JP4007664B2 - Liver function diagnostic agent - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a liver function diagnostic agent, and more particularly to a glycolytic system in which at least one specific carbon is labeled with ¹³ C and a liver function diagnostic agent containing a carboxylic acid constituting a citric acid cycle.
[0002]
[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.
[0003]
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.
[0004]
[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.
[0005]
[Means for Solving the Problems]
As a result of intensive studies to solve the above-mentioned problems, the present inventors administered a carboxylic acid constituting a glycolytic system in which at least one specific carbon is labeled with ¹³ C and a citric acid cycle, and breathed. The inventors have found that liver function can be accurately diagnosed by measuring the increase rate of ¹³ C concentration in CO ₂ , and have completed the present invention.
That is, the present invention is a liver function diagnostic agent comprising a glycolysis system in which at least one or more specific carbons are labeled with ¹³ C and a carboxylic acid constituting a citric acid cycle.
Hereinafter, the present invention will be described in detail.
[0006]
DETAILED DESCRIPTION OF THE INVENTION
The carboxylic acid constituting the glycolytic system and the citric acid cycle of the liver function diagnostic agent of the present invention is a carboxylic acid constituting the glycolytic system and the citric acid circuit in which at least one specific carbon is labeled with ¹³ C. is there. In the present invention, preferable examples of the carboxylic acid constituting the glycolytic system and the citric acid circuit include pyruvic acid, lactic acid, succinic acid, and citric acid, but are not limited thereto.
In addition, the carboxylic acid may be one in which at least one specific carbon is labeled. The carbon position to be labeled is not limited.
^{Since 13} C is a stable isotope, there is no danger of radiation exposure unlike radioisotopes, so there is no problem with the safety of this drug.
[0007]
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.
[0008]
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.
[0009]
The diagnostic agent for liver function of the present invention is a glycolytic system in which at least one or more specific carbons are labeled with ¹³ C and a carboxylic acid constituting a citric acid cycle (hereinafter referred to as labeled carboxylic acid) alone. Alternatively, it is mixed with an excipient or carrier and formulated into an oral preparation (tablet, capsule, powder, granule, liquid, etc.), injection or the like according to the administration route. 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, generally sterile water, physiological saline and various buffer solutions are desirable. Further, it can be used as an oral preparation as a lyophilized preparation, or it can be administered after being dissolved in an appropriate solvent for injection such as sterile water, physiological saline, electrolyte solution or the like at the time of administration.
[0010]
The content of the labeled carboxylic acid in the preparation varies depending on the kind of the preparation, but is usually 1 to 100% by weight, preferably 50 to 100% by weight. For example, in the case of an injection, it may be usually added so as to be 1 to 40% by weight. In the case of capsules, tablets, granules, and powders, the labeled carboxylic acid is about 10 to 100% by weight, preferably 50 to 100% by weight, and the balance is the carrier.
[0011]
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 1000 mg / kg body weight.
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.
[0012]
【Example】
The ¹³ C purity at the ¹³ C labeling position of the labeled carboxylic 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 aged 8-10 weeks were anesthetized by intraperitoneal administration of Nembutal (50 mg / kg) and then galactosamine hydrochloride (200 mg / ml saline solution) was intraperitoneally administered 0.6-1.2 g / kg [Koff, S et al., Proc. Soc. Exptl. Med 137, 696 (1971); Keppler, D. et al., Exp. Mol. Pathology, 9, 279 (1968); Testing and experimental methods for development, 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 Dry Chem FDC5500 [Fuji Photo Film Co., Ltd.].
[0013]
(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. The head was covered with a cap for exhalation, and a predetermined amount of labeled carboxylic 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).
[0014]
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.
[0015]
[Expression 1]
Δ ¹³ C (‰) = {( ¹³ C tmin− ¹³ C 0 min) / ¹³ C std} × 1000
[0016]
Example 2 ^1-13 C-lactate breath test normal rats (8 weeks old, total bilirubin value ≦ 0.6mg / dl, n = 4 ) and acute hepatitis rats (8 weeks old, total bilirubin value> 3.5 mg / dl and n = 4), physiological saline ^1-13 C-sodium lactate was dissolved in (mass Trace, Inc. purchased from) was administered 10 mg / kg from the femoral vein, according to the method described in example 1, in the exhaled CO ₂ The ¹³ C concentration increase rate (Δ ¹³ C (‰)) was measured.
[0017]
In healthy rats, ^1-13 C-sodium lactate to about 2 minutes after administration Δ ¹³ C (‰) value was rapidly increased, gradually decreased thereafter up to 20 minutes. On the other hand, in acute hepatitis rats, the Δ ¹³ C (‰) value increased until about 4 minutes after administration, but then gradually decreased until 20 minutes (FIG. 2).
The Δ ¹³ C (‰) value for 2 minutes after administration was 76.07 ± 5.56 ‰ in the acute hepatitis rat and 251.21 ± 26.15 ‰ in the healthy rat, and the acute hepatitis rat was very significant compared to the healthy rat (p <0.0001 ( ANOVA with Fischer LSD)).
[0018]
In addition, the slope of increase in Δ ¹³ C (‰) value from 1 to 2 minutes after administration was 43.41 ± 4.15 ‰ / min in acute hepatitis rats and 171.16 ± 22.39 ‰ / min in healthy rats. Compared to rats, it was very significant (p <0.0001 (ANOVA with Fischer LSD)).
Therefore, it is possible to detect hepatic dysfunction from the Δ ¹³ C (‰) value after a certain period of time after administration of 1 ¹³ C-sodium lactate or the slope of increase in Δ ¹³ C (‰) value after administration. is there.
[0019]
[Example 3] 3- ¹³ C-lactic acid breath test healthy rats (10 weeks old, total bilirubin value = 0.3 mg / dl, n = 3) and acute hepatitis rats (10 weeks old, total bilirubin value ≧ 2.5 mg / dl) , N = 6) was administered 50 mg / kg sodium ^13- C-lactate (purchased from mass Trace) dissolved in physiological saline from the femoral vein, and in exhaled CO ₂ according to the method described in Example 1. The ¹³ C concentration increase rate (Δ ¹³ C (‰)) was measured.
[0020]
In healthy rats, the Δ ¹³ C (‰) value increased rapidly until about 8 minutes after administration of 3- ¹³ C-sodium lactate, but remained almost constant until 20 minutes thereafter. On the other hand, in acute hepatitis rats, the Δ ¹³ C (‰) value continued to increase until 20 minutes after administration (FIG. 3).
The Δ ¹³ C (‰) value at 5 minutes after administration was 55.42 ± 10.84 ‰ in acute hepatitis rats and 124.48 ± 27.01 ‰ in healthy rats, and acute hepatitis rats were very significant compared to healthy rats (p <0.01 ( ANOVA with Fischer LSD)).
[0021]
The slope of the increase in Δ ¹³ C (‰) value from 10 to 15 minutes after administration was 19.15 ± 9.20 ‰ / 5 minutes in acute hepatitis rats and 0.81 ± 5.16 ‰ / 5 minutes in healthy rats. Was significantly larger (p <0.05 (ANOVA with Fischer LSD)) than healthy rats.
Therefore, it is possible to detect hepatic dysfunction from the Δ ¹³ C (‰) value after a certain period of time after administration of 3- ¹³ C-sodium lactate or the slope of increase in Δ ¹³ C (‰) value after administration. is there.
[0022]
Example 4 ^1-13 C-pyruvic acid breath test normal rats (8 weeks old, total bilirubin value ≦ 0.5mg / dl, n = 4 ) and acute hepatitis rats (8 weeks old, total bilirubin value> 3 mg / dl and n = 4), physiological saline ^1-13 C-sodium pyruvate was dissolved in (purchased from ICON Co.) was administered 20 mg / kg from the femoral vein, according to the method described in example 1, in the exhaled CO ₂ The ¹³ C concentration increase rate (Δ ¹³ C (‰)) was measured.
[0023]
In healthy rats, ^1-13 C-sodium pyruvate to about 4 min after administration Δ ¹³ C (‰) value was rapidly increased, decreased gradually thereafter until 14 minutes. On the other hand, in acute hepatitis rats, the Δ ¹³ C (‰) value increased rapidly until about 5 minutes after administration, but then gradually decreased until 14 minutes (FIG. 4).
[0024]
The Δ ¹³ C (‰) value for 4 minutes after administration was 234.23 ± 33.66 ‰ in acute hepatitis rats and 319.45 ± 21.16 ‰ in healthy rats, and acute hepatitis rats were very significant compared to healthy rats (p <0.01 ( ANOVA with Fischer LSD)).
Therefore, it is possible to detect a liver function disorder from the Δ ¹³ C (‰) value after a certain after administration of sodium ^1-13 C-pyruvate time.
[0025]
[Example 5] 3- ¹³ C-pyruvate breath test healthy rats (8 weeks old, total bilirubin value <0.5 mg / dl, n = 4) and acute hepatitis rats (8 weeks old, total bilirubin value> 3.5 mg /) dl, n = 4) was orally administered 20 mg / kg sodium 3- ¹³ C-pyruvate (purchased from ICON) dissolved in physiological saline, and in exhaled CO ₂ according to the method described in Example 1. The increasing rate of ¹³ C concentration (Δ ¹³ C (‰)) was measured.
[0026]
In healthy rats, the Δ ¹³ C (‰) value increased rapidly until about 7 minutes after administration of sodium 3- ¹³ C-pyruvate, but then remained almost constant until 20 minutes. On the other hand, in acute hepatitis rats, the Δ ¹³ C (‰) value increased rapidly until about 6 minutes after administration, but then gradually increased to 20 minutes (FIG. 5).
The Δ ¹³ C (‰) value at 7 minutes after administration was 160.20 ± 26.27 ‰ in acute hepatitis rats and 226.58 ± 26.56 ‰ in healthy rats, and acute hepatitis rats were significantly higher than healthy rats (p <0.05 (ANOVA with Fischer LSD)).
[0027]
The slope of increase in Δ ¹³ C (‰) from 10 to 20 minutes after administration was 26.35 ± 3.06 ‰ / 10 minutes in acute hepatitis rats and -0.28 ± 7.50 ‰ / 10 minutes in healthy rats. Rats were very significant (p <0.01 (ANOVA with Fischer LSD)) compared to healthy rats.
Therefore, it is possible to detect liver dysfunction from the Δ ¹³ C (‰) value after a certain period of time after administration of sodium 3- ¹³ C-pyruvate or the slope of increase in Δ ¹³ C (‰) value after administration. It is.
[0028]
[Example 6] 1,4- ¹³ C-succinic acid breath test healthy rats (10 weeks old, total bilirubin value ≦ 0.5 mg / dl, n = 3) and acute hepatitis rats (10 weeks old, total bilirubin value> 4 mg) / dl, n = 4) 1,4- ¹³ C-succinic acid (purchased from ICON) dissolved in physiological saline is administered at 4 mg / kg from the femoral vein, and exhaled according to the method described in Example 1. The increasing rate of ¹³ C concentration in CO ₂ (Δ ¹³ C (‰)) was measured.
[0029]
In both healthy and diabetic rats, the Δ ¹³ C (‰) value increased rapidly until 3 minutes after administration of 1,4- ¹³ C-succinic acid, but then gradually decreased until 20 minutes (FIG. 6).
The Δ ¹³ C (‰) value for 7 minutes after administration was 211.88 ± 10.19 ‰ in the acute hepatitis rat and 236.60 ± 5.93 ‰ in the healthy rat. The acute hepatitis rat was significantly higher than the healthy rat (p <0.05 (ANOVA with Fischer LSD)). Therefore, it is possible to detect liver dysfunction from the Δ ¹³ C (‰) value after a certain time after administration of 1,4- ¹³ C-succinic acid.
[0030]
[Example 7 ] 1,6- ¹³ C-citrate breath test Healthy rats (8 weeks old, total bilirubin value ≦ 0.6 mg / dl, n = 4) and acute hepatitis rats (8 weeks old, total bilirubin value> 3 mg) / dl, n = 4), 1,6- ¹³ C-citric acid (purchased from ICON) dissolved in physiological saline is administered at 5 mg / kg from the femoral vein, and exhaled according to the method described in Example 1. The increasing rate of ¹³ C concentration in CO ₂ (Δ ¹³ C (‰)) was measured.
[0031]
The Δ ¹³ C (‰) value of 15 minutes after administration of 1,6-citrate was 66.70 ± 1.10 ‰ in acute hepatitis rats and 74.54 ± 1.53 ‰ in healthy rats, which is much higher than that in healthy rats. Significantly low (p <0.001 (ANOVA with Fischer LSD)).
Therefore, it is possible to detect liver dysfunction from the Δ ¹³ C (‰) value after a certain time after administration of 1,6- ¹³ C-citric acid.
[0032]
[Formulation Example 1] (Injection)
To ^1-13 C-sodium lactate 10 parts by weight, 100 parts by weight of the total amount of physiological saline was added, and sterilization filtration using a lysis after Millipore filter this. The filtrate was taken in a vial and sealed to obtain an injection.
[0033]
[Formulation Example 2] (Internal solution)
^1-13 to C- sodium pyruvate 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.
[0034]
【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-lactic acid.
FIG. 3 shows an increase in exhaled ¹³ CO ₂ after administration of 3 ¹³ C-lactic acid.
FIG. 4 shows an increase in exhaled ¹³ CO ₂ after administration of 1 ¹³ C-pyruvic acid.
FIG. 5 shows an increase in exhaled ¹³ CO ₂ after administration of 3 ¹³ C-pyruvic acid.
FIG. 6 shows an increase in exhaled ¹³ CO ₂ after administration of 1,4- ¹³ C-succinic acid.

Claims (1)

^{1-13 C-lactate, 3-} 13 ^{C-lactate, 1-13} C-pyruvic ^{acid, 3-} 13 C-pyruvic ^{acid, 1,4} 13 C-succinic acid or ^1,6-13 C-citric acid, A diagnostic agent for liver function.

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