JP4007663B2 - Diabetes diagnostic agent - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a diagnostic agent for diabetes, and particularly to a diagnostic agent for diabetes containing lactic acid or citric acid in which at least one specific carbon is labeled with ¹³ C.
[0002]
[Prior art]
Test methods generally used for primary screening of diabetes diagnosis are urine glucose test and fasting blood glucose level test. Although these tests are simple and highly specific, they are low in sensitivity and negative in mild diabetic patients, so over 70% of patients are overlooked and are considered inadequate as a screening test for diabetes. (Sekikawa et al., Medical Practice 10:63, 1993). On the other hand, the glucose tolerance test used for the definitive diagnosis of diabetes requires side effects due to collection of a large amount of glucose, several hours of restraint, repeated blood sampling, and the physical burden on the subject is large and laborious. It is actually impossible to carry out as a screening test. In recent years, blood HbA1C and fructosamine tests that reflect the average blood glucose level over a certain period in the past have been introduced by some institutions as screening tests for diabetes. However, these methods are not sufficiently sensitive and specific to mild diabetes, and the problem of differences in measured values between facilities remains.
[0003]
On the other hand, blood glucose levels, HbA1C, and fructosamine tests are widely used in the management of diabetic patients and the determination of therapeutic effects. However, in mild diabetes, the blood glucose level decreases when hungry, so it is not a criterion for determination. In addition to the above-mentioned problems, HbA1C and fructosamine tests cannot be known until the next visit, so the problem is that instructions will be given to patients based on past test results. is there.
Under such circumstances, the burden on the test subject is small and the results can be obtained immediately and accurately, which is effective even when targeting diabetic patients for the diagnosis of diabetes, management of diabetic patients, and determination of therapeutic effects. The development of an inspection method that can be used is desired.
[0004]
[Problems to be solved by the invention]
An object of the present invention is to provide a diagnostic agent for diabetes that is effective even for mild diabetic patients and that can reduce the burden on the subject and can obtain accurate test results immediately.
[0005]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventors administered lactic acid or citric acid in which at least one specific carbon is labeled with ¹³ C, and the ¹³ C concentration in exhaled CO ₂ The inventors have found that diabetes can be accurately diagnosed by measuring the rate of increase in the amount of the protein, and have completed the present invention.
That is, the present invention is a diagnostic agent for diabetes containing lactic acid or citric 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 lactic acid or citric acid of the diagnostic agent for diabetes according to the present invention is lactic acid or citric acid in which at least one specific carbon is labeled with ¹³ C. 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 diagnostic agent for diabetes of the present invention is carried out by an exhalation test in which this is administered to a subject 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.) ) Diabetes is diagnosed from the data. Furthermore, evaluation by such a breath test is useful alone, but it is more preferable to make a comprehensive judgment in combination with blood glucose level, HbA1C level, fructosamine level and 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 diabetes according to the present invention is an oral preparation according to the administration route, wherein lactic acid or citric acid in which at least one specific carbon is labeled with ¹³ C is used alone or mixed with an excipient or carrier. (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, 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 labeled lactic acid or labeled citric acid in the preparation varies depending on the type of 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 lactic acid or labeled citric acid is about 10 to 100% by weight, preferably 50 to 100% by weight, and the balance is the carrier.
[0011]
The dosage of the diagnostic agent for diabetes of the present invention is required to be an amount that can confirm the increase in ¹³ CO _{2 in} exhaled breath by administration, and varies depending on the age, weight, and examination purpose of the patient. For example, the dosage per dose is For adults, it is about 1 to 1000 mg / kg body weight.
EXAMPLES The present invention will be specifically described below with reference to examples, but the scope of the present invention is not affected by these.
[0012]
【Example】
The ¹³ C purity at the ¹³ C labeling position of the labeled lactic acid or labeled citric 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 mildly diabetic rats Male Sprague-Dawley (SD) rats were purchased from Charles River, Japan. Newborn rats were purchased with the parent. The purchased rats were bred until use under conditions of 23 ± 2 ° C. and humidity of 55 ± 10%.
[0013]
Streptozotocin (STZ) administration to neonatal rats caused diabetes with insulin secretion deficiency (cell engineering separate volume, medical experiment manual series Diabetes research strategy Susumu Seino, Yoshikazu Oka, published by Shujunsha). STZ was administered at a dose of 90 mg / kg subcutaneously to rats on the second day after birth. STZ was dissolved in citrate buffer (pH 4.5), and the administration was completed within 5 minutes after dissolution. Two days later, blood was collected from the heart, blood glucose was measured at any time using Terumo Mediace (blood glucose measurement set), and individuals with a concentration of 275 mg / dl or more were selected. In these rats, the blood glucose level started to rise from 5 weeks after STZ administration, and the blood glucose level was elevated in almost all rats 7 weeks later. However, the increase in fasting blood glucose level is mild and almost normal.
According to the Japan Diabetes Society Standard (1982), fasting blood glucose level (venous whole blood) of 120 mg / dl or more is considered as diabetes (published by Diabetes Test Manual Yukio Shigeta, published by Nanedo). Therefore, the above insulin secretion deficient type diabetic rat, which is not regarded as diabetes from the fasting blood glucose test and has a fasting blood glucose level of less than 120 mg / dl (advanced blood glucose level of 250 mg / dl or more), was used as a mild diabetes model.
[0014]
(2) The following breath test was carried out on diabetic rats (8 weeks old) and healthy rats (8 weeks old) prepared in the ¹³ C-breath test (1).
Rats fasted overnight were anesthetized by intraperitoneal administration of Nembutal (50 mg / kg) and fixed on the operating table. Blood was collected from the tail vein and blood glucose level was measured using Terumo Mediace (blood glucose measurement set). The head was covered with a cap for exhalation and labeled lactic acid or labeled citrate was administered from the femoral vein. Stroke Pump [Variable Stroke Pump VS-500, (Ltd.) Shibata Scientific Industries] of sucked exhalation at a rate of about 100 ml / min using a directly ¹³ CO ₂ Analyzer EX-130S [(Ltd.) JASCO] 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).
[0015]
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.
[0016]
[Expression 1]
Δ ¹³ C (‰) = {( ¹³ C tmin− ¹³ C 0 min) / ¹³ C std} × 1000
[0017]
Example 2 3- ¹³ C-lactate breath test normal rats (8 weeks old, fasting blood glucose 67.8 ± 6.5mg / dl, n = 4) and diabetic rats (8 weeks old, at any time blood sugar 412.8 ± 71.7 mg / dl, fasting blood glucose 88.5 ± 17.2 mg / dl, the n = 4), purchased from dissolved 3- ¹³ C-sodium lactate (mass Trace Co. in physiological saline) was administered 50 mg / kg from the femoral vein, According to the method described in Example 1, the rate of increase in ¹³ C concentration (Δ ¹³ C (‰)) in exhaled CO ₂ was measured.
[0018]
In healthy rats, the Δ ¹³ C (‰) value increased rapidly until about 5 minutes after administration of 3- ¹³ C-sodium lactate, but then gradually increased to 20 minutes. On the other hand, in diabetic rats, the Δ ¹³ C (‰) value increased rapidly until about 7 minutes after administration, but then gradually decreased until 20 minutes (FIG. 2).
The Δ ¹³ C (‰) value at 10 minutes after administration was 202.08 ± 6.05 ‰ in diabetic rats and 130.30 ± 11.47 ‰ in healthy rats, and diabetic rats were very significant compared to healthy rats (p <0.0001 (ANOVA with Fischer LSD)).
[0019]
The slope from 2 to 4 minutes after administration was 96.83 ± 17.69 ‰ / 2 minutes in diabetic rats and 60.69 ± 6.80 ‰ / 2 minutes in healthy rats, and diabetic rats were significantly different from healthy rats (p <0.05). (ANOVA with Fischer LSD)).
Therefore, it is possible to diagnose diabetes 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. . Diabetes can also be diagnosed for mild diabetes that shows normal blood glucose levels on an empty stomach.
[0020]
[Example 3] 1,6- ¹³ C-citrate breath test healthy rats (8 weeks old, fasting blood glucose level 66.3 ± 9.6 mg / dl, n = 4) and diabetic rats (8 weeks old, occasional blood glucose level 376.5) ± 57.3 mg / dl, fasting blood glucose level 76.5 ± 16.6 mg / dl, n = 4), 1,6- ¹³ C-citric acid (purchased from ICON) dissolved in physiological saline from the femoral vein After administration of kg, 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.
The Δ ¹³ C (‰) value of 20 minutes after administration of 1,6- ¹³ C-citric acid was 54.38 ± 3.34 ‰ in diabetic rats and 67.49 ± 1.82 ‰ in healthy rats. Diabetic rats were compared to healthy rats, Very low (p <0.01 (ANOVA with Fischer LSD)).
[0021]
The slope from 10 to 14 minutes after administration was -10.45 ± 1.30 ‰ / 4 minutes in diabetic rats and -5.81 ± 0.92 ‰ / 4 minutes in healthy rats. (P <0.01 (ANOVA with Fischer LSD)).
Therefore, it is possible to diagnose diabetes from the Δ ¹³ C (‰) value after a certain time after administration of 1,6- ¹³ C-citric acid or the slope of increase of Δ ¹³ C (‰) value after administration It is. Diabetes can also be diagnosed for mild diabetes that shows normal blood glucose levels on an empty stomach.
[0022]
[Formulation Example 1] (Injection)
3- ¹³ C-to-sodium lactate 10 parts by weight, 100 parts by weight of the total amount of physiological saline was added, and sterilization filtration using a dissolved after Millipore filter it. The filtrate was taken in a vial and sealed to obtain an injection.
[0023]
[Formulation Example 2] (Internal solution)
Purified water was added to 10 parts by weight of 1,6- ¹³ C-citric acid to make a total amount of 100 parts by weight, and this was dissolved and then sterilized by filtration using a Millipore filter. The filtrate was taken in a vial and sealed to obtain an internal solution.
[0024]
【The invention's effect】
According to the present invention, there is provided a diagnostic agent for diabetes that is less burdensome on the subject, can immediately know accurate test results, and can be used safely without side effects. The diagnostic agent for diabetes of the present invention can screen even mild diabetic patients who exhibit normal blood glucose levels on an empty stomach, and can distinguish between healthy and diabetic patients even in situations where they are easily overlooked. Furthermore, it is useful for management of diabetic patients and determination of therapeutic effects.
[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 3 ¹³ C-lactic acid.

Claims (1)

Diabetes diagnostic agent containing 3- ¹³ C-lactic acid or 1,6- ¹³ C-citric acid .

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