JPH08259692A - Radioactive metal element-bonded polymer compound and radioactive medicine containing the same compound - Google Patents

Abstract

PURPOSE: To obtain a polymer compound bonding a radioactive metal element capable of manifesting high specific activities without causing deterioration and reduction in activities of a physiologically active substance and useful for radioactive medicines suitable for detection, treatment, etc., of diseases by bonding a radioactive metallic element through chelate bonds to a specific physiologically active substance-bonding polymeric compound. CONSTITUTION: This compound is a radioactive metallic element-bonding physiologically active substance obtained by bonding (A) a radioactive metallic element through chelate bonds to a physiologically active substance-bonding polymeric compound prepared by bonding the physiologically active substance (B2 ) to free amino groups in the reactive polymeric compound (B1 ). The compound is a compound prepared by bonding a polyamine compound (B<1> 1 ) having >=3 amino groups in the molecule which is polylysine having 500-1000000 molecular weight or a polymine having 500-100000 molecular weight through amide bonds (CONH) to a bifunctional ligand compound (B<2> 1 ) having carboxyl groups or reactive groups derived therefrom at a ratio of 2 molecules of the compound (B<2> 1 ) based on 1 molecule of the compound (B<1> 1 ).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a reactive polymer compound having an amino group and a bifunctional ligand and the use thereof, and particularly (a) an amino group capable of binding to a physiologically active substance and (b) The present invention relates to a reactive polymer compound having a bifunctional ligand capable of binding to a radioactive metal element and use of the reactive polymer compound as a radiopharmaceutical.

The reactive polymer compound of the present invention is a novel substance which has not been published in the literature, and is used to visualize specific organs, detect specific diseases,
It can be used for the production of stable radiopharmaceuticals labeled with radioactive metal elements, which are suitable for nuclear medicine applications such as kinetic examination of physiologically active substances and treatment of diseases.

[0003]

2. Description of the Related Art Conventionally, iodine-131 labeled physiologically active substances have been widely used in nuclear medicine for the purpose of imaging specific organs, detecting specific diseases, kinetic tests, and treating diseases using radioisotopes. It has been. For example, iodine-131 labeled human serum albumin used for visualizing the blood circulation system and dynamic tests, and iodine-1 for treating cancer.
31-labeled cancer-specific antibody and the like. However, since iodine-131 has a long half-life of about 8 days and emits beta rays in addition to gamma rays, it cannot be said to be suitable as a diagnostic agent. Further, also in application to therapy, iodine-131 has a drawback that it undergoes a deiodination reaction in a living body and is exposed to radiation to tissues other than the lesion.

Therefore, an attempt is made to obtain a useful radiopharmaceutical by introducing a radioactive metal element having more suitable physical properties into a physiologically active substance by a more suitable chemical method according to the purpose of nuclear medicine use. Is being continued. For example, the strong chelate-forming ability of various bifunctional ligand compounds such as diethylenetriaminepentaacetic acid (DTPA), 3-oxobutyral bis (N-methylthiosemicarbazonecarboxylic acid, and deferoxamine) to various metals, and their bifunctionality. Based on the reactivity of amino groups and carboxyl groups at the end of the ligand compound to various physiologically active substances,
A method of binding a radioactive metal element and a physiologically active substance via these bifunctional ligand compounds has been proposed.
The labeled compound obtained by these methods is relatively stable and retains the activity of a physiologically active substance, and thus is a drug of great interest in the field of nuclear medicine. However, the radiopharmaceuticals obtained by these methods are
Necessary for diagnosis and treatment when a physiologically active substance having a large molecular weight, such as fibrinogen having a molecular weight of approximately 340,000 or an immune antibody (IgG) having a molecular weight of approximately 150,000, which is used for thrombosis diagnosis and cancer diagnosis and treatment, is used There is a drawback that high specific activity cannot be obtained.

[0005]

DISCLOSURE OF THE INVENTION As a result of various researches conducted by the present inventors, a bioactive substance-binding polymer compound obtained by binding a bifunctional ligand compound and a bioactive substance to a polyamine compound has been found to be a radioactive metal element. It has been found that a radioactive metal element-binding compound in which a radioactive metal element is supported on the carrier is useful as a radiopharmaceutical that overcomes the above-mentioned drawbacks. This radiopharmaceutical has a large number of bifunctional ligands per molecule, which means that the number of radiometal elements bound per molecule cannot be corrected. It means much more than the use of itself. Then, by using the reactive polymer compound of the present invention, it has become possible to provide a radiopharmaceutical having a high specific activity without causing denaturation of the physiologically active substance and reduction in activity.
Generally, when a physiologically active substance having a large molecular weight is administered to the human body, it is desirable to make the dose as small as possible in consideration of its antigenicity. Therefore, it is extremely advantageous in this respect that the radiopharmaceutical obtained here has a high specific activity.

[0006]

The novel polymer compounds provided by the present invention are the following four types: The polyamine compound (1) having at least 3 amino groups in the molecule and the bifunctional ligand compound (2) are bound via the amide bond (-CONH-) in the ratio of the latter at least 2 molecules per one molecule. A reactive polymer compound (A) having at least one free amino group. 2. Polyamine compound (1) having at least 3 amino groups in the molecule and a physiologically active substance-bound polymer compound in which a physiologically active substance (3) is bound to at least one amino group
(B '). 3. A physiologically active substance-bound polymeric compound (B) comprising a physiologically active substance (3) bound to at least one free amino group present in the reactive polymeric compound (A). 4. A radioactive metal element-bonded polymer compound (C), which comprises a bioactive substance-bonded polymer compound (B) and a radioactive metal element (4) bound thereto via a chelate bond.

[0007]

[Function] The reactive polymer compound (A) is a polyamine compound
(1) and the bifunctional ligand compound (2) are bonded together. The polyamine compound (1) needs to have at least 3 amino groups in the molecule, and the larger the number of amino groups, the more preferable. At least two of those amino groups are consumed for binding with the bifunctional ligand compound (2), and at least one of the other amino groups remains free in the reactive polymer compound (A). Useful for binding to physiologically active substance (3). As described above, as the polyamine compound (1), the larger the number of amino groups present in the molecule, the more desirable it is.
For example, a high molecular polymer having a free amino group in its side chain is preferably used. The molecular weight may be appropriately selected in consideration of the physical properties and chemical properties of the physiologically active substance (3) to be bound later. As a specific example of the polyamine compound (1) which is preferably used, the molecular weight is about 500 to 1,000,000.
Polylysine, polyimine having a molecular weight of about 500 to 500,000, and the like.

On the other hand, the bifunctional ligand compound (2) (bifunctional chelating agent) forms a strong chelate bond to the radioactive metal element (4) and under relatively mild conditions. Those having a functional group capable of reacting with the amino group of the polyamine compound (1) (for example, a carboxyl group or a reactive group derived therefrom) are used. Specific examples of such a bifunctional ligand compound (2) include compounds represented by the formula:

Embedded image Diethylenetriaminepentaacetic acid cyclic acid anhydride represented by the formula:

Embedded image Examples include ethylenediaminetetraacetic acid succinimide and the like.

To produce the reactive polymer compound (A),
For example, polyamine compound (1) and bifunctional ligand compound
(2) may be reacted by a conventional method and purified by a conventional method such as dialysis method, salting-out method, gel filtration method, column chromatography and high performance liquid chromatography. The by-products and unreacted substances in the above reaction are followed by the reactive polymer compound (A) and the physiologically active substance.
The application of purification means is not particularly required as long as it has no inhibitory effect on the reaction with (3). Depending on the difference in the functional groups and reaction conditions of the bifunctional ligand compound (2), the polyamine compound
(1) The number of molecules of the bifunctional ligand compound (2) bonded to one molecule is different, but it is generally 2 or more, and particularly preferably 5 or more. However, at least one amino group in the polyamine compound (1) portion of the reactive polymer compound (A) obtained by this reaction should remain free due to the binding with the physiologically active substance (3). .

The reactive polymer compound is exemplified by the case where a commercially available polylysine (having a lysine unit of about 2 to 2000, preferably 2 to 500) is used as the polyamine compound (1).
The structure of (A) can be represented by a formula as follows:

Embedded image [In the formula, X is a residue obtained by removing a carbonyl group from the bifunctional ligand compound (2), p is an integer of 2 to about 2000, and q is 1 to about 20.
Represents an integer of 00. However, p + q is an integer of 3 to 2000. ]

Since the reactive polymer compound (A) has at least one free amino group in its molecule, it is treated with the physiologically active substance (3) via the amino group and with or without an appropriate cross-linking agent. The physiologically active substance-bound polymer compound (B) useful as a carrier for preparing a radiopharmaceutical can be provided by binding and purifying by a conventional means described above, if necessary.

The physiologically active substance (3) as used herein means a substance which accumulates in an appropriate organ or tissue or a specific lesion, or exhibits a specific behavior in response to a specific physiological condition. Specific examples of the physiologically active substance (3) include blood proteins
(E.g. human serum albumin, fibrinogen), enzymes (e.g. urokinase, streptokinase), hormones (e.g. adrenocorticotropic hormone, thyroid stimulating hormone), immune antibodies (e.g. F (a of IgG and fragments thereof).
b ') ₂ , Fab', Fab), antibiotics (eg bleomycin, mitomycin), neurotransmitters, sugars, fatty acids,
Examples include amino acids.

In order to bind the physiologically active substance (3) to the reactive polymer compound (A), it is necessary to use a suitable crosslinking agent such as carbodiimide, maleimide, active ester compound or glutaric aldehyde. It is preferable to carry out. The number of molecules of the physiologically active substance (3) introduced per molecule of the reactive polymer compound (A) varies depending on the crosslinking agent, reaction conditions and the like, but is usually 10 or less, particularly preferably 3 or less. The reactive polymer compound (A) thus produced and the physiologically active substance (3) conjugate, that is, the physiologically active substance-bonded polymer compound (B), is applied to the polymer substance as required by column chromatography. It may be purified by a conventional purification method such as a gel filtration method or a dialysis method.

The structure of the physiologically active substance-bound polymer compound (B) will be described by taking as an example the case where a commercially available polylysine (having a lysine unit of about 2 to 2000, preferably 2 to 500) is used as the polyamine compound (1). The formula is as follows:

[Chemical 4] [Wherein X is a residue obtained by removing a carbonyl group from the bifunctional ligand compound (2), Y is a residue of the physiologically active substance (3) or a physiologically active substance-crosslinking agent residue bond, and p is 2 to about 2000
, Q is an integer of 1 to about 2000, and r is an integer of 0 to about 2000. However, p + q and p + q + r are 3 to 2000 respectively
Is an integer. ]

The physiologically active substance-bonded polymer compound (B) is prepared by binding the polyamine compound (1) and the physiologically active substance (3), and the obtained physiologically active substance-bonded polymer compound is obtained.
It can also be prepared by binding the bifunctional ligand compound (2) to (B ′). The first-stage bond and the second-stage bond are performed in accordance with the above-described bond between the reactive polymer compound (A) and the physiologically active substance (3) and the bond between the polyamine compound (1) and the bifunctional ligand compound, respectively. I'll do it.

The physiologically active substance-bound polymer compound (B) is useful as a carrier for preparing radiopharmaceuticals. That is, the conjugate has a plurality of bifunctional ligand compounds (2) in the reactive polymer compound (A) portion, and thereby captures a plurality of radioactive metal elements (4). It is possible to provide a radiopharmaceutical having a very high amount of radioactive substance per unit of physiologically active substance (3) and a very high specific activity.

Although the physiologically active substance-bound polymer compound (B) as a carrier for preparing a radiopharmaceutical may be stored in the form of a solution, it is usually converted into a powder state by a freeze-drying method, a low temperature vacuum evaporation method and the like. Then, it is stored in a sterile manner, dissolved in sterile water, physiological saline, buffer solution or the like. The physiologically active substance-bound polymer compound (B) in a powdered state or after dissolution may optionally contain a pharmaceutically acceptable solubilizing agent (eg organic solvent), pH regulator (eg acid, base, buffer), stability. Agent
(Eg ascorbic acid), preservatives (eg sodium benzoate), isotonic agents (eg sodium chloride) and even reducing agents and oxidizing agents for adjusting the valence state of the radioactive metal element (4) Good.

The amount of the carrier for preparing the radiopharmaceutical used is such that the labeling rate of the finally produced radiopharmaceutical is high enough to cause no practical problems, and is in a pharmaceutically acceptable range. It is necessary. To prepare a radiopharmaceutical using a carrier for preparing a radiopharmaceutical, a carrier for radiopharmaceutical preparation which may contain the above-mentioned additive and a radioactive metal element (4) in an appropriate form are contacted in an aqueous medium. I'll do it. Usually, at least one of the two is made into an aqueous solution in advance, and the other is added to it. The radioactivity of the radioactive metal element (4) to be contacted is arbitrary, but it is such that sufficient information can be obtained when performing a nuclear medicine diagnosis, and the radiation exposure of the subject is possible. It is desirable that the radioactivity range be as low as possible. On the other hand, for the purpose of treatment, radioactivity is required so that the therapeutic effect is sufficiently obtained, and the range of radioactivity that minimizes radiation exposure to other normal organs and tissues. Is desirable.

The above-mentioned radioactive metal element (4) is a metal element having radioactivity, has physical characteristics and chemical characteristics suitable for nuclear medicine diagnosis and treatment, and has bifunctional coordination. A compound that can be easily captured by the ligand structure of the child compound (2) is used. Specific examples thereof include gallium-67 and gallium-6 as those used for diagnostic purposes.
8, thallium-201, indium-111, technetium-99m and the like, and yttrium-90, palladium-109, rhenium-186, gold-198, bismuth-212 and the like can be used for therapeutic purposes. . These are usually used in the form of salts, especially water-soluble salts, and are labeled by bringing them into contact with the physiologically active substance-bound polymer compound (B) in an aqueous medium. However, when the radioactive metal element (4) is in a valence state capable of forming a stable chelate complex (for example, gallium-67, indium-111), it is not necessary to add another reagent to the reaction system. However, if it is necessary to change the valence state to form a stable chelate complex (eg technetium-99m), then a reducing or oxidizing agent may need to be present in the reaction system. Examples of reducing agents include divalent tin salts (eg tin halide, tin sulfate, tin nitrate,
Tin acetate, tin citrate). Specific examples of the oxidizing agent include hydrogen peroxide. For example, when technetium-99m is used as the radioactive metal element (4), the physiologically active substance-bound polymer compound (B) is prepared in the form of a pertechnetate in the presence of a stannous salt as a reducing agent in an aqueous solvent. A technetium-99m labeled polymer compound can be prepared by treating with technetium-99m. In the above preparation, there is no particular limitation on the mixing order of the respective reagents, but it is usually preferable to avoid mixing the stannous salt and the pertechnetate first in an aqueous medium. The stannous salt is preferably used in an amount sufficient to reduce the pertechnetate.

To be useful as a radiopharmaceutical, the radiometal element-bound polymer compound (C) thus obtained must have a sufficient radioactivity and radioactivity concentration that enables diagnosis or treatment. is necessary. For example, when technetium-99m is used as the radioactive metal element (4), it is desirable to have a radioactivity concentration of 0.1 to 50 mCi per about 0.5 to 5.0 ml at the time of administration. Further, such a radioactive metal element-bound polymer (C) may be administered immediately after preparation, but it is preferable that it has such stability that it can be stored for a suitable time after preparation. Further, the radioactive metal element-bonded polymer compound (C) may optionally contain a solubilizing agent (eg organic solvent), pH regulator (eg acid, alkali, buffer), stabilizer (eg ascorbic acid), preservative. (Eg sodium benzoate), tonicity agent
(For example, sodium chloride) may be added.

Specific examples of the preparation of the radiometal element-bound polymer compound (C) useful as a radiopharmaceutical according to the present invention include polylysine as the polyamine compound (1) and diethylenetriamine penta as the bifunctional ligand compound (2). Acetic acid cyclic acid anhydride (CADTPA), physiologically active substance
The case where human serum albumin (HSA) is used as (3) and indium-111 ( ¹¹¹ In) is used as the radioactive metal element (4) will be described below. First, polylysine and CADTPA were bound to prepare a polylysine-diethylenetriaminepentaacetic acid (DTPA) conjugate, and this conjugate and HSA were coupled via N- (γ-maleimidobutyryloxy) succinimide (GMBS) to produce HSA. -A polylysine-DTPA conjugate is obtained. By contacting this conjugate with an aqueous solution containing ¹¹¹ In in the form of trivalent indium ions, a stable ¹¹¹ In-labeled HSA-polylysine-DTPA conjugate having high radioactivity is obtained.

The high-performance liquid chromatographic behavior of the labeled conjugate thus obtained is almost the same as that of HSA. The behavior of this labeled conjugate in rats is almost the same as that of the conventional iodine-131 labeled HSA. The specific activity of the ¹¹¹ In-labeled HSA-CADTPA conjugate obtained by the conventional method is about 7 mCi / mgHSA, whereas
¹¹¹ In-labeled HSA-polylysine-obtained by the present invention
That of the DTPA conjugate is greater than 35 mCi / mg HSA.

The radiopharmaceutical of the present invention is usually administered intravenously to the human body, but it is suitable for the physiologically active substance (3) part of the radiopharmaceutical to express its activity after administration. However, the present invention is not particularly limited to this as long as it is advantageous, and other appropriate methods may be adopted. As is clear from the above points, the carrier for preparing a radiopharmaceutical according to the present invention can provide a radiopharmaceutical having a high specific activity by an extremely simple method of bringing it into contact with an aqueous solution containing a radioactive metal ion. I can. Moreover, the obtained radiopharmaceuticals are the physiologically active substances that compose them (3)
It has the characteristic of substantially retaining the physiological activity derived from the part. Currently, as radiopharmaceuticals, not only those for the purpose of nuclear medicine diagnosis but also those for the purpose of treatment are known. The basic principle of therapeutic radiopharmaceuticals is based on the destruction of cells and tissues in diseased areas by radiation.
There are 31-labeled sodium iodide, gold-198 colloid used for malignant tumors on the inner surface of body cavities such as abdomen, chest, etc., and a PET ray emitting nuclide having a relatively short half-life is used. Recently, with the development of physiologically active substances such as monoclonal antibodies, which can be expected to be specifically accumulated in various lesions, they are targeted for beta-ray and alpha-ray emitting nuclides, electron capture and nuclear isomer transfer. The possibility of cancer treatment with radiopharmaceuticals labeled with radionuclides is suggested. The radioactive metal element-bound polymer compound (C) of the present invention meets such a therapeutic purpose, and in particular, since a large number of radioactive metal elements (4) can be bound per molecule, high radiation is achieved. There is an advantage that effective treatment due to radiation and high specific activity can be performed. Hereinafter, the present invention will be described more specifically with reference to examples.

[0024]

EXAMPLES The present invention will be described more concretely with reference to the following examples. Example 1 Preparation of Composition Containing Human Serum Albumin-Polylysine-Diethylenetriaminepentaacetic Acid (HSA-Poly Lys-DTPA) Conjugate (1) Acid buffer (pH = 8.
0) Dissolve in 10 ml, and add 148 mg of diethylenetriaminepentaacetic anhydride with stirring with a magnetic stirrer. After stirring overnight at room temperature for reaction, 0.05 ml of the reaction solution was taken and added to this solution in 0.1 M citrate buffer solution 0.4
5 ml, indium chloride ( ¹¹¹ In) 0.25 ml, (0.5 mCi)
Thin layer chromatography method (silica gel thin layer plate,
The number of molecules of diethylenetriaminepentaacetic acid (DTPA) bound to one molecule of polylysine was calculated by analysis with a 10% ammonium acetate solution 3: 1 solution). Rf of unreacted DTPA 0.5-0.6 Polylysine DTPA origin

The binding rate according to the above reaction conditions is 5.4 DTP.
A / polylysine. Polylysine obtained here-
DTPA (Poly (Lys-DTPA) 5.4) N-to 1.5 ml
(γ-maleimidobutyloxy) succinimide (GM
BS) dimethyl sulfoxide solution (33.6 mg / ml)
0.075 ml was added and the reaction was carried out at room temperature for 15 minutes while stirring with a magnetic stirrer. Next, 1.38 ml of this reaction intermediate was taken, and 0.5 ml of 90 mg / ml human serum albumin phosphate buffer (pH = 7.5) was added thereto, and the mixture was further reacted at room temperature overnight with stirring. Cut off reaction
The mixture was placed in a dialysis tube of 10,000 and dialyzed against a 1 M sodium chloride solution, and then unreacted polymer was removed and purified with a Sephadex G-75 column (22 × 300 mm) equilibrated with a physiological saline solution. Of the above operations, except for the measurement of the binding rate, all should be performed aseptically and all equipment used should be 18
Heat treatment at 0 ° C for 4 hours and burn with pyrodiene
It was washed with distilled water for injection and sterilized with an autoclave before use. The buffer solution was prepared by using distilled water for injection and sterilized by a filtration sterilization method using a membrane filter before use. The resin for column chromatography was washed with a dilute alkaline solution and then dealkalized with a physiological saline solution for injection. The purified human serum albumin-Poly (Lys-D
TPA) 5.4 1.1mg / ml of 0.45 ml was taken and DTPA
10 ^-6 mole / ml, 0.2 ml, 0.1M citrate buffer (pH)
= 6.0) 0.35 ml, indium chloride ( ¹¹¹ In) 2 mCi / m
Polymers bound to 1 molecule of human serum albumin were analyzed by electrophoresis under the following conditions after adding l and 0.5 ml.
The number of molecules was calculated.

Support: Cellulose acetate membrane Running buffer: 0.06M veronal buffer pH = 8.6 Running conditions: 1 mA / cm 20 minutes The binding rate under the above reaction conditions is about 1 molecule of Poly (Lys). -D
TPA) 5.4 / human serum albumin. The HSA-Poly Lys-DTPA obtained here was diluted with 0.1 M citrate buffer (pH = 6.0) to a concentration of 1 mg / ml, and 1 ml was dispensed into sterile vials while filtering with a membrane filter. Then, the intended composition was obtained.

Example 2 (HSA-Poly Lys-DTPA) -Preparation and Properties of ¹¹¹ In Injection (Biodistribution):-Commercial indium chloride ( ¹¹¹ In) injection into vial containing the composition obtained in Example 1. Liquid 2 mCi / ml (1.0 ml) was added to obtain the desired injection solution. The above operation is performed aseptically. When 25 μl of the injection solution obtained here was taken and analyzed by high performance liquid chromatography under the following conditions, the abundance of dimers was 1% or less, and unreacted polymer and DTPA were below the detection limit. . The retention time of the main component is about 23 minutes,
The average molecular weight calculated from a calibration curve obtained separately was about 7
It was 0.000.

Column: Toyo Soda TSK-2000SW
Column (0.75 × 60 cm) Eluent: 0.1 M citrate buffer pH = 6.0 Elution rate: 0.75 ml / min Further, 0.2 ml of the labeled substance was administered through the tail vein of SD female rats, and after administration 1 The distribution rate in the body after the elapse of time was measured. The results are shown in Table 1. HSA-DTPA- ¹¹¹ In de of conjugated directly DTPA human serum albumin as a control - showed data. As is clear from the results, Poly (Lys-D
Even when TPA) 5.4 was introduced into HSA, protein denaturation was not observed and almost the same distribution was observed.

[0029]

[Table 1] Rat biodistribution test Carrier HSA-Poly (Lys-DTPA) 5.4 * ¹ HSA-DTPA * ² organs Blood 83.0 88.0 Liver 9.4 9.0 Kidney 2.7 2.0 Lung 1.8 3.0 Bladder 0.7 0.7 Note: * 1 Unit:% / organ Indicate. * 2 Whole blood was 6.4% of body weight.

Example 3 Preparation of Composition Containing Human Serum Albumin-Polylysine-Diethylenetriaminepentaacetic Acid (HSA-Poly Lys-DTPA) (2):-HSA-Poly (Lys-obtained in the same manner as in Example 1) DT
PA) is diluted with 0.9% physiological saline to make the protein amount 2 mg / ml. Stannous chloride (Sn
Cl ₂ ) is added to 1 mM and a membrane filter-
The desired composition was obtained by dispensing 1.5 ml into sterile vials while filtering with. All the above operations were performed aseptically.

Example 4 Preparation and Properties of (HSA-Poly Lys-DTPA) -99mTc Injection: 20mCi / ml of commercially available sodium pertechnetate (99mTc) injection in a vial containing the composition obtained in Example 3. 1
ml (HSA-Poly Lys-DTPA) -99mTc injection was obtained. When the labeling rate of the labeled product obtained here was calculated by the thin layer chromatography method and the electrophoresis method shown in Example 1, a high value of 90% or more was obtained in each case.

Example 5 Preparation of Composition Containing Anti-Myosin Antibody Fab-Polylysine-Diethylenetriaminepentaacetic Acid (Fab-PolyLys-DTPA) Conjugate (3):-Poly (Lys-DTPA) 5.4 obtained in Example 1 Take 3 ml and add 3- (2-pyridyldithio) propionic acid N to this.
Diethylsulfoxide solution of hydroxysuccinimide ester (SPDP) (40 mg / ml, 0.12 ml) was added, and the mixture was reacted at room temperature for 35 hours while stirring with a magnetic stirrer. Mercaptoethanol 0.01 in the reaction solution
Add 3 ml and react for an additional 1 hour. The reaction solution was placed in a dialysis tube with a cutoff of 3,500, dialyzed against 0.04M phosphate buffer-1mM EDTA solution, and then equilibrated with the same buffer on a Sephadex G-25 column.
(22 × 300mm) to remove unreacted SPDP
-DTPA) -SH is obtained. Separately, 10 mg of anti-myosin antibody Fab was dissolved in 0.4M phosphate buffer pH = 7.0 to obtain 10 mg / ml solution, and N- (γ-
Maleimidobutyloxy) succinimide (GMBS)
4.2 mg / ml-DMSO solution 0.02 ml was added and the mixture was allowed to stand at room temperature for 1 hour.
Stir for 5 minutes to react. This reaction liquid was mixed with P prepared above.
oly- (Lys-DTPA) -SH, 1.7 × 10 ^-6 mole / m
l, 2.4 ml was added, and the mixture was stirred overnight at room temperature for reaction.
The reaction solution is placed in a dialysis tube with a cutoff of 10,000 and dialyzed against a 1M sodium chloride solution and then a 0.9% physiological saline solution. After dialysis, the reaction solution was Sephadex G-50 column (22 x 300 m) equilibrated with 0.9% physiological saline.
Unreacted Poly (Lys-DTPA) -SH can be removed by m) to purify the antibody Fab-Poly (Lys-DTPA). In addition to performing all the above operations aseptically, all the instruments and reagents used here were aseptically pyrogen-free (SPF) prepared by the method described in Example 1. The antibody Fab-Poly (Lys-DTPA) 0.8 mg / ml solution obtained here 0.3 ml
Take DTPA10 ^-7 mole / ml citrate buffer (pH =
6.0) 0.2 ml and indium chloride ( ¹¹¹ In) injection solution 2 mC
Add i / ml and 0.2 ml and analyze by electrophoresis under the following conditions:
The number of polymer molecules bound to one antibody molecule was calculated.

Support: Cellulose acetate membrane Running buffer: 0.06M veronal buffer pH = 8.6 Running conditions: 1 mA / cm, 20 minutes The binding rate obtained by the analysis of the above reaction conditions is It was 0.9 molecule Poly (Lys-DTPA) 5.4 / antibody Fab. The Fab-Poly (Lys-DTPA) thus obtained was used as a 0.1 M citrate buffer solution at a pH of 1 mg / ml so that the protein amount was 1 mg / ml.
The mixture was diluted with 6.0 and aseptically charged into a sterile vial through a membrane filter in an amount of 0.5 ml to obtain a desired composition.

[0034] Example 6 (Fab-Poly Lys-DTPA ) - 111 preparation and properties of In Injection: - commercial indium chloride ⁽¹¹¹ In) to a vial containing composition obtained in Example 5 Injection (2 mCi / 0.5 ml) was added to obtain the desired injection solution. The above operation was performed aseptically.
Twenty-five milliliters of the labeled product obtained here was taken and analyzed by high performance liquid chromatography under the following conditions. The abundance of dimers was 10% or less, unreacted polymer and DTPA
No radioactivity was detected in the fractions.

Column: Toyo Soda TSK-3000SW
Column (0.75 × 60 cm) Eluent: 0.1M citrate buffer pH = 6.0 Elution rate: 0.75 ml / min The retention time of the main component is about 23 minutes, calculated from a calibration curve obtained separately. Then, its molecular weight was 60,000. Antibody Fab-Poly (Lys-DTP) prepared by such a method
The antibody activity of A) ^-111 In was measured by a radiometric assay method using cardiac myosin as an antigen.
An affinity constant of ⁸ · M ⁻¹ was obtained. As is clear from the above results, the immune activity of the antibody was not lost even when Poly (Lys-DTPA) was introduced into the antibody Fab.

Example 7 Antitumor antibody 19-9 Fab'-polylysine-diethylenetriaminepentaacetic acid conjugate (19-9 Fab'-Poly Lys-D
Preparation of a composition containing TPA):-Poly (Lys-DTPA) 5.4,20 m obtained in Example 1
2.52 mg of N- (γ-maleimidobutyloxy) succinimide (GMBS) is added to 1.5 ml of g / ml phosphate buffer, and the mixture is reacted at room temperature for 15 minutes. 1.2 ml of this reaction solution contained 18.7 mg of antitumor antibody 19-9 Fab ',
04M phosphate buffer-1mM EDTA solution (pH = 6.0) 3.
5 ml was added and reacted at room temperature for 18 hours. Reaction solution is 1M
The solution was dialyzed against a saline solution and a 0.9% physiological saline solution, and further purified using a Sephadex G-75 column equilibrated with the physiological saline solution. All the above operations were performed aseptically, and all the instruments and reagents used were converted into SPF by the method described in Example 1 and used. Obtained here (19-9 Fab '
-Poly Lys-DTPA) was diluted with physiological saline to 0.5 mg.
/ Ml (protein amount) and dispensed into sterile vials to obtain the desired composition.

Example 8 (19-9 Fab'-Poly Lys-DTPA) -Preparation and properties of ¹¹¹ In injection: -Commercially available indium chloride ( ¹¹¹ In) injection in a vial containing the composition obtained in Example 7. An injection was obtained by aseptically adding 1 ml of (2 mCi / ml). The immunological activity of the antibody labeled by such a method was measured by an iminometric assay using beads immobilized with 19-9 antigen, and an affinity constant (Ka) of 3 × 10 ⁸ M ^-1 was obtained. Was given. By the way, Fab'-DTPA in which diethylenetriaminepentaacetic acid, which is a bifunctional group ligand, is directly bound to 19-9 Fab '
Also had a Ka value of about 3 × 10 ⁸ M ⁻¹ .

Example 9 Polyethyleneimine-diethylenetriaminepentaacetic acid (P
Preparation of EI-DTPA) conjugates: -Polyethyleneimine with side chains of average molecular weight about 70,000
(PEI) 10% aqueous solution was added to 0.2M phosphate buffer (pH 7.
Dilute with 8) to prepare a 0.1% aqueous solution. To this liquid was added 10 times the molar amount of diethylenetriaminepentaacetic acid cyclic acid anhydride, and the mixture was stirred overnight at room temperature. Then PEI
To measure the number of DTPA molecules bound per molecule, 200 μl of the reaction solution was taken, and 100 μl of 0.1M citrate buffer solution (pH 6.0) was added and mixed to obtain 2 mCi.
Labeling was performed by adding 100 μl of an aqueous solution of indium chloride ( ¹¹¹ In) of 1 ml / ml. One hour after labeling, the following thin layer chromatography
Of PEI-DTPA- ¹¹¹ In (near the origin) and free
¹¹¹ In-DTPA (Rf value 0.5 to 0.7) was separated, the radioactivity of each was measured, and the binding rate was calculated.

Thin layer plate: Silica gel G thin layer plate (manufactured by Merck) Developing solvent: methanol / 10% sodium acetate solution (1 /
1) Development time: about 1 hour As a result, in the above reactive polymer compound, PEI1
It was confirmed that 9 DTPAs were bound per molecule.

Measurement of the average molecular weight of the polyethyleneimine-diethylenetriaminepentaacetic acid (PEI-DTPA) conjugate: -The PEI-DTPA obtained in Example 9 was subjected to high performance liquid chromatography under the following conditions to measure the average molecular weight. did. Column: TSK-3000SW Solvent: 0.1 M citrate buffer pH = 6.0 Pressure: 380 psi Flow rate: 0.75 ml / min Absorption wavelength: 280 nm Retention time of PEI-DTPA in this system is about 24 minutes, retention of free DTPA The time was about 35 minutes. The average molecular weight of the above PEI-DTPA was calculated to be about 100,000 from the standard curve obtained using standard proteins of known molecular weight.

[0041]

As is apparent from the above, the polymer compound of the present invention, that is, the reactive polymer compound
(A), the physiologically active substance-bound polymer compound (B) and the radioactive metal element-bound polymer compound (C) are all novel substances, and the reactive polymer compound (A) and the physiologically active substance-bound polymer compound are In view of the chemical structural characteristics of (B), it is possible to provide a radioactive metal element-bound polymer compound (C) containing a relatively large number of radioactive metal elements per molecule and useful as a radiopharmaceutical.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Nobuo Ueda 2-11-11, Nagaura Ekimae, Sodegaura-cho, Kimitsu-gun, Chiba 14

Claims (2)

[Claims] 1. A bioactive substance-bound polymer in which a bioactive substance (3) is bound to at least one free amino group present in a reactive polymer compound (A) having at least one free amino group. Radioactive metal element-bonded polymer compound (C) obtained by binding radiometal element (4) to compound (B) through a chelate bond (provided that reactive polymer compound (A)
Is a polyamine compound (1) having at least 3 amino groups in a molecule selected from polylysine having a molecular weight of about 500 to 1,000,000 and polyimine having a molecular weight of about 500 to 500,000, and a carboxyl group or a derivative thereof. Bifunctional Ligand Compound Having Reactive Group
(2) is an amide bond (-CONH-) formed between the former amino group and the latter carboxyl group or a reactive group derived therefrom in a ratio of at least 2 molecules of the latter per 1 molecule of the former. It is formed by connecting through). 2. A bioactive substance-bound polymer in which a bioactive substance (3) is bound to at least one free amino group present in the reactive polymer compound (A) having at least one free amino group. A radiopharmaceutical containing a radiometal element-bonded polymer compound (C), which is formed by binding a radiometal element (4) to a compound (B) through a chelate bond (however,
The reactive polymer compound (A) has a molecular weight of about 500 to 1,
, 000,000 polylysine and molecular weight of about 500-5
Polyamine compound having at least 3 amino groups in a molecule selected from 00000 polyimines (1)
And a bifunctional ligand compound (2) having a carboxyl group or a reactive group derived therefrom, wherein the former amino group and the latter carboxyl group or the latter are present in the ratio of the latter at least 2 molecules per molecule of the former. An amide bond (-CONH-) formed with the derivatized reactive group
It is composed by connecting via).