JPS63267734A - Diagnosticum for tumor and inflammation - Google Patents

Abstract

PURPOSE:To obtain the titled diagnosticum consisting of a specific high polymer having affinity for organism. CONSTITUTION:A high polymer having affinity for organisms (e.g. alpha1 acidic glycoprotein, albumin, transferrin, polyethylene glycol or various synthetic peptides having <=100,000, preferably 10,000-70,000mol.wt.) is reacted with a given amount of a chelating agent [e.g. ethylenediaminetetraacetic acid + or -(EDTA) or diethylenetriaminepentaacetic acid anhydride (DTPA)] in a buffer solution such as HEPES buffer to give a high polymer-chelating agent binder. The binder is labeled with a gamma-ray radioactive metal (e.g. <67>Ga, <99m>Tc, <111>In, <125>I or <23>P) by metallic chelating reaction to give a diagnosticum for tumor and inflammation. The diagnosticum is a diagnosticum for intravenous administration to measure gamma-ray from tumor in the interior of organs by the use of a gamma-ray radioactive isotope by a scintiscanner.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a tumor diagnostic agent using a radioactive isotope, and more specifically, a tumor diagnostic agent using a radioactive isotope, and more specifically, a gamma-Me scintillation scanner or scintillator for measuring gamma-Me from a tumor inside an organ using a gamma-ray radioactive isotope. Gamma ray radioisotope for intravenous administration, ie, cancer imaging, is a method commonly used for cancer screening, and the method using the above-mentioned gamma ray radioisotope is one of them. For example, in gallium (Ga) scintigraphy, which is widely used clinically to diagnose solid cancers, citrate of 67Ga, a gamma-ray radioactive metal, is intravenously injected, and 48 to 72 hours later, the site of its accumulation is detected. The mechanism is that Ga ions introduced into the blood bind to the iron-binding site of blood proteins, especially transferrin, which has a molecular weight of about 90,000, and accumulate in cancer tissues. The radiation on the scintigram shows the accumulation of 67Ga-transferrin. However, 67Ga introduced into the blood
Io.

Not all of the 670a ions are bound to transferrin, but approximately 30-50% of the 670a ions remain in the free form, and these free 670a ions are mainly absorbed into bile or urine within the body after a few hours. Since it is excreted from the inside to the outside of the body, the utilization efficiency of 67(3a ions) is extremely low.

Therefore, it has been proposed to prepare antibodies specific to tumor cells and label them with a gamma ray radioactive isotope.

In particular, monoclonal antibodies against tumors bound to gamma-ray radioactive elements are currently being actively researched for clinical application. However, since monoclonal antibodies are usually foreign animal proteins, they have low biocompatibility within the human body and are easily removed from the blood by foreign body processing mechanisms such as macrophages. Moreover, in the second and subsequent administrations, the problem of side effects such as anaphylactic shock must be taken into consideration. Furthermore, even if human-derived monoclonal antibodies are produced, their molecular weight is as large as approximately 160,000, and their leakage from localized tumors is poor and they do not accumulate efficiently in tumors. That is, monoclonal antibodies do not accumulate in tumor tissues as efficiently as α1 acid glycoprotein or albumin, which has a molecular weight of about 40,000. The reason for this can be explained by the following findings regarding the vascular system of the present inventors.

That is, unlike normal tissue, at the local tumor site, new blood vessels are increasing and blood vessel permeability is restored, making it easy for various substances in the blood to leak into the extravascular space. In addition, once the polymers or oils leak into the cancerous tissue, they are not recovered due to the lack or scarcity of the lymphatic vessels that serve as the recovery route, and the leaked polymers or oils remain in the cancerous tissue for a long time. Stagnant. This phenomenon is
It becomes more noticeable over time, and under certain conditions more than 10 times the blood concentration accumulates within the tumor. This phenomenon is especially true when the molecular weight is about 1
It is noticeable in those with a value of 0,000 or less. Furthermore, such significant accumulation of polymers also occurs at sites of inflammation;
The difference between inflammation and a tumor is that inflammation occurs in normal tissue.

In addition to the above findings, the present inventors have discovered that in vitro, polymers, particularly biocompatible polymers having a molecular weight of about 1,000 to 1, OO, OOO, can be gamma-mediated by using a chelating agent.
By labeling with a radioactive isotope, we succeeded in creating a radioactive metal-labeled polymer that can be sensitively detected by a scintillation camera. In this case, because it has a lower molecular weight and better vascular permeability than GG, the young fruit that can be imaged is large, and by using albumin, which is inexpensive and available in large quantities, or synthetic polymers, inflammation can be reduced. It is possible to accumulate in the tumor site in a shorter time than potassium citrate etc. And thereby, tumors and inflammation sites can be easily detected. Furthermore, this method can use any divalent or bivalent radioactive metal, and if paramagnetic metals are used, hemodynamics can also be observed by nuclear magnetic resonance.

Biocompatible polymeric substances that can be used in the present invention include α1 acid glycoprotein, albumin, trans biopolymer, polyvinylpyrrolidone, polyethylene glycol, nutylene maleic acid copolymer, bilane copolymer, various synthetic peptides, etc. . In the present invention, these polymers generally have a molecular weight of about 100,000 or less, preferably a molecular weight of about 1 to 000.
0 to 70,000.

When the molecular weight is about 100,000 or more, the accumulation efficiency within tumor tissue or inflamed tissue is insufficient.

In the present invention, any known chelating agent can be used, but preferred is ethylenediaminetetraacetic acid (
EDTA), diethylenetriaminepentaacetic anhydride (
DTPA) or various derivatives thereof and other polyaminocarboxylic acids, particularly difunctional chelating agents.

Furthermore, in the present invention, the gamma ray radioactive isotope used to label the biocompatible polymeric substance includes the above-mentioned o 67
99 mT, 111 kn, 1251.13 for G a's candy
Conventional commer-radiating isotopes such as 11.32p, 51cr, 198Au, etc. can be used.

The gamma ray radioisotope-labeled polymer of the present invention can generally be prepared as follows. First, the above-mentioned biocompatible polymer and a predetermined amount of chelating agent are mixed in an appropriate buffer solution.
For example, polymer-
A chelating agent conjugate is obtained. This reaction generally occurs between the NF2 group in the polymer and the carboxylic acid group in the chelating agent, so the polymer material generally does not have functional groups such as NF2 groups like styrene-maleic acid copolymer. When using a polymer, it is necessary to introduce a functional group into the polymer in advance. Such a functional group can be easily introduced, for example, by using an amino group-containing substance having two or more NF2 groups in the molecule, such as L-lysine.

Furthermore, the polymer-chelating agent conjugate thus obtained can be labeled with a gamma-ray radioactive metal by a conventional metal chelation reaction.

The reactions for obtaining these series of radiometal-labeled polymers are clearly explained by the reaction equations of FIGS. 2 and 3 shown in connection with the Examples.

By intravenously injecting the thus obtained gamma-ray radioisotope-labeled polymer of the present invention at a dose equivalent to about 100 to 1.000 μC per body weight, clear sitigraphic images of tumors or inflamed sites can be obtained. Can be done.

Hereinafter, the present invention will be specifically explained with reference to Examples.

Example 1 (1) Reaction between serum protein and chelating agent Bovine serum albumin (BSA) was used as the serum protein, and DTPA anhydride was used as the chelating agent to be bound to the serum protein. Anhydrous 0.1mg
(equimolar amount) into a suitable vial, α1M
1 mf (i7) of HEPES buffer (pH 7,0) was added and rapidly dissolved.The reaction proceeded immediately, so stirred with a stirrer at room temperature for about 5 minutes.Unreacted DT
PA was removed by dialysis or gel filtration. Then, a sample was obtained by freeze-drying. The yield was over 90%.

In order to label or radioactivate the above DTPA-conjugated BSA, a radioactive metal was chelated as follows.

(2) DTPA formation B by chelation of radioactive metals
Labeling of SA DTPA-conjugated BSA obtained in (1) above was dissolved in HEPES buffer (pH 7, o) and the albumin concentration was 1 rr.
-E! Then, CrCl3 was added in an amount of 5 μC1 (5 μm), and the mixture was stirred overnight at room temperature (approximately 20°C) to chelate 51 Cr to the DTPA-modified BSA, followed by Sephadex G. -50 (0.1M acetic acid)
Purification was performed using IJum buffer (pHao). Each fraction was measured with an autogamma counter, and the 51Cr-DTPA BSA that appeared in the void volume and
Two peaks of Cr could be recognized (see Figure 1).

Thus, bovine serum albumin was radiometal labeled and 9
．． A radiometal labeled compound with a specific activity of 5X10 cpm/mg protein was obtained.

In a similar manner, ovomucoid, mouse serum albumin,
Mouse gamma globulin and human transferrin were radiolabeled with the above Cr. Their specific activities are 3,9 o5゜4.7 XI O5,0,9XI O5, respectively.
, 2,4 x 105 Qpm/mg. Note that 10 times the amount of mouse gamma globulin teha and DTPA was used.

A general formula showing the formation of these radiometal-labeled proteins is shown in FIG.

Example 2 (1) Reaction of synthetic polymer and chelating agent Styrene-maleic acid copolymer (styrene:maleic acid ratio =
1:1, average molecular weight 1600, hereinafter abbreviated as SMA) was used.

This SMA has D used as a chelating agent in its molecule.
Since there is no amino group to react with TPA anhydride, L having two amino groups in the molecule is prepared as follows.
- An amino group was introduced using lysine (L3'S).

First, 40 mg of SMA and water-soluble carbodiimide 1
-ethyl/L/-3-(3-dimethylaminopropyl)-
Carbodiimide hydrochloride (EDPC) 100
mg in the presence of an acid catalyst, 100 mg of L-lysine was added to the resulting reaction mixture, and the mixture was stirred at room temperature for 4 hours.
The product was obtained by reacting for a period of time. The resulting product (SMA-Lys) was then separated into G, Hoa v (Hoa
“Re, G, D, etc.”
, 247:2447-2448 (1967), purification on a Sephadex G-50 column gave 43 mg of purified SMA-LyS product.

The SMA-Lys product thus produced was prepared in Example 1-(
DTPA anhydride according to the procedure of Example 1-(2), and the resulting product was reacted with DTPA anhydride according to the procedure of Example 1-(2).
Labeled with Cr. The obtained 51Cr-DTPA-Lys
-SMA (7) Specific activity is 2,3 X 10 cm
p/mg protein teatuta.

A series of reactions for obtaining these Or-DTPA-Lys-SMAs is shown in FIG.

Example 3 Tumor accumulation experiment of the radioactive metal-labeled polymer of the present invention in an experimental tumor model 1 x 10 S-180 sarcomas were implanted into the ventral region of a mouse, and the tumor area reached a diameter of 8 to 1Q mm. Example 1
and various radioactive 51cr-labeled polymers (7) obtained in 2.
A total amount of I The accumulation rate per unit Durham (cpm x 100 of blood and tumor per gram/total cpm intravenously injected) is shown.

As is clear from Table 1, these radioactive metal-labeled polymers accumulate in tumor tissues at high concentrations. At this time, a longer range nuclide (67Ga) is used.

111, 99mTC, etc.), the tumor site and its size can be easily identified, and useful diagnosis of solid tumors can be made.

Example 4 Accumulation experiment of the radioactive metal-labeled polymer of the present invention at an inflamed site As an inflammatory lesion model, mice (ddY, 8 weeks old, 9 males, weighing approximately 30 g) were subjected to burn injuries and subjected to experiments. Burns are 7
The head of a 5-inch nail (thickness: 5 mm, length: 150 mm, head diameter: 12 mm) heated in a water bath at 0°C was pressed against the pre-pricked abdominal skin for about 10 seconds.

CR-labeled serum albumin 1 prepared in Example 1
0 μCi was dissolved in 50 μm of physiological saline, and this solution was injected by microsilling through the tail vein immediately after the burn. After decervication, tissues from the inflamed area and normal area were cut out, and the radioactivity of each was measured using an autogamma counter. As shown in Table 2, it is clear that the accumulation in inflamed areas is overwhelmingly higher than in normal areas.
The diagnostic utility of the invention is clearly demonstrated.

Normal skin 3.97 6.32 Burn area skin 53.85 69.71

[Brief explanation of the drawing]

Figure 1 shows Or-labeled DTPA-BS prepared in Example 1.
The radioactivity and absorption curves of A are shown. FIG. 2 is a reaction formula showing a series of reactions for obtaining the radiometal-labeled polymer of the present invention when various serum proteins are used as the biocompatible polymer. FIG. 3 is a reaction formula showing a series of reactions for obtaining the radiometal-labeled polymer of the present invention when a synthetic polymer is used as the biocompatible polymer. Figure 1 Franchise Shishi Nasibar Figure 2 DTPA Mutoki ↓ DTPA-8 Ar: /, white

Claims (4)

[Claims] (1) A diagnostic agent for tumors and inflammation consisting of a biocompatible polymer labeled with a radioactive metal via a chelating agent. (2) The diagnostic agent according to claim (1), wherein the radioactive metal is a gamma ray radioactive isotope. (3) Biocompatible polymer has a molecular weight of approximately 10,000 to 10
0,000. The diagnostic agent according to claim (1). (4) The diagnostic agent according to claim (1), wherein the biocompatible polymer is a serum protein or a synthetic polymer.