Radiodiagnostic agent for bacterial infections

Abstract

To provide means for more accurately performing the detection of a lesion with a radioactive diagnostic agent, in the diagnosis of a bacterial infection.SOLUTION: A radioactive diagnostic agent for a bacterial infection is completed by investigating, using various bacteria, the uptake of radioactive labeled compounds into bacterial cells in each growth phase, further examining the uptake rate of the radioactive labeled compounds into the bacterial cells, and discovering that a specific radioactive labeled compound shows a different accumulation tendency in each growth phase of various bacteria.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a radiodiagnostic agent for bacterial infections.

Background Art

[0002] Bacterial infections caused by various bacteria often threaten the healthy life of humans. Bacterial infections are caused by the infection of hosts by pathogenic bacteria, opportunistic infectious bacteria, etc., and their pathological conditions are diverse depending on the type of causative bacteria (Non-Patent Document 1).

[0003] The onset of bacterial infections is related to two factors: the pathogenic factors of bacteria and the host's infection defense ability (Non-Patent Document 2). Important pathogenic factors related to the infectivity of bacteria include factors involved in the adhesion and invasion of host tissues, various enzymes that induce tissue damage, toxins that disrupt physiological and immune functions, and the like. On the other hand, as infection defense, there are innate immune responses centered on phagocytosis of bacteria by neutrophils, macrophages, etc., and acquired immune responses that specifically eliminate bacteria antigen-specifically centered on T cells and B cells. Most bacteria are usually eliminated by these host immune responses and do not lead to the onset of infectious diseases. However, when the host immune response is weakened, the immune response to some pathogens is lacking, or when immune disruption by bacteria occurs, infectious diseases develop.

[0004] In severe acute infectious diseases such as severe streptococcal infections, in addition to the host immune response, rapid bacterial growth observed at the infection site and throughout the body is also a major problem. For example, necrotizing fasciitis is one of the symptoms of severe streptococcal infections (Non-Patent Document 1), and this symptom is accompanied by rapid growth of streptococci and fascial necrosis at the infection site, and elucidating the cause of this rapid streptococcal growth is a major issue. However, regarding the pathogenic factors of infectious bacteria, many studies have been conducted on the above-mentioned adhesion and invasion factors, various enzymes and toxins involved in tissue damage, etc., but there has been little progress in specific studies on the growth ability of bacteria at present.

[0005] Currently, the bacterial culture method is used for the examination of bacterial infections. However, since this method takes time until the test results are determined, it is not suitable for the diagnosis of severe infectious diseases with rapid progression of symptoms, high severity, and high mortality rate.

[0006] Patent Document 1 discloses a "radiological diagnostic agent for bacterial infections containing a radioisotope-labeled amino acid". However, it does not disclose or suggest the "radiological diagnostic agent for bacterial infections containing a radioactively labeled compound labeled with a radioactive isotope" of the present invention.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Summary of the Invention

Problems to be Solved by the Invention

[0009] An object of the present invention is to provide a means for more accurately detecting a lesion by a radioactive diagnostic agent in the diagnosis of a bacterial infectious disease.

Means for Solving the Problems

[0010] As a result of intensive studies, the present inventors examined the uptake of a radio-labeled compound into bacteria in each growth phase using Escherichia coli K-12 and EC-14-derived strains, Pseudomonas aeruginosa SR-24 strain, and Staphylococcus aureus SR-3637 strain, and further examined the uptake rate of the radio-labeled compound into the bacterial cells, and found that a specific radio-labeled compound shows different accumulation tendencies in each growth phase of Escherichia coli and pathogenic microorganisms of infectious diseases, thereby completing the present invention.

[0011] That is, the present invention is as follows. 1. A radiodiagnostic agent for bacterial infections, comprising a radioisotope-labeled radioactive labeling compound or a radioisotope-labeled amino acid. 2. The radiodiagnostic agent according to item 1 above, wherein the radioisotope is 99m-technetium ( 99m Tc), 123-iodine ( 123 I), 18-fluorine ( 18 F), 125-iodine ( 125 I), 67-gallium ( 67 Ga), 111-indium ( 111 In), 201-thallium ( 201 Tl), 3-hydrogen ( 3 H), 11-carbon ( 11 C), 15-oxygen ( 15 O), 32-phosphorus ( 32 P), 59-iron ( 59 Fe), 67-copper ( 67 Cu), 81m-krypton ( 81m Kr), 81-rubidium ( 81 Rb), 89-strontium ( 89 Sr), 90-yttrium ( 90 Y), 131-iodine ( 131 I), 133-xenon ( 133 Xe), 117m-samarium ( 117m Sm), 153-samarium ( 153 Sm), 186-rhenium ( 186 Re), 188-rhenium ( 188 Re), 212-bismuth ( 212 Bi), 213-bismuth ( 213 Bi), 211-astatine ( 211 At), 177-lutetium ( 177 Lu), 225-actinium ( 225 Ac) or 223-radium ( 223 Ra). 3. The radiodiagnostic agent according to item 1 or 2 above, wherein the radioactive labeling compound is 18 F-FDG, 123 I-BMIPP, 99m Tc-fuccinic acid, 99m Tc-GSA and / or 99m Tc-PMT. 4. The radioisotope-labeled amino acid is 125 I / 123 I-D-MIT, the radiodiagnostic agent according to item 1 or 2 above. 5. The radiolabeled compound is 18 F-FDG, the radiodiagnostic agent according to any one of items 1 to 3 above. 6. The radiolabeled compound is 123 I-BMIPP, the radiodiagnostic agent according to any one of items 1 to 3 above. 7. The radiolabeled compound is 99m Tc-phytate, the radiodiagnostic agent according to any one of items 1 to 3 above. 8. The radiolabeled compound is 99m Tc-GSA, the radiodiagnostic agent according to any one of items 1 to 3 above. 9. The radiolabeled compound is 99m Tc-PMT, the radiodiagnostic agent according to any one of items 1 to 3 above. 10. The bacterial infectious disease is an infectious disease caused by Escherichia coli, the radiodiagnostic agent according to any one of items 1 to 9 above. 11. The bacterial infectious disease is an infectious disease caused by pathogenic Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa or Streptococcus pyogenes, the radiodiagnostic agent according to any one of items 1 to 10 above. 12. A method for diagnosing or assisting in the diagnosis of a bacterial infectious disease, characterized by including the following steps: (1) A step of administering the radiodiagnostic agent according to any one of items 1 to 11 above. (2) A step of detecting the administered radiodiagnostic agent, and (3) A step of determining the presence, absence or progression of a bacterial infectious disease from the detection result. 13. Further including a step of detecting a radioisotope-labeled amino acid, the diagnostic method or diagnostic assistance method according to item 12 above. 14. A kit for diagnosing a bacterial infectious disease, containing the radiodiagnostic agent according to any one of items 1 to 11 above.

Advantages of the Invention

[0012] The present invention can provide a radiodiagnostic agent for bacterial infections. The radiodiagnostic agent can be used for early imaging diagnosis of bacterial infections before onset, during which the growth activity of bacteria is enhanced, in the course of the onset of infectious diseases from the invasion and infection of causative bacteria into the body to the enhancement of the growth activity of bacteria in the body and the onset of the disease.

Embodiments for Carrying Out the Invention

[0013] The present invention newly found and achieved the relationship between the growth ability of bacteria in the onset of bacterial infections and a radiolabeled compound, that is, the fact that the radiolabeled compound used for early diagnosis of cancer and the like is selectively taken up by microorganisms.

[0014] The present invention targets a radiodiagnostic agent for bacterial infections (hereinafter sometimes simply referred to as "radiodiagnostic agent") containing a specific radiolabeled compound. By accumulating the radiodiagnostic agent in the body of a subject (particularly, inside bacteria in the body), the radiodiagnostic agent of the present invention can detect even before onset when the growth activity of bacteria is enhanced earlier than the conventional method in the examination of bacterial infections.

[0015] (Radioactive tracer) A method of tracking the movement and distribution of a target substance by adding a radioisotope as a tracer and measuring its radioactivity is called a radioactive tracer method. As the radioactive tracer, 18 F-2-deoxy-2-fluoro-D-glucose ( 18 F-FDG) is currently used for cancer diagnosis (Non-Patent Documents 3 and 4).

[0016] (Growth phase of bacteria) Bacterial growth can be divided into four growth phases: the lag phase, the logarithmic growth phase, the stationary phase, and the death phase. The lag phase is a state in which bacterial growth has almost stopped, and it is a preparation period for bacteria to adapt to a new environment and start growing. In the logarithmic growth phase, the enzyme activity in the bacterial cells is most active, and bacteria grow vigorously. In the stationary phase, the growth rate of bacteria decreases due to nutrient deficiency and the accumulation of harmful metabolic products, and the death of bacteria begins. In the death phase, the number of viable bacteria decreases and the number of dead bacteria increases (Non-Patent Documents 2 and 5).

[0017] (Amino Acid Metabolism of Bacteria) For bacteria to grow and proliferate, it is essential to take in necessary nutrients from the outside world and use these nutrients to produce substances required for the synthesis of cell components and the acquisition of energy. The nutrients required for the growth and proliferation of bacteria are the same as those of higher organisms, including water, carbon sources, nitrogen sources, inorganic salts, growth factors, etc. (Non-Patent Document 2). Amino acids are essential for the maintenance of bacterial life as a nitrogen source, and amino acid transport functions are ubiquitous in bacterial cells (Non-Patent Document 6). Bacteria directly take in external amino acids into the cells and efficiently carry out protein synthesis, carbon source acquisition, and nitrogen metabolism necessary for growth and proliferation. In addition, bacteria take in various amino acids such as methionine, lysine, tyrosine, and histidine (Non-Patent Documents 7 to 10). The inventors of the present invention have shown a high accumulation of specific compounds containing amino acids that correlate with the growth process in Escherichia coli and pathogenic microorganisms of infectious diseases (Patent Document 1).

[0018] (Radioisotope) The radiolabeled compound contained in a radiodiagnostic agent is formed by binding a radioisotope to a compound. The radioisotope is not particularly limited, but examples include 99m-technetium ( 99m Tc), 123-iodine ( 123 I), 18-fluorine ( 18 F), 125-iodine ( 125 I), 67-gallium ( 67 Ga), 111-indium ( 111In), 201-Thallium( 201 Tl), 3-Hydrogen( 3 H), 11-Carbon( 11 C), 15-Oxygen( 15 O), 32-Phosphorus( 32 P), 59-Iron( 59 Fe), 67-Copper( 67 Cu), 81m-Krypton( 81m Kr), 81-Rubidium( 81 Rb), 89-Strontium( 89 Sr), 90-Yttrium( 90 Y), 131-Iodine( 131 I), 133-Xenon( 133 Xe), 117m-Samarium( 117m Sm), 153-Samarium( 153 Sm), 177-Lutetium( 177 Lu), 186-Rhenium( 186 Re), 188-Rhenium( 188 Re), 212-Bismuth( 212 Bi), 213-Bismuth( 212 Bi), 211-Astatine( 211 At), 225-Actinium( 225 Ac) and 223-Radium( 223 Ra) etc. can be mentioned.

[0019] (Radioactive Labeled Compound) The radioactive labeled compound contained in the radioactive diagnostic agent for bacterial infectious diseases of the present invention is not particularly limited. For example, galactosyl human serum albumin diethylenetriamine pentaacetic acid technetium( 99m Tc)( 99m Tc-GSA), N-pyridoxyl-5-methyltryptophan technetium( 99m Tc)( 99m Tc-PMT), phytic acid technetium( 99m Tc)( 99m Tc-phytic acid), dimercaptosuccinic acid technetium( 99m Tc)( 99m Tc-DMSA), mercaptoacetylglycylglycylglycine technetium( 99m Tc)(99m Technetium-99m mercaptoacetyltriglycine (Tc-MAG3), technetium pyrophosphate ( 99m Tc)( 99m Tc-PYP), technetium diethylenetriaminepentaacetate ( 99m Tc)( 99m Tc-DTPA), Technetium-99m ethyl cysteinate dimer( 99m Tc-ECD), 99m Tc-human serum albumin-diethylenetriaminepenta-acetic acid( 99m Tc-HSA-D), sodium pertechnetate ( 99m Tc)( 99m Tc-TcO4 - ), technetium methylene diphosphonate ( 99m Tc)( 99m Tc-MDP), 99m Tc-tetrofosmin( 99m Tc-TF), hexakis(2-methoxyisobutylisonitrile)technetium ( 99m Tc)( 99m Tc-MIBI), 15-(4-iodophenyl)-3(R,S)-methylpentadecanoic acid ( 123 I)( 123 I-BMIP), ioflupane ( 123 I)( 123 I-IFP), 3-iodobenzylguanidine ( 123 I)( 123 I-MIBG), 18 F-FDG, N-isopropyl- 123 I / 125 I-p-iodoamphetamine( 123 / 125 I-IMP), 123 I / 125 I-iomazenil( 123 / 125 I-IMZ), gallium citrate ( 67 Ga)( 67 Ga-citrate), indium pentetreotide ( 111 In)( 111 In-pentetoreotide) and thallium chloride ( 201 Tl)( 201Examples include TlCl).

[0020] (Amino acid) The amino acid to be radiolabeled is not particularly limited. For example, neutral amino acids such as D-alanine, D-methionine, Glycine, L-alanine, L-tyrosine, and L-methionine, acidic natural amino acids such as L-glutamic acid, basic natural amino acids such as L-lysine and L-histidine, etc. can be mentioned.

[0021] (Radiolabeled amino acid) The radiolabeled amino acid may be any one that can be administered in vivo to detect the pathogen of bacterial infectious diseases. As specific examples, 3 H, 18 F, 125 I or 123 Amino acids such as D-alanine, D-methionine, Glycine, L-tyrosine, L-methionine, acidic natural amino acids such as L-glutamic acid, L-lysine, and L-histidine, to which radioactive isotopes such as I are bound, salts thereof, and derivatives thereof, etc. can be mentioned. For example, [2,3- 3 H]-D-alanine labeled with radioactive tritium ( 3 H), [metyl- 3 H]-D-methionine, [2- 3 H]-glycine ( 3 H-Gly), [2,3- 3 H]-L-alanine ( 3 H-L-Ala), [ring3,5- 3 H]-L-tyrosine ( 3 H-L-Tyr), [methyl- 3 H]-L-methionine ( 3 H-L-Met), [2,3,4- 3 H]-L-glutamic acid ( 3 H-L-Glu), [4,5- 3H]-L-lysine( 3 H-L-Lys), [ring2,5- 3 H]-L-histidine( 3 H-L-His), radioactive iodine 125 I or 123 I-labeled 3- 125 I / 123 I]iodo-L-tyrosine( 125 I / 123 I-L-MIT(monoiodothyrosine)), 3- 125 I / 123 I]iodo-D-tyrosine( 125 I / 123 I-D-MIT), [5- 125 I / 123 I]-L-histidine( 125 I / 123 I-L-His), radioactive carbon (carbon 11: 11 C) -labeled [S-methyl- 11 C]-L-methionine and the like can be mentioned.

[0022] (Pharmaceutically acceptable carrier) The radiopharmaceutical of the present invention may contain, in addition to the radiolabeled compound, a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier means a pharmaceutically acceptable substance, composition or medium such as a liquid or solid excipient, diluent, lubricant, or solvent for encapsulating a substance. Each carrier must be "acceptable" in the sense that it is compatible with the other components of the formulation and not harmful to the recipient. Specific examples of pharmaceutically acceptable carriers include, for example, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; celluloses such as cellulose, sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; physiological saline; Ringer's solution; ethyl alcohol; polyanhydrides such as polyesters and polycarbonates, etc., but are not particularly limited. Those skilled in the art can appropriately select these carriers.

[0023] (Dosage form) When the radiopharmaceutical of the present invention is administered to a subject for the purpose of diagnosing a bacterial infection, it can be administered orally as a powder, granule, tablet, capsule, pill, liquid, etc., or parenterally as an injection, suppository, transdermal absorbent, inhalant, etc. The selection and manufacture of such dosage forms can be appropriately carried out by those skilled in the art using methods known per se.

[0024] (Bacterial infection) As used herein, the term "bacterial infectious disease" refers to an infectious disease caused by the growth of bacteria in a living body. The bacteria are not particularly limited, and examples include enteropathogenic Escherichia coli (EPEC), enteroinvasive Escherichia coli (EIEC), enterohemorrhagic Escherichia coli (EHEC), enterotoxigenic Escherichia coli (ETEC), enteroaggregative Escherichia coli (EAEC), Clostridium perfringens, Vibrio cholerae O1 and Vibrio cholerae O139, Corynebacterium diphtheriae, non-tuberculousis mycobacteria (NTM), Methicillin Resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Legionella spp., Serratia marcescens, Streptococcus pyogenes, and the like. Bacterial infectious diseases are not particularly limited, and examples include diarrheogenic Escherichia coli infection, Clostridium perfringens infection, cholera, diphtheria, nontuberculous mycobacteriosis, MRSA infection, Pseudomonas aeruginosa infection, Legionella pneumonia, Serratia infection, severe group A streptococcal infection, and the like.

[0025] (Kit for diagnosing bacterial infectious diseases) The present invention encompasses a kit for diagnosing bacterial infectious diseases, which contains a radiodiagnostic agent. The kit for diagnosing bacterial infectious diseases of the present invention may include instructions regarding the administration of each active ingredient. The instructions describe the administration schedule of each active ingredient in the method for diagnosing bacterial infectious diseases of the present invention as shown below. In particular, each active ingredient in the diagnostic kit for bacterial infections of the present invention is preferably an orally administrable tablet, but it can also be a sustained-release granule, syrup, fine granule, and / or injection.

[0026] (Method for diagnosing or assisting in the diagnosis of bacterial infections) The method for diagnosing or assisting in the diagnosis of bacterial infections using the radiodiagnostic agent of the present invention detects the accumulation of the radiodiagnostic agent in cells infected with the pathogen of bacterial infections. The diagnostic method or diagnostic assistance method includes: (1) a step of administering the radiodiagnostic agent of the present invention to a subject (test subject); (2) a step of detecting the administered radiodiagnostic agent; and (3) a step of determining the presence or absence or progression of bacterial infections based on the detection result. Furthermore, it may include the step of detecting the radioisotope-labeled amino acid described above.

[0027] (1) Regarding the method of administering the radiodiagnostic agent of the present invention, it can be administered in the same manner as other generally known radiological diagnostic agents. The administration method is not particularly limited, and examples include intravenous injection, subcutaneous injection, intradermal injection, and intramuscular injection. The timing of administration can be appropriately determined according to the condition of the subject (patient) and the situation of treatment or diagnosis.

[0028] (2) Regarding the method of detecting the administered radiodiagnostic agent, it is not particularly limited, and for example, it can be performed by imaging using a known method. For example, by detecting the radiation emitted from the compound using nuclear medicine images such as PET or SPECT, imaging of bacterial infections (bacterial growth in the patient's body) is possible.

[0029] (Radiation energy) The radiation energy of the radiodiagnostic agent of the present invention can be appropriately set to the radiation energy detectable in the step of (2). For example, in the case of PET imaging, it is necessary to have the radiation energy enabling PET imaging. For example, for the purpose of performing PET imaging on an adult, it is sufficient to have a radiation energy of 50 - 225 MBq at the time of use.

[0030] (3) Regarding this, the method for determining the presence or progression of a bacterial infection from the detection results is not particularly limited. For example, the signal intensity and / or signal distribution of the radioactivity detected in the subject are compared with the signal intensity and / or signal distribution of the radioactivity detected at the same site in a comparison subject (a comparison subject negative for bacterial infection) known not to have a bacterial infection after administration of the radiodiagnostic agent of the present invention, or with the signal intensity and / or signal distribution of the radioactivity detected at the same site in a comparison subject (a comparison subject positive for bacterial infection) known to have a bacterial infection after administration of the radiodiagnostic agent of the present invention, and the determination can be made based on this comparison. For example, if the signal intensity and / or signal distribution of the radioactivity detected in the subject are equal to or lower than the signal intensity and / or signal distribution of the radioactivity detected at the same site in a comparison subject negative for bacterial infection, it is determined that the subject is negative for bacterial infection. For example, if the signal intensity and / or signal distribution of the radioactivity detected in the subject are higher than the signal intensity and / or signal distribution of the radioactivity detected at the same site in a comparison subject negative for bacterial infection, it is determined that the subject is positive for bacterial infection. For example, if the signal intensity and / or signal distribution of the radioactivity detected in the subject are equal to or higher than the signal intensity and / or signal distribution of the radioactivity detected at the same site in a comparison subject positive for bacterial infection, it is determined that the subject is positive for bacterial infection. For example, if the signal intensity and / or signal distribution of the radioactivity detected in the subject are lower than the signal intensity and / or signal distribution of the radioactivity detected at the same site in a comparison subject positive for bacterial infection, it is determined that the subject is negative for bacterial infection.

[0031] (PET / SPECT) As a specific example of the method for diagnosing a bacterial infection using the radiodiagnostic agent of the present invention, the case of PET (Positron Emission Tomography) or SPECT (Single Photon Emission Computed Tomography) will be described. (1) Inject the radioactive diagnostic agent of the present invention into the subject intravenously. (2) Perform PET or SPECT imaging (or PET-CT or SPECT-CT imaging) 20 minutes to 1 hour after the injection. (3) If the accumulation of the labeled amino acid or the labeled compound in the PET or SPECT image is high, diagnose it as positive for bacterial infection.

[0032] Currently, many nuclear medicine diagnostic agents used clinically perform image diagnosis about 60 minutes after administration. The inventors of the present invention confirmed that the difference in the accumulation amount with human somatic cells of the D-form amino acid was large about 60 minutes after administration. On the other hand, it was confirmed that for the L-form amino acid, the difference in the accumulation amount between bacteria and cells was large at about 5 minutes after administration, similar to the D-form. Accordingly, if the radiation dose of the amino acid is measured at about 5 minutes (or 1 to 30 minutes, 2 to 15 minutes, 3 to 10 minutes) after administration of the L-form amino acid, and the radiation dose of the amino acid is measured at about 60 minutes (or 10 to 180 minutes, 20 to 120 minutes, 30 to 100 minutes) after administration of the D-form amino acid, diagnosis can be performed with high accuracy.

[0033] (Examples of diagnostic methods or diagnostic aid methods for bacterial infectious diseases) The diagnostic method for bacterial infectious diseases using the radioactive diagnostic agent of the present invention will be described based on examples. (1) To the subject, the radioactive diagnostic agent of the present invention, " 125 I / 123 I-D-MIT", " 11 C-L-Met" or " 11 C-D-Met" is injected intravenously. (2) Perform PET or SPECT imaging (or PET-CT or SPECT-CT imaging) 20 minutes to 1 hour after the injection. (3) From the PET or SPECT image, 125 I / 123 I-D-MIT, " 11 C-L-Met" or " 11 C-D-Met" is diagnosed as positive for bacterial infection when the accumulation is higher than the normal accumulation.

[0034] Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples at all.

Example

[0035] [Evaluation of Uptake of Radioactive Labeled Compounds into Bacterial Cells at Each Growth Phase] It was evaluated how much (accumulated) of which types of amino acids were taken up by bacteria at each growth phase of the induction phase, logarithmic growth phase, and stationary phase.

[0036] (Bacterial Strain Used and Preculture Conditions) In this example, a strain derived from E. coli K-12 was used as a model bacterium. As the medium for preculture, THY medium prepared by adding 0.2% Yeastextract (Becton, Dickinson and Company) to Todd’s Hewitt Broth (THB medium; Becton, Dickinson and Company) was used. The preculture conditions were set to 14 hours or more under conditions of 37°C and 5% CO2.

[0037] (Radioactive Labeled Compounds and Amino Acids Used) As radiopharmaceuticals 99m Tc-GSA, 99m Tc-PMT, 99m Tc-fuccinic acid, 99m Tc-DMSA, 99m Tc-MAG3, 99m Tc-PYP, 99m Tc-DTPA, 99m Tc-ECD, 99m Tc-HSA-D, 99m Tc-TcO4 - , 99m Tc-MDP, 99m Tc-TF, 99m Tc-MIBI, 123 I-BMIPP, 123 I-IFP, 123 I-MIBG, 18 F-FDG (concentration 4.5), 18 F-FDG (concentration 1.0), 18F-FDG (concentration 0.1), 123 / 125 I-IMP, 123 / 125 I-IMZ, 67 Ga-citrate, 111 In-pentetoreotide and 201 TlCl were used. As natural amino acids, L-methionine and L-alanine were selected, and as artificial amino acids, D-methionine and D-alanine were selected. As labeled amino acids, [methyl- 3 H]-L-methionine ( 3 H-L-Met), [2,3- 3 H]-L-alanine ( 3 H-L-Ala), [metyl- 3 H]-D-methionine ( 3 H-D-Met) and [2,3- 3 H]-D-Alanine ( 3 H-D-Ala) were used respectively.

[0038] (Comparison of accumulation of radiolabeled compounds in each growth phase) The strain derived from E. coli K-12 pre-cultured by the above method was inoculated with 400 μL of the bacterial solution into a 50 mL conical tube containing 10 mL of amino acid-free DMEM medium, and shake-cultured (main culture) at 37 °C and 160 rpm. Two hours after the start of culture (lag phase in the growth of the E. coli K-12-derived strain), six hours after (logarithmic growth phase), and twelve hours after (stationary phase), 1000 μL of the bacterial solution was aliquoted into 1.5 mL Eppendorf tubes. After pre-incubation at 37 °C for 10 min, 37 kBq of the radiolabeled compound or 7.4 kBq of the radiolabeled amino acid was administered to each tube, and it was taken up for 5 minutes while gently shaking in a water bath at 37 °C. Then, centrifugation was performed at 7000 G and 4 °C for 6 minutes, the supernatant was removed, and when the pellet was loosened, 1 mL of PBS was added, and the centrifugation operation was performed twice for washing. After the washing was completed, the supernatant was removed, the pellet was loosened, 1 mL of 0.1 M NaOH was added to lyse the E. coli, and then 500 μL was aliquoted therefrom, and the radioactivity was measured with a gamma counter. At each culture time, with the radioactivity administered to E. coli bacteria taken as 100%, the uptake rate (%ID) into strains derived from E. coli K-12 was evaluated.

[0039] (Results) The results of the radioactivity measurement are shown in Tables 1 and 2. From the results of Tables 1 and 2, changes were observed in the accumulation of each radiolabeled compound, and the accumulation tendency differed depending on the type of each radiolabeled compound. More specifically, GSA ( 99m Tc-GSA), PMT ( 99m Tc-PMT), fumaric acid ( 99m Tc-fumaric acid), BMIPP ( 123 I-BMIPP), and FDG ( 18 F-FDG) showed uptake equal to or greater than that of amino acids at each growth stage of the strain derived from E. coli K-12, and even greater uptake was observed during the induction period of the bacterial growth phase. Other radiolabeled compounds did not accumulate much in bacteria. GSA showed a stable accumulation amount at each growth stage, but showed the maximum accumulation amount during the logarithmic growth phase (6 hours after the start of culture) when the growth activity of the strain derived from E. coli K-12 was most active. Fumaric acid showed the maximum accumulation during the induction period (2 hours after the start of culture), which is the preparatory stage of the growth of the strain derived from E. coli K-12, and then showed a decreasing tendency. BMIPP and FDG showed a tendency for the accumulation amount to increase as they progressed to the induction period, logarithmic growth phase, and stationary phase accumulation amounts, and showed the maximum accumulation during the stationary phase (12 hours after the start of culture) when the growth activity of the strain derived from E. coli K-12 decreased and the number of dead bacteria began to increase. Also, by comparing the accumulation amounts of each radiolabeled compound in the strain derived from E. coli K-12, it was found that fumaric acid in the induction period, fumaric acid and BMIPP in the logarithmic growth phase, and BMIPP and FDG in the stationary phase had an uptake rate (%ID) of 10% or more and accumulated particularly much.

[0040] [Table 1]

[0041] [Table 2] ※Experimental method different from other agents.

[0042] According to this example, it was clarified that the clinically used radiolabeled compound exhibits high accumulation correlated with the growth process of Escherichia coli and pathogenic microorganisms of infectious diseases, and thus it can be used as an early imaging diagnostic method for infectious diseases. [Example]

[0043] [Evaluation of Uptake of Radioisotope-Labeled Amino Acids into Bacterial Cells at Each Growth Stage] For bacteria at each growth stage of the induction phase, logarithmic growth phase, and stationary phase 125 I-L-MIT as well as 125 I-D-MIT, the degree of uptake (accumulation) was evaluated.

[0044] [Bacterial Strains Used and Preculture Conditions] The model bacteria, the medium for preculture, and the preculture conditions are the same as in Example 1.

[0045] [Radioactive Labeled Amino Acids Used] For the 125 I-labeling of L-Tyr and D-Tyr, L-Tyr (Nacalai tesque) and D-Tyr (Nacalai tesque) were selected as labeling raw materials, and all other reagents were of special grade. For the 125 I-labeling reaction, a direct labeling method using chloramine-T as an oxidizing agent was carried out under carrier-free conditions. Then, separation and purification were performed using high-performance liquid chromatography (HPLC, Hitachi), and 125 I-L-MIT as well as 125 I-D-MIT were obtained.

[0046] [Labeling Method] The labeled raw materials L / D-Tyr and chloramine-T (Nacalai tesque) were each dissolved in 0.4 M and 0.05 M phosphate buffer (pH 6.2), and adjusted to 1×10 -7 mol / 100 μL and 1×10 -8 mol / 25 μL, respectively. 100 μL of the L / D-Tyr solution was added to carrier-free 125 I-NaI (5 MBq, PerkinElmer), and then 25 μL of the chloramine-T solution was added to initiate the reaction. 15 minutes after the start of the reaction, 25 μL of a solution obtained by diluting a saturated solution of sodium pyrosulfite (Wako Pure Chemical Industries) 10-fold with 0.05 M phosphate buffer (pH 6.2) was added to stop the reaction. The labeled 125 I-L-MIT and 125 I-D-MIT were analyzed by silica gel thin layer chromatography (TLC, Merck), and the labeling rate was evaluated using an auto well γ-counter (AccuFLEXγ7000, Aloka). In addition, methanol (≧99.5%, Sigma-Aldrich): acetic acid (Nacalai tesque) = 100:1 was used as the developing solvent for TLC.

[0047] (Separation and purification method) 125 I-L-MIT and 125 I-D-MIT were separated and purified using high-performance liquid chromatography (HPLC, Hitachi). The chromatogram was analyzed by PowerChrom (eDAQ). In addition, a normal phase column was hydrophilic interaction chromatography (HILIC, Cosmosil, Nacalaitesque). After separation and purification, nitrogen reflux was performed, and the collected 125 I-L-MIT and 125Acetonitrile (≥ 99.5%, Sigma-Aldrich) contained in I-D-MIT was removed.

[0048] (Comparison of accumulation of radiolabeled compounds in each growth phase) For the pre-cultured bacterial strains, main culture and radioactivity measurement were performed in the same manner as in Example 1. At each culture time, taking the radioactivity administered to E. coli bacteria as 100%, the uptake rate (%ID) into the strain derived from E. coli K-12 was evaluated.

[0049] (Results) The results of radioactivity measurement are shown in Table 3. From the results in Table 3, the radioiodine-labeled D-MIT is an artificial amino acid that can be labeled with the medical radionuclide 123 I and can be applied as an imaging diagnostic agent. It is a radioiodinated form of the nutrient L-tyrosine 125 No accumulation was observed in I-L-MIT, whereas the optical isomer 125 I-D-MIT, which is not originally necessary for life activities, showed significant accumulation.

[0050] [Table 3] [Examples]

[0051] [Evaluation using the strain derived from E. coli EC-14, a model of pathogenic bacteria] It was evaluated which types of amino acids and to what extent (accumulate) were taken up by bacteria in each growth phase of the induction phase, logarithmic growth phase, and stationary phase.

[0052] (Bacterial strains used and pre-culture conditions) In this example, the strain derived from E. coli EC-14, which is a model of pathogenic bacteria, was used as the model bacterium. As the medium for pre-culture, THY medium prepared by adding 0.2% Yeast extract (Becton, Dickinson and Company) to Todd’s Hewitt Broth (THB medium; Becton, Dickinson and Company) was used. The pre-pre-culture conditions were set at 37 °C and 5% CO2 for 9 hours or more, and the pre-culture conditions were set at 37 °C and 5% CO2 for 14 hours or more.

[0053] (Radioactive labeled compounds and amino acids used) As radiopharmaceuticals 99m Tc-GSA, 99m Tc-PMT, 99m Tc-fuccinic acid, 99m Tc-PYP, 99m Tc-DTPA, 99m Tc-ECD, 99m Tc-HSA-D, 99m Tc-TcO4 - , 99m Tc-MDP, 99m Tc-TF, 99m Tc-MIBI, 123 I-BMIPP, 123 I-MIBG, 18 F-FDG (concentration 4.5), 18 F-FDG (concentration 1.0), 18 F-FDG (concentration 0.1), 123 I-IMP, 67 Ga-citrate, 111 In-pentetoreotide and 201 TlCl were used. As natural amino acids, L-methionine and L-alanine were selected, and as artificial amino acids, D-methionine and D-alanine were selected. As labeled amino acids, [methyl- 3 H]-L-methionine ( 3 H-L-Met), [2,3- 3 H]-L-alanine ( 3 H-L-Ala), [metyl- 3 H]-D-methionine ( 3H-D-Met) and [2,3- 3 H]-D-Alanine( 3 H-D-Ala) were used respectively.

[0054] Also, for the 125 I-labeling, L-Tyr (Nacalai tesque) and D-Tyr (Nacalai tesque) were selected as the labeling raw materials, and all other reagents were of special grade. For the 125 I-labeling reaction, a direct labeling method using chloramine-T as the oxidant was carried out under carrier-free conditions. Then, separation and purification were performed using high-performance liquid chromatography (HPLC, Hitachi), and 125 I-L-MIT as well as 125 I-D-MIT were obtained.

[0055] (Labeling method) The labeling raw materials L / D-Tyr and chloramine-T (Nacalai tesque) were dissolved in 0.4 M and 0.05 M phosphate buffer (pH 6.2) respectively, and adjusted to 1×10 -7 mol / 100 μL and 1×10 -8 mol / 25 μL respectively. To carrier-free 125 I-NaI (5 MBq, PerkinElmer), 100 μL of the L / D-Tyr solution was added, then 25 μL of the chloramine-T solution was added to initiate the reaction. After 15 minutes from the start of the reaction, 25 μL of a solution obtained by diluting a saturated solution of sodium pyrosulfite (Wako Pure Chemical Industries) 10-fold with 0.05 M phosphate buffer (pH 6.2) was added to stop the reaction. The labeled 125 I-L-MIT and 125The analysis of I-D-MIT was performed by silica gel thin layer chromatography (TLC, Merck), and the labeling rate was evaluated using an auto well γ-counter (AccuFLEXγ7000, Aloka). In addition, methanol (≥99.5%, Sigma-Aldrich): acetic acid (Nacalai tesque) = 100:1 was used as the developing solvent for TLC.

[0056] (Separation and purification method) 125 I-L-MIT and 125 High-performance liquid chromatography (HPLC, Hitachi) was used for the separation and purification of I-L-MIT and I-D-MIT. The chromatogram was analyzed by PowerChrom (eDAQ). In addition, a normal phase column was used for hydrophilic interaction chromatography (HILIC, Cosmosil, Nacalaitesque). After separation and purification, nitrogen reflux was performed to remove the acetonitrile (≥99.5%, Sigma-Aldrich) contained in the collected I-L-MIT and I-D-MIT. 125 I-L-MIT and 125 I-D-MIT.

[0057] (Comparison of accumulation of radiolabeled compounds in each growth phase) The pre-cultured strain derived from E. coli EC-14 was inoculated with 400 μL of the bacterial solution into a 50 mL conical tube containing 20 mL of amino acid-free DMEM medium and cultured with shaking (main culture) at 37°C and 160 rpm. One hour after the start of cultivation (lag phase in the growth of the strain derived from E. coli EC-14), three hours after (logarithmic growth phase), and six hours after (stationary phase), 1000 μL of the bacterial solution was aliquoted into 1.5 mL Eppendorf tubes. 37 kBq of the radiolabeled compound or 7.4 kBq of the radiolabeled amino acid was administered to each tube, and it was allowed to be incorporated for 5 minutes while gently shaking in a water bath at 37 °C. Then, it was centrifuged at 7000 G for 10 minutes at 4 °C, the supernatant was removed, and when the pellet was loosened, 1 mL of PBS was added and centrifuged again. This operation was performed twice for washing. After the washing was completed, the supernatant was removed, the pellet was loosened, 1 mL of 0.1 M NaOH was added to lyse E. coli, and then 500 μL was aliquoted therefrom, and the radioactivity was measured with a gamma counter. At each cultivation time, taking the radioactivity administered to E. coli bacteria as 100%, the uptake rate (%ID) into the strain derived from E. coli EC-14 was evaluated.

[0058] (Results) The results of the radioactivity measurement are shown in Tables 4 and 5. From the results of Tables 4 and 5, changes were observed in the accumulation of each radiolabeled compound, and the accumulation tendency differed depending on the type of each radiolabeled compound. More specifically, GSA ( 99m Tc-GSA), PMT ( 99m Tc-PMT), HSA-D ( 99m Tc-HSA-D), phytic acid ( 99m Tc-phytic acid), BMIPP ( 123 I-BMIPP), and FDG ( 18 F-FDG) were not equivalent to amino acids in each growth phase of the strain derived from E. coli EC-14, but a lot of uptake of each radiolabeled compound was observed. Other radiolabeled compounds did not accumulate much in the bacteria. Fuchinic acid and HSA-D showed the maximum accumulation at the logarithmic growth phase (3 hours after the start of culture), where the growth activity of the strain derived from E. coli EC-14 was most active. GSA and PMT showed the maximum accumulation at the induction phase (1 hour after the start of culture), which is the preparatory stage for the growth of the strain derived from E. coli EC-14, and the accumulation once decreased during the logarithmic growth phase and tended to increase during the stationary phase (6 hours after the start of culture). BMIPP and FDG tended to reach a steady state in terms of the accumulation amount during the logarithmic growth phase and the stationary phase. Also, by comparing the accumulation amounts of each radiolabeled compound in the strain derived from E. coli EC-14, it was found that the uptake rate (%ID) of PMT was 3% or more during the induction phase, and the uptake rates (%ID) of BMIPP and FDG were 5% or more during the logarithmic growth phase and the stationary phase, indicating particularly high accumulation. On the other hand, radioiodine-labeled D-MIT 123 is an artificial amino acid that can be labeled with the medical radionuclide 125 I and can be applied as an imaging diagnostic agent. 125 No accumulation was observed with

[0059]

Table 4

[0060]

Table 5

Example

[0061] [Evaluation with Pseudomonas aeruginosa and Staphylococcus aureus] It was evaluated which types of amino acids and to what extent bacteria take up (accumulate) during each growth phase of the induction phase, logarithmic growth phase, and stationary phase.

[0062] (Bacterial species used and preculture conditions) In this example, Pseudomonas aeruginosa SR-24 strain and Staphylococcus aureus SR-3637 strain, which are pathogenic clinical isolates, were used. As the medium for preculture, THY medium obtained by adding 0.2% Yeast extract (Becton, Dickinson and Company) to Todd’s Hewitt Broth (THB medium; Becton, Dickinson and Company) was used. The pre-preculture conditions for Pseudomonas aeruginosa SR-24 strain were set to 9 hours or more under the conditions of 37 °C and 5% CO2, and the preculture conditions were set to 14 hours or more under the conditions of 37 °C and 5% CO2. In addition, the preculture conditions for Staphylococcus aureus SR-3637 strain were set to 14 hours or more under the conditions of 37 °C and 5% CO2.

[0063] (Radioactive labeled compound or radioactive labeled amino acid used) Both Escherichia coli K-12 strain and EC-14 strain showed high accumulation as radiopharmaceuticals. 123 I-BMIPP was used. In addition, for the 125 I labeling of L-Tyr and D-Tyr, L-Tyr (Nacalai tesque) and D-Tyr (Nacalai tesque) were selected as the labeling raw materials, and all other reagents were of special grade. The 125 I labeling reaction was carried out under carrier-free conditions by a direct labeling method using chloramine-T as an oxidizing agent. Then, separation and purification were performed using high-performance liquid chromatography (HPLC, Hitachi), and 125 I-L-MIT and 125 I-D-MIT were obtained.

[0064] (Comparison of accumulation of radioactive labeled compound or radioactive labeled amino acid in each growth phase) The Pseudomonas aeruginosa SR-24 strain and Staphylococcus aureus SR-3637 strain pre-cultured by the above method were each inoculated with 800 μL (P. aeruginosa SR-24 strain) and 1200 μL (S. aureus SR-3637 strain) of the bacterial solution into 50 mL conical tubes containing 20 mL of amino acid-free DMEM medium, and cultured with shaking (main culture) at 37 °C and 160 rpm. For the P. aeruginosa SR-24 strain, 1000 μL of the bacterial solution was aliquoted into 1.5 mL Eppendorf tubes at 1 hour after the start of culture (induction period in the growth of the P. aeruginosa SR-24 strain-derived strain), 3 hours after (logarithmic growth phase), and 6 hours after (stationary phase). For the S. aureus SR-3637 strain, 1000 μL of the bacterial solution was aliquoted into 1.5 mL Eppendorf tubes at 2 hours after the start of culture (induction period in the growth of the S. aureus SR-3637 strain-derived strain), 6 hours after (logarithmic growth phase), and 12 hours after (stationary phase). A radioactive labeled compound at 37 kBq or a radioactive labeled amino acid at 7.4 kBq was administered to each tube, and taken up for 5 minutes while gently shaking in a water bath at 37 °C. Then, centrifuged at 7000 G and 4 °C for 10 minutes, the supernatant was removed, and when the pellet was loosened, 1 mL of PBS was added, and the centrifugation operation was performed twice for washing. After completion of washing, the supernatant was removed, the pellet was loosened, 1 mL of 0.1 M NaOH was added to lyse the bacteria, and then 500 μL was aliquoted therefrom, and the radioactivity was measured with a gamma counter. At each culture time, with the radioactivity administered to the bacteria taken as 100%, the uptake rate (%ID) into the S. aureus SR-3637 strain and P. aeruginosa SR-24 strain was evaluated.

[0065] (Results) As a result of the radioactivity measurement, the accumulation rates in Pseudomonas aeruginosa SR-24 strain are shown in Table 6, and the accumulation rates in Staphylococcus aureus SR-3637 strain are shown in Table 7. From the results in Table 6 and Table 7, high accumulation of BMIPP ( 123 I-BMIPP) was observed in each of the pathogenic bacteria, Pseudomonas aeruginosa SR-24 strain and Staphylococcus aureus SR-3637 strain, and the accumulation rates were much higher than those in Escherichia coli K-12 strain and EC-14 strain. A radiolabeled compound that significantly accumulates in multiple pathogenic bacteria other than Escherichia coli was confirmed. On the other hand, radioiodinated D-MIT, an artificial amino acid that can be labeled with the medical radionuclide 123 I and can be applied as an imaging diagnostic agent, showed accumulation in Escherichia coli K-12 strain and EC-14 strain as shown in Examples 1 and 2, but no significant accumulation was observed in Pseudomonas aeruginosa SR-24 strain and Staphylococcus aureus SR-3637 strain. Thus, a radiolabeled compound that accumulates in specific bacterial species or strains but does not show accumulation in other bacterial species or strains can be used for differential diagnosis of the specific pathogenic bacteria.

[0066]

Table 6

[0067]

Table 7

[0068] This example clarified that the clinical radiolabeled compound shows high accumulation correlated with the growth process of Escherichia coli and pathogenic microorganisms of infectious diseases, and thus it can be used as an early imaging diagnostic method for infectious diseases.

Industrial Applicability

[0069] Early diagnosis of infectious diseases is very important for preventing the aggravation caused by infectious diseases. Since the clinical radiodiagnostic agent discovered this time is a radiolabeled compound that has already been used in the imaging diagnosis of pathological conditions such as cancer in clinical examinations, it can be immediately clinically available as an imaging diagnostic agent for infectious diseases and is expected to lead to global market expansion.

Claims

1. A radiodiagnostic agent for Escherichia coli infections, containing 99mTc-fuccinic acid.

2. A method of providing, as an indicator for diagnosing Escherichia coli infections, a determination result obtained from the following steps: (1) A step of administering a radiodiagnostic agent for Escherichia coli infections, containing 99mTc-fuccinic acid; (2) A step of detecting the administered radiodiagnostic agent; and (3) A step of determining the presence, absence or progression of Escherichia coli infections from the detection result.

3. A kit for diagnosing Escherichia coli infections, containing the radiodiagnostic agent according to Claim 1.

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