Patent Publication Number: US-2021190759-A1

Title: Composition for detecting hydrogen sulfide or measuring hydrogen sulfide concentration and composition comprising same as effective ingredient for diagnosing or imaging in vivo inflammation, tissues having hypoxic damage, or cancer

Description:
CROSS-REFERENCES TO RELATED APPLICATION 
     This patent application is the National Stage of International Application No. PCT/KR2019/011848 filed Sep. 11, 2019, which claims the benefit of priority from Korean Application No. 10-2018-0108234, filed Sep. 11, 2018, teachings of each of which are herein incorporated by reference in their entirety. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to a composition for detecting hydrogen sulfide or measuring a concentration of hydrogen sulfide, and a composition comprising the same as an active ingredient for diagnosing or imaging in vivo inflammation, tissues having hypoxic damage, or cancer. 
     2. Description of the Related Art 
     Hydrogen sulfide gas is caused by rotting proteins such as eggs and is famous for its bad smell. This gas is mainly generated from natural volcanoes, soil or water resources contaminated with organic matters, and may be generated from living organisms in a normal or disease status. In addition, hydrogen sulfide has been found to improve or worsen disease status depending on the type of disease in the animal body. Therefore, it is necessary to detect and quantify hydrogen sulfide in order to measure the pollution level of natural soil, air, and water resources, or to diagnose disease conditions in a living body. 
     In addition, hydrogen sulfide has been reported to be associated with numerous diseases such as various inflammatory responses in the human body, hypoxia, cardiovascular disease, stroke, angiogenesis, vasodilation, cancer, Down&#39;s syndrome, dementia, diabetes, and Huntington&#39;s disease. Therefore, its detection is very important for diagnosing various diseases and predicting the prognosis. 
     Accordingly, many methods such as methylene blue method, ion selective electrode method, monobromo obimane-HPLC method, thiobromobimane-GC method, and current detection method have been developed and reported to measure the hydrogen sulfide concentration in a sample (K R Olson, et al., Nitric Oxide (2014) 41; 11-26). However, since these methods simply measure the concentration of hydrogen sulfide in samples such as blood, it is impossible to know its distribution more accurately. 
     A method for detecting hydrogen sulfide by fluorescence was developed, making it possible to show the hydrogen sulfide generation site in an image. However, fluorescence is weak in permeability, so it cannot be used to detect or image hydrogen sulfide generated in humans or large animals. It can only be used to detect and image hydrogen sulfide generation in very small animals such as mouse and zebrafish, or in intracellular organs such as mitochondria (M D Hammers, et al., J Am Chem Soc (2015) 137:10216-10223; Y Chen, et al., Angew Chem Int Ed (2013) 52:1688-1691; K Sasakura, et al., J Am Chem Soc (2011) 133:18003-18005; AR Lippert, et al., J Am Chem Soc (2011) 133:10078-10080; N Kumar, et al., Coord Chem Rev (2013) 257:2335-2347). 
     A method of detection by low-temperature chemiluminescence has also been developed, but this method has a problem that is applicable only to small animals (J Cao, et al., Chem Sci (2015) 6:1979-1985). 
     Recently, a method for detecting hydrogen sulfide using a radioactive isotope that emits radiation with strong penetrating power has been developed. In this method,  64 Cu emitting positrons combinds with hydrogen sulfide to form insoluble  64 CuS and deposits on the hydrogen sulfide generation site to detect and image the radioactivity at this site (S Sarkar, et al., Angew Chem Int Ed (2016) 55:9365-9370; Korean Patent Publication No. 10-2017-0018121). The above method can image the hydrogen sulfide generation site deep in the human body with high resolution using PET (Positron Emission Tomography). However, since Cu-64 should be made by proton irradiation of Ni-64, an expensive target material, using an expensive cyclotron, the price is very expensive and there are many restrictions in use. 
     SUMMARY OF THE INVENTION 
     It is an object of the present invention to provide a composition for detecting hydrogen sulfide or measuring a concentration of hydrogen sulfide. 
     It is another object of the present invention to provide a composition for diagnosing or imaging inflammation, tissues having hypoxic damage, or cancer. 
     To achieve the above objects, in one aspect of the present invention, a composition for detecting hydrogen sulfide comprising a compound represented by formula 1 having alpha-hydroxy acid labeled with  99m Tc is provided: 
       O═ 99m Tc(O═CO − —CHO − —(CHR 1 ) m —CH 2 R 2 ) 2   [Formula 1]
 
     (In formula 1, 
     R 1  and R 2  are independently hydrogen or —OH; and 
     m is an integer of 0˜20). 
     In another aspect of the present invention, a preparation method of a composition for detecting hydrogen sulfide comprising a compound represented by formula 1 having alpha-hydroxy acid labeled with  99m Tc, containing a step of reacting  99m Tc with the alpha-hydroxy acid represented by formula 2 or an alkali metal or alkaline earth metal salt thereof is provided: 
       O═ 99m Tc(O═CO − —CHO − —(CHR 1 ) m —CH 2 R 2 ) 2   [Formula 1]
 
     (In formula 1, 
     R 1  and R 2  are independently hydrogen or —OH; and 
     m is an integer of 0˜20); 
       HOOC—CHOH—(CHR 1 ) m —CH 2 R 2   [Formula 2]
 
     (In formula 2, 
     R 1  and R 2  are independently hydrogen or —OH; and 
     m is an integer of 0˜20). 
     In another aspect of the present invention, a composition for measuring a concentration of hydrogen sulfide comprising a compound represented by formula 1 having alpha-hydroxy acid labeled with  99m Tc is provided: 
       O═ 99m Tc(O═CO − —CHO − —(CHR 1 ) m —CH 2 R 2 ) 2   [Formula 1]
 
     (In formula 1, 
     R 1  and R 2  are independently hydrogen or —OH; and 
     m is an integer of 0˜20). 
     In another aspect of the present invention, a composition for imaging a disease in which hydrogen sulfide is generated, comprising a compound represented by formula 1 is provided: 
       O═ 99m Tc(O═CO − —CHO − —(CHR 1 ) m —CH 2 R 2 ) 2   [Formula 1]
 
     (In formula 1, 
     R is independently hydrogen or hydroxyl group; and 
     m is an integer of 0˜20). 
     In another aspect of the present invention, a composition for diagnosing a disease in which hydrogen sulfide is generated, comprising a compound represented by formula 1 is provided: 
       O═ 99m Tc(O═CO − —CHO − —(CHR 1 ) m —CH 2 R 2 ) 2   [Formula 1]
 
     (In formula 1, 
     R is independently hydrogen or hydroxyl group; and 
     m is an integer of 0˜20). 
     In another aspect of the present invention, a kit for preparing a compound represented by formula 1 comprising the alpha-hydroxy acid represented by formula 2 or an alkali metal or alkaline earth metal salt thereof and an adjuvant is provided: 
       O═ 99m Tc(O═CO − —CHO − —(CHR 1 ) m —CH 2 R 2 ) 2   [Formula 1]
 
     (In formula 1, 
     R is independently hydrogen or hydroxyl group; and 
     m is an integer of 0˜20). 
       HOOC—CHOH—(CHR 1 ) m —CH 2 R 2   [Formula 2]
 
     (In formula 2, 
     R 1  and R 2  are independently hydrogen or —OH; and 
     m is an integer of 0˜20). 
     Advantageous Effect 
     The composition for detecting hydrogen sulfide or measuring a concentration of hydrogen sulfide according to the present invention, which comprises the compound represented by formula 1 ( 99m Tc-alpha-hydroxy acid) having alpha-hydroxy acid labeled with  99m Tc, enables the detection or concentration measurement of hydrogen sulfide in in-vitro and in-vivo levels and, as such, can be advantageously used for detecting hydrogen sulfide and measuring a concentration of hydrogen sulfide and furthermore for discovering biological roles of hydrogen sulfide in vivo, especially, for detecting, imaging, and quantitatively measuring hydrogen sulfide in a disease selected from the group consisting of angiogenesis, inflammation, cancer, Alzheimer&#39;s disease, cardiovascular ischemia, and cerebrovascular ischemia, or in hypoxic tissues. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a diagram showing the formula of alpha-hydroxy acid. 
         FIG. 2  is a set of graphs showing the results of measuring labeling efficiency by developing the  99m Tc-gluconate labeled with  99m Tc according to the method of Example 1 using D-gluconate as alpha-hydroxy acid by ITLC-SG using acetone and physiological saline. 
         FIG. 3  is a set of graphs showing the results of measuring labeling efficiency by developing the  99m Tc-glucoheptonate labeled with  99m Tc according to the method of Example 1 using D-glucoheptonate as alpha-hydroxy acid by ITLC-SG using acetone and physiological saline. 
         FIG. 4  is a set of graphs showing the results of measuring labeling efficiency by developing the  99m Tc-glucarate labeled with  99m Tc according to the method of Example 1 using D-glucarate as alpha-hydroxy acid by ITLC-SG using acetone and physiological saline. 
         FIG. 5  is a set of graphs showing the results of measuring labeling efficiency by developing the  99m Tc-citrate labeled with  99m Tc according to the method of Example 1 using citrate as alpha-hydroxy acid by ITLC-SG using acetone and physiological saline. 
         FIG. 6  is a set of graphs showing the results of measuring labeling efficiency by developing the  99m Tc-tartrate labeled with  99m Tc according to the method of Example 1 using L-tartrate as alpha-hydroxy acid by ITLC-SG using acetone and physiological saline. 
         FIG. 7  is a set of graphs showing the results of measuring labeling efficiency by developing the  99m Tc-glucuronate labeled with  99m Tc according to the method of Example 1 using D-glucuronate as alpha-hydroxy acid by ITLC-SG using acetone and physiological saline. 
         FIG. 8  is a graph showing the percentage of generating insoluble substances after the reaction of  99m Tc-alphs-hydroxy acid with NaHS and various active sulfides performed in Experimental Example 2. 
         FIG. 9  is a graph showing the amount of insoluble substances produced by reacting  99m Tc-alphs-hydroxy acid with NaHS of different concentrations performed in Experimental Example 3. 
         FIG. 10  is a set of SPECT/CT images obtained 1 hour after the administration of carrageenan to the sole of a mouse to induce inflammation and physiological saline to the other food and then  99m Tc-gluconate and  99m Tc-glucoheptonate performed in Experimental Example 5. 
         FIG. 11  is a set of images of the rat brain extracted, autoradiated and TTC stained 1 hour after the middle cerebral artery of a rat was occluded for hours, reperfused, and administered with  99m Tc-gluconate and [ 18 F]FDG simultaneously performed in Experimental Example 6. 
     
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     In one aspect of the present invention, a composition for detecting hydrogen sulfide comprising a compound represented by formula 1 having alpha-hydroxy acid labeled with  99m Tc is provided: 
       O═ 99m Tc(O═CO − —CHO − —(CHR 1 ) m —CH 2 R 2 ) 2   [Formula 1]
 
     (In formula 1, 
     R 1  and R 2  are independently hydrogen or —OH; and 
     m is an integer of 0˜20). 
     The alpha-hydroxy acid can be a compound represented by formula 2 below. 
       HOOC—CHOH—(CHR 1 ) m —CH 2 R 2   [Formula 2]
 
     (In formula 2, 
     R 1  and R 2  are independently hydrogen or —OH; and 
     m is an integer of 0˜20). 
     In addition, the alpha-hydroxy acid can be selected from the group consisting of D-gluconic acid, D-glucoheptonic acid, galactonic acid, D-glucaric acid, tartaric acid, citric acid, glycolic acid, D-lactic acid, L-lactic acid and D-glucuronic acid. 
     The composition for detecting hydrogen sulfide reacts with hydrogen sulfide to form an insoluble material, thereby enabling imaging. Thus, it can be used to detect hydrogen sulfide. 
     The composition for detecting hydrogen sulfide can detect hydrogen sulfide in the tissues or cells isolated from animal subjects. 
     The tissue or cell can be a tissue or cell in which hydrogen sulfide has been generated, or a tissue or cell of a disease selected from the group consisting of angiogenesis, inflammation, cancer, Alzheimer&#39;s dementia, cardiovascular ischemia, cerebrovascular ischemia and hypoxia in which hydrogen sulfide has been generated. In addition, the tissue or cell can be a tissue or cell of a disease selected from the group consisting of rheumatoid arthritis, non-rheumatic inflammatory arthritis, arthritis related to Lyme disease, pyelonephritis, nephritis, inflammatory osteoarthritis, meningitis, osteomyelitis, inflammatory bowel disease, appendicitis, pancreatitis, sepsis, inflammatory disease due to bacterial infection, myocardial infarction, heart ischemia, angina, angina pectoris, cardiomyopathy, endocarditis, arteriosclerosis, sepsis, diabetes, stroke, cirrhosis, asthma, Parkinson&#39;s disease, Alzheimer&#39;s disease, dementia, Down&#39;s syndrome, lung cancer, breast cancer, uterine cancer, ovarian cancer, liver cancer, brain cancer, prostate cancer, thyroid cancer, neuroendocrine tumor, stomach cancer, colon cancer, pancreatic cancer, bladder cancer, esophageal cancer and head/neck cancer. 
     The composition for detecting hydrogen sulfide can detect hydrogen sulfide in a disease selected from the group consisting of angiogenesis, inflammation, cancer, Alzheimer&#39;s dementia, cardiovascular ischemia, cerebrovascular ischemia and hypoxia. At this time, the disease can be a hydrogen sulfide-generated disease or hypoxic tissue. 
     Since the composition for detecting hydrogen sulfide of the present invention selectively detects only NaHS (hydrogen sulfide) among active sulfides, it can be effectively used for the detection of hydrogen sulfide (Experimental Example 2). 
     In another aspect of the present invention, the present invention provides a preparation method of a composition for detecting hydrogen sulfide comprising a compound represented by formula 1 having alpha-hydroxy acid labeled with  99m Tc, containing a step of reacting  99m Tc with the alpha-hydroxy acid represented by formula 2 or an alkali metal or alkaline earth metal salt thereof. 
       O═ 99m Tc(O═CO − —CHO − —(CHR 1 ) m —CH 2 R 2 ) 2   [Formula 1]
 
     (In formula 1, 
     R 1  and R 2  are independently hydrogen or —OH; and 
     m is an integer of 0˜20); 
       HOOC—CHOH—(CHR 1 ) m —CH 2 R 2   [Formula 2]
 
     (In formula 2, 
     R 1  and R 2  are independently hydrogen or —OH; and 
     m is an integer of 0˜20). 
     The alpha-hydroxy acid represented by formula 2 can be selected from the group consisting of D-gluconic acid, D-glucoheptonic acid, galactonic acid, D-glucaric acid, tartaric acid, citric acid, glycolic acid, D-lactic acid, L-lactic acid and D-glucuronic acid. 
     The alkali metal can be selected from the group consisting of lithium (Li), sodium (Na), potassium (K), rubidium (Rb) and cesium (Cs). 
     The alkaline earth metal can be selected from the group consisting of magnesium (Mg), calcium (Ca), strontium (Sr) and barium (Ba). 
     Specifically, the preparation method above is a method to obtain  99m Tc-labeled alpha-hydroxy acid, that is, a compound represented by formula 1, by synthesizing a technetium complex on the alpha-hydroxy acid represented by formula 2. 
     In the preparation method of the present invention,  99m Tc generated in a generator was used, but  99m Tc immediately released from the generator was present as a very stable form of pertechnetic acid with an oxidation number of +7, and thus was not labeled. Therefore, it is necessary to decrease the oxidation number by reducing it. At this time, the reducing agent can be used without limitation, as long as it is a commonly used reducing agent. In one embodiment of the present invention, SnCl 2  was used as the reducing agent, but not always limited thereto. 
     In order to easily and conveniently label the alpha-hydroxy acid with  99m Tc, stabilizers, excipients or buffers can further be used as other additives. SnCl 2 , ascorbic acid, gentisinic acid, calcium chloride, sodium chloride, sodium phosphate, mannitol, glucose, lactose, sodium ascorbate, and the like can be used as the other additives. 
     The preparation method according to the present invention was able to label the alpha-hydroxy acid with  99m Tc with a labeling efficiency of almost 100% (Experimental Example 1). 
     In another aspect of the present invention, the present invention provides a composition for measuring a concentration of hydrogen sulfide comprising a compound represented by formula 1 having alpha-hydroxy acid labeled with  99m Tc. 
       O═ 99m Tc(O═CO − —CHO − —(CHR 1 ) m —CH 2 R 2 ) 2   [Formula 1]
 
     (In formula 1, 
     R 1  and R 2  are independently hydrogen or —OH; and 
     m is an integer of 0˜20). 
     The alpha-hydroxy acid can be a compound represented by formula 2 below. 
       HOOC—CHOH—(CHR 1 ) m —CH 2 R 2   [Formula 2]
 
     (In formula 2, 
     R 1  and R 2  are independently hydrogen or —OH; and 
     m is an integer of 0˜20). 
     In addition, the alpha-hydroxy acid represented by formula 2 can be selected from the group consisting of D-gluconic acid, D-glucoheptonic acid, galactonic acid, D-glucaric acid, tartaric acid, citric acid, glycolic acid, D-lactic acid, L-lactic acid and D-glucuronic acid. 
     The composition for measuring a concentration of hydrogen sulfide reacts with hydrogen sulfide to form an insoluble material, so that the concentration of hydrogen sulfide can be measured. 
     The composition for measuring a concentration of hydrogen sulfide can measure the concentration of hydrogen sulfide in the tissues or cells isolated from animal subjects. 
     The tissue or cell can be a tissue or cell in which hydrogen sulfide has been generated, or a tissue or cell of a disease selected from the group consisting of angiogenesis, inflammation, cancer, Alzheimer&#39;s dementia, cardiovascular ischemia, cerebrovascular ischemia and hypoxia in which hydrogen sulfide has been generated. In addition, the tissue or cell can be a tissue or cell of a disease selected from the group consisting of rheumatoid arthritis, non-rheumatic inflammatory arthritis, arthritis related to Lyme disease, pyelonephritis, nephritis, inflammatory osteoarthritis, meningitis, osteomyelitis, inflammatory bowel disease, appendicitis, pancreatitis, sepsis, inflammatory disease due to bacterial infection, myocardial infarction, heart ischemia, angina, angina pectoris, cardiomyopathy, endocarditis, arteriosclerosis, sepsis, diabetes, stroke, cirrhosis, asthma, Parkinson&#39;s disease, Alzheimer&#39;s disease, dementia, Down&#39;s syndrome, lung cancer, breast cancer, uterine cancer, ovarian cancer, liver cancer, brain cancer, prostate cancer, thyroid cancer, neuroendocrine tumor, stomach cancer, colon cancer, pancreatic cancer, bladder cancer, esophageal cancer and head/neck cancer. 
     The composition for measuring a concentration of hydrogen sulfide can detect hydrogen sulfide in a disease selected from the group consisting of angiogenesis, inflammation, cancer, Alzheimer&#39;s dementia, cardiovascular ischemia, cerebrovascular ischemia and hypoxia. At this time, the disease can be a hydrogen sulfide-generated disease or hypoxic tissue. 
     The composition for measuring a concentration of hydrogen sulfide can be effectively used for measuring the concentration of hydrogen sulfide since the degree of formation of an insoluble substance changes according to the concentration of hydrogen sulfide (Experimental Example 3). 
     In another aspect of the present invention, the present invention provides a composition for imaging a disease in which hydrogen sulfide is generated, comprising a compound represented by formula 1. 
       O═ 99m Tc(O═CO − —CHO − —(CHR 1 ) m —CH 2 R 2 ) 2   [Formula 1]
 
     (In formula 1, 
     R is independently hydrogen or hydroxyl group; and 
     m is an integer of 0˜20). 
     The alpha-hydroxy acid can be a compound represented by formula 2 below. 
       HOOC—CHOH—(CHR 1 ) m —CH 2 R 2   [Formula 2]
 
     (In formula 2, 
     R 1  and R 2  are independently hydrogen or —OH; and 
     m is an integer of 0˜20). 
     In addition, the alpha-hydroxy acid can be selected from the group consisting of D-gluconic acid, D-glucoheptonic acid, galactonic acid, D-glucaric acid, tartaric acid, citric acid, glycolic acid, D-lactic acid, L-lactic acid and D-glucuronic acid. 
     The composition for imaging a disease in which hydrogen sulfide is generated reacts with hydrogen sulfide to form an insoluble material, so that hydrogen sulfide can be imaged. 
     The disease can be selected from the group consisting of angiogenesis, inflammation, cancer, Alzheimer&#39;s dementia, cardiovascular ischemia, cerebrovascular ischemia and hypoxia. 
     In addition, the disease can be selected from the group consisting of rheumatoid arthritis, non-rheumatic inflammatory arthritis, arthritis related to Lyme disease, pyelonephritis, nephritis, inflammatory osteoarthritis, meningitis, osteomyelitis, inflammatory bowel disease, appendicitis, pancreatitis, sepsis, inflammatory disease due to bacterial infection, myocardial infarction, heart ischemia, angina, angina pectoris, cardiomyopathy, endocarditis, arteriosclerosis, sepsis, diabetes, stroke, cirrhosis, asthma, Parkinson&#39;s disease, Alzheimer&#39;s disease, dementia, Down&#39;s syndrome, lung cancer, breast cancer, uterine cancer, ovarian cancer, liver cancer, brain cancer, prostate cancer, thyroid cancer, neuroendocrine tumor, stomach cancer, colon cancer, pancreatic cancer, bladder cancer, esophageal cancer and head/neck cancer. 
     The composition for imaging a disease in which hydrogen sulfide is generated according to the present invention can image the inflamed tissue in which hydrogen sulfide is generated, and the concentration increase can be known through whether the intake of the treated composition for imaging increases, so that not only imaging but also the increase in concentration can be confirmed. In addition, since the hydrogen sulfide concentration can be quantified and expressed numerically through fluorescence assay, it can be effectively used to measure the concentration of hydrogen sulfide in the inflamed tissue (Experimental Examples 4 and 5). 
     In addition, from the increase in the concentration of hydrogen sulfide in reperfusion after middle cerebral artery occlusion using the composition for imaging according to the present invention, it was confirmed that hydrogen sulfide was generated in the reperfused tissue. It was also confirmed that hydrogen sulfide detection, concentration measurement, and imaging in the reperfused tissue after middle cerebral artery occlusion were possible (Experimental Example 6). 
     In another aspect of the present invention, the present invention provides a composition for diagnosing a disease in which hydrogen sulfide is generated, comprising a compound represented by formula 1. 
       O═ 99m Tc(O═CO − —CHO − —(CHR 1 ) m —CH 2 R 2 ) 2   [Formula 1]
 
     (In formula 1, 
     R is independently hydrogen or hydroxyl group; and 
     m is an integer of 0˜20). 
     The alpha-hydroxy acid can be a compound represented by formula 2 below. 
       HOOC—CHOH—(CHR 1 ) m —CH 2 R 2   [Formula 2]
 
     (In formula 2, 
     R 1  and R 2  are independently hydrogen or —OH; and 
     m is an integer of 0˜20). 
     In addition, the alpha-hydroxy acid can be selected from the group consisting of D-gluconic acid, D-glucoheptonic acid, galactonic acid, D-glucaric acid, tartaric acid, citric acid, glycolic acid, D-lactic acid, L-lactic acid and D-glucuronic acid. 
     The composition for diagnosing a disease in which hydrogen sulfide is generated reacts with hydrogen sulfide to form an insoluble material, so that diagnosis of a disease in which hydrogen sulfide is generated is possible. 
     The disease can be selected from the group consisting of angiogenesis, inflammation, cancer, Alzheimer&#39;s dementia, cardiovascular ischemia, cerebrovascular ischemia and hypoxia. 
     In addition, the disease can be selected from the group consisting of rheumatoid arthritis, non-rheumatic inflammatory arthritis, arthritis related to Lyme disease, pyelonephritis, nephritis, inflammatory osteoarthritis, meningitis, osteomyelitis, inflammatory bowel disease, appendicitis, pancreatitis, sepsis, inflammatory disease due to bacterial infection, myocardial infarction, heart ischemia, angina, angina pectoris, cardiomyopathy, endocarditis, arteriosclerosis, sepsis, diabetes, stroke, cirrhosis, asthma, Parkinson&#39;s disease, Alzheimer&#39;s disease, dementia, Down&#39;s syndrome, lung cancer, breast cancer, uterine cancer, ovarian cancer, liver cancer, brain cancer, prostate cancer, thyroid cancer, neuroendocrine tumor, stomach cancer, colon cancer, pancreatic cancer, bladder cancer, esophageal cancer and head/neck cancer. 
     The composition for diagnosing a disease in which hydrogen sulfide is generated according to the present invention can image the inflamed tissue in which hydrogen sulfide is generated, and the concentration increase can be known through whether the intake of the treated composition for imaging increases, so that not only imaging but also the increase in concentration can be confirmed. In addition, since the hydrogen sulfide concentration can be quantified and expressed numerically through fluorescence assay, it can be effectively used not only for diagnosing inflammatory disease, but also for assessing progression (Experimental Example 5). 
     In addition, from the increase in the concentration of hydrogen sulfide in reperfusion after middle cerebral artery occlusion using the composition for imaging according to the present invention, it was confirmed that hydrogen sulfide was generated in the reperfused tissue. It was also confirmed that the reperfused tissue can be diagnosed after the middle cerebral artery occlusion (Experimental Example 6). 
     In another aspect of the present invention, the present invention provides a kit for preparing a compound represented by formula 1 comprising the alpha-hydroxy acid represented by formula 2 or an alkali metal or alkaline earth metal salt thereof and an adjuvant: 
       O═ 99m Tc(O═CO − —CHO − —(CHR 1 ) m —CH 2 R 2 ) 2   [Formula 1]
 
     (In formula 1, 
     R is independently hydrogen or hydroxyl group; and 
     m is an integer of 0˜20). 
       HOOC—CHOH—(CHR 1 ) m —CH 2 R 2   [Formula 2]
 
     (In formula 2, 
     R 1  and R 2  are independently hydrogen or —OH; and 
     m is an integer of 0˜20). 
     The alpha-hydroxy acid represented by formula 2 can be selected from the group consisting of D-gluconic acid, D-glucoheptonic acid, galactonic acid, D-glucaric acid, tartaric acid, citric acid, glycolic acid, D-lactic acid, L-lactic acid and D-glucuronic acid. 
     The adjuvant can be at least one selected from the group consisting of SnCl 2 , ascorbic acid, gentisinic acid, calcium chloride, sodium chloride, sodium phosphate, mannitol, glucose, lactose and sodium ascorbate. 
     The alkali metal can be selected from the group consisting of lithium (Li), sodium (Na), potassium (K), rubidium (Rb) and cesium (Cs). 
     The alkaline earth metal can be selected from the group consisting of magnesium (Mg), calcium (Ca), strontium (Sr) and barium (Ba). 
     The kit can include an adjuvant for easy and convenient labeling of alpha-hydroxy acid with  99m Tc. The adjuvant can be a stabilizer, an excipient or a buffering agent, and specifically, as the other additives, SnCl 2 , ascorbic acid, gentisinic acid, calcium chloride, sodium chloride, sodium phosphate, mannitol, glucose, lactose, sodium ascorbate, and the like can be used. 
     Specifically, the kit can be a freeze-dried kit including the alpha-hydroxy acid represented by formula 2 or an alkali metal or alkaline earth metal salt thereof and a reducing agent such as tin chloride as an adjuvant. At this time, in order to optimize the labeling efficiency, the kit can be prepared by adjusting the pH inside the kit to 3˜10, preferably 4˜7. 
       99m Tc is added to the kit so that it can be used by labeling alpha-hydroxy acid with  99m Tc within 1 hour at room temperature ˜100° C. 
     The kit can additionally contain an antioxidant and an excipient. At this time, the antioxidant is to prevent deterioration of the alpha-hydroxy acid labeled with a radioactive isotope by oxidation or radiolysis. Vitamin C or gentisic acid can be used as the antioxidant. The antioxidant and excipient can be contained about 0 to 500 mg per unit dosage of the kit. 
     The kit can be frozen or lyophilized in a sterilization container under inert gas environment. The kit can further include buffer sterilization vials, saline, syringes, filters, columns, and other auxiliary devices to prepare injections for use in hospitals. Such modifications are well known to those having ordinary skills in the art. 
     The kit can be used by labeling  99m Tc within 1 hour at room temperature ˜100° C. 
     The composition for detecting hydrogen sulfide or measuring a concentration of hydrogen sulfide according to the present invention, which comprises the compound represented by formula 1 ( 99m Tc-alpha-hydroxy acid) having alpha-hydroxy acid labeled with  99m Tc, enables the detection or concentration measurement of hydrogen sulfide in in-vitro and in-vivo levels and, as such, can be advantageously used for detecting hydrogen sulfide and measuring a concentration of hydrogen sulfide and furthermore for discovering biological roles of hydrogen sulfide in vivo, especially, for detecting, imaging, and quantitatively measuring hydrogen sulfide in a disease selected from the group consisting of angiogenesis, inflammation, cancer, Alzheimer&#39;s disease, cardiovascular ischemia, and cerebrovascular ischemia, or in hypoxic tissues. 
     In addition,  99m Tc is easier to supply than other radioactive isotopes and is competitive in price, so it has an economic advantage. 
     Hereinafter, the present invention will be described in detail by the following examples and experimental examples. 
     However, the following examples and experimental examples are only for illustrating the present invention, and the contents of the present invention are not limited thereto. 
     Example 1: Preparation of  99m Tc-Labeled Alpha-Hydroxy Acid ( 99m Tc-Alpha-Hydroxy Acid) 
     D-gluconate, D-glucoheptonate, D-glucarate, citrate, L-tartrate, and D-glucuronate were selected as the alpha-hydroxy acid, and each alpha-hydroxy acid was labeled with  99m Tc by the following method. All distilled water used for labeling was used after blowing with nitrogen gas for 1 hour. 10 μL of sodium ascorbate (25 mg/mL) was added to 100 μL of each 0.3 M alpha-hydroxy acid, to which 50 μL of SnCl 2 .2H 2 O (2.5 mg/mL in 0.05 M HCl) was added, followed by mixing well. 140 μL of  99m Tc (3 mCi) obtained from the generator was added thereto, which was placed at room temperature for 20 minutes, or heated at 100° C. for 10 minutes to label. 
     Experimental Example 1: Measurement of Labeling Efficiency 
     In order to measure the labeling efficiency of the  99m Tc-labeled alpha-hydroxy acid ( 99m Tc-alpha-hydroxy acid) prepared in Example 1, the following experiment was performed. The results of measuring the labeling efficiency of each  99m Tc-alpha-hydroxy acid are shown in  FIGS. 2-7 . 
     Particularly, the labeling efficiency was measured by ITLC (Instant Thin Layer Chromatography). After loading 1 to 5 μL of the sample at a position of 1 cm from the bottom of an ITLC plate having a length of 10 cm and a width of 1 cm, the plate was placed in a developing tank containing acetone or physiological saline and developed. Upon completion of the deployment, radioactivity was scanned using a Radio-TLC scanner. At this time, when developed with acetone, only the unlabeled  99m Tc went up along the solvent, and the labeled  99m Tc and the colloidal  99m Tc remained at the origin. When developed with physiological saline, the colloidal  99m Tc remained at the origin, and the unlabeled  99m Tc and the labeled  99m Tc went up along the solvent. 
     As shown in  FIGS. 2-7 , almost 100% of the labeling efficiency was shown for all alpha-hydroxy acids. 
     Experimental Example 2: Evaluation of Reaction of  99m Tc-Alpha-Hydroxy Acid with NaHS and Various Active Sulfides 
     In order to evaluate the reactivity of the  99m Tc-alpha-hydroxy acid according to the present invention with various active sulfides including NaHS (hydrogen sulfide), the following experiment was performed, and the results are shown in  FIG. 8  and Table 1. 
     Particularly, the  99m Tc-alpha-hydroxy acid prepared in Example 1 was diluted 5 times with physiological saline, and then 100 μL of each diluent was taken, to which 100 μL of each of 0.2 M sodium phosphate buffer (pH 7.4) containing 0.4 mM NaHS, 4 mM glutathione, 0.4 mM cysteine, 0.4 mM sodium sulfite, 0.4 mM sodium sulfate, 0.4 mM sodium thiosulfate, and 0.4 mM NONOate (NO generating reagent) was added, followed by reaction at 37° C. for 15 minutes. Then, ITLC was developed with physiological saline to determine the percentage of the radioactivity remaining at the origin. When developed with physiological saline, the radioactivity remaining at the origin was made of an insoluble substance and was deposited on the site. 
     Table 1 below shows the percentage of insoluble substances produced after the reaction between  99m Tc-labeled alpha-hydroxy acid and active sulfides. 
     
       
         
           
               
               
               
               
               
             
               
                   
                 TABLE 1 
               
               
                   
                   
               
               
                   
                   99m Tc- 
                   99m Tc- 
                   99m Tc- 
                   99m Tc- 
               
               
                   
                 gluconate 
                 glucoheptonate 
                 glucarate 
                 citrate 
               
               
                   
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 NaHS 
                 87.8 ± 4.3  
                 68.8 ± 5.7 
                 37.0 ± 4.7  
                 8.3 ± 1.0 
               
               
                 Glutathione 
                 12.0 ± 2.4  
                 12.1 ± 4.5 
                 13.4 ± 2.7  
                 7.5 ± 1.7 
               
               
                 Cysteine 
                 5.1 ± 0.6 
                  9.5 ± 0.8 
                 6.4 ± 1.7 
                 2.1 ± 0.2 
               
               
                 Sodium sulfite 
                 6.4 ± 0.7 
                 10.7 ± 1.7 
                 5.5 ± 0.8 
                 7.8 ± 0.7 
               
               
                 Sodium sulfate 
                 7.9 ± 1.5 
                 10.0 ± 1.3 
                 4.6 ± 0.5 
                 8.0 ± 0.5 
               
               
                 Thiosodium 
                 6.5 ± 1.0 
                  8.9 ± 0.5 
                 5.6 ± 0.3 
                 5.5 ± 0.1 
               
               
                 sulfate 
               
               
                 NO 
                 6.1 ± 1.0 
                  6.6 ± 0.9 
                 4.7 ± 0.7 
                 5.6 ± 0.4 
               
               
                 Buffer 
                 8.7 ± 1.8 
                 10.4 ± 2.4 
                 4.6 ± 0.5 
                 4.0 ± 0.1 
               
               
                   
               
            
           
         
       
     
     As shown in Table 1,  99m Tc-labeled gluconate, glucoheptonate, and glucarate reacted most with NaHS. Specifically,  99m Tc-gluconate produced 87.8±4.3% of insoluble substances, the most,  99m Tc-glucoheptonate produced 68.8±5.7% of insoluble substances, and  99m Tc-glucarate produced 37.0±4.7% of insoluble substances. On the other hand,  99m Tc-citrate produced 8.3±1.0% of insoluble substances, which was relatively low, but was the most reactive with hydrogen sulfide compared to other activated sulfides. Other  99m Tc-labeled alpha-hydroxy acids,  99m Tc-tartrate and  99m Tc-glucuronate prepared in Example 1 showed similar results to  99m Tc-citrate. 
     The results of Table 1 are summarized in a graph and shown in  FIG. 8 . 
     As shown in  FIG. 8 ,  99m Tc-labeled gluconate, glucoheptonate, and glucarate reacted only with NaHS to produce 37.0-87.8% of insoluble substances but not with other active sulfides, which means that the reaction with active sulfides other than hydrogen sulfide was very low. On the other hand,  99m Tc-citrate did not produce much insoluble substances even by NaHS. 
     The  99m Tc-labeled alpha-hydroxy acid according to the present invention produced less than 15% of insoluble substances by reacting with active substances other than NaHS, such as glutathione, cysteine, sodium sulfite, sodium sulfate, thiosodium sulfate, NO and phosphate buffer. The  99m Tc-labeled alpha-hydroxy acid did not appear in the image due to the low production of insoluble substances. 
     That is, it was confirmed that the  9m Tc-labeled alpha-hydroxy acid according to the present invention selectively detects only NaHS (hydrogen sulfide) among active sulfides, and thus it can be effectively used for the detection of hydrogen sulfide. 
     Experimental Example 3: Evaluation of Reaction Degree of  99m Tc-Alpha-Hydroxy Acid According to NaHS Concentration 
     In order to evaluate the reaction degree of the  99m Tc-alpha-hydroxy acid of the present invention according to the concentration of NaHS (hydrogen sulfide), the following experiment was performed, and the results are shown in  FIG. 9 . 
     Particularly, the  99m Tc-alpha-hydroxy acid prepared in Example 1 was diluted 5 times with physiological saline, and then 100 μL of each diluent was taken, to which 100 μL of 0.2 M sodium phosphate buffer (pH 7.4) containing 0-0.4 mM NaHS was added, followed by reaction at 37° C. for 15 minutes. Then, ITLC was developed with physiological saline to determine the percentage of insoluble substances. 
     As shown in Table 9,  99m Tc-gluconate reacted with hydrogen sulfide to produce the most insoluble substances, and it was confirmed that the production of insoluble substances increased as the concentration increased at the concentrations below 0.1 mM. However, equilibrium was reached at the concentrations above 0.1 mM. 
     It was confirmed that  99m Tc-glucoheptonate gradually increased the production of insoluble substances as the concentration of hydrogen sulfide increased. 
     It was also confirmed that  99m Tc-glucarate gradually increased the production of insoluble substances as the concentration of hydrogen sulfide increased. 
     On the other hand, it was confirmed that  99m Tc-citrate hardly produced insoluble substances. 
     From the above results, it was confirmed that the production of insoluble substances by the  99m Tc-alpha-hydroxy acid of the present invention varied depending on the concentration of hydrogen sulfide, and thus, it can be effectively used for measuring the concentration of hydrogen sulfide. 
     Experimental Example 4: Observation of Imaging of Inflamed Tissue where Hydrogen Sulfide Produced 
     It has been reported that hydrogen sulfide was generated in the inflamed tissue induced by the administration of carrageenan (Li L, Bhatia M, Zhu Y Z, et al. FASEB J. (2005) 19:1196-1198; Bhatia M, Sidhapuriwala J, Moochhala S M, et al. Br J Pharmacol. (2005) 145:141-144). Accordingly, the following experiment was performed to confirm whether the imaging of the inflamed tissue where hydrogen sulfide was generated was possible when the  99m Tc-alpha-hydroxy acid according to the present invention was administered. 
     &lt;4-1&gt; Imaging of Inflamed Tissue 
     30 μL of physiological saline containing 1% carrageenan was injected into the right hind paw of a mouse, and 30 μL of physiological saline was injected into the left hind paw of the mouse. After 4 hours, 300 μCi of  99m Tc-gluconate or  99m Tc-glucoheptonate labeled in Example 1 was injected into the tail vein of the mouse. After 1 hour, the hind paws were taken with SPECT-CT, and the results are shown in  FIG. 10 . 
     As shown in  FIG. 10 , both images showed the results of high radioisotope intake in the feet in which inflammation was induced. 
     In other words, both  99m Tc-gluconate and  99m Tc-glucoheptonate showed that the radioactivity of the inflamed area administered with carrageenan was clearly higher than that of the area administered with physiological saline. Therefore, it was proved that it is possible to image the inflamed area where hydrogen sulfide was produced. 
     &lt;4-2&gt; Evaluation of Hydrogen Sulfide Concentration in Inflamed Area 
     In order to confirm that the concentration of hydrogen sulfide in the inflamed area was higher than the concentration of hydrogen sulfide in the normal area, it was measured as follows using a method described in the literature (A D Ang, A Konigstorfer, G I Giles, M Bhatia. Adv Biol Chem, 2012, 2:360-365). After 4 hours, the mouse was euthanized with carbon dioxide gas, and the ankle was cut and the weight was measured. 500 μL of 50 mM sodium carbonate buffer (pH 9) chilled with ice was added thereto and homogenized for 30 seconds gently, 1 minute 30 seconds moderately, and seconds vigorously. After centrifugation at 1200×g for 5 minutes, the supernatant was obtained, to which 400 μL of a mixed solution of 350 μL of 1% zinc acetate and 50 μL of 1.5 M sodium hydroxide was added, followed by mixing well. After centrifugation at 1200×g for 5 minutes, the supernatant was discarded. To the precipitated pellet, 1 mL of distilled water saturated with nitrogen was added, which was mixed by vortexing for 1 minute. After centrifugation at 1200×g for 5 minutes, the supernatant was discarded. 160 μL of 25 mM sodium hydroxide solution containing 1 g/L of ascorbic acid was added to the pellet and mixed well. 20 μL of 47.5 mM DMPD dissolved in 7.2 M hydrochloric acid and 20 μL of 80 mM FeCl 3  dissolved in 1.2 M hydrochloric acid were added thereto and vortexed for 10 seconds. After reacting at room temperature for 15 minutes, absorbance was measured at 665 nm. A standard quantification curve was drawn with a standard sample measured by the same method, and then quantified. 
     As a result of the quantification, the concentration of hydrogen sulfide in the paws treated with physiological saline was 19.8±4.4 μM (n=3), whereas the concentration of hydrogen sulfide in the paws treated with carrageenan was 43.7±3.5 μM (n=3), which was more than doubled. Therefore, it was confirmed that the increased intake of  99m Tc-gluconate and  99m Tc-glucoheptonate was correlated with the increase of hydrogen sulfide concentration. 
     The  99m Tc-alpha-hydroxy acid according to the present invention can image the inflamed tissue in which hydrogen sulfide is generated, and the concentration increase can be known through whether the intake of the treated  99m Tc-alpha-hydroxy acid increases, so that not only imaging but also the increase in concentration can be confirmed. In addition, since the hydrogen sulfide concentration can be quantified and expressed numerically through fluorescence assay, it can be effectively used to measure the concentration of hydrogen sulfide in the inflamed tissue. 
     Experimental Example 5: Observation of Imaging of Mouse Brain Reperfused after Middle Cerebral Artery Occlusion 
     It has been reported that the concentration of hydrogen sulfide in the brain increased 12 hours after the blood flow in the mouse brain was blocked and reperfused (Ren C, Du A, Li D, et al. Brain Res. (2010) 1345:197-205). Accordingly, the following experiment was performed to confirm whether the concentration of hydrogen sulfide can be measured and imaged in the reperfused mouse brain after middle cerebral artery occlusion when the  99m Tc-alpha-hydroxy acid according to the present invention was administered, and the results are shown in  FIG. 11 . 
     Particularly, a reperfusion model after middle cerebral artery occlusion was constructed according to the method of Koizumi et al. (Koizumi J, Yoshida Y, Nakazawa T, et al. Jpn J Stroke (1986) 8:1-8). The mouse was anesthetized by intramuscular injection of ketamine (80 mg/kg) and the carotid artery, the internal carotid artery, and the external carotid artery were separated in order by incising the skin of the neck. Then, a hole was made in the external carotid artery with a 27 gauge needle, and a nylon 4.0 thread coated with silicone was inserted about 17 mm to close the middle cerebral artery, and after 2 hours, the nylon thread was removed again to resume blood flow. The skin of the mouse was sutured and recovered for 12 hours, and then 1 mCi of the  99m Tc-gluconate prepared in Example 1 and 1 mCi of [ 18 F]FDG were mixed and administered to the tail vein of the mouse. After 1 hour, the mouse was anesthetized with ether and the brain was extracted by dissecting the skull. Coronal sections of 1 mm thick were made with the extracted brain, frozen, and exposed to a BAS2500 image plate (Fuji Film Co.) for 20 minutes in a −20° C. freezer. The sections were left for 20 hours to attenuate the radioactivity of  18 F and exposed to the image plate for 24 hours in the freezer. The exposed tissues were stained in 1% 2,3,5-tetrazolium chloride (TTC) solution. In  FIG. 11 , TTC staining represents the living brain tissues, [ 18 F]FDG represents the degree of glucose metabolism, and  99m Tc-gluconate represents the generation site of hydrogen sulfide. 
     As shown in  FIG. 11 , the living tissues were stained red by TTC. [ 18 F]FDG image showed glucose metabolism, and it can be seen that it almost overlapped with the red area stained by TTC.  99m Tc-gluconate was ingested in the damaged area after reperfusion because it was ingested at the boundary area rather than completely dead brain tissues and the normal brain tissues, and it can be estimated that hydrogen sulfide generation was high in that area. 
     From the above results, it was confirmed that hydrogen sulfide was generated in the reperfused tissue, as the concentration of hydrogen sulfide increased in the reperfused tissue after middle cerebral artery occlusion. 
     In addition, it was confirmed that hydrogen sulfide detection, concentration measurement, and imaging of reperfused tissue after middle cerebral artery occlusion were possible using the  99m Tc-alpha-hydroxy acid according to the present invention. 
     Experimental Example 6: Confirmation of  99m Tc Accumulation in Tissue Administered with  99m Tc-Alpha-Hydroxy Acid 
     The following experiment was performed to confirm whether the  99m Tc was accumulated when the  99m Tc-alpha-hydroxy acid according to the present invention was administered to the tissue containing hydrogen sulfide. 
     Particularly, NaHS was dissolved in matrigel at the concentration of 1.7 mg/mL and injected subcutaneously into the back of a BALB/c mouse by 50 μL, and 1 mCi of  99m Tc-gluconate was injected into the tail vein. One hour later, the matrigel was collected, weighed, and the radioactivity was measured. Using the results, the ingested amount (% ID/g) was calculated for the injected amount per tissue weight. 
     The composition for detecting hydrogen sulfide or measuring a concentration of hydrogen sulfide according to the present invention, which comprises the compound represented by formula 1 ( 99m Tc-alpha-hydroxy acid) having alpha-hydroxy acid labeled with  99m Tc could selectively detect only hydrogen sulfide among various active sulfides, and the degree of generation of insoluble substances was changed according to the concentration of hydrogen sulfide. 
     Using the composition of the present invention, the concentration of hydrogen sulfide could be measured, imaging of the inflamed tissue in which hydrogen sulfide was generated, and the increase in the concentration of the generated hydrogen sulfide could be confirmed, and the hydrogen sulfide concentration could be quantified and expressed numerically through fluorescence assay. In addition, it was confirmed that hydrogen sulfide detection, concentration measurement, and imaging of reperfused tissue after middle cerebral artery occlusion were possible using the composition of the present invention. 
     Therefore, the composition for detecting hydrogen sulfide or measuring a concentration of hydrogen sulfide according to the present invention, which comprises the compound represented by formula 1 ( 99m Tc-alpha-hydroxy acid) having alpha-hydroxy acid labeled with  99m Tc, enables the detection or concentration measurement of hydrogen sulfide in in-vitro and in-vivo levels and, as such, can be advantageously used for detecting hydrogen sulfide and measuring a concentration of hydrogen sulfide and furthermore for discovering biological roles of hydrogen sulfide in vivo, especially, for detecting, imaging, and quantitatively measuring hydrogen sulfide in a disease selected from the group consisting of angiogenesis, inflammation, cancer, Alzheimer&#39;s disease, cardiovascular ischemia, and cerebrovascular ischemia, or in hypoxic tissues. 
     In addition,  99m Tc is easier to supply than other radioactive isotopes and is competitive in price, so it has an economic advantage. 
     INDUSTRIAL APPLICABILITY 
     The composition for detecting hydrogen sulfide or measuring a concentration of hydrogen sulfide according to the present invention, which comprises the compound represented by formula 1 ( 99m Tc-alpha-hydroxy acid) having alpha-hydroxy acid labeled with  99m Tc, enables the detection or concentration measurement of hydrogen sulfide in in-vitro and in-vivo levels and, as such, can be advantageously used for detecting hydrogen sulfide and measuring a concentration of hydrogen sulfide and furthermore for discovering biological roles of hydrogen sulfide in vivo, especially, for detecting, imaging, and quantitatively measuring hydrogen sulfide in a disease selected from the group consisting of angiogenesis, inflammation, cancer, Alzheimer&#39;s disease, cardiovascular ischemia, and cerebrovascular ischemia, or in hypoxic tissues.