JPH03505726A - Method for preparing metal-radioisotope-labeled protein - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Method for preparing metal-radioisotope-labeled proteins The present invention is suitable for use in diagnostic or therapeutic purposes. metal-radioisotope-labeled protein or protein system used for Concerning methods for preparing substances. The invention also relates to novel compounds for use in this method.

Radioisotope-labeled compounds are used for diagnostic tests, such as the morphology and function of internal organs. It is used to investigate whether there are abnormalities or lesions in the body. For this purpose, A composition containing a radioactive substance is administered to a patient, for example in the form of an injection solution. T-line power Using a suitable detector such as a camera, an image of the organ in which the radioactive material has been incorporated is obtained. or lesions are created by recording or scanning radioactivity.

Radioactively labeled biological macromolecules, especially proteins and protein-based substances, e.g. blood cells, immunoglobulins, glycopeptides, monoclonal antibodies such as antimyosin or monoclonal antibodies against tumor antigens, other fluorescent antibodies suitable for localization purposes, e.g. or double-chain urokinase, tissue plasminogen activator, plasmin, etc. Other plasmin derivatives, such as miniplasmin, are promising substances for diagnostic use. Ru. Some proteins have broad organ-specific targets and are Reacts with strong selectivity with biopolymers present in the body after being administered to Can be done. A good example of such a reaction is between an antigen and an antibody in the body or an antibody flag. selective reaction with ment.

Various metal-radioactive isotopes can be used with tumor-selective biopolymers such as glycopeptides. Combined with mycin, it can be used successfully to suppress tumors and is useful in radiotherapy. It becomes a powerful tool.

These polymers deliver the necessary amount of radiation to the tumor, i.e. metal- It serves as a carrier for transporting radioactive isotopes.

Disadvantages of direct labeling of proteins or protein-based substances with metal-radioisotopes There are two points. First of all, the The biologically active sites of proteins are easily blocked by this reaction and biopolymers The normal functioning of the body is disrupted. In addition, the relationship between radioactive isotopes and polymers Affinity is often insufficient, and even if a binding is formed, it is limited by physiological conditions. It has low stability under conditions and tends to lose its integrity, so even if administered, it cannot be used for diagnostic purposes. (the protein's action in the body can no longer be tracked), and even therapeutic use. (If the radiation dose is not delivered to the target area and is returned to the target area, it becomes useless) (This would put an undesirable radiation burden on other parts of the body.)

In order to alleviate this disadvantage, European Patent Application No. 237150 In this method, a protein substance having disulfide bonds is first treated with a disulfide reducing agent such as disulfide. treatment with thiothreitol and then the reduced state in which the mercapto group is liberated. Protein-based substances and radioactive isotopes such as Tc-99m-tartrate or Tc-9 A method of specifically reacting with 9m-glucoheptonate has been proposed. this The disadvantage of the method is that the protein requires cleavage of the disulfide bond to obtain the desired mercapto group. The point is that a reduction process is performed that "cleaves" the quality. Damage to protein molecules occurs. It becomes easier to

A biopolymer ( In recent years, many reports have been published regarding proteins (usually proteins or protein-based substances). . Recent patents in this field include U.S. Pat. No. 511550, No. 4652440 and No. 4678667, European Special Patent Application No. 83129, No. 173629 and No. 188256, Netherlands Patent Application No. Patent Application No. 8204108, PCT Question 85103231 and WO3 There is 6103010.

Even with such modification methods, the biological operation of the original polymer is preserved as much as possible. Of course, it is indispensable. This means that metal-radioactive isotopes interact with proteins. The chelator or coupling agent used for binding is not very bulky. This should not be the case, especially for relatively low-molecular proteins. It means that. In addition, proteins that are often highly sensitive or Protein-based substances can be used during the labeling process, especially when combined with chelators or coupling agents. In the chemical reaction, damage conditions that adversely affect the properties of the polymer should be avoided as much as possible. must be kept from being exposed. Long incubation, high temperature treatment exposure to organic solvents or pH conditions significantly different from physiological pH, oxidizing agents or reducing All reactions in the presence of base agents must be avoided as much as possible. . As mentioned above, one type of coupling agent is to combine proteins or protein-based substances with strong It must be able to bind firmly, and it must also be able to bind firmly to metals and radioactive isotopes. No.

If the binding is not fully maintained under physiological conditions, i.e. the radioactive isotope When it disassociates in the flow and is transported by other blood particles to non-target parts of the body, Radioactive substances impose an undesirable radiation load on the tissues of the site, and the amount Even therapeutically effective doses cause severe tissue damage.

The storage stability of the labeled polymer is low, and the half-life of the metal-radioactive isotope is also short. Label proteins or proteins that can be used immediately by the user. In such cases, where it is often impossible to keep quality materials on hand, If the user performs labeling with radioisotopes in the clinic or clinic laboratory, This is why the kit is called a “kit”. In this way, various reaction components are provided to the user. The user can use the key provided Radioactively labeled proteins or protein-based substances can be obtained from These operations eliminate the need for tedious separation and purification steps. It goes without saying that it must be as simple as possible. Label effect rate or label (label rate also plays an important role. Loss of expensive materials) In addition, unconverted feedstock may be removed from the product if the label is incomplete. Users usually carefully dispose of radioactive products under sterile conditions, as they must be thoroughly removed. must be refined into

The chelators or coupling agents described in the above-mentioned patent documents meet the above requirements. There is still much to be desired. For example, US Patent No. 447 No. 9930, No. 4511550, No. 4678667 and European Patent Publication No. The bulk of the chelator used in Application No. 188256 is relatively large.

US Patent No. 4,652,440, European Patent Application No. 173,629, PC In the manner described in T-Applications-085103231 and 11o86103010 is used in the coupling reaction process for coupling a chelator to a protein. conditions are used that damage the protein. US Patent No. 4479 No. 930 and PCT Application No. 085103231. The strength of the bond between the protein and the metal-radioactive isotope is not sufficient. How to label The method is often laborious and requires subsequent purification of the labeled protein. (U.S. Patent No. 4,652,440, European Application No. 83129, Patent Application No. 188256, Netherlands Patent Application No. 8204108, PCT Issue @ WO36103010 shows this). Due to incomplete labeling Additional purification steps may be required (U.S. Patent No. 4479). No. 930, U.S. Patent No. 4,652,440, PCT Issue @ 8,510,3231 and and PCT entry 1!186103010 indicate this).

The object of the present invention is to produce metal-radioisotope-labeled proteins for use in diagnosis or therapy. or to provide a method for preparing protein-based substances; Alternatively, a coupling agent that couples a radioactive isotope to a protein-based substance and a protein A protein complex is created by reacting with a substance or a protein-based substance, and then this complex is The content involves reacting the body with radioactive isotopes to form a radioactive isotope complex. However, in this method, the preparation method described in the above-mentioned patent document does not These disadvantages may not occur at all, or may be of much lesser magnitude. It is something.

The object is to provide at least substantially the following general formula (1) in its preparation: % formula % (1) X is a hydrogen atom or a suitable protecting group, R is an optionally substituted branch having 1 to 10 carbon atoms, preferably 1 to 4 carbon atoms. or unbranched hydrocarbon radicals (more preferably one or more alkyl standards which may have a terror atom), Y is at least a terminal reactive group capable of reacting with a functional group of a protein or proteinaceous substance. ] a thio compound represented by, or having 5 to 7 ring atoms, having the formula (I) (where X and Y together represent a protein or Cyclic condensation of thio compounds (forming a reactive group that reacts with the functional group of a substance) using a coupling agent consisting of the product or a water-soluble salt of this condensation product. This is achieved by

Examples of suitable protecting groups X for mercapto groups include acetyl, trifluoro Cetyl group, hydroxyacetyl group, carboxyacetyl group, acetamidomethyl group, benzoyl group.

Protects benzyl groups, benzoylaminomethyl groups, and other sulfur atoms from being oxidized Examples include groups suitable for

In the method of the invention, in a radiolabeled amount, i.e. a diagnostic or therapeutic amount. Examples of metal-radioisotopes suitable for use include Tc-99m, Re-1 86, Re-188°Cu-67, Pb-203, Pb-212, Ga-67, Ga-68, B1-212. As-72, As-77, In-111, In-1 13,RL! -97, Y-90, Ag 111°Pd-109, Sm153. "y'b-175, Lu-177° and Gd-159. These Among radioactive isotopes, Tc-99m, Pb-203, Ga-67, Ga-68°A s-72, In-111, In-113 and Ru-97 are not used for diagnostic purposes. It will be done. Radioisotopes other than these are particularly useful in therapeutically active compositions.

In the complex-forming reaction, the desired radioactive isotope is in salt form, preferably compared to Weak chelators - e.g. pyrophosphate.

Phosphonates, or polyphosphonates, oxynates, carboxylates, Droxycarboxylate, aminocarboxylate, enolate or the like in the form of a chelate bound to a mixture of Added. In the latter case, the desired complex is such that the sulfur atom of the thio compound is metal - Formed by the principle of ligand exchange to form a strong chelate bond with radioactive isotopes. Ru.

Derivatization of proteins or protein-based substances, i.e., reaction with coupling agents, is a widespread process. It can be carried out under suitable conditions. These conditions apply to the selected coupling agent and label pair. The amount may be changed as appropriate depending on the type of protein or fluorescent substance to be detected. reaction medium The pH of is preferably between 6.5 and 9, more preferably between 7 and 8.5. stomach Incubation temperature has a negative effect on the physical structure of proteins or proteinaceous materials. It is fine as long as it does not exceed the temperature. The preferred temperature is room temperature. time of incubation The time ranges from a few minutes to a few hours, but the preferred time is 20 minutes to 2 hours. .

Coupling agent and protein or pallor substance, each polypeptide molecule A complex consisting of one or more coupling molecules is formed. Polypepti The larger the molecule, the more potentially reactive sites it has, so it is possible to bind to it. The number of coupled molecules also increases. For example, immunoglobulin The preferred ratio is 5 moles of coupling agent to 1 mole of protein; For Bumin, the preferred ratio is 2.5 to 1. For larger proteins, The ratio of coupling agent typically ranges from about 0.5 to about 20 moles of coupling agent per mole of protein. . However, in the case of small proteins or protein-based substances, the Aggregation of peptide chains is achieved by contacting the coupling agent at a low ratio. ton). For example, this ratio for somatostatin is 0 ．． 1 to 1 is preferable. For small proteins or protein fragments, About 0.01 mole to about 2 moles of coupling agent per mole of white matter or proteinaceous material. Let's do it.

A preferred coupling agent in the method of the present invention has the following general formula (Ir)Y'-R'- -3' -X (II 'J (here X is as specified before, and 2 R' is an optionally substituted branched or unbranched group having 1 to 10 carbon atoms. Hydrocarbon radical (one or more heteroatoms selected from 0, N and S) and/or may have one or more NH groups), Y' is an isocyanate atom group, formyl group, orthohalonitrophenyl group, diazonium group, isothi Oceanate group.

Epoxy group, trichloro-3-)riazinyl group, ethyleneimino group, chlorosulfate Honyl group, alkoxycarbimidoyl group, substituted or unsubstituted alkylcarbonyl group carbonyl group, substituted or unsubstituted alkylcarbonylimidazolyl group , or sulfonated or non-sulfonated N-hydroxy-succinimide ester A thio compound represented by a group (succinimidoxycarponyl group), 5 or or a formula (■) having 5 to 7 ring atoms (where X and Y together represent the following group; Bonyl group, carbimidoyl group, N-alkylcarbimidoyl group, N-hydroxy N-alkoxy-rubimidoyl group or N-alkoxy-rubimidoyl group) or a water-soluble salt of this condensation product.

When R' is a substituted hydrocarbon radical, the substituent may include a metal- Can chelate with radioisotopes, metal-polymerization for radioisotopes Select from groups that form redentate ligands (polydentate ligands). These groups Examples of protected or unprotected mercapto groups, carxyl groups, hydroxyl groups, etc. There are syl groups, amino groups, etc.

In the above definition, the alkyl group preferably has 1 to 4 carbon atoms. Yes.

Water-soluble salts include salts with various acids, such as hydrochloric acid, sulfuric acid, phosphoric acid, perchloric acid, organic Examples include salts with acids (for example, citric acid and tartaric acid).

As a cyclic coupling agent, the following general formula (I[[)X is as defined above, R# is unsubstituted or branched or unsubstituted with one or more XS groups. A branched alkyl group (1 to 10 carbon atoms), R'' represents an alkyl group having 1 to 4 carbon atoms], or Its water-soluble salts are suitable.

Preferred examples of the compound represented by the formula (I[[) are It is.

Particularly preferred are the following general formula (■): Rz, Rz', R3, R:l', R 4, R-', R5 and R5' are the same or each independently hydrogen Atom, aalkyl5. xs-alkyl group, alkylthioalkyl group.

Alkylcarbonylthioalkyl group, aminoalkyl group.

N-alkylaminoalkyl group, N-alkylcarbonyl-aminoalkyl group or or N, -N-dialkylaminoalkyl group (X has the same meaning as above, The number of carbon atoms in the kill group is 1 to 4), n is 0 or 1, R6 is a hydrogen atom or an alkyl group (1 to 6 carbon atoms) Examples include novel compounds or water-soluble salts thereof.

Its stability, ease of handling, selectivity for protein free NH groups, coupling In view of the fact that the reaction proceeds easily without producing undesirable by-products, The following general formula (V) R1 and n are as defined above, P is 0 or 1, q is O or 1, Z is a hydrogen atom, C=Ca alkyl group, mercapto group, C8-04 alkylthio group , or c, -CS alkanoylthio group], or a water-soluble salt thereof is preferred.

A protein complex (conj uga te) formed using this coupling agent ) is suitable for complex formation with metal-radioisotopes, especially Tc-99m. Ru. All other than unsubstituted 2-iminothiolane and 2-iminothiacyclohexane Examples of 2-iminothio compounds include alkyl-substituted 2-iminothiolanes. (e.g. 2-N-methyliminothiolane, 4-methyl-2-iminothiolane, 2-N-methyliminothiolane, -N-tert-butyliminothiolane and 4,4-dimethyl-2-iminothi orane), and iminothiolane with thio or amino functionality (e.g. 4- Mercaptomethyl-2-iminothiolane, 4-acetylthiomethyl-2-imino Thiolane, 4-ethylthiomethyl-2-iminothiolane and 4-methylamino methyl-2-iminothiolane).

As is clear from the examples, 4-acetylthiomethyl-2- When iminothiolane is used, protein can be obtained with 100% yield by technetium-99m. is labeled.

In 1978, Jue et al. used 2-iminothiolane to modify proteins (o+o (BiochemistryVo) l, 1? , No. 25.1978.5399-5406).

Perham and Thomas further mercurylated proteins modified in a similar manner. reported that the structure of the protein was elucidated by reacting it with a compound (J, Mo1. B iol, (1971) 62.415-418). Proteins modified using this method Preparation of metal-radioisotope labeled proteins using Until now, there have been no reports on the excellent biological properties of labeled proteins. It was.

Used as a coupling agent in the preparation of labeled proteins or protein-based substances Substituted 2-iminothiolane and 2-iminothiacyclohexane are new compounds. Ru. Therefore, the present invention is based on the above general formula (■) (each symbol has the above meaning, but all All substituents, Rz, Rz', Rs, Rx', Ra, R4', Rs.

(excluding cases where R, r and R6 are hydrogen) be.

More specifically, these new compounds have the general formula (V) above (each symbol represents the above formula (V)). It has significance, but when P and q are both 0, R6 is a C, -C, alkyl group ).

The present invention uses the above-mentioned novel compound as a coupling agent to produce proteins or fluorescent proteins. It also relates to a protein complex obtained by reacting with a system substance.

The present invention further provides metal-radioisotope-labeled proteins obtained by the above-described method. White matter or proteinaceous substances; as well as pharmacologically acceptable carrier substances and metals. - radiopharmaceutical combinations containing radioisotope-labeled proteins or protein-based substances; It also applies to products. Solutions of labeled proteins or protein-based substances are radiopharmaceutical compositions. 1. Used as is. If necessary, purify this solution further or or added with a pharmacologically acceptable carrier substance, preferably physiological saline. Additionally, it is prepared into a form more suitable for intravenous or subcutaneous injection. Places like this It is necessary to guarantee that the protein will not be damaged even when subjected to treatment. be.

It is also important that this solution be sterile when administered intravenously or subcutaneously. Of course.

In carrying out radiodiagnostic procedures, the above-mentioned compositions may be used, if necessary, in a pharmacologically acceptable manner. 100% for warm-blooded animals after dilution with a liquid, preferably physiological saline. Administer at a dose of μCi to 30 mCi, then record the radiation emitted from the body. Record (measure).

When using this composition for radiotherapy, as mentioned above, it is necessary to use a suitable metal for the label reaction. Select genus-radioisotope. Dilute with pharmacologically acceptable liquid if necessary. , administered to warm-blooded animals in an amount effective for tumor suppression.

Because the radiopharmaceutical compositions of the present invention are easy and simple to prepare, they can be easily and conveniently prepared by users. It can be easily prepared. Therefore, as mentioned above, the present invention is applied to a so-called "kit". However, this kit contains (1) at least substantially a thio compound or or a cyclic condensation product thereof and a protein or protein system. Protein complex preparations obtained by reaction with substances (dry state may also be used), (2) Metals - a solution of a salt or chelate of a radioactive isotope, and (3) optionally (1); It consists of instructions for use with prescriptions for reacting (2).

As mentioned above, in this complex formation reaction, the desired radioactive isotope is For coalescence, preferably relatively weak chelators such as birophosphates, phosphonates, etc. or polyphosphonates, oxynates, carboxylates, vitroxica Ruboxylates, aminocarpoxylate enolates or mixtures thereof. It is added as a combined chelate (the reaction is carried out in a neutral medium). radioisotope 8-Hydroxyquinoline or its derivatives are suitable as chelators for body solutions. Conductor dicarboxylic, polycarboxylic or hydroxycarboxylic acids such as oxalic acid, Malonic acid, succinic acid, maleic acid.

Orthofter hydrofluoric acid. Malic acid, lactic acid, tartaric acid, citric acid.

Ascorbic acid, salicylic acid or derivatives of these acids; birophosphate; phosphoric acid nates or polyphosphonates, such as methylene diphosphonate, hydroxyethyl tylene diphosphonate or hydroxymethylene diphosphonate; or enola For example, β-diketones (acetylacetone, furoylacetone, thenoylacetone) (benzoyl aceone, dibenzoylmethane, trobolone, etc.) or These are the enolates of diketone derivatives. 8-Hydroxyquinoline. quest acid.

Tartaric acid, ascorbic acid, glucoheptonic acid or their derivatives or acetate Ruacetone appears to be particularly suitable because indium-III or Chelation of these chelators with radioactive isotopes such as lead-203 can be carried out in a suitable medium. , preferably in an aqueous buffer and at physiological pH, readily reacts with said protein complex. Accordingly, the desired radioisotope complex can be produced in high yield and with high efficiency by ligand exchange. This is because it forms with high purity. Injimu~llll1ro passed for this purpose, An aqueous buffered solution of boronate (used to form the desired complex) is added to the iodine solution. -Loppa Patent Application No. 131327 (incorporated as a reference) . As for Ki No. 1-, the components mentioned in (1) above and the instruction manual are supplied as a set. However, since the metal-radioactive isotope solution in (2) has a limited lifespan, it must be purchased separately. Supplied.

Another, but same, (and highly preferred embodiment) kit of the invention includes: (1) The above-mentioned thio compound or a cyclic form thereof By reacting a coupling agent consisting of a condensation product with a protein or protein-based substance. The obtained protein complex preparation (which may be in a dry state), (2) the above chelator and reducing agent, and (3) optionally (1) and (2) in pertechnetate solution. Instructions for use with prescription for reacting with technetium-99m. This composition A reducing agent such as dithionite or primary dithionite is used to reduce pertechnetate. Contains tin ions. This kit is used for the preparation of Tc-99m-labeled drug compositions. It is intended that

Pertechnetate solutions are available from molybdenum-technetium generators. can be easily obtained by the user from With Re-186 or Re-188 Similar kits are used to prepare labeled drug compositions, but they also Berurenate fJ solution is mixed with a suitable reducing agent, such as dithionite or primary Reduced by tin ions. If necessary, as specified in (1) and (2) above. The components together are a combination unless they repel each other. The protein complex may be adversely affected by any of the components specified in (2). This is because they do not receive it.

In this type of single component (monocomponent t->kit), the combined It is best to lyophilize each component, but this single-component kit allows the user to easily freeze-dry the components. It is very suitable for reactions using radioactive isotope solutions.

In yet another highly preferred embodiment, the kit of the present invention comprises the following components: (1) at least substantially the aforementioned thio compound; (2) Optionally, the components described in (1) (these (preferably kept in one vial) and protein (supplied separately to the user). ) and technetium-99m or perrhenium as pertechnetate solution Reaction formulation with rhenium-186 or rhenium-188 as an acid solution Instructions for use with . By using this so-called “multi-purpose kit”, users can Labeling any of the proteins with radioactive technetium or rhenium is not possible. (by forming the desired protein complex).

In a related aspect to this last kit, the kit of the invention comprises the following components: consists of; (1) the above coupling agent; (2) a metal-radioisotope salt or chelate; Instructions for use of the solution and (3) optionally the reaction formulation of (1) and (2).

Metal reducing agents, such as Sn (TI), Fe (II), Cu (I), Ti ( HI) or Sb(III) are preferred as reducing agents for the above kits. , Sn (II) is optimal. The amount of reducing agent has no effect on the results. It is a generally known fact that (PCT application WO37104164, (See page 7 of the application record).

Surprisingly, metal reducing agents are present in very small quantities, i.e. in protein complexes or protein complexes. 0.1 to 10 μg of metal per square inch of white matter, preferably a small amount of 1 to 4 μg However, not only is it sufficient, but using such a small amount of reducing agent It was observed that the labeling efficiency and stability of labeled products were significantly improved. to this This will be explained in detail with reference to Examples.

Component (1) of the kit may be in solution, for example in physiological saline or in a buffered solution. However, it is preferably supplied in a dry state, for example in a freeze-dried state. for injection When used as an ingredient, it must be sterilized. Therefore, the components are in a dry state. If the condition is present, use sterile physiological saline as the solvent.

Optionally, these components include acolpine reference, gentisic acid, or their derivatives. Stabilization may be achieved using stabilizing agents such as salts of acids, or with other adjuvants, such as glucose. -s, lactose.

Fillers such as mannitol may also be added.

The present invention will be explained in detail with reference to the following examples.

Kogyo Madarajo -Human albumin at a concentration of 5 tag/Ili of rubumin in Mino-O7 Solution (0,0M) in HCf) (pH 8,0) 0.51d Used as raw material. Add 0.5M 2-iminothiolane solution (1M to this solution) 25μ of ethanolamine (in HCl) (pH 8,0) was added. at room temperature Incubated for 1 hour. Protein paste NH, 2-iminothiolane is attached to the base Multiple coupling yields one extra 13H group. with 2-iminothiolane The progress of the reaction was measured by determining the number of -SH groups before and after the reaction.

-3H group was calculated by Grassetti et al. (“The Deter+m1natio n of Th1ols and of Total Glutathione inHuman Blood Using 6.6’-Dithiodini cotintc Ac1d (CPOS)’in Biochen+1cal Medicine; pp, 149453 (1975). ) according to the method It was. The principle of this measurement is as follows.One CPDS is a thiol (SH- group). The reaction produces 6-mercaptonicotinic acid (6-MNA) and disulfide.

6-MNA was measured by a spectrophotometer at 344 nm, and then about the raw material. Calculate the molar concentration of thiol. The number of 3H groups in proteins is 2-iminothiolane It is 1°57 before the modification and 25.92 after the modification.

1 ball side 1 2--Nienoshi and Atamichichi'' and Tc7 Ae Kushi Kohebutone Nidan Nishoshin- Human albumin solution at a concentration of 5 B/d (0.0 M triethanolamine-HC#! Medium) (pH 8,0) 2.5d was used as the raw material. This solution contains 0. 5M 2-iminothiolane solution (1M triethanolamine-H in l) (p H8,0) was added. 0 reaction lli incubated for 1.5 hours at room temperature was purified by gel chromatography (5 ephadex G 25) and further Sample 1- was left unpurified.

Both purified and unpurified modified albumin were treated with Tc-99m glucoheptonate. 0.2 d (corresponding to 4.5 mC1). Incubate for 20 minutes at room temperature Tc-99m labeling of both samples at the time of incubation and after 2 hours of incubation. was analyzed. Analysis was performed using gel chromatography using 5ephadex G25. I went there.

The labeling rate of purified modified albumin was 40.8% after 20 minutes of incubation. It is 85.6% after 2 hours of incubation.

The labeling rate of unpurified modified albumin was 88.7% after 20 minutes of incubation. It is 93.2% after 2 hours of incubation.

n master To the blood Lord ffl Cause Z two changes otsuiro A Lord lzuyu C-, llL sentence lkp Tamayodo 591 Purified product of Tc-99m label sample obtained in Example 2 Excess DTPA, (diethylenetriaminepentaacetic acid) was added to.

The ratio of DTPA to protein (weight unit) was 5ets. DTPA and Tc-99 The mixed sample of m-labeled albumin was incubated for 48 hours at room temperature. albu The degree of binding of Tc-99m to min was measured using a gel using 5ephadex G25. Measured by chromatography. The degree of binding is 97. 3% (corrected for radioactive decay). This means that the albumin label will disappear. This shows that the loss is only 2.7%.

1!1l- ) Two Sakakushi J Sat Onishakuyo-4” - CuLf11 Yuy Industrial Coupling and Tc-99m Gu Human Ig solution (0.06M ) reethanolamide 7-HCp, medium) (pH 8,0) 2.5ad as the raw material. Used. Add 0.5M 2-iminothiolane solution (IM) to this solution/-) Li7mi7-HCj! Medium) (pH 8,0) 25. crf was added. 1 at room temperature Incubated for hours. Then Tc-99m glucoheptonate 2d (22 4mC1) was added to reaction mixture 2.51d and incubated for 30 minutes.

According to analysis using gel chromatography, the label ratio is 73.6%.

Implementation day 2-Iminothiolane's production of globulin ゛　　　　　　　　　　　　　　-sa” 6-j2i: Two-chain 9mj-on and the combination of Σbutonate ・ Concentration 10+wg/at? 7)! : ToTg solution (0.06M To + J x Ri, / - in Rumidy HCJ2) (pH 8,0) ld was used as the raw material.

Add 0.18M 2-iminothiolane solution (1M triethanolamine-H Ci! , medium) (pH 8-0).

After incubating for 1 hour at room temperature, divide the reaction mixture into two samples of 5 d each. divided. Add 1 m (10 mCi) of rc 99+u glucoheptone to one sample. To the other sample, Tc-99m tartrate 1 relative (corresponding to 10m Ci) was added. equivalent) was added. Incubate for 45 minutes and then gel chromatograph the reaction. It was analyzed using 5ephadex G25. Tc-99m gluco The labeling rate with heptonate was only 17%, but with Tc-99m tartaric acid. In the case of salt, it was quite high, at 58%.

Control tests performed on unmodified 1g samples did not allow for labeling. Ta.

Implementation team day 2-Iminothiolane-I Glob 1-1Tc-m-Bell's A solution containing 1mg of Ig per blood sample (0.06M) reethanolamide - in HCjE) (pH 8,0) 0.1d to 0.07M 2-iminothiolane solution ( 5 μi of 1 M triethanolamine in HCl (pH 8,0) was added. At room temperature , and reacted for 1 hour. Add Tc-99m tartrate 0.5d (42,7mC1) Then, the mixture was incubated for 1 hour at room temperature. As a result of the analysis, the label ratio is 7. It was 2.7%. After 2 hours, the unpurified reaction mixture was diluted with 0 and 1 d each. Two samples were collected, and human serum 095d was added to one sample (0, 1d). However, 0.5 - of physiological saline was added to other samples (0, 1d). has been modified However, 0.5 d of human serum was added to 1 g sample containing Tc-99m tartrate. It was used as a link test. After 24 hours of incubation at room temperature, gel clot was applied to each sample. The degree of binding of Tc-99m to Ig was analyzed using matography. Tc-9 The degree of binding with 9m2-iminothiolane modified Ig was 91%, but with physiological saline The same value of 91% was also shown when incubated with .

Unmodified Ig shows a degree of non-specific binding (binding to serum proteins) of 45% in serum. did. Incubation in serum does not result in separation of Tc-99m from proteins. We can conclude that there is no.

Disaster 1 hindrance 1 2-(S/-Pf'-7"rlffi, Tc-99m1 and Tc- M Rukoheto Iji'-Bell Tari,, Komen Globlin's 1 spit foot Immune activity is measured by actinic chromatography, and for this purpose, goats are first Ig fluxigon, an anti-human Ig obtained from h) Covalently bonded to agarose (small spheres). Injection of rnl using conjugate A small column was created in a syringe. Twenty columns are at neutral pH It binds approximately 0.6 μg of human 1 g. If the column is natural (untreated) Ig, In addition, albumin was eluted with PBS (phosphate buffer solution) and activated. For albumin, the column retention rate showed an optimal value (60-80%), but for albumin, it showed an optimal value (60-80%). No retention was observed (5%) with 0.1M glucine. The column was washed with a solution (0.15M NaCl in pH 2.4). immunity Activity was calculated as a percentage of Tc-99m-labeled Ig sample by the following formula: be done.

I of Example 5 modified with 2-iminothiolane and labeled with Tc-99m tartrate. g (1:10 ratio) showed 99.4% immunoactivity against the original Ig. . In the case of labeling with Tc-99m glucoheptonate, the value is 94.9%. there were.

royal family lonely ( Monochrome by 3/-Ft-ton and Tc-99m? A bell-stopping monoclonal antibody obtained from mice and human CEA ( used against carcinoembryonic antigen). This is labeled and used to detect colon tumors. It is something that can be done. This antibody is called Pariai4. 1mg Parlas+ A solution containing CPBS in diphosphate buffer (pH 7,6) O, Id 0.077 M2-im) f orane solution (in 1M triethanolamine HCf) (pH 8 ,0) 5 μl was added. The reaction was allowed to proceed at room temperature for 1 hour. 71°99m tartrate solution Add 0,511 N (equivalent to 8 mCi) and incubate for 1.5 hours at room temperature. Ta. As a result of gel chromatography analysis, the labeling rate was 79.5%. Ta.

Hushidomame 2-Imino-f-f-7-ITc-99m' T-<le-mo-low Rule 2: ``CEA (carcinoembryonic anti-inflammatory agent) is used as an antigen for measuring immune activity. The Elisa test is used. For the measurement procedure, please refer to Lam5 There are reports of drop and others (J, lm5uno1. Methods 39+ 293-405 (1980) + “Tmnunoper-oxidase pro cedure to detect monoclonal antibody s against cell 5 surface ar+tigen, Qua nt+, tation of binding and staining of 1ndioidual cells’). Phosphate buffer P B S (Ph CEA in oshate-buffered 5aline) (bovine serum albumin) in a 96-well microtiter plate. Add the ink to the wells of a Microtiter plate at room temperature. It was incubated. Parlam 4 antibody (in PBS) was diluted with 1% BSA (1/100 dilution) and diluted stepwise. After incubation, the wells are PB After washing three times with S (0,05% Tween 20), rabbit anti-mouse Ig (diluted to 1/400) and treated with peroxidase. incu After washing 3 times with PBS (0.05% Tseen 20), the substrate (0-phenylenediamine 1 mg/0.1 M acetic acid 1-1 pH 5,0) was added. After a color reaction occurred, absorbance was measured at 482n* using a spectrophotometer.

The following samples were compared; (1) Modified Parlas+4 (PM4) of Example 8, (2) Tc 99 Modified PM4 labeled with m-tartrate (Example 8), (3) Unmodified Parla m4 (presence of Shikashi Tc99m tartrate), (4) ascites (ascit Parlam 4 in es 1iquid).

From the graph in Figure 1, it can be seen that 2-iminothiolane modification and Tc-99m tartrate It can be seen that the label has no detectable effect on immune activity. In the graph, "ABS" indicates absorbance, and "v, f" indicates dilution factor.

The relationship between the curve and the sample is as follows; (1) '------' Pbarmacon 4 (P M 4) , modified with 2-iminothiolane (2) 0-, ---, modified with OPM4.2-iminothiolane, Tc-99m tartar Label with acid salt (3)→−−−−−+　PM4+Tc-99m tartrate (4)　x-”-x PM4 Ascites! Arashi Kuyu ) Niino Aufu ``Otohie 2 sides and 2'',,b2LL3-Quen commonly Yorubell Albumin solution (0.06M) at a concentration of 5 mg/d Reethanolamizy HCI! , medium) (pH 8,0) 2.5d was used as the raw material. 0.5M in this solution 2-iminothiolane solution (in 1M triethanolamine-HCl) (pH 8.0 ) 10μβ was added.

Incubate for 65 minutes at room temperature. Take 1d of the reaction solution and apply gel chromatography It was purified by 5ephadex G25. Thus, the concentration is 3.3 1.5 d of modified albumin of vag/d is obtained. P b-203 Kukon 1 m of acid salt (equivalent to 0.5 mC1) was added and incubated for 45 hours at room temperature. . According to analysis by gel chromatography, the labeling rate is 75%.

Unmodified albumin showed no labeling rate under the same labeling conditions. It was.

Upper folding fan In-111 Toro Posanju Nijo≦dan Ig solution (0.06M) at a concentration of 10 mg/d in Lieta, Norami 7-HCI ) (pH 8,0) 3d was used as a raw material. Add 0.5M 2-imide to this solution. Notiolane solution (1M triethanolamine in HCf) (pH 8,0) 25μ 2 was added. The reaction was allowed to proceed at room temperature for 1 hour. Take 0.5 d of the reaction mixture and transfer it to Guelph. It was purified by filtration (5 ephadex G 25). Thus, about 5 A modified Tgm containing a modified protein of tag. I got 5d. To this, In, -11 1 toporonate Q, 3 m (equivalent to 0.3 m Ci) was added. unrefined Similarly, for the modified 1g0.5yJ, In-111 Tropolonate 0.3- We broke up. After incubation for 30 minutes at room temperature, gel chroma) The labeling rate was measured. This rate is 1 for both purified and unpurified modified Ig. It showed a value of 00%. Under the same conditions, the labeling rate of unmodified Ig was 41%.

Inu Shido 11 iJAJ! Made by LLmu 200 μg of 2-iminothiolane and 5 mg of 5n (n) (as tin tartrate) Combined and placed in a vial, both bath components were lyophilized.

This versatile kit can be used to label all types of proteins. example For example, add 2 d of saline containing 1 g of 2001I to this and mix the whole thing. pertechnetate (physiological saline) at 10 mCt (370 MBq). 0.5-medium). Incubate for 10 minutes at room temperature, then Measure the labeling rate by elution using an ephadex G 25 column do. Its value is 91°9%.

Implementation Team Yu”, Kei Ingredients ``Tenmu L Milk 2 Main Y Stop 1'' J Yu Ying 11 (1st 1)'' Oli Gongko Goes Emperor 1 Plate Precept Human Ig was modified with 2-iminothiolane in the same manner as in Example 5. modified protein 11g of tartaric acid, 5g of tartaric acid, and 2ttg of 5n(If) (as chloride). I put it in a vial.

This Kira i was labeled using technetium-99rn; -0.5d of pertechnetate obtained from a technetium generator (0.5mC1) was placed in a vial and incubated at room temperature for 20 minutes.

The labeling rate was 99.2%.

This labeled product was used to localize the infection process in tumor-bearing mice. Ru. For this purpose, the product (i.e. Radiopharmacon) is (5 taphyloccus aureus 8-12 (100 10,000 units) intravenously into the infected right thigh muscle. After intravenous injection, 1. At 6 and 24 hours, a scintigram was taken and compared with that of the corresponding part of the left non-infected thigh. did. Results (Re L ratio, infected right thigh and uninfected left thigh) ratio) is shown below.

As a comparison, radiophar*acon, which is often used for the same purpose, i.e. A similar test was conducted using gallium-67 citrate. The results are from 4 test animals. This is the average value for

The difference between both substances is obvious.

imperial donation ■earth 4 bells of tin Ig 1■ modified with 2-iminothiolane and prepared in the same manner as in Example 5 was mixed with 5n (II) (as tin tartrate) in a vial. Pertechne Tate (5 mCi; 185 MBq) (0.5 d in physiological saline) by The mixture was incubated at room temperature for 20 minutes. 5ephadex The labeling rate was measured by elution with a G25 column, and a value of 93.8% was obtained.

Similarly tested in the presence of 12.5 μg of 5n(II) (as tin tartrate), In that case, the labeling rate was only 74.9%.

Real m 2 Ki Furnace One of the two vials For the latter, modified Ig 1++ prepared by the method of Example 5 using 2-iminothiolane. +g of tartaric acid and 20dg of tartaric acid and 5n (If) (as tin chloride) on the other side. Put in.

Both components are in a freeze-dried state.

The kit was labeled as follows; i.e. with tartaric acid and 5n(II). Add 5 d of saline to the vial.

Place the resulting solution into a vial with modified Ig and mix the entire solution. Add pertechnetate and incubate as in Example 13.

For this Pharmacon, use a mouse in exactly the same manner as in Example 13. A comparison was made with gallium-67 citrate.

The results (R:L ratio) are shown in the table below.

Results are average values for 4 animals.

Even in this test, the differences with known products are clear.

Implementation ±1 day −−−−Le Ouf's I Albumin a) 9.28 g of refined methylallyl chloride via CaC1t-tube Air was passed through for 90 minutes. Then HBr was passed through it for 3 hours while cooling. generate 1-Bromo-3-chloro-2-methylpropane in water, KzCOs-solution, After washing with water, the product was imaged under reduced pressure and identified by NMR spectrum. Yield 1 5.5g.

b) Dissolve 1.43 g of NaCN in 2.5 d of water, add 10 d of ethanol and a 5 g of the compound obtained in ) was added. Reflux the reaction mixture at approximately 85°C for 20 hours. and then treated as follows; evaporation of ethanol, addition of methylene chloride and Na Br filtration, drying, and vacuum image. Target substance i.e. 3-chloro-2-methylpropyl Lucyanide (identified by NMR spectrum) was obtained in a yield of 1.75 g.

c) Add 0.534 g of potassium thioacetate (KSAC) to anhydrous dimethyl sulfoxide. Stir 0.5 g of the compound obtained in b) in a solution dissolved in DMSO>0.7d. Added while stirring. After stirring at room temperature for 1 hour, water was added to the reaction mixture and diethyl The ether layer was washed with concentrated NaCl solution and dried. diethyl The ether was removed by evaporation and the product was imaged under reduced pressure.

3-Acetylthio-2-methylpropyl cyanide (NMR-spectrum and IR- Spectral identification) was obtained in a yield of 0.365 g.

d) 0.77 g of the product in c) was mixed with 2.14 d of anhydrous methanol and 7,6 N The mixture was stirred for 48 hours in 2.95 d of HCf/methanol for cyclization. meta The nol/HCI was evaporated off and diethyl ether was added after cooling. 3- Imino-4-methylthiolane crystals were filtered off and washed with diethyl ether. Melt Point 175-177°C.

Using the obtained 2-imino-4-methylthiolane, 2-iminothiolane was prepared in Example 1. Human albumin was modified in a manner similar to that described for 5n (n ) and 5 μg of sodium tartrate (in physiological saline), then Tc-99m. Pertechnetate 0.5d (in physiological saline) (Habo 5mCi (185MBq) ) and labeled with Tc-99m. The labeling rate was 96.9%.

SILLE -Ishirohekin It was obtained in the same manner as in Example 16 using ω-bromobutyl cyanide as a raw material.

Example 1 Using 2-iminothiacyclohexane with human albumin having a melting point of 182°C was modified in the same manner as in Example 16, and labeled using Tc-99m in the same manner as in Example 16.

The labeling rate was 83.4%.

Hugoban construction worker 2 N-tert-buti ni 4’;< / ++ -y 7 (7)” snoring number a) Add 19 g of tert-butylamine and 28 g of triethylamine to 400 d of dichloromethane. 100 g of 4-chlorobutyl chloride in a solution dissolved in ethyl ether A solution of rd in diethyl ether was added dropwise with stirring. After refluxing for 1 hour The mixture was poured into water, the organic layer was separated, and the aqueous layer was extracted with diethyl ether.

The organic layers were combined, washed sequentially with water and saturated NaCl, and dried. Evaporate the solvent Then, the yield of 4-chloro-N-tert-butylbutyric acid amide was 40.8 g. Obtained. Purification by recrystallization from methanol, loading 62.5-64°C0b) A solution of 7.1 g of the above amide dissolved in 300 g of toluene was stirred and refluxed. Reni P, S,. 17.8 g was added little by little. After refluxing for 60 hours and cooling The mixture was poured into saturated NaHCO3 solution. Separate the organic layer and add saturated NaHCOs The solution was washed twice with 1 solution of saturated NaCl and dried. When solvent leaks, the title The compound, 2-N-tert-butyliminothiolane, was obtained as a yellow liquid. . Boiling point: 115-117°C/40mmHg, product is TR spectrum, NMR Identified by spectrum and mass spectrum.

Elemental analysis values: C60, 81% (calculated value 61.09), H9, 73% (calculated value 9. 61), 320.20% (calculated value 20.39) C) This 2-N-tert-butyl Human albumin was modified using luminothiolane in the same manner as in Example 1, and Example It was labeled with Tc-99m in the same manner as No. 16. Labeling rate 91.2%.

Implementation■ee Earth Nim Upper Next Mu 11 Engineering 2 "2" Iminochi Mori Otsu 9 Onriku Ee Hibikiku ■ Kakin Risshiri a) A solution of 356 g of triphenylphosphine dissolved in 12 parts of carbon tetrachloride is dissolved in 7 parts of carbon tetrachloride. 217 g of 2-isopropyl-5-hydroxymethyl A solution of -1,3-dioxane dissolved in 11 carbon tetrachloride was added dropwise. This mixture The material was refluxed and after about 1 hour cooled (in a water bath), filtered and distilled. Dilute the residue ether and then filtered. After leaking the filtrate, the residue was image-purified. .

Objective! , 2-isopropyl-5-chloromethyl-1,3-dioxane is a colorless Obtained as a liquid. Boiling point 84-94"C (mixture of cis-trans isomers, NMR ).

b) 2-isopropyl-5-chloromethyl-1,3-dioxane 183g, water 2 A mixture of 5 g and 5 d of concentrated hydrochloric acid was refluxed overnight. Cool and diethyl ether After adding 2-chloromethyl-1,3-propanediol was extracted with water. water layer was washed with diethyl ether and evaporated. Dissolve the residue in acetone and dehydrate. , evaporated. A colorless liquid (identified by IR and NMR) was obtained as a product.

C) Put 16.2g of powdered NaCN into 100-〇〇MSO and heat it to 140°C. To this was added 32.8 g of the above 2-chloromethyl-1,3-propanediol (etc. of DMSO) was added. During the addition process the temperature is about 160-170℃ Rise to ℃.

After cooling to room temperature, the mixture was diluted with 2-3 volumes (volume) of acetone and Filtered with SOa. The filtrate was subjected to vacuum extraction and the DMSO leaked out. 400- Extract twice with acetone to obtain the desired 2-cyanomethyl-1,3-propanediol. - I got J. After dehydration and leakage of solvent, the desired substance was obtained in a yield of 23 g. . Boiling point: 200°C 10,005 Hg, identified by IR and NMR.

d) 2-cyanomethyl-1,3-propanediol 17°5g2 Triethyl bene 1.3 g of ammonium chloride and aqueous NaOH solution (30%) 1 A mixture consisting of 50d was stirred and cooled, and 60g of tosyl chloride was added to it. A solution dissolved in Zen 400d was added. This mixture was stirred overnight. organic layer and The aqueous layer was separated and washed with diethyl ether. Combine organic layers and make neutral with water Washed, dried and evaporated until dry. 2-cyanomethyl-J,3-bis(toshi) 62 g (as a liquid) of fluoroxypropane were obtained. IR, NMR and mass Identified by spectrum.

e) 62 g of 2-cyanomethyl-1,3-bis(tosyloxy)propane in D MSO250d! Add 36g of potassium thioacetate to 200d of DMS. It was added dropwise to a solution containing O while stirring. Stir for 1 hour, then add DMSO. After addition, the mixture was poured into ice water. Extract with diethyl ether and color The organic layer was washed with NaOH aqueous solution, water and 2 saturated NaCj solutions (in medium). until it became sexual). After dehydration and distillation, the target substance 2-cyanomethyl-1. 3-Bis(acetylthio)propane is a pale yellow liquid (boiling point: 146-150"C) 10,50,1 force Hg) in a yield of 20 g. Identified by IR and NMR.

f) 6.3 g of 2-cyanomethyl-1,3-bis(acetylthio)propane A solution of 20g of HCf dissolved in methanol is added to a solution of water and methanol. solution (2.1 mmol of HCf relative to the solvent of Ig) was added. Reflux for 1.5 hours Afterwards, the solution was evaporated. Title compound, 4-mercaptomethyl-2-iminothiola (HCff salt) was obtained in a yield of 4.9 g. This liquid compound is IR,’HN It was identified by MR and CNMR.

I3CNMR (75.4MHz)"δ: 26.9 (CH, SH).

4 i, 9 (CHzS), 45.0 (CHz), 46.1 (CH).

205.9 (C=N).

When the hydrochloride salt is treated with a base, it is easily converted to the free iminothiolane compound.

g) 3 g of 2-cyanomethyl-1,3-bis(acetylthio)propane (Example 19, (e)) and 1.8 g of methanol (obtained by Tetrahydrofurane of 307 HCf gas was passed for 0.5 hour through a solution (temperature 40-55°C) consisting of . The solution was evaporated and diethyl ether was added to the residue. Suction filter the precipitate , washed with diethyl ether and dried. Second target substance, 4-acetylthiome Chill-2-iminothiolane (hydrochloride) Melting point: 186-188℃ (decomposition)) is 60 A yield of 0.0 cm was obtained. Identified by 'HNMR and 'CNMR. This compound is also a free base. It can be converted to .

h) 4-mercaptomethyl-2-iminothiolane and Modification of human albumin with 4-acetylthiomethyl-2-iminothiolane and implementation Labeled with Tc-99m as in Example 16.

The labeling rates are 82.8% and 100%, respectively.

Fruit 11tt1 2- Fc fugument by iminothione small reformed imperial engineering and -m゛　　　 -Bell Fc fragment in 0.06 M triethanolamine Hc2 at 10a+g/ d(7) concentration: 0:) (pH 8,0) used as a raw material. child 0.18M 2-iminothiolane solution (1M triethanolamine 5 units of HCl2) (pH 8,0) were added. Incubate for 1 hour at room temperature Then, using gel chromatography (5ephadex G25) Unreacted 2-iminothiolane was separated. Tc-99m tartrate 0.5 d(1 (equivalent to 0 mCi) was added and incubated at room temperature for 0.5 hours. gel chroma According to topography analysis, the labeling rate was 91.5%.

Conducted on 1st Saturday kTc of F ab') Fugumen by 2-iminothione -99m' - Bell concentration 10mg/d (0,06M)'J-1-ta/-)Li7 Mi7HCI! The F(ab’)z fragment solution (pH 8,0) of used as. Add 0.18M 2-iminothiolane solution (IM trichloride) to this solution. Ethanolamine HcI! Medium) (pH 8,0) 5 uE was added. 1 hour at room temperature After incubation, gel chromatography (5ephadex G 25 ) to separate unreacted 2-iminothiolane. Tc-99m tartrate 0. 5 d (equivalent to 10 mCi) was added and incubated at room temperature for 0.5 hours.

According to gel chromatography analysis, the labeling rate is 92.8%.

1/] Procedural amendment a (method) to

Claims (1)

[Claims] 1. A compound represented by the following general formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (here, Are R2, R2', R3, R3', R4, R4', R5 and R5' the same? , or each independently a hydrogen atom, an alkyl group, an XS-alkyl group, an alkyl ruthioalkyl group, alkylcarbonylthioalkyl group, aminoalkyl group, N -alkylaminoalkyl group, N-alkylcarbonylaminoalkyl group, or represents an N,N-dialkylaminoalkyl group, X is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, n is 0 or 1, R6 represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, provided that R2, R2', R 3. If R3', R4, R4', R5 and R5' are all hydrogen atoms, (excluding) 2. Water-soluble salt of the compound of claim 1 3. The compound of claim 1 represented by the following formula; ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (here, R6 and n are the same as defined in claim 1, P is 0 or 1, q is 0 or 1, Z is a hydrogen atom, C1-C4 alkyl group, mercapto group, C2-C4 alkylthio group, or a C2-C5 alkanoylthio group, provided that when P and q are 0, R6 is an alkyl group having 1 to 6 carbon atoms) 4. A water-soluble salt of the compound of claim 3 5. A protein containing a protein or protein-based substance that has reacted with the compound according to claims 1 to 4. protein complex 6. Coupling that couples a radioactive isotope to a protein or protein-based substance The binding agent reacts with proteins or protein-based substances to form a protein complex, and this protein A radioactive isotope complex is formed by forming a complex with a radioactive isotope and a radioactive isotope. diagnostic or therapeutic metal-radioisotope radioisotope In the method for producing Bell protein or protein-based substance, the coupling agent is one of the following: General formula: Y-R-S-X 〔here, X is a hydrogen atom or a suitable protecting group, R is an optionally substituted branched or unbranched group having 1 to 10 carbon atoms. A hydrocarbon radical (which may have one or more heteroatoms), Y is at least one terminal reactive group that can react with a functional group of a protein or protein-based substance thio compound represented by ], or Formula 1 (where X and Y together form a protein or protein) having 5 to 7 ring atoms. of thio compounds (forming reactive groups that can react with functional groups of system substances) A compound selected from the group consisting of a cyclic condensation product or a water-soluble salt of this condensation product diagnostic or therapeutic metal-radioisotope-label characterized in that it is Method for producing proteins or protein-based substances 7. The coupling agent has the following general formula; Y'-R'-S'-X 〔here, X is a hydrogen atom or a suitable protecting group, R' is an optionally substituted branched or unbranched group having 1 to 10 carbon atoms. hydrocarbon radical (one or more hetero radicals selected from 0, N and S) and/or one or more NH groups), Y' is an isosyl Anato group, formyl group, orthohalonitrophenyl group, diazonium group, iso Thiocyanate group, epoxy group, trichloro-S-triazinyl group, ethylene Mino group, chlorosulfonyl group, alkoxycarbimidoyl group, substituted or unsubstituted alkylcarbonyloxycarbonyl group, substituted or unsubstituted alkylcarboxylate Nylimidazolyl group, or sulfonated or non-sulfonated N-hydroxysulfonate succinimide ester group (succinimide-oxycarbonyl group)] or a thio compound of formula (II) having 5 to 7 ring atoms, where X and Y together becomes the following group; carbonyl group, carbimidoyl group, N-alkylcarbimidoyl group, N-hydroxycarbimidoyl group or N-alkoxycarbimidoyl group a cyclic condensation product of thio compounds represented by 7. The method according to claim 6, wherein the method is a water-soluble salt of. 8. The coupling agent has the following general formula; ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ 〔here, X is a hydrogen atom or a suitable protecting group, R'' is unsubstituted or branched or substituted with one or more XS groups; An unbranched alkyl group (1 to 10 carbon atoms), R''' represents an alkyl group having 1 to 4 carbon atoms], or or a water-soluble salt thereof. 9. The coupling agent has the following formula; ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (where X represents a hydrogen atom or an appropriate protecting group) The method described in claim 8. 10. The coupling agent has the following formula; ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (Here, R3, R4, R5 are the same as in claim 1) The method according to claim 6 11. Claim 1 wherein the coupling agent is 2-iminothiolane or a water-soluble salt thereof. Method described in 0 12. The coupling agent is 2-iminothiacyclohexane or a water-soluble salt thereof. The method according to claim 6 13. Claim 6, wherein the coupling agent is a compound selected from the compounds of Claim 1. How to write 14. The coupling agent is 4-methyl-2-iminothiolane, 2-N-tert- Butyliminothiolane, 4-mercaptomethyl-2-iminothiolane and 4 -A compound selected from the group consisting of acetylthiomethyl-2-iminothiolane. The method according to claim 13, 15. Metal-radioisotope-label obtained using the method according to claims 6 to 14 protein or proteinaceous substance 16. a pharmaceutically acceptable liquid carrier material; Radiopharmaceutical combinations containing metal-radioisotope-labeled proteins or protein-based substances In the composition, the composition comprises metal- Radioactive isotope-labeled protein or protein-based substance radiopharmaceutical composition 17. After diluting the composition according to claim 16 with a pharmaceutically acceptable liquid, warm-blooded animals 100μCi to 30mCi per 70kg body weight, preferably 0.5mC It is specially designed to administer at a dose of 10 mCi from i to 10 mCi and measure the radiation emitted from the body. Radiological diagnostic testing method 18. The composition of claim 16 is optionally diluted with a pharmaceutically acceptable liquid. After treatment, the drug should be administered to the animal in an amount effective to inhibit tumors or other pathologies. Method of radiotherapy for characteristic warm-blooded animals 19. A kit for preparing a radiopharmaceutical composition comprising the following components; (1) the method of claim 6; A protein complex obtained by reacting a coupling agent with a protein or protein-based substance. product (can be dried), (2) a solution of metal-radioisotope salt or chelate; (3) optionally, (1) Instructions for use with reaction prescription for ) and (2) 20. Radiopharmaceuticals consisting of: Composition preparation kit; (1) The coupling agent of claim 6 is added to a protein or protein-based Protein complex preparations obtained by reacting with substances (dry state may also be used), (2) a chelator and a reducing agent (which may be together); (3) optionally a chelator and a reducing agent; Cunetium-99m (pertechnetate solution), or rhenium-188 or rhenium Reacting Ni-186 (both perurenate solutions) with components (1) and (2) instructions for use with prescription; 21. A kit for preparing a radiopharmaceutical composition comprising the following components; (1) the method of claim 6; coupling agents (which may be in dry form), chelators and reducing agents, (2) If desired, the components of (1) and protein and technetium-99m (pel technetate solution), or rhenium-186 or rhenium-188 (both petroleum 22. Instructions for use with a prescription for reacting with renate solution); 22. coupling agent Is 2-iminothiolane, 2-iminothiacyclohexane, and its water-soluble salts? The kit according to claims 19 to 21, which is selected from 23. Claims 19 to 21, wherein the coupling agent is a compound according to Claim 1. kit, 24. The coupling agent is 4-methyl-2-iminothiolane, 2-N-tert- Butyliminothiolane, 4-mercaptomethyl-2-iminothiolane and 4- It is selected from the group consisting of acetylthiomethyl-2-iminothiolane. The kit according to claim 23.