Radiopositron emission nuclide C11 labeled compound and radioactive composition

Abstract

The present invention addresses the problem of providing a compound that can be used as a tracer that can detect the infiltration of lymphocytes into cardiac muscle tissue by means of positron emission tomography (PET) or the like. A compound labeled with radiopositron emission nuclide C11 ([11C]) 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, and R7 is a linear or branched C1-6 alkyl group having a radiopositron emission nuclide C11 [11C], and other than a hydrogen atom, and R1 is a hydrogen atom, and R2 is an integer of 1-3, or a pharmaceutically acceptable salt of the compound labeled with the radiopositron emission nuclide C11 [11C], or a pharmaceutically acceptable salt of the compound labeled with the radiopositron emission nuclide C11 [11C]. And R8 represents a hydrogen atom or a linear or branched alkyl group having 1-6 carbon atoms).

Description

Technical Field

[0001] This specification discloses radioactive positron-emitting carbon-11 labeled compounds, or pharmaceutically acceptable salts thereof, and radioactive compositions thereof. Background Technology

[0002] Myocarditis is an inflammatory disease primarily affecting the myocardium. It accounts for approximately 10% of sudden cardiac deaths, and about 30% of myocarditis cases progress to chronic myocarditis (dilated cardiomyopathy). The mortality rate of myocarditis is 22%, with fulminant myocarditis reaching 43%. According to non-patent literature 1, myocarditis can be classified histologically into lymphocytic myocarditis, giant cell myocarditis, eosinophilic myocarditis, and granulomatous myocarditis. It is claimed that viral infections such as Coxsackievirus and adenovirus are the most common causes of lymphocytic myocarditis. Giant cell myocarditis, eosinophilic myocarditis, and granulomatous myocarditis are reportedly complications of cardiotoxic substances, drugs, autoimmune diseases, and systemic diseases. As for drugs, anthracyclines, fluorouracil, immune checkpoint inhibitors, and coronavirus vaccines have been reported to cause myocarditis. Regarding immune checkpoint inhibitors, it has been reported that myocarditis occurred in 0.5% of cases treated with anti-PD-1 antibody alone, 2.4% of cases treated with anti-PD-L1 antibody alone, and 3.3% of cases treated with anti-CTLA4 alone. In lesions suspected of being caused by immune checkpoint inhibitors, images of tissue infiltration dominated by T lymphocytes were confirmed. Furthermore, in cases of myocarditis induced by immune checkpoint inhibitors, the mortality rate reaches 46% when the mortality from heart failure and cardiac-related causes are combined (Non-Patent Literature 2).

[0003] Existing technical documents

[0004] Non-patent literature

[0005] Non-patent literature 1: Kociol.RD et al: Circulation. 2020; 141

[0006] Non-Patent Literature 2: Mahmood, SS: J Am Coll Cardiol 2018; 71: 1755-64 Summary of the Invention

[0007] The problem the invention aims to solve

[0008] In particular, fulminant myocarditis is characterized by rapid deterioration, hemodynamic abnormalities, and severe acute myocardial damage. Even after inflammation subsides, cardiac function may not recover, leading to progression to dilated cardiomyopathy and a worse prognosis. Therefore, it is crucial to diagnose myocarditis promptly and initiate primary-drug-based therapy as soon as possible.

[0009] For the diagnosis and treatment of myocarditis, myocardial biopsy is necessary to examine the myocardial tissue. However, myocardial biopsy is highly invasive and may cause complications such as cardiac tamponade. Furthermore, the biopsy site is limited to the right ventricular septum, thus increasing the likelihood of false negatives.

[0010] One objective of this invention is to provide a compound that serves as a tracer capable of detecting lymphocyte infiltration into myocardial tissue using methods such as positron emission tomography (PET).

[0011] Solution for solving the problem

[0012] The present invention may include the following embodiments.

[0013] Item 1. A radioactive positron-emitting nuclide carbon-11 labeled compound represented by the following general formula (I) 11 [C] labeled compound) or its pharmaceutically acceptable salt,

[0014]

[0015] In general formula (I), R 1 It is a chlorine atom.

[0016] m is an integer from 1 to 3.

[0017] R 2 R 3 R 4 R 5 R 6 and R 7 At least one of them is a straight-chain or branched alkyl group having 1 to 6 carbon atoms, wherein the aforementioned alkyl group has a radioactive positron-emitting nuclide carbon-11. 11 C], and in addition to that, hydrogen atoms,

[0018] R 8 It is a hydrogen atom, or a straight-chain or branched alkyl group having 1 to 6 carbon atoms.

[0019] Item 2. As described in Item 1 above 11 C-labeled compounds or pharmaceutically acceptable salts thereof, wherein the aforementioned [ 11 C] Labelled compounds are represented by the following general formula (II),

[0020]

[0021] In general formula (II), R 2 R 3 R 4 R5 R 6 R 7 and R 8 Same as above.

[0022] Item 3. As described in Item 1 above 11 C-labeled compounds or pharmaceutically acceptable salts thereof, wherein the aforementioned [ 11 C] The labeled compound is represented by the following formula (1) or (2),

[0023] ;

[0024] .

[0025] Item 4. A radioactive composition comprising the foregoing [described in Item 1]. 11 [C] labeling compounds or their pharmaceutically acceptable salts.

[0026] Item 5. The radioactive composition according to Item 4, used for detecting lymphocytic inflammatory diseases.

[0027] Item 6. The radioactive composition according to Item 5, wherein the aforementioned lymphocytic inflammatory disease is myocarditis or myelitis (wherein, myocarditis does not include autoimmune myocarditis with myosin as an antigen).

[0028] Item 7. The radioactive composition according to Item 5, wherein the aforementioned myocarditis is myocarditis that depends on the binding of Vcam-1 and VLA-4.

[0029] Item 8. The radioactive composition according to Item 5, wherein the aforementioned myocarditis is myocarditis caused by an immune checkpoint blocker.

[0030] Item 9. The radioactive composition according to Item 4, used to determine the therapeutic effect of myocarditis.

[0031] Item 10. The radioactive composition according to Item 4, used for predicting the therapeutic effect of myocarditis.

[0032] Item 11. The radioactive composition according to Item 4, used to determine in patients with myocarditis or suspected myocarditis the following: predicting the therapeutic effect achieved by the VLA-4 blocking substance, and administering the treatment to patients determined to be effective by the treatment.

[0033] The effects of the invention

[0034] A compound that can be provided as a tracer capable of detecting lymphocyte infiltration into myocardial tissue by means of methods such as positron emission tomography (PET). Attached Figure Description

[0035] Figure 1 Showing [ 11 Manufacturing scheme for C]HCA2969.

[0036] Figure 2 Showing the manufactured [ 11 Chromatogram of C]HCA2969.

[0037] Figure 3 Showing the manufactured [ 11 The long-term stability of C]HCA2969.

[0038] Figure 4 Macroscopic images of the heart on days 12, 19, and 25, as well as low- and high-magnification images of hematopoietic staining of the tissues.

[0039] Figure 5 The results of population analysis of cells in myocardial tissue obtained through single-cell analysis are shown.

[0040] Figure 6 The expression of Vcam-1 on CTL, day 8, day 14, day 21, and day 25 is shown.

[0041] Figure 7 (A) Shows the schedule of the adhesion experiment. (B) Shows the results of Western blot analysis on Vcam-1 expression. (C) Shows the adhesion of TNFα-dependent cardiac fibroblasts and monocytes.

[0042] Figure 8 (A) Shows the experimental schedule. (B) Shows the inhibitory effect of siVcam-1 on Vcam-1 gene expression. (C) Shows the adhesion of cardiac fibroblasts and monocytes.

[0043] Figure 9 (A) Shows the dosing schedule for Vcam-1 and VLA-4 neutralizing antibodies. (B) Shows the left ventricular circumference shortening (%fractional shortening; FS) measured by echocardiography for all mice in the CTL and neutralizing antibody groups. (C) Shows box plots of left ventricular end-diastolic diameter and left ventricular circumference shortening on day 28 for the same mice as in (B). (D) Shows the Kaplan-Meier curves for the CTL and neutralizing antibody dosing groups.

[0044] Figure 10 (A) Shows the dosing schedule for the neutralizing antibodies. (B) Shows the Kaplan-Meyer curves for each group.

[0045] Figure 11 (A) Shows wild-type mice (WT), wild-type mice, and MRL-Pdcd1 mice at 3 weeks postnatal age. - / - F1 (hetero: MRL-Pdcd1) produced by mating - MRL-Pdcd1 - / - (B) shows the %FS value of (homo). (C) shows the results of dividing the homo group into a high %FS group (good cardiac function group: n=11) and a low %FS group (decreased cardiac function group: n=20). (D) shows the dosing schedule of the VLA-4 neutralizing antibody.

[0046] Figure 12 Clinical data for the CTL group, VLA-4 group, and normal EF group are shown.

[0047] Figure 13 The Kaplan-Mayer curves for the CTL group (symbol a) and the VLA-4 group (symbol b) are shown.

[0048] Figure 14 The Kaplan-Mayer curves for the CTL group (symbol a), VLA-4 group (symbol b), and normal EF group (symbol c) are shown.

[0049] Figure 15 This shows the MRL-Pdcd1 - / - Kaplan-Mayer curves for each group during VLA4 inhibitor administration. In the figure, symbol a represents the AJM300(+) group and symbol b represents the AJM300(-) group.

[0050] Figure 16 This shows the MRL-Pdcd1 - / - Heart rate and left ventricular systolic capacity in each group during VLA4 blocker administration: %Fractional Shortening (%) results.

[0051] Figure 17 The study showed the serum concentrations of soluble Vcam-1 in 3 healthy individuals (CTL), 4 individuals with Covid-related myocarditis, 8 individuals with lymphocytic myocarditis (Lym), 6 individuals with eosinophilic myocarditis (Eosi), and 3 individuals with giant cell myocarditis (Giant).

[0052] Figure 18 The serum sVcam-1 concentrations in patients with acute and recovery phases of lymphocytic myocarditis are shown.

[0053] Figure 19 : Figure 21 (A) shows [ 11Radiation dose at various addition levels of C]HCA2969. Figure 21 (B) shows each 1nM[ 11 The radiation dose of C]HCA2969.

[0054] Figure 20 The following shows the results for two MRL-Pdcd1 - / - Mice used [ 11 PET / CT images taken by C]HCA2969.

[0055] Figure 21 The use of [[] was shown in two wild-type mice. 11 PET / CT images taken by C]HCA2969.

[0056] Figure 22 This demonstrates the use of [[] in a rat model of autoimmune myocarditis. 11 PET / CT images taken by C]HCA2969.

[0057] Figure 23 The schedule for the administration of anti-VLA-4 neutralizing antibody in a rat model of autoimmune myocarditis is shown.

[0058] Figure 24 The schedule for AJM300 dosing experiments in a rat model of autoimmune myocarditis is shown.

[0059] Figure 25 (A) Shows the left ventricular systolic capacity in the Placebo (placebo) group, the AJM300 administration group, and the neutralizing antibody administration group. (B) Shows the heart rate distribution in the Placebo group, the AJM300 administration group, and the neutralizing antibody administration group.

[0060] Figure 26 : Figure 26 (A) shows wild-type mice (WT) and PD1 - / - Mouse (PD1) - / - Examples of PET / CT imaging. Figure 26 (B) is a box plot that quantifies the aggregation of the cardiac portion in each group.

[0061] Figure 27 : Figure 27 (A) shows in Figure 26 The box plot of (B) shows the myocardial tissue images of individuals with solid circles marked on the box plot. Figure 27 (B) shows in Figure 26 The box plot of (B) shows the myocardial tissue images of individuals with dashed circles.

[0062] Figure 28The SUV median (Bq / ml) and SUV mean (Bq / ml) are shown for the negative control (Control) group and the EAE model group (EAE). Figure 28 (A) is the median value for SUVs (Bq / ml). Figure 28 (B) is the average value of SUVs (Bq / ml).

[0063] Figure 29 The results of the well counter are shown.

[0064] Figure 30 The %ID / g values ​​of each individual in the EAE model group and the negative control group (CTL group) are shown.

[0065] Figure 31 HE-stained images of the spinal cords of individuals in the EAE model group are shown. Detailed Implementation

[0066] 1. Radioactive positron-emitting carbon-11 labeled compounds, or pharmaceutically acceptable salts thereof.

[0067] One embodiment involves a radioactive positron-emitting carbon-11 labeled compound (hereinafter also referred to as "[ 11 "C" labeled compound or its pharmaceutically acceptable salt.

[0068] [ 11 C] The compound is labeled as a compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof:

[0069] .

[0070] (where R is in the formula) 1 It is a chlorine atom.

[0071] m is an integer from 1 to 3.

[0072] R 2 R 3 R 4 R 5 R 6 and R 7 At least one of them is a straight-chain or branched alkyl group having 1 to 6 carbon atoms, wherein the aforementioned alkyl group has a radioactive positron-emitting nuclide carbon-11. 11 [C], and in addition to that, there are hydrogen atoms.

[0073] R 8 It is a hydrogen atom, or an alkyl group with 1 to 6 carbon atoms, either straight or branched.

[0074] m is preferably 2.

[0075] As R 2 R 3 R 4 R 5 R 6 R 7 and R 8 Alkyl groups with 1 to 6 carbon atoms, either straight or branched, can be listed as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, neopentyl, isopentyl, sec-pentyl, tert-pentyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,3-dimethylbutyl, and 2,2-dimethylbutyl.

[0076] The alkyl group having 1 to 6 carbon atoms, whether straight or branched, is preferably methyl, ethyl, n-propyl, or isopropyl, and more preferably methyl.

[0077] [ 11 C] is preferably the carbon of the methyl group.

[0078] R 2 R 3 and R 7 Any one, any two, or all of them are preferably hydrogen atoms.

[0079] As R 8 Preferably, it is a hydrogen atom or a methyl group, more preferably a hydrogen atom.

[0080] As [ 11 C] Labelled compounds, preferably compounds represented by the following general formula (II):

[0081]

[0082] (where R is in the formula) 2 R 3 R 4 R 5 R 6 R 7 and R 8 Same as above).

[0083] [ 11 C] The labeled compound is more preferably a compound represented by the following formula (1) or (2):

[0084] ,

[0085] .

[0086] As [ 11Salts of compounds labeled C can include inorganic acid salts such as hydrochloride, nitrate, hydrobromide, and sulfate; organic carboxylic acid salts such as acetate and trifluoroacetate; sulfonates such as methanesulfonate; or metal salts such as sodium salt, potassium salt, calcium salt, and magnesium salt; and salts of alkaline substances such as ammonium salt.

[0087] 2.[ 11 C] Method for manufacturing labeled compounds

[0088] [ 11 The labeled compound can be manufactured according to the following manufacturing method:

[0089]

[0090] In the above scheme, R 1 m, R 2 R 3 R 4 R 5 R 6 R 7 and R 8 Same as general formula (I).

[0091] (1) For example, using a medical cyclotron (CYPRIS HM-18, Sumitomo), with N2 gas as the target, through 14 N(p, α) 11 C nuclear reaction produces [ 11 C]CO2. The obtained [ 11 [C]CO2 is introduced into the gas-phase iodomethane synthesis unit (C-GPC-100, Sumitomo) to synthesize highly reactive [ 11 C] Trifluoromethanesulfonate ([ 11 C]CH3OTf).

[0092] (2) [ 11 [C]CH3OTf was introduced into a reaction vessel containing an acetone solution of compound A dissolved at a concentration of approximately 0.2 mg / mL to 1.5 mg / mL, and an alkali such as sodium hydroxide at a final concentration of approximately 0.003 mol / L to 0.008 mol / L, and bubbled. This reaction yielded compound B.

[0093] (3) Add sodium hydroxide or other alkali to the bubbling liquid obtained in (2) at a final concentration of about 0.01 mol / L to 0.03 mol / L, and carry out a deprotection reaction for about 10 minutes at room temperature (about 20℃ to 27℃). Through this reaction, compound C is obtained.

[0094] (4) From the deprotected liquid obtained in (3), [the following preparations were made by HPLC] 11 C]HCA2969.

[0095] Preferably, [ 11 The labeled compound can be manufactured according to the following manufacturing method:

[0096] .

[0097] In this scheme, in step (2) of the above manufacturing method, as a preferred method for compound A, compound QD is used as the starting material, thereby obtaining compound B as a preferred method in step (2). 11 C]AJM300. Next, in step (3), the preferred form of compound C is obtained. 11 C]HCA2969.

[0098] 3. Includes [ 11 C] Radioactive compositions of labeled compounds or pharmaceutically acceptable salts thereof

[0099] One implementation involves including [ 11 C] Radioactive compositions of labeled compounds or pharmaceutically acceptable salts thereof (hereinafter also referred to as "radioactive compositions").

[0100] The radioactive composition can be in a dry state or in a liquid state dissolved in a solvent. Examples of solvents include ethanol, mixtures of ethanol and water (ethanol content greater than 0% and less than 99.5%), and N,N-dimethylformamide.

[0101] The radioactive composition can be administered at a radiation dose of approximately 1 MBq / kg to 500 MBq / kg, preferably 3 MBq / kg to 100 MBq / kg.

[0102] The radioactive composition can be used to detect lymphocytic inflammatory diseases. Preferably, lymphocytic inflammatory diseases for which VLA4 blockers show efficacy are lymphocytic inflammatory diseases. More preferably, examples of lymphocytic inflammatory diseases include myocarditis, encephalomyelitis, ulcerative colitis, Crohn's disease, and heart transplant rejection. Even more preferably, examples of lymphocytic inflammatory diseases include myocarditis and encephalomyelitis.

[0103] In this manual, "myocarditis" refers to an inflammatory disease with the myocardium as the primary site of lesion. Based on clinical course, myocarditis includes acute myocarditis and chronic myocarditis. Furthermore, acute myocarditis includes fulminant myocarditis and other types of myocarditis. Chronic myocarditis includes dilated cardiomyopathy with ventricular hypertrophy and other types of chronic myocarditis.

[0104] In addition, in histological classification, myocarditis includes lymphocytic myocarditis, giant cell myocarditis, eosinophilic myocarditis, and granulomatous myocarditis. Lymphocytic myocarditis has been reported to be caused by viral infections. The causative viruses are mostly enteroviruses, including Coxsackievirus (Group A or B), echovirus, and poliovirus; hepatitis viruses (A or C); influenza viruses (A or B); RS virus; mumps virus; measles virus; dengue virus; yellow fever virus; chikungunya virus; rabies virus; HIV; vaccinia virus; herpes zoster virus; cytomegalovirus; herpes simplex virus; Epstein-Barr virus; measles virus; adenovirus; and parvovirus. Giant cell myocarditis and eosinophilic myocarditis have been reported to be caused by bacterial infections. Eosinophilic myocarditis has been reported to be caused by fungal infections. Granulomatous myocarditis is often associated with infections such as rickettsia, spirochetes, protozoan infections, and parasitic infections; drug or chemical ingestion; allergic reactions and autoimmune diseases; Kawasaki disease; sarcoidosis; radiation exposure; and heatstroke. Drugs that can 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.

[0105] Myocarditis preferably excludes autoimmune myocarditis that uses myosin as an antigen. Furthermore, myocarditis preferably excludes eosinophilic myocarditis.

[0106] As for myocarditis, lymphocytic myocarditis is preferred. Furthermore, as for myocarditis, myocarditis that depends on the binding of Vcam-1 and VLA-4 is preferred. More preferably, myocarditis caused by immune checkpoint inhibitors is preferred.

[0107] In this specification, VLA-4 is a protein also known as integrin subunit alpha 4 or CD49D, which, in the human case, is a protein expressed by a gene registered with the National Center for Biotechnology Information (NCBI) as Gene ID: 3676. VLA-4 is a ligand for vascular cell adhesion molecule 1 (VCAM1; CD106; INCAM-100).

[0108] The detection of myocarditis using radioactive compositions will detect the presence of [[] in the radioactive composition. 11 [C] is used as a tracer in vivo. 11C] It can be detected by using a PET (positron emission tomography) examination (hereinafter also referred to as "PET examination"). PET examinations can be performed according to known methods. In addition, PET examinations can include positron emission tomography-only examinations, PET-CT examinations combined with CT examinations, PET-MRI examinations combining PET and MRI, etc.

[0109] The radioactive composition can be used to determine the therapeutic effect of myocarditis. The therapeutic effect can be determined by: (1) obtaining a first PET image using the radioactive composition at a first moment for a patient suspected of having myocarditis, or a patient with myocarditis; (2) treating the aforementioned patient with myocarditis; (3) obtaining a second PET image using the radioactive composition at a second moment after the first moment, during or after the treatment of myocarditis; (4) [the radioactive composition is then used to determine the therapeutic effect of the radioactive composition in the first and second PET images]. 11 The aggregation comparison of [C]; and (5) in the second PET examination image [ 11 If the aggregation of [C] is less than that in the first PET scan image, it is considered to have a therapeutic effect, and / or if [C] is less in the second PET scan image. 11 If the aggregation of C] is equal to or better than that of the first PET scan image, it is considered that there is no therapeutic effect.

[0110] In addition, as one embodiment, the radioactive composition can be used to predict the therapeutic effects achieved by using VLA-4 blocking substances.

[0111] For example, in patients suspected of having myocarditis, or patients with myocarditis, PET images obtained using a radioactive composition are used to confirm the presence of [a radioactive component in the heart]. 11 If the aggregation of [C] is observed, it can be determined that treatment with VLA-4 blocking substances for the aforementioned patients is effective. Additionally, for patients suspected of having myocarditis or those with myocarditis, PET images obtained using radioactive compositions, if no [C] is confirmed in the cardiac region... 11 If the aggregation of C] is observed, it can be determined that treatment using VLA-4 blocking substances is ineffective for the aforementioned patients.

[0112] Myocarditis is usually diagnosed through endocardial biopsy to obtain myocardial tissue, and the diagnosis is confirmed by the images of this tissue. Therefore, a myocarditis patient is someone who has been diagnosed with myocarditis. A suspected myocarditis patient is someone who, although not yet diagnosed, has been suggested to have myocarditis based on examinations such as electrocardiogram; pulse; echocardiography (tomographic images, left ventricular diameter shortening rate, left ventricular ejection fraction, and other cardiac function evaluations); blood tests for CRP, AST, LDH, CK-MB, cardiac troponin (T or I), sVcam-1; chest X-ray; and cardiac MRI.

[0113] Treatment for myocarditis may include administering VLA-4 blocking agents and discontinuing medications that cause myocarditis.

[0114] Examples of substances that can block VLA-4 include anti-VLA-4 antibodies or fragments containing its antigen-binding domain.

[0115] In this specification, 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 can be either a polyclonal antibody or a monoclonal antibody. Both polyclonal and monoclonal antibodies can be appropriately prepared using methods known to those skilled in the art. When the antibody is a monoclonal antibody, it can be a chimeric antibody, humanized antibody, or human antibody prepared using known methods. Furthermore, the aforementioned antibody can be antibody fragments such as Fab, F(ab)2, biantibodies, scFv, small antibodies, peptides, or mimetibodies. Preferably, the anti-VLA-4 antibody is a neutralizing antibody. For example, anti-VLA-4 antibodies include InVivoMAb anti-mouse / human VLA-4 (CD49d) (clone No. PS / 2, Cat No. BE0071, Bio X Cell) and vedolizumab, as described in the literature: Ferreira, EFB et al (Current Medicinal Chemistry, 2021, 28, 5884-5895).

[0116] In this specification, the VLA-4 antagonist can be either a competitive antagonist of VLA-4 (competitive antagonist type) or a non-competitive antagonist of VLA-4 (non-competitive antagonist type). A non-competitive antagonist type is preferred. The VLA-4 antagonist can be a compound or a peptide.

[0117] Examples of VLA-4 antagonists include methylcarbenatate (International Publication No. 2016 / 051828); SB683699 / firategrast, R411 / valategrast, CT7758 / CDP323, DS-70, and TBC3486 as described in Ferreira, EFB et al (Current Medicinal Chemistry, 2021, 28, 5884-5895); PN-943 and MORF-057 as described in Slack, RJ (Nature Reviews Drug Discovery volume 21, pages 60-78 (2022)); BIO1211 and BIO5192 as described in Baiula, M et al (Front Chem .2019 Jul 9;7:489); and Ohkuro, M et al. ER464195-01 is described in al(Nat Commun. 2018May 17;9(1):1982ER464195-01); E6007 is an analog of ER464195-01. PN-943 is a peptide.

[0118] As VLA-4 blocking agents, RNA molecules that target VLA-4 mRNA can be listed. There are no restrictions on the aforementioned RNA molecules as long as they target VLA-4 mRNA and can inhibit its expression. As for the aforementioned RNA, at least one RNA selected from the group consisting of siRNA, shRNA, miRNA, and antisense RNA that has the function of degrading the target mRNA and / or inhibiting the translation of the target mRNA can be listed. The sequences of these RNA molecules can be appropriately designed based on information about the base sequence of the aforementioned target gene using methods known to those skilled in the art. Furthermore, the RNA molecule can be produced using known methods, or commercially available RNA molecules can be obtained and used. For example, as an antisense RNA for VLA-4, ATL1102 described in the literature: Limmroth, V. et al (Neurology 83 November 11, 2014). RNA molecules or vectors capable of expressing these RNA molecules can be listed.

[0119] Vectors capable of expressing RNA molecules targeting VLA-4 mRNA are not particularly limited as long as they can express RNA molecules that inhibit the expression of the VLA-4 protein in vivo or within cells. Examples include hairpin RNA expression vectors. Hairpin RNA expression vectors contain at least the following: a sense strand DNA sequence downstream of a promoter sequence suitable for short RNA expression, such as the U6 promoter, having the same sequence as the sense strand of the target mRNA (wherein, uracil in the mRNA is replaced by thymine); a circular base sequence that forms a loop structure after transcription; an antisense strand DNA sequence that can bind complementaryly to the entire or a portion of the aforementioned sense strand DNA sequence; and a terminator sequence. Examples of vectors include plasmid vectors, adeno-associated virus (AAV) vectors, adenovirus vectors, retroviral vectors, and lentiviral vectors.

[0120] Examples of VLA-4 blocking agents include genome editing systems targeting the VLA-4 gene. There are no restrictions on genome editing systems targeting the VLA-4 gene, as long as they can induce recombination within the VLA-4 gene in an individual. Examples include CompoZr zinc finger nuclease (ZFN) systems, TAL effector nuclease (TALEN) systems, and clustered regularly spaced short palindromic repeats / CRISPR-associated protein 9 (CRISPR / Cas9) systems. CRISPR / Cas9 systems are preferred. Vector-based CRISPR / Cas9 systems are preferred. In vector-based CRISPR / Cas9 systems, the nucleic acid encoding CRISPR and the nucleic acid encoding Cas9 can be on different vectors, or they can be on the same vector. The promoter used to activate CRISPR is not particularly limited, but the U6 promoter is preferred. The promoter used to activate Cas9 is not particularly limited, but promoters that induce expression in mammalian cells, such as cytomegalovirus promoters, are preferred. For CRISPR / Cas9 systems, commercially available vectors such as pX330-U6-Chimeric_BB-CBh-hSpCas9 are preferred.

[0121] There are no restrictions on the VLA-4 gene-targeting sequence that is integrated into a CRISPR sequence, as long as it can be integrated into the guide RNA (also known as gRNA or crRNA) via the CRISPR / Cas9 system and transcribed to recombine with the VLA-4 gene. Typically, a sequence of about 20 bases upstream of the 5' flanking region of the "NGG" base sequence within the VLA-4 gene can be selected as the VLA-4 gene-targeting sequence. VLA-4 gene target sequences can be designed using publicly available design tools such as Optimized CRISPR design tool (Massachusetts Institute of Technology, ZhangLab website (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)), Cas9 design (http: / / cas9.cbi.pku.edu.cn (Peking University)), CRISPRdirect (http: / / crispr.dbcls.jp (University of Tokyo)), and CRISPR-P (http: / / cbi.hzau.edu.cn / crispr / (Huazhong Agricultural University)).

[0122] Example

[0123] The following embodiments illustrate the invention in more detail. However, the invention is not limited to these embodiments.

[0124] In addition, all animal experiments in this embodiment were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Experimentation Committee of Osaka University.

[0125] 1.[ 11 Manufacturing of C]HCA2969

[0126] To manufacture, follow these steps: 11 C]HCA2969. The synthesis scheme is shown in... Figure 1 .

[0127] (1) Using a medical cyclotron (CYPRIS HM-18, Sumitomo), with N2 gas as the target, through...14 N(p, α) 11 C nuclear reaction produces [ 11 C]CO2. The obtained [ 11 [C]CO2 is introduced into the gas-phase iodomethane synthesis unit (C-GPC-100, Sumitomo) to synthesize highly reactive [ 11 C] Trifluoromethanesulfonate ([ 11 C]CH3OTf).

[0128] (2) [ 11 C]CH3OTf was introduced into a reaction vessel containing 500 μL of 1 mg / mL HCA2969 precursor (QD) / acetone solution and 3 μL of 1 mol / L NaOH and bubbled.

[0129] (3) Add 100 μL of 0.1 mol / L NaOH to the bubbling liquid obtained in (2) and carry out a deprotection reaction at room temperature for 10 minutes.

[0130] (4) Add 1 mL of water for injection to the deprotected liquid obtained in (3), mix thoroughly, and then inject the total volume into HPLC to prepare [ 11 C]HCA2969. A representative HPLC chromatogram is shown in... Figure 2 .

[0131] (5) Dilute the preparation solution with water for injection, maintaining its total volume in the solid phase extraction column (HLB column). 11 In C]HCA2969, the HLB column was pretreated with 10 mL of ethanol and 20 mL of water for injection.

[0132] (6) Keep the [in (5)] 11 After washing the HLB column of C]HCA2969 with water for injection to remove impurities, ethanol was used to [ 11 C]HCA2969 was recovered and added to the product vial. Physiological saline was added to the product vial to bring the final ethanol concentration to approximately 10%. The recovered [[ 11 C]HCA2969 solution was used as the final formulation.

[0133] (7) A portion of the final formulation was taken out and subjected to quality testing. The results of the quality testing were obtained under irradiation conditions of 25 μA for 30 minutes. 11 Using CO2 as the starting material, approximately 2 GBq of [ 11 [C]HCA2969. Furthermore, its radiochemical purity was confirmed to be consistently high, exceeding 90% immediately after synthesis and even after 90 days. The results are presented below. Figure 3 .

[0134] 2. Single-cell analysis using a rat model of autoimmune myocarditis

[0135] (1) Rats with autoimmune myocarditis

[0136] An autoimmune myocarditis rat model was established by administering porcine cardiac myosin (purified from porcine heart by our company) at 10 mg / ml and an adjuvant (CFA 10 mg / ml; Chondrex, 7002) to male Lewis rats (8 weeks old). The day of administration of porcine cardiac myosin was designated as day 0. In this model, myocarditis peaked at day 21. Figure 4 Macroscopic images of the heart and hematopoietic staining of tissues are shown at low and high magnification on days 12, 19, and 25. On day 12, inflammatory cells begin to infiltrate the myocardial tissue, and stronger inflammatory cell infiltration is observed on day 19. On day 25, myocardial tissue destruction due to inflammation is observed. Eosinophilic infiltration was not observed at any stage of the disease.

[0137] (2) Population analysis of cells in myocardial tissue based on single-cell analysis

[0138] Single-cell analysis was performed as follows: rat heart cells were dispersed using the enzymes (Liberase™ (Roche, 5401119001 / Liberase DH (Roche, 5401054001)) and analyzed using Chromium (10x Genomics). Figure 5 The results are shown. In the figure, "CTL" represents the control group without the antigen, and "Myocarditis day…" indicates the number of days since administration of porcine cardiac myosin. On day 21, the number of T cells and neutrophils increased, while the number of vascular endothelial cells decreased relatively. Additionally, the number of monocytes and macrophages increased significantly, but the number of NK cells decreased, and B cells were not identified. Eosinophils were not detected. Furthermore, unlike CTL, the activated cardiac fibroblast population increased.

[0139] Figure 6 The expression of Vcam-1 at each CTL, day 8, day 14, day 21, and day 25 is shown. Vcam-1 expression was observed in vascular endothelial cells and in activated cardiac fibroblasts (Postn... + / FAP + Vcam-1 was also highly expressed in the sample.

[0140] 3. In vitro mononuclear cell adhesion experiment using cardiac fibroblasts

[0141] This experiment was conducted to determine whether stimulation by TNFα, an inflammatory cytokine, alters the expression of Vcam-1, and also to determine whether TNFα stimulation alters the adhesion between cardiac fibroblasts and monocytes.

[0142] (1) Culture conditions and adhesion conditions

[0143] Figure 7 (A) shows the schedule for the adhesion experiment.

[0144] For cardiac fibroblasts, the hearts of newborn rats were dispersed using type 2 collagenase (Worthington Biochemical Corporation, LS004176), and the isolated fibroblasts were cultured in 96-well plates. For splenic mononuclear cells, spleens were harvested from wild-type mice, homogenized, and monocyte fractions were collected using Optiprep (Serumwerk Bernburg, 1893) based on specific gravity separation. In adhesion assays, cardiac fibroblasts were cultured in 96-well plates for 48 hours, followed by TNFα + / - medium exchange and a further 24 hours of culture. Splenic mononuclear cells stained with fluorescent dyes (Hoechst 33342 (Dongren Chemical, 341-07901) or Cytored (Dongren Chemical, 342-08531)) were added and incubated at 37°C for 30 minutes. After washing six times with PBS(-) to remove non-adherent monocytes, adherent monocytes were counted using an In CellAnalyzer 6000 (GE Healthcare Life Sciences).

[0145] TNFα (Peprotech, 300-01A) was added to the wells at a final concentration of 5–15 ng / ml.

[0146] Western blotting confirmed that Vcam-1 induction was caused by TNFα. The primary antibody for Western blotting was the anti-Vcam1 antibody EPR5047 (Abcam, ab134047). For the assay, HRP-labeled anti-rabbit antibody was used, and the luminescent reagent was Chemi-Lumi One L (Nacalai tesque, 07880-70). GAPDH was used as an internal control in the Western blotting.

[0147] (2) Results

[0148] The results of the protein blotting are shown in Figure 7(B). Western blot analysis was repeated for each sample. Low levels of Vcam-1 expression were observed up to 0 hours after TNFα addition. However, expression increased further at 12 and 24 hours after TNFα addition. Additionally, Figure 7 (C) is a camera image obtained from the In Cell Analyzer 6000. For example... Figure 7 As shown in (C), the adhesion between cardiac fibroblasts and monocytes is enhanced due to stimulation by TNFα.

[0149] 4. In vitro adhesion experiment of cardiac fibroblasts and monocytes using siRNA.

[0150] To confirm whether Vcam-1 directly adheres to cardiac fibroblasts and monocytes, siRNA was used and cardiac fibroblasts with V-cam-1 knocked down were used to confirm adhesion to monocytes.

[0151] (1) Experimental conditions

[0152] Figure 8 (A) shows the schedule for this experiment.

[0153] The culture and adhesion experiments of cardiac fibroblasts were performed in the same manner as described in 3.(1) above.

[0154] Regarding Vcam-1 siRNA (Silencer) TM Select pre-designed siRNA (siRNA IDs 129593, Thermo; also known as siVcam-1), and use Lipofectamine at a final concentration of 30 nM. TM RNAiMAX Transfection Reagent (Thermo, 13778075) was transfected into cardiac fibroblasts seeded in wells 2 hours prior. Adhesion assays with monocytes were performed 72 hours after seeding the cardiac fibroblasts in the wells. Silencer was used as a control for siRNA (siCTL). TM Select Negative Control No. 1 siRNA (Thermo, 4390843). The transfection amount was set to be equal to that of siVcam-1. In addition, TNFα was added at a rate of 5 ng / ml 46 hours after transfection.

[0155] The protein blotting of Vcam-1 was performed in the same manner as described in 3.(1) above.

[0156] In the detection of monocytes, the In Cell Analyzer 6000 (GE Healthcare Life Sciences) was used to count the adhering monocytes.

[0157] (2) Results

[0158] like Figure 8 As shown in (B), Vcam-1 signaling was detected in cardiac fibroblasts transfected with siCTL, but not in cardiac fibroblasts transfected with siVcam-1. This result indicates that siVcam-1 inhibits Vcam-1 expression in cardiac fibroblasts. Adhesion to monocytes was confirmed using this experimental system. The results are shown in… Figure 8 (C). In the case of cells transfected with siVcam-1, monocyte adhesion was reduced. On the other hand, in the case of cells transfected with siCTL, monocyte adhesion was confirmed. This result indicates that the adhesion of cardiac fibroblasts to monocytes is facilitated by Vcam-1.

[0159] 5. Validation of the improved prognostic effect of the combined use of Vcam-1 neutralizing antibody and VLA-4 neutralizing antibody in myocarditis.

[0160] According to the literature: Int Immunol. 2010 Jun;22(6):443-52, the spontaneous-onset systemic lupus erythematosus mouse model MRL-Fas lpr / lpr Compared with PD-1 knockout mice C57BL / 6-Pdcd1 - / - MRL-Pdcd1 was obtained through mating. - / - This is a spontaneous lymphocytic myocarditis model mouse with a 96% incidence of myocarditis between 4 and 8 weeks of age. Furthermore, the survival rate at 10 weeks of age is approximately 30%, significantly lower than that of wild-type mice. Using this MRL-Pdcd1... - / - The following myocarditis experiment was performed.

[0161] (1) Combined administration of Vcam-1 neutralizing antibody and VLA-4 neutralizing antibody

[0162] 13 MRL-Pdcd1 - / -The subjects were divided into two groups: a control group (CTL: n=7) and a group receiving a combination of Vcam-1 neutralizing antibody and VLA-4 neutralizing antibody (neutralizing antibody group: n=7). Starting from day 14 postnatally, the CTL group received intraperitoneal administration of saline, while the neutralizing antibody group received both 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 dosage for each was set at 10 μg / g. The dosing schedule was as follows: Figure 9 As shown in (A), the procedures were performed at 3-day intervals from day 14 to day 26 after birth. Echocardiography was performed on day 14 and day 28 after birth. In addition, after obtaining echocardiographic data on day 28 after birth, blood and heart samples were collected from all mice.

[0163] (2) Results

[0164] Figure 9 (B) shows the left ventricular ventricular diameter rate (%fractional shortening; FS) measured by echocardiography for all mice in the CTL group and the neutralizing antibody group. In the CTL group, ventricular contractility was significantly decreased at day 28 compared to day 14. On the other hand, in the neutralizing antibody group, the decrease in ventricular contractility was mild compared to the CTL group. Figure 9 (C) shows Figure 9 Box plots of left ventricular end-diastolic diameter [LVDd (mm)] and left ventricular diameter shortening rate [%FS (%)] on day 28 are shown in (B). No significant difference in LVDd was observed between the CTL group and the neutralizing antibody group (p=0.22). On the other hand, the %FS was significantly higher in the neutralizing antibody group (p=0.0103). Figure 9 Figure (D) shows the Kaplan-Mayer curves for the CTL group and the neutralizing antibody group. In the figure, symbol a represents the CTL group and symbol b represents the neutralizing antibody group. In the study up to day 28 after birth, the number of individuals dying on day 20 after birth increased in the CTL group. On the other hand, no deaths were observed in the neutralizing antibody group. The difference between the two groups was statistically significant at p=0.033 in the log-rank analysis. This result indicates that combined administration of Vcam-1 and VLA-4 neutralizing antibodies can improve mortality from myocarditis.

[0165] 6. The effect of VLA-4 neutralizing antibodies

[0166] To investigate which of the Vcam-1 neutralizing antibodies and the VLA-4 neutralizing antibodies helps reduce mortality from myocarditis, the following study was conducted.

[0167] (1) Antibody administration

[0168] 29 MRL-Pdcd1 - / - The study was divided into four groups: control (CTL: n=12), Vcam-1 group (n=5) receiving only Vcam-1 neutralizing antibody, VLA-4 group (n=6) receiving VLA-4 neutralizing antibody, and Vcam-1 / VLA-4 group (n=6) receiving a combination of Vcam-1 and VLA-4 neutralizing antibody.

[0169] The dosage of each antibody is set to be the same as that in 4.(1) above. For example... Figure 10 As shown in (A), the dosing schedule for neutralizing antibodies is the same as in 4.(1) above.

[0170] (2) Results

[0171] Figure 10 Figure (B) shows the Kaplan-Meier curves for each group. In the figure, symbol a represents the CTL group, symbol b represents the Vcam-1 group, symbol c represents the Vcam-1 / VLA-4 group, and symbol d represents the VLA-4 group. In the study up to day 28 after birth, in the CTL group (symbol a), the number of individuals dying on day 19 after birth increased. In the Vcam-1 group (symbol b), the number of individuals dying from day 20 after birth also increased. On the other hand, no deaths were observed in the Vcam-1 / VLA-4 group (symbol c). In the VLA-4 group (symbol d), one individual died on day 18, but the other individuals survived to day 28 after birth. This result suggests that VLA-4 neutralizing antibodies contribute to improving the prognosis of myocarditis.

[0172] 7. Effects of VLA-4 neutralizing antibodies in mice with impaired cardiac function

[0173] Figure 11 (A) shows wild-type mice (WT) at 3 weeks postnatal, compared with MRL-Pdcd1. - / - F1 (hetero: MRL-Pdcd1) produced by mating - MRL-Pdcd1 - / -The %FS value of (homo) was measured. The %FS value of the WT group was above 75%, with little bias, showing a small difference between 25% and 100%. On the other hand, both the homo and hetero groups had individuals with high and low %FS values, exhibiting a relatively wide distribution overall.

[0174] Therefore, as Figure 11 As shown in (B), the homologous 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) without VLA-4 neutralizing antibody treatment and a VLA-4 treatment group (VLA-4 group) with VLA-4 neutralizing antibody treatment to study the effect of the VLA-4 neutralizing antibody. The cutoff value for the good cardiac function group (normal EF group) and the decreased cardiac function group was set at 70%. Groups with a cutoff value above 70% were classified as the good cardiac function group, and groups with a cutoff value below 70% were classified as the decreased cardiac function group. Based on this, according to... Figure 11 The VLA-4 neutralizing antibody was administered according to the schedule shown in (C). Body weight (BW), pulse rate (HR), and %FS values ​​for each group are shown in [the table / image / image]. Figure 12 There were no significant differences in any of the parameters between the CTL group and the VLA-4 group.

[0175] Figure 13 The Kaplan-Mayer curves for the CTL group (symbol a) and the VLA-4 group (symbol b) are shown. The log-rank analysis results show p=0.0285, indicating a significantly longer survival rate in the VLA-4 group.

[0176] Figure 14 Kaplan-Mayer curves are shown for survival of the normal EF group (symbol c), which was supplemented with additional curves. The normal EF group, which consisted of patients with good cardiac function and who had not received VLA-4 neutralizing antibodies, had significantly lower survival rates compared to the VLA-4 group (log-rank analysis results, p = 0.0275).

[0177] These results indicate that administration of VLA-4 neutralizing antibodies to individuals with myocarditis improves the prognosis of myocarditis, regardless of individual cardiac function data. Furthermore, this demonstrates that administration of VLA-4 neutralizing antibodies to individuals with myocarditis is effective in the prevention or treatment of myocarditis.

[0178] 8. The effects of administering VLA4 blockers

[0179] To investigate whether administering AJM300 (methylcartastat), one of the VLA4 blockers, could help reduce mortality from myocarditis, the following study was conducted.

[0180] (1) Administration of AJM300

[0181] 44 MRL-Pdcd1 - / - The patients were divided into two groups: the AJM300(-) group (n=13) without AJM300 administration and the AJM300(+) group (n=12) with AJM300 administration.

[0182] For the AJM300 group (+), AJM300 1% was administered in the mixed feed from 2 weeks after birth until death.

[0183] In addition, 9 animals in the AJM300(-) group and 10 animals in the AJM300(+) group underwent echocardiographic cardiac function evaluation at 28 days of age, were euthanized, and heart and blood samples were collected.

[0184] (2) Results

[0185] Figure 15 The Kaplan-Mayer curves for each group are shown. In the figure, symbol a represents the AJM300(+) group, and symbol b represents the AJM300(-) group. Compared with the AJM300(-) group, the AJM300(+) group had a significantly longer survival (log rank analysis p=0.0022). This result indicates that administration of VLA4 blockers helps improve the prognosis of myocarditis.

[0186] Figure 16 The results show the heart rate and left ventricular systolic capacity (%Fractional Shortening) measured by echocardiography. The AJM300(+) group showed significantly improved bradycardia compared to the AJM300(-) group (t-test, p=0.0231). Furthermore, the AJM300(+) group showed significantly improved left ventricular systolic capacity compared to the AJM300(-) group (t-test, p=0.0231). These results indicate that administration of VLA4 blockers helps improve cardiac function.

[0187] 9. Concentration of soluble Vcam-1 in human serum

[0188] The serum concentration of soluble Vcam-1 (sVcam-1) was compared between healthy individuals and patients with myocarditis.

[0189] Results from 3 healthy individuals (CTL), 4 individuals with Covid-related myocarditis, 8 individuals with lymphocytic myocarditis (Lym), 6 individuals with eosinophilic myocarditis (Eosi), and 3 individuals with giant cell myocarditis (Giant) are presented in... Figure 17In all types of myocarditis, including lymphocytic myocarditis, elevated serum sVcam-1 concentrations were observed compared to healthy individuals. This result indicates that the VLA-4 / Vcam-1 pathway is associated with the pathogenesis of myocarditis.

[0190] 10. Changes in serum sVcam-1 levels during myocarditis

[0191] The serum sVcam-1 concentrations of three patients with lymphocytic myocarditis were compared during the acute and recovery phases.

[0192] The results are shown in Figure 18 In the acute phase, serum sVcam-1 concentrations were very high in all three patients. Figure 17 The comparison of sVcam-1 concentrations in the serum of healthy individuals shows that the three patients also improved to the levels of healthy individuals during the recovery period.

[0193] These results indicate that the VLA-4 / Vcam-1 pathway is associated with myocarditis.

[0194] 11.[ 11 Evaluation of the binding affinity of C]HCA2969 to VLA-4 in mouse monocytes

[0195] Evaluation was conducted using monocytes recovered from mice. 11 The binding affinity of C]HCA2969 to VLA-4.

[0196] (1) Method

[0197] 2 × 10⁶ mononuclear cells were recovered from the spleen of a mouse. 6 The [synthetic] and the above 1. 11 C]HCA2969 was added to buffer (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 incubation at room temperature for 20 minutes, the cells were washed twice with buffer (10 mM Tris pH 7.5, 0.1% BSA, 150 mM NaCl, 2 mM MnCl2, 2 mM D-glucose). The radiation dose was measured using a scintillation counter for the washed cells.

[0198] (2) Results

[0199] Figure 19 (A) shows [ 11 Radiation dose at various addition levels of C]HCA2969. Figure 19 (B) shows the [ ] per 1 nM11 The radiation dose of HCA2969 was determined by plotting a clear saturation curve in (A) and calculating the equilibrium dissociation constants Kd (0.16 nM) and Bmax (81.85 Bq) using Scatchard analysis in (B).

[0200] 12. Used [ 11 C]HCA2969 Detection of Myocarditis

[0201] For the MRL-Pdcd1 produced in step 5 above - / - In wild-type mice, on days 25-26, under anesthesia with inhaled isoflurane, […] 11 Approximately 1-10 MBq of C]HCA2969 tracer was administered via tail vein and followed by PET / CT imaging.

[0202] Figure 20 Two MRL-Pdcd1 are shown. - / - Results in mice: In all cases, tracer accumulation in the heart was confirmed.

[0203] Figure 21 Results are shown for two wild-type mice. No tracer accumulation in the heart was observed in either mouse.

[0204] 13. Used [ 11 Detection of autoimmune myocarditis using C]HCA2969

[0205] For the rat model of autoimmune myocarditis established in section 2 above, during the third week after the peak of myocarditis induction, under isoflurane inhalation anesthesia, […]. 11 Approximately 1-10 MBq of C]HCA2969 tracer was administered via tail vein for PET / CT imaging.

[0206] like Figure 22 As shown, similar to non-induced myocarditis rats (left: labeled CTL), even in autoimmune myocarditis model rats (right: labeled myocarditis), no tracer accumulation in the heart was confirmed.

[0207] like Figure 23 , Figure 24As shown, the above-mentioned anti-VLA-4 neutralizing antibody or AJM300 administration experiments were conducted on rats with an autoimmune myocarditis model. The study groups consisted of a Placebo group (n=5), an AJM300 administration group (n=4), and a neutralizing antibody administration group (n=5). The Placebo group was fed a mixture of Placebo (0.43% Metolose + 0.14% Ac-Di-Sol) from 3 days before myocarditis induction to 3 weeks after induction. The AJM300 administration group was fed a diet containing 1% AJM300 from 3 days before myocarditis induction to 3 weeks after induction. The neutralizing antibody administration group 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 systolic capacity and heart rate were evaluated on day 21 after myocarditis induction. Figure 25 (A) shows the left ventricular contractile capacity of the Placebo group, the AJM300 administration group, and the neutralizing antibody administration group. Figure 25 (B) shows the heart rate distribution in the Placebo group, the AJM300 administration group, and the neutralizing antibody administration group. Figure 25 As shown, in rats with an autoimmune myocarditis model, no significant differences in cardiac function were found among the Placebo group, the AJM300 administration group, and the neutralizing antibody administration group. Neither AJM300 nor the anti-VLA-4 neutralizing antibody showed any improvement in cardiac function.

[0208] By combining the results of 12 above, it is shown that [ 11 The C]HCA2969 tracer was observed to accumulate in the myocardium in a myocarditis model in which the therapeutic effects of AJM300 and anti-VLA-4 neutralizing antibodies could be expected.

[0209] 14. [used] 11 Determination of the therapeutic effect of C]HCA2969

[0210] Next, PD1 knockout mice (MRL-Pdcd1) were used. - / - Mice: PD1 - / - Mice), using [ 11 C]HCA2969 tracer was used to evaluate the therapeutic effect of myocarditis.

[0211] (1) Method

[0212] Prepare PD1 in 4-week-old wild-type mice, placebo group, AJM300 administration group, and anti-VLA-4 neutralizing antibody administration group. - / -Mouse groups. The placebo group was fed a normal diet, while the AJM300-treated group was fed a diet containing 1% AJM300 from day 14 to day 28 post-feeding. The anti-VLA-4 neutralizing antibody-treated group was fed a normal diet, and the anti-VLA-4 neutralizing antibody (10 mg / kg) was administered intraperitoneally on days 14 and 21 post-feeding.

[0213] On day 28 from the start of feeding, all mice were anesthetized with isoflurane (1-3% / 2L O2), and injected via the tail vein. 11 C]HCA2969 tracer (1~10 MBq: 10 μg of unlabeled HCA2969 containing 10 MBq of PET tracer). PET / CT imaging was performed 10 minutes after injection.

[0214] (2) Results

[0215] The results are shown in Figure 26 and Figure 27 . Figure 26 (A) shows wild-type mice (WT) and PD1 - / - Mouse (PD1) - / - Examples of PET / CT imaging. PD1 - / - In mice, [the following was confirmed in the heart region] 11 Aggregation of C]HCA2969 tracer. Figure 26 (B) is a box plot that quantifies the aggregation of the cardiac portion in each group.

[0216] PD1 - / - In the placebo group of mice, [[] was confirmed to be present. 11 Aggregation of the C]HCA2969 tracer. On the other hand, in the AJM300 administration group and the anti-VLA-4 neutralizing antibody administration group, aggregation was lower compared with the placebo group.

[0217] Based on this result, it is believed that, 11 C]HCA2969 tracer can determine the therapeutic effects of AJM300 and anti-VLA-4 neutralizing antibodies.

[0218] Figure 27 (A) shows in Figure 26 The box plot of the placebo group (B) shows the myocardial tissue image of the individual with the solid circle marked on it. This individual is [ 11 Individuals with particularly high aggregation of the C]HCA2969 tracer. Figure 27 (B) shows in Figure 26 The box plot of the placebo group (B) shows the myocardial tissue image of the individual marked with a dashed circle. This individual is [ 11 Individuals with particularly low aggregation of the C]HCA2969 tracer. Figure 27 In (A), mononuclear cell infiltration was confirmed in the tissue, indicating strong inflammation of the myocardium. On the other hand, in Figure 27 In (B), no signs of inflammation were observed. Based on this result, it is believed that [ 11 The C]HCA2969 tracer specifically accumulates in myocardial inflammation.

[0219] 15. [used] 11 Diagnosis of VLA-4 positive lymphocyte-related diseases other than myocarditis using C]HCA2969 tracer

[0220] The experimental autoimmune encephalitis (EAE) model (mouse encephalitis model: EAE mice) was used to evaluate […]. 11 The availability of C]HCA2969 in the diagnosis of VLA-4 positive lymphocyte-related diseases other than myocarditis.

[0221] 15-1.[ 11 Observation of the aggregation of C]HCA2969 tracer signal to the affected area

[0222] (1) Production of EAE mice (immunization)

[0223] 40 mg of H37RA tuberculosis toxin (BD) was suspended in 10 ml of Freund's incomplete adjuvant (BD) to prepare complete Freund's adjuvant (CFA). MOG peptide (MOG 35-55, biosynthesis) was dissolved in physiological saline to a concentration of 1 mg / ml. For each 9-week-old male C57BL / 6J mouse, 100 μl of CFA and 100 μl of MOG solution were mixed / suspended in a glass syringe to prepare an emulsion. The prepared turbid emulsion solution was subcutaneously injected into both groins at a rate of 200 μl per mouse (day 0 of immunization). Then, on the day of emulsion administration, each mouse was intraperitoneally administered 100 μl of a solution of pertussis toxin (LBL) dissolved in PBS (4 μg / ml), followed by a second intraperitoneal administration 48 hours later. Starting from day 0 of immunization, record the disease score of EAE.

[0224] In this model, inflammatory cell infiltration and paralysis of the spinal cord appear within approximately one week, peak within two weeks, and show improvement after four weeks. Inflammation is more prevalent in the spinal cord than in the brain, and the inflammation originates from the lumbar region, thus paralysis begins in the tail and hind limbs.

[0225] In the following experiment, mice with paralysis symptoms and an EAE disease score of 0.5 or higher on day 15 post-immunization were used.

[0226] (2) PET / CT imaging

[0227] On day 15 post-immunization, mice were anesthetized with isoflurane (1-3% / 2L O2), and the isoflurane was injected via the tail vein. 11 C]HCA2969 tracer (1~10 MBq: 10 μg of unlabeled HCA2969 containing 10 MBq of PET tracer). PET / CT imaging was performed 10 minutes after injection, 20 minutes later. Regarding imaging, a region of interest (ROI) was defined in the thoracic-lumbar spinal cord region. 11 The C]HCA2969 tracer signal aggregation is counted to calculate the median SUV value (Bq / ml) and the average SUV value (Bq / ml).

[0228] 3. Results

[0229] Figure 28 The median SUV (Bq / ml) and mean SUV (Bq / ml) are shown in the negative control (Control) group and the EAE model group (EAE). Figure 28 (A) is the median value for SUVs (Bq / ml). Figure 28 (B) represents the average SUV value (Bq / ml). High values ​​were confirmed in the EAE model group. 11 The aggregation of the C]HCA2969 tracer signal indicates that the present invention [ 11 The C]HCA2969 tracer can also detect inflammation in tissues other than the heart.

[0230] 15-2.[ 11 Observation of the entry of C]HCA2969 tracer into the affected area

[0231] Using EAE model mice, observations were conducted [ 11 The C]HCA2969 tracer enters the spinal cord and brain.

[0232] (1) Method

[0233] For the EAE model established in 17-1. above, mice exhibiting paralytic symptoms with a disease score of 0.5 or higher on day 15 post-immunization were administered the drug according to the method described in 17-1.(2). 11 C]HCA2969 tracer. Mice were euthanized 30 minutes after administration, and spinal cord and brain tissue were collected. A portion of the collected tissue was used to determine the radiation dose using a well counter, and another portion was used for HE staining.

[0234] (2) Results

[0235] Figure 29 The results of well-drilled counter measurements are shown. A significant increase in tracer signal accumulation was confirmed in the spinal cord in the EAE model group compared to the negative control group (CTL group). Increased accumulation was also observed in the brain. Inflammatory cell infiltration centered on the spinal cord occurs in the EAE model; therefore, this accumulation is considered to reflect inflammatory cell infiltration.

[0236] Figure 30 The %ID / g values ​​for each individual in the EAE model group and the negative control group (CTL group) are shown. Figure 31 HE-stained images of the spinal cords of individual individuals in the EAE model group are shown. Figure 30 and Figure 31 In the diagram, individuals marked with an asterisk are those identified in the spinal cord. 11 Individuals showed aggregation of the C]HCA2969 tracer. This was confirmed in the spinal cord. 11 In individuals with aggregates of the tracer C]HCA2969, lymphocyte infiltration was confirmed in spinal cord tissue by HE staining in EAE1, EAE2, EAE5, and EA6.

[0237] These results indicate that, 11 The C]HCA2969 tracer can evaluate not only the myocardium but also inflammation in other tissues with lymphocyte infiltration.

Claims

1. A radioactive positron-emitting nuclide carbon-11 labeled compound represented by the following general formula (I), namely [ 11 [C] Labelling compound or its pharmaceutically acceptable salt In general formula (I), R 1 It is a chlorine atom. m is an integer from 1 to 3. R 2 R 3 R 4 R 5 R 6 and R 7 At least one of them is a straight-chain or branched alkyl group having 1 to 6 carbon atoms, said alkyl group having a radioactive positron-emitting nuclide of carbon-11. 11 C], and in addition to that, hydrogen atoms, R 8 It is a hydrogen atom, or a straight-chain or branched alkyl group having 1 to 6 carbon atoms.

2. The [ ] as described in claim 1 11 C] labeling compound or its pharmaceutically acceptable salt, wherein... The [ 11 C] Labelled compounds are represented by the following general formula (II), In general formula (II), R 2 R 3 R 4 R 5 R 6 R 7 and R 8 Same as above.

3. The [ ] as described in claim 1 11 C] labeling compound or its pharmaceutically acceptable salt, wherein... The [ 11 C] The labeled compound is represented by the following formula (1) or (2), ； 。 4. A radioactive composition comprising the [method / composition] of claim 1. 11 [C] labeling compounds or their pharmaceutically acceptable salts.

5. The radioactive composition according to claim 4, used for detecting lymphocytic inflammatory diseases.

6. The radioactive composition according to claim 5, wherein, The lymphocytic inflammatory diseases mentioned are myocarditis or myelitis, among which myocarditis with myosin as an antigen does not include autoimmune myocarditis.

7. The radioactive composition according to claim 5, wherein, The myocarditis described is a type of myocarditis that depends on the binding of Vcam-1 and VLA-4.

8. The radioactive composition according to claim 5, wherein, The myocarditis mentioned is myocarditis caused by immune checkpoint blockers.

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

10. The radioactive composition according to claim 4, used for predicting the therapeutic effect of myocarditis.

11. The radioactive composition according to claim 4, used to determine in patients with myocarditis or suspected myocarditis the following: predicting the therapeutic effect achieved by the VLA-4 blocking substance, and administering the treatment to patients determined to be effective by the treatment.

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