JPS6353201B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a polymer compound having at least one free formyl group, particularly a polymer compound having at least one free formyl group, which is formed by bonding a formyl group-containing polysaccharide derivative and an amino group-containing bifunctional ligand compound. Concerning molecular compounds. 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 dynamic diseases, dynamic examination of physiologically active substances, and treatment of diseases. It is possible to provide a radioactive drug labeled with a metal element. Physiologically active substances labeled with iodine-131 have conventionally been widely used for non-invasive nuclear medicine diagnosis for the purpose of depicting specific organs, detecting specific diseases, and testing dynamics. 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, iodine-131 has a long half-life of about 8 days, and emits beta rays in addition to gamma rays, which are useful for nuclear medicine diagnosis, so it has the drawback of subjecting the subject to a large amount of radiation exposure. 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-99m-labeled human serum albumin, indium-111-labeled bleomycin, and the like are known to be obtained by a labeling method in which a radioactive metal salt is directly applied to a physiologically active substance. Furthermore, the strong chelate-forming ability of difunctional ligand compounds such as diethylenetriaminepentaacetic acid (DTPA), 3-oxobutyral bis(N-methylthiosemicarbazone)carboxylic acid, and deferoxamine for various metals, and the amino groups at the terminals of these compounds. Based on the reactivity of the carboxyl group to various physiologically active substances, methods for bonding radioactive metals and physiologically active substances via these bifunctional ligand compounds have also been proposed. The labeled compounds obtained by these methods are relatively stable and retain the activity of physiologically active substances, so they are very interesting drugs for nuclear medicine diagnostic purposes. However, the radioactive diagnostic agents obtained by these methods use bioactive substances with large molecular weights, such as fibrinogen, which has a molecular weight of about 340,000, and IgG, which has a molecular weight of about 160,000, which are used for thrombosis diagnosis and cancer diagnosis, respectively. In this case, the disadvantage is that it cannot obtain the high specific radioactivity required for diagnosis. As a result of various studies to solve this drawback, the present inventors first developed a polymer compound in which an amino group-containing bifunctional ligand compound and an amino group-containing physiologically active substance were bonded to dialdehyde starch. They were particularly successful (see Japanese Patent Application Laid-open Nos. 105002/1982 and 106425/1987). This polymer compound has a large number of ligands per molecule, which means that the number of radioactive metal ions bound per molecule is greater than that of conventional bifunctional ligand compounds. This means that there are significantly more. 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. As a result of further research based on the above findings,
It has been discovered that even when other formyl group-containing polysaccharide derivatives are used in place of dialdehyde starch, radiopharmaceuticals with high specific radioactivity can be obtained without causing denaturation or activity reduction of physiologically active substances. Ta. In general, dialdehyde starch has a wide molecular weight distribution and a network structure, so the number of repeating structures tends to make it difficult for bifunctional ligand compounds to bind effectively. If a compound having a narrow distribution and a linear structure is used, a large number of bifunctional ligand compounds can be effectively bonded. The present invention was completed based on the above findings, and its gist is that polysaccharide derivatives (excluding dialdehyde starch) having at least three formyl groups in the molecule () and amino groups The containing bifunctional ligand compound () is bonded via a methyleneimino bond (-CH=N-) or a methyleneamine bond (-CH ₂ NH-) at a ratio of at least 2 molecules of the latter per 1 molecule of the former. Polymeric compounds () having at least one free formyl group, especially (a) repeating units 2-
1000 dialdehyde amylose or dialdehyde dextran and (b) deferoxamine or 3-
The oxobutyral bis(N-methylthiosemicarbazone) carboxylic acid/diamine condensate forms a methyleneimine bond (-CH=N-) or a methyleneamine bond (-CH ₂ NH-) at a ratio of at least two molecules of the latter per one molecule of the former. It consists of a polymeric compound having at least one free formyl group bonded through. The above-mentioned polymer compound ( ) which is the object of the present invention is composed of a polysaccharide derivative ( ) and a bifunctional ligand compound ( ) bonded together. It is necessary for the polysaccharide derivative () to have at least three formyl groups in the molecule, and the larger the number of formyl groups, the more preferable. At least two of these formyl groups are useful for bonding with the difunctional ligand compound (), and at least one other remains free in the polymer compound () and is later added to the polymer compound (). ) to bind physiologically active substances (). Polysaccharide derivatives () are obtained, for example, by treating polysaccharide, which may be appropriately substituted, with an oxidizing agent (e.g., sodium periodate), in principle one or two per saccharide unit. Those having a formyl group can be used. The polysaccharide may be oricosaccharite, but as understood from the purpose of the present invention, pentosan, hexo-
Preferred are higher polysaccharides such as san, polyglucosamine, polyuronic acid, glycosaminoglycan, glycouronoglycan, and heterohexosane. Specific examples include amylose, amylopectin, dextran, cellulose, inulin, pectic acid, pullulan, etc., and mixtures and dehydrated condensates thereof may also be used. Generally, those having saccharide units of 3000 or less, particularly 1000 or less are desirable. The difunctional ligand compound () has an amino group that forms a strong chelate bond with the radioactive metal element () and can react with the formyl group of the polysaccharide derivative () under relatively mild conditions. What you have is used. Specific examples of such bifunctional ligand compounds () include deferoxamine (Merck Index, 9th edition, p. 374 (1976)), formula: (In the formula, R ¹ and R ² each represent hydrogen, C ₁ - C ₃ alkyl or phenyl.) Patent application No. 54920), formula: (In the formula, R ³ and R ⁴ are each hydrogen or C ₁ ~
C ₃ alkyl, n represents an integer of 0 to 3; ) 1-(p-aminoalkyl)phenylpropane-1,2-dione-bis(thiosemicarbazone) derivatives (Australia Patent No. 533722). Even if the substance itself does not have an amino group, if it has a group or structure that can easily form an amino group or an amino group-containing structure, as long as it has the property of capturing radioactive metal elements, this may also be used. Furthermore, after forming an amino group or an amino group-containing structure, it can be used as a bifunctional ligand compound (). For example, an amino group can be easily introduced into a substance having a carboxyl group by reacting it with an alkylene diamine.
In the present invention, it can be used as one type of difunctional ligand compound (). Specific examples of such difunctional ligand compounds () include diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), and dimercaptoacetylethylenediamine (Fritzberg).
et al.: Journal of Nuclear Medicine (J. Nucl. Med., 23 , 917 (1982)) and bisaminoethanethiol (Fritzberg et al.: J. Nucl.
Med., 25 , 916 (1984)), _Cyclan (Keiring _et al.):
Journal of Nuclear Medicine (J.
Nucl.Med.), 23 , 917 (1982)) _.
Ligand, N,N′-bis(2-hydroxyethyl)ethylenediamine (Wagner Jr. et al.: Proceedings of
The International Symposium on
Technetium in Chemistry and Nuclear Medicine, Padova, Italy (Proceedings of the International Symposium)
on Technetium in Chemistry and Nuclear
Medicine, Padova, Italy.) p. 161 (1982)), the N ₂ O ₂ ligand, formula: (In the formula, R ⁵ , R ⁶ , R ⁷ , R ⁸ and R ⁹ are hydrogen or
Examples include 2-propionaldehyde-bis(thiosemicarbazone) derivatives (US Pat. No. 4,287,362) represented by (C ₁ -C ₃ alkyl). To produce the polymer compound (), 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; If necessary, this methyleneimine bond may be reduced to convert it into a methyleneamine bond. The above condensation reaction may be carried out by any conventional means employed to condense a formyl group and an amino group, and the above reduction reaction may also be carried out by any conventional means employed to convert a methyleneimine bond into a methyleneamine bond. For example, by using metal hydrides such as sodium borohydride. During the above reduction reaction, a compound in which the formyl group in the saccharide 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 the polysaccharide derivative () varies depending on the reaction reagents, reaction conditions, etc., but is generally 5 or more, preferably 10 or more. However, at least one formyl group in the polysaccharide derivative () portion of the polymer compound () obtained by this reaction should remain free for bonding with the physiologically active substance (). Taking as an example the case where an amylose-derived polysaccharide derivative () is used, the structure of the polymer compound () is shown as a formula as follows: (In the formula, X is the residue obtained by removing the amino group from the bifunctional ligand compound (), Y is -CHO or -
CH ₂ OH, P represents an integer of 2 to 1000, and q, r, s and t each represent an integer of 0 to 1000. however,
q+r+s+t is an integer from 2 to 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 itself. The number of saccharide units is usually 2 to 1000, particularly preferably 2 to 500. The polymer compound () thus produced may be purified, if necessary, by a conventional purification method applied to polymer substances, such as column chromatography, gel filtration, or dialysis. Since the polymer compound () of the present invention has at least one free formyl group in its molecule, it binds to a physiologically active substance () having an amino group via the formyl group, and becomes a carrier for preparing radiopharmaceuticals. can be provided. The term "biologically active substance" as used herein refers to a substance that accumulates in an appropriate organ or tissue or a specific lesion, or exhibits a unique behavior in response to a specific physiological state, and the behavior of such a substance in a living body. By tracking this, it is possible to obtain diagnostically useful information. Generally, many physiologically active substances themselves have an amino group, but not only such physiologically active substances but also substances that do not themselves have an amino group 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. Since the reaction between a formyl group and an amino group proceeds under relatively mild conditions, it can be advantageously utilized in the present invention. Specific examples of physiologically active substances include blood proteins (e.g. human serum albumin, fibrinogen), enzymes (e.g. urokinase, streptokinase), hormones (e.g. thyroid stimulating hormone, parathyroid hormone), immune antibodies (e.g. IgG and its F(ab) ₂ ′ of the fragment, Fab′,
Fab), monoclonal antibodies, antibiotics (for example, bleomycin, kanamycin), saccharides, fatty acids, amino acids, etc. In order to bind a physiologically active substance ( ) to the polymer compound ( ) of the present invention, for example, the polymer compound ( ) and the physiologically active substance ( ) are condensed to form a bond between the formyl group of the former and the amino group of the latter. A methyleneimine bond may be formed, and if necessary, this methyleneimine bond may be reduced to convert it into a methyleneamine bond. Both the condensation reaction and the reduction reaction may be carried out by conventional methods. The number of molecules of the physiologically active substance introduced per molecule of the polymer compound () varies depending on the reaction reagent, reaction conditions, etc., but is usually 10 or less, preferably 3 or less. The conjugate of the polymer compound () and the physiologically active substance () produced here is subjected to column chromatography, gel filtration, or gel filtration, where the polymer is applied as necessary.
It may be purified by conventional purification methods such as dialysis. This polymer compound () - physiologically active substance ()
The conjugates are useful as carriers for the preparation of radiopharmaceuticals, as described above. That is, in the conjugate, a plurality of bifunctional ligand compounds () are present in the polymer compound (), which makes it possible to capture a plurality of radioactive metal elements (). A conjugate that captures a plurality of radioactive metal elements () can itself be used as a radiopharmaceutical. Here, the radioactive metal element () is a metal element that has radioactivity, has physical or chemical properties suitable for nuclear medicine diagnosis and treatment, and has a difunctional ligand compound (). Those that can be easily captured by the atomic structure are used. Specific examples include gallium-67, gallium-68, thallium-201, indium-111, and technetium-99m.
These are usually used in the form of salts, especially water-soluble salts, and are labeled by contacting them with the polymer compound ()-physiologically active substance () conjugate in an aqueous medium. However, if the radioactive metal element () is in a valence state that allows it to form a stable chelate complex (e.g., potassium-67, indium-111), there is no need for other reagents to be present in the reaction system; If it is necessary to change the valence state to form a specific chelate complex (eg, technetium-99m), it may be necessary to have a reducing or oxidizing agent present in the reaction system. As the reducing agent, divalent tin salts (for example, tin halides, tin sulfate, tin nitrate, tin acetate,
tin citrate). For example, when using technetium-99m as the radioactive metal element (), the polymer compound ()-bioactive substance () conjugate is prepared in the form of pertechnetate in the presence of a stannous salt as a reducing agent in an aqueous medium. Technetium-99m labeled polymeric compound () by treatment with technetium-99m
- Biologically active substance () conjugates can be prepared. In the above preparation, there are no particular restrictions on the order in which the reagents are mixed, but it is usually 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 pertechnetate. In order for the thus obtained labeled polymer compound ()-physiologically active substance () conjugate to be useful as a radiodiagnostic agent, it must have sufficient radioactivity and radioactivity concentration to enable diagnosis. is necessary. For example, when technetium-99m is used as the radioactive metal element, it is desirable to have a radioactivity concentration of 0.1 to 50 mCi per approximately 0.5 to 5.0 ml upon administration. Although such a labeled polymer compound ()-physiologically active substance () conjugate may be administered immediately after preparation, it is desirable that it has sufficient stability to withstand storage for an appropriate period of time after preparation. Furthermore, the labeled polymer compound ()-physiologically active substance () conjugate may be supplemented with PH regulators (e.g., acids, bases, buffers), stabilizers (e.g., ascorbic acid), and isotonic agents (e.g., chloride), if necessary. Sodium) etc. may be added. An example of a radiopharmaceutical obtained using the polymer compound of the present invention () is dialdehyde amylose as a polysaccharide derivative (), deferoxacin as a bifunctional ligand compound (), human serum albumin as a physiologically active substance (), The case where gallium-67 is used as the radioactive metal element (2) will be explained as follows. First, a dialdehyde amylose-deferoxamine conjugate is prepared by condensing dialdehyde amylose with deferoxamine and then reducing it as necessary. After condensing human serum albumin with this conjugate, reducing it as necessary. By doing so, a human serum albumin-dialdehyde amylose-deferoxamine conjugate is obtained. By contacting this conjugate with an aqueous solution containing gallium-67 in the form of trivalent gallium ions, stable and highly radioactive gallium-67-labeled human serum albumin.
A dialdehyde amylose-deferoxamine conjugate is obtained. The electrophoretic behavior of this labeled conjugate is exactly the same as that of human serum albumin. In addition, the distribution of this labeled conjugate in rats was different from that of conventional iodine.
Almost the same as 131-labeled human serum albumin.
The half-life of the blood concentration is approximately 30 hours, and the half-life of the blood concentration of iodine-131-labeled hyde serum albumin (5.7
time). That is, it can be understood that the labeled conjugate according to the present invention is useful for depicting cardiac pool images. The human serum albumin-dialdehydroamylose-deferoxamine conjugate according to the present invention (Sample 1) and the product in which human serum albumin and deferoxane were directly bound by the conventional method (Sample 2)
When we compared the labeling ability for 1 mCi of gallium-67 with the following results, we obtained the following results.

[Table] As shown in Table 1, the carrier prepared according to the present invention (Sample 1) has a human serum albumin content of 0.52
When using mg, 100% of 1 mCi of gallium-67 can be labeled in 1 hour, which is a practical labeling time, whereas conventional non-radioactive carriers can only label 15% under similar conditions. , 3.5 mg is required to obtain 100% labeling rate. As is clear from the above results, by using the polymer compound () of the present invention, it is possible to prepare a radiopharmaceutical with high specific radioactivity. EXAMPLES The present invention will be explained in more detail with reference to Examples below. Example 1 Production of dialdehyde amylose-deferoxamine conjugate (condensate): - Dialdehyde amylose (hereinafter abbreviated as DAA) (10 mg) having an average molecular weight of 2900 was dissolved in water (0.4 ml). This solution will be referred to as Solution A. Separately, add deferoxamine (hereinafter abbreviated as DFO) (34 mg) to water (0.6 ml).
and an equimolar amount (5.5 mg) of triethylamine was added to this solution. This solution will be referred to as Solution B.
After stirring Solution B at room temperature for 10 minutes, it was added to Solution A and stirred at room temperature for 30 minutes. In order to purify the produced DAA-DFO conjugate, the following gel chromatography was performed. Carrier: Cephadex G-50 Solvent: 0.03M phosphate buffer, PH7.0 Column size: 2.2 cm in diameter, 30 cm in height Flow rate: 47 ml/hr The DAA-DFO conjugate was eluted in 70 to 100 ml.
Unreacted DFO was eluted in 110-130 ml. DAA−
The target polymer compound can be obtained by freeze-drying 70 to 100 ml of the eluate containing the DFO conjugate. When this polymer compound was analyzed by high performance liquid chromatography under the following conditions, the retained volume was 24.7 ml. Note that free DFO was not detected (the retention volume of DFO in this system was 29.2 ml). Column: TSK-3000SW Solvent: 0.2M phosphate buffer, PH7.0 Pressure: 380psi Flow rate: 0.75ml/min Absorption wavelength: 280nm Example 2 Production of dialdehyde amylose-deferoxamine conjugate (condensation reductant) :- DAA (10 mg) with average molecular weight 2900 in water (0.4 ml)
dissolved in This solution will be referred to as Solution A. Separately DFO
(38 mg) was dissolved in water (0.6 ml) and an equimolar amount (5.5 mg) of triethylamine was added to this solution. This solution will be referred to as Solution B. After stirring Solution B at room temperature for 10 minutes, it was added to Solution A and stirred at room temperature for 30 minutes. Sodium borohydride (2.4 mg) was added to this, and the mixture was reduced with stirring at room temperature for about 1 hour. generated
In order to purify the DAA-DFO conjugate, the following gel chromatography was performed. Carrier: Cephadex G-50 Solvent: 0.03M phosphate buffer, PH7.0 Column size: 2.2 cm in diameter, 30 cm in height Flow rate: 47 ml/hr The DAA-DFO conjugate was eluted in 75 to 100 ml.
Unreacted DFO was eluted in 110-130 ml. DAA
The target polymer compound can be obtained by freeze-drying 75 to 100 ml of the eluate containing the -DFO conjugate. When this polymer compound was analyzed by high performance liquid chromatography under the following conditions, the retained volume was 25.5 ml. Note that free DFO was not detected (the retention volume of DFO in this system was 30.4
ml). Column: TSK-3000SW Solvent: 0.2M phosphate buffer, PH7.0 Pressure: 380psi Flow rate: 0.75ml/min Absorption wavelength: 280nm Fe() and DFO form a 1:1 complex, and the absorption wavelength is 420nm.
It has maximum absorption in . 420n of Fe()−DFC complex
Emax at m was 2.63×10 ³ . Dissolve known amounts of the above polymer compounds in water, DFO and Fe()
Sufficient ferric chloride solution was added to form a 1:1 complex, and after standing for 1 hour, the absorbance at 420 nm was measured. As a result, it was confirmed that 11.5 DFOs were bound per DAA molecule in the above polymer compound. Therefore, the average molecular weight of the above-mentioned polymer compound is calculated to be about 10,000. Example 3 Production of conjugate (condensation reductant) of dialdehyde amylose-3-oxobutyral bis(N-methylthiosemicarbazone) carboxylic acid amine derivative: -3-oxobutyral bis(N-methylthiosemicarbazone)carvone Acid (hereinafter abbreviated as KTS) (132 mg) was dissolved in anhydrous dioxane (5 ml), and after cooling to around 10°C, tri-n-butylamine (0.12 ml) and isobutyl chloroformate (64 μ) were added. The mixture was stirred at the same temperature for about 50 minutes to obtain a mixed acid anhydride. Separately, N-tert-butyloxycarbonyl-1,
A solution of 6-hexanediamine (104 mg) dissolved in anhydrous dioxane (2 ml) was prepared, and this solution was added to the mixed acid anhydride solution and stirred at around 10°C for about 15 hours. A carbonyl-1,6-hexanediamine condensate was obtained. Concentrated hydrochloric acid (1 to 2 drops) was added to this condensate solution to lower the pH to 2, thereby removing the N-tert-butyloxycarbonyl group to obtain a solution of KTS-hexanediamine condensate. After adding this to a solution of DAA (83 mg) in dimethyl sulfoxide (5 ml), sodium borohydride (17.2 mg) was added, and the mixture was reacted at room temperature for about 3 hours.
A solution of KTS conjugate was obtained. The above solution was placed in an ordinary dialysis tube and dialyzed for 30 hours by a conventional method to remove unreacted reagents, and then freeze-dried to obtain the desired polymer compound. The maximum absorption of hexanediamine-KTS conjugate is
It was confirmed that it exists at a wavelength of 334 nm and its Emax is 4.37×10 ⁴ . The above polymer compound was dissolved in water to a concentration of 3 mg/ml, and water was used as a control to give a concentration of 334n.
Absorbance was measured at m. As a result, it was confirmed that 10.5 KTS were bound to each DAA molecule. Therefore, the average molecular weight of the above polymer compound is approximately
It is calculated as 7000. Example 4 Preparation of dialdehyde dextran-deferoxamine conjugate (condensation reductant): - Dissolve DFO (2.8 g) in water (30 ml), add equimolar amount (432 mg) of triethylamine to this solution,
Stirred at room temperature for 10 minutes. This was added to a solution of dialdehyde dextran (hereinafter abbreviated as DAD) (1 g; formyl group content 5.1 μmol/mg) in water (40 ml) and stirred for 15 minutes at room temperature. Sodium borohydride (167 mg) was added to this, and the mixture was reduced with stirring at room temperature for about 1 hour. In order to purify the produced DAD-DFO conjugate, it was introduced into a dialysis tube, dialyzed against water for 3 days, and then subjected to gel chromatography as described below. Support: Cephadex G-50 Solvent: Water Column size: Diameter 4.5 cm, height 50 cm Flow rate: 2.5 ml/min The DAD-DFO conjugate was eluted in 300 to 450 ml, and unreacted DFO was eluted in 550 to 600 ml. Eluted.
The target polymer compound can be obtained by freeze-drying the eluate containing the DAO-DFO conjugate. When this polymer compound is analyzed by high performance liquid chromatography under the following conditions, the retention volume is
It was 27.3ml. 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 Sodium Chloride-
Hydrochloric acid buffer, PH7.4 Pressure: 100Kg/cm ² Flow rate: 1.0ml/min Absorption wavelength: 280nm The above examples are intended to illustrate the invention and do not limit its scope in any way. It's not something.

Claims (1)

[Claims] 1 (a) dialdehyde amylose or dialdehyde dextran with 2 to 1000 repeating units and (b) deferoxamine or 3-oxobutyral bis(N-methylthiosemicarbazone) carboxylic acid.
The diamine condensate has a methyleneimine bond (-
CH=N-) or methyleneamine bond (-
at least one bonded via CH ₂ NH−)
A polymer compound with 1 free formyl groups.