the metalloporphyrin, the metal-hydrogen sulfide bond spontaneously undergoes homolytic cleavage, resulting in the single-electron reduction of the central metal. When the metal atom is iron, it is reduced from ferric (Fe3+) to ferrous (Fe2+). In vivo, the ferrous porphyrin complex forms an oxygen complex with oxygen molecules. This oxygen complex undergoes an auto-oxidation reaction, being reduced back to the original ferric (Fe3+), which can then re-bind with hydrogen sulfide molecules, potentially achieving a catalytic detoxification effect. That is, the faster the auto-oxidation reaction, the higher the potential effect as a detoxifying agent. The auto-oxidation rate can be controlled, for example, through the linker structure of the cross-linked cyclodextrin dimers. The more atoms involved, the longer the distance between the cyclodextrin dimers, and the easier it is for the central metal atom of the porphyrin to contact water molecules, thus often accelerating the auto-oxidation rate. In water with a pH of 7, the rate of auto-oxidation reaction is preferably within 30 hours, and more preferably within 5 hours.

[0115] In addition to the structures described in the examples, the cyclodextrin dimer / water-soluble metalloporphyrin inclusion complex that satisfies the above points and is effective in detoxifying hydrogen sulfide poisoning in vivo may also have the following structures.

[0116]

[0117] The parameters of each hemoCD compound are shown in Table 1 below.

[0118] Table 1

[0119]

[0120] Next, the decomposition rate of hydrogen sulfide by each hemoCD was investigated. The decomposition rate of hydrogen sulfide was determined according to the following steps: In the presence of trifluoroacetic acid, an amount equivalent to more than 40 equivalents of N,N-dimethyl-p-p-phenylenediamine sulfate and more than 60 equivalents of ferric chloride(III) were added to a solution containing hydrogen sulfide. The concentration of methylene blue generated from the reaction with hydrogen sulfide was calculated based on the absorbance at 665 nm. Assuming that the concentration of methylene blue was equal to the concentration of hydrogen sulfide in the solution, the decomposition rate of hydrogen sulfide was determined based on the change in hydrogen sulfide concentration over time, with or without hemoCD. The results were... Figure 4 The results are shown in the diagram. Furthermore, the half-life of each hemoCD auto-oxidation reaction studied according to the method shown in this specification was compared with the half-life of the hemoglobin auto-oxidation reaction. Figure 5 The diagram shows this. Additionally, the binding rates (kJ / kJ) of hydrogen sulfide with each hemoCD were investigated. on ) and dissociation velocity (k off The binding rate (kJ / kJ) of hemoCD with hydrogen sulfide. on The determination was performed according to the following steps. At least 50 equivalents of sodium hydrogen sulfide were added to the solution relative to the amount of hemoCD present. Immediately after addition, the absorbance at 410 nm was measured over time, and the rate constant k was determined by approximate calculation based on the following formula.

[0121]

[0122] Where t is the time elapsed after the addition, [A] is the absorbance at 410 nm at a certain time t, and [A]0 is the absorbance at 410 nm before the addition.

[0123] k was determined for different sodium bisulfide concentrations. A graph was plotted between k and sodium bisulfide concentration, and the binding rate (k0) was calculated based on the slope of the approximate straight line. on Furthermore, the dissociation velocity (k) is calculated according to the following relationship. off The result was in Figure 6 As shown in the image.

[0124]

[0125] Wherein, K is the binding constant between hemoCD and hydrogen sulfide. The binding constant K between hemoCD and hydrogen sulfide is determined according to the following steps: An aqueous solution of sodium hydrogen sulfide is titrated in an aqueous hemoCD solution at pH 7.0 and 25°C. The UV-Vis absorption spectra of hydrogen sulfide coordinated with hemoCD are measured, and the changes in absorbance at any wavelength before and after hydrogen sulfide coordination are plotted against the concentration of sodium hydrogen sulfide. K is calculated by analyzing the obtained titration curve using the software "SPANA" through a Sequential 1:1 Complex Formation System.

[0126] like Figure 4 As shown, it can be seen that the consumption rate of hydrogen sulfide is significantly increased in the presence of hemoCD compared to the condition without hemoCD. Furthermore, hemoCD-I consumes hydrogen sulfide at a faster rate than hemoCD-P. This indicates that, as... Figure 5 As shown, the oxygen adduct of hemoCD-I has low stability, therefore Figure 3 The catalytic decomposition of hydrogen sulfide shown can proceed more efficiently. Furthermore, as... Figure 6 As shown, the binding rate of hemoCD with hydrogen sulfide decreases as the pH of the aqueous solution increases. This is because, in the higher pH region, hydrogen sulfide (H2S) dissociates into hydrogen sulfide ions (HS-H2S). - This leads to a decrease in the actual effective hydrogen sulfide concentration. Among these, hemoCD-I exhibits a higher binding rate in the near-physiological pH range of 7.0 to 7.4 compared to hemoCD-P. This is because hemoCD-I has a near-physiological pKa of 7.7, and within the pH range of 7.0 to 7.4, water molecules constitute a larger proportion of the sixth ligand. Since water molecules readily exchange with hydrogen sulfide, hemoCD-I exhibits a greater binding rate within this near-physiological pH range. It should be noted that the acid dissociation constant of the coordinated water of hemoCD-P was determined using the method described in this paper, and the result is 5.5.

Claims

1. A hydrogen sulfide antidote, comprising as an active ingredient an inclusion complex consisting of a cyclodextrin dimer containing a water-soluble metalloporphyrin represented by the following chemical formula (1), In the formula, n can be the same or different, representing any number from 1, 2, and 3; R can be the same or different, representing any one of -OCH3 group, -OH group, or hydroxypropyl group; L can be the same or different, representing amide bond, thioether bond, amine bond, or -(CH2) bond. k - represents any of the hydrocarbon groups, wherein, k represents any number from 1, 2, 3, 4, 5; J represents any one of pyridine or imidazole substituted with one or more straight-chain or branched hydrocarbon groups, wherein the hydrocarbon group has any one of 1, 2, 3, 4, 5 carbon atoms; L is bonded to one carbon atom of the one or more straight-chain or branched hydrocarbon groups in J.

2. The hydrogen sulfide antidote according to claim 1, wherein, The cyclodextrin dimer represented by chemical formula (1) is the cyclodextrin dimer represented by the following chemical formula (9) or chemical formula (10). In the above general formula (9), R represents the protecting group protecting the hydroxyl group of the cyclodextrin; m can be the same or different, representing integers from 1 to 2 respectively; n can be the same or different, representing integers from 1 to 3 respectively; In the above general formula (10), R represents the protecting group of the hydroxyl group of the cyclodextrin; p can be the same or different, representing integers from 1 to 2 respectively; q can be the same or different, representing integers from 1 to 3 respectively.

3. The hydrogen sulfide antidote according to claim 1, wherein, Water-soluble metalloporphyrins are represented by the following chemical formula (2). In the formula, A represents any one of anionic substituents, neutral or cationic functional groups; Z represents any one of Fe or Co.

4. The hydrogen sulfide antidote according to claim 3, wherein, In chemical formula (2), A represents sulfonyl (-SO3) - ), sulfonamide group, amino group, nitro group, carboxyl group, hydrogen atom, chlorine atom, hydroxyl group, or, representing any one 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, representing integers from 1 to 200 respectively, and R in chemical formulas (3) to (4) may be the same or different, representing hydrogen atom or hydrocarbon group respectively. 。 5. The hydrogen sulfide antidote according to claim 1, wherein, In the inclusion complex formed by the inclusion of a water-soluble metalloporphyrin by a cyclodextrin dimer represented by chemical formula (1), the half-life of the auto-oxidation reaction represented by the following reaction formula (11) carried out at pH 7.0 and 25°C is 0.01 h to 5 h. In the formula, Z represents the metal atom in the water-soluble metal porphyrin; L represents the valence of Z during the ionization process.

6. The hydrogen sulfide antidote according to claim 1, wherein, The acid dissociation constant of the coordinating water of the inclusion complex formed by the inclusion of water-soluble metal porphyrin by the cyclodextrin dimer represented by chemical formula (1) is 5 to 9.

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

8. A method for manufacturing a pharmaceutical composition for treating and / or preventing hydrogen sulfide poisoning, comprising: The mixture comprises the cyclodextrin dimer represented by chemical formula (1) according to claim 1, and the water-soluble metalloporphyrin represented by chemical formula (2) according to claim 3.

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

10. A hydrogen sulfide antidote, comprising as an active ingredient an inclusion complex consisting of a water-soluble metalloporphyrin encapsulated by a cyclodextrin dimer represented by the following chemical formula (1) or (12), In formula (1) or formula (12), n can be the same or different, representing any number from 1, 2, and 3 respectively; R can be the same or different, representing any one of -OCH3 group, -OH group, and hydroxypropyl group respectively; L can be the same or different, representing amide bond, thioether bond, amine bond, and -(CH2) bond respectively. k - represents any of the hydrocarbon groups, wherein, k represents any number from 1, 2, 3, 4, 5; J represents any one of pyridine or imidazole substituted with one or more straight-chain or branched hydrocarbon groups, wherein the hydrocarbon group has any one of 1, 2, 3, 4, 5 carbon atoms; L is bonded to one carbon atom of the one or more straight-chain or branched hydrocarbon groups in J.

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