Radioactive positron-emitting nuclide carbon-11 labeled compound and radioactive composition

Abstract

The present invention addresses the problem of providing a compound that can act as a tracer to detect lymphocyte infiltration into myocardial tissue using positron emission tomography (PET) or the like. The problem is solved by a radioactive positron-emitting nuclide carbon-11 labeled compound ([11C] labeled compound) represented by general formula (I), or a pharmaceutically acceptable salt thereof. (In the formula, R1 is a chlorine atom. m is an integer of 1-3. At least one of R2, R3, R4, R5, R6, or R7 is a linear or branched C1-6 alkyl group, the alkyl group has radioactive positron-emitting nuclide carbon-11 [11C], and the rest are hydrogen atoms. R8 is a hydrogen atom or a linear or branched C1-6 alkyl group.)

Description

Radioactive positron-emitting nuclide carbon-11 labeled compounds and radioactive compositions

[0001] Disclosed herein are radioactive positron-emitting nuclide carbon-11 labeled compounds, or pharmaceutically acceptable salts thereof, radioactive compositions.

[0002] Myocarditis is an inflammatory disease primarily affecting the myocardium. It accounts for approximately 10% of sudden deaths, and approximately 30% of myocardial cases progress to chronic myocarditis (dilated cardiomyopathy). The mortality rate for myocarditis is 22%, with fulminant myocarditis reaching 43%. According to Non-Patent Document 1, myocarditis is classified into lymphocytic, giant cell, eosinophilic, and granulomatous myocarditis based on histological characteristics. Lymphocytic myocarditis is often caused by viral infections such as coxsackievirus and adenovirus. Giant cell, eosinophilic, and granulomatous myocarditis are often associated with cardiotoxic substances, drugs, autoimmune diseases, and systemic diseases. Drugs such as anthracyclines, fluorouracil, immune checkpoint inhibitors, and COVID-19 vaccines have been reported to cause myocarditis. In particular, with regard to immune checkpoint inhibitors, myocarditis has been reported in 0.5% of patients receiving anti-PD-1 antibodies alone, 2.4% of patients receiving anti-PD-L1 antibodies alone, and 3.3% of patients receiving anti-CTLA4 antibodies alone. T-lymphocyte-dominant tissue infiltration has been observed in lesions of myocarditis thought to be caused by immune checkpoint inhibitors. Furthermore, when myocarditis develops due to immune checkpoint inhibitor administration, the combined mortality rate from heart failure and cardiogenic causes reaches 46% (Non-Patent Document 2).

[0003] Kociol. RD et al: Circulation. 2020;141Mahmood, SS: J Am Coll Cardiol 2018;71:1755-64

[0004] In particular, fulminant myocarditis progresses rapidly, causing hemodynamic collapse, and if acute myocardial damage is severe, cardiac function may not recover even after inflammation has subsided, leading to a progression to dilated cardiomyopathy and a worsening prognosis. Therefore, it is advisable to diagnose myocarditis promptly and begin treatment with this drug as soon as possible.

[0005] To diagnose myocarditis and select treatment options, myocardial tissue must be examined by myocardial biopsy pathology. However, myocardial biopsy is highly invasive and may result in complications such as cardiac tamponade. Furthermore, because the biopsy site is limited to the right ventricular septum, there is a high possibility of false-negative results.

[0006] An object of the present invention is to provide a compound that can be used as a tracer that can detect lymphocyte infiltration into myocardial tissue by positron emission tomography (PET) or the like.

[0007] The present invention may include the following embodiments: Item 1. A radioactive positron-emitting nuclide carbon-11 labeled compound represented by the following general formula (I) ([ １１ C] labeled compound), or a pharmaceutically acceptable salt thereof: (In the formula, R １ is a chlorine atom. m is an integer of 1 to 3. R ２ , R ３ , R ４ , R ５ , R ６ , and R ７ At least one of the alkyl groups is a linear or branched alkyl group having 1 to 6 carbon atoms, and the alkyl group is a radioactive positron-emitting nuclide, carbon-11[ １１ C], and the rest are hydrogen atoms. ８ is a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms.) Item 2. The above [ １１ Item 3. The method according to Item 1, wherein the labeled compound is represented by the following general formula (II): １１ C-labeled compound, or a pharmaceutically acceptable salt thereof: (In the formula, R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , and R ８ The same as above.) Item 3. The above [ １１ Item 3. The method according to Item 1, wherein the labeled compound is represented by the following formula (1) or (2): １１ C-labeled compound, or a pharmaceutically acceptable salt thereof: : Item 4. The method according to item 1, １１ Item 5. A radioactive composition comprising a [C]-labeled compound, or a pharmaceutically acceptable salt thereof. Item 5. The radioactive composition according to Item 4, which is used to detect a lymphocytic inflammatory disease. Item 6. The radioactive composition according to Item 5, wherein the lymphocytic inflammatory disease is myocarditis or myelitis (however, autoimmune myocarditis caused by myosin as an antigen is excluded from myocarditis). Item 7. The radioactive composition according to Item 5, wherein the myocarditis develops in a manner dependent on the binding of Vcam-1 to VLA-4. Item 8. The radioactive composition according to Item 5, wherein the myocarditis is caused by an immune checkpoint inhibitor. Item 9. The radioactive composition according to Item 4, which is used to assess the therapeutic effect of myocarditis. Item 10. The radioactive composition according to Item 4, which is used to predict the therapeutic effect of myocarditis. Item 11. Item 5. The radioactive composition according to Item 4, which is used to predict the therapeutic effect of a VLA-4 inhibitor on patients with myocarditis or patients suspected of having myocarditis, and to determine whether to administer said treatment to patients for whom said treatment is determined to be effective.

[0008] It is possible to provide a compound that can serve as a tracer that enables lymphocyte infiltration into myocardial tissue to be detected by positron emission tomography (PET) or the like.

[0009] [ 11 The production scheme of [C]HCA2969 is shown. 11 The chromatogram of the prepared [C]HCA2969 is shown. 11[C] Stability of HCA2969 over time is shown. Macroscopic images of the heart on days 12, 19, and 25, as well as low- and high-magnification images of hematopoietic stained tissue are shown. Results of single-cell analysis of cell populations within myocardial tissue are shown. Vcam-1 expression in CTLs on days 8, 14, 21, and 25 is shown. (A) Schedule of adhesion experiments is shown. (B) Western blotting results for Vcam-1 expression are shown. (C) TNFα-dependent adhesion between cardiac fibroblasts and monocytes is shown. (A) Schedule of experiments is shown. (B) The effect of siVcam-1 on Vcam-1 gene expression is shown. (C) Adhesion between cardiac fibroblasts and monocytes is shown. (A) Administration schedule for Vcam-1 neutralizing antibody and VLA-4 neutralizing antibody is shown. (B) Left ventricular fractional shortening (% fractional shortening; FS) measured by echocardiography for all mice in the CTL and neutralizing antibody groups. (C) Box plots of left ventricular end-diastolic diameter and left ventricular fractional shortening for the same mice as in (B) on day 28. (D) Kaplan-Meier curves for the CTL and neutralizing antibody-administered groups. (A) Neutralizing antibody administration schedule. (B) Kaplan-Meier curves for each group. (A) Wild-type mice (WT), wild-type mice, and MRL-Pdcd1 mice at 3 weeks of age. - / - The F1 (hetero: MRL-Pdcd1 - ), MRL-Pdcd1 - / - (B) The homo group was divided into a high %FS group (good cardiac function group: n=11) and a low %FS group (poor cardiac function group: n=20). (C) The administration schedule of VLA-4 neutralizing antibodies is shown. Clinical data for the CTL group, VLA-4 group, and normal EF group are shown. Kaplan-Meier curves for the CTL group (symbol a) and VLA-4 group (symbol b) are shown. Kaplan-Meier curves for the CTL group (symbol a), VLA-4 group (symbol b), and normal EF group (symbol c) are shown. MRL-Pdcd1 - / -The Kaplan-Meier curves for each group when a VLA4 inhibitor was administered to MRL-Pdcd1 are shown. In the figure, symbol a indicates the AJM300(+) group, and symbol b indicates the AJM300(-) group. - / - The results of heart rate and left ventricular contractility (%Fractional Shortening) in each group when a VLA4 inhibitor was administered are shown. The soluble Vcam-1 concentrations in the serum of three healthy subjects (CTL), four patients with Covid-related myocarditis (Covid-related), eight patients with lymphocytic myocarditis (Lym), six patients with eosinophilic myocarditis (Eosi), and three patients with giant cell myocarditis (Giant) are shown. The serum sVcam-1 concentrations in patients with lymphocytic myocarditis during the acute and recovery phases are shown. Figure 21 (A) shows the [ 11 C] The radiation dose for each amount of HCA2969 added is shown in Figure 21 (B). 11 The radiation dose per 1 nM of [C]HCA2969 is shown. 11 C]HCA2969 was used in two wild-type mice. 11 PET / CT images taken using [C]HCA2969 in a rat model of autoimmune myocarditis. 11 C] PET / CT images taken using HCA2969 are shown. Schedule of an experiment in which an anti-VLA-4 neutralizing antibody was administered to a rat autoimmune myocarditis model is shown. Schedule of an experiment in which an AJM300 was administered to a rat autoimmune myocarditis model is shown. (A) Left ventricular contractility is shown for the placebo group, the AJM300 administration group, and the neutralizing antibody administration group. (B) Distribution of heart rates is shown for the placebo group, the AJM300 administration group, and the neutralizing antibody administration group. Figure 26 (A) shows the relationship between wild-type mice (WT) and PD1 - / - Mouse (PD1 - / -) shows an example of PET / CT imaging. Figure 26(B) shows a boxplot quantifying cardiac accumulation in each group. Figure 27(A) shows myocardial tissue images of individuals marked with solid circles in the boxplot for the placebo group in Figure 26(B). Figure 27(B) shows myocardial tissue images of individuals marked with dotted circles in the boxplot for the placebo group in Figure 26(B). Figure 28 shows SUV median (Bq / ml) and SUV mean (Bq / ml) for the negative control (Control) group and the EAE model group (EAE). Figure 28(A) shows SUV median (Bq / ml), and Figure 28(B) shows SUV mean (Bq / ml). Figure 29 shows the results of well counter measurements. Figure 30 shows the %ID / g for each individual in the EAE model group and the negative control (CTL) group. Figure 31 shows HE-stained images of the spinal cord for each individual in the EAE model group.

[0010] 1. A radioactive positron-emitting nuclide carbon-11 labeled compound, or a pharmaceutically acceptable salt thereof. In some embodiments, a radioactive positron-emitting nuclide carbon-11 labeled compound (hereinafter, "carbon-11 labeled compound") is used. １１ [C] labeled compound), or a pharmaceutically acceptable salt thereof. １１ C] The labeled compound is a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof: . (In the formula, R １ is a chlorine atom. m is an integer of 1 to 3. R ２ , R ３ , R ４ , R ５ , R ６ , and R ７ At least one of the alkyl groups is a linear or branched alkyl group having 1 to 6 carbon atoms, and the alkyl group is a radioactive positron-emitting nuclide, carbon-11[ １１ C], and the rest are hydrogen atoms. ８ is a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms.

[0011] m is preferably 2.

[0012] R ２ , R ３ , R ４ , R ５, R ６ , R ７ , and R ８ Examples of the linear or branched alkyl group having 1 to 6 carbon atoms in the formula (I) include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, a sec-butyl group, an isobutyl group, a tert-butyl group, an n-pentyl group, a neopentyl group, an isopentyl group, a sec-pentyl group, a tert-pentyl group, an n-hexyl group, a 2-methylpentyl group, a 3-methylpentyl group, a 2,3-dimethylbutyl group, and a 2,2-dimethylbutyl group.

[0013] The linear or branched alkyl group having 1 to 6 carbon atoms is preferably a methyl group, an ethyl group, an n-propyl group, or an isopropyl group, and more preferably a methyl group.

[0014] [ １１ C] is preferably a carbon atom of a methyl group.

[0015] R ２ , R ３ , and R ７ It is preferred that any one, any two, or all of the above are hydrogen atoms.

[0016] R ８ is preferably a hydrogen atom or a methyl group, and more preferably a hydrogen atom. １１ C] As the labeled compound, a compound represented by the following general formula (II) is preferred: (In the formula, R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , and R ８ Same as above.

[0017] [ １１ C] The labeled compound is more preferably a compound represented by the following formula (1) or (2): , .

[0018] [ １１C] Examples of salts of the labeled compound include inorganic acid salts such as hydrochloride, nitrate, hydrobromide, and sulfate; organic carboxylic acid salts such as acetate and trifluoroacetate; sulfonate salts such as methanesulfonate; metal salts such as sodium salt, potassium salt, calcium salt, and magnesium salt; and salts of basic substances such as ammonium salt.

[0019] 2. [ １１ C] Method for producing labeled compound [ １１ C] The labeled compound can be prepared according to the following preparation scheme:

[0020] In the above scheme, R １ , m, R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , and R ８ is the same as in general formula (I).

[0021] (1) For example, a medical cyclotron (CYPRIS HM-18, Sumitomo) was used, and N ２ Targeting gas １４ N(p, α) １１ C nuclear reaction [ １１ C]CO ２ The obtained [ １１ C]CO ２ was introduced into a gas phase methyl iodide synthesis apparatus (C-GPC-100, Sumitomo) and the highly reactive [ １１ C] trifluoromethanesulfonate ([ １１ C]CH ３ OTf) is synthesized.

[0022] (2) [ １１ C]CHOTf is introduced into a reaction vessel containing an acetone solution of compound A dissolved to a concentration of, for example, about 0.2 mg / mL to 1.5 mg / mL and a base such as sodium hydroxide at a final concentration of about 0.003 mol / L to 0.008 mol / L, and the mixture is bubbled. Compound B is obtained by this reaction.

[0023] (3) A base such as sodium hydroxide is added to the bubbling solution obtained in (2) so that the final concentration is about 0.01 mol / L to 0.03 mol / L, and a deprotection reaction is carried out at room temperature (about 20°C to 27°C) for about 10 minutes, yielding Compound C.

[0024] (4) From the solution obtained in (3) after deprotection, [ １１ C] HCA2969 is collected.

[0025] Preferably, １１ C] The labeled compound can be prepared according to the following preparation scheme: .

[0026] In this scheme, compound QD is used as a starting material as a preferred embodiment of compound A in step (2) of the above-mentioned production method, and in step (2), compound B, which is a preferred embodiment of compound B, [ １１ Subsequently, in step (3), a preferred embodiment of compound C, [C]AJM300, is obtained. １１ C]HCA2969 is obtained.

[0027] 3. [ １１ [C] A radioactive composition comprising a labeled compound or a pharmaceutically acceptable salt thereof. １１ The present invention relates to a radioactive composition (hereinafter also simply referred to as "radioactive composition") comprising a C]-labeled compound or a pharmaceutically acceptable salt thereof.

[0028] The radioactive composition may be in a dry state or in a liquid state dissolved in a solvent. Examples of the solvent include ethanol, a mixture of ethanol and water (the ethanol content is greater than 0% and less than 99.5%), and N,N-dimethylformamide. The radioactive composition can be administered at a radiation dose of about 1 MBq / kg to 500 MBq / kg, preferably about 3 MBq / kg to 100 MBq / kg.

[0029] The radioactive composition can be used to detect lymphocytic inflammatory diseases. Lymphocytic inflammatory diseases are preferably those for which a VLA4 inhibitor is effective. More preferably, lymphocytic inflammatory diseases include myocarditis, encephalomyelitis, ulcerative colitis, Crohn's disease, and heart transplant rejection. Even more preferably, lymphocytic inflammatory diseases include myocarditis and encephalomyelitis.

[0030] As used herein, "myocarditis" refers to an inflammatory disease primarily affecting the myocardium. Myocarditis is classified based on the clinical course and includes acute myocarditis and chronic myocarditis. Acute myocarditis also includes fulminant myocarditis and myocarditis other than fulminant myocarditis. Chronic myocarditis includes dilated cardiomyopathy accompanied by ventricular hypertrophy and chronic myocarditis other than dilated cardiomyopathy.

[0031] In addition, in terms of histological classification, myocarditis includes lymphocytic myocarditis, giant cell myocarditis, eosinophilic myocarditis, granulomatous myocarditis, etc. Viral infection has been reported as the etiology of lymphocytic myocarditis. Causative viruses include enterovirus infections, including coxsackievirus (group A or B), echovirus, and poliovirus; hepatitis virus (type A or C); influenza virus (type A or B); respiratory syncytial virus; mumps virus; measles virus; dengue fever virus; yellow fever virus; chikuningia virus; rabies virus; HIV virus; vaccinia virus; varicella-zoster virus; cytomegalovirus; herpes simplex virus; Epstein-Barr virus; measles virus; adenovirus; and parvovirus. Giant cell myocarditis, eosinophilic myocarditis, and giant cell myocarditis have been reported to be caused by bacterial infections. Eosinophilic myocarditis has been reported to be caused by fungal infections. Granulomatous myocarditis is said to often occur in association with infectious diseases such as rickettsial infections, spirochetal infections, protozoan infections, and parasitic infections; ingestion of drugs or chemicals; allergies, autoimmune diseases; Kawasaki disease; sarcoidosis; radiation exposure; and heatstroke. Drugs that may induce myocarditis include anthracyclines, fluorouracil, immune checkpoint inhibitors, and coronavirus vaccines. Immune checkpoint inhibitors include anti-PD-1 antibodies, anti-PD-L1 antibodies, and anti-CTLA4 antibodies.

[0032] Myocarditis preferably does not include autoimmune myocarditis caused by myosin as an antigen, and preferably does not include eosinophilic myocarditis.

[0033] The myocarditis is preferably lymphocytic myocarditis. Also, the myocarditis is preferably myocarditis that develops dependently on the binding of Vcam-1 and VLA-4. More preferably, the myocarditis is caused by an immune checkpoint inhibitor.

[0034] As used herein, VLA-4 is a protein also known as integrin subunit alpha 4 or CD49D, which in humans is expressed from the gene registered with the National Center for Biotechnology Information as Gene ID: 3676. VLA-4 is a ligand for vascular cell adhesion molecule 1 (VCAM1; CD106; INCAM-100).

[0035] The detection of myocarditis by a radioactive composition is carried out by detecting the [ １１ C] is used as a tracer in the body. １１ [C] can be detected by an examination using PET (positron emission tomography) (hereinafter also referred to as "PET examination"). PET examinations can be performed according to known methods. PET examinations may include an examination using only positron emission tomography, a PET-CT examination in which PET is combined with a CT examination, a PET-MRI examination in which PET and MRI are combined, and the like.

[0036] The radioactive composition can be used to determine the effectiveness of treatment for myocarditis. The effectiveness of treatment can be determined by: (1) obtaining a first PET examination image using the radioactive composition on a patient suspected of having myocarditis or a patient with myocarditis at a first time point; (2) treating the patient for myocarditis; (3) obtaining a second PET examination image using the radioactive composition on the patient at a second time point after the first time point during or after the treatment for myocarditis; and (4) determining the [ ] of the heart part in the first PET examination image and the second PET examination image. １１ (5) comparing the accumulation of [C] in the second PET examination image; １１ and determining that the treatment is effective when the accumulation of [C] in the second PET examination image is less than that in the first PET examination image, and / or determining that the treatment is effective when the accumulation of [C] in the second PET examination image is less than that in the first PET examination image. １１ C] is equal to or greater than that in the first PET examination image, the therapeutic effect can be determined to be ineffective.

[0037] In one embodiment, the radioactive composition can be used to predict the therapeutic effect of a VLA-4 inhibitor. For example, a PET scan image using a radioactive composition is obtained for a patient suspected of having myocarditis or a patient with myocarditis, and [ １１ If accumulation of [C] is observed, it can be determined that treatment with a VLA-4 inhibitor is effective for the patient. In addition, a PET scan image using a radioactive composition is obtained for a patient suspected of having myocarditis or a patient with myocarditis, and [C] is detected in the heart. １１ If no accumulation of VLA-4 is observed, it can be determined that treatment with a VLA-4 inhibitor is not effective for the patient.

[0038] Myocarditis is generally diagnosed by collecting myocardial tissue by endomyocardial biopsy and making a definitive diagnosis based on tissue images. Therefore, a patient with myocarditis is intended to be a patient who has been definitively diagnosed with myocarditis. A patient suspected of having myocarditis is intended to be a patient who has not been definitively diagnosed but whose possible myocarditis has been suggested by tests such as an electrocardiogram; pulse; echocardiogram (cross-sectional images; cardiac function evaluations such as left ventricular fractional shortening and left ventricular ejection fraction); blood measurements of CRP, AST, LDH, CK-MB, cardiac troponin (T or I), and sVcam-1; chest X-ray; and cardiac MRI.

[0039] Treatment of myocarditis includes, for example, administration of a VLA-4 inhibitor, discontinuation of use of the drug causing the myocarditis, and the like.

[0040] An example of a VLA-4 inhibitor is an anti-VLA-4 antibody, or a fragment thereof containing the antigen-binding domain.

[0041] As used herein, the term "anti-VLA-4 antibody" is not limited as long as it can specifically bind to the VLA-4 protein and inhibit its functional expression. The antibody may be either a polyclonal antibody or a monoclonal antibody. Both polyclonal and monoclonal antibodies can be appropriately prepared by those skilled in the art using known methods. When the antibody is a monoclonal antibody, it may be a chimeric antibody, a humanized antibody, or a human antibody prepared using known methods. Furthermore, the antibody may be Fab, F(ab), or the like. ２The anti-VLA-4 antibody may be an antibody fragment such as a diabody, scFv, minibody, peptibody, or mimetibody. Preferably, the anti-VLA-4 antibody is a neutralizing antibody. Examples of anti-VLA-4 antibodies include InVivoMAb anti-mouse / human VLA-4 (CD49d) (clone No. PS / 2, Cat No. BE0071, Bio X Cell) and vedolizumab described in Ferreira, EFB et al. (Current Medicinal Chemistry, 2021, 28, 5884-5895). As used herein, the VLA-4 antagonist may be either a type that competitively inhibits VLA-4 (competitive inhibitory type) or a type that non-competitively inhibits VLA-4 (non-competitive inhibitory type). Preferably, it is a non-competitive inhibitory type. The VLA-4 antagonist may be a compound or a peptide.

[0042] Examples of VLA-4 antagonists include carotegrast methyl (Publication No. WO 2016 / 051828); SB683699 / Firategrast, R411 / Valategrast, CT7758 / CDP323, DS-70, and TBC3486 described in Ferreira, EFB et al. (Current Medicinal Chemistry, 2021, 28, 5884-5895); PN-943 and MORF-057 described in Slack, RJ (Nature Reviews Drug Discovery volume 21, pages 60-78 (2022)); and Baiula, M et al. (Front Chem. 2019 Jul. 2019). 9;7:489), BIO1211 and BIO5192; ER464195-01 described in Ohkuro, M et al. (Nat Commun. 2018 May 17;9(1):1982ER464195-01); and E6007, an analog of ER464195-01. PN-943 is a peptide.

[0043] An example of a VLA-4 inhibitor is an RNA molecule that targets VLA-4 mRNA. The RNA molecule is not limited as long as it targets VLA-4 mRNA and inhibits VLA-4 mRNA expression. Examples of the RNA include at least one RNA molecule selected from the group consisting of siRNA, shRNA, miRNA, and antisense RNA, which has the effect of degrading the target mRNA and / or inhibits the translation of the target mRNA. The sequences of these RNA molecules can be appropriately designed by those skilled in the art using known techniques based on the base sequence of the target gene. Furthermore, the RNA molecule may be prepared using known techniques, or commercially available RNA molecules may be obtained and used. For example, an example of a VLA-4 antisense RNA is ATL1102, described in the literature: Limmroth, V. et al. (Neurology 83, November 11, 2014). Examples include RNA molecules or vectors capable of expressing the RNA molecule.

[0044] The vector capable of expressing an RNA molecule targeting VLA-4 mRNA is not particularly limited as long as it is capable of expressing an RNA molecule that inhibits the expression of the VLA-4 protein in an individual's body or cells. Examples include hairpin RNA expression vectors. A hairpin RNA expression vector includes, downstream of a promoter sequence suitable for expressing short-stranded RNA, such as a U6 promoter, at least: a sense strand DNA sequence having the same sequence as the sense strand of the target mRNA (except that uracil in the mRNA is replaced with thymine); a loop sequence that forms a loop structure after transcription; an antisense strand DNA sequence that can bind in whole or in part in a complementary manner to the sense strand DNA sequence; and a terminator sequence. Examples of vectors include plasmid vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, retrovirus vectors, and lentivirus vectors.

[0045] Examples of VLA-4 inhibitors include genome editing systems that target the VLA-4 gene. Genome editing systems that target the VLA-4 gene are not limited, as long as they are systems that can cause recombination within the VLA-4 gene in an individual's body. Examples include the CompoZr Zinc Finger Nuclease (ZFN) system, the TAL effector nuclease (TALEN) system, and the Clustered regularly interspaced short palindromic repeats / CRISPR associated protein 9 (CRISPR / Cas9) system. Preferably, the CRISPR / Cas9 system uses a vector. In a CRISPR / Cas9 system using a vector, the nucleic acid encoding CRISPR and the nucleic acid encoding Cas9 may be on different vectors or may be on a single vector. The promoter for driving CRISPR to function is not particularly limited, but the U6 promoter is preferred. The promoter for driving Cas9 to function is not particularly limited, but a promoter expressed in mammalian cells, such as a cytomegalovirus promoter, is preferred. Commercially available vectors such as the pX330-U6-Chimeric_BB-CBh-hSpCas9 vector can be used as the CRISPR / Cas9 system.

[0046] The sequence that targets the VLA-4 gene (hereinafter also referred to as "VLA-4 gene target sequence") to be incorporated into the CRISPR sequence is not limited as long as it is a sequence that can be incorporated into guide RNA (also referred to as gRNA or crRNA) by the CRISPR / Cas9 system, transcribed, and recombines the VLA-4 gene. Generally, it is said that the VLA-4 gene target sequence can be a sequence of about 20 bases upstream of the 5'-side of the base sequence "NGG" present in the VLA-4 gene. The VLA-4 gene target sequence was identified using the Optimized CRISPR design tool (Massachusetts Institute of Technology, ZhangLab webpage (http: / / crispr.mit.edu / )), E-CRISP (http: / / www.e-crisp.org / E-CRISP / (German Cancer Research Center)), ZiFiT Targeter (http: / / zifit.partners.org / ZiFit / (Zing Finger Consortium)), and Cas9 design (http: / / cas9.cbi.pku.edu.cn (Peking University)), CRISPRdirect (http: / / crispr.dbcls.jp (University of Tokyo)), CRISPR-P (http: / / cbi.hzau.edu.cn / crispr / (Hua Zhong Agricultural University)) and the like. Can be designed using known design tools published.

[0047] The present invention will be described in more detail below with reference to examples, but the present invention should not be construed as being limited to these examples.

[0048] All animal experiments in this example were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and with the approval of the Animal Experimentation Committee of Osaka University.

[0049] 1. [ 11 C]HCA2969 Production 11 [C]HCA2969 was prepared. The synthetic scheme is shown in Figure 1.

[0050] (1) Using a medical cyclotron (CYPRIS HM-18, Sumitomo), N2 gas was used as the target. 14 N(p, α) 11 C nuclear reaction [ 11 C]CO 2 The obtained [ 11 C]CO2 was introduced into a gas-phase methyl iodide synthesis apparatus (C-GPC-100, Sumitomo) and reacted with the highly reactive [ 11 C]trifluoromethanesulfonate ([ 11 C]CH3OTf) was synthesized.

[0051] (2) [ 11 [C]CH3OTf was introduced into a reaction vessel containing 500 μL of a 1 mg / mL HCA2969 precursor (QD) / acetone solution and 3 μL of 1 mol / L NaOH, followed by bubbling.

[0052] (3) 100 μL of 0.1 mol / L NaOH was added to the bubbling solution obtained in (2), and a deprotection reaction was carried out at room temperature for 10 minutes.

[0053] (4) 1 mL of water for injection was added to the deprotected solution obtained in (3), and mixed thoroughly. The entire amount was then injected into HPLC. 11 A representative HPLC chromatogram is shown in Figure 2.

[0054] (5) Dilute the collected solution with water for injection, and apply the entire amount to a solid-phase extraction column (HLB column) [ 11 The HLB column was preconditioned with 10 mL of ethanol and 20 mL of water for injection.

[0055] (6) In (5) [ 11 C] The HLB column loaded with HCA2969 was washed with water for injection to remove impurities, and then ethanol was added to [ 11 C]HCA2969 was collected in a product vial. Physiological saline was added to the product vial so that the final concentration of ethanol was about 10%. 11 C]HCA2969 solution was used as the final formulation.

[0056] (7) A portion of the final formulation was sampled and subjected to quality testing. As a result of the quality testing, the product obtained under irradiation conditions of 25 μA and 30 minutes was 11 Starting from [C]CO2, approximately 2 GBq of [ 11 The radiochemical purity of [C]HCA2969 was confirmed to be stable and high, exceeding 90% immediately after synthesis and even 90 days after synthesis. The results are shown in Figure 3.

[0057] 2. Single-Cell Analysis Using a Rat Model of Autoimmune Myocarditis (1) Rat Model of Autoimmune Myocarditis The rat model of autoimmune myocarditis was established by administering 10 mg / ml of pig cardiac myosin (autopurified from pig hearts) and adjuvant (CFA 10 mg / ml; Chondrex, 7002) to Lewis rats (male, 8 weeks old). The day of pig cardiac myosin administration was designated Day 0. In this model, myocarditis reached its peak on Day 21. Figure 4 shows macroscopic images of the heart on Days 12, 19, and 25, as well as low- and high-magnification images of hematopoietic stained tissue. Infiltration of inflammatory cells into the myocardial tissue began on Day 12, and was more severe on Day 19. By Day 25, inflammation-induced myocardial tissue destruction was observed. Eosinophil infiltration was not observed at any stage of the disease.

[0058] (2) Analysis of cell populations within myocardial tissue by single-cell analysis. Single-cell analysis was performed by dissociating rat hearts with an enzyme (Liberase™ (Roche, 5401119001 / Liberase DH (Roche, 5401054001)) and analyzing the cells using Chromium (10x Genomics). The results are shown in Figure 5. In the figure, "CTL" indicates the control without antigen administration, and "Myocarditis day..." indicates the number of days since administration of Pig Cardiac Myosin. On day 21, the numbers of T cells and neutrophils increased, while the number of vascular endothelial cells relatively decreased. Furthermore, the numbers of monocytes and macrophages significantly increased, while the number of NK cells decreased and no B cells were detected. Eosinophils were also not detected. Furthermore, unlike CTLs, the activated cardiac fibroblast population increased.

[0059] Figure 6 shows the expression of Vcam-1 in each CTL on days 8, 14, 21, and 25. Vcam-1 expression was observed in vascular endothelial cells, but it was also highly expressed in activated cardiac fibroblasts (Postn+ / FAP+).

[0060] 3. In vitro mononuclear cell adhesion experiment using cardiac fibroblasts This experiment was conducted to determine whether stimulation with the inflammatory cytokine TNFα alters Vcam-1 expression and whether TNFα stimulation alters the adhesiveness between cardiac fibroblasts and mononuclear cells.

[0061] (1) Culture and Adhesion Conditions Figure 7(A) shows the schedule for the adhesion experiment. Cardiac fibroblasts were isolated from hearts removed from neonatal rats after cell dispersion with collagenase type 2 (Worthington Biochemical Corporation, LS004176) and cultured in 96-well plates. Splenic mononuclear cells were obtained by removing spleens from wild-type mice, homogenizing them, and then gravity-separating them using Optiprep (Serumwerk Bernburg, 1893). For adhesion experiments, cardiac fibroblasts were cultured in 96-well plates for 48 hours, followed by a TNFα + / - medium change and further culture for 24 hours. Fluorescently stained splenic mononuclear cells (Hoechst 33342 [Dojin Chemical Co., Ltd., 341-07901] or Cytored [Dojin Chemical Co., Ltd., 342-08531]) were added and incubated for 30 minutes at 37°C. After washing six times with PBS(-) to remove non-adherent mononuclear cells, adherent mononuclear cells were counted using an In Cell Analyzer 6000 (GE Healthcare Life Sciences).

[0062] TNFα (Peprotech, 300-01A) was added to the wells to a final concentration of 5 to 15 ng / ml.

[0063] The induction of Vcam-1 by TNFα was confirmed by Western blotting. Anti-Vcam1 antibody EPR5047 (Abcam, ab134047) was used as the primary antibody for Western blotting. HRP-labeled anti-rabbit antibody was used for detection, and Chemiluminescent reagent Chemiluminescent reagent Chemiluminescent reagent Chemiluminescent reagent L (Nacalai tesque, 07880-70) was used as the internal control. Western blotting of GAPDH was performed.

[0064] (2) Results The results of Western blotting are shown in Figure 7(B). Western blotting was performed in duplicate for each sample. Vcam-1 expression was observed even 0 hours after the addition of TNFα. However, expression increased 12 and 24 hours after the addition of TNFα. Figure 7(C) shows images taken with the In Cell Analyzer 6000. As shown in Figure 7(C), stimulation of cardiac fibroblasts with TNFα strengthened their adhesion to monocytes.

[0065] 4. In vitro adhesion experiment of cardiac fibroblasts and monocytes using siRNA To confirm the direct adhesion of Vcam-1 to cardiac fibroblasts and monocytes, cardiac fibroblasts in which V-cam-1 was knocked down using siRNA were used, and adhesion to monocytes was examined.

[0066] (1) Experimental Conditions The experimental schedule is shown in Figure 8(A). The cardiac fibroblast culture and adhesion experiment were carried out in the same manner as in 3.(1) above.

[0067] Vcam-1 siRNA (Silencer™ Select Pre-Designed siRNA, siRNA ID s129593, Thermo Scientific; also referred to as siVcam-1) was transfected into cardiac fibroblasts at a final concentration of 30 nM using Lipofectamine™ RNAiMAX Transfection Reagent (Thermo, 13778075) 2 hours after seeding. Mononuclear cell adhesion experiments were performed 72 hours after seeding. Silencer™ Select Negative Control No. 1 siRNA (Thermo, 4390843) was used as an siRNA control (siCTL). The transfection dose was the same as that of siVcam-1. TNFα was added at 5 ng / ml 46 hours after transfection. Western blotting of Vcam-1 was performed as described in 3.(1) above.

[0068] For detection of mononuclear cells, adherent mononuclear cells were counted using an In Cell Analyzer 6000 (GE Healthcare Life Sciences).

[0069] (2) Results As shown in Figure 8(B), Vcam-1 signaling was detected in cardiac fibroblasts transfected with siCTL, but not in those transfected with siVcam-1. This indicates that siVcam-1 suppresses Vcam-1 expression in cardiac fibroblasts. Using this experimental system, we examined adhesion to monocytes. The results are shown in Figure 8(C). Mononuclear cell adhesion was reduced in siVcam-1-transfected cells. In contrast, mononuclear cell adhesion was observed in siCTL-transfected cells. This indicates that adhesion between cardiac fibroblasts and monocytes is mediated by Vcam-1.

[0070] 5. Verification of the effect of combined use of Vcam-1 and VLA-4 neutralizing antibodies on improving the prognosis of myocarditis. According to the literature report, Int Immunol. 2010 Jun;22(6):443-52, the MRL-Pdcd1- / - mouse model, a spontaneous systemic lupus erythematosus mouse model, was crossed with the PD-1 knockout C57BL / 6-Pdcd1- / - mouse. This mouse model spontaneously develops lymphocytic myocarditis in 96% of cases between 4 and 8 weeks of age. Furthermore, the survival rate at 10 weeks of age was approximately 30%, significantly lower than that of wild-type mice. The following myocarditis experiments were performed using this MRL-Pdcd1- / - mouse model.

[0071] (1) Combined Administration of Vcam-1 and VLA-4 Neutralizing Antibodies. Thirteen MRL-Pdcd1- / - mice were divided into two groups: control (CTL: n = 7) and those receiving combined Vcam-1 and VLA-4 neutralizing antibodies (neutralizing antibody group: n = 7). Starting on day 14 of age, the CTL group received intraperitoneal administration of saline, while the neutralizing antibody group received combined Vcam-1 and VLA-4 neutralizing antibodies. The Vcam-1 neutralizing antibody used was InVivoMAb anti-mouse CD106 (VCAM-1) (clone No. M / K-2.7, Cat No. BE0027, Bio X Cell). The VLA-4 neutralizing antibody used was InVivoMAb anti-mouse / human VLA-4 (CD49d) (clone No. PS / 2, Cat No. BE0071, Bio X Cell). The dose of each compound was 10 μg / g. The administration schedule was as shown in Figure 9 (A), starting from postnatal day 14, every three days until postnatal day 26. Echocardiography was performed on postnatal days 14 and 28. After obtaining the echocardiographic data on postnatal day 28, blood samples were taken and hearts were sampled from all mice.

[0072] (2) Results. Figure 9(B) shows the left ventricular fractional shortening (%FS) measured by echocardiography in all mice in the CTL and neutralizing antibody groups. Ventricular contractility was significantly reduced in the CTL group on day 28 of life compared with day 14 of life. In contrast, the neutralizing antibody-treated group showed a milder reduction in ventricular contractility than the CTL group. Figure 9(C) shows boxplots of the left ventricular end-diastolic diameter [LVDd (mm)] and left ventricular fractional shortening [%FS (%)] on day 28 of life for the mice shown in Figure 9(B). No significant difference in LVDd was observed between the CTL and neutralizing antibody-treated groups (p=0.22). On the other hand, %FS was significantly higher in the neutralizing antibody-treated group (p=0.0103). Figure 9(D) shows Kaplan-Meier curves for the CTL and neutralizing antibody-treated groups. In the figure, symbol a indicates the CTL group, and symbol b indicates the neutralizing antibody group. In an examination up to 28 days after birth, the number of individuals who died increased from the 20th day after birth in the CTL group. On the other hand, no individuals died in the neutralizing antibody-administered group. The difference between the two groups was significant, with p=0.033 in the log-rank analysis. This indicates that combined administration of Vcam-1 and VLA-4 neutralizing antibodies improves the mortality rate due to myocarditis.

[0073] 6. Effect of VLA-4 neutralizing antibody The following study was carried out to determine whether Vcam-1 neutralizing antibody or VLA-4 neutralizing antibody contributes to reducing the mortality rate from myocarditis.

[0074] (1) Antibody administration: 29 MRL-Pdcd1 mice - / - The mice were divided into four groups: a control (CTL: n = 12), a Vcam-1 group (n = 5) receiving only a Vcam-1 neutralizing antibody, a VLA-4 group (n = 6) receiving a VLA-4 neutralizing antibody, and a Vcam-1 / VLA-4 group (n = 6) receiving a combination of a Vcam-1 neutralizing antibody and a VLA-4 neutralizing antibody. The doses of each antibody were the same as those described in 4.(1) above. As shown in Figure 10(A), the administration schedule for the neutralizing antibodies was also the same as that described in 4.(1) above.

[0075] (2) Results Figure 10(B) shows the Kaplan-Meier curves for each group. In the figure, symbol a indicates the CTL group, symbol b indicates the Vcam-1 group, symbol c indicates the Vcam-1 / VLA-4 group, and symbol d indicates the VLA-4 group. In the analysis up to 28 days after birth, the number of individuals who died increased from day 19 after birth in the CTL group (symbol a). The number of individuals who died also increased from day 20 after birth in the Vcam-1 group (symbol b). In contrast, no individuals died in the Vcam-1 / VLA-4 group (symbol c). In the VLA-4 group (symbol d), one individual died on day 18, but the remaining individuals survived until day 28 after birth. These results suggest that VLA-4 neutralizing antibodies contribute to the improved prognosis of myocarditis.

[0076] 7. Effect of VLA-4 neutralizing antibodies on mice with impaired cardiac function Figure 11(A) shows the %FS values ​​of 3-week-old wild-type mice (WT), F1 mice (hetero: MRL-Pdcd1-) crossed with wild-type mice and MRL-Pdcd1- / - mice, and MRL-Pdcd1- / - mice (homo). The %FS value of the WT group was over 75%, with little variation between the first and fourth quartiles. In contrast, the %FS values ​​of the homo and hetero groups were broadly distributed, with some individuals showing high and some showing low values.

[0077] Therefore, as shown in Figure 11(B), the homo group was divided into a high %FS group (good cardiac function group: n = 11) and a low %FS group (decreased cardiac function group: n = 20). The decreased cardiac function group was further divided into a control group (CTL group) that did not receive VLA-4 neutralizing antibodies and a VLA-4-treated group (VLA-4 group) that received VLA-4 neutralizing antibodies to examine the effects of VLA-4 neutralizing antibodies. The cutoff value for the normal EF group and the decreased cardiac function group was set at 70%, with groups above 70% classified as the good cardiac function group and groups below 70% classified as the decreased cardiac function group. VLA-4 neutralizing antibodies were then administered according to the schedule shown in Figure 11(C). Body weight (BW), pulse rate (HR), and %FS values ​​for each group are shown in Figure 12. There were no significant differences in any of the parameters between the CTL and VLA-4 groups.

[0078] Kaplan-Meier curves for the CTL group (symbol a) and the VLA-4 group (symbol b) are shown in Figure 13. Log-rank analysis revealed a p=0.0285 result, indicating that the VLA-4 group had a significantly longer survival rate.

[0079] Figure 14 shows a Kaplan-Meier curve with the survival curve for the normal EF group (symbol c) added. The normal EF group had good cardiac function and was not administered a VLA-4 neutralizing antibody, but the survival rate was significantly lower than that of the VLA-4 group (log-rank analysis, p = 0.0275).

[0080] These results suggest that administering VLA-4 neutralizing antibodies to individuals with myocarditis improves the prognosis of myocarditis, regardless of the cardiac function data of each individual. They also suggest that administering VLA-4 neutralizing antibodies to individuals with myocarditis is effective in preventing or treating myocarditis.

[0081] 8. Effect of VLA4 inhibitor administration The following study was conducted to determine whether administration of AJM300 (carotegrast methyl), a VLA4 inhibitor, contributes to reducing the mortality rate due to myocarditis.

[0082] (1) AJM300 administration Forty-four MRL-Pdcd1- / - mice were divided into two groups: the AJM300(-) group (n = 13) that did not receive AJM300, and the AJM300(+) group (n = 12) that received AJM300. The AJM300(+) group was fed a 1% AJM300 diet from 2 weeks after birth until death.

[0083] In addition, nine mice in the AJM300(-) group and ten mice in the AJM300(+) group underwent echocardiographic evaluation of cardiac function at 28 days of age, and were then euthanized to collect heart and blood samples.

[0084] (2) Results Figure 15 shows the Kaplan-Meier curves for each group. In the figure, symbol a indicates the AJM300(+) group, and symbol b indicates the AJM300(-) group. The AJM300(+) group had a significantly longer survival time than the AJM300(-) group (Log rank analysis, p=0.0022). This indicates that administration of a VLA4 inhibitor contributes to improving the prognosis of myocarditis.

[0085] Figure 16 shows the results of heart rate and left ventricular contractility (%Fractional Shortening) measured by echocardiography. Bradycardia was significantly improved in the AJM300(+) group compared to the AJM300(-) group (t-test p=0.0231). Furthermore, left ventricular contractility was significantly improved in the AJM300(+) group compared to the AJM300(-) group (t-test p=0.0231). These results demonstrate that administration of a VLA4 inhibitor contributes to the improvement of cardiac function.

[0086] 9. Human serum soluble Vcam-1 concentrations. Serum concentrations of soluble Vcam-1 (sVcam-1) were compared between healthy individuals and patients with myocarditis. Results for three healthy individuals (CTL), four patients with Covid-related myocarditis, eight patients with lymphocytic myocarditis (Lym), six patients with eosinophilic myocarditis (Eosi), and three patients with giant cell myocarditis (Giant) are shown in Figure 17. Serum sVcam-1 concentrations were elevated in all types of myocarditis, including lymphocytic myocarditis, compared with healthy individuals. This suggests that the VLA-4 / Vcam-1 pathway is involved in the development of myocarditis.

[0087] 10. Changes in serum sVcam-1 levels during myocarditis pathology We compared serum sVcam-1 levels in three patients with lymphocytic myocarditis during the acute and recovery phases. The results are shown in Figure 18. Serum sVcam-1 levels in the three patients were very high during the acute phase. As can be seen from a comparison with serum sVcam-1 levels in healthy subjects shown in Figure 17, the levels in the three patients improved to healthy levels during the recovery phase. This suggests that the VLA-4 / Vcam-1 pathway is involved in myocarditis.

[0088] 11. [11 C] Evaluation of binding of HCA2969 to VLA-4 in mouse mononuclear cells. 11 C] The binding of HCA2969 to VLA-4 was evaluated.

[0089] (1) Method: 2 × 10 mononuclear cells collected from mouse spleens 6 and the compound synthesized in 1. above. 11 [C]HCA2969 was added to a buffer solution (10 mM Tris pH 7.5, 0.1% BSA, 150 mM NaCl, 2 mM MnCl2, 2 mM D-glucose) at concentrations of 0.025 nM, 0.1 nM, 0.4 nM, 1.6 nM, 6.4 nM, and 25.6 nM, respectively, and mixed. After mixing, the cells were incubated at room temperature for 20 minutes and then washed twice with buffer (10 mM Tris pH 7.5, 0.1% BSA, 150 mM NaCl, 2 mM MnCl2, 2 mM D-glucose). After washing, the radioactivity of the washed cells was measured using a scintillation counter.

[0090] (2) Results Figure 19(A) shows 11 C] The radiation dose for each amount of HCA2969 added is shown in Figure 19(B). 11 C] The radiation dose per 1 nM of HCA2969 is shown. (A) A clear saturation curve was obtained, and (B) Scatchard analysis yielded an equilibrium dissociation constant Kd of 0.16 nM and a Bmax of 81.85 Bq.

[0091] 12. [ 11 Detection of myocarditis using [C]HCA2969 MRL-Pdcd1 prepared in 5 above - / - On days 25-26, the mice were anesthetized with isoflurane inhalation. 11 Approximately 1-10 MBq of [C]HCA2969 tracer was administered via the tail vein, and PET / CT imaging was performed. - / - The results for two mice are shown. In both cases, accumulation of the tracer in the heart was observed. Figure 21 shows the results for two wild-type mice. In both cases, accumulation of the tracer in the heart was not observed.

[0092] 13. [ 11 Detection of autoimmune myocarditis using [C]HCA2969. The rat model of autoimmune myocarditis prepared in 2. above was used in the rats at the peak of myocarditis, 3 weeks after induction, under isoflurane inhalation anesthesia. 11 Approximately 1-10 MBq of [C]HCA2969 tracer was administered via the tail vein, and PET / CT imaging was performed.

[0093] As shown in FIG. 22, no accumulation of the tracer in the heart was observed in the autoimmune myocarditis model rats (right: labeled "myocarditis") as well as in the rats without myocarditis induction (left: labeled "CTL").

[0094] As shown in Figures 23 and 24, the above-mentioned anti-VLA-4 neutralizing antibody or AJM300 administration experiment was conducted on rats with autoimmune myocarditis. The study groups consisted of a placebo group (5 animals), an AJM300-administered group (4 animals), and a neutralizing antibody-administered group (5 animals) administered with an anti-VLA-4 neutralizing antibody. The placebo group was fed a diet containing placebo (0.43% Metolose + 0.14% Acdyzol) from 3 days before induction of myocarditis until 3 weeks after induction. The AJM300-administered group was fed a diet containing 1% AJM300 from 3 days before induction until 3 weeks after induction. The neutralizing antibody-administered groups received 5 mg / kg of neutralizing antibody intravenously at the time of myocarditis induction (0w), 1 week after induction (1w), 2 weeks after induction (2w), and 3 weeks after induction (3w). Left ventricular contractility and heart rate were evaluated 21 days after myocarditis induction. Figure 25(A) shows the left ventricular contractility in the placebo group, AJM300-administered group, and neutralizing antibody-administered group. Figure 25(B) shows the distribution of heart rates in the placebo group, AJM300-administered group, and neutralizing antibody-administered group. As shown in Figure 25, no significant differences in cardiac function were observed among the placebo group, AJM300-administered group, and neutralizing antibody-administered group in the autoimmune myocarditis model rats, and AJM300 and anti-VLA-4 neutralizing antibody showed no effect on improving cardiac function.

[0095] By combining the results of 12. above, 11 [C]HCA2969 tracer was shown to accumulate in the myocardium in a myocarditis model in which the therapeutic effects of AJM300 and anti-VLA-4 neutralizing antibodies are expected.

[0096] 14. [ 11 Next, we evaluated the therapeutic effect of PD1 knockout mice (MRL-Pdcd1 - / - Mouse: PD1 - / - Use the mouse to 11 The therapeutic effect of [C]HCA2969 tracer on myocarditis was evaluated.

[0097] (1) Methods: PD1 expression was measured in 4-week-old wild-type mice, placebo mice, AJM300-treated mice, and anti-VLA-4 neutralizing antibody-treated mice. - / - Mice were prepared in groups. The placebo group was fed normal chow, while the AJM300-treated group was fed chow mixed with 1% AJM300 from day 14 to day 28 after the start of breeding. The anti-VLA-4 neutralizing antibody-treated group was fed normal chow, and anti-VLA-4 neutralizing antibody (10 mg / kg) was administered intraperitoneally on days 14 and 21 after breeding.

[0098] On the 28th day after the start of breeding, each mouse was anesthetized with isoflurane (1-3% / 2L O2) and 11 [C]HCA2969 tracer (1–10 MBq: 10 μg of unlabeled HCA2969 contains 10 MBq of PET tracer) was injected via the tail vein. 20 min after injection, 10 min of PET / CT imaging was performed.

[0099] (2) Results The results are shown in Figures 26 and 27. Figure 26(A) shows the results of wild-type mice (WT) and PD1 - / - Mouse (PD1 - / - ) PET / CT imaging example of PD1 - / - In mice, the heart 11 Figure 26(B) shows box plots quantifying the accumulation of the cardiac tracer in each group.

[0100] PD1 - / - In the placebo group of mice, [ 11 Accumulation of the [C]HCA2969 tracer was observed. However, accumulation was lower in the AJM300 and anti-VLA-4 neutralizing antibody groups compared to the placebo group.

[0101] From this result, [ 11It was considered that the [C]HCA2969 tracer was able to assess the therapeutic effects of AJM300 and anti-VLA-4 neutralizing antibody.

[0102] Figure 27(A) shows the myocardial tissue image of the individual marked with a solid circle in the box plot of the placebo group in Figure 26(B). 11 C]HCA2969 tracer accumulation was particularly high. Figure 27(B) shows the myocardial tissue image of the individual marked with a dotted circle in the box plot of the placebo group in Figure 26(B). This individual [ 11 C] This is an individual with particularly low accumulation of HCA2969 tracer. In Figure 27(A), mononuclear cell infiltration was observed in the tissue, and there was strong inflammation in the myocardium. On the other hand, in Figure 27(B), no signs of inflammation were observed. From these results, [ 11 The [C]HCA2969 tracer appeared to accumulate specifically in myocardial inflammation.

[0103] 15. [ 11 Diagnosis of VLA-4 positive lymphocyte-associated diseases other than myocarditis using the [C]HCA2969 tracer. Using the Experimental Autoimmune Encephalitis (EAE) model (mouse encephalomyelitis model: EAE mice), we investigated the efficacy of the [C]HCA2969 tracer in the diagnosis of VLA-4 positive lymphocyte-associated diseases other than myocarditis. 11 The availability of [C]HCA2969 was evaluated.

[0104] 15-1. 11Observation of accumulation of [C]HCA2969 tracer signal in the affected area. (1) Generation of EAE mice (immunization). Complete Freund's Adjuvant (CFA) was prepared by suspending 40 mg of H37RA Mycobacterium tuberculosis toxin (BD) in 10 ml of incomplete Freund's Adjuvant (BD). MOG peptide (MOG 35-55, Biosynthesis) was dissolved in saline to a concentration of 1 mg / ml. Nine-week-old male C57BL / 6J mice were mixed and suspended in a glass syringe with 100 μl of CFA and 100 μl of MOG solution per mouse. The resulting cloudy emulsion was injected subcutaneously into both groins (200 μl per mouse) (immunization day 0). Subsequently, 100 μl of pertussis toxin (LBL) dissolved in PBS (4 μg / ml) was intraperitoneally administered to each mouse on the day of emulsion administration and again 48 hours later. EAE disease scores were recorded from day 0 of immunization.

[0105] In this model, inflammatory cell infiltration into the spinal cord and paralysis symptoms appear in approximately one week, peak at two weeks, and improve after four weeks. Inflammation is predominant in the spinal cord rather than the brain, and because inflammation occurs from the lumbar spinal cord side, paralysis develops in the tail and hind limbs.

[0106] In the following experiments, mice that showed paralytic symptoms of EAE with a disease score of 0.5 or higher on day 15 after immunization were used.

[0107] (2) PET / CT Imaging On the 15th day after immunization, each mouse was anesthetized with isoflurane (1-3% / 2L O2) and 11 [C]HCA2969 tracer (1-10MBq: 10μg of unlabeled HCA2969 contains 10MBq of PET tracer) was injected via the tail vein. 20 minutes after injection, PET / CT imaging was performed for 10 minutes. The region of interest (ROI) was set in the thoracic to lumbar spinal cord, and [ 11The accumulation of [C]HCA2969 tracer signal was counted, and SUV median (Bq / ml) and SUV mean (Bq / ml) were calculated.

[0108] (3) Results Figure 28 shows the SUV median (Bq / ml) and SUV mean (Bq / ml) for the negative control (Control) group and the EAE model group (EAE). Figure 28(A) shows the SUV median (Bq / ml), and Figure 28(B) shows the SUV mean (Bq / ml). Both were higher in the EAE model group. 11 [C]HCA2969 tracer signal accumulation was observed, and therefore, the [ 11 The [C]HCA2969 tracer was shown to be capable of detecting inflammation in tissues other than the heart.

[0109] 15-2. 11 Observation of uptake of [C]HCA2969 tracer into the affected area Using EAE model mice, we observed [C]HCA2969 tracer uptake into the spinal cord and brain. 11 C]HCA2969 tracer uptake was observed.

[0110] (1) Methods For the EAE model prepared in the above 17-1., mice showing paralytic symptoms of EAE with a disease score of 0.5 or more on the 15th day after immunization were treated with the method described in the above 17-1. (2). 11 The mice were then euthanized 30 minutes after administration, and the spinal cord and brain were collected. A portion of the collected tissue was measured for radioactivity using a well counter, and a portion was subjected to HE staining.

[0111] (2) Results Figure 29 shows the results of measurements using the Well Counter. The EAE model group showed a significant increase in tracer signal accumulation in the spinal cord compared to the negative control group (CTL group). Accumulation also tended to increase in the brain. Because inflammatory cell infiltration occurs primarily in the spinal cord in the EAE model, this accumulation was thought to reflect inflammatory cell infiltration.

[0112] Figure 30 shows the %ID / g of each individual in the EAE model group and the negative control group (CTL group). Figure 31 shows HE-stained images of the spinal cord of each individual in the EAE model group. Individuals marked with an asterisk in Figures 30 and 31 have [ 11 C]HCA2969 tracer accumulation was observed in the spinal cord. 11 In EAE1, EAE2, EAE5, and EA6 individuals in which accumulation of the [C]HCA2969 tracer was observed, lymphocyte infiltration was observed in the spinal cord tissue by HE staining.

[0113] From these results, 11 The [C]HCA2969 tracer was shown to be capable of assessing inflammation associated with lymphocyte infiltration not only in the myocardium but also in other tissues.

Claims

1. A radioactive positron-emitting nuclide carbon-11 labeled compound represented by the following general formula (I) ([ １１ C] labeled compound), or a pharma- ceutically acceptable salt thereof: (In the formula, R １ is a chlorine atom. m is an integer from 1 to 3. ２ , R ３ , R ４ , R ５ , R ６ , and R ７ At least one of the alkyl groups is a linear or branched alkyl group having 1 to 6 carbon atoms, and the alkyl group is a radioactive positron-emitting nuclide, carbon-11[ １１ C], and the rest are hydrogen atoms. ８ is a hydrogen atom or a linear or branched alkyl group having 1 to 6 carbon atoms.

2. The above [ １１ The labeled compound according to claim 1, wherein the labeled compound is represented by the following general formula (II): １１ C] A labeled compound, or a pharma- ceutically acceptable salt thereof: (In the formula, R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , and R ８ Same as above.) 3. The above [ １１ The labeled compound according to claim 1, wherein the labeled compound is represented by the following formula (1) or (2): １１ C] A labeled compound, or a pharma- ceutically acceptable salt thereof: ; .

4. The above-mentioned [ １１ C] A radioactive composition comprising a labeled compound, or a pharma- ceutically acceptable salt thereof.

5. The radioactive composition according to claim 4, which is used to detect lymphocytic inflammatory diseases.

6. The radioactive composition according to claim 5, which is a lymphocytic inflammatory disease, myocarditis or myelitis (however, myocarditis caused by myosin as an antigen excludes autoimmune myocarditis).

7. The radioactive composition according to claim 5, wherein the myocarditis develops depending on the binding of Vcam-1 to VLA-4.

8. The radioactive composition according to claim 5, wherein the myocarditis is myocarditis caused by an immune checkpoint inhibitor.

9. The radioactive composition according to claim 4, which is used to assess the therapeutic effect of myocarditis.

10. The radioactive composition according to claim 4, which is used to predict the therapeutic effect of myocarditis.

11. The radioactive composition according to claim 4, which is used to predict the therapeutic effect of a VLA-4 inhibitor on a patient with myocarditis or a patient suspected of having myocarditis, and to determine whether or not to administer said treatment to a patient for whom said treatment is deemed effective.

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