JPH07116142B2 - Novel ethylenediaminetetraacetic acid derivative - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel ethylenediaminetetraacetic acid derivative. The ethylenediaminetetraacetic acid derivative compound of the present invention is a novel substance not yet published in the literature, and is useful as a diagnostic or therapeutic substance using a radiometal nuclide-labeled antibody.

[0002]

2. Description of the Related Art Currently, diagnostic imaging is showing great power in the analysis of lesion properties and extent of progress in the medical field.
It is one of the indispensable techniques. Typically,
Chest and abdominal X-ray examination, ultrasonic diagnosis (US), X-ray computed tomography (X-ray CT), computed tomography using nuclear magnetic resonance (MRI) or nuclear medicine diagnosis using radionuclides There is a law.

Of these, the US and CT methods are the most common methods for diagnosing malignant tumors (cancers), but there is a limit to early diagnosis that catches the occurrence at an extremely early stage and qualitative diagnosis that distinguishes benign from malignant. There is.

Recently, a technique using a cancer-specific monoclonal antibody has been adopted, and not only diagnosis but also treatment research has been conducted. For example, so-called "missile therapy", in which a drug such as an anticancer drug bound to an antitumor monoclonal antibody is administered and accumulated in the tumor site and the tumor is selectively destroyed by the drug, is a typical example. .

A radiometal nuclide having a short half-life is used in place of this drug, and it is accumulated in the tumor site for image diagnosis,
A method of destroying tumor cells by irradiation from the inside of the body has also been attempted as a therapeutic method. When diagnosis is performed by this method, a very early tumor in which an antigen has appeared can be clearly captured, and an earlier treatment can be performed. Further, since it is a cancer-specific image diagnosis, it is also excellent in diagnosing the quality of tumors.

When radionuclides are used for diagnosis and treatment, the most important problem is selective accumulation on tumors.
That is, it has good exclusivity from other normal tissues, which is always required for diagnosis and treatment.

[0007]

The present invention will be described in a little more detail as follows. The following conditions must be considered when developing a diagnostic or therapeutic method using a radiometal nuclide-labeled antibody.

(A) Improving the efficiency of binding between the metal ligand and the antibody. B) Rapid and stable complex formation between antibody-binding ligands and metals. C) Suppression of nonspecific accumulation of radioactive metal-labeled antibody.

If any of these conditions is not satisfied, application to diagnosis and treatment becomes difficult. In other words, it is no exaggeration to say that the success or failure of this methodology is determined by the superiority or inferiority of the labeling method, since the distribution dynamics in the body greatly differ depending on the labeling method of the radionuclide.

For example, since it is a necessary condition to use a radioisotope having a short half-life as a radionuclide, it is necessary to carry out operations such as labeling by complex formation and purification of labeled antibody as simply as possible and in a short time. There is a need for high efficiency labeling.

Regarding the above-mentioned (c), many activated antibodies currently used have a problem of non-specific accumulation in normal organs, particularly in the liver. Therefore, in developing a radiometal-labeled antibody, it is an important task to minimize nonspecific accumulation of the radiometal-labeled antibody.

Anhydrous diethylenetriaminepentaacetic acid (hereinafter abbreviated as “DPTAanhyd.”) Which has been widely used in research.
Antibody labeling method or antibody labeling method using a compound developed by Marys or Gunsaw (US Pat. No. 4,634,58).
No. 6, and U.S. Pat. Known and clear diagnosis,
Treatment was difficult. In addition, adverse effects on the specimen or the human body due to residual radioactivity are unavoidable.

[0013] In order to increase the efficiency of elimination from the body, antibody labeling in which an antibody and a radioactive metal ligand are bound via a metabolic spacer has been studied, but a complete solution has not been reached yet.

[0014]

Means for Solving the Problems As a result of intensive studies to find a compound satisfying the above three conditions, the present inventors have found a novel ethylenediaminetetraacetic acid derivative.

That is, the novel compound of the present invention has the general formula (I)

[Chemical 3] (In the formula, n is an integer of 2 to 20) and is a novel ethylenediaminetetraacetic acid derivative.

The compound represented by the general formula (I) has the following general formula (II)

[Chemical 4] The compound represented by the following general formula (III)

[Chemical 5] (In the formula, n is in the range of 2 to 20).

Table 1 shows specific examples (compound symbols A to H) of the compound of the present invention represented by the general formula (I) thus synthesized and elemental analysis values of the compound. All of these compounds of the present invention are white to slightly brown powdery substances.
In addition, as a radionuclide for labeling the compound of the present invention, indium-111, technetium-99m, gallium-, which are conventionally known as radioisotopes having a short half-life, are used.
67, yttrium-90, etc. are used.

[0017]

The compound of the present invention represented by the above general formula (I) is
Since the maleimide group binds to the antibody and ethylenediaminetetraacetic acid coordinates with the metal, the distance between the antibody and the coordination metal can be changed by selecting n and using carbon chains of various lengths.

[0018]

[Effect of the invention] As shown in the following examples, ¹¹¹ In-labeled A7 monoclonal antibody was prepared using the compound of the present invention,
When experiments were carried out in mice, the larger the value of n in the general formula (I), the less the nonspecific accumulation of ¹¹¹ In-labeled antibody, and the best result was obtained when n = 10.

DT widely used with n = 10 compounds of the present invention
Comparing the ¹¹¹ In-labeled A7 monoclonal antibodies prepared using PA Anhyd. With each other, it is apparent that the compound of the present invention has less non-specific accumulation of ¹¹¹ In-labeled antibodies and higher tumor accumulation. The significant result that the image can be drawn in a short time was obtained.

Further, the compound of the present invention in which n = 10 is DTPA anh
It has also been found that the binding efficiency to the antibody is higher than that of yd., it is added stoichiometrically, the complex formation rate with metal is fast, and time-consuming purification such as chromatography is unnecessary. It was From the above, the compound of the present invention is extremely effective as a composition for diagnostic imaging.

[0021]

[Examples] Next, more specifically, the method for synthesizing the compound of the present invention (Examples 1 to 3), the radiometal nuclide labeling method for proteins using the same (Example 4), and diagnostic imaging The method (Example 5) will be described, but the present invention is not limited to the following Examples unless the gist thereof is exceeded. In the following examples, various characteristics were measured by the following methods.

(1) Thin layer chromatography (TLC) Stationary phase: Silica gel 60 manufactured by Merck, 2 × 7 cm Developing solution: 25% ammonia water / methanol / n-butanol / acetic acid / water = 1/1/4/1 / 1 (Volume ratio) Detection: UV absorption (254 nm) Measurement value: Transfer rate Rf.

(2) Nuclear magnetic resonance spectrum ( ¹ H-NMR) Measuring apparatus: Bruker Co., AC200 Sample concentration: 5% by weight Reference substance: TMS (tetramethylsilane) Measured value: chemical shift δ (ppm) .

(3) Infrared absorption spectrum (IR) Measuring device: Hitachi, Hitachi 270-30 Measuring method: KBr method.

(4) Mass spectrum (FAB-MS) Measuring device: JEOL Ltd., JMS-AX 505W method: Fast atom bombardment type ionization Measured value: (MH) ⁻ .

Example 1 Synthesis of Compound (B) 1.1 g of 4-maleimidobutanoic acid was dissolved in 10 ml of chloroform, cooled to 0 ° C. and 0.68 g of N, N-dicyclohexylcarbodiimide was added. After stirring at 0 ° C. for 2 hours and then at room temperature for 4 hours, the by-produced dicyclohexylurea was filtered off, and the filtrate was concentrated to dryness.

On the other hand, 0.795 g of 1- (4-aminobenzyl) ethylenediamine-N, N, N, N-tetraacetic acid was dissolved in 10 ml of N, N-dimethylformamide, and the dry matter was added at room temperature. After stirring for 4 hours, the mixture was concentrated to dryness. Chloroform was added to the residue for crystallization, and the crystals were collected by filtration. The yield was 1.1 g, and the yield was 98%.

The properties of the obtained compound were as follows.

TLC: Rf = 0.75 ¹ H-NMR (D ₂ O) δppm (TMS): 1.85 to 4.04 (m, 19H), 6.
76 (s, 2H), 7.23 ~ 7.39 (dd, 4H) IR (cm ^-1 ): 3450, 1710, 1610, 1530 FAB-MS: 561 (MH) ^-

Example 2 Synthesis of Compound (D) 1.27 g of 6-maleimidohexanoic acid was dissolved in 10 ml of chloroform, cooled to 0 ° C., and 0.68 g of N, N-dicyclohexylcarbodiimide was added. After stirring at 0 ° C. for 2 hours and at room temperature for 4 hours, by-product dicyclohexylurea was filtered off,
The filtrate was concentrated to dryness.

On the other hand, 0.795 g of 1- (4-aminobenzyl) ethylenediamine-N, N, N, N-tetraacetic acid was dissolved in 10 ml of N, N-dimethylformamide, and the dry matter was added at room temperature. After stirring for 4 hours, the mixture was concentrated to dryness. Chloroform was added to the residue for crystallization, and the crystals were collected by filtration. Yield 0.91g, 77% yield
Met.

TLC: Rf = 0.78 ¹ H-NMR (CD ₃ OD) δppm (TMS): 1.13 to 3.94 (m, 23H),
6.77 (s, 2H), 7.17 ~ 7.52 (dd, 4H) IR (cm ^-1 ): 3480, 1720, 1610, 1530 FAB-MS: 589 (MH) ^-

Example 3 Synthesis of compound (G) 1.27 g of 11-maleimidoundecanoic acid was added to 10 ml of chloroform.
, 0.50 g of N, N-dicyclohexylcarbodiimide was added. After stirring at 0 ° C. for 2 hours and at room temperature for 4 hours, by-product dicyclohexylurea was filtered off,
The filtrate was concentrated to dryness.

On the other hand, 0.596 g of 1- (4-aminobenzyl) ethylenediamine-N, N, N, N-tetraacetic acid was dissolved in 10 ml of N, N-dimethylformamide, and the dry solid was added to it at room temperature. After stirring for 4 hours, the mixture was concentrated to dryness. Chloroform was added to the residue for crystallization, and the crystals were collected by filtration. The yield was 0.8 g, which was 81%.

TLC: Rf = 0.80 ¹ H-NMR (DMSO-d ₆ ) δppm (TMS): 1.10-3.60 (m, 33H),
6.99 (s, 2H), 7.08 ~ 7.50 (dd, 4H) IR (cm ^-1 ): 3450, 1710, 1608, 1540

Example 4 Radioactive metal nuclide labeling of protein using compounds (B), (D) and (G) 100 μl of monoclonal antibody A7 solution (30 mg / ml) and 2 μl of 2-mercaptoethanol were mixed at room temperature, After reacting for 30 minutes, the antibody fraction was separated by gel filtration (Sephadex G-50). For elution, a 0.1M phosphate buffer solution (pH 6.0) (hereinafter abbreviated as "PB") was used.

Compound (B) 0.52 mg, Compound (D) 0.55 m
g and 1,22 mg of the compound (G) were dissolved in 1 ml of PB, and 5 μl of each solution and 40 μl of the monoclonal antibody A7 (30 mg / ml) dissolved in PB were mixed and reacted at room temperature for 2 hours. After the completion, 20 μl of 0.3 M acetic acid-0.03 M citrate buffer (pH 5.0) was added to each.

0.5 ml of 0.3M acetic acid-0.03M citrate buffer (pH 5.0) was added to 1 ml of indium chloride-111 (In-111C13, manufactured by Nippon Mediphysics Co., Ltd.). 0.5 ml of them was added to each of the above reaction products and reacted at room temperature for 30 minutes.
After completion, the protein (antibody) fraction was separated by gel filtration high performance liquid chromatography. The radioactivity labeling rate and the specific activity were as shown in Table 2.

[0039]

[Table 2]

Example 5 Biodistribution of radionuclide-labeled antibody in tumor-bearing animals The monoclonal antibody A7 specifically reacts with human colon cancer. Compounds (B), (D), (G) in nude mice in which a human colon cancer cell line (LS-180) was subcutaneously transplanted into the thigh
We also examined the time course of biodistribution of radionuclide-labeled A7 using conventional DTPA anhyd.

Nude mice were subjected to the experiment about 3 weeks after the LS-180 transplantation, when the seed tumor became the size of the little finger. In-111 labeled A7 50 μCi labeled with each compound was intravenously injected from the tail vein of a nude mouse, 6, 24, 48, respectively.
After 72 and 96 hours, the distribution of radioactivity in the body was imaged with a gamma camera equipped with a pinhole collimator.

FIG. 1 shows the images after 6, 24 and 72 hours. The transplanted tumor of the thigh shown by the arrow in the image is clearly drawn with time, but the contrast with the normal tissue radioactivity in the background of the abdomen (mainly liver) is in the case of the compound (G), It was the best compared to the other three. In the early image 6 hours after administration, the DTPA anhy
d., compound (B), (D), (G).

[Brief description of drawings]

FIG. 1 is a photograph showing the distribution of radioactivity in nude mice by a gamma camera. The row shows the type of compound,
The columns show the time, and the arrows show the site of the seed tumor implanted in the thigh. In the row of compounds, n = 3 is the compound (B) and n = 5
Corresponds to the compound (D), and n = 10 corresponds to the compound (G).

[Table 1]

Claims (2)

[Claims] 1. A general formula: (In the formula, n is an integer of 2 to 20) A novel ethylenediaminetetraacetic acid derivative. 2. A general formula: (In the formula, n is an integer of 2 to 20) A radionuclide-labeled compound labeled with indium-111, technetium-99m, gallium-67 or yttrium-90 via a novel ethylenediaminetetraacetic acid derivative represented by .