JP2007223952A - DIAGNOSTIC IMAGING PROBE FOR DISEASE OF AMYLOID beta PROTEIN ACCUMULATION - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a diagnostic imaging probe for diseases of amyloid β protein accumulation including Alzheimer's disease. <P>SOLUTION: 6-[2-(Fluoro)ethoxy]-2-[2-(2-dimethylaminothiazol-5-yl)ethenyl]benzoxazole labeled by a radioactive nuclide, preferably a positron emitting nuclide, especially<SP>11</SP>C is used as a diagnostic imaging probe for diseases of amyloid β protein accumulation. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a diagnostic probe compound BF-227 for a disease in which amyloid β protein accumulates, which is labeled with a radionuclide, preferably a positron emitting nuclide, particularly ¹¹ C, and a diagnostic imaging composition and kit comprising the same.

  The diseases in which amyloid accumulates include various diseases characterized by the deposition of insoluble fibrillar protein (amyloid) in various organs and tissues in the body, and include Alzheimer's disease and Down's syndrome. Of these, Alzheimer's disease (AD) is currently regarded as one of the most difficult diseases to treat, and accurate early diagnosis is desired.

  Alzheimer's disease (AD) is considered as one of the most difficult diseases to treat at present, and is a disease for which an accurate early diagnosis is desired. Alzheimer's disease is a disease characterized by progressive dementia that occurs mainly from presenescence to old age. Pathologically, it is characterized by global atrophy of the cerebrum, marked degeneration and loss of nerve cells, neurofibrillary tangles and appearance of senile plaques. It is known that the greatest risk factor of dementia represented by Alzheimer's disease is aging. Therefore, the increase in the number of patients accompanying the increase in the aging population is particularly remarkable in Japan, the United States, and European countries, which are aging societies. Yes.

  In Japan, the number of Alzheimer's disease patients is estimated to be about 1 million, and it is expected that the number of patients will increase as the population ages. Since the cost of Alzheimer's disease patients is considered to exceed 2.5 million yen per patient including the cost of nursing care, we have already paid socio-economic costs exceeding 2.5 trillion yen in Japan. Become. In Alzheimer's disease, it is now common knowledge in the world that adding treatment before or as soon as possible of the symptoms of dementia has a great medical economic effect. However, at present, it is extremely difficult to accurately diagnose Alzheimer's disease at these stages.

  There are various current methods for diagnosing Alzheimer's disease. In Japan, methods such as Hasegawa, ADAS, and MMSE are generally used to quantitatively evaluate the decline in cognitive function of individuals suspected of Alzheimer's disease. Diagnostic methods (MRI, CT, etc.) are used in an auxiliary manner. However, these diagnostic methods are not sufficient to determine the disease, and a definite diagnosis requires a biopsy of the brain before birth (biopsy) and a histopathological examination of the postmortem brain. Thus, despite strenuous research on Alzheimer's disease diagnostic methods, there has not been much progress. Many studies have shown that the neurodegeneration characteristic of Alzheimer's disease has already begun long before the first clinical symptoms appear (in the long case about 40 years ago). In this disease, it is known that the pathological image in the brain has already progressed to an irreversible state when the family or clinician surrounding the patient notices the first clinical symptoms. Considering the progression characteristics of the disease states and the rapid increase in the number of patients as described above, the necessity and significance of an accurate early diagnosis of Alzheimer's disease are extremely great.

  The histopathology of Alzheimer's disease is represented by two main features. Senile plaques and neurofibrillary tangles. The former main component is amyloid β (Aβ) protein having a β sheet structure, and the latter is hyperphosphorylated amyloid β protein. A definitive diagnosis of Alzheimer's disease relies on the appearance of these pathological features in the patient's brain.

  Amyloid β protein is characteristic of diseases in which amyloid accumulates, including Alzheimer's disease, and is closely related. Therefore, detection of amyloid β protein having a β sheet structure in the body, particularly in the brain, as a marker is one of important diagnostic methods for diseases in which amyloid accumulates, particularly Alzheimer's disease. For the purpose of diagnosing diseases in which amyloid accumulates such as Alzheimer's disease, a search for a substance that specifically binds to and stains amyloid β protein in the body, particularly in the brain, has been performed. Examples of such substances include Congo Red (see Non-Patent Document 1) and Thioflavin S (see Non-Patent Document 2), Thioflavin T (see Non-Patent Document 3), Chrysamine G and its derivatives (see Patent Documents 1 and 2), and the like. However, there are many problems in terms of binding specificity to amyloid β protein, blood-brain barrier permeability, solubility, toxicity, and the like.

  There is known a disease in which a protein in the brain itself is caused by a β-sheet structure. In Alzheimer's disease, amyloid β protein and tau protein have a β sheet structure, and it is considered that these proteins themselves are part of the pathogenesis or pathogenesis. Yankner et al. Reported for the first time that amyloid β protein exerts neurocytotoxicity by adopting a β sheet structure (see Non-Patent Document 4). Thereafter, many additional tests were conducted, and it was confirmed that amyloid β protein having a β sheet structure has neurotoxicity. Thus, since neurotoxicity is observed in amyloid β protein and tau protein having a β sheet structure, early detection of these leads to accurate diagnosis and treatment of diseases.

  So far, for the study of Alzheimer's disease or diagnosis using biopsy or autopsy samples, brain sections have been prepared from Alzheimer patients and stained. However, the conventional methods have many unsuitable points for early diagnosis before birth and have a heavy burden on patients.

In recent years, diagnostic imaging using a probe labeled with a radionuclide has come to be used as a diagnostic means that has high detection sensitivity and accuracy of a target substance, is noninvasive, and has a light burden on a patient. The general requirements for diagnostic imaging probes and detection means are that they can be diagnosed in vivo, have little damage to the patient (especially noninvasive), have high detection sensitivity, and have an appropriate half-life. (The labeled probe preparation time and diagnosis time are appropriate). Therefore, recently, positron emission tomography (PET) using γ-rays exhibiting high detection sensitivity and substance permeability or computer tomography (SPECT) using γ-ray emitting nuclides has been used. Among these, PET detects two γ-rays radiated from the positron emitting nuclide in the opposite direction by a coincidence counting method with a pair of detectors, so that information excellent in resolution and quantification can be obtained. .
International Patent Application PCT / US96 / 05918 Specification International Patent Application PCT / US98 / 07889 Specification Puchtler et al., Journal of Histochemistry and Cytochemistry, 10, 35, 1962 Puchtler et al., Journal of Histochemistry and Cytochemistry, 77, 431, 1983 LeVine, Protein Science, 2, 404-410, 1993 Yankner et al., Science, 245, 417-420, 1989

  Therefore, it is a probe for diagnostic imaging of diseases in which amyloid β protein accumulates such as Alzheimer's disease, and has high specificity for amyloid β protein, high blood-brain barrier permeability, high solubility, low toxicity, etc. It was an object of the present invention to develop a probe having the following characteristics and appropriately labeled.

The inventors have identified 6- [2- (fluoro) ethoxy] -2- [2- (2-dimethylaminothiazol-5-yl) ethenyl] benzoxazole (hereinafter referred to as “BF-227”). Excellent specificity, such as high specificity to amyloid β protein, high blood-brain barrier permeability (brain migration), and extremely high safety, and low baseline fluctuation during actual use It was confirmed to have Furthermore, the present inventors have also confirmed that radionuclides, preferably positron emitting nuclei, particularly BF-227 labeled with ¹¹ C, are very suitable for diagnostic imaging using PET for amyloid β protein accumulation by PET. . BF-227 is a compound having the structure shown below.

で示される、アミロイドβ蛋白が蓄積する疾患の画像診断プローブ化合物；
（２）陽電子放出核種が¹¹Ｃである、（１）記載の化合物；
（３）下式：

で示される、（２）記載の画像診断プローブ化合物；
（４）（１）〜（３）のいずれかに記載の画像診断プローブ化合物を含む、アミロイドβ蛋白が蓄積する疾患の画像診断用組成物；
（５）（１）〜（３）のいずれかに記載の画像診断プローブ化合物を含む、アミロイドβ蛋白が蓄積する疾患の画像診断用キット
に関するものである。 Therefore, the present invention
(1) Labeled with a positron emitting nuclide, the following formula:

A diagnostic imaging probe compound for a disease in which amyloid β protein is accumulated;
(2) The compound according to (1), wherein the positron emitting nuclide is ¹¹ C;
(3) The following formula:

A diagnostic imaging probe compound according to (2),
(4) A composition for diagnostic imaging of a disease in which amyloid β protein accumulates, comprising the diagnostic imaging probe compound according to any one of (1) to (3);
(5) The present invention relates to a diagnostic imaging kit for a disease in which amyloid β protein accumulates, comprising the diagnostic imaging probe compound according to any one of (1) to (3).

  According to the present invention, a disease in which amyloid β protein accumulates such as Alzheimer's disease can be image-diagnosed with high sensitivity and specificity using PET or SPECT, and the burden on the patient can be reduced.

A PET diagnostic probe for a disease in which amyloid β protein of the present invention accumulates is a radionuclide, preferably a positron emitting nuclide, especially BF-227 labeled with ¹¹ C, or a salt or solvate thereof if present. It is. The salt of BF-227 is preferably a pharmaceutically acceptable salt. The solvate of BF-227 is also preferably a pharmaceutically acceptable solvate. Unless otherwise specified in this specification, "BF-227", "compound of this invention" or "6- [2- (fluoro) ethoxy] -2- [2- (2-dimethylaminothiazol-5-yl)" References to “ethenyl] benzoxazole” (including those represented by the structural formula) include BF-227 and salts and solvates thereof where possible.

  The labeled BF-227 of the present invention can be synthesized by various methods known to those skilled in the art. Further, as shown in the examples, an appropriate precursor may be synthesized by a known method and labeled.

In the present invention, BF-227 or a salt thereof that specifically binds in vivo to amyloid β protein (also referred to herein as “Aβ protein” or “Aβ”) in a subject in a disease in which amyloid β protein accumulates, Solvates are used labeled with radionuclides, preferably positron emitting nuclides, especially ¹¹ C. BF-227 is extremely specific for Aβ protein, and senile plaques (including diffuse senile plaques) are clearly stained. The term “disease in which Aβ accumulates” in this specification refers to a disease whose main feature is accumulation of Aβ protein in the brain. Examples of diseases that can be diagnosed using Aβ protein, that is, senile plaques, as markers are Alzheimer's disease and Down's syndrome. Etc.

  General requirements for diagnostic imaging probes and detection means include: in vivo diagnosis, low patient damage (especially low or non-toxic, non-invasive), detection sensitivity For example, it has a high half-life (appropriate label probe preparation time and diagnosis time are appropriate), and the baseline fluctuation is small during actual measurement.

Recently, PET or SPECT using γ rays exhibiting high detection sensitivity and substance permeability has been used for various diagnoses and examinations. PET is preferable because two γ-rays radiated from the positron emitting nuclide are detected in the opposite direction by a coincidence counting method with a pair of detectors, and information excellent in resolving power and quantitativeness can be obtained. For PET, probe compounds labeled with positron emitting nuclides such as ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸ F, ⁶² Cu, ⁶⁸ Ga, and ⁷⁶ Br are used. Among positron emitting nuclides, ¹¹ C, ¹³ N, ¹⁵ O, and ¹⁸ F are preferably used from the viewpoints of appropriate half-life and ease of labeling. ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸ F can be used to label BF-227. Especially labeling of preferred BF-227 is ¹¹ C.

For SPECT, γ-ray emitting nuclides such as ^99m Tc, ¹¹¹ In, ⁶⁷ Ga, ²⁰¹ Tl, ¹²³ I, and ¹³³ Xe are used as labels. BF-227 can be labeled with these labels.

Generally, these nuclides are produced by devices called cyclotrons or generators. A person skilled in the art can select a production method and apparatus according to the production nuclide. The nuclides thus produced can be used to label BF-227. When using a nuclide such as ¹¹ C having a moderately short half-life, a desired nuclide is obtained from an (ultra) small cyclotron installed in a facility such as a hospital, and BF-227 is placed at a desired position by a method known in the art. The label can be used immediately for diagnosis, examination, treatment and the like.

The label may be introduced at any position of BF-227. When 11C is used as a label, any carbon of BF-227 may be substituted with ¹¹ C, and preferably one of the two methyl groups of the dimethylamino group is ¹¹ C. For example, when ¹⁸ F is used as a label, the fluorine at the end of BF-227 can be ¹⁸ F.

Sufficient time course for binding and decay to amyloid β protein by administering to the subject BF-227 labeled with a radionuclide, preferably positron emitting nuclide, especially BF-227 labeled with ¹¹ C Later, the examination site can be examined by PET or SPECT. Sufficient time for binding and disintegration to amyloid β protein varies depending on factors such as the type of disease, the condition of the subject, the examination site, etc., and those skilled in the art can easily determine this. Administration of the labeled BF-227 to the subject may be local or systemic. The route of administration includes intradermal, intraperitoneal, intravenous, arterial, or spinal fluid injection or infusion, but can be selected depending on factors such as the type of disease, the nuclide used, the compound used, the condition of the subject, and the examination site. . Known means and methods can be used for this purpose, and can be appropriately selected according to factors such as the type of disease, the state of the target, and the site to be examined.

The dose of BF-227 labeled with a radionuclide varies depending on the type of disease, age of the subject, physical condition, sex, degree of disease, examination site, and the like. In particular, it is necessary to pay close attention to the exposure dose of the subject. Incidentally, the amount of radioactivity of BF-227 labeled with ¹¹ C is usually in the range of 3.7 to 3.7 gigabecrel, and preferably 18 to 740 megabecquerel.

The present invention also provides a diagnostic imaging composition for a disease in which amyloid β protein accumulates, comprising BF-227 labeled with a radionuclide. Preferably, the composition of the present invention comprises a positron radionuclide, especially BF-227 labeled with ¹¹ C and a pharmaceutically acceptable carrier. The form of the diagnostic imaging composition of the present invention is preferably a form that can be injected or infused for that purpose. Accordingly, the pharmaceutically acceptable carrier is preferably a liquid, and is an aqueous solvent such as potassium phosphate buffer, physiological saline, Ringer's solution, distilled water, or polyethylene glycol, vegetable oils, ethanol, glycerin, dimethyl sulfone. There are non-aqueous solvents such as, but not limited to, xylene and propylene glycol. The mixing ratio of the carrier and BF-227 labeled with the radionuclide can be appropriately selected according to the application site, detection means, etc., but is usually a ratio of 100,000 to 1 to 2 to 1, preferably 1. The ratio is 10: 1 to 10: 1. In addition, the composition of the present invention further includes known antibacterial agents (for example, antibiotic agents), local anesthetics (for example, procaine hydrochloride, dibucaine hydrochloride, etc.), buffers (for example, Tris-HCl buffer, hepes buffer, etc.), An osmotic pressure regulator (for example, glucose, sorbitol, sodium chloride, etc.) and the like may be contained.

Furthermore, the present invention provides a diagnostic imaging kit for a disease in which amyloid β protein accumulates, comprising a radionuclide, preferably a positron emitting nuclide, particularly BF-227 labeled with ¹¹ C, as an essential component. Usually, kits are labeled BF-227, the solvent for dissolving it, buffer, osmotic pressure adjusting agent, antibacterial agent, local anesthetic agent, etc. It is a collection of things in a container.

  In the above kit, the labeled BF-227 may be provided as a solid such as a lyophilized powder, or may be provided by dissolving in a suitable solvent. The solvent may be the same as the carrier used in the above-described composition of the present invention. In addition, each component such as a buffer, an osmotic pressure regulator, an antibacterial agent, and a local anesthetic may be the same as that used in the above-described composition of the present invention. Various containers for storing each component can be selected as appropriate. For example, the container may be made of a light-shielding material, or may be shaped like a vial or a syringe for convenient administration to a patient. The kit may appropriately include instruments necessary for diagnosis, for example, an instrument used for a syringe, an infusion set, or an image diagnostic apparatus to be used. In general, instructions are attached to the kit.

Furthermore, the present invention provides an image of a disease in which amyloid β protein accumulates, characterized by using a radionuclide, preferably a positron emitting nuclide, particularly BF-227 labeled with ¹¹ C or a salt or solvate thereof as a probe. Provide a diagnostic method.

  As described above, Congo Red or Thioflavin S, which has been researched and developed conventionally, stains both amyloid β protein and neurofibrillary tangles, but BF-227 is highly specific for amyloid β protein. Therefore, BF-227 is useful for, for example, image diagnosis of diseases in which amyloid β protein accumulates and research of such diseases. In addition, many conventional compounds such as Congo Red or Thioflavin S do not specifically bind to diffuse senile plaques, but BF-227 binds to it. Amyloid β (Aβ) protein, the main component of senile plaques in Alzheimer's disease, is thought to start accumulating long before the disease develops (at least 10 years before dementia manifests) This initial accumulation image is considered to be diffuse senile plaque. Since BF-227 also binds to diffuse senile plaques, it is extremely useful for early detection and diagnosis of diseases in which amyloid β protein accumulates, such as Alzheimer's disease, by combining with image diagnostic methods.

  Procedures and methods for diagnosing a disease in which amyloid β protein accumulates using BF-227 labeled with a radionuclide are known in the art. The subject is rested, preferably administered BF-227 labeled by injection or infusion as a liquid formulation, and image data is obtained after time for binding and disintegration to Aβ. Various means and methods known in the art can be used for image data analysis. In the case of using PET, it is preferable to perform image data analysis using a standardized uptake value (SUV) method, a distribution volume ratio (DVR) method, a DVR method using a Logan method, or the like.

  The present invention will be described in more detail and specifically with reference to the following examples. However, the examples should not be construed as limiting the present invention.

  Various properties of BF-227 were examined. The compound used was not labeled. The test items were: (a) β structure recognition test, (b) staining test for amyloid β protein on brain section of Alzheimer's disease patient, (c) acute toxicity test, and (d) brain migration test. These test methods and procedures are described below.

(A) β Structure Recognition Test (1) Amyloid β protein (purchased from Peptide Institute) was dissolved in phosphate buffer (pH 7.4) and allowed to stand at 37 ° C. for 4 days.
(2) 50 μl of Congo red dissolved in the same buffer was dispensed into a 96-well microplate (final concentration 0.1 μM).
(3) 100 μl of amyloid β protein was added (final concentration 5 μM) and left for 30 minutes.
(4) 100 μl of BF-227 dissolved in the same buffer was added (final concentration 10 μM) and left for 60 minutes.
(5) Using a fluorescence microplate reader (manufactured by Molecular Devices, fmax), the measurement was performed at the optimum excitation and measurement wavelength, which had been measured in advance.
(6) Fluorescence in the presence of BF-227, amyloid β protein and Congo red is A, B is in the presence of test compound and Congo red, C is in the presence of BF-227 and amyloid β protein, and BF-227 The degree of β structure recognition of BF-227 was calculated by the following equation, where D was a single substance.
BF-227β structure recognition degree (%) = {(A−B) / (C−D)} × 100
(7) It can be said that the higher the β structure recognition degree, the higher the binding specificity of the compound for amyloid β protein.

(B) Staining test of amyloid β protein on brain sections of Alzheimer's disease patients (1) Brain specimens in temporal lobes or hippocampus of patients and normal elderly who were pathologically confirmed as Alzheimer's disease were used.
(2) The paraffin-embedded brain tissue was sliced at a thickness of 6 μm or 8 μm, spread on a slide glass, and dried. The paraffin brain sections were deparaffinized in the order of xylene 10 minutes × 2, 100% ethanol 15 minutes × 2, 95% ethanol 15 minutes × 2, and washing with running water 10 minutes.
(3) As a pretreatment for staining with the compound of the present invention, a treatment for removing autofluorescence by lipofuscin was performed. First, the deparaffinized section was immersed in a 10% formalin solution for 60 minutes, washed with PBS for 5 minutes, and then immersed in a 0.25% KMnO ₄ solution for 90 minutes. After washing with PBS for 2 minutes × 2 times, it was immersed in a 0.1% K ₂ S ₂ O ₅ / oxalic acid solution for about 30 seconds, and further washed with PBS for 2 minutes × 3 times.
(4) About 150 μl of 100 μM BF-227 solution dissolved in 50% ethanol was dropped and reacted for 10 minutes. After 5 times in tap water, 3-5 times in 50% ethanol to quickly separate, then soaked in PBS for 60 minutes, sealed with Fluor Save Reagent (Calbiochem) and fluorescent microscope (Nikon, Eclips E800). Images were taken with a digital camera (Plaroid PDMCII).
As a control, an immunostaining test of amyloid β protein on a brain section of an Alzheimer's disease patient was performed according to the following procedure.
(1) After the deparaffinization step in (b), washing was performed in distilled water for 2 minutes × 2 and the tissue was surrounded with immunopen, and then about 150 μl of formic acid was dropped and allowed to stand at room temperature for 5 minutes. After washing with tap water for 5 minutes, it was immersed in cold PBS-Tween 20 for 2 minutes, and then approximately 150 μl of 0.05% trypsin solution was dropped and reacted at 37 ° C. for 15 minutes.
(2) After washing with cold PBS-Tween 20 for 5 minutes × 2 times in an ice bath, drop 2 drops of blocking serum and react at 37 ° C. for 30 minutes to remove excess water, then amyloid β protein About 150 μl of 6F / 3D (DAKO, diluted 1:50), which is a specific antibody, was dropped and reacted at 37 ° C. for 1 hour.
(3) After further washing with cold PBS-Tween 20 for 2 minutes × 5 times, 2 drops of anti-mouse IgG (H + L), goat and biotin-binding solution were added dropwise, reacted at 37 ° C. for 1 hour, and 2 times with cold PBS-Tween 20 After washing 3 times for 3 minutes, 2 drops of ABC solution (streptavidin-biotin-peroxidase complex solution) was dropped and allowed to stand for 30 minutes. Again, after washing with cold PBS-Tween 20 for 2 minutes × 3 times, DAB solution (10 mg of DAB dissolved in 20 ml of 0.05 mol / l Tris-HCl buffer and 100 μl of 3% hydrogen peroxide solution added immediately before use) About 150 μl of the solution was dropped to obtain a sufficient color, and then washed with distilled water for 1 minute to stop the reaction.

(C) Acute toxicity test The acute toxicity of BF-227 was examined by intravenous administration using mice. Crj: CD1 male mice were used as 4 mice per group (the average body weight of each group was 31 to 32 g). BF-227 was dissolved in a mixture of 1N HCl, polyethylene glycol 400 and distilled water, or dissolved in DMSO, diluted with distilled water, administered via the tail vein, and observed up to 7 days thereafter.

(D) Brain transportability test BF-227 was intravenously administered to mice, and brain transportability in vivo was measured.
(1) Mice used 30-40 g (7 weeks old, n = 3) of Slc: ICR (Japan SLC).
(2) BF-227 is dissolved in a mixture of 1N HCl, polyethylene glycol 400 and DMSO, dissolved in DMSO or ethyl alcohol, diluted with purified water, injected through the tail vein, and ether anesthetized 2 minutes after administration. Below, blood was collected from the abdominal aorta using a heparin-treated syringe, and brain samples were collected.
(3) The blood was centrifuged at 4 ° C. and 14,000 rpm for 10 minutes after blood collection, and the supernatant was stored as plasma at −80 ° C. The brain (including cerebellum) was stored at −80 ° C. after sampling.
(4) At the time of use, the plasma was dissolved, diluted with purified water, added to a conditioned C18 solid phase extraction cartridge (bond elute C18, 200 mg, Varian), and eluted with methyl alcohol. Alternatively, after dissolution, a diethyl ether / cyclohexane mixture was added and shaken, and then centrifuged to dispense the oil layer.
(5) When the brain was in use, the wet weight was measured, physiological saline was added, and homogenization was performed. The homogenate was centrifuged for 10 minutes, and the supernatant was added to a conditioned C18 solid-phase extraction cartridge and eluted with methyl alcohol. Alternatively, after measuring the wet weight, the mixture was homogenized by adding diethyl ether / cyclohexane mixture, shaken, and then centrifuged to dispense the oil layer.
(6) Absorption and fluorescence were detected using high performance liquid chromatography.
(7) For each plasma and brain, the BF-227 content (% ID (injected dose) / ml or g) in the plasma or brain relative to the dose was determined.

  Various characteristics of BF-227 obtained by the above test were as follows.

  (A) The β structure recognition degree of BF-227 is about 1.2 times that of thioflavin T (conventionally specific to Aβ), indicating extremely high specificity of BF-227 for Aβ. .

  (B) The staining properties of BF-227 (FIG. 1 left panel) and anti-Aβ antibody 6F / 3D immunostaining (FIG. 1 right panel = adjacent section of FIG. 1 left panel) in brain sections of Alzheimer's disease patients were compared. BF-227 was found to stain senile plaques (wedge-shaped arrowheads) and diffuse senile plaques (dotted circles) stained with anti-Aβ antibody 6F / 3D.

(C) In general, a single intravenous administration of 1 × 10 ⁻¹² to 1 × 10 ⁻⁵ mg / kg is used as a total dose of positron-labeled and unlabeled compounds for human PET imaging, and in many cases Intravenous administration of 1 × 10 ⁻¹⁰ to 1 × 10 ⁻⁷ mg / kg is used. The maximum tolerated dose of BF-227 is 10 mg / kg or more, and considering the total amount of compounds required at the time of PET imaging, there is a gap of at least 100,000 times between them. It is considered a very safe compound as a probe for photographing.

  (D) The content in the brain 2 minutes after administration of BF-227 was 7.9% ID / g. It is considered that 0.5% ID / g or more is sufficient for the brain migration of a compound for PET or SPECT targeting the central nervous system. In that sense, BF-227 is a compound with extremely high brain migration.

  Thus, BF-227 was found to be a highly safe compound with high specificity for amyloid β protein, high blood-brain barrier permeability (brain migration), and high safety.

で示される前駆体化合物６−［２−（フルオロ）エトキシ］−２−［２−（２−メチルアミノチアゾール−５−イル）エテニル］ベンゾキサゾール（以下、「ＴＨＫ−００１」と称する）のメチルアミノ基の水素を、以下に説明するメチル化反応により［¹¹Ｃ］ＣＨ₃とすることにより、［¹¹Ｃ］ＢＦ−２２７を得た。 Synthesis under formula BF-227 labeled with Example ^{1 11 C ([11 C]} BF-227):

Of the precursor compound 6- [2- (fluoro) ethoxy] -2- [2- (2-methylaminothiazol-5-yl) ethenyl] benzoxazole (hereinafter referred to as “THK-001”) [ ¹¹ C] BF-227 was obtained by changing the hydrogen of the methylamino group to [ ¹¹ C] CH ₃ by a methylation reaction described below.

THK-001 was synthesized as described below.

(1) Synthesis of 3 2 (10.0 g, 99.9 mmol) was dissolved in tetrahydrofuran (20 ml), (Boc) ₂ O (27.5 ml, 119.9 mmol) was added, and the mixture was stirred at room temperature for 16 hours. The solvent of the reaction solution was distilled off under reduced pressure, and n-hexane was added to the obtained crystals and pulverized to obtain 3 (19.25 g, 96%) as colorless crystals.

mp: 181.5-182.5 ° C, ¹ H NMR (CDCl ₃ ) δ 1.60 (9H, s), 6.88 (1H, d, J = 3.7Hz), 7.38 (1H, d, J = 3.7Hz), 11.9 (1H, s), IR (Nujol) 1713 cm ^-1 , APCI-MS m / z: 201 [M + H] ⁺

(2) Synthesis of 4 3 (10.0 g, 49.9 mmol) was dissolved in tetrahydrofuran (100 ml), t-butoxypotassium (6.72 g, 59.9 mmol) was added with stirring at 0 ° C., and at the same temperature. The mixture was stirred for 10 minutes, iodomethane (4.66 ml, 74.9 mmol) was added, and the mixture was stirred at room temperature for 16 hours. The insoluble matter in the reaction solution was filtered off, the solvent in the filtrate was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane / ethyl acetate = 9, 1), and 4 (6.53 g, 61%). ) Was obtained as a colorless solid.

mp: 51-52 ° C, ¹ H NMR (DMSO) δ 1.53 (9H, s), 3.47 (3H, s), 7.25 (1H, d, J = 3.5Hz), 7.45 (1H, d, J = 3.5Hz ), IR (Neat) 1734 cm ^-1 , APCI-MS m / z: 215 [M + H] ⁺

(3) Synthesis of 5 To a solution of diisopropylamine (5.12 ml, 36.4 mmol) in tetrahydrofuran (50 ml) under an argon atmosphere at −60 ° C. or lower, a 1.58 M n-butyllithium / n-hexane solution (23.0 ml). , 36.4 mmol) was added dropwise, and the temperature was gradually raised to 0 ° C. Next, a solution of 4 (7.08 g, 33.1 mmol) in tetrahydrofuran (10 ml) was added dropwise at −70 ° C. or lower, and the mixture was stirred at −78 ° C. for 1 hour. At the same temperature, a solution of N-formylmorpholine (4.98 ml, 49.6 mmol) in tetrahydrofuran (10 ml) was added all at once, and the mixture was stirred at the same temperature for 30 minutes. Cold water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The extract was washed with water and dried, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane / ethyl acetate = 10-4) to obtain 5 (6.40 g, 80%) as a yellow solid.

mp: 80-81 ° C, ¹ H NMR (DMSO) δ 1.55 (9H, s), 3.52 (3H, s), 8.38 (1H, s), 9.90 (1H, s), IR (Nujol) 1714, 1659 cm ^-1 , APCI-MS m / z: 243 [M + H] ⁺

(4) Synthesis of 7 To a solution of 6 (1.20 g, 8.1 mmol) synthesized in literature method 1) in tetrahydrofuran (30 ml) was added 2-fluoroethanol (1.04 ml, 17.7 mmol) and triphenylphosphine (4 .64 g, 17.7 mmol) was added, and diisopropyl azodicarboxylate (3.48 ml, 17.7 mmol) was added dropwise with stirring at room temperature, followed by stirring at room temperature for 16 hours. The solvent of the reaction solution was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (n-hexane / ethyl acetate = 1) to obtain 7 (1.50 g, 96%) as a colorless oil.

¹ H NMR (DMSO) δ 2.56 (3H, s), 4.29 (2H, dt, J = 30.1, 3.7Hz), 4.77 (2H, dt, J = 47.9, 3.7Hz), 6.96 (1H, dd, J = 8.7, 2.3Hz), 7.33 (1H, d, J = 2.3Hz), 7.54 (1H, d, J = 8.7Hz), APCI-MS m / z: 196 [M + H] ⁺
1) Fujita, S., Koyama, K., Inagaki, Y .; Synthesis, 1982, 68.

(5) Synthesis of 8 To a solution of diisopropylamine (1.51 ml, 10.7 mmol) in tetrahydrofuran (40 ml) under an argon atmosphere at −60 ° C. or lower, a 1.58 M n-butyllithium / n-hexane solution (6.78 ml). , 10.7 mmol) was added dropwise, and the temperature was gradually raised to 0 ° C. Next, a solution of 7 (1.90 g, 9.7 mmol) in tetrahydrofuran (10 ml) was added dropwise at −70 ° C. or less, and the mixture was stirred at −78 ° C. for 1 hour. At the same temperature, a solution of 5 (3.06 g, 12.6 mmol) in tetrahydrofuran (10 ml) was added dropwise and stirred at the same temperature for 30 minutes. Cold water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The extract was washed with water and dried, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane / ethyl acetate = 1) to obtain 8 (2.26 g, 53%) as a pale orange oil.

¹ H NMR (DMSO) δ 1.51 (9H, s), 3.40 (3H, s), 4.28 (2H, dt, J = 30.0, 3.7Hz), 4.76 (2H, dt, J = 47.7, 3.7Hz), 5.33 (1H, m), 6.02 (1H, d, J = 5.3Hz), 6.97 (1H, dd, J = 8.7, 2.3Hz), 7.27 (1H, s), 7.34 (1H, d, J = 2.3Hz) , 7.56 (1H, d, J = 8.7Hz), APCI-MS m / z: 438 [M + H] ⁺

(6) Synthesis of 9 Triethylamine (4.33 ml, 20.7 mmol) was added to a solution of 8 (2.26 g, 5.2 mmol) in dichloromethane (30 ml), and methanesulfonyl chloride (0.800 ml, 10 ml) was stirred with ice cooling. .3 mmol) was added dropwise and stirred at room temperature for 30 minutes. Cold water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The extract was washed with water and dried, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate) and recrystallized from ethyl acetate / n-hexane to obtain 9 (1.61 g, 74%) as orange crystals.

mp: 194-195 ° C, ¹ H NMR (DMSO) δ 1.56 (9H, s), 3.49 (3H, s), 4.33 (2H, dt, J = 29.9, 3.5Hz), 4.78 (2H, dt, J = 47.7, 3.5Hz), 6.77 (1H, d, J = 16.1Hz), 7.02 (1H, dd, J = 8.7, 2.2Hz), 7.37 (1H, d, J = 2.2Hz), 7.62 (1H, d, J = 8.7Hz), 7.84 (1H, d, J = 16.1Hz), 7.87 (1H, s), IR (Nujol) 1695, 1632 cm ^-1 , APCI-MS m / z: 420 [M + H] ⁺

(7) Synthesis of 1 (THK-001) 9 (1.00 g, 2.4 mmol) was suspended in dichloromethane (24 ml), and trifluoroacetic acid (6 ml) was added dropwise with stirring under ice-cooling. Stir for hours. Ice pieces were added to the reaction solution, an aqueous potassium carbonate solution was added to adjust to pH = 8, and the mixture was extracted with ethyl acetate. The extract was washed with water and dried, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate) and recrystallized from ethyl acetate to obtain 1 (633 mg, 83%) as yellow crystals.

mp: 234-236 ° C, ¹ H NMR (DMSO) δ 2.89 (3H, d, J = 4.6Hz), 4.30 (2H, dt, J = 30.2, 3.9Hz), 4.77 (2H, dt, J = 47.8, 3.9Hz), 6.28 (1H, d, J = 15.7Hz), 6.97 (1H, dd, J = 8.7, 2.4Hz), 7.31 (1H, d, J = 2.4Hz), 7.51 (1H, s), 7.54 (1H, d, J = 8.7Hz), 7.74 (1H, d, J = 15.7Hz), 8.20 (1H, q, J = 4.6Hz), IR (Nujol) 3240, 1637, 1618 cm ^-1 , APCI- MS m / z: 320 [M + H] ⁺

Next, a synthesis scheme of [ ¹¹ C] BF-227 from THK-001 is shown.

[ ¹¹ C] CO ₂ was synthesized by irradiating N 2 gas (containing 0.5% O ₂ gas) with a 12 MeV proton beam accelerated by a cyclotron HM12 (manufactured by Sumitomo Heavy Industries) for 30 minutes. Then using the MicroLab methyl iodide synthesis system (GE Co.) was synthesized ^[11 C] CH ₃ I from ^[11 C] CO _2, by passing a heated AgOTf packed column (about 190 ° C.) ^[11 C] CH ₃ I was converted to [ ¹¹ C] CH ₃ OTf. Then, THK-001 (about 1 mg) is dissolved in DMSO (450 μL), and helium gas containing [ ¹¹ C] CH ₃ OTf is poured into a solution to which 2M NaOH aqueous solution (7 μL) is added at room temperature. Then, ¹¹ C-labeled methylation reaction was performed. To this reaction solution, 5% AcOH (ethanol solution, 150 μL) was added and stirred well. The mixture was subjected to semi-preparative HPLC (column: YMC-Pack Pro C18 RS (10 × 250 mm) + guard column (10 × 30 mm), mobile phase: A radioactive peak derived from [ ¹¹ C] BF227 eluting at about 9-10 minutes was collected over EtOH / MeCN / 20 mM NaH ₂ PO ₄ = 15/40/45, flow rate: 6.0 mL / min). The radiochemical yield determined from preparative HPLC chromatogram was 50% or more. The [ ¹¹ C] BF-227-containing fraction was diluted with distilled water (about 30 mL) and loaded onto a Sep-Pak tC18 cartridge (manufactured by Waters). After washing the cartridge with distilled water, [ ¹¹ C] BF— 227 was eluted. Appropriate amounts of 25% ascorbic acid aqueous solution and 5% polysorbate 80 ethanol solution were added to this EtOH eluate, and the solvent was distilled off with a rotary evaporator while heating at 80 ° C. The resulting [ ¹¹ C] BF-227-containing radioactive residue was obtained. Dissolved in physiological saline and sterilized by filtration through a 0.22 μm filter. The radiochemical purity of the [ ¹¹ C] BF-227-containing physiological saline was 95% or more, and the average specific activity was about 170 GBq / μmol.

Example 2 Clinical Trial Using [ ¹¹ C] BF-227 An exploratory clinical trial was conducted with the approval of the Ethics Committee of Tohoku University School of Medicine. The committee approval number is 2005-52 (as of June 20, 2005) for studies using normal healthy individuals, and 2005-186 (as of October 18, 2005) for studies using Alzheimer's disease patients. ).

括弧内はそれぞれ個々の年齢およびＭＭＳＥスコアを示す。
ＭＭＳＥ：Mini-Mental State Examination（Folstein MF et al.: Mini-mental state". A practical method for grading the cognitive state of patients for the clinician. J Psychiat Res 12 :189-198. 1975）。 Normal healthy individuals were recruited in the Department of Functional Pharmacology, Graduate School of Medicine, Tohoku University, and Alzheimer's disease patients were recruited in the Department of Respiratory Medicine and Advanced Kampo Medicine. All participants were confirmed to be normal by performing chest radiographs, electrocardiograms, blood biochemical tests, etc. as general medical examinations, and in order to evaluate the presence or absence of cerebrovascular disorders and the degree of brain atrophy. An MRI scan was performed. Seven normal healthy individuals who were confirmed to have normal cognitive function by neuropsychological tests, including 3 healthy young people and 4 healthy elderly people, and NINCDS-ADRDA (National Institute of Neurologic and Communicative Disorders and Stroke-Alzheimer's Disease and Related Disorders Association criteria (McKahann G et al .: Clinical diagnosis of Alzheimer's disease-report of the NINCDS-ADRDA Work Group under the auspieces of Department of Health and Human Services Task Force on Alzheimer's disease-. Neurology 34: 939 1984) and 4 patients diagnosed with “probable Alzheimer's disease” participated in an exploratory clinical trial (Table 1).

Each age and MMSE score are shown in parentheses.
MMSE: Mini-Mental State Examination (Folstein MF et al .: Mini-mental state ". A practical method for grading the cognitive state of patients for the clinician. J Psychiat Res 12: 189-198. 1975).

The average and standard deviation were calculated according to the following formula.

PET imaging of exploratory clinical trial participants was performed according to the following procedure.
As the PET scanner, SET-2400W (manufactured by Shimadzu Corporation) was used in the three-dimensional mode. The subject was in the supine position on the bed of the PET scanner, the head was fixed to the head holder, and then a transmission scan using an external radiation source for absorption correction was performed for 8 minutes. Subsequently, [ ¹¹ C] BF-227 5-10 mCi was rapidly administered intravenously from a venous catheter previously secured in the upper right arm vein, and dynamic imaging was performed for 90 minutes from the start of administration. The imaging protocol was 29 frames in total: 30 seconds X5 frames, 60 seconds X5 frames, 150 seconds X5 frames, 300 seconds X14 frames. The left forearm was heated, a catheter was placed in the arterial brachial vein, and continuous blood collection was performed simultaneously with the start of imaging. Blood sampling time is 20 seconds, 40 seconds, 1 minute, 1 minute 20 seconds, 1 minute 40 seconds, 2 minutes, 2 minutes 20 seconds, 2 minutes 40 seconds, 3 minutes, 4 minutes, 5 minutes, 7 minutes after the start of administration. 30 seconds, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, total 22 times. Three times after 30 minutes, 5 ml was used for metabolic analysis, and other times were 1.5 ml. The collected blood sample was transferred to a heparin-containing blood collection tube, centrifuged at 3000 rpm for 3 minutes, only the plasma sample was separated, and then the radioactivity in blood at each time point was measured by a gamma counter. After applying attenuation correction and metabolite correction, a time activity curve in plasma was calculated and used as an input function in quantitative calculations.

  Image analysis was performed according to the following procedure. An SUV (Standardized Uptake Value) image was created by a PET measurement program manufactured by Shimadzu Corporation. Further, the dynamic image data and the image obtained by averaging each frame were converted to the ANALYZE format for processing with commercially available software. The format of each individual's MRI T1-weighted image (cross section, slice thickness 2 mm) was also converted. Next, using the registration function of SPM2 software (Welcome Department of Cognitive Neurology, London, UK), the MRI T1-weighted image was converted to match the PET added image, and the slice was cut out. Subsequently, the converted MRI image is converted into a Dr. View / LINUX software (manufactured by Asahi Kasei Information System), frontal lobe, temporal lobe, parietal lobe, occipital lobe, anterior cingulate gyrus, striatum, thalamus, medial temporal lobe, bridge, cerebellar cortex A region of interest (ROI) was set in each white matter region. Thereafter, the ROI information set on the MRI is read onto the PET dynamic image, and Dr. Radioactivity in each region and each time frame was measured using View / LINUX software. Quantitative analysis was performed using the radioactivity in each brain region measured by the above processing and the input function of the plasma sample. For quantitative analysis, Logan J et al .: (1990) Graphical analysis of reversible radioligand binding from timeactivity measurements applied to N-11C-methyl]-()-cocaine PETstudies in human subjects. J Cereb Blood Flow Metab 10: 7407, 1990 and Logan J. Graphical analysis of PET data applied to reversible and irreversible tracers. Nucl Med Biol 27: 661670, 2000). Using the cerebellar cortex where senile plaques (or fibrotic amyloid β protein) do not accumulate as a reference region, the distribution volume ratio (DVR) of each region was calculated and a DVR calculation image using the Logan method was created. .

  SUVs in the cerebellar cortex region where senile plaques (or amyloid β protein) are known not to accumulate in Alzheimer's disease are shown in FIG. 2, and the DVR and Logan methods for each region created using the cerebellar cortex region as a reference region (standard) The DVR (Logan DVR) of each region used and the DVR (Logan DVR) calculation image using the Logan method are shown in FIGS. 3, 4 and 5, respectively.

  According to many studies so far, senile plaques (or amyloid β protein) in Alzheimer's disease hardly accumulate in the cerebellar cortex region, but mainly in the cerebral cortex (frontal lobe, temporal lobe, parietal lobe, occipital lobe, frontal zonal). (Braak H, Braak E: Neuropthological Stageing of Alzheimer-related changes. Acta Neuropathol 82. 239-259.1991).

As shown in FIG. 2, the [ ¹¹ C] BF-227 SUV in the cerebellar cortex region showed little difference between healthy elderly and Alzheimer's disease patients. This indicates that senile plaques (or amyloid β protein) are not accumulated in this region in both healthy elderly and Alzheimer's disease patients.

As shown in FIG. 3, DVRs in the frontal lobe, temporal lobe, parietal lobe, occipital lobe, frontal cingulate gyrus, and posterior cingulate gyrus of healthy young and healthy elderly who created the cerebellar cortex region as a reference region (standard) It was almost 1. This indicates that senile plaques (or amyloid β protein) are hardly accumulated in these regions of healthy young people and healthy elderly people.
On the other hand, DVR in the frontal lobe, temporal lobe, parietal lobe, occipital lobe, anterior cingulate gyrus, and posterior zonal gyrus of Alzheimer's disease patients was clearly higher than those in healthy young and healthy elderly.

  As shown in FIG. 4, the Logan DVR in the frontal lobe, temporal lobe, and parietal lobe of healthy young people and healthy elderly people who created the cerebellar cortex region as a reference region (standard) was almost 1. This indicates that senile plaques (or amyloid β protein) are hardly accumulated in these regions of healthy young people and healthy elderly people.

  On the other hand, Logan DVR in the frontal lobe, temporal lobe, and parietal lobe of Alzheimer's disease patients was clearly higher than those in healthy young people and healthy elderly people. When the threshold value of Logan DVR in the frontal lobe, temporal lobe, and parietal lobe was 1.1, both the sensitivity (Sensitivity) and specificity (specificity) for diagnosing Alzheimer's disease were 100%.

When comparing representative examples of Logan DVR calculation images among healthy young people, healthy elderly people, and Alzheimer's disease patients created using the cerebellar cortex region as a reference region (standard) as shown in FIG. There was no difference between Alzheimer's patients.
On the other hand, between healthy subjects and Alzheimer's disease patients, as shown in FIG. 3 and FIG.

As described above, a method for diagnosing a disease in which amyloid β protein accumulates using PET using [ ¹¹ C] BF-227 as a probe, particularly Alzheimer's disease, and an SUV method using PET using [ ¹¹ C] BF-227 as a probe A method for diagnosing a disease in which amyloid β protein accumulates, particularly Alzheimer's disease, a method for diagnosing a disease in which amyloid β protein accumulates by the DVR method using PET using [ ¹¹ C] BF-227 as a probe, In addition, a method for diagnosing a disease in which amyloid β protein accumulates by the Logan DVR method using PET using [ ¹¹ C] BF-227 as a probe, particularly Alzheimer's disease, can obtain high sensitivity and specificity.

  The present invention can be used in the field of diagnostic agents, particularly in the field of diagnostic agents for diseases in which amyloid β protein accumulates, such as Alzheimer's disease, and in the field of research for diseases in which amyloid β protein accumulates.

FIG. 1 shows a comparison of staining properties of BF-227 (left panel) and anti-Aβ antibody 6F / 3D immunostaining (right panel, adjacent section on left panel) in brain sections of Alzheimer's disease patients. The wedge-shaped arrow indicates senile plaques stained with the anti-Aβ antibody 6F / 3D, and the dashed circle indicates diffuse senile plaques. FIG. 2 is a graph showing the SUV (Standardized Uptake Value) of [ ¹¹ C] BF-227 in the cerebellar cortex of healthy elderly people and Alzheimer's disease patients. FIG. 3 is a graph showing DVR (Distribution Volume Ratio) in each region of healthy young people, healthy elderly people, and Alzheimer's disease patients when the cerebellar cortex is used as a reference region. FIG. 4 is a graph showing Logan DVR (Distribution Volume Ratio) in each region of healthy young people, healthy elderly people, and Alzheimer's disease patients when the cerebellar cortex is used as a reference region. FIG. 5 is a Logan DVR (Distribution Volume Ratio) calculation image of a healthy young person (36 years old), a healthy elderly person (69 years old), and an Alzheimer's disease patient when the cerebellar cortex is used as a reference region.

Claims (5)

Labeled with a positron emitting nuclide, the following formula:

A diagnostic imaging probe compound for diseases in which amyloid β protein accumulates, Positron emitting nuclide is ¹¹ C, a compound according to claim 1. The following formula:

The diagnostic imaging probe compound of Claim 2 shown by these.   A diagnostic imaging composition for a disease in which amyloid β protein accumulates, comprising the diagnostic imaging probe compound according to any one of claims 1 to 3.   A diagnostic imaging kit for a disease in which amyloid β protein accumulates, comprising the diagnostic imaging probe compound according to any one of claims 1 to 3.

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