JPS60186502A - Radioactive pharmaceutical and high polymer for preparation thereof - Google Patents

Abstract

PURPOSE:A novel high polymer, obtained by bonding a formyl group-containing polysaccharide derivative to an amino group-containing physiologically active substance and a bifunctional ligand compound, and useful as a carrier for preparing a radioactive pharmaceutical having high specific radioactivity. CONSTITUTION:A compound is obtained by bonding 1 molcule polysaccharide derivative having 3 or more formly groups in the molecule except dialdehyde starch, e.g. amylose or amylopectin, to >=2 molecules bifunctional ligand compound containing -NH2 and >=1 molecule physiologically active substance containing -NH2, e.g. blood protein, enzyme or hormone, through -CH=N- or -CH2NH- bonds. Examples of the bifunctional ligand compound include compounds of the formula (R<1> and R<2> are H, 1-3C alkyl or phenyl). EFFECT:The aimed radioactive pharmaceutical is obtained by chelating the above- mentioned compound with a radioactive metal element without deteriorating or reducing the activity of the physiologically active substance.

Description

[Detailed Description of the Invention] The present invention provides radiopharmaceuticals and polymer compounds for their production, particularly a formyl group-containing polyzaracalite derivative, in which a physiologically active substance containing an amino acid and a difunctional ligand compound containing an amino acid are bonded. The present invention relates to a radioactive polymer compound useful as a carrier for drug preparation, and a labeled polymer compound useful as a radiopharmaceutical, which is made by bonding a radioactive metal element to the polymer compound.

The polymer compound of the present invention is a new substance that has not been described in any literature, and has stable radioactivity suitable for nuclear medicine applications such as visualization of specific organs, detection of specific diseases, dynamic examination of physiologically active substances, and treatment of diseases. It is possible to provide metal-labeled radiopharmaceuticals.

BACKGROUND ART Conventionally, physiologically active substances labeled with iodine-131 have been widely used as radiopharmaceuticals for the purpose of depicting specific organs, detecting specific diseases, and testing the dynamics of physiologically active substances.

Examples include iodine-131-labeled human serum albumin, which is used for depicting the blood circulation system and dynamic examination, and iodine-131-labeled fibrinogen, which is used for detecting blood clots. However, although iodine-131 has a long half-life of about 8 days, which is advantageous for radiotherapy, it emits beta rays in addition to gamma rays, which are useful for nuclear medicine diagnosis, so patients are exposed to large amounts of radiation. The shortcomings of providing this have been pointed out.

Therefore, attempts are being made to obtain useful radiodiagnostic agents by introducing radioactive metals having physical properties more suitable for nuclear medicine diagnosis into physiologically active substances by other methods.

For example, technetium-99 can be obtained by a labeling method in which a radioactive metal salt is directly applied to a physiologically active substance.
Known examples include m-labeled human serum altumin and indium-111-labeled bleomycin. Furthermore, the strong chelate-forming ability of difunctional ligand compounds such as diethylenetriaminepentaacetic acid (DTPA), 3-oxobutyralpis (N-methylthiosemicarbazone) carboxylic acid, and defene-1noxamino to various metals, and the terminal terminals of these compounds. Based on the reactivity of the amino group and carboxyl group to various physiologically active substances, a method of binding radioactive metals and physiologically active substances via bifunctional interposition compounds has also been proposed.

The labeled compounds obtained by these methods are relatively stable and retain the activity of physiologically active substances, making them very interesting drugs for nuclear medicine diagnostic purposes. The radioactive diagnostic agents obtained by these methods are
When using physiologically active substances with large molecular ends, such as fibrinogen, which is used for frame diagnosis and can diagnosis, and has a molecular weight of about 340,000 each, or +gG, which has a molecular weight of about 160,000, the high molecular weight required for diagnosis is The drawback is that specific radioactivity cannot be obtained.

The present invention has been devised (J) to resolve this drawback 4-
Succeeded in developing a polymer compound in which a functional ligand compound and an amino M-containing physiologically active substance were combined (Japanese Patent Application Laid-Open No.
Specification No. 9-105692 and JP-A-59-1064
(See specification No. 25). This polymer compound has a large number of ligands per molecule, and this means that the number of radioactive metal ions bound per molecule is 1 compared to conventional bifunctional ligand compounds. ．． -Means a lot of things in ζdan. It has been discovered that by using this polymer compound, a radioactive diagnostic agent with high specific radioactivity can be obtained without causing denaturation of physiologically active substances or reduction in activity.

Based on the above knowledge, we further investigated the results and found that using other formyl group-containing derivatives instead of noaldehyde (J borizanocuff (1) also causes denaturation and activity reduction of physiologically active substances. It has been discovered that radiopharmaceuticals with high specific radioactivity can be obtained without any nuisance.Generally, when administering a physiologically active substance with a large molecular weight to humans, the dose should be kept as low as possible if its antigenicity is taken into consideration. It is desirable to use small amounts.

Therefore, it is extremely advantageous in this respect that the radiopharmaceutical obtained here has high specific radioactivity. In addition, noaldehyde starch has a wide molecular weight distribution and a network structure, so the number of repeating structures may make it difficult for bifunctional intersite compounds to bind effectively. If a compound having a narrow molecular weight distribution and a linear structure is used, a large number of bifunctional ligand compounds can be effectively bound, and therefore a product with high specific radioactivity can be easily obtained.

The present invention has been completed based on the above findings, and the gist of the invention is to provide for phorisanocalite derivatives (excluding dialdehyde debunk) (■) having at least three polmyls in the molecule. The amino group-containing bifunctional coordinating gold compound (■) and the Amihamipuriku 11 logical substance (■) are combined with at least two molecules of the bifunctional coordinating compound (III) per molecule of the polyzanocalide derivative (11). A polymer compound (1) comprising at least one molecule of the physiologically active substance (IV) with 1ζ bond via a methylene-imine bond (-C1l, N) J:8 (j methyleneamine bond (-C1l, Ni1) and the polysaccharide derivative (■), the bifunctional ligand compound (■) and the physiologically active substance (IV) are added to the polysaccharide derivative (■) per molecule of the polysaccharide derivative (■).
■) At least two molecules of the physiologically active substance (■) have a methyleneimine bond (-CH-N-
) or methyleneamine bond (CH2NH ) to a labeled polymer compound (1), in which a radioactive P1 metal nodile ( ) is bonded via a chelate bond.

The above-mentioned polymer compound (1), which is the object of the present invention, is a difunctional ligand compound (■
) and a physiologically active substance (■), which is a biologically active substance Vt < hemi polyzanolyte derivative (II) bifunctional ligand compound (■) conjugate.

It is necessary that the polysaccharide derivative (11) has at least three formyl groups in the molecule, and it is preferable that the number of formyl groups is greater. Those Q) formyl Jl (at least 2 of them are bifunctional interposition r・compound (Ill)
At least one of the molecules is useful for binding with the physiologically active substance (1v).

The polyzasocharide derivative (11) (J, for example, can be obtained by treating appropriately substituted borizanocalite with an oxidizing agent (for example, sodium periodate), in principle one or more polyzanocalide derivatives at each 111-position of the zanocalide. For the purpose of the present invention, it is understood that formyl may be used. Preferably, higher-order polyzaccalai IS such as zamin, poly(nonacid, glycosaminocrypnon, glyco1-uronoglypone, hetero-1-sozan), specific examples include amylose, amylobectin, digistran, and cell. -sugar, inulin, pectic acid, pullulan, etc., and may be a mixture or a dehydrated product thereof.

is preferably 3000 or more, especially l(]00 or more and 1.0 or more).

Bifunctional position 1'・Compound (11) (J, radiation 14
It forms a strong sharp bond with 1 metallic lunate (V), and under relatively mild conditions, polyzaracaride derivatives (
(2) Use a compound that has an amino group that can react with the formyl group. Specific examples of such bifunctional interstitial compounds (■) include deferoxamine (Merck Index, 9th edition, p. 374 (1976)), formula: (wherein R1
and R2 each represent hydrogen, C1-C3 alkyl or phenyl. ) 3 aminomethylene 2.4
Benotannoone His(thiosemicarbazone) derivative (European Patent Application No. 54920), formula.

(In the formula, R2 and R4 are each hydrogen or C1-0
, alkyl and 11 represent an integer of 0 to 3. ) 1-(p-aminoalkyl)phenylbropan-1,2
-dione-bis(thiosemicarbazone) derivatives (Australian Patent No. 533722) and the like.

Even if the substance itself does not contain an amino group, it can easily be used as an amino group or a group or structure that can form an amino group-containing structure. As long as it has the quality, this also forms an amino group or an amino group-containing structure, and then the amino base a
It can be used as a difunctional ligand compound. For example, an amino group can be easily introduced into a compound having a carboxyl group by reacting it with an alkylene diamine, and is used as a type of difunctional ligand compound (■) in the present invention. You can. Specific examples include diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), dimercaptoacetylethylenediamine (Fritzberg et al.: J, Nucl.
．． Med. , 23, 917 (1982)) and hisaminoethanethiol (Fritzberg et al.: J, Nu
Cl.
): N represented by +, ligand, N, N゛-bis(2
Hydroxyethyl)ethylenediamine (Wagner
Jr. et al.: Proceedings of the I
International Symposium on
Technetium in Chemistry
nf Nuclear Medicine, Padov
a Italy, p. 161 (1982)), N2O3 ligand, formula: (wherein R5, R6, R7, R
Examples include 2-propionaldehyde-bis(thiosemicarpazone) derivatives (US Pat. No. 4,287,362), in which 8 and R9 are hydrogen or C1-C3 argyl.

Physiologically active substances (■) are substances that accumulate in appropriate organs or tissues or specific lesions, or that exhibit characteristic behavior in response to specific physiological conditions, and the behavior of such substances in vivo. The ability to obtain diagnostic or therapeutically useful information and effects by tracking each patient should be used. Generally, many physiologically active substances have an amino group by themselves, but even if such a physiologically active substance does not have an amino group by itself, it can be treated with an appropriate method. It is possible to use those into which an amino group or an amino group-containing structure is introduced.

Specific examples of physiologically active substances (■) include blood proteins (e.g., human serum albumin, fibrinogen), enzymes (e.g., urokinase, strebtokinase), hormones (e.g., thyroid-stimulating hormone, parathyroid gland hormone), and immune system. Antibodies (e.g. 1gC and its fragments F(a
b3) 2', Fab', Fab), monoclonal antibodies, antibiotics (e.g. pleomycin, kanamycin)
, sugars, fatty acids, amino acids, etc.

To produce the polymer compound (1), for example, a polysaccharide derivative (■) and a bifunctional ligand compound (■) are condensed to form a methyleneimine bond between the formyl group of the former and the amino group of the latter. Then, if necessary, reduce this methyleneimine bond to convert it into a methyleneamine bond, and then condense the obtained polysaccharide derivative (■) bifunctional ligand compound (■) conjugate with the physiologically active substance (■). to form a methyleneimine bond between the polmyl group present in the former polysaccharide derivative (■) moiety and the amino group of the latter, and if necessary, this methyleneimine bond may be reduced to convert it into a methyleneamine bond. .

In addition, polysaccharide derivatives (■) and physiologically active substances (
(2) is condensed to form a methyleneimine bond between the formyl group of the former and the amino group of the latter, and if necessary, this methyleneimine bond is reduced and converted to a methyleneimine bond, and the obtained poly A saccharide derivative (■) physiologically active substance (■) conjugate and a bifunctional ligand compound (■) are condensed to form a formyl group present in the former polysaccharite derivative (■) portion and an amino group of the latter. A methyleneimine bond is formed, and if necessary, this methyleneimine bond may be reduced to convert into a methyleneamine bond.

The condensation reaction in each of the above methods may be carried out by any conventional means employed for condensing a polmyl group and an amino group. Reduction reactions are also carried out by conventional means employed in converting methyleneimine bonds into methyleneamine bonds, for example by using metal hydrides such as sodium hydride. Both the condensation reaction and the reduction reaction described above can proceed under relatively mild conditions, so even if the physiologically active substance (1v) is relatively unstable, it will not affect the biological activity of the biologically active substance (1v). The polymer compound (1), that is, the physiologically active substance (
(2)-Polysaccharide derivative (11) Bifunctional ligand compound (2) A conjugate can be produced.

Due to differences in reaction reagents, reaction conditions, etc., a bifunctional ligand compound (■
) and the number of molecules of the physiologically active substance (■) are different, but the number of molecules of the bifunctional ligand compound (■) is generally 5 or more, preferably 10 or more, and the number of biologically active molecules (■) The number of molecules of is generally 10 or less, especially 3
Either the bifunctional ligand compound (■) or the physiologically active substance (■) may be bonded to the polysaccharite derivative (■) first, but the physiologically active substance (■) to be bonded later may be The reaction should be carried out in such a way that a suitable number of free polmyl groups remain for attachment of the active substance (■) or the difunctional ligand compound (■).

Various conjugates obtained during the above production method and the polymer compound (1) finally obtained can be processed by column chromatography, high performance liquid chromatography, gel filtration, etc., which are applied to polymer substances as necessary. It may be purified by applying conventional purification methods such as dialysis and dialysis.

Taking as an example the case where a polysaccharide derivative (11) derived from amylose is used, it is first combined with a bifunctional ligand compound (■) and then reduced, and then the resulting polysaccharide derivative (11) is Saccharide derivative (■) Bifunctional interfunctional compound (■) The conjugate is condensed with physiologically active substance (■) and then reduced to produce the desired polymer compound (1), that is, physiologically active substance (■) −Polyzaccharite′ derivative (
II) The formula for producing a conjugate of a bifunctional ligand compound (■) is as follows (wherein, X is a residue obtained by removing the amino group from the bifunctional ligand compound (■), Y is a residue obtained by removing the amino group from the physiologically active substance (■), Z is CHO or -CH2OH, p is an integer of 2 to 1000, q and s are each 1 to 100
The integer of 0, r and t each represent an integer of 0 to 1000. However, q|r and q|s|t are each 2 to
It is an integer of 1000. ).

In the above formula, dialdehyde amylose (■a) is a chain polymer substance obtained by partially or totally oxidizing amylose with an oxidizing agent such as periodic acid. It is commercially available as such.

The number of saccharide units is usually 2 to 1000, particularly preferably 2 to 500.

The polymer compound (1) of the present invention, that is, the physiologically active substance (
■), polysaccharide derivative (■), bifunctional ligand compound (■) conjugate is useful as a carrier for preparing radiopharmaceuticals. That is, the polymer compound (■
) has a plurality of bifunctional ligand compound (■) moieties, which makes it possible to capture a plurality of radioactive metal elements (V). The labeled polymer compound that captures a plurality of radioactive metal elements (■) in this way is itself used as a radiopharmaceutical.

Here, the radioactive metal element (V) is a metal element that has radioactivity, has physical or chemical properties suitable for nuclear medicine diagnosis and treatment, and is a difunctional ligand compound (
Those that can be easily captured by the ligand structure (ii) to form a stable chelate complex may be used. As a specific example, gallium (
17, potassium-68, thallium-201, indium-111, technetiuno, 94h11, etc., and those used for therapeutic purposes include copper-67, inotrium-90, paranochosim-1F+ tl, Rennie's Beta-emitting nuclides such as Zino, -186, gold-1
Examples include alpha-ray nuclides such as bismuth-212 and bismuth-212. These are usually used in the form of salts, especially water-soluble salts, and are labeled by contacting them with the polymer compound (1) in an aqueous medium.
) is in a valence state capable of forming a stable chelate complex (for example, gallium-67, indium-111
), it is not necessary to have other reagents present in the reaction system, but if it is necessary to change the valence state to form a stable chelate complex (e.g., technetium-9
9m), it may be necessary to have a reducing agent or oxidizing agent present in the reaction system. Examples of reducing agents include divalent tin salts (e.g. tin halides, tin sulfate, tin nitrate, tin acetate,
tin citrate).

For example, as a radioactive metal element (V), technetium-
When using 99m, the technetium-99m-labeled polymeric compound (1) is prepared by treating the polymeric compound (1) with technetium-99m in the form of batechnetate in the presence of a stannous salt as a reducing agent in an aqueous medium. can be prepared. In the above preparation, there are no particular restrictions on the order of mixing the reagents, but it is generally desirable to avoid mixing the stannous salt and pertechnetate in the aqueous medium first. The stannous salt is preferably used in an amount sufficient to reduce vertechnetate.

In order for the thus obtained labeled polymer compound (1) to be useful as a radiopharmaceutical, such as a radiodiagnostic agent or a radiotherapeutic agent, it must have sufficient radioactivity and radioactivity concentration to enable diagnosis and treatment. It is necessary to have For example, in the case of a radioactive diagnostic agent that uses technetium-99m as the radioactive metal element (■), approximately 0.5 to 5.
It is desirable to have a radioactivity concentration of 0.1 to 50 mCi per 0 ml. In addition, such labeled polymer compounds (
Although 1) may be administered immediately after preparation, it is desirable that the drug has sufficient stability to withstand storage for an appropriate period of time after preparation.

The carrier for preparing a radiopharmaceutical comprising the polymer compound (1) of the present invention may be stored in the form of a solution, but it is usually converted into a powder state by freeze-drying, low-temperature reduction, evaporation, etc., and then stored. Before use, it is dissolved in sterile water, physiological saline, buffer solution, etc. If necessary, solubilizing agents (e.g. organic solvents), pH adjusting agents (e.g. acids, bases, buffers), stabilizers (e.g. ascorbic acid), preservatives (e.g. sodium benzoate), isotonic agents (e.g. sodium chloride), ) etc. may be blended. Further, in order to adjust the valence state of the radioactive metal element (V) as described above, a reducing agent or an oxidizing agent may be added. In view of its use as a carrier for the preparation of radiopharmaceuticals, all of these additives must be pharmaceutically acceptable.

The amount of carrier for preparing radiopharmaceuticals must be such that the labeling rate of the final manufactured radiopharmaceutical is high enough to cause no practical problems, and it must also be within a pharmaceutically acceptable range. .

To prepare a radiopharmaceutical using the carrier for preparing a radiopharmaceutical, a composition comprising the carrier for preparing a radiopharmaceutical, if necessary, the additives described above, and a radioactive substance in the appropriate form described above. The metal element (V) may be brought into contact with the metal element (V) in an aqueous medium. Usually, at least one of the two is made into an aqueous solution in advance, and then the other is added thereto. The radioactivity of the radioactive metal element (V) to be brought into contact is arbitrary, but when carrying out nuclear medicine diagnosis, it is necessary to ensure that the radioactivity is such that sufficient information can be obtained and that it is possible to be exposed to radiation for a long time. It is desirable that the radioactivity range be as low as possible. On the other hand, when the purpose is treatment, it is necessary to have enough radioactivity to obtain a sufficient therapeutic effect, and at the same time to keep the radioactivity within a range that minimizes radiation exposure to other normal organs and tissues. It is desirable that there be.

To administer the radiopharmaceutical of the present invention to humans:

Usually, it is carried out intravenously, or as long as the physiologically active substance (■) part of the radiopharmaceutical is suitable or advantageous for expressing its activity after injection, it is not particularly limited to this. Instead, the appropriate method for the pond may be adopted.

-J- As is clear from the description, the carrier for preparing radiopharmaceuticals according to the present invention provides radiopharmaceuticals with high specific radioactivity by an extremely simple method of contacting with an aqueous solution containing radioactive metal ions. I can do it. In addition, the obtained radiopharmaceutical has the characteristic that it substantially retains the physiological activity derived from the physiologically active substance (■) portion constituting it.

Currently, radiopharmaceuticals are known not only for the purpose of nuclear medicine diagnosis but also for therapeutic purposes.

The basic principle of therapeutic radiopharmaceuticals is based on the destruction of cells and tissues in diseased areas by radiation. Practical examples include iodine I31-labeled sodium iodide used for goiter, abdomen, chest, etc. Examples include gold-198 colloid, which is used to treat malignant tumors on the inner surface of body cavities, and beta-ray-emitting nuclides with relatively short half-lives are used. Recently, with the development of monoclonal antibodies and other physiologically active substances that can be expected to accumulate specifically in various cancer lesions, these can be combined with beta- and alpha-emitting nuclides, electron capture,
The possibility of cancer treatment using radiopharmaceuticals labeled with nuclides that undergo nuclear isomerism has been suggested. The polymer compound (1) of the present invention is compatible with such therapeutic purposes,
In particular, since it is possible to bind a large number of radioactive metal elements (V) per molecule, it has the advantage of being able to provide effective treatment with high radioactivity and high specific radioactivity.

EXAMPLES The present invention will be explained in more detail with reference to Examples below.

Example 1 Manufacture of human serum albumin-dialdehyde amylose-deferoxamine conjugate (1): Deferoxamine (hereinafter abbreviated as DFO) (15 mg) was added to a 0.5 mg solution at pH 7.0.
The mixture was dissolved in 03M phosphate buffer (1 ml), triethylamine (99% solution) (3.2 μl) was added thereto, and the mixture was stirred at room temperature for about 5 minutes. Add 25 mg of dialdehyde amylose (hereinafter abbreviated as DAA) dissolved in water to this solution.
/ml solution (1 ml) was added and stirred at room temperature for 30 minutes. This solution will be referred to as Solution A. Separately human serum albumin (
100aV) was dissolved in 0.03M phosphate buffer (1 ml), and this solution was designated as Solution B. Add solution A (2N) to B at room temperature.
The mixture was added to the solution and stirred at the same temperature for about 6 hours to react. After the reaction is complete, add 24% to 1M sodium chloride solution at room temperature.
After time dialysis, Sephacryl S200 (column: diameter 2.2 cm,
Column chromatography was performed with a height of 50 cm). By lyophilizing this eluate, human serum albumin-DAA- as a carrier for radiopharmaceutical preparation was prepared.
A DFO conjugate was obtained.

Example 2 Production of human serum-albumin-dialdehyde amylose deferoxamine conjugate (2): DFO (15 mg) was dissolved in 0.03 M phosphate buffer (1 ml), and triethylamine (99% solution) ( 3.2 μl) and stirred at room temperature for about 5 minutes.

To this solution was added a 25 mg/ml solution of DAA dissolved in water (
1 ml) was added thereto, and the mixture was stirred at room temperature for 30 minutes. This solution will be referred to as Solution A. Separately human serum albumin (100mg)
was dissolved in 0.03M phosphate buffer (1 ml) and used as Solution B. Solution A (2 ml) was added to solution B at room temperature, and the mixture was stirred and reacted at the same temperature for about 6 hours. Sodium borohydride (1.5 mg) was added to this reaction solution, and the mixture was reduced with stirring at room temperature for about 1 hour.

After the reaction is complete, add 2% to 1M sodium chloride solution at room temperature.
After dialysis for 4 hours, Sephacryl S200 (column: diameter 2.2 cm
, height 50 cm). By freeze-drying this eluate, human serum albumin-D as a carrier for preparing radiopharmaceuticals was prepared.
An AA-DFO conjugate was obtained.

DFO and human serum albumin in the above conjugate were quantified by filter paper electrophoresis. That is, a solution containing potassium-671mCi as gallium citrate was added to the reaction solution after the completion of the reduction reaction (solution before the purification step) for labeling. This gallium-67 labeled solution was subjected to electrophoresis (31 nΔ/port, 30 minutes) using veronal buffer (pH 8, 6) as a developing solution and cellulose acetate as an electrophoresis membrane, and then run using a radiochromatography scanner. mediated. The peak of radioactivity is 1 cm on the positive side and 1.5 cm on the negative side from the original line.
cm and 3 and 5 cm on the negative side, and were identified as gallium-67-labeled human serum albumin DAA-DFO conjugate, gallium, 67-labeled DAA, BFO conjugate, and gallium, 67-labeled DFO, respectively. From a comparison of the radioactivity values of each peak, it was found that the number of DFO molecules per human serum albumin molecule in the conjugate obtained in this example was 1.
The number of molecules within DAA was calculated to be 1.5 and 0.9.

Example 3 Preparation of human serum albumin-dialdehyde amylose 3-oxobutyralbis(N-methylthiosemicarbazone)carboxylic acid:hexanoneamine condensate conjugate: 3-oxobutyralhis(N-notylthiosemicarbasono)carboxylic acid ( (hereinafter abbreviated as KTS) (132m
g) in anhydrous dioxane (5 ml) and cooled to around 10°C, tri-n-butylamine (0,12n
1), isobutyl chloroformate (64 μl) was further added, and the mixture was stirred at the same temperature for about 50 minutes to obtain a mixed acid anhydride solution. Separately N tert-butyloxycarbonyl-
A solution of 1,6-hexanediamine (104 mg) dissolved in anhydrous dioxane (2 ml) was prepared, and this solution was added to the above mixed acid anhydride solution and stirred at around 10°C for about 15 hours. -Butyloxycarbonyl-1,6-hexanediamine condensate was obtained. Concentrated hydrochloric acid (1 to 2 drops) was added to this condensate solution to adjust the pH to 2, thereby removing the N-tert-butyloxycarbonyl group to obtain a KTS:hexanediamine condensate.

DAA (83 mg) was dissolved in dimethyl sulfoxide (5 ml).
To this solution was added the solution of the KTS:hexanediamine condensate, and the mixture was reacted at room temperature for about 3 hours to dissolve DAA-
A KTS:hexanediamine condensate conjugate solution was obtained. This solution (5 ml) was added to a solution of human serum albumin (50 mg) in 0.03 M phosphate buffer (pH 70) (50 ml), and reacted with stirring at room temperature for about 3 hours. Sodium borohydride (12.9 mg) was added to this reaction solution,
Stirring was continued for about 1 hour. After the reaction was completed, the reaction solution was placed in a regular dialysis tube and dialyzed against 0.03M phosphate buffer overnight. After dialysis, Sephacryl S200 (column: diameter 2.0 mm) was used with 0.03 M phosphate buffer as eluent.
2 cm, 50.0 cm) was performed. By freeze-drying this solution, a human serum albumin DAA KTS:hexanediamine condensate conjugate as a carrier for radiopharmaceutical preparation was obtained.

Example 4 Production of antimionone antibody fragment Fab dialdehyde amylose deferoxamine conjugate: 0.9% aqueous sodium chloride solution (8.1 m
g/ml) (1 ml), add DAA 0.03M phosphate buffer solution (9.5 mg/ml) (0.2 ml),
Stirred for 30 minutes at room temperature. This solution will be referred to as Solution A. Separately, DFO (30 mg) was dissolved in water (1 ml), triethylamine (about 99% solution) (12 μl) was added thereto, and the mixture was stirred at room temperature for about 5 minutes.

This solution will be referred to as Solution B. Add solution B (0.3ml) to solution A at room temperature.
) and stirred at the same temperature for about 4 hours to react. Sodium borohydride (2.5 mg) was added to this reaction solution, and the mixture was reduced with stirring at room temperature for about 1 hour. After completion of the reaction, dialysis was performed for 24 hours at room temperature against a 1M sodium chloride-0.5% glucose solution, and then TSK3000SW (column: diameter 0.75cm, height 60cm) was prepared using 0.03M phosphate buffer as an eluent. A solution containing an antimyosin antibody fragment Fab-DAA-DFO conjugate as a carrier for radiopharmaceutical preparation was obtained.

Example 5 Preparation of human fibrinogen-dialdehyde amylose-deferoxamine conjugate: Human fibrinogen (20 mg) was dissolved in 0.03 M phosphate buffer (2 ml) and added to a concentration of 10 mg/m
1 of DAA solution (60 μl) was added and stirred for about 30 minutes at room temperature. This solution will be referred to as Solution A. Separately DFO (68m
g) in water (1 ml), triethylamine (99
% solution) (17.5 μl) was added and reacted for 5 minutes at room temperature. This solution will be referred to as Solution B. Add B solution to A solution (88μl)
was added and stirred at room temperature for 5 hours to react. Sodium borohydride (1 mg) was added to this reaction solution, and the mixture was reduced with stirring at room temperature for about 1 hour. After the reaction was completed, it was dialyzed against 1M sodium chloride and 0.5% glucose solution for 24 hours, and then T
SK4000SW (column: diameter 0.75cm, height 6
High performance liquid chromatography was performed using a radiopharmaceutical preparation carrier (human fibrinogen DA).
ADFO conjugate) was obtained.

Example 6 Production of human fibrinogen-dialdehyde dextran deferoxamine conjugate: Dialdehyde dextran (hereinafter abbreviated as DAD) (127 mg) was added to 0.01
M phosphate buffer 0.15M sodium chloride aqueous solution (5m
DFO (370 mg) was dissolved in this. After adding triethylamine (78.9 μl), 1
Stirred for 20 minutes at 0-15°C. Human fibrinogen (400mg) in 0.01M phosphate buffer 0.15M
It was dissolved in an aqueous sodium chloride solution (40 ml), the above solution was added thereto, and the mixture was stirred at 10 to 15°C for 2 hours. To this reaction mixture was added sodium borohydride (12.3 mg).
was added and stirred at 10-15°C for 1 hour. The reaction mixture was mixed with 0-b 35M citric acid solution in water. Dialysis for 311 hours? Next, take the silver solution eluent.
- (Sepharose CL6B (column: diameter 4.4 cm,
Column chromatography was performed with a height of 100 cm. The eluate was diluted with a 0.01 M glucose-0.35 M sodium citrate aqueous solution to give a human fibrinogen concentration of 1 mg/ml, and the concentration was adjusted to 30 mM by adding sodium ascorbate. Human fibrinogen-DAD as a carrier for preparing radiopharmaceuticals was prepared by putting 3 ml of the obtained solution into a vial and freeze-drying it.
-DFO conjugate was obtained.

Example 7 Gallium-67 labeled radiopharmaceutical (Gallium-67
Production of labeled human serum albumin-dialdehyde amylose-deferoxamine conjugate: Gallium-67lmCi was added as potassium citrate to the radiopharmaceutical preparation carrier (human serum albumin-DAA-DFO conjugate) produced by the method of Example 2. solution containing (1 μl
) and gallium-67 labeled radiopharmaceutical (gallium-67 labeled human serum albumin-DAA-DFO).
A conjugate) was obtained. The gallium-67-labeled radiopharmaceutical obtained in this example is a very pale yellow clear solution with a pH of about 7.0.

This gallium-67 labeled radiopharmaceutical was subjected to electrophoresis as described in Example 2.

Radioactivity is detected as a single peak at a location 1 cm on the positive side from the original line, and the position of this radioactivity peak is
The color band coincided with that of human serum albumin by 3R. From this result, the labeling rate of the gallium-67-labeled radiopharmaceutical was approximately 100%, and no difference was observed in its charge state from that of human serum albumin.

In addition, the potassium 67-labeled radiopharmaceutical (0.
2 ml) and add several S, D. The drug was administered intravenously to female rats, and the time-course changes in the concentration in music and the distribution behavior in the body were investigated. The uptake rate at each measurement time immediately after administration was
Shown in the table.

The appearance of distribution in the body was almost the same as that of conventional iodine-131-labeled human serum albumin.

However, it was shown that the decay rate of the concentration in the dish was considerably greater for iodine-131-labeled human serum albumin than for the gallium-67-labeled radiopharmaceutical in this example.

Example 8 Production of technetium-99m-labeled radiopharmaceutical (technetium-99m [labeled human albumin, dialdehyde amylose-3, oxobutyral hiss (N-methane diamine) carboxylic acid hexanediamine condensate conjugate]: A carrier for the preparation of radiopharmaceuticals (human serum albumin) prepared by the method of Example 3)
DAA KTS: hegizanone amine condensate conjugate) (1
00 mg) was dissolved in water (IfiOMρ) in which dissolved oxygen was removed by blowing nitrogen gas, and added to a 1 mM stannous chloride solution (10 ml) and further sodium ascolhinate (10 ml).
0.6 g) was added and completely dissolved. This solution (1,
5 ml) of physiological saline (1.5
ml) to obtain a technetium-99m-labeled radiopharmaceutical (technetium-99m-labeled human serum albumin DAA KTS: hexanediamine condensate conjugate). The technetium-99m-labeled radiopharmaceutical obtained in this example is a very pale pale yellow clear liquid.

Example 9 Gallium 67-labeled radiopharmaceutical (Gallium 67
Production of labeled antimyosin antibody fragment Fab (dialdehyde amylose deferoxamine conjugate): Carrier for radiopharmaceutical preparation produced by the method of Example 4 (antimyosin antibody fragment Fab DAA D
A solution (1 ml) containing 1 mCi of gallium 67 as gallium citrate was added to a solution (approximately 1 mg/ml) (1 ml) of gallium-67 labeled radiopharmaceutical (gallium-67 labeled antimyosin antibody fragment Fab). DAA DFO conjugate) was obtained.

This gallium-67 labeled radiopharmaceutical was subjected to electrophoresis as described in Example 2. Radioactivity was detected as a single peak at a location 1.5 cm on the negative side from the original line,
Moreover, the position of this radioactivity peak coincided with the color band of the anothymionone antibody fragment Fab produced by Pump-3R.

In addition, the above gallium-67 labeled radiopharmaceutical has 0.
The mixture was diluted with 03M phosphate buffer to adjust the antimyosin antibody fragment Fab concentration to 0.05 μg/ml. Next, add this diluted solution (1 ml) to a concentration of 50 μg/
ml of myosin solution (antigen) (0.15 ml) was added and reacted at room temperature for 1.5 hours. Then this reaction solution (
A human fibrinogen solution (0.1 ml) with a concentration of 7.5 mg/ml was added to the mixture (0.3 ml), and after gentle shaking, radioactivity was measured. Furthermore, 18% polyethylene glycol solution (0.3 ml) was added to this solution, stirred for 10 seconds, and then centrifuged at 4000 rpm for 20 minutes at 4°C. After washing the sediment three times, radioactivity was measured. From the ratio of both radioactivities, the binding constant: Ka-2.
5×10°M1 was obtained. Since the binding constant when indium-111 was directly labeled with antimyosin antibody fragment Fab via diethylenetriaminepentaacetic acid was 2×10°M1, it was confirmed that this product retained sufficient physiological activity.

Example 10 Gallium 67-labeled radiopharmaceutical (Gallium 67
Labeled human fibrinogen dialdehyde amylose
Production of deferoxamine conjugate): A solution containing gallium-67 ImCi as gallium citrate (1 ml) was added to the radiopharmaceutical preparation carrier (human fibrinogen DAA DFO conjugate) (1 ml) produced by the method of Example 5. , potassium-67 labeled radiopharmaceutical (gallium-67 labeled human fibrinogen)
DAA DFO conjugate) was obtained.

This gallium-67 labeled radiopharmaceutical was subjected to electrophoresis as described in Example 2. Radioactivity is detected as a single peak at a location 0.5 cm on the negative side from the original line,
Moreover, the position of this radioactivity peak coincided with the color band of human fibrinogen produced by Pump-3R.

In addition, the above gallium-67 labeled radiopharmaceutical has 0.
A 0.1 M sodium diethylbarbinolate hydrochloride buffer (pH 7.3) containing 0.5% calcium chloride was added to adjust the human fibrinogen concentration to 1 mg/ml. Further, 100 units/ml thrombin (0.1 ml) was added to this solution, and the mixture was left in a water bath for 30 minutes. After completely removing the generated fibrin clot,
The coagulation ability of the gallium-67-labeled radioactive drug of this example was measured by counting and measuring the radioactivity in the fibrin clot and the liquid from which the fibrin coagulation had been removed. % clotting ability.

Gore means that dialdehyde starch bound with deferoxamine is used as a polymer compound and has a physiological activity equal to or higher than that of unused human fibrinogen (Japanese Patent Application Laid-Open No. 59-106425). .

Example 11 Gallium-67 labeled radiopharmaceutical (Gallium-67
Production of labeled human serum albumin dialdehyde amylose deferoxamine conjugate: Carrier for radiopharmaceutical preparation produced in Example 2 (human serum albumin
DAA DFO conjugate) was dissolved in distilled water for injection, a solution (1 ml) containing gallium-67ImCi as gallium citrate was added, and the mixture was left at room temperature for 1 hour to obtain a gallium-67-labeled radiopharmaceutical (sample 1). . In addition, human serum albumin and DFO were directly bound by a conventional method, and gallium-67 was bound to the resulting radiopharmaceutical preparation carrier to obtain a gallium-67-labeled radiopharmaceutical (sample 2). The labeling rate of Sample 1 and Sample 2 was measured according to the method described in Example 7. Display the results on a second screen.

From the results in Table 2, the gear of the present invention (sample 1) is as follows:
When using human serum albumin (0.52 mg),
While 100% of lmCi of gallium-67 can be labeled in a practical labeling time of 1 hour, conventional carriers can only be labeled 115% under similar conditions (A', 100% labeling rate). It can be seen that human serum albumin (3.5 mg) is required to obtain .

In addition, after appropriately attenuating the radioactivity of the gallium-67 labeled radiopharmaceutical produced as described above, S, D
, 0 per 100 g body weight for each group of 5 male and female rats.
．． 1 ml (equivalent to 60 times the planned human dose) was administered intravenously, and each animal was observed, but no abnormalities were observed, including the results of examination of each organ after autopsy.

Example 12 Gallium-67 labeled radiopharmaceutical (Gallium-67
Production of labeled human fibrinogen-dialdehyde dextran-deferoxamine conjugate): Adding gallium 672mC as gallium citrate to the radiopharmaceutical preparation carrier (human fibrinogen-DAD-DFO conjugate) produced by the method of Example 6
A solution (2 ml) containing i was added to obtain a gallium-67-labeled radiopharmaceutical (gallium-67-labeled human fibrinogen DAD-DFO conjugate). This solution was pale yellow and transparent and had a pH of 7.8.

This gallium-67 labeled radiopharmaceutical was subjected to electrophoresis as described in Example 2. Radioactivity was detected as a single peak at a location 0.5 cm on the negative side from the original line, and the position of this radioactivity peak coincided with the color band of human fibrinogen produced by Pump 3R. From this result, the labeling rate of the gallium-67-labeled radiopharmaceutical was almost 100%, and no difference was observed in its charge state from that of human fibrinogen.

A 0.1 M sodium diethylbarbinolate hydrochloride buffer (pH 7.3) containing 0.05% calcium chloride was added to the above gallium 67-labeled radiopharmaceutical to adjust the fibrinogen concentration to 1 mg/ml.

Thrombin (100 units/ml) (0.1 ml) was added and left in a water bath for 30 minutes. After completely removing the generated fibrin clot, the clotting ability of the gallium-67 labeled radiopharmaceutical of this example was measured by counting the fibrin clot and the radioactivity in the liquid from which the fibrin clot was removed. 8 for human fibrinogen, which is
It showed a coagulation ability of 4%.

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Claims (1)

[Claims] 1. An amino group-containing bifunctional ligand compound and an amino group-containing physiologically active substance for polysaccharide derivatives having at least three formyl groups in the molecule (excluding dialdehyde starch) is the polysaccharide derivative 1
A methyleneimine bond (CH-N) or a methyleneamine bond (CH
A polymer compound formed by bonding via 2NH). 2. For polysaccharide derivatives having at least three polmyl groups in the molecule (excluding dialdehyde starch), an amino group-containing bifunctional ligand compound and an amino group-containing physiologically active substance are added to the polysaccharide derivative 1.
A methyleneimine bond (-CH=N-) or a methyleneamine bond (
A labeled polymer compound in which radioactive metal nosine is bonded to a polymer compound formed by bonding via CH2NH-) via a chelate bond.