JPH0568452B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a nuclear medicine radiodiagnostic agent comprising insulin labeled with radioactive iodine.

As is well known, insulin is an endocrine hormone secreted from the pancreatic Langerhans islet beta cells, and is a protein with a molecular weight of 5807. In the blood,
Very small amounts of insulin (30 μIU/ml of plasma in normal people) are present at any given time. Insulin can be administered subcutaneously to lower normal blood sugar levels to 100.
Since it has the effect of lowering mg/dl to about 65 mg/dl, it is used as a diabetes treatment drug. Furthermore, iodine-labeled insulin has already been put to practical use in the radioimmunoassay method for measuring blood insulin concentration in the category of determining the pathological condition of diabetes. Unlike this, the diagnostic agent of the present invention measures the absorption and distribution of radioactive iodine-labeled insulin into each organ and tissue after intravenous injection, and the subsequent dynamics of excretion of radioactive insulin and its metabolites from the body. Visualization using nuclear medicine techniques enables early diagnosis of various diseases. The knowledge that the pharmacokinetics of insulin, using radioactive iodine as an indicator, enables the early diagnosis of various diseases, especially cancer, its metastasis, and hyperglycemia, is completely new and will dramatically improve the effectiveness of various nuclear medicine methods. It has the advantage that it can contribute to expanding the range of diagnosis, increasing accuracy, and shortening the period of diagnosis.

In the radioactive diagnostic agent of the present invention, insulin containing a radioactive iodine atom in its molecule, which is used as the main component, can be produced by introducing radioactive iodine into insulin. The radioactive iodine referred to herein includes ¹²³ I, ¹²⁵ I and ¹³¹ I. Furthermore, insulin includes human insulin, bovine insulin, pig insulin, and the like. The introduction method includes the chloramine T method, the enzyme method, the direct introduction method, etc. In other words, a compound that generates I ^- ions (e.g. NaI) is introduced in the presence of chloramine T, or
Two methods are used: ^one in which insulin is acted on in the presence of H ₂ O ₂ and lactoperoxidase ^, and another in which ¹²³ .

The radioactivity contained in the radiodiagnostic agent of this invention is:
It is desirable that the amount be such that sufficient information can be obtained when performing a nuclear medicine diagnosis and that the radiation exposure of the subject be as low as possible, but in general, about 0.05-10 mCi/ml is appropriate.

The radioactive diagnostic agent of the present invention may contain, as necessary, commonly used buffers, acids or alkalis for pH adjustment, isotonicity agents, preservatives, etc., in addition to insulin, which contains radioactive iodine atoms in its molecules. I can do it.

The method for administering the radiodiagnostic agent of this invention is as follows:
Administration is generally done intravenously.

Next, the manufacturing method, usage, and effects of the radioactive diagnostic agent of the present invention will be illustrated by manufacturing examples and examples.

Manufacturing example 1 Synthesis of radioiodine-labeled insulin.

As radioactive iodine, ¹²⁵ I-NaI (for protein labeling) purchased from Amashyam Japan and ¹²⁵ I-NaI purchased from Nihon Medi-Physics Co., Ltd. were used.

¹²⁵ I-Insulin synthesis method.

First, prepare an alkaline aqueous solution (PH8-10) containing 1mCi/0.01ml of ¹²⁵ I-NaI, and
By method, tyrosine ¹²⁵ I in insulin protein
Labeled with. i.e. borate buffer (0.1M)
0.1 ml of 125 ^I -NaI solution, 0.05 ml of 125 I-NaI solution, insulin aqueous solution (Actrapid MC manufactured by Novo Pharmaceutical Co., Ltd. (40 IU/ml)
Make a homogeneous mixture of 0.5 ml of porcine purified insulin, and add chloramine T solution (1 mg/ml) to this mixture.
ml), stirred for 15 seconds under ice-cooling, and then added 0.02ml of Chloramine T solution and stirred.
After 15 seconds, add sodium metabisulfite aqueous solution (1
The reaction was immediately stopped by adding 0.03 ml of 20 mg/ml). To this, 0.1 ml of 100 mg/ml potassium iodide aqueous solution was added to stop the reaction and chloramine T
completely stopped the excessive effects of

Immediately pour these mixtures into Cephadex G-25.
Only insulin labeled with radioactive iodine was fractionated by passing through a 0.3M glycine-0.45% sodium chloride solution column. Its specific activity is
It was 500μCi/2IU insulin.

Method for synthesizing ¹²⁵ I or ¹³¹ I-insulin.

The method for synthesizing ¹²⁸ I-insulin was followed.
When insulin was labeled using ¹²³ I and ¹³¹ I-Nal according to the above synthesis method, a labeled product completely similar to that described above could be synthesized.

Formulation Example 1 The ¹²⁵ I-insulin solution obtained in Production Example 1 was diluted with physiological saline to 50 μCi/ml to obtain a radioactive diagnostic agent of the present invention.

Example 1 Absorption, distribution, and excretion (ADME) of radioactivity in the rat body after injecting radioactive iodine ^125- labeled insulin into the tail vein of the rat.

Although nuclear medicine techniques are excellent for short-term, painless, and non-restrictive diagnosis in humans, there is an alternative method for small animals such as rats that uses whole-body techniques with extremely high photographic resolution. Macro autoradiography is often used. In this example, the visible radioactivity distribution was also recorded entirely by this method.

First, 5 μCi/0.1 ml of radioactive iodine-labeled insulin was injected into the tail vein of 5-week-old male Wistar rats, and they were killed by ether anesthesia at 10 minutes, 30 minutes, 1 hour, 3 hours, and 5 hours. It was immediately frozen in liquid nitrogen, and thin sections with a thickness of 20 μm were prepared using a Leitz 1400 large sliding microtome, and the sections were freeze-dried under reduced pressure. Sakura MARG was applied closely to the dried sections and exposed for about 4 weeks in a dark box, and then the film was subjected to photo processing specified by Sakura to obtain whole-body macroautoradiography.
The photographic darkening density of autoradiography is completely correlated with the radioactivity density. The distribution after administration showed specific distributions in each of the three periods: early, middle, and late.

1 Distribution in the early stage High radioactivity was recorded in the kidneys, atria, ventricles, various arteries, lungs, and liver. The brain and pancreas are
There was no significant radioactivity distribution, and rather high radioactivity was recorded in muscle tissue.

2 Mid-term distribution Radioactivity was low in the atria, ventricles, inferior aorta, and large and small veins in the liver, which are considered to indicate the presence of radioactivity in the blood, but the distribution of radioactivity concentration among each organ and tissue was Comparisons were similar to the previous period of administration.

3 Late distribution 3 to 5 hours after administration, the overall blackening concentration is
Faded. In particular, a clear concentration dilution was observed in intermediate concentration groups such as skeletal muscle, cardiac muscle, lung, and liver, which had shown high concentrations until the middle stage. On the other hand, the radioactivity concentration remained in the arteries connected to the left atrium, including the aorta, inferior aorta, and carotid artery, with almost no difference from the mid-term distribution concentration. During this period, radioactivity in the blood was absorbed into organs and tissues according to their needs, and the excess was excreted from the liver into bile and excreted into the gastrointestinal tract and kidneys. In particular, a specific distribution in the cortex and medulla of the kidney was noted.

In addition, in this example, alloxan was administered intravenously in advance to induce hyperglycemia. Glucose loading was performed in order to make the difference in symptoms from normal rats noticeable, and 1 hour later, 125I-insulin (5 mCi/
head), and a whole-body macroautoradiograph was created 30 minutes later.

As a result, radioactivity in the kidneys and liver of the hyperglycemic rats group was significantly lower than that of the control group, and if the 125I-insulin distribution was imaged using a cinch camera in a nuclear medicine clinical setting, there would be a clear difference. It is clear that pathological conditions can be diagnosed.

Additionally, the urine of hyperglycemic rats is characterized by the excretion of high radioactivity, probably due to the weak affinity of 125I-insulin in the kidneys, and this is also an extremely effective guideline for identifying the cause of complex hyperglycemia. It is clear that

As seen in the above examples, by intravenously injecting the radioactive diagnostic agent (radioactive iodine-labeled insulin) according to the present invention, it is possible to know the distribution of radioactivity in the body and the changes in its concentration in each organ and tissue. As a result, it has become possible to diagnose various diseases specific to arterial blood vessels and diseases not only in the main vascular system but also in organs and tissues at an early stage without pain or damage.

Example 2 Distribution and excretion in the body of B-16 melanoma transplanted _C57 black mice.

24 hours and 12 days after transplantation of B-16 melanoma (black skin cancer), 5 μCi/0.1 ml of radioactive ¹²⁵ I-labeled insulin was administered to the tail vein of black mice, and the radioactivity distribution in the body was measured 20 minutes later. Visualized by macroautoradiography. 24 hours after cancer transplantation, when the skin is inverted at the subcutaneous tissue, the inoculated black cancer cell mass is tightly adhered to the subcutaneous tissue, and the inoculated area is covered with several layers of connective tissue; Since no significant increase in volume was apparently observed, it could not be assumed that cancer cell proliferation was significant within 24 hours after inoculation. However, even in such lesions, ¹²⁵ I-labeled insulin showed a very specific intralesional distribution.

The radioactivity distribution 24 hours after inoculation clearly showed a high concentration of ¹²⁵ I-labeled insulin specifically at the site of contact with the inoculated cancer cells, which was in marked contrast to the concentration in the tissue around the contact site. This may be due to the high energy demands and concomitant significant acceleration of glycolytic turnover at the site of inoculation cancer contact. In this way, the specific concentration of radioactivity at the lesion site highly and reliably increases the accuracy of the initial diagnosis of the progress of cancer lesions, and the radioactive diagnostic agent according to the present invention can be used to treat cancer, which has been desired for a long time. This proves that it has utility as an early diagnostic agent. Twelve days after inoculation, a large, capsule-shaped black mass, which is characteristic of this cancerous pathology, was formed, and the outer capsule was covered with rather strong connective tissue, exhibiting a unique morphology that was extremely rich in blood vessels. Quiet cell clusters and some necrosis were already observed in the center of the lesion. In the lesion 12 days after cancer inoculation (late-stage cancer), only the newly formed cancer cells within the envelope around the lesion (the active part where cell division is still connected) show a strong radioactivity distribution pattern. Ta. This was in good agreement with the distribution of ¹⁴ C-radioactivity in the same lesion area 1 hour after injection of ¹⁴ C-2-thymidine into the tail vein according to a separate experiment.

Example 3 Use as a biometric diagnostic agent (B-16) A tumor-bearing mouse ( _C57 black) 24 hours after being inoculated with melanoma was anesthetized with ethyl ether and frozen using liquid nitrogen. The left side of the corpse was taken as a cross-section, and a thin section containing the left kidney (each
20 μm) and thin sections showing midline cross sections (20 μm each)
I created many.

The preparation was carried out almost according to Ulberg's original method using a cryomicrotome with the help of Scotch tape #615 at -15°C. The obtained sections were freeze-dried under reduced pressure. Radioactive iodine is added to this section.
0.005 ml of 2 mCi/0.1 ml of ¹²⁵ I-labeled insulin was sprayed using a microsprayer, allowed to stand at 37°C for 1 minute, thoroughly washed with water, and air-dried. This ¹²⁵ I
When the treated section was brought into close contact with a high-sensitivity X-ray film and exposed for two days, the X-ray film was photographically processed, and extremely strong photographic darkening was observed at the site of cancer contact. This fact indicates that the diagnostic agent of the present invention has specific affinity for host-side localities such as cancer contact sites and carcinogenic sites. This means that
If carcinogenesis or metastasis cannot be determined but there is a possibility, cancerousness can be diagnosed in vitro from simple frozen sections prepared by removing a portion of the site (biopsy) for biological examination, or from plasma or serum. The diagnostic agent of the present invention has been proven to be useful as a testing reagent.

In addition, we performed the same treatment as above on hyperglycemic rats, which were induced by intraperitoneal injection of alloxan at 25 mg per 100 g of body weight at 5 weeks of age, to examine the specificity of in vitro diagnosis. The results showed that compared to the control group, the effect on the liver was
The affinity of ¹²⁵ I-insulin was extremely low, and the difference was particularly noticeable in the glucose-loaded group. From these results, it was found that the diagnostic agent of the present invention has a remarkable effect as a diagnostic agent for performing in vitro diagnosis from simple frozen sections prepared by biopsy, etc., as described above. .

In all three examples above, as radiolabeled iodine
It is clear that using ¹²³ I, ¹³¹ I, etc. would have a similar effect (because these are isotopes), but when we added all the experiments, we found that
It became clear that ¹³¹ I was inferior in terms of photographic resolution.

From these Examples, it became clear that the usefulness of the diagnostic agent of the present invention is remarkable and unprecedented, especially when used as an early cancer diagnostic agent and a liver function diagnostic agent.

Claims (1)

[Scope of Claims] 1. A radioactive nuclear medicine diagnostic agent for diagnosing cancer or its metastasis or hyperglycemia, the main component of which is insulin containing a radioactive iodine atom in its molecule. 2. The radioactive diagnostic agent according to claim 1, wherein the radioactive iodine atom is ¹²⁵ I. 3. The radioactive diagnostic agent according to claim 1 or 2, which is determined by measuring the distribution of radioactivity in the body or its transition, or the radioactivity concentration in biopsy material or urine. 4. The radioactive diagnostic agent according to claim 3 for diagnosing cancer or its metastasis by measuring the distribution of radioactivity in the body or its transition, or the radioactivity concentration in a biopsy material. 5. The radioactive diagnostic agent according to claim 3 for diagnosing hyperglycemia by measuring the distribution of radioactivity in the body or its transition, or the radioactivity concentration in a biopsy material. 6. The radioactive diagnostic agent according to claims 1 to 3 for diagnosing liver function or renal function in hyperglycemia. 7. The radioactive diagnostic agent according to claims 1 to 3, which is used by measuring the radioactivity concentration in urine for diagnosing the pathological condition of hyperglycemia. 8. The radioactive diagnostic agent according to claims 1 to 3, which is obtained by measuring the radioactive concentration in a biopsy specimen brought into contact with the diagnostic agent.