JP4007653B2 - Diabetes diagnostic agent - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a diagnostic agent for diabetes, and more particularly to a diagnostic agent for diabetes containing a fatty 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 the collection of a large amount of glucose, restraints for several hours, 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 patients with diabetes 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 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-mentioned problems, the present inventors administered a fatty acid in which at least one specific carbon is labeled with ¹³ C, and the rate of increase in ¹³ C concentration in exhaled CO _2. As a result, it was found that diabetes can be accurately diagnosed, and the present invention has been completed.
That is, the present invention is a diagnostic agent for diabetes comprising 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 diagnostic agent for diabetes of the present invention is a fatty acid in which at least one specific carbon is labeled with ¹³ C.
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.
[0007]
The fatty acid used in the present invention preferably includes, but is not limited to, acetic acid, octanoic acid, palmitic acid, oleic acid, linoleic acid, linolenic acid and the like.
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.
[0008]
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 (‰)) over a certain period of 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.
[0009]
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.
[0010]
The diagnostic agent for diabetes according to the present invention comprises a fatty acid in which at least one or more specific carbons are labeled with ¹³ C (hereinafter referred to as labeled fatty acid) alone or mixed with an excipient or carrier, and is used as an administration route. Accordingly, it is formulated into oral preparations (tablets, capsules, powders, granules, liquids, etc.) and injections. 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 generally 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.
[0011]
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.
[0012]
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 2000 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.
[0013]
【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 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 reared until use under conditions of 23 ± 2 ° C. and humidity of 55 ± 10%.
[0014]
Streptozotocin (STZ) administration to neonatal rats caused diabetes of insulin secretion failure type (separate cell engineering, 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 on the second day of life. STZ was dissolved in citrate buffer (pH 4.5), and 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 after 7 weeks. However, the increase in fasting blood glucose level is mild and almost normal.
[0015]
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 (Diabetes Test Manual, supervised by 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.
[0016]
(2) The following breath test was carried out on diabetic rats (8 or 9 weeks old) and healthy rats (8 or 9 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). 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.
[0018]
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
[0019]
[Example 2] 1-1 ¹³ C-acetic acid breath test healthy rats (8 weeks old, fasting blood glucose level 73.8 ± 5.1 mg / dl, n = 4) and diabetic rats (8 weeks old, occasional blood glucose level 398.3 ± 57.5 mg) / dl, fasting blood glucose 75.0 ± 8.3 mg / dl, the n = 3), purchased from dissolved in physiological saline ^1-13 C-acetate (mass Trace, Inc.) were administered 10 mg / kg from the femoral vein, performed 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.
[0020]
In healthy rats, the Δ ¹³ C (‰) value increased rapidly until about 5 minutes after administration of 1 ¹³ C-acetic acid, but then remained almost constant until 20 minutes. On the other hand, in diabetic rats, the Δ ¹³ C (‰) value increased rapidly until about 4 minutes after administration, but then gradually decreased until 20 minutes (FIG. 2).
The Δ ¹³ C (‰) value for 3 minutes after administration was 193.09 ± 25.69 ‰ in diabetic rats and 117.50 ± 8.74 ‰ in healthy rats. Diabetic rats were significantly more significant (p <0.01 (ANOVA with Fischer LSD)).
[0021]
In addition, the slope from 10 minutes to 20 minutes after administration was -31.25 ± 13.00 ‰ / 10 minutes in diabetic rats and -4.67 ± 8.49 ‰ / 10 minutes in healthy rats, and diabetic rats were significantly different from healthy rats (p. <0.05 (ANOVA with Fischer LSD)).
Thus, ^1-13 C-fixed time after administration acetate Δ ¹³ C (‰) value or the slope of increase of Δ ¹³ C (‰) value after administration, it is possible to diagnose diabetes. Diabetes can also be diagnosed for mild diabetes that shows normal blood glucose levels on an empty stomach.
[0022]
Example 3 ^1-13 C-oleic acid breath test normal rats (9 weeks old, fasting blood glucose 74.8 ± 9.8mg / dl, n = 4) and diabetic rats (9 weeks old, at any time blood sugar 402.5 ± 31.9 mg / dl, fasting blood glucose 83.5 ± 4.4mg / dl, n = 4 in), was emulsified with Tween 20 (0.1%) saline ^1-13 C-oleic acid (purchased from ICON Co.) 70 mg / kg was administered from the femoral vein, and the rate of increase in ¹³ C concentration (Δ ¹³ C (‰)) in exhaled CO ₂ was measured according to the method described in Example 1.
[0023]
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 diabetic rats, the Δ ¹³ C (‰) value increased rapidly until about 8 minutes after administration, but then gradually decreased until 20 minutes (FIG. 3).
The Δ ¹³ C (‰) value at 8 minutes after administration was 132.93 ± 7.09 ‰ in diabetic rats and 86.51 ± 14.72 ‰ in healthy rats. Diabetic rats were very significant compared to healthy rats (p <0.01 with Fischer LSD)).
[0024]
In addition, the slope from 10 minutes to 20 minutes after administration was -12.79 ± 3.72 ‰ / 10 minutes in diabetic rats and 13.61 ± 4.58 ‰ / 10 minutes in healthy rats, and diabetic rats were very significant compared to healthy rats ( p <0.001 (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 1 ¹³ C-oleic acid 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.
[0025]
Example 4 ^1-13 C-palmitic acid breath test normal rats (9 weeks old, fasting blood glucose 74.8 ± 7.1mg / dl, n = 4) and diabetic rats (9 weeks old, at any time blood sugar 422.8 ± 42.0 mg / dl, fasting blood glucose 80.8 ± 5.1mg / dl, n = 4 ) which was emulsified with Tween 20 (0.2%) saline ^1-13 C-palmitic acid (mass Trace, Inc. purchased from) Was administered at 50 mg / kg from the femoral vein, and the rate of increase in ¹³ C concentration (Δ ¹³ C (‰)) in exhaled CO ₂ was measured according to the method described in Example 1.
[0026]
In healthy rats, the Δ ¹³ C (‰) value continued to increase until 20 minutes after administration of 1 ¹³ C-palmitic acid. On the other hand, in diabetic rats, the Δ ¹³ C (‰) value increased rapidly until about 5 minutes after administration, but then gradually decreased until 20 minutes (FIG. 4).
The slope from 10 to 20 minutes after administration was -4.40 ± 2.21 ‰ / 10 minutes in diabetic rats and 1.43 ± 1.33 ‰ / 10 minutes in healthy rats, and diabetic rats were very significant compared to healthy rats (p < 0.01 (ANOVA with Fischer LSD)).
[0027]
Therefore, it is possible to diagnose diabetes from the slope of increase in Δ ¹³ C (‰) value after administration of 1 ¹³ C-palmitic acid. Diabetes can also be diagnosed for mild diabetes that shows normal blood glucose levels on an empty stomach.
[0028]
[Example 5] 1-1 ¹³ C-octanoic acid breath test healthy rats (9 weeks old, fasting blood glucose level 78.3 ± 4.7 mg / dl, n = 4) and diabetic rats (9 weeks old, occasional blood glucose level 440.3 ± 38.1) mg / dl, fasting blood glucose 89.8 ± 9.4mg / dl, n = 4 ) which was emulsified with Tween 20 (0.2%) saline ^1-13 C-octanoic acid (mass Trace, Inc. purchased from) Was administered at 30 mg / kg from the femoral vein, and the rate of increase in ¹³ C concentration (Δ ¹³ C (‰)) in exhaled CO ₂ was measured according to the method described in Example 1.
[0029]
In healthy rats, the Δ ¹³ C (‰) value increased rapidly until about 5 minutes after administration of 1 ¹³ C-octanoic acid, but then remained almost constant until 20 minutes. On the other hand, in diabetic rats, the Δ ¹³ C (‰) value increased rapidly until about 5 minutes after administration, but then gradually decreased until 20 minutes (FIG. 5).
The slope from 10 to 20 minutes after administration was -20.55 ± 7.75 ‰ / 10 minutes in diabetic rats and -4.76 ± 4.41 ‰ / 10 minutes in healthy rats. Diabetic rats were significantly more significant than healthy rats (p <0.05). (ANOVA with Fischer LSD)).
[0030]
Therefore, it is possible to diagnose diabetes from the slope of increase in Δ ¹³ C (‰) value after administration of 1 ¹³ C-octanoic acid. Diabetes can also be diagnosed for mild diabetes that shows normal blood glucose levels on an empty stomach.
[0031]
[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.
[0032]
[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.
[0033]
【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 1 ¹³ C-acetic acid.
FIG. 3 shows an increase in exhaled ¹³ CO ₂ after administration of 1 ¹³ C-oleic acid.
FIG. 4 shows an increase in exhaled ¹³ CO ₂ after administration of 1 ¹³ C-palmitic acid.
FIG. 5 shows an increase in exhaled ¹³ CO ₂ after administration of 1 ¹³ C-octanoic acid.

Claims (1)

^{1-13 C-acetate, 1-13} C-oleic ^acid, diabetes diagnostic agent comprising a fatty acid selected from the group consisting of ^1-13 C-palmitic acid and ^1-13 C-octanoic acid.

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