Antidotes, antidote kits, and novel inclusion complexes

Abstract

The present invention addresses the problem of providing a detoxifying agent and a detoxifying agent kit both for detoxifying CO and HCN in a living body simultaneously.　Provided is a detoxifying agent for removing carbon monoxide and hydrogen cyanide from a body, the detoxifying agent being characterized by containing, as active ingredients, a first inclusion complex having such a structure that a first cyclodextrin dimer represented by general formula (1) includes a first water-soluble metal porphyrin having a bivalent central metal and a second inclusion complex having such a structure that a second cyclodextrin dimer represented by general formula (2) includes a first water-soluble metal porphyrin having a bivalent central metal, and also characterized by further containing a reducing agent if necessary.

Description

Technical Field

[0001] The present invention relates to an antidote and an antidote kit, and more particularly, to an antidote having an antidotal effect against carbon monoxide and hydrogen cyanide, an antidote kit for preparing this antidote for use, and a novel inclusion complex that can be used in the antidote.

Background Art

[0002] Poisoning deaths due to inhalation of toxic gases are the leading cause of death in fires. The main toxic components in combustion gases are carbon monoxide (CO) and hydrogen cyanide (HCN). These gas components strongly bind to in-vivo heme proteins such as hemoglobin in the blood, myoglobin in muscles, and cytochromes in the inner mitochondrial membrane, thereby inhibiting aerobic respiration.

[0003] Currently, the treatment method for CO poisoning is only oxygen (O2) ventilation. This is a method of expelling CO out of the body by introducing high-purity O2 gas, but it is difficult to remove CO infiltrated into tissues by this method. CO poisoning frequently causes sequelae such as cognitive impairment even after recovery.

[0004] HCN is much more toxic than CO. In Japan, amyl nitrite is used for the treatment of HCN poisoning. Amyl nitrite oxidizes hemoglobin in the blood from iron (II) to iron (III), and the hemoglobin in the iron (III) state strongly binds to cyanide ions, thus reducing toxicity. However, this method impairs the original O2 transport function of hemoglobin, and is not suitable for fire gas poisoning that causes oxygen deficiency.

[0005] In a fire scene, CO is generated by incomplete combustion of wood, and HCN gas is generated by combustion of synthetic fibers such as acrylic fibers. Both are extremely toxic, and there is no method in the world to detoxify these gases simultaneously.

[0006] By the way, the present inventor has hitherto proposed various artificial hemoglobin model complexes using various cyclodextrin dimers.

[0007] For example, we have proposed an oxygen infusion solution for artificial blood and a carbon monoxide scavenger containing, as an active ingredient, an inclusion complex in which a predetermined cyclodextrin dimer encapsulates a water-soluble metal porphyrin (see Patent Documents 1 and 2). Furthermore, a cyanide detoxifier containing an inclusion complex, which is formed by encapsulating a predetermined cyclodextrin dimer with a water-soluble metal porphyrin, as an active ingredient has been proposed (see Patent Document 3).

[0008] However, we have yet to propose a practical method for simultaneously detoxifying CO and HCN in living organisms. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Patent No. 4803631 [Patent Document 2] Japanese Patent Publication No. 2010-194475 [Patent Document 3] Patent No. 5619500 [Overview of the project] [Problems that the invention aims to solve]

[0010] Therefore, the object of the present invention is to provide an antidote, an antidote kit, and a novel inclusion complex for simultaneously detoxifying CO and HCN in living organisms. [Means for solving the problem]

[0011] The inventors of this invention conducted thorough research to solve the aforementioned problems. The inclusion complex described in Patent Document 2 exhibits high bonding affinity to CO when the oxidation state of the central metal atom is divalent. In Example 1 of Patent Document 2, the central iron is reduced from trivalent to divalent to prepare a carbon monoxide scavenging agent (see paragraph 0033 of the specification of Patent Document 2). On the other hand, the inclusion complex described in Patent Document 3 has a high bonding affinity to hydrogen cyanide when the oxidation state of the central metal atom is trivalent. In Example 2 of Patent Document 3, an inclusion complex in the iron(III) state is also prepared as a cyanide detoxifier (see paragraph 0050 of the specification of Patent Document 3). Furthermore, it is described that when a reducing agent is added after adsorbing cyanide ions onto this cyanide detoxifier, the cyanide ions are rapidly desorbed due to the reduction from iron(III) to iron(II) (see paragraph 0054 of the specification of Patent Document 3).

[0012] Thus, since the inclusion complex described in Patent Document 2 and the inclusion complex described in Patent Document 3 have different oxidation states of the central metal atom when adsorbing toxic gases, it appears at first glance that the two technologies cannot be combined. However, the inventors focused on the fact that the oxidation state of the inclusion complex at the time of administration is not necessarily the same as the oxidation state of the inclusion complex in vivo. They came up with a novel idea to combine the cyclodextrin dimers that are the raw materials for the inclusion complexes of Patent Document 2 and Patent Document 3, a water-soluble metal porphyrin, and a reducing agent. When they actually conducted experiments, they confirmed that CO and HCN could be detoxified simultaneously. Specifically, when the inclusion complexes were administered in a divalent state by combining them with a reducing agent, the inclusion complex of Patent Document 1 was not oxidized in vivo and could adsorb carbon monoxide in its divalent state and excrete it from the body. On the other hand, the inclusion complex of Patent Document 3 was oxidized in vivo to a trivalent state and could adsorb cyanide and excrete it from the body.

[0013] This invention was completed based on the aforementioned findings. That is, the first antidote according to the present invention (hereinafter referred to as "the antidote 1 of the present invention") is an antidote for removing carbon monoxide and hydrogen cyanide in the body, and a first cyclodextrin dimer represented by the following general formula (1) encapsulates a first water-soluble metal porphyrin with a divalent central metal to form a first inclusion complex (hereinafter sometimes referred to as "hemoCDP(II)"), and a second inclusion complex which is a novel inclusion complex formed by a second cyclodextrin dimer represented by the following general formula (2) encapsulating a first water-soluble metal porphyrin with a divalent central metal (hereinafter sometimes referred to as "hemoCDI(II)") are included as active ingredients.

[0014]

Chemical formula

[0015]

Chemical formula

[0016] (In the general formula (2), R represents a protecting group for protecting the hydroxyl group of cyclodextrin, p represents an integer of 1 to 2, and q represents an integer of 1 to  3.)

[0017] In order for the above-mentioned antidote 1 to have a divalent central metal that is easily oxidized and maintain a stable state for a long time, a reducing agent may be mixed.

[0018] The first antidote kit according to the present invention (hereinafter referred to as "the antidote kit 1 of the present invention") is an antidote kit for preparing the antidote 1 of the present invention as needed, and the first cyclodextrin dimer represented by the general formula (1) is a third inclusion complex formed by inclusion of a water-soluble metal porphyrin having a divalent or higher central metal, and an inclusion complex mixed aqueous solution containing a fourth inclusion complex formed by inclusion of a water-soluble metal porphyrin having a divalent or higher central metal by the second cyclodextrin dimer represented by the general formula (2) or its raw material, and a reducing agent.

[0019] The second antidote according to the present invention (hereinafter referred to as "the antidote 2 of the present invention") is an antidote for removing carbon monoxide and hydrogen cyanide in the body, and the first cyclodextrin dimer represented by the general formula (1) is a novel inclusion complex formed by inclusion of a second water-soluble metal porphyrin having a trivalent central metal, a fifth inclusion complex (hereinafter sometimes referred to as "hemoCDP(III)"), and the second cyclodextrin dimer represented by the general formula (2) is characterized by containing, as active ingredients, a sixth inclusion complex (hereinafter sometimes referred to as "hemoCDI(III)") formed by inclusion of a second water-soluble metal porphyrin having a trivalent central metal.

[0020] The second antidote kit according to the present invention (hereinafter referred to as "the antidote kit 2 of the present invention") is an antidote kit for preparing the antidote 2 of the present invention as needed, and is characterized by containing an inclusion complex mixed aqueous solution containing the fifth inclusion complex (hemoCDP(III)) and the sixth inclusion complex (hemoCDI(III)) or its raw material.

Effects of the Invention

[0021] According to the antidotes 1 and 2 of the present invention, CO and HCN in the living body can be detoxified simultaneously by a single administration. The antidote kits 1 and 2 of the present invention are excellent in storage stability, and can prepare the antidote simply and quickly, and can be used even at a highly urgent site.

Brief Description of the Drawings

[0022] [Figure 1] This figure shows the measurement results of the change in the ultraviolet-visible absorption spectrum over time due to the auto-oxidation of the first inclusion complex (hemoCDP(II), Reference Example 1) in Test Example 1. [Figure 2] This figure shows the measurement results of the change in the ultraviolet-visible absorption spectrum over time due to the auto-oxidation of the second inclusion complex (hemoCDI(II), Reference Example 2) in Test Example 1. [Figure 3] This figure shows the measurement results of the change in the ultraviolet-visible absorption spectrum over time due to auto-oxidation of hemoCD-Twins (Example 1), which is a mixture of two inclusion complexes in Test Example 1. [Figure 4] This figure shows the results of measuring the change in the ultraviolet-visible absorption spectrum when CO gas and NaCN were sequentially added to hemoCD-Twins (Example 1), which is a mixture of two inclusion complexes, in Test Example 2. [Figure 5] This figure shows the results of measuring the change in the ultraviolet-visible absorption spectrum when NaCN and CO gas were sequentially added to hemoCD-Twins (Example 1), which is a mixture of two inclusion complexes, in Test Example 2. [Figure 6] This figure shows three ultraviolet-visible absorption spectra: the ultraviolet-visible absorption spectrum of the urine of mice administered the first inclusion complex, hemoCDP(II) (Reference Example 1), in Test Example 3; the ultraviolet-visible absorption spectrum after introducing CO gas into the urine; and the ultraviolet-visible absorption spectrum after adding a reducing agent. [Figure 7] This figure shows three ultraviolet-visible absorption spectra: the ultraviolet-visible absorption spectrum of the urine of mice administered hemoCDI(II) (Reference Example 2), the second inclusion complex, in Test Example 3; the ultraviolet-visible absorption spectrum after introducing CO gas into the urine; and the ultraviolet-visible absorption spectrum after adding a reducing agent. [Figure 8]In Test Example 3, this figure shows three ultraviolet-visible absorption spectra: the ultraviolet-visible absorption spectrum of the urine of mice administered hemoCD-Twins (Example 1), a mixture of hemoCDP(II) and hemoCDI(II); the ultraviolet-visible absorption spectrum after introducing CO gas into the urine; and the ultraviolet-visible absorption spectrum after adding a reducing agent. [Figure 9] This figure shows the results of quantifying the amount of hemoCD-Twins excreted in the urine of rats administered hemoCD-Twins (Example 1) from the ultraviolet-visible absorption spectrum (excretion amount per hour) in Test Example 4. [Figure 10] This figure shows the results (cumulative value of excretion) of the urinary excretion of hemoCD-Twins from the ultraviolet-visible absorption spectrum of the urine of rats administered hemoCD-Twins (Example 1) in Test Example 4. [Figure 11] This figure shows the change in heart rate of rats after administration of hemoCD-Twins (Example 1) in Test Example 5. [Figure 12] This figure shows the change in blood pressure in rats after administration of hemoCD-Twins (Example 1) in Test Example 5. [Figure 13] In Test Example 6, the chromatogram shows the changes in blood concentration in rats after administration of hemoCD-Twins (Example 1). [Figure 14] This figure shows the time course of blood concentration in rats after administration of hemoCD-Twins (Example 1) in Test Example 6. [Figure 15] This is an explanatory diagram of the test method for Test Example 7. [Figure 16] This figure shows the survival curves of rats after administration of NaCN and / or CO in Test Example 7. [Figure 17] This figure shows the behavioral observation results of rats after administration of NaCN and / or CO in Test Example 7. [Figure 18] This is an explanatory diagram of the test method for Test Example 8. [Figure 19]This figure shows the survival curves of rats in Test Example 8, comparing the group that received hemoCD-Twins (Example 1) after administering 0.15 mg of NaCN and 5000 ppm of CO, with the group that did not receive the treatment. [Figure 20] This figure shows the behavioral observation results of rats in Test Example 8, comparing the group that received hemoCD-Twins (Example 1) after administering 0.15 mg of NaCN and 5000 ppm of CO, with the group that did not receive the drug. [Figure 21] This is an explanatory diagram of the test method for Test Example 9. [Figure 22] This figure shows the survival curves of rats in Test Example 9, comparing the group that received hemoCD-Twins (Example 1) after administering 0.10 mg of NaCN and 5000 ppm of CO, with the group that did not receive the treatment. [Figure 23] This figure shows the behavioral observation results of rats in Test Example 9, comparing the group that received hemoCD-Twins (Example 1) after administering 0.10 mg of NaCN and 5000 ppm of CO, with the group that did not receive the drug. [Figure 24] This figure shows the blood pressure changes in rats in Test Example 10, comparing a group that received hemoCD-Twins (Example 1) after administering 0.15 mg of NaCN and 5000 ppm of CO, with a group that did not receive the drug. [Figure 25] This figure shows the change in CO-Hb over time in rats in Test Example 11, comparing a group that received hemoCD-Twins (Example 1) after being administered 2000 PPM of CO with a group that did not receive the drug. [Figure 26] This figure shows the time course of CN- concentration in the blood of rats in Test Example 12, comparing a group that received hemoCD-Twins (Example 1) after administering 5 mg of NaCN per kg of body weight, with a group that did not receive the drug. [Figure 27] This figure shows the time course of blood concentration in rats after administration of hemoCD-Twins (Example 1) in Test Example 13. [Figure 28]In Test Example 14, after administering hemoCD-Twins (Example 1) to healthy rats via the femoral vein, the amount of hemoCD-Twins contained in urine collected every 10 minutes was quantified by UV-vis, and the urinary clearance profile was plotted as the ratio of detected hemoCD-Twins to the administered dose against the urine collection time. [Figure 29] In Test Example 15, the hemoCD-Twins (Example 1) was administered via the femoral vein over 30 minutes, and this figure shows the changes in heart rate and blood pressure over time before and after the administration. [Figure 30] In Test Example 16, healthy mice were administered hemoCD-Twins (Example 1) via the femoral vein over 30 minutes, and the amount of creatinine in the blood collected 24 hours later was measured. This figure shows the results compared to a case where PBS was administered instead of hemoCD-Twins. [Figure 31] This figure illustrates the process in which, in Test Example 17, an aqueous solution containing hemoCDP(III), a novel inclusion complex of the present invention, is administered to a mouse, and urine is collected 30 minutes later. [Figure 32] This figure shows the results of spectral analysis of CO-bound compounds, Fe(II) oxygen-bound compounds (oxy), and Fe(III) oxidized compounds (met) in urine collected 30 minutes after administering an aqueous solution containing hemoCDP(III), the novel inclusion complex of the present invention, to mice in Test Example 17. [Figure 33] This figure illustrates the process in Test Example 18, where mice are administered CO at 2000 PPM for 15 minutes, and then, 15 minutes later, an aqueous solution containing the novel fifth inclusion complex of the present invention and PBS are administered to the mice, and the blood CO-Hb concentration is measured every 10 minutes for each. [Figure 34] In Test Example 18, mice were administered 2000 PPM of CO for 15 minutes, and then 15 minutes later, an aqueous solution containing the novel fifth inclusion complex of the present invention and PBS were administered to the mice. This figure shows a comparison of the results of measuring the blood CO-Hb concentration at 10-minute intervals for each. [Modes for carrying out the invention]

[0023] Preferred embodiments of the antidote 1 and antidote kit 1 according to the present invention will be described in detail below, but the scope of the present invention is not limited to these descriptions, and other embodiments may be modified as appropriate without impairing the spirit of the present invention.

[0024] [First cyclodextrin dimer] The first cyclodextrin dimer is represented by the following general formula (1).

[0025] [ka]

[0026] In the general formula (1) above, R represents a protecting group that protects the hydroxyl group of cyclodextrin, m represents an integer from 1 to 2, and n represents an integer from 1 to 3.

[0027] The cyclodextrin dimer represented by the general formula (1) can be produced, for example, by tosyling and epoxidizing cyclodextrin, then methylating the hydroxyl group of the cyclodextrin, and finally bonding the methylated cyclodextrin with a linker molecule, as described in Patent Document 1. By protecting the hydroxyl groups of cyclodextrin with methyl groups beforehand, it is possible to prevent the pores of the cyclodextrin from hardening due to hydrogen bonding caused by the hydroxyl groups, which would otherwise make it difficult for water-soluble metal porphyrins to be encapsulated in the pores of the cyclodextrin dimer. In addition to the methyl group, other examples of protecting group R in the general formula (1) include the ethyl group, acetyl group, and hydroxypropyl group.

[0028] As the cyclodextrin used as the raw material for the cyclodextrin dimer represented by the general formula (1) above, any of α-cyclodextrin, β-cyclodextrin (n=2), or γ-cyclodextrin may be used, but it is preferable to use β-cyclodextrin as the raw material because it readily encapsulates water-soluble metal porphyrins, and to set the linker molecule to m=1.

[0029] [Second cyclodextrin dimer] The second cyclodextrin dimer is represented by the following general formula (2).

[0030] [ka]

[0031] In the general formula (2) above, R represents a protecting group that protects the hydroxyl group of cyclodextrin, p represents an integer from 1 to 2, and q represents an integer from 1 to 3.

[0032] The cyclodextrin dimer represented by the general formula (2) can be produced, for example, by tosyling and epoxidizing cyclodextrin, then methylating the hydroxyl group of the cyclodextrin, and finally bonding the methylated cyclodextrin with a linker molecule, as described in Patent Document 3. By protecting the hydroxyl groups of cyclodextrin with methyl groups beforehand, it is possible to prevent the pores of the cyclodextrin from hardening due to hydrogen bonding caused by the hydroxyl groups, which would otherwise make it difficult for water-soluble metal porphyrins to be encapsulated in the pores of the cyclodextrin dimer.

[0033] As the cyclodextrin used as the raw material for the cyclodextrin dimer represented by the general formula (2) above, any of α-cyclodextrin, β-cyclodextrin (q=2), or γ-cyclodextrin may be used, but it is preferable to use β-cyclodextrin as the raw material because it readily encapsulates water-soluble metal porphyrins, and to set the linker molecule to p=1. The protecting group R in the general formula (2) is not limited to a methyl group, but may also be an ethyl group, an acetyl group, a hydroxypropyl group, or the like.

[0034] [First water-soluble metal porphyrin] The first water-soluble metal porphyrin is an organic compound having a cyclic structure formed by the combination of four pyrrole molecules, with a divalent metal ion M1 coordinated to the central nitrogen atom, and possessing water solubility. While not particularly limited, examples of those represented by the following general formulas (3) or (4) are preferred.

[0035] [ka]

[0036] [ka]

[0037] In the general formulas (3) and (4) above, R1 and R2 represent either a carboxyl group, a sulfonyl group, or a hydroxyl group, respectively, and M1 is Fe 2+ Mn 2+ Co 2+ Zn 2+ It represents one of the following.

[0038] In particular, among the compounds represented by the general formula (3), 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin iron complex is preferred, and among the compounds represented by the general formula (4), 5,15-bis(3,5-dicarboxylatophenyl)-10,20-diphenylporphyrin iron complex is preferred. These compounds may be synthesized by known methods, or commercially available products (e.g., Frontier Scientific, Tokyo Chemical Industry Co., Ltd., etc.) may be used as is.

[0039] In the antidote 1 of the present invention, the central metals of the first and second inclusion complexes are divalent, but either divalent or trivalent metals can be used as the central metal of the water-soluble metal porphyrin used to prepare this antidote. When a water-soluble metal porphyrin with a trivalent central metal is used as a raw material for the antidote, it can be reduced entirely to divalent metal by using it together with a reducing agent. Since trivalent metals are generally more stable, it is convenient to use a water-soluble metal porphyrin with a trivalent central metal as a raw material for preparing the antidote.

[0040] [Reducing agent] By using a reducing agent, the central metal of the inclusion complex can be reduced to the divalent state. Specific examples of reducing agents include sodium dithionite (Na2S2O4), sodium ascorbate, dithiothreitol, mercaptoethanol, cysteine, and glutathione.

[0041] [Antidote 1] The antidote 1 of the present invention can be prepared using the first and second cyclodextrin dimers described above, a water-soluble metal porphyrin, and a reducing agent, as follows.

[0042] The first and second cyclodextrin dimers and the first water-soluble metal porphyrin are all water-soluble. When the first and second cyclodextrin dimers and the first water-soluble metal porphyrin are mixed in an aqueous medium, an aqueous solution of inclusion complexes can be obtained, which contains a first inclusion complex with a divalent central metal, formed by the inclusion of the first water-soluble metal porphyrin by the first cyclodextrin dimer, and a second inclusion complex with a divalent central metal, formed by the inclusion of the first water-soluble metal porphyrin by the second cyclodextrin dimer. Furthermore, as a water-soluble metal porphyrin, a water-soluble metal porphyrin with a central metal of trivalent or higher can also be used. In such cases, the antidote 1 of the present invention can be prepared by first preparing an aqueous solution of the inclusion complex by mixing the first and second cyclodextrin dimers with a water-soluble porphyrin, and then adding and mixing a reducing agent thereto to divalentize the central metals of the first and second inclusion complexes.

[0043] The antidote 1 of the present invention can also be prepared, for example, by sequentially or simultaneously adding the first and second cyclodextrin dimers, water-soluble metal porphyrin, and reducing agent components to an aqueous medium. Another possible method involves dissolving each component in an aqueous medium to create aqueous solutions, and then mixing these solutions together.

[0044] Furthermore, some of the raw materials may be mixed in advance to obtain a preliminary mixture (for example, a mixture of powdered components before dissolving them in an aqueous medium), and this preliminary mixture may then be mixed with the other raw materials. Thus, various patterns are possible for the preparation method of antidote 1 and the order in which each component is mixed, and the method is not limited to any one of them.

[0045] The stoichiometric ratio of the first and second cyclodextrin dimers to the water-soluble metal porphyrin is 1:1 (molar ratio) for water-soluble metal porphyrin : (total amount of the first and second cyclodextrin dimers), but it is not limited to this ratio, and the total amount of the first and second cyclodextrin dimers may be added in excess of the water-soluble metal porphyrin. The basic ratio of the first cyclodextrin dimer to the second cyclodextrin dimer is 1:1 (molar ratio), but if carbon monoxide removal is a priority, a larger proportion of the first cyclodextrin dimer may be added. Conversely, if hydrogen cyanide removal is a priority, a larger proportion of the second cyclodextrin dimer may be added.

[0046] The reducing agent should be added in an amount sufficient to reduce the central metal of each inclusion complex to the divalent state. Furthermore, an excess amount of reducing agent may be used. Even if an excess amount of reducing agent remains in antidote 1, the auto-oxidation of the second inclusion complex will proceed once it is administered into the body and diluted, and by the time it is excreted in the urine, it will be completely oxidized. This can be seen from the test results of Test Example 3 described later, in particular the test results for hemoCDI(II) (corresponding to the second inclusion complex in the antidote of the present invention) (see also Figure 7). Therefore, even when an excess amount of reducing agent is used, it is not essential to remove the residual reducing agent before administering antidote 1.

[0047] The antidote 1 of the present invention includes not only aqueous solutions obtained by mixing and dissolving each of the above-mentioned components in an aqueous medium, or solutions with their concentrations appropriately adjusted, but also solutions processed into a desired dosage form together with other active ingredients or additives. There are no particular restrictions on the method of administration or the amount; it should be determined appropriately according to the dosage form, the patient's age, weight, and the severity of the disease.

[0048] [Antidote Kit 1] The antidote kit 1 of the present invention is a kit for preparing the antidote 1 of the present invention on a case-by-case basis, and comprises an aqueous solution of inclusion complexes containing the third inclusion complex and the fourth inclusion complex, or a raw material thereof, and a reducing agent. The following explains each of these points.

[0049] <Antidote kit 1 containing an aqueous solution of inclusion complex and a reducing agent> As described above, when the first and second cyclodextrin dimers and a water-soluble metal porphyrin having a central metal of 2 valence or higher are mixed in an aqueous medium, an aqueous solution of inclusion complexes 1 containing the third and fourth inclusion complexes can be obtained. In this aqueous solution of inclusion complexes, the central metal of the water-soluble metal porphyrin is divalent or higher. Therefore, the antidote kit of the present invention may include this inclusion complex mixed aqueous solution 1 as a component of the antidote kit 1. Then, by mixing this inclusion complex aqueous solution 1 with the reducing agent, which is another component of the antidote kit 1, the above-mentioned antidote 1 can be easily prepared.

[0050] <Antidote kit 1 containing raw materials and reducing agent for inclusion complex aqueous solution> The raw materials for preparing the inclusion complex mixed aqueous solution 1 include, for example, the first and second cyclodextrin dimers, the water-soluble metal porphyrin components (which may also be aqueous solutions), or a preliminary mixture thereof (for example, a mixture of the powdered components before dissolving them in an aqueous medium). The first and second cyclodextrin dimers and the water-soluble metal porphyrin are all available as powders, are stable at room temperature, and can be stored for a long period of time in powder form. Furthermore, the inclusion complex mixture aqueous solution 1 is also stable at room temperature and can be stored for a long period of time. Therefore, in terms of long-term storage stability, it does not matter whether the raw materials for preparing the inclusion complex aqueous solution are in powder or aqueous solution form. The antidote 1 described above can be easily prepared by mixing the raw materials of this inclusion complex aqueous solution together with the reducing agent, which is another component of the antidote kit 1.

[0051] <Ratio of each ingredient> Regarding the mixing ratio of each component, it is preferable that the appropriate amount is pre-adjusted in the antidote kit 1, as this eliminates the need for on-site weighing and concentration adjustment, allowing for the rapid preparation and use of the antidote 1. However, the present invention is not limited to this, and the amount of each component used may be adjusted during the preparation of the antidote 1, rather than pre-adjusting the appropriate amount in the antidote kit 1.

[0052] [Use of Antidote 1 and Antidote Kit 1] The antidote 1 and antidote kit 1 of the present invention are • It simultaneously captures CO and HCN within the body and excretes them in the urine. • It can be synthesized in large quantities at a relatively low cost. • The raw materials and their solutions are highly stable and can be stored at room temperature for more than one year. • Allows for easy and quick task preparation, making it suitable for use even in urgent situations. • A single dose provides immediate relief. • It does not remain in the body, making it highly safe. It has the following advantages. Taking advantage of these benefits, the antidote 1 and antidote kit 1 of the present invention are expected to be widely used as new assets, for example, 1) emergency treatment at fire scenes, 2) suppression of sequelae from fire gas poisoning, and 3) acquisition of fire gas tolerance through pre-administration.

[0053] 1) Emergency medical treatment at a fire scene When antidote 1 of the present invention is administered to an animal, it rapidly captures CO and HCN within the body and excretes them in the urine within one hour. The raw materials for antidote 1 are typically powder reagents, and since they are stable at room temperature for more than one year even when dissolved in physiological saline, they can be stored. When administered to patients whose level of consciousness has decreased due to smoke inhalation at a fire scene, it is expected to improve the survival rate and recovery rate from acute poisoning.

[0054] 2) Suppression of sequelae from fire gas poisoning The sequelae induced by residual CO / HCN in the body can be suppressed by continuous intravenous administration of the antidote 1 of the present invention. Brain dysfunction caused by fire gases can be suppressed by the antidote 1 of the present invention, and since residual CO / HCN levels can be measured by analyzing components in urine, it is also possible to adjust the dosage and determine when to discontinue administration.

[0055] 3) Acquisition of fire gas resistance through pre-administration Because the antidote 1 of the present invention binds CO / HCN more strongly than heme proteins in the body, pre-administration can temporarily provide resistance to fire gases. It is believed that pre-vaccination of emergency medical personnel can reduce accidents caused by accidental inhalation of smoke.

[0056] Preferred embodiments of the antidote 2 and antidote kit 2 according to the present invention will be described in detail below, but the scope of the present invention is not limited to these descriptions, and other embodiments may be modified as appropriate without impairing the spirit of the present invention.

[0057] [Antidote 2] Antidote 2 contains the novel inclusion complexes hemoCDP(III) and hemoCDI(III). In antidote 2, the first cyclodextrin dimer and the second cyclodextrin dimer can be the same as those used in antidote 1.

[0058] [Second water-soluble metal porphyrin] The second water-soluble metal porphyrin is an organic compound having a cyclic structure formed by the combination of four pyrrole molecules, with a trivalent metal ion M2 coordinated to the central nitrogen, and possessing water solubility. While not particularly limited, examples of those represented by the following general formulas (5) or (6) are preferred.

[0059] [ka]

[0060] [ka]

[0061] In the general formulas (5) and (6) above, R1 and R2 represent one of a carboxyl group, a sulfonyl group, or a hydroxyl group, respectively, and M2 is Fe 3+ Co 3+ It represents one of the following.

[0062] [Antidote Kit 2] The antidote kit 2 of the present invention is a kit for preparing the antidote 2 of the present invention on demand, and includes an aqueous solution 2 of a mixed inclusion complex containing the fifth inclusion complex and the sixth inclusion complex, or its raw materials. The following explains each of these points.

[0063] <Antidote Kit 2 containing an aqueous solution of inclusion complexes> As described above, when the first and second cyclodextrin dimers and a water-soluble metal porphyrin with a trivalent central metal are mixed in an aqueous medium, an aqueous solution 2 of the inclusion complex mixture containing the fifth and sixth inclusion complexes can be obtained. This inclusion complex aqueous solution 2 is stable at room temperature and can be stored for a long period of time because the central metal is trivalent. Therefore, the antidote kit 2 of the present invention may include this inclusion complex mixed aqueous solution 2 as a component of the antidote kit 2.

[0064] <Antidote kit 2 containing inclusion complex mixed aqueous solution 2> The raw materials for preparing the inclusion complex mixed aqueous solution 2 include, for example, the first and second cyclodextrin dimers, the second water-soluble metal porphyrin with a trivalent central metal (which may also be in aqueous solution form), and a preliminary mixture thereof (for example, a mixture of the powdered components before dissolving them in an aqueous medium). The first and second cyclodextrin dimers, and the second water-soluble metal porphyrin, are all available as powders, are stable at room temperature, and can be stored for long periods in powder form. Furthermore, the aqueous solution of the inclusion complex is also stable at room temperature and can be stored for long periods. Therefore, in terms of long-term storage stability, it does not matter whether the raw materials for preparing the inclusion complex mixed aqueous solution 2 are in powder form or aqueous solution form.

[0065] <Ratio of each ingredient> Regarding the mixing ratio of each component, it is preferable that the appropriate amount is pre-adjusted in the antidote kit 2, as this eliminates the need for on-site weighing and concentration adjustment, allowing for the rapid preparation and use of the antidote 2. However, the present invention is not limited thereto, and the amount of each component used may be adjusted during the preparation of the antidote 2 without pre-adjusting the appropriate amount in the antidote kit 2.

[0066] [Use of Antidote 2 and Antidote Kit 2] The antidote 2 and antidote kit 2 of the present invention are • It simultaneously captures CO and HCN within the body and excretes them in the urine. • It can be synthesized in large quantities at a relatively low cost. • The raw materials and their solutions are highly stable and can be stored at room temperature for more than one year. • Allows for easy and quick task preparation, making it suitable for use even in urgent situations. • A single dose provides immediate relief. • It does not remain in the body, making it highly safe. It has the following advantages. Taking advantage of these benefits, the antidote 2 and antidote kit 2 of the present invention are expected to be widely used as new assets, for example, 1) emergency treatment at fire scenes, 2) suppression of sequelae from fire gas poisoning, and 3) acquisition of fire gas tolerance through pre-administration.

[0067] 1) Emergency medical treatment at a fire scene When the antidote 2 of the present invention is administered to an animal, it rapidly captures CO and HCN within the body and excretes them in the urine within one hour. The raw materials for the antidote 2 are typically powder reagents, and since they are stable at room temperature for more than one year even when dissolved in physiological saline, they can be stored. When administered to patients whose level of consciousness has decreased due to smoke inhalation at a fire scene, it is expected to improve the survival rate and recovery rate from acute poisoning.

[0068] 2) Suppression of sequelae from fire gas poisoning The sequelae induced by residual CO / HCN in the body can be suppressed by continuous intravenous administration of the antidote 2 of the present invention. Brain dysfunction caused by fire gases can be suppressed by the antidote 2 of the present invention, and since residual CO / HCN levels can be measured by analyzing components in urine, it is possible to adjust the dosage and determine when to discontinue administration.

[0069] 3) Acquisition of fire gas resistance through pre-administration Because the antidote 2 of the present invention binds CO / HCN more strongly than heme proteins in the body, pre-administration can temporarily provide resistance to fire gases. It is believed that pre-vaccination of emergency medical personnel can reduce accidents caused by accidental inhalation of smoke.

[0070] Preferred embodiments of the antidote 1, 2 and antidote kit 1, 2 according to the present invention will be described in detail below. However, the scope of the present invention is not limited to these descriptions, and other embodiments may be modified as appropriate without impairing the spirit of the present invention. [Examples]

[0071] The antidote, antidote kit, and novel inclusion complex according to the present invention will be described below using examples, but the present invention is not limited to these examples.

[0072] [Reagents, etc.] The following reagents were used in the examples. As the first cyclodextrin dimer, one synthesized according to the synthesis method for cyclodextrin dimer (CD2) described in Patent Document 1 (see paragraphs 0016 to 0020 and Figure 1 of the specification of Patent Document 1). Hereinafter, it will be abbreviated as "Py3CD" or "P". The specific chemical structure of Py3CD corresponds to the case in the general formula (1) above where all R are methyl groups, m is 1, and n is 2. As the second cyclodextrin dimer, one synthesized according to the synthesis method for cyclodextrin dimer (Im3CD) described in Patent Document 3 (see paragraphs 0033 to 0047 and Figure 1 of the specification of Patent Document 3). Hereinafter, it will be abbreviated as "Im3CD" or "I". The specific chemical structure of Im3CD corresponds to the case in the general formula (2) above where all R are methyl groups, p is 1, and q is 2. The water-soluble metal porphyrin used was a 5,10,15,20-tetrakis(4-sulfonatofinyl)porphyrin iron(III) complex synthesized by known methods. This compound is also available commercially (e.g., from Frontier Scientific). Hereinafter, it will be abbreviated as "Fe(III)TPPS" or "F". Sodium dithionite (Na2S2O4) was used as the reducing agent. Hereafter, it will simply be referred to as the "reducing agent." Furthermore, ultraviolet-visible absorption spectra were measured using a spectrophotometer (UV-2600i, UV-2450, manufactured by Shimadzu Corporation).

[0073] [Example 1] Fe(III)TPPS(F), Py3CD(P), and Im3CD(I) were mixed in a molar ratio of 2:1:1 and added to physiological saline to prepare an aqueous solution of inclusion complexes. To prepare 10 mL of a 3.5 mM aqueous solution of the inclusion complexes, the amounts used were 40 mg of Fe(III)TPPS, 60 mg of Py3CD, and 60 mg of Im3CD. To prepare 10 mL of 7.0 mM and 14 mM aqueous solutions of the inclusion complexes, the amounts of each compound added were doubled and quadrupled as appropriate. The aqueous solution of inclusion complexes prepared in this manner contains an inclusion complex formed by Py3CD inclusion of Fe(III)TPPS (corresponding to the fifth inclusion complex (hemoCDP(III)) in the present invention) and an inclusion complex formed by Im3CD inclusion of Fe(III)TPPS (corresponding to the sixth inclusion complex (hemoCDI(III)) in the present invention). Next, an excess reducing agent was added to reduce the central iron of the two inclusion complexes contained in the mixed aqueous solution from iron(III) to iron(II), thereby obtaining the first inclusion complex (hemoCDP(II)) and the second inclusion complex (hemoCDI(II)) in the present invention. The amount of reducing agent added was standardized at 5 mg / mL (approximately 28 mM) regardless of the concentration of the mixed aqueous solution of inclusion complexes. It should be noted that the dose of sodium dithionite used in this study (50 mg / kg body weight) is significantly lower than the value indicating oral toxicity of sodium dithionite (2500 mg / kg body weight). This was designated as Antidote 1 of Example 1. The antidote 1 of Example 1 may be referred to as "hemoCD-Twins" below.

[0074] [Reference example 1] Fe(III)TPPS and Py3CD were mixed in a molar ratio of 1:1 and added to physiological saline to prepare an aqueous solution of the inclusion complex. To prepare 10 mL of a 3.5 mM aqueous solution of the inclusion complex, 40 mg of Fe(III)TPPS and 120 mg of Py3CD were used. To prepare 10 mL of 7.0 mM and 14 mM aqueous solutions of the inclusion complex, the amounts of each substance added were doubled and quadrupled as appropriate. Next, an excess reducing agent was added to reduce the central iron of the inclusion complex contained in the aqueous solution of the inclusion complex from iron(III) to iron(II). This was used as the antidote for Reference Example 1. The antidote in Reference Example 1 consists of the first inclusion complex in the present invention and may hereafter be referred to as "hemoCDP(II)".

[0075] [Reference example 2] Fe(III)TPPS and Im3CD were mixed in a molar ratio of 1:1 and added to physiological saline to prepare an aqueous solution of the inclusion complex. To prepare 10 mL of a 3.5 mM aqueous solution of the inclusion complex, 40 mg of Fe(III)TPPS and 120 mg of Im3CD were used. To prepare 10 mL of 7.0 mM and 14 mM aqueous solutions of the inclusion complex, the amounts of each substance added were doubled and quadrupled as appropriate. Next, an excess reducing agent was added to reduce the central iron of the inclusion complex contained in the aqueous solution of the inclusion complex from iron(III) to iron(II). This was used as the antidote for Reference Example 2. The antidote in Reference Example 2 consists of the sixth inclusion complex in the present invention and may hereafter be referred to as "hemoCDI(II)".

[0076] [Test Example 1: Measurement of the rate of auto-oxidation reaction of the central metal in inclusion complexes] The hemoCDP(II) synthesized in Reference Example 1 was diluted with physiological saline, and the time course of the ultraviolet-visible absorption spectrum was observed at a cell concentration of 4.0 × 10⁻⁶ M under physiological conditions (pH 7.4, 37°C). The results are shown in Figure 1. Figure 1 also shows the change in absorbance over time at the peak wavelength in the upper right. This change in absorbance was analyzed based on a first-order reaction rate equation.

[0077]

number

[0078] Here, A0 represents the absorbance at the start of the measurement, A∞ represents the absorbance at the end of the measurement, At represents the absorbance at 422 nm at time t, and kobs is the pseudo-first-order rate constant in the auto-oxidation reaction. The obtained spectral changes were analyzed by the curve fitting method based on equation (1), and kobs and the half-life t1 / 2 were calculated. These values ​​are also shown in Figure 1.

[0079] Similarly, for hemoCDI(II) synthesized in Reference Example 2, we observed the change in its ultraviolet-visible absorption spectrum over time and calculated the auto-oxidation reaction rate constant kobs and half-life t1 / 2. The results are shown in Figure 2.

[0080] Furthermore, the time-dependent changes in the ultraviolet-visible absorption spectrum were similarly observed for the hemoCD-Twins synthesized in Example 1. The results are shown in Figure 3. This change in absorbance was analyzed using the two-phase curve fitting method.

[0081]

number

[0082] Here, A0 represents the absorbance at the start of the measurement, A∞ represents the absorbance at the end of the measurement, At represents the absorbance at 422 nm at time t, and kfast and kslow are the respective reaction rate constants for the auto-oxidation reaction of the two components. The obtained spectral changes were analyzed by the curve fitting method based on equation (2), and kfast, kslow, and half-life t were analyzed. 1 / 2 The result was calculated.

[0083] As shown in Figure 1, under physiological conditions (pH 7.4, 37°C), the iron(II) half-life of hemoCDP(II) was 5.0 hours, while that of hemoCDI(II), as shown in Figure 2, was 36 minutes. It has been previously reported that at 25°C, the half-life of hemoCDP(II) is 30.1 hours and that of hemoCDI(II) is 3 hours (Inorg. Chem. 2006; Chem. Asian J. 2006). Furthermore, the auto-oxidation reaction of hemoCD-Twins could be analyzed using a second-order rate equation, and the two rate constants (kfast and kslow) were consistent with those measured for hemoCDP(II) and hemoCDI(II) alone, respectively. Therefore, it was found that in the hemoCD-Twins synthesized in Example 1, the physical properties of hemoCDP(II) and hemoCDI(II) were mixed without interference, and their oxidation rates were the same as those of each individual.

[0084] [Test Example 2: Verification of CO and CN scavenging ability by inclusion complexes] The hemoCD-Twins synthesized in Example 1 were diluted, and the ultraviolet-visible absorption spectrum was measured at a cell concentration of 4.9 × 10⁻⁶ M (spectrum a in Figure 4). Then, CO gas was introduced, and the spectral change was observed (spectrum b in Figure 4). Furthermore, NaCN (0.2 mM) was added, and the spectral change was observed (spectrum c in Figure 4).

[0085] Furthermore, the experiment was conducted with the order of addition of CO and NaCN reversed. Specifically, the hemoCD-Twins solution synthesized in Example 1 was diluted, and the ultraviolet-visible absorption spectrum was measured at a cell concentration of 4.5 × 10⁻⁶ M (spectrum a in Figure 5). Subsequently, NaCN (0.2 mM) was added, and the spectral change was observed (spectrum b in Figure 5). Finally, CO gas was introduced, and the spectral change was observed (spectrum c in Figure 5).

[0086] When CO is added to hemoCD-Twins, hemoCDP(II) in the iron(II) state binds to the CO. On the other hand, hemoCDI(II) undergoes auto-oxidation upon dilution and is oxidized to iron(III), exhibiting a considerably strong CN scavenging ability. The hemoCD-Twins aqueous solution shows scavenging ability for both CO and CN, and it was found that it scavenges both regardless of which component is added first.

[0087] [Test Example 3: Spectral analysis of urine after administration of inclusion complex to mice] Mice were administered intraperitoneally (200 μL) hemoCDP(II) (7 mM) synthesized in Reference Example 1, hemoCDI(II) (7 mM) synthesized in Reference Example 2, and hemoCD-Twins (14 mM) synthesized in Example 1. The absorption spectrum of the urine excreted 30 minutes later was measured. Subsequently, to determine the iron(II) / iron(III) ratio of the inclusion complex excreted in the urine, CO gas was introduced and the spectrum was measured. A reducing agent (Na2S2O4) was then added and the spectrum was measured again.

[0088] From the three spectra mentioned above, the proportions of oxygen (oxy) complexes, CO complexes, and iron(III) complexes (met complexes) in the inclusion complex in the solution can be calculated. Specifically, since CO gas does not react with iron(III), each complex is detected in each spectral measurement as follows. (a) Initial spectral measurement: Detection of oxy, CO, and met complexes. (b) Spectral measurement after introducing CO gas to (a): CO complex and met complex detected. (c)(b) Spectral measurement after addition of reducing agent: CO complex detected The ratio of the met complex can be calculated from the spectral change from (b) to (c), and furthermore, the ratio of the oxy complex to the CO complex can be calculated from the spectral change from (a) to (b). The results of the three spectral measurements for hemoCDP(II) synthesized in Reference Example 1, hemoCDI(II) synthesized in Reference Example 2, and hemoCD-Twins synthesized in Example 1 are shown in Figures 6-8, along with the calculated proportions of each complex.

[0089] The proportion of the complex released as iron(II) was 83% for hemoCDP(II) in Reference Example 1 and 12% for hemoCDI(II) in Reference Example 2. These results correspond to their respective auto-oxidation rates, indicating that the main component released was in a state capable of binding to CO and cyanide ions. Furthermore, the mixture hemoCD-Twins consisted of 49% iron(II) and 51% iron(III), showing that iron(II) and iron(III) were released together, indicating a state effective for simultaneous CO / HCN detoxification.

[0090] [Test Example 4: Verification of urinary excretion of antidote using rats] Rats weighing 300 ± 15 g were administered hemoCD-Twins (3.5 mM, 2 mL) from Example 1 via the femoral vein over 30 minutes, and the subsequent urinary excretion was quantified by ultraviolet-visible absorption spectroscopy. The urinary excretion over time is shown in Figure 9, and the cumulative urinary excretion is shown in Figure 10. As shown in Figures 9 and 10, it was found that approximately 80% of hemoCD-Twins were excreted in the urine 3 hours after administration.

[0091] [Test Example 5: Measurement of heart rate and blood changes in rats after administration of antidote 1] HemoCD-Twins (3.5 mM, 2 mL) from Example 1 was administered intrafemorally to rats weighing 300 ± 15 g over 30 minutes, and the subsequent heart rate and blood pressure were measured using "Bio Amps" (ADINSTRUMENTS). The results are shown in Figures 11 and 12, respectively. The administration was performed at time 0 min, and although both heart rate and blood pressure changed slightly immediately after administration, they quickly returned to steady state. This indicates that hemoCD-Twins administration does not affect heart rate or blood pressure.

[0092] [Test Example 6: Measurement of changes in blood antidote concentration in rats after administration of antidote 1] HemoCD-Twins (3.5 mM, 2 mL) from Example 1 was administered to rats weighing 300 ± 15 g via the femoral vein over 30 minutes. 200 μL of blood was collected from the jugular vein at 10, 20, 30, 40, 50, 60, 75, 90, 120, 150, and 180 minutes. Each blood sample was centrifuged at 12000 rpm for 5 minutes at 4°C, and 100 μL was taken from the supernatant plasma. 50 μL of methanol was added to this plasma to precipitate the proteins. After standing at 4°C for 15 minutes, the plasma was centrifuged at 12000 rpm for 15 minutes. Subsequently, 100 μL of the supernatant was taken and diluted to 1 mL with PBS (phosphate buffer). Sodium dithionite (Na2S2O4) and carbon monoxide were added to this solution to prepare the analytical sample. The subsequent changes in blood concentration were measured using an absolute calibration curve method with HPLC (size exclusion column). The analysis was performed using an Erich SEC70 10×300 gel filtration column at a flow rate of 0.5 mL / min, and elution was confirmed with an absorber at 422 nm. The results are shown in Figures 13 and 14. The results shown in Figures 13 and 14 indicate that the blood concentration rises and falls rapidly, and that it is quickly cleared from the blood into the urine.

[0093] [Test Example 7: Verification of the toxicity of CO and HCN using mice] As shown in Figure 15, the survival rate and behavioral recovery rate after administration were investigated in three groups of mice: one group administered 0.15 mg of NaCN (oral) and 5000 ppm of CO (n=10), one group administered only 0.15 mg of NaCN (n=7), and one group administered only 5000 ppm of CO (n=6). The results are shown in Figures 16 and 17, respectively.

[0094] As can be seen from the survival curve in Figure 16, the survival rate was high with administration of NaCN alone or CO alone, but all patients died within 30 minutes when both were administered. As shown in the behavioral observations in Figure 17, recovery from incapacity was observed when CO and NaCN were administered alone, but recovery from incapacity was not observed when both were administered. Based on the above, the additive toxic effects of CO and cyanide were confirmed.

[0095] [Test Example 8: Verification of the effect of administering antidote 1 to CO / HCN poisoned mice 1] As shown in Figure 18, mice were administered 0.15 mg of NaCN (oral administration) and 5000 ppm of CO., and then, 5 minutes after CO aspiration, hemoCD-Twins synthesized in Example 1 were administered intraperitoneally (14 mM, 200 μl). The subsequent survival rate and behavioral recovery rate were then examined (n=9). A group that did not receive hemoCD-Twins (n=10) was used as a comparison group, and the results are shown in Figures 19 and 20, respectively.

[0096] As shown in Figures 19 and 20, mice administered 0.15 mg of NaCN and placed in 5000 ppm of CO became immobile and died within 30 minutes. However, approximately 80% of mice administered hemoCD-Twins 5 minutes after CO aspiration survived (Figure 19), and behavioral recovery was observed about 15 minutes after administration (Figure 20). Based on the above, it can be concluded that administering hemoCD-Twins to co-occurring CO / HCN poisoning has a significant therapeutic effect.

[0097] [Test Example 9: Verification of the effect of administering antidote 1 to CO / HCN-poisoned mice (Part 2)] The experiment was conducted in the same manner as in Experiment 8, except that the NaCN dose was set to 0.1 mg (Figure 21). In this case, the mortality rate decreased even in the non-administered group (Figure 22), but the immobile state persisted for a long time (Figure 23). In the hemoCD-Twins administered group, all mice survived and showed rapid behavioral recovery, demonstrating a rapid therapeutic effect against immobility caused by CO / HCN poisoning.

[0098] [Test Example 10: Observation 1 after administration of antidote 1 to a rat model of simultaneous CO / HCN poisoning] In the same manner as in Test Example 7, hemoCD-Twins (14 mM, 2 mL) from Example 1 was administered via femoral vein over 30 minutes to rats simultaneously poisoned with NaCN 0.15 mg (oral administration) and CO 5000 ppm, and blood pressure was subsequently measured using "Bio Amps" (ADINSTRUMENTS). Additionally, PBS (2 mL) was administered via femoral vein over 30 minutes to rats weighing 300 ± 15 g, and blood pressure was subsequently measured using "Bio Amps" (ADINSTRUMENTS). Figure 24 shows the time course of blood pressure in rats administered hemoCD-Twins and rats administered PBS. As shown in Figure 24, in rats simultaneously poisoned with CO / HCN, blood pressure rapidly decreased due to the simultaneous poisoning, but in rats administered hemoCD-Twins, blood pressure immediately returned to a steady state upon administration of hemoCD-Twins.

[0099] [Test Example 11: Observation of a rat model of CO poisoning after administration of antidote 1] Figure 25 shows the time course of CO-Hb (carbon monoxide hemoglobin) in the blood of a group of rats (n=3) administered CO2000 ppm as experimental animals, compared with the time course of CO-Hb in the blood when PBS was administered instead of hemoCD-Twins for comparison. Figure 25 shows that hemoCD-Twins also exhibits a detoxification effect on rats poisoned by CO alone.

[0100] [Test Example 12: Observation of CN-single poisoning rat model after administration of antidote 1] In a study using rats as experimental animals, Figure 26 shows the time course of blood CN concentration after administration of hemoCD-Twins to a group of rats (n=3) administered 5 mg NaCN per kg of body weight, and for comparison, the time course of blood CN concentration when PBS was administered instead of hemoCD-Twins. Figure 26 shows that hemoCD-Twins also exerts a detoxification effect on rats poisoned with CN alone.

[0101] [Test Example 13: Blood concentration of hemoCD-Twins] Two mL of a PBS solution containing 3.5 mM hemoCD-Twins was administered via the femoral vein of rats (average 300 g, anesthetized with isoflurane) over 30 minutes. Blood was then collected from the carotid artery at regular intervals, and the plasma was obtained by centrifugation. The hemoCD-Twins content in the plasma was quantified using size exclusion chromatography. The quantification results were plotted against time to obtain the blood concentration profile shown in Figure 27. Figure 27 shows that hemoCD-Twins are almost completely eliminated in about 100 minutes.

[0102] [Test Example 14: Urinary clearance of hemoCD-Twins] Two mL of a PBS solution containing 14 mM hemoCD-Twins was administered via the femoral vein of rats (average 300 g, isoflurane anesthetized) over 10 minutes. Urine was then collected from the bladder at regular intervals. The amount of hemoCD-Twins in the collected urine was quantified by UV-vis spectroscopy. The percentage of detected hemoCD-Twins relative to the administered dose was plotted against the urine collection time to obtain the urinary clearance profile shown in Figure 28. Figure 28 shows that hemoCD-Twins administered into the bloodstream of rats is 100% excreted in the urine after 120 minutes. In other words, hemoCD-Twins, once ingested, is not broken down in the body and is quantitatively excreted in the urine, thus having no effect on the rat's body and demonstrating high safety.

[0103] [Test Example 15: Blood Pressure and Heart Rate Monitoring] Two mL of a PBS solution containing 3.5 mM hemoCD-Twins was administered via the femoral vein of rats (average 300 g, anesthetized with isoflurane) over 30 minutes. During this time, the rats' blood pressure and heart rate were measured using Bio Amps (AD Instruments Ltd), and the results are shown in Figure 29. Figure 29 shows that both blood pressure and heart rate change slightly immediately after administration, but quickly return to steady-state levels. Therefore, it can be seen that the administration of hemoCD-Twins does not affect heart rate and blood pressure.

[0104] [Test Example 16: Evaluation of nephrotoxicity of hemoCD-Twins] 0.2 mL of a PBS solution containing 14 mM hemoCD-Twins was administered intraperitoneally to mice (average 20 g, unanesthetized). A control group receiving PBS (0.2 mL) was also prepared simultaneously. After 24 hours, blood was collected from the mice, and serum creatinine concentration was quantified using LabAssay Creatinine (Fujifilm Wako, Japan). The results are shown in Figure 30. Figure 30 shows that the blood creatinine level after hemoCD-Twins administration was almost the same as the creatinine level after PBS administration, indicating that hemoCD-Twins is non-nephrotoxic and highly safe.

[0105] [Test Example 17: Verification of the CO detoxification effect of the novel fifth inclusion complex 1] Similar to Reference Example 1, Fe(III)TPPS and Py3CD were mixed in a molar ratio of 1:1 and added to physiological saline to form a novel fifth inclusion complex (central metal M2 = Fe 3+ An aqueous solution containing ) was obtained. Next, as shown in Figure 31, mice were inhaled CO gas (5000 ppm), and 5 minutes later, 200 μL of the aqueous solution (novel fifth inclusion complex: 7 mM) was administered intraperitoneally. Urine collected 30 minutes later was measured in the same manner as in Test Example 3. The proportions of CO conjugates, Fe(II) oxygen conjugates (oxy), and Fe(III) oxidized forms (met) were calculated using the molar extinction coefficient, and the results are shown in Figure 32. Figure 32 shows that approximately 82% (CO form + oxy form) is excreted in the urine as reduced iron(II). In other words, it can be seen that the fifth inclusion complex, even if the central metal is trivalent, is reduced in the body and exerts a CO detoxification effect.

[0106] [Test Example 18: Verification of the CO detoxification effect of the novel inclusion complex, the fifth inclusion complex, Part 2] As shown in Figure 33, rats were inhaled CO gas (2000 ppm) for 15 minutes, and then 2 mL of the aqueous solution (novel fifth inclusion complex: 10 mM) was administered intravenously at a rate of 15 mL / min after 15 minutes. CO-Hb levels in the blood were measured every 10 minutes thereafter. In addition, rats were inhaled CO gas (2000 ppm), and then 2 mL of PBS was administered intravenously at a rate of 15 mL / min after 15 minutes. CO-Hb levels in the blood were measured every 10 minutes thereafter. The results are shown in comparison in Figure 34. Figure 34 shows that the novel fifth inclusion complex exhibits a CO detoxification effect even when the central metal is trivalent.

[0107] The inventors of the present invention initially believed that the first inclusion complex hemoCDP(II) had the ability to detoxify CO, but the novel fifth inclusion complex hemoCDP(III) did not. However, from the above test examples 15-18, it was found that the novel fifth inclusion complex hemoCDP(III) can be reduced in the body and become capable of detoxifying CO.

[0108] Similarly, while it was initially thought that the sixth inclusion complex hemoCDI(III) had the ability to detoxify cyanide, the novel second inclusion complex hemoCDI(II) did not. However, it was discovered that the novel second inclusion complex hemoCDI(II) can be oxidized in the body and become capable of detoxifying cyanide. Therefore, it was found that the combination drug hemoCD-Twins can be administered simply by having both components at the same valency, and that it functions as an antidote that can simultaneously detoxify CO and cyanide in the body.

Claims

1. An antidote for removing carbon monoxide and hydrogen cyanide from the body, comprising as active ingredients a fifth inclusion complex in which a first cyclodextrin dimer represented by the following general formula (8) encloses a second water-soluble metallic porphyrin having Fe³⁺ as its central metal, and a sixth inclusion complex in which a second cyclodextrin dimer represented by the following general formula (9) encloses a second water-soluble metallic porphyrin having Fe³⁺ as its central metal, An antidote characterized in that the second water-soluble metal porphyrin is represented by the following general formula (10) or (11). 【Transformation 8】 (In the above general formula (8), R represents a protecting group selected from the group consisting of a methyl group, an ethyl group, an acetyl group, and a hydroxypropyl group; m represents an integer from 1 to 2; and n represents an integer from 1 to 3.) 【Chemistry 9】 (In the above general formula (9), R represents a protecting group selected from the group consisting of a methyl group, an ethyl group, an acetyl group, and a hydroxypropyl group; p represents an integer from 1 to 2; and q represents an integer from 1 to 3.) 【Chemistry 10】 【Chemistry 11】 (In the above general formulas (10) and (11), R1 and R2 represent sulfonyl groups, and M2 represents Fe³⁺.)

2. An antidote kit for preparing the antidote described in Claim 1, An antidote kit characterized by comprising an aqueous solution of inclusion complexes or its raw materials, comprising a fifth inclusion complex formed by the inclusion of a second water-soluble metal porphyrin by a first cyclodextrin dimer represented by the general formula (8), and a sixth inclusion complex formed by the inclusion of a second water-soluble metal porphyrin by a second cyclodextrin dimer represented by the general formula (9).

3. An antidote for removing carbon monoxide and hydrogen cyanide from the body, comprising: an active ingredient comprising: a first inclusion complex in which a first cyclodextrin dimer represented by the following general formula (1) encloses a first water-soluble metal porphyrin having Fe²⁺ as its central metal; a second inclusion complex in which a second cyclodextrin dimer represented by the following general formula (2) encloses a first water-soluble metal porphyrin having Fe²⁺ as its central metal; and a reducing agent, An antidote characterized in that the first water-soluble metal porphyrin is represented by the following general formula (3) or (4). 【Chemistry 1】 (In the above general formula (1), R represents a protecting group selected from the group consisting of a methyl group, an ethyl group, an acetyl group, and a hydroxypropyl group; m represents an integer from 1 to 2; and n represents an integer from 1 to 3.) 【Chemistry 2】 (In the above general formula (2), R represents a protecting group selected from the group consisting of a methyl group, an ethyl group, an acetyl group, and a hydroxypropyl group; p represents an integer from 1 to 2; and q represents an integer from 1 to 3.) 【Transformation 3】 【Chemistry 4】 (In the above general formulas (3) and (4), R1 and R2 represent sulfonyl groups, and M1 represents Fe²⁺.)

4. A carbon monoxide detoxification inclusion complex characterized in that a cyclodextrin dimer represented by the following general formula (12) is inclusion of a second water-soluble metal porphyrin having Fe³⁺ as its central metal, and the second water-soluble metal porphyrin is represented by the following general formula (13) or (14). 【Chemistry 12】 (In the above general formula (12), R represents a protecting group selected from the group consisting of a methyl group, an ethyl group, an acetyl group, and a hydroxypropyl group; p represents an integer from 1 to 2; and q represents an integer from 1 to 3.) 【Chemistry 13】 【Chemistry 14】 (In the above general formulas (13) and (14), R1 and R2 represent sulfonyl groups, and M2 represents Fe³⁺.)

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