JP4007647B2 - Liver function diagnostic agent - Google Patents

Description

0000
BACKGROUND OF THE INVENTION
The present invention relates to a liver function diagnostic agent, and more particularly to a liver function diagnostic agent containing a fatty acid in which at least one specific carbon is labeled with ¹³ C.
[0001]
[Prior art]
For screening for hepatic dysfunction, blood biochemical tests that quantify enzymes such as transaminases (GPT and GOT), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) in blood are generally performed. These enzymes leak into the blood from liver tissue during liver injury. Among them, GPT and GOT are enzymes mainly present in the liver and are excellent indicators for sensitive detection of liver damage because they are significantly higher in liver function disorders while being low in normal blood levels. . However, when chronic hepatitis or liver cirrhosis in which liver function is remarkably reduced, the amount of enzyme in the liver tissue is reduced, so the amount of leaked enzyme is also reduced, and although the degree of injury is high, it does not show a high value (Today's medical CD-ROM Vol.6, published by Medical School). In addition, since it takes time for the leaked enzyme to disappear from the blood, it may show a high value even if it recovers from liver dysfunction at the time of the test. Therefore, quantification of these enzymes is insufficient as a test method for evaluating the degree of liver dysfunction.
[0002]
In particular, during liver surgery, it is very important to evaluate the extent of liver damage and liver function (Gastrointestinal Practice 1 Diagnostic approach to liver damage, edited by Akiyuki Okubo, published by Bunkodo). Serum bilirubin measurements and ICG tolerance tests are mainly used to evaluate the degree of liver damage and liver function. However, each of these test methods has its own problems (Gastrointestinal Practice 1 Diagnostic approach to liver damage, edited by Akiyuki Okubo, published by Bunkodo). For example, an increase in serum bilirubin does not necessarily mean a decrease in liver function, and it is difficult to follow a process in which the liver function changes drastically in a short period of time such as after surgery. The ICG tolerance test competes with bilirubin when ICG is taken up by hepatocytes, so reliable results cannot be obtained when bilirubin is elevated. Under such circumstances, there is a demand for means capable of safely and simply evaluating the degree of liver damage and liver function at the time of examination regardless of the condition of the subject.
[0003]
[Problems to be solved by the invention]
The subject of this invention is providing the liver function diagnostic agent which can evaluate liver function safely and simply irrespective of a test subject's state.
[0004]
[Means for Solving the Problems]
The present inventors have result of intensive studies to solve the above problems, administering a fatty acid labeled with carbon atoms of at least one or more specific positions is ¹³ C, the rate of increase of ¹³ C levels in exhaled CO ₂ It has been found that liver function can be accurately diagnosed by measurement, and the present invention has been completed.
[0005]
That is, the present invention is a liver function diagnostic agent containing a fatty acid in which at least one specific carbon is labeled with ¹³ C.
Hereinafter, the present invention will be described in detail.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
The fatty acid of the liver function diagnostic agent of the present invention is a fatty acid in which at least one specific carbon is labeled with ¹³ C.
[0007]
Fatty acids are constituents of fats and oils and are contained in food. It is also a component added as a nutrient source to infusions. Furthermore, since ¹³ C is a stable isotope, unlike radioisotopes, there is no risk of radiation exposure, so there is no problem with the safety of this drug.
[0008]
The fatty acid used in the present invention preferably includes acetic acid, octanoic acid, palmitic acid, oleic acid, linoleic acid, linolenic acid and the like, but is not limited thereto.
In addition, the fatty acid may be any fatty acid labeled with at least one specific carbon. The carbon position to be labeled is not limited.
[0009]
The test using the liver function diagnostic agent of the present invention is performed by an exhalation test in which the subject is administered once or continuously, and an increase in ¹³ C concentration in exhaled CO ₂ after the administration is measured. Specifically, the ¹³ C concentration in exhaled CO ₂ after administration is measured, and the rate of increase in ¹³ C concentration in exhaled CO ₂ after a certain period of time (eg, 5 minutes, 10 minutes, 15 minutes) after administration ( Δ ¹³ C (‰)), or the rate of increase in ¹³ C concentration in exhaled CO ₂ (Δ ¹³ C (‰)) up to a certain time after administration (rise slope, change in slope, peak time, etc.) ) To evaluate liver function. Furthermore, evaluation by such a breath test is useful alone, but it is more preferable to make a comprehensive judgment in combination with a bilirubin value or the like.
[0010]
Here, measurement of ¹³ C concentration in exhaled CO ₂ is performed by gas chromatography-mass spectrometry (GC-MS), infrared spectroscopy, mass spectrometry, photoelectric acoustic spectroscopy, NMR (nuclear magnetic resonance) method. be able to.
[0011]
The liver function diagnostic agent of the present invention comprises the above-mentioned fatty acid in which at least one specific carbon is labeled with ¹³ C (hereinafter referred to as labeled fatty acid) alone or mixed with an excipient or carrier, and the route of administration. Depending on the dosage form, it is formulated into oral preparations (tablets, capsules, powders, granules, liquids, etc.), injections and the like. The excipient or carrier may be any one that is conventionally used in the art and is pharmaceutically acceptable, and the type and composition thereof are appropriately changed depending on the administration route and administration method. For example, water is used as the liquid carrier. As the solid carrier, cellulose derivatives such as hydroxypropyl cellulose, organic acid salts such as magnesium stearate, and the like are used. In the case of injections, it is desirable to emulsify by adding an emulsifier to liquids for intravenous administration such as sterilized water, physiological saline, various buffer solutions, and electrolyte solutions. It can also be administered as an oral preparation as a lyophilized preparation.
[0012]
The content of the labeled fatty acid in the preparation varies depending on the kind of preparation, but is usually 1 to 100% by weight, preferably 50 to 100% by weight. For example, in the case of an injection, a labeled fatty acid may be added so that it is usually 1 to 40% by weight. In the case of capsules, tablets, granules, and powders, the labeled fatty acid is about 10 to 100% by weight, preferably 50 to 100% by weight, and the balance is the carrier.
[0013]
The dose of the diagnostic agent for liver function of the present invention is required to be an amount capable of confirming an increase in ¹³ CO _{2 in} exhaled breath by administration, and varies depending on the patient's age, body weight, and examination purpose. In the case of adults, it is about 1 to 2000 mg / kg body weight.
[0014]
The agent for diagnosing liver function of the present invention can be used for liver diseases such as cirrhosis, chronic hepatitis, acute hepatitis, and liver cancer, diagnosis of liver damage, and evaluation of liver function before and after surgery.
EXAMPLES The present invention will be specifically described below with reference to examples, but the scope of the present invention is not limited to these examples.
[0015]
【Example】
The ¹³ C purity at the ¹³ C labeling position of the labeled fatty acid used in the present invention is 99% or more. Unless otherwise specified, special grade reagents were used.
[Example 1] Method of breath test
(1) Preparation of acute hepatitis rats Male Sprague-Dawley (SD) rats were purchased from Charles River, Japan as test animals. The purchased rats were reared until use under conditions of 23 ± 2 ° C. and humidity 55 ± 10%. The rats were anesthetized by intraperitoneal administration of Nembutal (50 mg / kg) and then galactosamine hydrochloride (200 mg / ml saline solution) was intraperitoneally administered 0.6 to 1.2 g / kg [Koff, S. et al., Proc. Soc. Exptl. Med 137, 696 (1971); Keppler, D. et al., Exp. Mol. Pathology, 9, 279 (1968); (by disease) Model animal production and new drug development Tests and experimental methods for this, supervised by Masaharu Uchi, 126 (1993)]. Two days later, blood was collected from the tail vein, and the serum was separated, and glutamic pyruvic transaminase activity (GPT) value and total bilirubin value were measured by Fuji Drychem FDC5500 [Fuji Photo Film Co., Ltd.].
[0016]
(2) ¹³ C-Breath test
The following exhalation tests were performed on acute hepatitis rats and healthy rats prepared in (1).
Rats fasted overnight were anesthetized by intraperitoneal administration of Nembutal (50 mg / kg) and fixed on the operating table. A cap for exhalation of breath was placed on the head, and a predetermined amount of labeled fatty acid was administered from the femoral vein. Using a stroke pump [Variable Stroke Pump VS-500, Shibata Kagaku Kogyo Co., Ltd.], aspirated air is sucked at a rate of about 100 ml / min, and the ¹³ CO ₂ Analyzer EX-130S [JASCO Corporation] It was introduced into the flow cell. A perma pure dryer (MD-050-12P, Perma Pure INC.) Was installed between the cap for exhalation suction and the stroke pump to remove water vapor in the exhalation (Fig. 1).
[0017]
¹³ Data output from the CO ₂ analyzer is A / D converted and imported into a personal computer (Apple Power Macintosh 8500). Using the data processing software Lab VIEW (National Instruments), 10 points of data are averaged every 100 msec at 5-second intervals. Then, a continuous measurement ¹³ C-expiration test was conducted by converting into ¹³ Cat%, Δ ¹³ C (‰), and carbon dioxide concentration (%). The converted data was displayed on the screen in real time and then saved on the hard disk. During the breath test, rectal temperature was monitored and maintained at 37 ± 0.5 ° C. with a small animal body temperature controller TR-100 (Fine Science Tools INC.). The carbon dioxide concentration in the exhaled breath was maintained at 3 ± 0.5%. At the end of the experiment, the rats were sacrificed with an excess of Nembutal.
Δ ¹³ C (‰) was calculated from the ¹³ C concentration ( ¹³ C tmin) in the exhaled CO ₂ at each time point and the ¹³ C concentration ( ¹³ C std) of the CO ₂ standard gas by the following equation.
[Expression 1]
Δ ¹³ C (‰) = {( ¹³ C tmin− ¹³ C 0 min) / ¹³ C std} × 1000
[0018]
For Example 2 ^1-13 C-oleic acid breath test normal rats (total bilirubin value ≦ 0.7mg / dl, n = 4 ) and acute hepatitis rats (total bilirubin value ≧ 2.4mg / dl, n = 4 ), Twin ¹³ (0.2%) and 1-13C-oleic acid (purchased from ICON) emulsified with physiological saline were administered at 70 mg / kg from the femoral vein, and exhaled CO ₂ according to the method described in Example 1. The increase rate of ¹³ C concentration (Δ ¹³ C (‰)) was measured.
[0019]
In healthy rats, ^1-13 C-oleic acid up to 20 minutes after the administration Δ ¹³ C (‰) values continued to increase. On the other hand, in acute hepatitis rats, the Δ ¹³ C (‰) value increased until about 8 minutes after administration, but remained almost constant until 20 minutes thereafter (FIG. 2).
[0020]
The Δ ¹³ C (‰) value of 20 minutes after administration was 89.80 ± 10.44 ‰ in acute hepatitis rats and 119.81 ± 2.14 ‰ in healthy rats, and acute hepatitis rats were very significant compared to healthy rats (p <0.01 ( ANOVA with Fischer LSD)).
[0021]
In addition, the slope of increase in Δ ¹³ C (‰) from 10 to 20 minutes after administration was -1.79 ± 1.58 ‰ / 10 minutes in acute hepatitis rats and 18.22 ± 2.94 ‰ / 10 minutes in healthy rats. Rats were significantly smaller (p <0.0001 (ANOVA with Fischer LSD)) compared to healthy rats.
[0022]
Therefore, it is possible to detect liver dysfunction from the Δ ¹³ C (‰) value after a certain time after administration of 1 ¹³ C-oleic acid or the slope of increase of Δ ¹³ C (‰) value after administration. is there.
[0023]
For Example 3 ^1-13 C-octanoic acid breath test normal rats (total bilirubin value ≦ 0.7mg / dl, n = 4 ) and acute hepatitis rats (total bilirubin value> 2.5mg / dl, n = 4 ), Twin 20 emulsified with the ^1-13 C-octanoic acid (mass Trace, Inc. purchased from) was administered 30 mg / kg from the femoral vein at (0.2%) saline, according to the method described in example 1, exhaled CO The increase rate of ¹³ C concentration in ₂ (Δ ¹³ C (‰)) was measured.
[0024]
In both healthy rats and acute hepatitis rats, the Δ ¹³ C (‰) value increased rapidly until about 5 minutes after administration of 1 ¹³ C-octanoic acid, but then gradually decreased until 20 minutes (FIG. 3).
[0025]
The Δ ¹³ C (‰) value for 2 minutes after administration was 12.71 ± 2.00 ‰ in acute hepatitis rats and 8.37 ± 1.42 ‰ in healthy rats. Acute hepatitis rats were significantly different from normal rats (p <0.05 (ANOVA with Fischer LSD)).
[0026]
The slope of increase in Δ ¹³ C (‰) from 10 to 20 minutes after administration was −5.48 ± 1.33 ‰ / 10 minutes in acute hepatitis rats, and −2.21 ± 0.29 ‰ / 10 minutes in healthy rats. Was very significant (p <0.01 (ANOVA with Fischer LSD)) compared to healthy rats.
[0027]
Therefore, it is possible to detect liver dysfunction from the Δ ¹³ C (‰) value after a certain time after administration of 1 ¹³ C-octanoic acid or the slope of increase of Δ ¹³ C (‰) value after administration. is there.
[0028]
[Example 4] 1 to ¹³ C-palmitic acid breath test healthy rats (total bilirubin value ≦ 0.7 mg / dl, n = 4) and acute hepatitis rats (total bilirubin value ≧ 4 mg / dl, n = 4) 20 emulsified with the ^1-13 C-palmitic acid (mass Trace, Inc. purchased from) was administered 50 mg / kg from the femoral vein at 0.4% saline, according to the method described in example 1, exhaled CO ₂ The increase rate of ¹³ C concentration (Δ ¹³ C (‰)) was measured.
[0029]
The Δ ¹³ C (‰) value at 15 minutes after administration was 12.22 ± 0.36 ‰ in acute hepatitis rats and 13.67 ± 0.41 ‰ in healthy rats. Acute hepatitis rats were very significant compared to healthy rats (p <0.01 ( ANOVA with Fischer LSD)).
[0030]
Therefore, it is possible to detect liver dysfunction from the Δ ¹³ C (‰) value after a certain time after administration of 1 ¹³ C-palmitic acid.
[0031]
Example 5 ^1-13 C-acetate breath test normal rats (total bilirubin value ≦ 0.8mg / dl, n = 4 ) and acute hepatitis rats (total bilirubin value> 2mg / dl, n = 4 ) for a physiological saline was dissolved in water ^1-13 C-acetate (mass Trace, Inc. purchased from) was administered 10 mg / kg from the femoral vein, according to the method described in example 1, the rate of increase in ¹³ C concentration in exhaled CO ₂ (Δ ¹³ C (‰)) was measured.
[0032]
The Δ ¹³ C (‰) value of 20 minutes after administration was 144.05 ± 3.81 ‰ in acute hepatitis rats and 153.55 ± 1.94 ‰ in healthy rats, and acute hepatitis rats were very significant compared to healthy rats (p <0.01 ANOVA with Fischer LSD)).
[0033]
Therefore, it is possible to detect liver dysfunction from the Δ ¹³ C (‰) value after a certain time after administration of 1 ¹³ C-acetic acid.
[0034]
[Formulation Example 1] (Injection)
Pre-sterilized 1 ¹³ C-oleic acid, physiological saline, and polysorbate 80 were aseptically mixed with 10 parts by weight, 89 parts by weight, and 1 part by weight, respectively, and emulsified with an ultrasonic homogenizer. This emulsion was taken in a vial and sealed to obtain an injection.
[0035]
[Formulation Example 2] (Internal solution)
To ^1-13 C-acetate 10 parts by weight, 100 parts by weight of the total amount added to purified water to sterilizing filtration using a dissolved after Millipore filter it. The filtrate was taken in a vial and sealed to obtain an internal solution.
[0036]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, the liver function diagnostic agent which a test subject's physical burden is small, can know an exact test result immediately, can be used safely without a side effect is provided. The diagnostic agent for liver function of the present invention is useful for evaluating liver function at the time of examination.
[Brief description of the drawings]
FIG. 1 shows a schematic diagram of a method of collecting rat breath.
FIG. 2 shows an increase in exhaled ¹³ CO ₂ after administration of 1 ¹³ C-oleic acid.
FIG. 3 shows an increase in exhaled ¹³ CO ₂ after administration of 1 ¹³ C-octanoic acid.

Claims (1)

^1-13 C-oleic ^{acid, 1-13} C-octanoic ^{acid, 1-13} C-palmitic acid or ^1-13 C-liver function diagnosis agent containing acetic acid.

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