JPH11171797A - Diagnostic agent for diabetes - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a diagnostic agent for diabetes capable of instantaneously knowing accurate test results with a small body load of a subject and safely usable without adverse side effects. SOLUTION: This diagnostic agent for diabetes contains a saccharide selected from the group consisting of galactose, fructose and xylose having at least one or more specific sites labeled with<13> C or the diagnostic agent for the diabetes contains a starch containing glucose having at least one or more specified sites labeled with the<13> C as a constituent saccharide. The diagnostic agent for diabetes is capable of discriminating even a healthy person and a patient suffering from the diabetes even under conditions readily overlooking the diabetes.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a diagnostic agent for diabetes, and more particularly to a diagnostic agent for diabetes containing a sugar selected from the group consisting of galactose, fructose and xylose labeled with ¹³ C at least one carbon at a specific position. Or a diagnostic agent for diabetes comprising starch containing glucose whose at least one specific position carbon is labeled with ¹³ C as a constituent sugar.

[0002]

2. Description of the Related Art Test methods generally used for primary screening of diabetes diagnosis are a urine sugar test and a fasting blood glucose test. Although these tests are simple and have high specificity, they are low in sensitivity and negative in mildly diabetic patients, so more than 70% of patients are overlooked and are considered to be insufficient as screening tests for diabetes. Yes (Sekikawa
Et al., Medical Practice 10:63, 1993). On the other hand, the glucose tolerance test used for the definitive diagnosis of diabetes requires the side effects of collecting a large amount of glucose, the restriction of several hours, the necessity of repeated blood sampling, and the physical burden on the subject is large. It is practically impossible to perform 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 tests are not sufficiently sensitive or specific to mild diabetes,
At present, there still remains the problem of differences in measured values between facilities.

[0003] On the other hand, blood glucose, HbA1C, and fructosamine tests are widely used for management of diabetic outpatients and determination of therapeutic effects. However, in the case of mild diabetes, the blood glucose level decreases on an empty stomach, and thus is not a criterion for determination. In addition, the HbA1C and fructosamine tests have the drawback that, in addition to the above-mentioned problems, the results cannot be known until the next visit, and patients will be instructed based on past test results. is there. Under such circumstances, it is effective even when targeting mild diabetic patients for diagnosing diabetes, managing diabetic patients, and judging the therapeutic effect, the burden on the subjects is small, and the results are obtained immediately and accurately. It is hoped that a new testing method will be developed.

[0004]

SUMMARY OF THE INVENTION An object of the present invention is to provide a diabetic diagnosis which is effective even for a mild diabetic patient, has a small burden on a subject, and can immediately obtain an accurate test result. To provide an agent.

[0005]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that at least one carbon at a specific position is labeled with ¹³ C from galactose, fructose, and xylose. A sugar selected from the group consisting of
Alternatively, a starch containing glucose whose at least one specific carbon is labeled with ¹³ C as a constituent sugar is administered, and exhaled C
The present inventors have found that diabetes can be diagnosed accurately by measuring the rate of increase in the concentration of ¹³ C in O ₂ , and have completed the present invention. That is, the present invention provides at least one
A galactose in which the carbon at the specific position is labeled with ¹³ C,
It is a diagnostic agent for diabetes containing a sugar selected from the group consisting of fructose and xylose, or a diagnostic agent for diabetes containing starch having at least one carbon at the specific position labeled with ¹³ C as a constituent sugar. Hereinafter, the present invention will be described in detail.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The galactose, fructose, xylose or starch of the diagnostic agent for diabetes of the present invention is galactose, fructose, xylose or starch in which at least one carbon at a specific position is labeled with ¹³ C. The carbon position to be labeled is not limited. ^{Since 13} C is a stable isotope, unlike radioisotopes, there is no danger of radiation exposure, so there is no problem with the safety of this drug.

The test using the diagnostic agent for diabetes of the present invention is carried out by a single or continuous administration to a subject, and a breath test for measuring an increase in the concentration of ¹³ C in exhaled CO ₂ after the administration. Specifically, the ¹³ C concentration in the exhaled CO ₂ after the administration is measured, and a certain time after the administration (for example, 5 minutes, 10 minutes, 15 minutes)
After the elapse, the rate of increase of the ¹³ C concentration in the exhaled CO ₂ (Δ ¹³
C (‰)), or the change over time (increase slope, change in slope, peak time, etc.) of the rate of increase of ¹³ C concentration (Δ ¹³ C (‰)) in exhaled CO ₂ up to a certain time after administration. Diagnose diabetes from the data. Further, the evaluation by the breath test is useful alone, but the blood glucose level, HbA1C level,
It is more preferable to make a comprehensive judgment in combination with a fructosamine value or the like.

Here, the measurement of the ¹³ C concentration in the exhaled CO ₂ is performed by gas chromatography-mass spectrometry (GC-MS), infrared spectroscopy, mass spectrometry, photoelectric acoustic spectroscopy, NMR (nuclear magnetic resonance) Can be done by law.

[0009] The diagnostic agent for diabetes of the present invention comprises galactose, fructose, xylose (hereinafter, referred to as labeled galactose, labeled fructose, labeled xylose), or at least one of the above-mentioned at least one carbon at the specific position labeled with ¹³ C The above starch containing glucose labeled with ¹³ C at the specific position (hereinafter referred to as “labeled starch”) alone or mixed with an excipient or carrier may be used as an oral preparation (tablet) depending on the administration route. , Capsules, powders, granules, liquids, etc.), injections and the like. The excipient or carrier may be any of those conventionally used in the art and pharmaceutically acceptable.
It is appropriately changed depending on the administration route and administration method. For example,
Water is used as the liquid carrier. As a solid carrier,
Cellulose derivatives such as hydroxypropyl cellulose and organic acid salts such as magnesium stearate are used. In the case of injections, sterile water, physiological saline,
Various buffers are desirable. In addition, it can be used as an oral preparation as a lyophilized preparation or an appropriate solvent for injection at the time of administration,
For example, it can be administered by dissolving it in a liquid for intravenous administration such as sterilized water, physiological saline, or an electrolyte solution.

[0010] The content of labeled galactose, labeled fructose, labeled xylose, or labeled starch in the preparation is as follows:
Although it depends on the type of preparation, it is usually 1 to 100% by weight, preferably 50 to 100% by weight. For example, in the case of an injection, it may be added so as to be usually 1 to 40% by weight. In the case of capsules, tablets, granules and powders, the amount of labeled galactose, labeled fructose, labeled xylose or labeled starch is about 10 to 100% by weight, preferably 50 to 100% by weight, with the balance being a carrier.

The dose of the diagnostic agent for diabetes of the present invention must be sufficient to confirm the increase of ¹³ CO _{2 in} the exhaled breath due to the administration, and varies depending on the age, weight, and purpose of the test of the patient. The dose is about 1 to 2000 mg / kg body weight for adults. Hereinafter, the present invention will be described specifically with reference to Examples, but the scope of the present invention is not affected by these.

[0012]

EXAMPLES The ¹³ C purity of the labeled galactose, labeled fructose or labeled xylose used in the present invention at the ¹³ C labeled position is 99% or more. The ¹³ C purity at the ¹³ C-labeled position of the labeled starch is about 98%. Other special grade reagents were used unless otherwise specified. Example 1 Breath Test Method (1) Preparation of Mildly Diabetic Rat Male Sprague-Dawley (SD) rats were purchased from Charles River Japan. Newborn rats were purchased together with the nursery parents. The purchased rats were bred under conditions of 23 ± 2 ° C. and 55 ± 10% humidity until use.

Streptozotocin (STZ) administration to neonatal rats caused insulin-deficiency-type diabetes mellitus (Cell Engineering Supplement, Medical Experiment Manual Series Diabetes Research Strategy Susumu Kiyono, Yoshikazu Oka, published by Shujunsha). Two days after birth, rats were subcutaneously administered with 90 mg / kg of STZ. STZ was dissolved in a citrate buffer (pH 4.5), and administration was completed within 5 minutes after dissolution. Two days later, blood is collected from the heart and Terumo Mediace (blood glucose measurement set)
The blood glucose level was measured at any time using, and individuals at 275 mg / dl or more were selected. In these rats, the blood sugar level starts to increase at any time from 5 weeks after the STZ administration, and after 7 weeks, almost all rats show a high blood sugar level as needed. However, the increase in fasting blood glucose level is mild and almost normal. Japanese Diabetes Association standards
According to (1982), a fasting blood glucose level (whole venous blood) of 120 mg / dl or more is considered to be diabetes (managed by Diabetes Test Manual, supervised by Yukio Shigeta, published by Nankodo). Therefore, the above-mentioned insulin-deficient type diabetic rats, which are not regarded as diabetic from the fasting blood glucose level test and have a fasting blood glucose level of less than 120 mg / dl (250 mg / dl or more at any time), were used as a mild diabetes model.

(2) ¹³ C-breath test The following breath test was performed on the diabetic rats (8-9 weeks old) and the healthy rats (8-9 weeks old) prepared in (1). (2) -1 Intravenous administration Rats fasted overnight were intraperitoneally administered with Nembutal (50 mg /
kg) and fixed on an operating table. Blood was collected from the tail vein, and the blood glucose level was measured using Terumo mediace (blood glucose measurement set). A cap for exhalation suction was put on the head, and a predetermined amount of labeled galactose or labeled fructose was administered from the femoral vein. Using a stroke pump [Variable Stroke Pump VS-500, Shibata Kagaku Kogyo Co., Ltd.], inhale the exhaled air at a rate of about 100 ml / min.
¹³ CO ₂ analyzer EX-130S [Nihon Bunko Co., Ltd.] was introduced into the flow cell. A perm pure dryer (MD-050-12) is placed between the exhalation suction cap and the stroke pump.
P, Perma Pure INC.) Was installed to remove water vapor in the breath.

[0015] The data output from the ¹³ CO ₂ analyzer is subjected to A / D conversion and then converted to a personal computer (Apple Po
wer uptake Macintosh 8500), the data processing software Lab VIEW (National Instruments) integrated average data of 100msec every 10 points at 5 second intervals using, ¹³ C atom
%, Δ ¹³ C (‰), and carbon dioxide concentration (%) were converted into a continuous measurement ¹³ C-breath test. The converted data was displayed on the screen in real time and saved on the hard disk. During the breath test, the rectal temperature is monitored and the small animal temperature controller TR-100 (Fine Science Tools INC.)
To maintain the temperature at 37 ± 0.5 ° C. In addition, the concentration of carbon dioxide gas during inhalation expiration was maintained at 3 ± 0.5%. At the end of the experiment, the rats were sacrificed by administering an excess amount of Nembutal. Note that Δ ¹³
C (‰) is the concentration of ¹³ C in exhaled CO ₂ at each time point ( ¹³ C tmin)
It was calculated from the following equation from the ¹³ C concentration ( ¹³ C std) of the CO ₂ standard gas.

[0016]

[Equation 1] Δ ¹³ C (‰) = ｛( ¹³ C tmin- ¹³ C 0 min) / ¹³ C s
td｝ × 1000

(2) -2 Oral administration A fasted rat was fixed in a rat holder for a micro-irradiation apparatus without anesthesia, and blood was collected from a tail vein.
Blood glucose was measured using Terumo Mediace (blood glucose measurement set). Using a stroke pump [Variable Stroke Pump VS-500, Shibata Kagaku Kogyo Co., Ltd.], exhaled air is sucked at a speed of about 100-300 ml / min, and ¹³ CO2
₂ Introduced into the flow cell of the analyzer EX-130S [JASCO Corporation]. A perma pure dryer (MD-050-12P, Perma Pur
e INC.) was installed to remove the water vapor in the breath. The rat was once taken out of the rat holder in a state where the carbon dioxide concentration was stable, and labeled xylose or labeled starch was administered into the stomach in a predetermined amount using a probe for oral administration.

The data output from the ¹³ CO ₂ analyzer is subjected to A / D conversion, and then converted to a personal computer (Apple Po
wer uptake Macintosh 8500), the data processing software Lab VIEW (National Instruments) integrated average data of 100msec every 10 points at 5 second intervals using, ¹³ C atom
%, Δ ¹³ C (‰), and carbon dioxide concentration (%) were converted into a continuous measurement ¹³ C-breath test. The converted data was displayed on the screen in real time and saved on the hard disk. In addition, the concentration of carbon dioxide gas during inhalation expiration was maintained at 3 ± 0.5%. At the end of the experiment, the rats were sacrificed by administering an excess amount of Nembutal. Note that Δ ¹³ C (‰) was calculated by the above equation.

[0019] Example 2 ^1-13 C-galactose breath test normal rats (9 weeks old, fasting blood glucose 74.8 ± 5.1mg / dl, n =
4) and diabetic rats (9-week-old, occasional blood glucose 423.3 ± 8
7.5 mg / dl, fasting blood glucose level 94 ± 18.5mg / dl, n = 4 in), purchased from dissolved in physiological saline ^1-13 C-galactose (ICON Co.) was administered 100 mg / kg from the femoral vein, According to the method described in Example 1, the rate of increase of the ¹³ C concentration in the exhaled CO ₂ (Δ
¹³ C (‰)) was measured. In both healthy rats and diabetic rats, Δ ¹³ C (‰) values continued to increase up to 30 minutes after administration of 1- ¹³ C-galactose (FIG. 1).

The Δ ¹³ C (‰) value at 20 minutes after administration was 64.90 ± 5.75 ° in diabetic rats and 37.31 ± 3.37 ° in healthy rats, and the diabetic rats were significantly more significant (p < 0.001 (ANOVA with Fischer LSD)).
The slope from 5 minutes to 15 minutes after administration was 35.53 ± 4.34 ‰ / 10 minutes in diabetic rats and 20.59 ± 2.26 in healthy rats.
‰ / 10 minutes, and the diabetic rat is compared with the healthy rat,
It was very significant (p <0.01 (ANOVA with Fischer LSD)). Therefore, the Δ ¹³ C (‰) value after a certain period of time after administration of 1- ¹³ C-galactose, or the Δ ¹³ C
(Ii) Diabetes can be diagnosed from the slope of the increase in the value. Diagnosis of this diabetes can also be made for mild diabetes that shows normoglycemia on an empty stomach.

Example 3 2- ¹³ C-galactose breath test Healthy rats (9 weeks old, fasting blood glucose 63.8 ± 13.4 mg / dl, n
= 4) and diabetic rats (9 weeks old, occasional blood glucose 371.5 ± 6
3.4mg / dl, fasting blood glucose 86.5 ± 22.6mg / dl, n = 4)
2- ¹³ C-galactose (purchased from ICON) dissolved in physiological saline was administered at 100 mg / kg from the femoral vein, and the increase rate of ¹³ C concentration in exhaled CO ₂ (Δ ¹³ C (‰)) was measured. Δ ¹³ C (‰) until 20 minutes after administration of 2- ¹³ C-galactose in both healthy rats and diabetic rats
The values continued to increase (FIG. 2).

The Δ ¹³ C (‰) value at 20 minutes after administration was 29.68 ± 8.55 ° in diabetic rats and 15.00 ± 1.63 ° in healthy rats, and the diabetic rats were significantly (p <
0.05 (ANOVA with Fischer LSD)). Therefore, Δ ¹³ C after a certain time after administration of 2- ¹³ C-galactose
(‰) Diabetes can be diagnosed from the values. Diagnosis of this diabetes can also be made for mild diabetes that shows normoglycemia on an empty stomach.

[0023] Example 4 ^1-13 C-fructose breath test normal rats (8 weeks old, fasting blood glucose 75.3 ± 4.1mg / dl, n =
4) and diabetic rats (8 weeks old, occasional blood glucose 498.5 ± 7
5.1 mg / dl, fasting blood glucose 99.3 ± 11.8 mg / dl, n = 4)
100 mg / kg of 1- ¹³ C-fructose (purchased from ICON) dissolved in physiological saline was administered via the femoral vein, and the increase rate of ¹³ C concentration in exhaled CO ₂ (Δ ¹³ C (‰)) was measured. In both healthy rats and diabetic rats, Δ ¹³ C (‰) until 20 minutes after administration of 1- ¹³ C-fructose
The values continued to increase (FIG. 3).

The Δ ¹³ C (‰) value at 15 minutes after the administration was 83.51 ± 7.30 ° in diabetic rats and 63.90 ± 7.42 ° in healthy rats, and the diabetic rats were significantly more significant (p < 0.01 (ANOVA with Fischer LSD)).
The slope from 2 minutes to 7 minutes after administration was 47.87 ± 8.34 ‰ / 5 minutes in diabetic rats and 29.07 ± 4.08 ‰ in healthy rats.
/ 5 min, and the diabetic rats were significantly (p <0.05 (ANOVA with Fischer LSD)) larger than the healthy rats. Thus, ^1-13 C-fructose specific time after administration Δ ¹³ C (‰) value or the slope of increase of Δ ¹³ C (‰) value after administration, it is possible to diagnose diabetes.
Diagnosis of this diabetes can also be made for mild diabetes that shows normoglycemia on an empty stomach.

Example 5 2- ¹³ C-Fructose Breath Test Healthy Rats (8 weeks old, fasting blood glucose 75 ± 3.1 mg / dl, n =
4) and diabetic rats (8 weeks old, occasional blood glucose level 533.5mg / d
l, fasting blood sugar level 86 mg / dl, n = 2), 100 mg / kg of 2- ¹³ C-fructose (purchased from ICON) dissolved in physiological saline was administered through the femoral vein, as described in Example 1. According to the method, the rate of increase of the ¹³ C concentration in the exhaled CO ₂ (Δ ¹³ C (‰)) was measured. In healthy rats, Δ ¹³ C (‰) values continued to increase up to 20 minutes after administration of 2- ¹³ C-fructose. In diabetic rats, Δ ¹³ C (‰) values increased sharply up to about 8 minutes after administration, but then gradually increased up to 20 minutes (FIG. 4).

The Δ ¹³ C (‰) value at 20 minutes after administration was 94.68 ‰ in diabetic rats and 58.75 ± 2.73 ‰ in healthy rats, and the diabetic rats were significantly more significant (p <
0.01 (ANOVA with Fischer LSD)). The slope from 2 minutes to 6 minutes after administration was 47.66 in diabetic rats.
‰ / 4 min, 21.4 ± 1.84 ‰ / 4 min in healthy rats, and the diabetic rats were significantly more significant (p <
0.01 (ANOVA with Fischer LSD)). Therefore, Δ ¹³ after a certain time after administration of 2- ¹³ C-fructose
Diabetes can be diagnosed from the C (‰) value or the slope of the increase in Δ ¹³ C (‰) value after administration. Diagnosis of this diabetes can also be made for mild diabetes that shows normoglycemia on an empty stomach.

[0027] Example 6 ^1-13 C-xylose breath test normal rats (9 weeks old, fasting blood glucose 75.5 ± 2.1mg / dl, n =
4) and diabetic rats (9 weeks old, occasional blood glucose 370.3 ± 3
9.0 mg / dl, fasting blood glucose level 77 ± 9.1mg / dl, n = 4 ) which was dissolved in distilled water ^1-13 C-xylose (purchased from ICON Ltd.) 3
00 mg / kg orally and according to the method described in Example 1,
The rate of increase of the ¹³ C concentration in the exhaled CO ₂ (Δ ¹³ C (）)) was measured. The Δ ¹³ C (‰) value 30 minutes after administration of 1- ¹³ C-xylose was 23.40 ± 7.607.6 in diabetic rats and 6.8 in healthy rats.
2 ± 3.02 ‰, which was significantly (p <0.05 (ANOVA with Fischer LSD)) higher in diabetic rats than in healthy rats.

The slope at 20 to 30 minutes after the administration is 12.80 ± 2.18 ° / 10 minutes in diabetic rats and 4.61 ± 1.40 ° / 10 minutes in healthy rats. Significant (p <0.01 (ANOVA with Fi
scher LSD)). Therefore, it is possible to diagnose diabetes from the Δ ¹³ C (‰) value after a certain period of time after the administration of 1- ¹³ C-xylose or the slope of the increase of the Δ ¹³ C (‰) value after the administration. Diagnosis of this diabetes can also be made for mild diabetes that shows normoglycemia on an empty stomach.

[Example 7] U- ^13C- starch breath test healthy rats (9 weeks old, fasting blood glucose level 57.7 ± 4.5 mg / dl, n =
3) and diabetic rats (9 weeks old, occasional blood glucose 398.3 ± 2
Example 2 Oral administration of U- ^13C- starch (purchased from Chlorella Industry) dissolved in distilled water at 30 mg / kg to 4.2 mg / dl, fasting blood glucose 76.5 ± 7.9 mg / dl, n = 4) According to the method described in 1, the rate of increase of the ¹³ C concentration in the exhaled CO ₂ (Δ ¹³ C
(‰)) was measured. Both healthy and diabetic rats have U-
^The Δ ¹³ C (‰) value continued to increase until 20 minutes after the administration of ¹³ C-starch (FIG. 5).

The Δ ¹³ C (‰) value 30 minutes after administration is 144.50 ± 23.2727 in diabetic rats and 213.89 ± 4.66 ‰ in healthy rats.
Diabetic rats were significantly significantly lower (p <0.01 (ANOVA with Fischer LSD)) than healthy rats. Therefore, Δ ¹³ after a certain time after administration of U- ¹³ C-starch
Diabetes can be diagnosed from the C (‰) value. Diagnosis of this diabetes can also be made for mild diabetes that shows normoglycemia on an empty stomach.

[0031] For Formulation Example 1 (Injection) ^1-13 C-galactose 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 placed in a vial and sealed to obtain an injection.

[0032] For Formulation Example 2 (oral liquid) ^1-13 C-fructose 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 placed in a vial and sealed to obtain an oral solution.

[0033]

According to the present invention, there is provided a diagnostic agent for diabetes which can be used safely without any side effects, with a small physical burden on the subject, an accurate test result being immediately known. The diabetic diagnostic agent of the present invention can screen even mild diabetic patients who show normoglycemia on an empty stomach, and can distinguish healthy subjects from diabetic patients even under conditions that are easily overlooked. Furthermore, it is useful for management of outpatients with diabetes and determination of therapeutic effects.

[Brief description of the drawings]

[1] ^1-13 C-after galactose administration in the exhaled ¹³ CO ₂
Indicates an increase.

FIG. 2: Expired ¹³ CO ₂ after administration of 2- ¹³ C-galactose
Indicates an increase.

[3] ^1-13 C-after fructose administration in the exhaled ¹³ CO ₂
Indicates an increase.

FIG. 4: ¹³ CO _{2 in} expiration after administration of 2- ¹³ C-fructose
Indicates an increase.

FIG. 5 shows the increase of ¹³ CO ₂ in breath after administration of U- ¹³ C-starch.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Kunihiko Shibata, Inventor 104-1-405, Takidaicho, Funabashi City, Chiba Prefecture

Claims (2)

[Claims] (1) at least one carbon at a specific position is ¹³ C
A diagnostic agent for diabetes comprising a sugar selected from the group consisting of galactose, fructose, and xylose labeled with: 2. The method according to claim 2, wherein at least one or more specific carbon atoms are ¹³ C
A diagnostic agent for diabetes comprising a starch comprising glucose labeled as a constituent sugar.