Radioactive fluorine-labeled diagnostic imaging agents for measuring drug-metabolizing enzyme activities

Abstract

To provide a 18F-labeled mequitazine derivative that has a high labeling rate of 18F and also has high drug stability after labeling.SOLUTION: The present invention discloses a compound represented by formula (I) (where Y- is an anion); a test drug for measuring metabolism containing the compound; and a method for producing the compound that includes reacting a compound represented by formula: 18F-CH2-Y (where Y is a group detachable as an anion) with mequitazine. The test drug measures drug metabolism by measuring the amount of radioactive metabolite of the compound as biliary excretion.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a radioactive fluorine-labeled diagnostic imaging agent for measuring drug-metabolizing enzyme activity. [Background technology]

[0002] After a drug is ingested, most of it is metabolized into other compounds by drug-metabolizing enzymes. It is known that the activity of these drug-metabolizing enzymes varies from person to person, and the amount of metabolites varies depending on the level of activity. Therefore, even if the drug is administered at the same dose, there are individual differences in the efficacy and side effects. By measuring the individual differences in the activity of these drug-metabolizing enzymes, it is thought that it is possible to determine the dosage of a drug that is tailored to the individual, achieving the required efficacy while minimizing side effects. In addition, determining the dosage based on the measurement of drug-metabolizing enzyme activity is thought to be very effective from the perspective of evidence-based personalized medicine, which has become increasingly important in recent years.

[0003] Mequitazine (10-[(3RS)-1-azabicyclo[2.2.2]oct-3-ylmethyl]-10H-phenotiazine) is a drug used clinically as an antihistamine. It has also been reported to be a substrate drug for CYP2D6, which is a representative drug-metabolizing enzyme and is considered to show relatively large individual differences among CYP molecular species (Non-Patent Document 1).

[0004] Patent Document 1 states: Formula (II):

[0005] [ka]

[0006] (In the formula, X 2 represents a radionuclide or a chelating moiety for labeling a radionuclide, Y - represents an anion.) It is disclosed that a mequitazine derivative represented by the formula: is useful as a test agent for measuring drug metabolism function, and the radioactive nuclide is 18-fluorine ( 18 F) is listed. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2016-69311 A (claims 1 and 3) [Non-patent literature]

[0008] [Non-Patent Document 1] K. Nakamura, et al.; Oxidation of histamine H1 antagonist mequitazine is catalyzed by cytochrome P450 2D6 in human liver microsomes, J. Pharmacol. Exp. Ther. 1998 Feb; 284(2): 437-442. Summary of the Invention [Problem to be solved by the invention]

[0009] The present inventors have aimed to quantify real-time drug-metabolizing enzyme activity by nuclear medicine imaging diagnosis, and have discovered a compound having a nuclide for positron emission tomography (PET), which is represented by the formula (II) X 2 but 18 F-CH2-, and Y - Compounds in which the compound is a bromide ion (hereinafter referred to as 18 F-FMQ) was synthesized. 18 The F labeling rate was low, and the drug stability after labeling was also unsatisfactory.

[0010] The object of the present invention is to 18 High F labeling rate and excellent drug stability after labeling 18The present invention aims to provide an F-labeled mequitazine derivative. [Means for solving the problem]

[0011] Means for Solving the Problems The present inventors have conducted extensive research in order to solve the above problems, and as a result have completed the present invention.

[0012] That is, the gist of the present invention is as follows. (1) The following formula (I): [ka] (In the formula, Y - represents an anion.) A compound represented by the formula: (2) A test agent for measuring metabolic function, comprising the compound according to (1) above. (3) The test agent according to (2) above for measuring drug metabolism function by measuring the amount of a radioactive metabolite of the compound according to (1) above excreted in the bile. (4) The following formula: 18 F-CH2-Y (In the formula, Y represents a group that can be eliminated as an anion.) A method for producing the compound according to (1) above, comprising reacting a compound represented by the following formula: with mequitazine. Effect of the Invention

[0013] According to the present invention, 18 High F labeling rate and excellent drug stability after labeling 18 F-labeled mequitazine derivatives can be provided and real-time drug metabolizing enzyme activities can be quantified by nuclear medicine imaging. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 shows an HPLC chromatogram of 18F-FMQ during purification. [Diagram 2] FIG. 2 is an HPLC chromatogram of the purity test of 18F-FMQ. [Diagram 3]FIG. 3 is an HPLC chromatogram of 18F-FMMQ during purification. [Figure 4] FIG. 4 is an HPLC chromatogram of the purity test of 18F-FMMQ. [Diagram 5] FIG. 5 shows the results of in vitro metabolite analysis. [Figure 6] FIG. 6 shows the results of an in vitro metabolic inhibition study. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The compound of the present invention represented by the formula (I) is a histamine H1 antagonist mequitazine, which is metabolized by the CYP2D6 enzyme, and has a radionuclide attached to the nitrogen atom of the azabicyclo[2.2.2]octane ring. 18 It introduces F.

[0016] The compound represented by the formula (I) satisfies the conditions that (i) it is metabolized in the liver, (ii) only the metabolites are excreted in the bile, and (iii) the metabolites are radioactive. Therefore, metabolic functions can be captured by imaging.

[0017] In the formula (I), Y - Examples of the anion represented by the formula (I) include bromide ion, chloride ion, iodide ion, fluoride ion, methanesulfonate ion, paratoluenesulfonate ion, trifluoromethanesulfonate ion, and tetrafluoroborate ion, and preferably bromide ion.

[0018] The compound represented by formula (I) is The following formula: 18 F-CH2-Y (In the formula, Y represents a group that can be eliminated as an anion.) The compound can be prepared by reacting a compound represented by the formula: with mequitazine.

[0019] For example, in the formula (I), Y - Compounds in which the compound is a bromide ion (hereinafter referred to as18 F-FMMQ) can be produced as follows.

[0020] [ka]

[0021] That is, 18 It can be prepared by reacting F-fluoromethyl bromide with mequitazine at 110° C. for 10 minutes.

[0022] 18 Instead of F-fluoromethyl bromide, 18 F-fluoromethyl chloride, 18 F-fluoromethyl iodide, 18 F-fluoromethyl tosylate, 18 Radionuclides such as F-fluoromethyl mesylate 18 Compounds containing F can also be used. 18 F-fluoromethyl bromide is used to convert Y in formula (I). - After synthesis of a compound in which is a bromide ion, the bromide ion can be converted to another counter anion such as a chloride ion or an iodide ion by ion exchange or the like.

[0023] The diagnostic agent of the present invention enables non-invasive imaging when measuring metabolic functions in tissues deep within the human body. Furthermore, even if multiple metabolites exist after metabolism by a specific metabolic enzyme, metabolic enzyme activity can be imaged.

[0024] The administration routes of the test agent of the present invention include intravenous, intradermal, subcutaneous, oral, transmucosal, and rectal administration.

[0025] The dosage form of the test agent of the present invention may be appropriately selected from injections, liquids, tablets, etc., as long as it is a dosage form suitable for the administration route, and may further contain a pharma- ceutically acceptable carrier or additives generally used in the technical field according to the dosage form, so long as they do not impair the action and effect of the present invention. As additives, for example, colorants, preservatives, flavoring agents, aroma improvers, taste improvers, sweeteners, stabilizers, and other pharma- ceutically acceptable additives may be contained.

[0026] The dosage of the test agent of the present invention may be appropriately determined depending on the administration method, the compound to be administered, and the age, sex, and weight of the patient. EXAMPLES

[0027] The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited to the following examples.

[0028] Example 1 18 Synthesis of F-labeled mequitazine derivatives The medical cyclotron (Sumitomo Heavy Industries, HM12S) and multipurpose synthesizer (Sumitomo Heavy Industries, Cupid) at the Hamamatsu University School of Medicine were used to synthesize PET nuclides. 18 The synthesis was carried out at F.

[0029] 1. 18 Synthesis of F-FMQ 18 F-FMQ was synthesized via the three-step reaction shown below. (1) First stage 18 Synthesis of F-α-fluoro-α'-bromo-p-xylene (Zuoquan Zhao, et al.; Highly efficient one-pot labeling of new phosphonium cations with fluorine-18 as potential PET agents for myocardial perfusion imaging, Mol. Pharm. 2014 Nov 3; 11(11): 3823-3831) 18F ion production raw material target water ( 18 After irradiation with 30 μA of 10000 Hz ... 18 O(p,n) 18 Obtained by F reaction 18 Target water containing F ions was passed through a Sep-Pak QMA cartridge (Waters, WAT023525). 18 F ions were adsorbed. 18 F ions were eluted into the reaction vessel with a mixed solution of 1.5 mg / 0.3 mL of K2CO3 (SIGMA-ALDRICH, 367877-50G) aqueous solution and 2.0 mg / 1.0 mL of K.222 (Merck Millipore, 8.10647.0001) in CH3CN solution.

[0030] The eluate was dried at 110°C, and then 1 mL of anhydrous CH3CN was added and concentrated to dryness to remove the solvent. A 1.0 mg / 0.5 mL solution of α,α'-dibromo-p-xylene (TCI, D0216) in CH3CN was added to the reaction vessel thus obtained and reacted at 100°C for 10 minutes, after which the solvent was concentrated to dryness.

[0031] [ka]

[0032] (2) Second step: synthesis of 18F-FMQ Obtained 18 To F-α-fluoro-α'-bromo-p-xylene was added a 3.2 mg / 0.5 mL solution of mequitazine in CH3CN, and the mixture was reacted at 110°C for 10 minutes.

[0033] [ka]

[0034] (3) Third stage: Removal of by-products Since the Br-compounds produced during the reaction made it difficult to purify the target compound, they were removed by the Gabriel reaction using phthalimide potassium salt (TCI, P0403) (T. Takahashi, et al.; Improved synthesis of pure [18F]fluoro-compounds for PET studies from bromo-compounds, Appl. Radiat. Isot. 2003 May; 58(5): 557-566).

[0035] After the second reaction was completed, 2.7 mg / 0.5 mL of phthalimide potassium salt DMF (Kanto Chemical, 11392-25) solution was added and reacted for 10 minutes at 110° C. Then, 0.5 mL of 20 mM phosphate buffer (pH 7.3) was added and introduced into HPLC, and the RI peak detected at a retention time of 13.0-13.5 minutes was fractionated.

[0036] After HPLC separation, the mixture was purified by solid-phase extraction to replace the solvent with high-concentration methanol. The separation and solid-phase extraction conditions are shown in Tables 1 and 2, respectively. After solid-phase extraction, the ethanol was concentrated under an Ar stream and diluted with physiological saline.

[0037] [Table 1]

[0038] [Table 2]

[0039] 2. 18 Purity and stability testing of F-FMQ Refined 18 The purity test of F-FMQ was performed using HPLC (Shimadzu). The detectors used were UV (SPD-20A, Shimadzu) and RI (BIOSCAN), and the chromatogram was analyzed using LabSolutions (Shimadzu). The purity test conditions are shown in Table 3.

[0040] [Table 3]

[0041] 3. 18 Synthesis of F-FMMQ 18 F-FMMQ was synthesized via the two-step reaction shown below. (1) First stage 18 Synthesis of F-fluoromethyl bromide (Production and quality control of PET radiopharmaceuticals - A guide to synthesis and clinical use -, 5th ed., PET Chemistry Workshop, Kazuhiro Takahashi / Jun Toyohara / Kentaro Hatano, 1st edition published on February 6, 2015, p.80-84) 18 F ion production raw material target water ( 18 After irradiation with 30 μA of 10000 Hz ... 18 O(p,n) 18 Obtained by F reaction 18 Target water containing F ions was passed through the Sep-Pak QMA cartridge. 18 F ions were adsorbed. 18 F ions were eluted into the reaction vessel with a mixed solution of 1.5 mg / 0.3 mL K2CO3 aqueous solution and 2.0 mg / 1.0 mL K.222 CH3CN solution.

[0042] The eluate was dried at 110°C, and then 1mL of anhydrous CH3CN was added and concentrated to dryness to remove the solvent. 1mL of anhydrous CH3CN mixed with 100μL of dibromomethane (Nacalai Tesque, 22413-62) was added to the first reaction vessel thus obtained, and the reaction was carried out at 110°C for 5 minutes.

[0043] [ka]

[0044] (2) Second stage 18 Synthesis of F-FMMQ After the reaction in the first reaction vessel was completed, four Sep-Pak Silica cartridges (Waters, WAT020520) were connected in series under a helium gas flow (50 mL / min), and the cartridge outlet was evacuated while RI monitoring was performed. After the RI monitor detected radioactivity at the silica outlet, the circuit to the second reaction vessel was opened, and the gas was bubbled into the second reaction vessel containing a 3.2 mg / mL solution of mequitazine in CH3CN that had been prepared in advance. 18 F-fluoromethyl bromide was collected. Bubbling was stopped when the RI count in the second reaction vessel reached a maximum.

[0045] After reacting at 110°C for 10 minutes, 1 mL of 20 mM phosphate buffer (pH 7.3) was added and introduced into HPLC, and the RI peak detected at a retention time of 10-11 minutes was fractionated. The fractionation conditions are shown in Table 1. 18 The HPLC separation conditions were the same as those for F-FMQ. The solution after HPLC separation was evaporated at 110°C and recovered with 1.0 mL of physiological saline.

[0046] [ka]

[0047] 4. 18 Purity and stability testing of F-FMMQ Refined 18 The purity test of F-FMMQ was performed using HPLC (Shimadzu). The detectors used were UV (SPD-20A, Shimadzu) and RI (BIOSCAN), and the chromatogram was analyzed using LabSolutions (Shimadzu). The purity test conditions are shown in Table 4.

[0048] [Table 4]

[0049] 5.Results (1) 18 Synthesis of F-FMQ 18The HPLC chromatogram of F-FMQ purification is shown in Figure 1. 18 The retention time of F ions was 2.5 min, with by-products at 5 and 6 min, and 13.5 min. 18 The labeling rate was calculated from the area under each RI peak, and the labeling rate was 47.9 ± 5.4% (n = 3). 18 F-FMQ was obtained.

[0050] (2) 18 Purity and stability testing of F-FMQ The HPLC chromatogram of the purity test is shown in FIG. 18 The peak of F-FMQ was confirmed at 5 minutes. 18 For F ions, an RI peak was observed at 2.5 minutes under the same conditions. 18 No RI peaks other than F-FMQ were observed. The area ratio under these peaks indicates that the radiochemical purity is 95% or higher. 18 F-FMQ was purified.

[0051] In addition, in the stability test at room temperature, the drug was stored in physiological saline after separation. 18 It was found that the purity of F-FMQ decreased by approximately 30% after 2 hours over time, but no decomposition was observed during storage in ethanol without concentrating the physiological saline solution, regardless of the time elapsed.

[0052] (3) 18 Synthesis of F-FMMQ 18 The HPLC chromatogram of F-FMMQ purified is shown in Figure 3. 18 The retention time of F ions was 2.5 min, 6.5 min was the first stage product, and 10.0 min was the 18 The labeling rate was calculated from the area under each RI peak, and the labeling rate was 82.6 ± 2.6% (n = 3). 18 F-FMMQ was obtained.

[0053] (4) 18 Purity and stability testing of F-FMMQ The HPLC chromatogram of the purity test is shown in FIG. 18 The peak of F-FMMQ was confirmed at 7.5 minutes. 18 For F ions, an RI peak was observed at 2.5 minutes under the same conditions. 18 No RI peaks other than F-FMMQ were observed. The area ratio under these peaks indicates that the radiochemical purity is very high, at 99% or more. 18 F-FMMQ was purified.

[0054] In addition, in the stability test at room temperature, the drug was stored in physiological saline after separation. 18 F-FMMQ was confirmed to be extremely stable, with a radiochemical purity of 99% or more even one hour after isolation.

[0055] 6. Discussion In this example, we used the PET nuclide of mequitazine as a new radiopharmaceutical to quantify drug-metabolizing enzyme activity. 18 F labeling was attempted.

[0056] The compound synthesized in this example 18 F-FMQ was synthesized with a labeling rate of 47.9 ± 5.4% (n = 3) and a radiochemical purity of 95% or more. 18 We succeeded in purifying F-FMQ. However, when stored in saline at room temperature, 30% of the labeled substance was decomposed within 2 hours. Although the decomposition was suppressed by adding ethanol, it is necessary to consider a method of preserving it for a short period of time. 18 F-FMMQ was synthesized with a labeling rate of 82.6 ± 2.6% (n = 3) and a radiochemical purity of over 99%, which is extremely high. 18 F-FMMQ was successfully purified. Furthermore, no decomposition of the label was observed up to one hour at room temperature, demonstrating its high short-term stability.

[0057] (Example 2) In vitro metabolite analysis and in vivo metabolite analysis 18 In vitro and in vivo metabolite analysis was performed using F-FMMQ. 1.In vitro metabolite analysis In an in vitro study, the following mixture was reacted for 60 minutes and analyzed. Mouse liver homogenate (SCID mouse) · NADPH generation system (energy source for CYP) Phosphate buffer ·Sign RI( 18 F-FMMQ) To confirm the involvement of CYP in metabolic reactions, purified water was added instead to samples to which the NADPH generating system was not added. (Time course measurement)

[0058] [Table 5]

[0059] Preparation of NADPH generating system (1)133.3mM Glucose-6-phosphate 18.2mg / milliQ 536μL (2)12.2mM β-NADP+ 4.5 mg / milliQ 482.4μL (3) 53.6 μL of 1M MgCl2 (4)1U / mL Glucose-6-phosphate dehydrogenase 10.72μL [{(1)+(2)}+(3)]+(4) l8 F-FMMQ metabolite analysis confirmed the production of radioactive metabolites by CYP in mouse liver homogenates (see M1 and M2 in FIG. 5).

[0060] 2. In vitro metabolic inhibition study In an in vitro study, the following mixture was reacted for 60 minutes and analyzed. Mouse liver homogenate (SCID mouse) · NADPH generation system (energy source for CYP) Phosphate buffer -Specific inhibitors of each CYP ·Sign RI(18 F-FMMQ) The proportion of metabolites in each reaction was calculated (if there were multiple metabolites, the total was calculated), and the inhibition rate was calculated based on the NADPH(+) sample to which no inhibitor was added.

[0061] [Table 6]

[0062] The results are shown in Figure 6. A significant inhibitory effect was observed when paroxetine, which has a specific inhibitory effect on CYP2D6, was administered.

[0063] 3.In vivo study (imaging) In the in vivo study, regions of interest were set in the captured images and accumulation was compared. 18 Paroxetine, a specific inhibitor of CYP2D6, was administered intraperitoneally 1 hour before F-FMMQ administration, and comparisons were made between normal mice and mice loaded with the inhibitor (a model of reduced CYP2D6 activity). The target organs were the liver, bile excretion (small intestine, gallbladder, upper intestine), and kidney.

[0064] Imaging showed that paroxetine loading increased hepatic accumulation and reduced biliary excretion within 30 minutes of administration in scid mice.

[0065] 4. In vivo study (distribution in the body) In an in vivo study, the distribution in the body after administration was compared. 18 Paroxetine, a specific inhibitor of CYP2D6, was administered intraperitoneally one hour before administration of F-FMMQ, and the effects were compared between normal mice and mice loaded with the inhibitor (a model with reduced CYP2D6 activity).

[0066] In terms of biodistribution, paroxetine loading in ddY mice increased accumulation in the liver (significance level 0.05 at 5 and 10 minutes), and decreased accumulation in the gallbladder (significance level 0.05 at 10 and 15 minutes) and small intestine (including contents: significance level 0.01 at 15 minutes). These results showed the same tendency as in the scid mouse imaging.

[0067] 5. Ex vivo study (scid mice 1-3: normal, scid mice 4-6: paroxetine challenge) 18 The mice were sacrificed 15 minutes after administration of F-FMMQ, and the gallbladder and liver were removed. The liver was homogenized by adding 3 times the wet weight (approximately 100 mg) of Krebs-Ringer phosphate buffer (KRPB). The supernatant was collected by centrifugation (4°C, 15,000 rpm, 5 minutes). 200 μL of ethanol was added to 250 μL of the supernatant, and then 50 μL of perchloric acid solution was added. The supernatant was collected again by centrifugation (4°C, 15,000 rpm, 5 minutes) and analyzed by HPLC. The gallbladder was diluted with 10 μL of ethanol and analyzed by HPLC together for scid mice 1-3 and scid mice 4-6. Because it was below the detection limit of the gamma counter attached to the HPLC, the waste liquid was collected at 30-second intervals and measured for 1 minute each using an autowell gamma counter.

[0068] To CYP2D6 inhibition model mice loaded with CYP2D6 specific inhibitor paroxetine l8 F-FMMQ was administered, and the radioactive substances in the liver and bile extracted 15 minutes later were analyzed. Compared to normal mice, the amount of unchanged substance was increased in the liver, and the accumulation of radioactive metabolites in the bile was reduced.

[0069] From the above results 18 F-FMMQ was shown to be useful as a new imaging diagnostic agent that can detect CYP2D6 activity in the liver as a change in the time-dependent accumulation curve in the gallbladder.

Claims

1. The following formula (I): 【Chemistry 1】 (In the formula, Y － represents an anion.) A compound represented by the formula:

2. A test agent for measuring metabolic function, comprising the compound according to claim 1.

3. 3. The diagnostic agent according to claim 2, for determining drug metabolic function by measuring the amount of a radioactive metabolite of the compound according to claim 1 excreted in the bile.

4. Secondary formula: １８ F-CH ２ -Y (In the formula, Y represents a group which can be eliminated as an anion.) 2. A process for preparing the compound of claim 1, comprising reacting a compound of the formula: with mequitazine.

Images