JPH11147842A - Diabetic diagnostic - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a diabetic diagnostic having a small load on a light diabetic patient, capable of instantly preparing an accurate examination result, by making the diagnostic include a specific aminopyrine. SOLUTION: This diabetic diagnostic contains an aminopyrine (N,N- dimethyl-<13> C-aminopyrine), in which a carbon atom at the 4-position of dimethylamino group is labeled with<13> C, a phenacetin (ethoxy-1-<13> C-phenacetin) in which a carbon atom at the 1-position of ethoxy group is labeled with<13> C or a methacetin (methoxy-<13> C-methacetin) in which a carbon atom of methoxy group is labeled with<13> C. The content of labeled aminopyrine, labeled phenacetin and labeled methacetin in the preparation is preferably 50-100 wt.%. In the case of injection, the content is 1-40 wt.% and in the case of capsule, tablet, etc., the content is preferably 50-100 wt.%. A dose is, for example, 1-2,000 mg/kg weight per adult daily.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the diabetes diagnostic agent, details 4-position of aminopyrine carbon dimethylamino group is labeled with ¹³ C, phenacetin carbon of 1-position of the ethoxy groups were labeled with ¹³ C, Or a diagnostic agent for diabetes comprising methacetin labeled with ¹³ C at the carbon atom of the methoxy group.

[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 inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, the aminopyrine and the ethoxy group in which the carbon of the dimethylamino group at the 4-position is labeled with ¹³ C are described. Diabetes is diagnosed correctly by administering phenacetin labeled with ¹³ C or methacetin labeled with ¹³ C with the carbon of the methoxy group, and measuring the increase rate of ¹³ C concentration in exhaled CO _2. They have found that they can do this and have completed the present invention.

Namely, the present invention provides 4-position of aminopyrine carbon dimethylamino group is labeled with ¹³ C, phenacetin carbon of 1-position of the ethoxy groups were labeled with ¹³ C, or carbon ¹³ C methoxy groups It is a diagnostic agent for diabetes containing methacetin labeled with. Hereinafter, the present invention will be described in detail.

[0007]

Aminopyrine diabetes diagnostic agent of the embodiment of the present invention is the carbon of the 4-position of the dimethylamino group is labeled with ¹³ C aminopyrine (N, N-dimethyl - ¹³ C-aminopyrine) is, phenacetin ethoxy The carbon at the 1-position of the group is phenacetin labeled with ¹³ C (ethoxy-1- ¹³ C-phenacetin), and methacetin is methacetin labeled with ¹³ C at the carbon of the methoxy group (methoxy- ¹³ C-methacetin). is there. N, N-dimethyl- ^13C- aminopyrine, ethoxy-1-
^{The structures of 13} C-phenacetin and methoxy- ¹³ C-methacetin are shown below.

[0008]

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[0009]

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[0010]

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[0013] Since ¹³ C is a stable isotope, unlike radioisotopes, there is no danger of radiation exposure, and there is no problem in the safety of the present drug. The test using the diabetes diagnostic agent of the present invention is carried out by a single or continuous administration of the agent 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, ¹³ % of the exhaled CO ₂ after administration
The C concentration is measured, and a certain time after administration (for example, 5 minutes, 10 minutes, 1 minute)
5 min) Increase rate of ¹³ C concentration in exhaled CO ₂ after elapse (Δ ¹³ C (‰)) or exhaled C up to a certain time after administration
Diagnosis of diabetes is made from data on the time-dependent change (increase in slope, change in slope, peak time, etc.) of the rate of increase of ¹³ C concentration in O ₂ (Δ ¹³ C (‰)). Furthermore, the evaluation by such a breath test is useful alone, but the blood glucose level, Hb
It is more preferable to judge comprehensively in combination with A1C value, fructosamine value and 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, photoacoustic spectroscopy, NMR (nuclear magnetic resonance). Can be done by law. The diagnostic agent for diabetes according to the present invention comprises aminopyrine labeled with ¹³ C at the dimethylamino group at the 4-position, phenacetin labeled at ¹³ C at the 1-position of the ethoxy group, or ¹³ ppm at the methoxy group. C
Methacetin (hereinafter, referred to as labeled aminopyrine, labeled phenacetin, or labeled methacetin) alone or mixed with an excipient or carrier, and oral preparations (tablets, capsules, powders, granules) depending on the administration route , Liquids, etc.), injections and the like. The excipient or carrier may be any of those conventionally used in the art and pharmaceutically acceptable. The type and composition of the excipient or carrier are appropriately changed depending on the administration route and administration method. For example, water is used as the liquid carrier. As the solid carrier, a cellulose derivative such as hydroxypropylcellulose, an organic acid salt such as magnesium stearate and the like are used.
In the case of injections, sterile water, physiological saline and various buffers are generally desirable. Moreover, it can also be used as an oral preparation as a freeze-dried preparation.

The content of labeled aminopyrine, phenacetin or methacetin in the preparation varies depending on the type of preparation, but is usually 1 to 100% by weight, preferably 50 to 1% by weight.
00% by weight. For example, in the case of injection, usually 1 to 40
What is necessary is just to add so that it may be set to weight%. In the case of capsules, tablets, granules, and powders, the content of labeled aminopyrine, labeled phenacetin, or labeled methacetin is about 10 to 100.
%, Preferably 50 to 100% by weight, with the balance being the 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 air due to the administration, and varies depending on the patient's age, body weight and test purpose. 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.

[0015]

EXAMPLES The ¹³ C purity at the ¹³ C-labeling position of the labeled aminopyrine, phenacetin or methacetin used in the present invention is 99% or more. 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 (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 (2) -1 Intravenous administration The following breath test was performed on the diabetic rat (8 weeks old) and the healthy rat (8 weeks old) prepared in (1). Intraperitoneal administration of Nembutal (50 mg / k
Anesthetized in g) 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 aminopyrine was administered from a femoral vein.
Stroke pump [Variable stroke pump VS-
500, Shibata Scientific Industry Co., Ltd.]
Suction at the speed of in, and use the ¹³ CO ₂ analyzer EX as it is
-130S [Nippon Bunko Co., Ltd.] flow cell. Perm Pure Dryer (MD-050-12P, Perma Pure INC.) Between the exhalation suction cap and the stroke pump
Was installed to remove the water vapor in the breath.

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.

[0019]

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

(2) -2 Oral administration Healthy rats (8- or 9-week-old) and diabetic rats (8- or 9-week-old) fasted overnight were placed in a rat holder for a micro-irradiation apparatus without anesthesia. After fixation, blood was collected from the tail vein, and the blood glucose level was measured using Terumo Mediace (blood glucose measurement set). Stroke pump [Variable
Using a stroke pump VS-500, Shibata Kagaku Kogyo Co., Ltd.], the exhaled air is suctioned at a speed of about 100 to 300 ml / min, and directly introduced into the flow cell of the ¹³ CO ₂ analyzer EX-130S [JASCO Corporation]. did. A perma pure dryer (MD-050-12P, P
erma Pure INC.) was installed to remove 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 a predetermined amount of labeled phenacetin or labeled methacetin was administered into the stomach 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. 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 (‰) was calculated from the above equation.

Example 2 N, N-Dimethyl- ¹³ C-aminopyrine breath test Healthy rats (8 weeks old, fasting blood glucose level 68.8 ± 4.0 mg / dl, n =
4) and diabetic rats (8 weeks old, ad hoc blood glucose 414.5 ± 2
9.9mg / dl, fasting blood glucose 94.8 ± 10.0mg / dl, n = 4)
N, N-dimethyl- ^13C- aminopyrine (purchased from ICON) dissolved in physiological saline was administered at 40 mg / kg through the femoral vein, and the concentration of ^{13C in} exhaled CO _{2 was} measured according to the method described in Example 1. The rate of increase (Δ ¹³ C (‰)) was measured.

In healthy rats, the value of Δ ¹³ C (‰) sharply increased up to about 3 minutes after administration of N, N-dimethyl- ¹³ C-aminopyrine, but gradually increased until 20 minutes thereafter. On the other hand, in diabetic rats, the Δ ¹³ C (‰) value rapidly increased until about 5 minutes after administration, but remained almost constant until 20 minutes thereafter (FIG. 1).
The Δ ¹³ C (‰) value 20 minutes after administration was 193.62 in diabetic rats.
± 22.21 ‰ and 92.2 ± 12.64 ‰ in healthy rats, and the diabetic rats were significantly more significant (p <
0.001 (ANOVA with Fischer LSD)).

The slope from 1 minute to 2 minutes after administration is as follows:
95.68 ± 16.33 ‰ / min in diabetic rats, 4 in healthy rats
1.16 ± 8.89 ‰ / min, indicating that the diabetic rats were significantly more significant (p <0.01 (ANOVA with Fischer
LSD)). Thus, N, N-dimethyl - ¹³ C-
Diabetes can be diagnosed based on the Δ ¹³ C (‰) value after a certain period of time after aminopyrine administration or the slope of 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.

Example 3 Ethoxy-1- ¹³ C-phenacetin breath test Healthy rats (9 weeks old, fasting blood glucose 72 ± 8.9 mg / dl, n =
4) and diabetic rats (9 weeks old, occasional blood glucose level 504.7 ± 5
5.0 mg / dl, fasting blood glucose 101.7 ± 3.3 mg / dl, n = 3)
Ethoxy-1- ¹³ C-phenacetin (purchased from ICON) suspended in 0.5% sodium carboxymethylcellulose aqueous solution was orally administered at 90 mg / kg, and the ¹³ C concentration in exhaled CO ₂ according to the method described in Example 1. Increase rate (Δ ¹³ C
(‰)) was measured.

In both healthy rats and diabetic rats, the value of Δ ¹³ C (値) continued to increase until 30 minutes after administration of ethoxy-1- ¹³ C-phenacetin. (FIG. 2). The Δ ¹³ C (‰) value 30 minutes after administration was 119.85 ± 13.87 ‰ in diabetic rats and in healthy rats.
70.99 ± 12.34 ‰, and the diabetic rats were significantly more significant (p <0.01 (ANOVA with Fischer
LSD)).

The slope from 10 minutes to 15 minutes after administration is 30.88 ± 5.62 ‰ / 5 minutes in diabetic rats and 16.3 ± 4.47 ‰ / 5 minutes in healthy rats. p <0.05 (ANOVA with Fischer LS
D)) was big. Therefore, ^ethoxy-1-13 C-constant time after the phenacetin administration Δ ¹³ C (‰) value or the slope of increase of Δ ¹³ C (‰) values after administration, is possible to diagnose diabetes is there. The diagnosis of diabetes
Diagnosis can also be made for mild diabetes that shows normoglycemia on an empty stomach.

Example 4 Methoxy- ¹³ C-methacetin breath test Healthy rats (8 weeks old, fasting blood glucose 86 ± 2.9 mg / dl, n =
4) and diabetic rats (8 weeks old, ad hoc blood glucose 418.5 ± 2
4.9 mg / dl, fasting blood glucose 90.3 ± 6.6 mg / dl, n = 4)
Methoxy- ¹³ C-methacetin suspended in 5% sodium carboxymethylcellulose aqueous solution (purchased from CIL)
Was orally administered at a dose of 90 mg / kg, and the increase rate of the ¹³ C concentration in the exhaled CO ₂ (Δ ¹³ C (‰)) was measured according to the method described in Example 1.

From 12 minutes after the administration of methoxy- ^13C- methacetin
The slope at 17 minutes is -8.40 ± 7.67 ‰ / diabetic rat
5 minutes, 16.05 ± 7.71 ‰ / 5 minutes in healthy rats, and the diabetic rats were significantly more significant (p <0.01) than the healthy rats.
(ANOVA with Fischer LSD)). Therefore, it is possible to diagnose diabetes from the slope of the increase in Δ ¹³ C (‰) value after administration of methoxy- ¹³ C-methacetin.
Diagnosis of this diabetes can also be made for mild diabetes that shows normoglycemia on an empty stomach.

[Preparation Example 1] (Injection) 10 parts by weight of N, N-dimethyl- ^13C- aminopyrine was added with physiological saline to make the total amount 100 parts by weight, and after dissolution, it was removed using a Millipore filter. The bacteria were filtered. The filtrate was placed in a vial and sealed to obtain an injection. [Formulation Example 2] (Internal liquid) Purified water was added to 10 parts by weight of N, N-dimethyl- ^13C- aminopyrine to make the total amount 100 parts by weight, and after dissolving, the mixture was sterilized and filtered using a Millipore filter. The filtrate was placed in a vial and sealed to obtain an oral solution.

[0031]

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]

FIG. 1 shows the increase of ¹³ CO ₂ in exhaled air after administration of N, N-dimethyl- ¹³ C-aminopyrine.

FIG. 2 shows the increase in exhaled ¹³ CO ₂ after administration of ethoxy-1- ¹³ C-phenacetin.

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

Claims (3)

[Claims] 1. A diabetes diagnostic agent comprising aminopyrine labeled with ¹³ C at the dimethylamino group at the 4-position. 2. A diagnostic agent for diabetes comprising phenacetin labeled with ¹³ C at the 1-position of the ethoxy group. 3. A diagnostic agent for diabetes comprising methacetin in which the carbon of a methoxy group is labeled with ¹³ C.