JPH0345723B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to a reactive polymer compound, and more particularly, to a reactive polymer compound in which dialdehyde starch and a bifunctional ligand compound are bonded. The compound of the present invention is a novel compound that has not been described in any literature, and is a stable radioactive metal that can be applied to nuclear medicine applications for the purpose of depicting specific organs, detecting specific diseases, and testing the dynamics of physiologically active compounds. Used in the production of labeled radioactive diagnostic agents. (Prior Art) Currently, various radiolabeled compounds have been developed for non-invasive nuclear medicine diagnosis, and their clinical utility as radiodiagnostic agents has been highly recognized. in general,
After introduction into the body, radiolabeled compounds exhibit specific accumulation in specific organs or diseased areas, or
It is required that specific dynamics correspond to various physiological conditions in the living body. Conventionally, as a radioactive metal used in a labeled compound, in addition to iodine-131 formed by a covalent bond, radioactive metals formed by a coordinate bond or the like have also been frequently used. For example, technetium-99m, gallium-67, indium-111, etc. These radioactive metals exhibit physical properties more suitable for nuclear medical diagnosis. Furthermore, attempts have been made to use human serum albumin, bleomycin, fibrinogen, etc. as physiologically active compounds to be labeled, and to directly introduce radioactive metals such as gallium-67 into these compounds. However, the chelate-forming properties of these bioactive compounds are not necessarily high, the stability of the prepared labeled compounds after administration to the body is low, and furthermore, the behavior of radioactivity in the body and the behavior of the bioactive compound do not match, etc. In applications intended for medical diagnosis,
It's not something to be satisfied with. As used herein, the term "bioactive compound" refers to a compound that exhibits specific accumulation in a specific organ or disease site, or exhibits specific dynamics corresponding to various physiological conditions in the body. By tracking its internal behavior,
Refers to a compound that can provide useful information for various diagnoses. If a radioactive metal with excellent physical properties can be stably introduced into such a physiologically active compound without impairing the physiological activity of the compound, extremely useful applications in nuclear medicine diagnosis are expected. Recently, methods using difunctional ligand compounds have been proposed. That is, this method utilizes the strong chelate-forming ability of a bifunctional ligand compound with respect to various metals and the reactivity of the unterminated groups (amino group and carboxyl group) of such a ligand compound with respect to physiologically active compounds. It consists of bonding a radioactive metal and the physiologically active compound via this ligand compound, and the ligand compound includes diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetriacetic acid (EDTA), 3-oxobutyralbis (N-methylthiosemicarbazone)carboxylic acid, deferoxamine, 3-aminomethylene-2,4-pentanedione bis(thiosemicarbazone) derivative, 1
-(p-aminoalkyl)phenylpropane-1,
2-dione-bis(N-methylthiosemicarbazone) derivatives have been reported (GE
Krejcarek, Biochemical & Biophysical
Research Communication 77 2.581−585
1977; C.S. Leung, Int.J.Appl.Radiation&
Isotope 29 687-692 1978, JP-A-56-34634,
Unexamined Japanese Patent Publication No. 56-125317, Unexamined Japanese Patent Application No. 57-102820, Patent Application No. 1987-102820
-157372). According to these methods, first 2
A non-radioactive carrier is prepared by binding a functional ligand compound and a physiologically active compound, and this carrier is supplemented with a radioactive metal. The obtained labeled compound is relatively stable and retains the activity of a physiologically active compound, and is a very interesting drug as a radiodiagnostic agent in nuclear medicine diagnosis. (Problems to be Solved by the Invention) However, the biggest drawback of these known radioactive diagnostic agents using bifunctional ligand compounds is that human fibrinogen (molecular weight approximately 340,000) for blood clot diagnosis is used as a physiologically active compound. When using something with a large molecular weight, such as IgG (molecular weight approximately 160,000) for cancer diagnosis, the proportion of radioactive metals decreases relatively, and as a result, the radioactivity level necessary for diagnosis cannot be obtained. There is no such thing. One solution to this problem is to bond a large number of bifunctional ligand compounds to one molecule of a physiologically active compound,
Next, a method of obtaining a compound with high specific radioactivity by coordinating a radioactive metal to each of these bifunctional ligand compounds is considered. However, this method is not preferable because it results in the denaturation of the physiologically active compound, or its activity is reduced or eliminated. On the other hand, when using bioactive compounds with large molecular weights, it is generally desired to minimize the dose from the viewpoint of their antigenicity, and for this reason, it is desirable to use bioactive compounds to replace conventional bifunctional ligand compounds. The emergence of such compounds for radiodiagnostic agents with high specific radioactivity has been desired. (Means for Solving the Problems) As a result of investigations from various viewpoints in order to solve the above problems, the present inventors have developed a new method in which dialdehyde starch and a bifunctional ligand compound are combined. They succeeded in developing a high-molecular compound. That is, in the present invention, one molecule of dialdehyde starch having at least three free aldehyde groups and at least two molecules of an amino group-containing bifunctional ligand compound are -
The present invention provides a reactive polymer compound having at least one free aldehyde group bonded via a CH=N- group or a -CH2 _- NH- group. According to the compound of the present invention, a radioactive diagnostic agent with high specific radioactivity can be obtained without denaturing the physiologically active compound or reducing its activity. (Action of the Invention) The reactive polymer compound of the present invention is a compound having a large number of metal forming sites (ligands) as well as an unconverted aldehyde group capable of binding to a physiologically active compound. That is, this compound is characterized in that the number of radioactive metal ions that can be bound per molecule is significantly larger than that of conventional simple bifunctional ligand compounds. Therefore, even if a relatively small number of the polymer compounds of the present invention are bound to one molecule of a physiologically active compound, it is possible to bind a much larger amount of radioactive metal per molecule of the physiologically active compound than in conventional methods. can. Therefore, according to the novel compound of the present invention, a desired radioactive diagnostic agent with high specific radioactivity can be obtained without causing denaturation or reduction in activity of the physiologically active compound. (Detailed Description of the Invention) The reactive polymer compound of the present invention is composed of a combination of dialdehyde starch and a bifunctional ligand compound. The dialdehyde starch that can be used in the present invention is
It is necessary to have at least three aldehyde groups, and the higher the number, the better. At least two of these aldehyde groups are utilized for binding with the bifunctional ligand compound, and at least one of the others remains free in the target new compound and is used for binding with the physiologically active compound. used for. Such dialdehyde starch is a chain polymer compound produced by oxidizing starch or the like as a raw material with a suitable oxidizing agent such as periodate, and is commercially available. The starch typically has two aldehyde groups as functional groups in the unit molecule, and the number of repeating units is 2 to 1000, preferably
10-500. Bifunctional ligand compounds that can be used in the present invention are:
strong chelation sites with various radioactive metals,
Any compound having an amino group capable of bonding with an aldehyde group under relatively mild conditions may be used. Compounds having a carboxyl group instead of such an amino group can also be used in the present invention by converting the carboxyl group into an amino group with hexanediamine or the like and giving it the ability to bond with an aldehyde group under mild conditions. I can do it. Such bifunctional ligand compounds include deferoxamine, 3-aminomethylene-2,4-pentanedione-bis(thiosemicarbazone) derivatives, and 1-(p-aminoalkyl)phenylpropane-1,2-dione. - Amino-terminated bifunctional ligand compounds such as bis(thiosemicarbazone) derivatives, as well as diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetriacetic acid (EDTA),
Examples include difunctional ligand compounds that can be derived into amino-terminated compounds such as 3-oxobutyral bis(N-methylthiosemicarbazone)carboxylic acid. To produce the reactive polymer compound of the present invention,
For example, dialdehyde starch and a difunctional ligand compound are condensed to form a -CH=N- bond between the aldehyde group of the former and the amino group of the latter, and if necessary, this bond is reduced to -CH- Just convert it to NH- bond. The above condensation reaction may be carried out by a conventional method employed for condensing an aldehyde group and an amino group, and the above reduction reaction may also be carried out by a conventional method, for example by using a metal hydride such as sodium borohydride. It will be done. During the above reduction reaction, a compound in which the aldehyde group in the repeating unit is partially reduced may be produced as a by-product, but this does not impede the object of the present invention. The number of molecules of the bifunctional ligand compound bonded to one molecule of dialdehyde starch varies depending on the reaction reagents, reaction conditions, etc., but it is 2 or more, typically 5 or more, especially 10 or more. preferable. However, at least one aldehyde group in the dialdehyde starch moiety of the reactive polymer substance obtained by this reaction should remain free for bonding with the physiologically active compound. Next, a typical method for producing the polymer compound of the present invention will be explained according to the following reaction route. In the formula, X is an amino group-reactive residue of a difunctional ligand compound, R is -CHO or -CH ₂ OH, and p is 2-
An integer of 1000, k, l, m and n are each from 0 to
Represents an integer of 1000. However, k+l+m+n is 2 to 1000. As shown in the reaction route, the aldehyde group of dialdehyde starch is reacted with the amino group of a bifunctional ligand compound to obtain the target compound (1), which is further reduced with sodium borohydride to obtain the target compound (1). 2)
get. In the reduction step to obtain the target product (2), a compound in which some of the aldehyde groups in the dialdehyde starch are reduced is produced as a by-product, but as described above, this does not impede the object of the present invention in any way. Target products (1) and (2) can be purified by column chromatography, gel filtration, dialysis, etc., which are purification methods usually applied to polymer compounds. The reactive polymer compound of the present invention can be attached with a radioactive metal label by binding a physiologically active compound to the dialdehyde starch moiety of the compound and capturing a radioactive metal to the bifunctional ligand moiety of the compound. It can be used as a compound for a radioactive diagnostic agent, and as a physiologically active compound, for example, human serum albumin, bleomycin, fibrinogen, etc. can be used, and as a radioactive metal, for example, gallium-67, technetium-99m, indium-111, etc. can be used. I can do it. Next, a specific example of a radioactive diagnostic agent obtained using the reactive polymer compound of the present invention will be described. First, the compound of the present invention, in which the difunctional ligand compound is deferoxamine, is allowed to act on human fibrinogen in the presence of triethylamine, and then further reduced with sodium borohydride as necessary. A condensate of a high molecular weight compound and human fibrinogen (hereinafter referred to as non-radioactive chiaria) is obtained. By contacting this condensate with an aqueous solution containing gallium-67 in the form of trivalent gallium ions, an extremely stable gallium-67-labeled fibrinogen derivative with high specific radioactivity is obtained. The electrophoretic behavior of this labeled derivative is exactly the same as that of human fibrinogen, and the physiological activity, or clotting ability, of the labeled derivative maintains almost the same clotting ability as human fibrinogen. ing. Furthermore, the distribution of this labeled derivative in rats is exactly the same as that of conventional iodine-131 labeled fibrinogen. When considered together with the above coagulation ability test results,
Labeled derivatives using the compounds of the invention have proven useful for the purpose of thrombus detection. A non-radioactive carrier using a compound of the present invention,
The labeling ability for 1 mCi of gallium-67 was investigated. As a control, the conventional method (Japanese Patent Application Laid-open No. 125317-1983)
A compound in which deferoxamine and fibrinogen were directly bound together was used. The result below is the first
Shown in the table.

[Table] As shown in Table 1, when using 1 mg of fibrinogen, the non-radioactive carrier of the compound of the present invention can label 100% of 1 mCi of gallium-67 in 1 hour, which is the practical labeling time. can. In contrast, in the control, under similar conditions
Not only can only 17.0% be labeled, but even with as much as 25.1 mg of fibrinogen, only 83.5% can be labeled. From the above results, by using the compound of the present invention, it is possible to produce a gallium-67-labeled fibrinogen derivative with high specific radioactivity, and this labeled substance can be used in nuclear medicine diagnosis for the purpose of detecting blood clots. It has been shown to be highly suitable for the application, thus proving the usefulness of the new compounds of the present invention. (Example) Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto. Example 1 Production of dialdehyde starch-deferoxamine condensate 1 g of dialdehyde starch (oxidation degree 80%) (hereinafter referred to as DAS) having an average molecular weight of 7000 was dissolved in 40 ml of water. This solution will be referred to as Solution A. Separately, dissolve 2.4 g of deferoxamine (hereinafter referred to as DFO) in 30 ml of water,
388 mg (equimolar amount) of triethylamine in this solution
added. This solution will be referred to as Solution B. Solution B at room temperature
The mixture was stirred for 10 minutes, added to Solution A, and further stirred at room temperature for 15 minutes. The produced DAS-DFO condensate was purified by gel chromatography as described below. Support: Sephadex G-50 Solvent: Water Column size: Diameter 4.5 cm Height 50 cm Flow rate: 25 ml/min The DAS-DFO condensate was eluted from 270 to 430 ml.
Unreacted DFO was eluted in 550-600 ml. DAS−
The target polymer compound was obtained by freeze-drying 270 to 430 ml of the eluate containing the DFO condensate. When this polymer compound is analyzed by high performance liquid chromatography under the following conditions, the retention volume is
It was 26.1ml. Note that free DFO was not detected (the retention volume of DFO in this system was 32.8 ml). Column: TSK-3000SW Solvent: 0.05M Tris-0.15M saline/hydrochloric acid buffer
PH7.4 Pressure: 100Kg/cm ² Flow rate: 1.0ml/min Absorption wavelength: 280nm Example 2 Production of dialdehyde starch-deferoxamine condensate reduced product 1 g of DAS (oxidation degree 80%) with an average molecular weight of 7000 was mixed with water.
Dissolved in 40ml. This solution will be referred to as Solution A. Separately,
2.4 g of DFO was dissolved in 30 ml of water, and 388 mg (equimolar amount) of triethylamine was added to this solution. This solution will be referred to as Solution B. Stir solution B for 10 minutes at room temperature, and
added to the liquid. This solution was stirred at room temperature for 15 minutes.
Furthermore, sodium borohydride 167 was added to the reaction solution.
mg was added thereto, and reduction was carried out with stirring at room temperature for about 1 hour. The produced DAS-DFO condensate was purified by gel chromatography as described below. Support: Sephadex G-50 Solvent: Water Column size: Diameter 4.5 cm Height 50 cm Flow rate: 2.5 ml/min The DAS-DFO condensate is eluted in 300 to 450 ml, and unreacted DFO is eluted in 550 to 600 ml. It was done.
The target polymer compound was obtained by freeze-drying 300 to 450 ml of the eluate containing the condensation reductant of DAS-DFO. When this polymer compound is analyzed by high performance liquid chromatography under the following conditions, the retention volume is
It was 26.9ml. Note that free DFO was not detected. (The retention volume of DFO in this system is 32.8 ml). Column: TSK-3000SW Solvent: 0.05M Tris-0.15M saline/hydrochloric acid buffer
PH7.4 Pressure: 100Kg/ ^cm2 Flow rate: 1.0ml/min Absorption wavelength: 280nm Example 3 Amine derivative/condensation reduction product of dialdehyde starch-3-oxobutyral bis(N-methylthiosemicarbazone)carboxylic acid Production of 3-oxobutyral bis(N-methylthiosemicarbazone)carboxylic acid (hereinafter referred to as KTS) 132mg
was dissolved in 5 ml of anhydrous dioxane, cooled to around 10°C, 0.12 ml of tri-n-butylamine and 64 μ of isobutyl chloroformate were added, and stirred at the same temperature for about 50 minutes to obtain an acid anhydride mixture. Ta. Separately, N-tert-butyloxycarbonyl-
104 mg of 1,6-hexanediamine was dissolved in 2 mg of anhydrous dioxane, the resulting solution was added to the above acid anhydride mixture, stirred at around 10°C for about 15 hours, and KTS
-N-tert-butyloxycarbonyl-1,6-
A hexanediamine condensate was obtained. By adding 1 to 2 drops of concentrated hydrochloric acid to this condensate solution to lower the pH to 2, the N-tert-butyloxycarbonyl group, which is a protecting group for the amino group, was removed, and a KTS-hexanediamine condensate solution was obtained. Add this solution to DAS200
After adding 17.2 mg of sodium borohydride to a solution of 5 ml of dimethyl sulfoxide, the mixture was reacted at room temperature for about 3 hours to obtain a DAS-hexanediamine-KTS solution. After the reaction was completed, the mixed solution was placed in a conventional dialysis tube and dialyzed for 30 hours using a conventional method to remove unreacted reagents, and then freeze-dried to obtain the desired polymer compound. Example 4 DFO in DAS-DFO condensate (Example 2)
Quantification of Fe() and DFO form a 1:1 complex, and 420n
It has maximum absorption at m. Fe()−DFO complex
The absorbance (εmax) at 420 nm was 2.63×10 ³ . A known amount of the condensation reductant obtained in Example 2 was dissolved in water, and a sufficient amount of FeCl ₃ solution was added to form a 1:1 complex of DFO and Fe( ). After this mixture was allowed to stand for 1 hour, the absorbance at 420 nm was measured. In the above manner, the measured
It was confirmed that 19.6 DFOs were bound in one DAS molecule. The average molecular weight of the condensation reductant obtained in Example 2 is calculated to be approximately 18,000 based on the above data. Example 5 Quantification of KTS residues contained in the amine derivative/condensation reductant of DAS-KTS (Example 3) The maximum absorption of the KTS-hexadiamine derivative is
Exists at a wavelength of 334 nm, and has an absorbance (εmax) of 4.37
It was confirmed that ×10 ⁴ . Therefore, the KTS residue in the condensate obtained in Example 3 was quantified by the following method. The condensate produced in Example 3 was dissolved in water, and 3
The concentration was mg/ml. This solution was added to water as a control.
Absorbance was measured at 334 nm. As a result, it was confirmed that 11.2 KTS were bound per DAS molecule. Furthermore, the average molecular weight of the condensate of Example 3 was calculated to be 11,500.

Claims (1)

[Scope of Claims] 1. One molecule of dialdehyde starch having at least three aldehyde groups and at least two molecules of an amino group-containing bifunctional ligand compound capable of capturing radioactive metals, the former's aldehyde group and the latter's amino group (a) At least one aldehyde group capable of reacting with the amino group of the amino group-containing physiologically active compound, which is bonded so that a -CH=N- group or a -CH2 _- NH- group is formed between , (b) A complex for preparing a radioactive metal carrier, which has a bifunctional ligand structure capable of capturing a radioactive metal. 2 The number of repeating units of dialdehyde starch is 2 or more
1000. 3. Claim 1, wherein a dialdehyde starch and an amino group-containing bifunctional ligand compound are condensed between the aldehyde group of the former and the amino group of the latter to form a -CH=N- group. or the complex according to item 2. 4 Dialdehyde starch and an amino group-containing bifunctional ligand compound are condensed between the aldehyde group of the former and the amino group of the latter to form a -CH=N- group, which is further reduced to form -CH ₂ -NH- group is formed in claim 1 or 2.
Complexes described in Section. 5 The amino group-containing bifunctional ligand compound is diethylenetriaminepentaacetic acid, ethylenediaminetriacetic acid,
3-oxobutyral bis(N-methylthiosemicarbazone)carboxylic acid, deferoxamine, 3
- selected from the group consisting of aminomethylene-2,4-pentanedione bis(thiosemicarbazone) derivatives and 1-(p-aminoalkyl)phenylpropane-1,2-dione-bis(thiosemicarbazone) derivatives. Claim 1 or 2
Complexes described in Section.