JP6024939B2 - Drug metabolism function measurement method - Google Patents

Description

  The present invention relates to a method for measuring drug metabolism function using a metabolite such as a drug.

  Cytochrome P450 is an oxidase having a molecular weight of about 45,000 to 60,000, which is present in almost all living organisms from bacteria to plants and mammals, and is a major first-phase reaction enzyme in foreign body (drug) metabolism. It was named cytochrome P450 (P450) in the meaning of a dye that binds to carbon monoxide in a reduced state and exhibits an absorption maximum at 450 nm. Cytochrome P450 is a general term for the hydroxylase family and is often referred to as CYP (ship) for short. It plays many roles because it hydroxylates various substrates. Although known as an enzyme that detoxifies in the liver, it is also involved in reactions necessary for normal activities of organisms, such as biosynthesis of steroid hormones, fatty acid metabolism, and plant secondary metabolism. It is common for cytochrome P450 to hydroxylate a substrate using an electron donor such as NADPH and oxygen. Cytochrome P450 is abundant in the endoplasmic reticulum in the cell, and a part thereof is present in the mitochondria. In animals it is often found in the liver and is particularly well studied.

  In people with low cytochrome P450 metabolic capacity, the blood concentration increases and exceeds the therapeutic range when taken in the usual dosage regimen, whereas in people with high metabolic capacity, the blood concentration is in the therapeutic range. There is a risk that the therapeutic effect will not be obtained. If a person with low metabolic capacity of cytochrome P450 continuously takes the same amount as a person with normal metabolic capacity, there is a risk that the drug accumulates and the blood concentration rises.

  In determining an appropriate dose of a drug or examining a patient's metabolic abnormality, it is very important to measure a drug metabolic function by cytochrome P450.

  Patent Document 1 discloses a method for measuring drug metabolizing enzyme activity in which a drug metabolizing enzyme such as cytochrome P450 is allowed to act and then the resulting product (metabolite) is measured by an immunochemical technique.

  However, in the method described in Patent Document 1, it is necessary to prepare an antibody for each metabolite, and when the metabolite is unknown, it is difficult to prepare an antibody.

Patent Document 2 discloses CYP2C19-related metabolic ability by determining the relative amount of ¹³ CO ₂ exhaled by a subject after intravenous or oral administration of a ¹³ C-labeled cytochrome P450 2C19 isozyme (CYP2C19) substrate compound by breath analysis. A method of measuring is disclosed.

However, the method described in Patent Document 2 is based on a dealkylation reaction (de-O-methyl reaction), and it is necessary to label ^13- C in the breath by labeling the O-methyl group that undergoes metabolism. Yes, the scope of application is limited.

  In addition, various radiopharmaceuticals are known as radioactive compounds to be administered in vivo. However, radiopharmaceuticals conventionally administered in vivo are premised on not being metabolized in order to exert their actions and effects. So far, analysis of radioactive metabolites has not been performed.

JP 2004-69491 A Special table 2010-502194 gazette

  An object of the present invention is to provide a method for measuring a function of drug metabolism that can be applied even if the structure of the metabolite is unknown and has a wide application range.

The gist of the present invention is as follows.
(1) A method for measuring a drug metabolic function, comprising analyzing a radioactive metabolite of the radioactive compound contained in a sample of a subject who has been administered a radioactive compound.
(2) The method according to (1) above, wherein the drug metabolism function is a drug metabolism function by cytochrome P450.
(3) The method according to (1) above, wherein the radioactive compound is a radioactive amino acid.
(4) The method according to (3) above, wherein the radioactive amino acid is a radioactive label of a natural amino acid and / or an unnatural amino acid.
(5) The method according to any one of (1) to (4), which is performed to determine an appropriate dose of a drug.
(6) The method according to any one of (1) to (4), which is performed to examine a metabolic abnormality of a subject.
(7) The method according to any one of (1) to (6), wherein the subject's sample is a sample obtained from blood, serum or plasma.
(8) A test agent for measuring a drug metabolic function by cytochrome P450 containing N-isopropyl-p-iodoamphetamine or a salt thereof, or a labeled form thereof.

  The method for measuring drug metabolism function of the present invention can be applied even if the structure of the metabolite is unknown, and has a wide range of applications.

FIG. 1 shows a reaction time dependent decrease in the concentration of N-isopropyl-p-iodoamphetamine hydrochloride (IMP) in the presence of human liver microsomes and NADPH. FIG. 2 shows the results of the IMP elimination reaction with 12 types of recombinant human CYPs. FIG. 3A shows the result of the disappearance reaction of IMP by CYP2C19. FIG. 3B shows the result of the disappearance reaction of IMP by CYP1A1. FIG. 4 shows the effect of CYP2C19 ^* 2 / ^* 2 on the disappearance of IMP. FIG. 5 shows the time course of peak area in liquid chromatography of ¹²⁵ I-N-isopropyl-p-iodoamphetamine hydrochloride ( ¹²⁵ I-IMP) -derived radiometabolite. FIG. 6 shows the rate of incorporation of ¹⁴ C-L-Met and ¹⁴ C- / ³ HD-Met into proteins in mouse pancreas, liver, and kidney. FIG. 7 shows the abundance ratios of ¹⁴ C-L-Met and ¹⁴ C-D-Met in the tissue of the liver and kidney.

  The radioactive compound used in the method for measuring a drug metabolic function of the present invention is not particularly limited as long as it is metabolized in vivo to produce a radioactive metabolite. For example, it is metabolized by cytochrome P450 to produce a radioactive metabolite. And radioactive labels of natural amino acids and / or unnatural amino acids.

Examples of the radioactive compound that is metabolized by cytochrome P450 to produce a radioactive metabolite include, for example, ¹²³ IN-isopropyl-p-iodoamphetamine ( ¹²³ I-IMP) or a salt thereof commercially available as a cerebral blood flow diagnostic agent, For example, hydrochloride is mentioned.

  In addition, since compounds that can serve as substrates are known for each molecular species of cytochrome P450 (CYP), radioactive compounds corresponding to these compounds can be synthesized and used by conventional methods.

Specific examples of CYP molecular species and compounds that can be substrates are shown below.
(CYP1A2)
amitriptyline, caffeine, clomipramine, clozapine, cyclobenzaprine, estradiol, fluvoxamine, haloperidol, imipramine, mexiletine, naproxen, olanzapine, ondansetron, acetaminophen, propranolol, riluzole, ropivacaine, tacrine, theophylline veruine miton
(CYP2B6)
bupropion, cyclophosphamide, efavirenz, ifosphamide, methadone, sorafenib
(CYP2C8)
amodiaquine, cerivastatin, paclitaxel, repaglinide, sorafenib, torsemide
(CYP2C9)
Non-steroidal anti-inflammatory drugs: diclofenac, ibuprofen, lornoxicam, meloxicam, S-naproxen, piroxicam, suprofen
Oral hypoglycemic agent: tolbutamide, glipizide
Angiotensin II inhibitor: losartan, irbesartan
Sulfonylurea drugs: glyburide, glibenclamide, glipizide, glimepiride, tolbutamide
Others: amitriptyline, celecoxib, fluoxetine, fluvastatin, glyburide, nateglinide, phenytoin-4-OH2, rosiglitazone, tamoxifen, torsemide, S-warfarin
(CYP2C19)
Proton pump inhibitors: lansoprazole, omeprazole, pantoprazole, rabeprazole
Antiepileptic drugs: diazepam, phenytoin, S-mephenytoin, phenobarbitone
Others: amitriptyline, carisoprodol, citalopram, chloramphenicol, clomipramine, clopidogrel, cyclophosphamide, hexobarbital, imipramine, indomethacin, R-mephobarbital, moclobemide, nelfinavir, nilutamide, primidone, progesterone, proguanil, farporineol, proguanil, propranolol
(CYP2D6)
tamoxifen
β-blockers: carvedilol, S-metoprolol, propafenone, timolol
Antidepressants: amitriptyline, clomipramine, desipramine, fluoxetine, imipramine, paroxetine
Antipsychotics: haloperidol, perphenazine, risperidone, thioridazine, zuclopenthixol
Others: alprenolol, amphetamine, aripiprazole, atomoxetine, bufuralol, chlorphenimipramine, chlorpromazine, codeine, debrisoquine, dexfenfluramine, dextromethorphan, donepezil, duloxetine, encainide, flecainide, fluvoxamine, lidocaine, metoclopr oxycodone, perhexiline, phenacetin, phenformin, promethazine, propranolol, sparteine, tramadol, venlafaxine
(CYP2E1)
Anesthetics: enflurane, halothane, isoflurane, methoxyflurane, sevoflurane
Others: acetaminophen, aniline, benzene, chlorzoxazone, ethanol, N, N-dimethylformamide, theophylline
(CYP3A4, 5, 7)
Macrolide antibiotics: clarithromycin, erythromycin (not 3A5)
Antiarrhythmic drug: quinidine (not 3A5)
Benzodiazepines: alprazolam, diazepam, midazolam, triazolam
Immunomodulator: cyclosporine, tacrolimus (FK506)
Anti-HIV drugs: indinavir, nelfinavir, ritonavir, saquinavir
Gastrointestinal motility improver: cisapride
Antihistamines: astemizole, chlorpheniramine, terfenadine
Calcium antagonists: amlodipine, diltiazem, felodipine, lercanidipine, nifedipine, nisoldipine, nitrendipine, verapamil
HMG-CoA reductase inhibitors: atorvastatin, cerivastatin, lovastatin, simvastatin
Steroids: estradiol, hydrocortisone, progesterone, testosterone
Others: alfentanil, aprepitant, aripiprazole, boceprevir, buspirone, caffeine, cilostazol, cocaine, dapsone, dexamethasone, dextromethorphan, docetaxel, domperidone, eplerenone, fentanyl, finasteride, gleevec, haloperidol, iriperamine, haloperidol, note ondansetron, pimozide, propranolol, quetiapine, quinine, risperidone, salmeterol, sildenafil, sirolimus, sorafenib, sunitinib, tamoxifen, taxol, telaprevir, terfenadine, torisel, trazodone, vincristine, zaleplon, olpi

  Natural amino acids are easily metabolized, and unnatural amino acids are not easily metabolized. Therefore, by analyzing a radiometabolite contained in a sample of a subject who has been administered a radioactive label of a natural amino acid and / or a non-natural amino acid, a drug metabolic function can be measured, and an appropriate dose of the drug can be determined. It can be determined or examined for metabolic abnormalities in the subject.

  In the method for measuring a drug metabolic function of the present invention, one or more radioactive compounds serving as substrates are used depending on the purpose.

  The form of the radioactive metabolite that is the analysis target may be any of an organic substance, an inorganic substance, and an ion, but an organic substance is preferable in terms of ease of analysis. The metabolite of a radioactive amino acid in the present invention includes a protein in which the radioactive amino acid is incorporated.

Examples of the radionuclide used for synthesizing the radioactive compound used in the measurement method of the present invention include tritium ( ³ H), 11-carbon ( ¹¹ C), 14-carbon ( ¹⁴ C), and 15-oxygen ( ¹⁵ O). 18- fluorine ⁽¹⁸ F), 32- phosphorus ⁽³² P), 59-iron ⁽⁵⁹ Fe), 67- copper ⁽⁶⁷ Cu), 67- gallium ⁽⁶⁷ Ga), ^81m- krypton ^(81m Kr), 81 - rubidium ⁽⁸¹ Rb), 89- Sutoronchimu ⁽⁸⁹ Sr), 90- yttrium ⁽⁹⁰ Y), ^99m- technetium ^(99m Tc), 111- indium ⁽¹¹¹ In), 123- iodine ⁽¹²³ I), 125- iodine ⁽¹²⁵ I), 131- iodine ⁽¹³¹ I), 133- xenon ⁽¹³³ Xe), ^117m- tin ^(117m Sn), 153- Samarium ( ¹⁵³ Sm), ^186- rhenium ( ¹⁸⁶ Re), ^188- rhenium ( ¹⁸⁸ Re), 201-thallium ( ²⁰¹ Tl), ^212- bismuth ( ²¹² Bi), 213-bismuth ( ²¹³ Bi) and 211-astatin ( ²¹¹ At).

  Examples of the administration route of the radioactive compound in the present invention include intravenous, intradermal, subcutaneous, oral, transmucosal, and rectal administration. Examples of the subject's sample include blood, serum, plasma, urine, saliva or other body fluid, preferably blood, serum, plasma.

  The administration form of the radioactive compound may be appropriately selected from injections, solutions, tablets and the like as long as it is a dosage form suitable for the administration route, and is pharmaceutically acceptable as long as the action and effect of the present invention are not impaired. It may further comprise additives commonly used in the art depending on the carrier or dosage form. As an additive, for example, a colorant, a preservative, a flavoring agent, an aroma improving agent, a taste improving agent, a sweetening agent, a stabilizer, and other pharmaceutically acceptable additives can be contained.

  The dose of the radioactive compound may be appropriately determined depending on the administration method, the compound to be administered, the age, sex and body weight of the patient.

  In the measurement method of the present invention, the radioactive metabolite of the radioactive compound contained in the sample of the subject administered with the radioactive compound is analyzed. The analysis of the radiometabolite can be performed, for example, by separating the radio compound of the parent compound (unmodified) and the radio metabolite and measuring the radioactivity of the radio metabolite.

  A fat-soluble radioactive compound is often metabolized to produce a water-soluble radioactive metabolite. In such a case, for example, by octanol extraction method, the radioactive compound of the parent compound (unmodified) and the radioactive metabolism are obtained. Things can be separated. In addition, the radioactive compound of the parent compound (unchanged) and the radioactive metabolite can also be separated by thin layer chromatography, paper chromatography, or the like.

  According to the measurement method of the present invention, various metabolic abnormalities such as CYP abnormalities can be found by analyzing radioactive metabolites contaminated in general radiodiagnostic drugs. By seeing, without examining genes for each CYP molecular species, a metabolic profile can be obtained for the CYP family as a whole, and an appropriate dose of the drug can be determined.

  Since the measurement method of the present invention analyzes a radiometabolite, it can exert a sufficient effect even at a very low dose as compared with the case of analyzing an unlabeled metabolite. It can also be easily distinguished from things.

N-isopropyl-p-iodoamphetamine (IMP) is selectively metabolized by CYP2C19. Therefore, IMP or a salt thereof, or a labeled form thereof can be used as a test agent for measuring a drug metabolic function by CYP, particularly CYP2C19. As described above, ¹²³ I-N-isopropyl-p-iodoamphetamine is obtained from the point that a sufficient effect can be obtained at a low dose, and safety has been confirmed so as to be marketed as a diagnostic agent for cerebral blood flow. It is preferable to use ( ¹²³ I-IMP) or a salt thereof.

As a salt of IMP or ¹²³ I-IMP, a pharmaceutically acceptable salt is preferable. For example, inorganic salts such as hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, nitric acid, pyrosulfuric acid, and metaphosphoric acid. Examples thereof include salts with acids or organic acids such as citric acid, benzoic acid, acetic acid, propionic acid, fumaric acid, maleic acid, and sulfonic acid (for example, methanesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid).

  Examples of the label used for the IMP label include, in addition to the above-described radionuclide labeling, isotope labeling and fluorescence labeling.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further more concretely, the scope of the present invention is not limited to these Examples.

(Example 1) Analysis of metabolites of N-isopropyl-p-iodoamphetamine hydrochloride by CYP2C19 (Purpose)
¹²³ I-N-isopropyl-p-iodoamphetamine hydrochloride ( ¹²³ I-IMP) is clinically used for diagnosis of cerebral blood flow by single photon emission tomography (SPECT). ¹²³ I-IMP is a rare diagnostic agent that undergoes metabolism in the human body. The first stage of metabolism ¹²³ I-p-iodo be amphetamines is the generation of, also subsequently ¹²³ I-p-iodobenzoic acid, is reported to be metabolized to more ¹²³ I-p-iodo-hippuric acid Yes. However, there is no knowledge of drug metabolizing enzymes involved in these metabolic reactions so far. Therefore, in this experiment, the involvement of cytochrome P450 (CYP) in IMP metabolism was examined.

(Method)
N-isopropyl-p-iodoamphetamine hydrochloride (IMP) and unlabeled p-iodobenzoic acid were used. Using human liver microsomes as an enzyme source, it was examined whether or not the metabolism of IMP to p-iodobenzoic acid occurred in an NADPH-dependent manner. At this time, the presence or absence of IMP and the degree thereof were also examined. Recombinant human CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5 were used to examine the contribution of each CYP molecular species in IMP metabolism.
I. Reagent disodium hydrogen phosphate, 12 water (Wako Pure Chemical Industries)
Potassium dihydrogen phosphate (Wako Pure Chemical Industries)
EDTA · 2Na (Wako Pure Chemical Industries)
Glucose-6-phosphate (Wako Pure Chemical Industries)
β-NADP ⁺ (Wako Pure Chemical Industries)
Glucose-6-phosphate dehydrogenase
MgCl ₂ · _{6H 2} O (Wako Pure Chemical Industries, Ltd.)
Perchloric acid (60%) (Wako Pure Chemical Industries)
p-Chlorobenzoic acid (Wako Pure Chemical Industries)
Acetonitrile (Wako Pure Chemical Industries)
Dibutylamine phosphate solution (D4 reagent) (Waters)
BD Ultrapool 150 Donor Pooled Human Liver Microsome (Nippon Becton Dickinson)
Special grade glycerin (Wako Pure Chemical Industries)

II. Reagent preparation Microsomes Upon delivery, the microsomes are suspended in 250 mM sucrose and the protein concentration is 20 mg / mL. Once melted, 125 μL of glycerol was added to 500 μL of microsome (protein concentration was 16 mg / mL). The solution was dispensed into Eppenes and stored at -80 ° C, and the required amount was melted during the reaction.

2.0.167 mM EDTA / 0.33 M NaK phosphate buffer (pH 7.4)
(1) A 0.5 M potassium dihydrogen phosphate aqueous solution (MW 136.09, 34.02 g / 500 mL) was added to a 0.5 M disodium hydrogen phosphate aqueous solution (MW: 358.14, 177.07 g / 1000 mL). A 5M NaK phosphate buffer (pH 7.4) was prepared.
(2) A 0.5 mM EDTA aqueous solution was prepared (MW 372.24, 37.22 mg / 200 mL).
(3) 0.5 mM EDTA aqueous solution: 0.5 M NaK phosphate buffer (pH 7.4) = 1: 2 mixed, 0.167 mM EDTA / 0.33 M NaK phosphate buffer (pH 7.4) It was.

3. NADPH production system (prepared when used)
(1) A 1M MgCl ₂ aqueous solution was prepared (MW: 203.30, 10.17 g / 50 mL).
(2) 18.2 mg of Glucose-6-phosphate was dissolved in 536 μL of purified water.
(3) 4.5 mg of β-NADP ⁺ was dissolved in 482.4 μL of purified water.
(4) Glucose-6-phosphate dissolved in (2) and β-NADP ⁺ in (3) are mixed, and 53.6 μL of 1M MgCl ₂ aqueous solution and (1) Uglucose-6-phosphate dehydrogenase (1000 U / 1 mL) 10.72 μL was added to obtain an NADPH generation system.

III. Experimental method 1. Method using index of decrease in parent compound (IMP) Reaction solution was 0.05 mM EDTA, 100 mM NaK phosphate buffer (pH 7.4), glycerin-added human liver microsome corresponding to 0.64 mg / mL protein, NADPH production system (0 .5 mM β-NADP ⁺ , 5 mM Glucose-6-phosphate, 1 U / mL Glucose-6-phosphate dehydrogenase, 5 mM MgCl ₂ ) and 0.5 μM IMP were mixed to give 250 μL (i). As controls, the NADPH generation system was replaced with purified water (ii), and the microsomes and NADPH generation system were replaced with purified water (iii). The reaction was started by the addition of IMP, and after the reaction at 37 ° C., 50 μL perchloric acid aqueous solution was added to stop the reaction. The reaction time was 10, 20, and 60 minutes. After stopping the reaction, 30 μL of 20 μM p-chlorobenzoic acid aqueous solution as an internal standard substance was added and centrifuged at 15000 rpm and 20 ° C. for 5 minutes. 100 μL of the supernatant was analyzed by liquid chromatography.
Table 1 shows the composition and operation per specimen.

The analysis conditions are shown below.
Column: CAPCELL PAK C18 SG120, 4.6 x 250 mm (manufactured by Shiseido)
Column temperature: 40 ° C
In mobile phase A, 330 mL of acetonitrile and 670 mL of water were mixed, and 10 mL of dibutylamine phosphate solution (D4 reagent) was added.
In mobile phase B, 900 mL of acetonitrile and 100 mL of water were mixed, and 10 mL of dibutylamine phosphate solution (D4 reagent) was added.
Elution conditions: mobile phase A: mobile phase B = 100: 0 → 78: 22 (0 min. → 16 min.) Gradient, then equilibrated with mobile phase A: mobile phase B = 100: 0 for 9 minutes.
Flow rate: 0.8 mL / min.
Detection (UV): IMP was detected at 232 nm, metabolite p-iodobenzoic acid was detected at 248 nm, and internal standard substance p-chlorobenzoic acid was detected at 235 nm.
* IMP was detected at about 6.3 minutes, metabolite p-iodobenzoic acid at about 15.6 minutes, and internal standard substance p-chlorobenzoic acid at about 12.6 minutes under these conditions.

2. Method using increase in metabolite (p-iodobenzoic acid) as an indicator The reaction solution was 0.05 mM EDTA, 100 mM NaK phosphate buffer (pH 7.4), glycerin-added human liver microsome corresponding to 0.64 mg / mL protein, A NADPH production system (0.5 mM NADP ⁺ , 5 mM Glucose-6-phosphate, 1 U / mL Glucose-6-phosphate dehydrogenase, 5 mM MgCl ₂ ) and 1000 μM IMP were mixed to give 250 μL. Controls were experimental method 1. As well as. The reaction was started by the addition of IMP, and after the reaction at 37 ° C., 50 μL perchloric acid aqueous solution was added to stop the reaction. The reaction time was 10, 60, 120 minutes. For the operation and analysis method after the reaction stop, the experimental method 1. Is the same.
The composition per specimen is determined according to the experimental method 1. 5 μM IMP was changed to 10 mM IMP, and the others were performed in the same manner.

(Results and discussion)
When IMP (0.5 or 1 μM) was incubated in the presence of human liver microsomes and NADPH, production of p-iodobenzoic acid was not observed. The concentration of IMP decreased in a reaction time-dependent manner in the presence of NADPH (FIG. 1), revealing that IMP was metabolized in an NADPH-dependent manner by enzymes in human liver microsomes. Experiments with recombinant human CYP found that IMP was metabolized exclusively by CYP2C19 (FIGS. 2 and 3). The Km of IMP disappearance reaction by CYP2C19 was 8.6 μM, and Vmax was 9.7 nmol / min / nmol CYP. When human liver microsomes lacking CYP2C19 (CYP2C19 ^* 2 / ^* 2) are used, the rate of disappearance of IMP is about 50% when using human liver microsomes expressing wild-type CYP2C19 (FIG. 4). It was clarified that the metabolism of IMP depends on the polymorphism of CYP2C19. As a result of IMP being metabolized by CYP2C19, an unknown peak was detected in the HPLC chromatogram. The peak area increased in proportion to the reaction time in proportion to the decrease of the IMP peak.

(Conclusion)
As described above, it was found that IMP is metabolized by CYP2C19. Therefore, it was found that the drug metabolic function by cytochrome P450 can be measured by using IMP or its label.

Example 2 Analysis of metabolites of ¹²⁵ I-N-isopropyl-p-iodoamphetamine hydrochloride by CYP2C19 ¹²⁵ I-N-isopropyl-p- in place of N-isopropyl-p-iodoamphetamine hydrochloride (IMP) The experiment was conducted in the same manner as in Example 1 except that iodoamphetamine hydrochloride ( ¹²⁵ I-IMP) was used. The results are shown in Table 2 and FIG.

¹²⁵ I-IMP was also metabolized by CYP2C19 in the same manner as IMP. Therefore, it was found that the drug metabolic function by cytochrome P450 can be measured by using ¹²⁵ I-IMP.

Example 3 Analysis of Radioactive Metabolites of Radioiodine Labeled Amino Acids (1) Preparation of Radioiodine Labeled Tyrosine
¹²⁵ I-3-iodo-D-tyrosine ( ¹²⁵ ID-MIT) and ¹²⁵ I-3-iodo-L-tyrosine ( ¹²⁵ IL-MIT) were prepared as follows.
¹²⁵ I-NaI was purchased from Amersham. ¹²⁵ ID-MIT and ¹²⁵ IL-MIT were produced by the chloramine-T method using D- and L-tyrosine as raw materials, respectively. That is, 10 μl of 0.05 M phosphate buffer solution (pH 6.2) in which chloramine-T 2.0 × 10 ⁻⁸ mole (Aldrich) was dissolved was added to 1.0 × 10 ⁻⁸ mole of the labeling raw material and no carrier. ¹²⁵ I-NaI (7,4-37 MBq) was added to 35 μl of 0.4 M phosphate buffer solution (pH 6.2). The reaction solution was allowed to stand for 2 minutes, and a 10-fold diluted solution of a saturated aqueous sodium pyrosulfite solution was added to stop the reaction. Purification of radioiodine-labeled MIT was performed by Sephadex LH-20 (Pharmacia) column chromatography (10 × 200 mm, eluent; ethyl acetate: methanol: 2N aqueous ammonia = 40: 10: 4). The eluted labeled amino acid fractions were collected, the solvent was completely distilled off under reduced pressure, and the residue was dissolved in physiological saline and used in the following experiments.

(2) Deiodination reaction using mouse tissue homogenate After killing and dissecting two ddY male mice weighing about 25 g, Krebs-Ringer phosphoric acid was immediately removed from the liver and kidney and cooled to a certain amount of tissue with water. A buffer solution (pH 7.4) was added and homogenized. Add 20 μl (1.1 × 10 ⁻¹³ mole, 7.4 kBq) of ¹²⁵ I-D-MIT or ¹²⁵ I-L-MIT to 100 μl of each ice-cold homogenate, and incubate at 37 ° C. or 4 ° C. After minutes, trichloroacetic acid was added to a final concentration of 5%. After centrifugation, the supernatant was analyzed by silica gel thin layer chromatography (Merck, Art. 5553, developing solvent; methanol: acetic acid = 100: 1, methanol: 10% aqueous ammonia acetate = 10: 1). The results after 5 minutes of incubation are shown in Table 3.

¹²⁵ I-D-MIT was hardly subjected to deiodination at both 37 ° C. and 4 ° C. temperature conditions. On the other hand, ¹²⁵ IL-MIT was almost deiodinated when incubated at 37 ° C.

(3) Mouse biodistribution experiment and metabolite analysis
¹²⁵ ml of ¹²⁵ I-D-MIT and ¹²⁵ IL-MIT physiological saline solutions (1.6 × 10 ⁻¹³ mole, 11.1 kBq) were added to two ddY male mice weighing approximately 25 g in the tail vein. After certain time, it was sacrificed by ether anesthesia. Blood was collected by cardiac puncture using a heparinized syringe, and then the liver and kidney were removed.

  50 mg of each tissue was precisely weighed, 450 μl of a bicarbonate buffer solution was added, and homogenized. This fixed amount was diluted to 1.9 ml, and 0.1 ml of 100% trichloroacetic acid solution was added and mixed. After centrifugation, the supernatant was analyzed by silica gel thin layer chromatography (Merck, Art. 5553, developing solvent; methanol: acetic acid = 100: 1, methanol: 10% aqueous ammonia acetate = 10: 1). The results after 10 minutes of incubation are shown in Table 4.

In any tissue, ¹²⁵ I-L-MIT was easily metabolized (enzymatic deiodination), and a large amount of free iodine as a radiometabolite was detected, compared to ¹²⁵ I-D-MIT. This tendency was more prominent in urine.

As described above, ¹²⁵ I-D-MIT and ¹²⁵ I-L-MIT differ greatly in the ease of metabolism, and by utilizing these tendencies, differences in the amount of radioactive metabolites due to individual differences and pathological changes can be obtained. It was found that the drug metabolic function can be measured by measuring.

Example 4 Analysis of Radioactive Metabolites of Radioactive Natural / Non-natural Amino Acids 5 weeks old ddY male mice were injected with labeled L- / D-methionine (Met) via tail vein and sacrificed 10 minutes later. The pancreas, liver and kidney were removed. The extracted organ was homogenized by adding a bicarbonate buffer. To this homogenate, 100% trichloroacetic acid (Nacalai tesque) was added to a final concentration of 5% and mixed. The precipitate fraction is collected on a glass filter (GC-50, Toyo), washed with ice-cold 5% trichloroacetic acid, and then heat treated at 150 ° C. for 1 hour to immobilize the protein and measure its radioactivity. This was evaluated as the rate of incorporation into the protein fraction.

  Further, the homogenate to which the above-mentioned trichloroacetic acid was added was centrifuged, the supernatant was spotted on a thin layer chromatography, developed with a developing solvent (butanol: acetic acid: water = 4: 1: 1), and an imaging plate (BAS- SR2025 (Fuji Film) was exposed and analyzed using BAS5000 (Fuji Film) and evaluated as the unchanged substance remaining rate in the acid-soluble fraction.

Regarding D-Met, in the pancreas, since the pancreas of the biodistribution experiment was analyzed, ³ H body for double tracer was used. Also, the liver, because of using ¹⁴ C-dedicated imaging plate for evaluation of the metabolic stability in acid-soluble fraction with respect to ^{kidney, [S-methyl- 14 C]} -D-Met (14 C-D-Met; American Radiolabeled Chemicals) was used. Therefore, the notation of D-Met in radiometabolite analysis is ¹⁴ C− / ³ H-D-Met.

FIG. 6 shows the incorporation rate of ¹⁴ C-L-Met and ¹⁴ C- / ³ HD-Met into proteins in mouse pancreas, liver, and kidney 10 minutes after administration of the tracer. ¹⁴ C-L-Met showed a protein incorporation rate of about 25 to 50% of the tissue accumulation rate, whereas ¹⁴ C- / ³ HD-Met was about 3% compared with ¹⁴ C-L-Met. And it decreased significantly.

FIG. 7 shows the abundance ratios of ¹⁴ C-L-Met and ¹⁴ C-D-Met in the tissue when the tissue accumulation rate of liver and kidney, which are typical metabolic tissues, is 100%. Show. ^{In 14} C-D-Met, about 80% was present as an unchanged form, whereas in ¹⁴ C-L-Met, no more than about 40% of the unchanged form was present. It was confirmed that 50% was present in the protein fraction and about 25-50% was present as a radiometabolite.

Claims (5)

A radioactive metabolite of the radioactive label contained in a sample selected from blood, serum, plasma, urine and saliva of a subject administered with a radioactive label of a natural amino acid and a radioactive label of an unnatural amino acid , A method for measuring a drug metabolic function, comprising analyzing in order to determine an appropriate dose or to examine a metabolic abnormality of a subject. The method according to claim 1 , wherein the subject's sample is a sample obtained from urine. The method according to claim 1 or 2 , wherein the method is carried out to determine an appropriate dose of the drug. The method of Claim 1 or 2 performed in order to test | inspect a subject's metabolic abnormality. Radiometabolism of the N-isopropyl-p-iodoamphetamine contained in a sample selected from blood, serum, plasma, urine and saliva of a subject who has been administered N-isopropyl-p-iodoamphetamine labeled with a radionuclide A method for measuring a function of drug metabolism, comprising analyzing an object to examine abnormalities in metabolism by CYP2C19 in a subject .

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