JPS60126231A - Radioactie diagnostic agent - Google Patents

Abstract

PURPOSE:To provide the titled diagnostic agent containing insulin having ratioactive iodine atom in the molecule, as a main component, and useful for the diagnosis of cancer, its metastasis, or hepatic function including hyperglycemia by the determination of the distribution of the radioactivity in the body. CONSTITUTION:A nucleomedical radioactive diagnostic agent composed mainly of insulin containing radioactive iodine atom (e.g. <123>I, <125>I, <131>I) in the molecule. Various diseases such as cancer, its metastasis, or hepatic function disorder including hyperglycemia, etc. can be diagnosed by injecting the insulin labeled with the radioactive iodine, and inspecting the absorption and distribution of the radioactive iodine in the organs or tissues or the secretion of the radioactive insulin or its metabolite by a nucleomedical means. The introduction of the radioactive iodine in insulin is carried out by chloramine-T process or enzymatic process, etc., and its radioactivity is usually about 0.05-10mCi/ml.

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 Landerhans islet beta cells, and has a molecular weight of 5.
,807 protein. There is a very small amount of (
30 μIU/plasma III for a normal person! , ) insulin is always present. By subcutaneously administering insulin, the normal blood sugar level of 100-g/dj2 can be reduced by 6
Since it has the effect of lowering the blood pressure to about 5 mg/d,j, it is used as a therapeutic drug for diabetes. 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 pathology of diabetes.

Differently from this, the diagnostic agent of the present invention focuses on the UV/UV distribution of radioactive iodine-labeled insulin in each organ and deep blue tissue after intravenous injection, and the radioactive insulin and its metabolites derived therefrom. 6-Radioactive iodine-labeled insulin, which enables early diagnosis of various diseases by visualizing the dynamics of external excretion using nuclear medicine techniques, can be used for this purpose, as well as the radioactive iodine method described above. The knowledge that the pharmacokinetics of insulin as an indicator enables the early diagnosis of various diseases is completely new, and will dramatically expand the range of various nuclear medicine diagnoses, improve accuracy, and shorten the time period. It has the advantage that it can contribute to shortening the length.

In the radioactive diagnostic agent of the present invention, insulin containing a radioactive iodine atom in its molecule, which is used as a main component, can be produced by introducing radioactive iodine into insulin. The radioactive iodine referred to here includes 1:3I, 1
Includes 25I and 1311. Insulin includes human insulin, bovine insulin, porcine insulin, and the like. Introducing methods include the chloramine T method, enzyme method, direct introduction method, etc. In other words, ■ A compound that generates − ions (e.g. Na1) is allowed to act on insulin in the presence of chloramine T or in the presence of H202 and lactoperoxidase. method, and Te' to 'H
+23Xe generated by irradiation with e is added to insulin,
A method is used in which the material is allowed to stand for several hours and decays into 123I.

It is desirable that the radioactivity contained in the radiodiagnostic agent of the present invention be in an amount that allows sufficient information to be obtained when carrying out nuclear medicine diagnosis, and also in an amount that minimizes the radiation exposure of the subject. 1 is generally 0.05-05-1O/
Approximately mj2 is appropriate.

In addition to insulin containing a radioactive iodine atom in its molecule, the radioactive diagnostic agent of this invention may contain commonly used buffers, pH adjusting acids or alkalis, isotonic agents, preservatives, etc. as necessary. I can do it.

The radioactive diagnostic agent of this invention is generally administered 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.

Radioactive iodine was purchased from Ama Jam Japan.
NaI (for protein labeling) and 1251 NaI purchased from Nippon Mediphysics were used.

Method for synthesizing I25I-insulin.

First, prepare an alkaline aqueous solution (1+H8~1O) containing 12J-NaI of ] mC110,01-, and use the chloramine T method to determine the tyrosine 125 in insulin protein.
Labeled with I. That is, oxalic acid buffer (0.1M)
0.1m, e, +25I-NaI solution 0', 05J, insulin aqueous solution (Actra manufactured by Novo Pharmaceutical Co., Ltd.
Prepare a homogeneous mixture of pid MC (401U/J J: Purified Insulin) 0.5m and (:), and add Lyolamin T solution (Ill1g/ml) (7) to this mixture.
0, t: +2- was added, stirred for 15 seconds under water cooling, further stirred by adding 0.02 m, j of talolamine solution, and after 15 seconds, metabisulfite (1 g) /J, )(7) 0,03 mj2
Okaete, I stopped the reaction immediately. “10 for this
0 mg/m (!, potassium iodide aqueous solution (), 1
- was added to stop the reaction and completely stop the excessive action of chloramine T.

Immediately add these mixtures to Cephadex G-25 (1,
Only insulin labeled with radioactive iodine was fractionated by passing through a 3M glycine-0.45% sodium chloride solution column. Its specific activity was 500 μCi/2 IU insulin.

+25■ or 1311-method for synthesizing insulin.

The method for synthesizing 125I-insulin was followed.

According to the above synthesis method, 1231 and 13'I Na,l
When insulin was labeled using this method, a labeled product identical to that described above could be synthesized.

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

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

Nuclear medicine techniques are excellent for short-term diagnosis of humans, bear pain, and non-restrictive diagnosis, but in the case of small animals such as rats, there is an alternative method that has excellent photographic resolution. Whole body macroautoradiography is commonly used. In this example, the visible radioactivity distribution was also recorded entirely by this method.

First, 5μC was added to the tail vein of Wistar male rat 5.
110,in+,e radioactive iodine-labeled insulin was injected at 10 minutes, 30 minutes, 1 hour, 3 hours, and 5 hours, anesthetized with ether, immediately frozen in liquid nitrogen, and placed on a Life 1400 large-scale slide. 20 μm Pi[7] thin sections were prepared using a microtome, and the sections were freeze-dried under reduced pressure.

The dried sections were closely covered with Sakura M A RG and exposed for about 4 weeks in a dark box, and then the film was subjected to Sakura-specified photographic processing to obtain whole-body macroautoradiography. The photographic darkening density of autoradiography is completely correlated with the radioactivity density. The distribution after injection shows a specific distribution in the early, middle, and late stages.1) Distribution in the early stageHigh radioactivity was found in the kidneys, atria, ventricles, various arteries, lungs, and liver. was recorded. The brain and pancreas did not have a significant radioactivity distribution, and slightly high radioactivity was recorded in the muscle tissue.

2) Distribution in the mid-term The radioactivity was low in the atria, ventricles, vena cava, and large and small veins in the liver, which are thought to indicate the presence of radioactivity in the blood. Comparisons of distribution were made in the early and near-period of administration.

3) Late distribution 3-5 hours after administration, the overall darkening density faded.

In particular, skeletal muscle and cardiac muscle, which showed high concentrations until the middle stage,
Clear concentration dilution was observed in intermediate concentration groups such as lungs and liver. On the other hand, there was almost no difference from the mid-term distribution concentration, and the radioactivity concentration remained in arteries including the aorta connected to the left atrium, the operative aorta, and the carotid artery. 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 into the gastrointestinal tract and from the kidneys. In particular, a specific distribution in the renal cortex and medulla was noted.

As shown in the above example, 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. 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 C5T black mice transplanted with B-16 melanoma.

5 μC of radioactive iodine-125I-labeled insulin in the tail vein of black mice at 24 hours and 121 hours after implantation with B-16 melanoma (black skin cancer).
1. (!) was administered, and the distribution of radioactivity in the body 20 minutes later was visualized by whole-body macroautoradiography. 24 hours after cancer transplantation, the inoculated black cancer cell mass was observed to be solid in the subcutaneous tissue when the skin was inverted. The inoculated area was in close contact with the subcutaneous tissue, and the inoculated area was covered with several layers of connective tissue.However, no significant increase in the volume of the black area was observed, indicating that the proliferation of cancer cells was due to inoculation. It was not estimated that it was noticeable in the 24 hours after vaccination.On the other hand, 12 days after inoculation, a large capsule-shaped black mass, which is characteristic of this cancer pathology, was formed, and the outer capsule was made of somewhat strong connective tissue. It showed a unique morphology that was coated and extremely vascularized.

Quiet cell clusters and some necrosis were already observed in the center of the lesion. 125I is effective against progressive proliferation of such focal areas.
- Labeled insulin showed a very specific intralesional distribution.

First, the distribution 24 hours after inoculation clearly showed a high concentration of radioactivity 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 is,
This may be due to the high energy demands and concomitant remarkable acceleration of glycolytic turnover at the site of inoculation cancer contact. In this way, the specific concentration of radioactivity at the lesion site increases the accuracy and reliability of the initial diagnosis of the progress of cancer lesions, and the radioactive diagnostic agent according to the present invention has been desired for a long time. This proves that it is useful as an agent for early diagnosis of cancer. Cancer inoculation 121
In the later (late-stage cancer) lesions, only the newly formed cancer cell group (the active area where cell division is still connected) within the envelope around the lesion area showed a strong radioactivity distribution pattern. This was in good agreement with the 140-radioactivity distribution in the same lesion area 1 hour after injection of C-2-thymidine into the tail vein, as determined by a separate experiment.

Example 3 Use as a biopsy diagnostic agent (B-16) Tumor-bearing mice 24 hours after inoculation with Mela 7-ma (
C67 Black) were frozen using liquid nitrogen after death under ethyl ether anesthesia. As a cross section of the left side of the corpse,
A large number of thin sections (20 μm each) containing the left kidney and many thin sections (20 μm each) showing midline cross sections were prepared.

Created using Scotch tape # according to Habo-Ullberg's original method.
Using a cryomicrotome with the help of 615 -15
Repetition was carried out at ℃. The obtained sections were freeze-dried under reduced pressure. This section was sprayed with 0.005 J of radioactive iodine 125I-labeled insulin 2 tnc i/0, 1- using a microsprayer, left at 37°C for 1 minute, thoroughly washed with water, and air-dried. This 125I-treated section was brought into close contact with a high-sensitivity X-ray film, and after exposure between 2 and 3 seconds, the X-ray film was photographically processed and extremely strong photographic darkening was observed at the cancer contact site. 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 risk, cancerousness can be diagnosed in vitro using simple frozen sections, plasma, or serum prepared by removing a portion of the site for biological examination (biopsy). The diagnostic agent of the present invention has been proven to be useful as a testing reagent.

In addition, 1 alloxan was added to 5 withdrawals of Wistar male rats.
Hyperglycemic rats induced by intraperitoneal injection at 25m8/00g body weight were treated in the same way as above to examine the specificity of in vitro diagnosis.
Compared to the control group, the affinity of 125I-insulin to the liver was particularly marked, and the difference was particularly remarkable in the glucose-loaded group.These results demonstrate that the diagnostic agent of the present invention Similarly to the above, it was found to have a remarkable effect as a diagnostic agent for in vitro diagnosis using simple frozen sections prepared by biopsy.

In all of the above three examples, 123I, as radiolabeled iodine,
It is clear that using 131■ etc. will have a similar effect (because these are isotopes), but when all experiments were additionally performed, it was found that 1311 is inferior in photographic resolution. became clear.

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

Patent applicant Bioscience Research Institute Co., Ltd. Agent Patent attorney Aoyama Aoyama and 1 other person signed the procedural amendment) ■, Small case indication 2, Name of invention radioactive diagnostic agent 3, Person making the amendment Relationship with the case Patent application Person address: 340 Minauchi, Shirai-cho, Ingan-gun, Chiba Prefecture, 2 names: Bioscience Research Institute 4, Agent address: 7, Honmachi Building, 2-10 Honmachi, Higashi-ku, Osaka City, Osaka Prefecture;
Contents of the amendment (a) As per the appendix in the scope of claims.

(b) Insert the following passage between lines 5 and 6 on page 9 of the description in the detailed description of the invention. Glucose loading was performed for the purpose of highlighting the difference in symptoms from normal rats for those with glycemia, and 1 hour later, 125I-insulin (5 mCi/head) was administered.
Create a whole-body macroautoradiograph after 0 minutes.

As a result, radioactivity in the kidneys and liver of the hyperglycemic rat group was significantly lower than that in the control group, and if the 1251-insulin distribution was imaged using a nuclear medicine clinical scinticamera, there would be a clear difference. It is clear that pathological conditions can be diagnosed.

In addition, the urine of hyperglycemic rats is characterized by high radioactivity excretion, perhaps due to the renal affinity of 125I-insulin, which is also extremely useful for identifying the cause of complex hyperglycemia. It is clear that this will serve as a guideline. ] (Attachment) [Claim 1 (1) A nuclear medicine radioactive diagnostic agent whose main component is insulin containing a radioactive iodine atom in its molecule.

(2) Radioactive iodine atoms are 123■, 12sl or 1
31■ The radioactive diagnostic agent according to claim 1.

(3) The radioactive diagnostic agent according to claim 1 or 2, which is for measuring the distribution of radioactivity in the body or its transition.

(4) The radioactive diagnostic agent according to claim 1 or 2, which is used for radioimmunoassay of a substance with specific affinity in serum, plasma, or biopsy material.

(5) Claim 1, which is used for diagnosing cancer or its metastasis, or liver function, including hyperglycemia, by measuring the distribution of radioactivity in the body or the concentration in a biopsy material. Or the radioactive diagnostic agent according to item 2.

(6) It is used to identify patients with hyperglycemia by measuring the amount of radioactivity excreted in urine.
The radioactive diagnostic agent according to claim 1 or 2.

Claims (5)

[Claims] (1) A nuclear medicine radioactive diagnostic agent whose main component 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 12'll, +251 or +311. (3) The radioactive diagnostic agent according to claim 1 or 2, which is for measuring the distribution of radioactivity in the body or its transition. (4) The radioactive diagnostic agent according to claim 4 or 2, which is used for radioimmunoassay of a substance with specific affinity in serum, plasma, or biopsy material. (5) Measurement of radioactivity distribution in the body A radioactive diagnostic agent used for diagnosing cancer or its metastasis or liver function including hyperglycemia by measuring the concentration in a biopsy material.