METHOD FOR PREPARING A METAL-RADIONUCLIDE-LABELLED PROTEIN

Abstract: Disclosed is a method of preparing a metal-radionuclide-labelled protein or proteinaceous material, which is intended for diagnostic or therapeutic applicaton, by reacting a protein or proteinaceous material with a bifunctional agent for coupling the radionuclide to the protein or a proteinaceous material, a conjugate being formed by reaction between bifunctional agent and free amino or mercapto groups in the protein or proteinaceous material, and by then complexing radionuclide with the conjugate. The bifunctional agent used consists of a thio compound of the general formula: Y - R - S - X, wherein X is an optionally halogenated alkanoyl group having 2-5 carbon atoms, or an optionally substituted benzoyl group; R is an optionally substituted hydrocarbon radical having 1-10 carbon atoms; and Y is at least one terminal group capable of reacting with a free amino group or mercapto group in the protein or proteinaceous material. Further disclosed is the labelled protein or proteinaceous material thus obtained, and a kit for preparing a radiopharmaceutical composition.

Description: Method for preparing a metal - radionuc1ide - labelled protein The invention relates to a method of preparing a metal- radionuclide - labelled protein or proteinaceous materia which is intended for diagnostic or therapeutic application. Radionuclide- labelled compounds may be used for diagnostic examination, for example, into deviations in shape and function of internal organs and into the presence and location of pathological processes in the body. For this purpose, a composition in which the radioactive compound is present, is administered to the patient, for example, in the form of an injectable liquid. By means of suitable detection apparatus, for example, a gamma camera, pictures of, e.g. , the organ or the pathological process in which the radioacti ve compound'has been incorporated can be obtained by recording the emitted radiation ("scanning"). Radioactive- labelled biological materials, in particular proteins and proteinaceous materials, e.g. , blood cells, immunoglobulins , glycopeptides , monoclonal antibodies like anti yosin and monoclonals against tumour antigens, peptides, aminofunctions -containing hormones like somatosta- tin and ACTH, and other proteins suitable for this purpose, such as plasmin and plasmin derivatives, e.g. , iniplasmin and tissue plasminogen activator, present interesting perspectives for diagnostic application. Certain proteins have a very large target organ specificity and, after having been introduced into the patient's body, can react very selectively with biological macromolecules present therein; good example thereof is the selective reaction of antibodies or antibody fragments with antigens present in the body. SUBSTITUTE SHEET Various metal- radionuclides , provided they are bound to tumour-selective biological macromolecules, such as the glycopeptide bleomycin, can be used successfully for controlling tumours, and thus form a powerful tool in radiotherapy. The macromolecules used thus serve as vehicles for the transportation of the desired radiation dose, i.e. , the metal- radionuclide , to the tumour to be exposed to radiation. The direct labelling of a protein or a proteina¬ ceous material with a metal-radionuclide has two disadvan¬ tages. First, the biologically active site of the protein necessary for a good target organ specificity or selectivi¬ ty, may easily be blocked by this reaction, so that the normal behaviour of the biological macromolecule is distur¬ bed. In addition, the affinity between metal-radionuclide an macromolecule often is insufficient, as a result of which th formed bond is not sufficiently stable to remain intact unde physiological conditions. The administered material then is no longer useful, neither as diagnostic - the behaviour of the protein in the body can no longer be traced - nor as therapeutic - the radiation dose is no longer transported to the desired site but causes an undesired radiation burden elsewhere ! In order to mitigate these disadvantages, it is suggested in European Patent Application 237150 and in PTC Patent Application WO 87/04164 to treat proteinaceous substances which comprise disulphide bonds with a sulphide reducing agent *" for example, dithiothreitol , prior to reacting the reduced proteinaceous substance, which now comprises free mercapto groups, specifically with radionuc¬ lide species, for example, with Tc-99m- tartrate or -glucohep tonate. The disadvantage of this method is the reductive SUBSTITUTESHEET treatment of the protein, in which the protein is "unfolded" by breaking the disulphide bonds to the desired mercapto groups. Damage to the protein molecules may then easily occur . In the past, few years a great number of patent publications have appeared in which biological macromolecu¬ les, usually proteins or proteinaceous substances, have been described which comprise chelating groups for a bond with th desired metal-radionuclide . Recent patent publications in this field are the United States Patent Specifications (US) nos. 4479930, 4511550, 4652440, 4652519 and 4678667, the European Patent Applications (EP) nos. 83129, 173629 and 188256, The Netherlands Patent Application (NL) 8204108 and the PCT Patent Applications WO 85/03231 and WO 86/03010. Of course, the biological behaviour of the original macromolecule must be maintained as well as possibl by this modification. This means that the chelator or the bifunctional agent with which the metal-radionuclide is boun to the protein, may not to be too bulky, certainly not when used for comparatively small protein molecules. Moreover, th usually extremely sensitive protein or proteinaceous materia must be exposed as little as possible to damaging conditions during the coupling with chelator or bifunctional agent, which may adversely influence the properties of the macromo¬ lecule. Long-lasting incubations, treatments at elevated temperatures, the presence of organic solvents or conditions of acidity differing from the physiological pH , reactions in the presence of oxidizing or reducing agents, all these treatments should be avoided as much as possible. As already stated hereinbefore, the selected bifunctional agent should ensure a strong bond between the protein or proteinaceous material on the one hand and the metal-radionuclide on the I..-* * * ^* *> - - * ' * •* «■ *- - • *** "- * other. In case the bond does not remain intact under physiological conditions, i.e. , the radionuclide comes loose in the bloodstream and can be be transported to undesired sites in the body by other particles in the blood, the radioactive material may cause an undesired radiation burden for the tissue at those sites, and may even seriously damage the tissue there if present in therapeutically. effective quantities. Furthermore, in connection with the often poor shelf life of the labelled macromolecule and/or the short* half-life of the metal-radionuclide used, it is often not possible to place the ready- for-use labelled protein or proteinaceous material at the user's disposal. In such cases the user himself will perform the labelling reaction with th radionuclide in the clinic or the clinical laboratory, for which purpose the various reaction components are then offered to him in a so-called "kit". It will be obvious that the operations to be carried out and which have to be performed under aseptic conditons must be as simple as possible (so a minimum number of reaction steps and reaction components and no laborious separation or purification) , in order to enable the user to prepare the radioactive- labelled protein or proteinaceous material with the auxiliary means available to him from the supplied kit. The labelling efficiency or yield also plays an important part. Apart from the loss of valuable material, unconverted starting material must be removed from the resulting product, in the case of incomplete labelling, as a result of which a laborious purification of a radioactive product under aseptic conditi¬ ons must ususally be carried out by the user. The chelators or bifunctional agents described in the above-mentioned patent publications are not satisfactory with regard to one or more of the requirements mentioned hereinbefore. For example, a comparatively bulky chelator is SUBSTITUTE SHEET used in US 4479930, US 4511550, US 4652519, US 4678667 and 188256. In order to couple the said chelator to the protein, conditions which are damaging to the protein during the coupling reaction are applied in the methods described in US 4652440, EP 173629, WO 85/03231 and WO 86/03010. The bond between protein and metal - radionuclide is not sufficiently strong in the proteins labelled according to US 4479930 and WO 85/03231. In many cases the labelling method is laborious and purification afterwards of the labelled protein is necessary: US 4652440, EP 83129, EP 188256, NL 8204108, WO 86/03010. Sometimes the labelling also is incomplete to such an extent that as a result of this an extra processing step is necessary: US 4479930, US 4652440, WO 85/03231 and WO 86/03010. It is suggested in WO 87/04164 to use S-acetylmer capto succinic anhydride (SAMSA) as bifunctional agent to prepare a technetium- labelled antibody conjugate by reaction with TcN(H l) . However, in this method an extra reduction step is necessary to form an -SH group which is capable of reacting with the said technetium compound. Quite recently a Patent Application was published namely WO 89/07456, in which 2 - iminothiolane and related compounds are used as a bifunctional agent for coupling metal-radionuclides to proteins. This agent indeed has considerable advantages over the already known agents, because it can very readily be reacted with the protein, in which the formed protein conjugate can react with the radionuclide in one single reaction step. This latter reaction directly provides the desired labelled protein conjugate without disturbing by-products. It has been found, however, that upon labelling certain proteins according to the method described in WO 89/07456, sometimes polymerisation occurs, even under the for this reaction usual conditions which are poor in oxygen. It is the object of the present invention to provide a method of preparing a metal-radionuclide-labelled protein or proteinaceous material which is intended for diagnostic or therapeutic application, by reacting a protein or a proteinaceous material with a bifunctional agent for coupling the radionuclide to the protein or proteinaceous material, a protein conjugate being formed by reaction between bifunctional agent and free amino groups or mercapto groups in the protein or the proteinaceous material, and by then complexing the radionuclide with the conjugate thus formed to a radionuclide complex, in which on the one hand the advantages of the method described in WO 89/07456 are maintained, but on the other hand the signalized disadvantag does not occur. This object can be achieved by performing the coupling reaction with a bifunctional agent which consists at least substantially of a thio compound of the general formula Y - R - S - X (I) wherein X is a halogenated or non-halogenated alkanoyl group having 2-5 carbon atoms or a substituted or non-substituted benzoyl group; R is an optionally substituted hydrocarbon radical having 1-10 carbon atoms; and Y is at least one terminal reactive group which is capable of reacting with a free amino group or mercapto group in the protein or the proteinace¬ ous material ; and by then reacting the formed protein conjugate with the SUBSTITUTE SHEET radionuclide . Metal -radionuclides suitable for use in the method according to the invention are Tc-99m, Re-186, Re- 188, As-72 and As-77. Of these radionuclides Tc-99m and As - 72 may be used for diagnostic purposes, the other radionu¬ clides are paricularly useful in therapeutically active compositions . The derivatisation of the protein or the protein ceous material, i.e. , the reaction with the bifunctional agent, may be carried out in a very simple manner in a neutral medium (pH between 6.5 and 8) and at room tempera¬ ture. In the complex formation reaction the desired radionu- elide is presented to the protein conjugate in the form of salt or preferable in the form of a chelate bound to comparatively weak chelators, for example, a pyrophosphate, phosphonate or a polyphosphonate , an oxinate, a carboxylate, a hydroxycarboxylate , an aminocarboxylate , an enolate or a mixture thereof, likewise in a neutral medium. In the latter case the desired complex is formed via the principle of ligand exchange, in which the sulphur atom of the thio compound forms a strong chelate bond with the metal - radionu¬ clide . The use of a mercapto-protecting group X is very important because for that reason polymerisation of the protein conjugate during the preparation is avoided. It has been found surprisingly that an extra reduction step to form free mercapto groups before carrying out the reaction with the radionuclide is not necessary. The formed protein conjugate may directly be labelled with the desired metal- radionuclide , which means a considerable advantage in particular when supplied in the form of a kit. Examples of EET suitable protecting groups X for the mercapto group are: acetyl, halogenated acetyl and substituted or non-substitu¬ ted benzoyl, in which in particular eleetrons -withdrawing groups, such as nitro, halogen and sulpho are to be conside- red as substituents . For the method according to the invention may be used more in particular a bifunctional agent which consists at least substantially of a thio compound of the general formula Y' - R - S - X (II) wherein X and R have the meanings given hereinbefore; and Y' is an isocyanate group, a formyl group, a diazonium group, an isothiocyanato group, an ' epoxyethylene group, a trichloro- s - triazinyl group, an ethylene imino group, a halocarbonyl group, a halosulphonyl group, a maleimido group, sulphonated or non-sulphonated alkylcarbonyl- oxycarbonyl group, a sulphonated or non-sulphona ted alkylcarbonyliminocarbonyl group, a 2,4- dinitrophenoxycarbonyl group, or a sulphonated o non-sulphonated nitrogen-containing heterocyclic five- or six-membered ring which is bound to R with the ring nitrogen via a carbonyl group or oxycarbonyl group and which is substituted in th ortho position with an oxo function or a thioxo function; or wherein R and Y' together form an epoxyethylene group, a sulphonated or non-sulphonated alkylcarbonyl- oxycarbonyl group, or a sulphonated or non- sulphonated alkylcarbonyliminocarbonyl group. Examples of alkylcarbonyloxycarbonyl groups and of alkylcarbonyliminocarbonyl groups are radicals derived SUBSTITUTE SHEET from succinic anhydride and succinimide, respectively, As stated hereinbefore, examples of suitable protective groups X are: acetyl, halogenated acetyl such as trifluoroacetyl , and benzoyl whether or not substituted with nitro, halogen or sulp-ho. Of the above-mentioned substituent Y' the following groups are to be preferred: a 2,4-dinitro- phenoxycarbonyl group or a sulphonated or non-sulphonated nitrogen-containing heterocyclic five- or six-membered ring which is bound to R with the ring-nitrogen via a carbonyl group or oxycarbonyl group and which is substituted in the ortho position with an oxo function or a thioxo function. Particularly suitable has proved to be a bifuncti onal agent of the general formula O O II II C ■ R' c - ■ cz- (IV) wherein Z is a hydrogen atom or a fluorine atom; R' is a methylene group or an ethylene group; and Y' ' 'is a sulphonated or non- sulphonated succini- mido-oxy group or a 2 - thioxo - thiazolidin- 3 -yl group . The last-mentioned preferred compound according to the invention is excellently suitable for use in the preparation of labelled proteins or proteinaceous materials. It has been found surprisingly that the use of an extra reduction step preceding the radioactive labelling of the protein -conjugate formed with this preferred compound is not only superfluous but is even undesired. The labelling efficiency is considerably higher without a reduction, as will become apparent from the specific examples. As already explained hereinbefore, this means a considerable advantage SUBSTITUTE SHEET in particular when supplied in a kit, because the desired labelled product can be obtained directly in a high purity with one single labelling reaction. It has been found in addition that the last-mentioned bifunctional agent can react with the amino functions of the protein selectively and without damage to the protein molecule. Examples of bifunctional agents included in the last-mentioned general formula IV may be presented by the following formulae: wherein Z has the meaning given hereinbefore, A is hydrogen or an alkali- (or ammonium- )sulphonate group, and n. is 1 or 2. Duncan et al . (Anal. Biochem. 132. 1983, 68-73) and Fujii et al . (Chem. Parm. Bull, lid) , 1985, 362-367) have described the use of the above compounds for introdu¬ cing mercapto groups into proteins. The thus modified proteins may be used for the preparation of protein-protein conjugates, i.e. , for the coupling of the modified protein to another protein. However, the use of the proteins modifie in this manner for the preparation according to the present invention of metal-radionuclide-labelled proteins and the favourable biological properties of the labelled proteins thus obtained were not known. The invention also relates to a metal-radionu- clide- labelled protein or proteinaceous material obtained by using the method as described hereinbefore, and to a radiopharmaceutical composition which comprises, in addition to a pharamaceutically acceptable liquid carrier material, a metal-radionuclide- labelled protein or proteinaceous material. The resulting solution of the labelled protein or SUBSTITUTE SHEET proteinaceous material may be used directly as a radiopharm ceutical composition. If necessary, the solution may be brought into a form which is better suitable for intravenou or subcutaneous administration, for example, by the additio of a pharmaceutically acceptable liquid carrier material, preferably a physiological saline solution. Of course it should be ensured that the protein is not damaged during the said treatment. Naturally, the solution should be sterile for intravenous or subcutaneous administration. For carrying out a radiodiagnostic examination the composition, as described hereinbefore, optionally after dilution with a pharmaceutically acceptable liquid, prefera¬ bly a physiological saline solution, may be administered to warmblooded living being in a quantity from lOO Ci to 30 mCi, preferably from 0.5 to 10 mCi, per 70 kg of body weight after which the radioactive radiation emitted by the living being is recorded. If the composition is to be used for a radiothe- rapeutic treatment, a suitable metal- radionuclide should be selected for the labelling reaction, as indicated hereinbe- fore. Upon use, the composition, optionally after dilution with a pharmaceutically acceptable liquid, is administered to a warmblooded living being in a quantity effective for combating or controlling tumours. Since the radiopharmaceutical composition according to the invention can so easily and simply be prepared, said preparation can particularly readily be carried out by the user himself. The invention therefore also relates to a so-called "kit", as described hereinbe- SUBSTITUTE SHEET fore, comprising (1) in an optionally dry condition a composition of a protein conjugate, which is formed by reaction of a protein or a proteinaceous material with a bifunctional agent, consisting at least substantially of a thio compound as defined hereinbefore, (2) a solution of a salt or chelate of a metal-radionuclide , and (3) instructi¬ ons for use with a prescription for reacting the ingredients present in the kit. A protein hereafter is to be understood to mean a protein including a proteineous material. As state hereinbefore, for this complexing reaction the desired radionuclide is preferably presented to the protein conjugate in the form of a chelate bound to comparatively weak chela¬ tors, for example, a pyrophosphate, and phosphonate or polyphosphonate , an oxinate, a carboxylate, a hydroxycar- boxylate, an aminocarboxylate , an enolate or a mixture there¬ of, in which the reaction can take place in a neutral medium. Examples of suitable chelators for the radionuclide are 8- hydroxyquinoline or derivatives thereof; dicarboxylic acids, polycarboxylic acids or hydroxycarboxylic acids, for example, oxalic acid, malonic acid, succinic acid, maleic acid, orthophthalic acid, malic acid, lactic acid, tartaric acid, citric acid, ascorbic acid, salicylic acid or derivatives of these acids; pyrophosphates ; phosphonates or polyphosphona- tes, for example, methylene diphosphonate , hydroxyethylene diphosphonate or hydroxymethylene diphosphonate; or enolates, for example with a β-diketone such as acetyl acetone, furoyl acetone, thenoyl acetone, benzoyl acetone, dibenzoyl methane, tropolone or derivatives of these diketones. 8-Hydroxyquin- oline, citric acid, tartaric acid, ascorbic acid, glucohep- tonic acid or a derivative thereof, or acetyl acetone, are t be considered as particularly suitable as chelators, because it has been found that a chelate of a radionuclide with one of these chelators in a suitable medium, preferably a buffered aqeuous solution, easily reacts at a physiological SUBSTITUTE SHEET pH with a protein conjugate as defined hereinbefore, the desired radionuclide complex being formed by ligand exchange in a high yield and purity. The supplied kit may also comprise the constituents mentioned sub (1) with instruction for use, whereas the solution of the metal - radionuclide defined sub (2) , having a limited shelf life, may be supplie to the user separately. In another likewise extremely favourable embodi- ment the kit according to the invention is equipped so as to comprise the following ingredients: (1) in an optionally dry condition a composition of a protein conjugate, which is formed by reaction of a protein with a bifunctional agent consisting at least substantially of a thio compound as defined hereinbefore; (2) a chelator as described hereinbe¬ fore and a reducing agent; and (3) instructions for use with a prescription for reacting the ingredients of the kit with technetium- 99m in the form of a pertechnetate solution. The composition should comprise a reducing agent to reduce the pertechnetate , for example, a dithionite or stannous ions. Such a kit is intended for the preparation of a Tc-99m- labelled pharmaceutical composition. The pertechnetate solution can simply be obtained by the user from a molybde¬ num- technetium generator available to him. A similar kit may be used for the preparation of a pharmaceutical composition labelled with Re-186 or Re-188, in which the perrhenate solution must also be reduced with a suitable reducing agent, for example, a dithionite or stannous ions. If desired, the ingredients defined above sub (1) and (2) may be combined, provided they are compatible. Such a kit, in which the combined ingredients are preferably lyophilized, is extremel suitable for being reacted by the user with the radionuclide solution in a simple manner. In again another, likewise excellently suitable embodiment the kit according to the invention is equipped so as to comprise (1) in an optionally dry condition a bifuncti¬ onal agent, consisting at least substantially of a thio compound as defined hereinbefore, as well as a chelator as described hereinbefore and a reducing agent, and (2) instructions for use with a prescription for reacting the ingredients mentioned sub (1) , which are preferably accomoda ted in one vial, with a protein which is separately supplied to the user, and then with technetium- 99m in the form of a pertechnetate solution or with rhenium-186 or rhenium-188 in the form of a perrhenate solution. By means of this so-calle "multipurpose" kit the user can hence label any desired protein available to him with radioactive technetium or rhenium, in which the protein conjugate required therefor is hence formed intermediately. In an embodiment related to the last-mentioned kit, the kit according to the invention comprises (1) a bifunctional agent as defined hereinbefore, (2) a solution of a salt or chelate of a metal-radionuclide , and (3) instructions for use with a prescription for reacting the ingredient sub (1) with a protein and then with the ingre¬ dient mentioned sub 2. A metallic reducing agent, for example, Sn(II), Fe(II), Cu(I), Ti(III) or Sb(III), is preferably used as a reducing agent for the kits mentioned hereinbefore; Sn(II) is excellently suitable. It has been found that an extremely small quantit of metallic reducing agent, namely from 0.1 to lO -tg of meta per mg of protein conjugate or protein, preferably 1-4 -g pe mg, will suffice. SUBSTITUTE SHEET The constituent of the above-mentioned kits stated sub (1) may be supplied as a solution, for example, in the form of a physiological saline solution, or in some buffer solution or other, but is preferably ' present in a d condition, for example, in a lyophilized condition. When u as a component for an injection liquid it should be steril in which, if the constituent is present in a dry condition, the user should use a sterile physiological saline solutio as a solvent. If desired, the above-mentioned constituent be stabilised in the usual manner with suitable stabiliser or may comprise other auxiliary means like fillers, e.g, glucose, lactose, raannitol, and the like. The invention will now be described in greater detail with reference to the ensuing specific example. EXAMPLE Modification of immunoglobulin with SATA and labelling with technetium-99m. In this experiment a direct labelling is compare with a labelling preceded by a reducing with hydroxylamine . Starting material is 1 ml of humane IgG (immuno¬ globulin G) in a concentration of 20 mg/ml in 15 mM of an ammonium bicarbonate buffer solution. N-succinimidyl-S -acet thioacetate (SATA) is dissolved in the same buffer solution and added to the IgG solution in a molar ratio of 25 : 1 (SATA : IgG). After incubating at room temperature for 30 m the non-reacted SATA is removed from the reaction mixture b means of gel chromatography (Sephadex® G25) . The purified modified IgG fraction is divided into four fractions of 0.5 ET ml. The first fraction ("A") is labelled directly with technetium- 99m by adding successively 0.25 ml of tin tartra solution (approximately 5 mg of Sn-II) and 0.5 ml of Tc-99m pertechnetate solution (6.7 mCi). The other fractions are first treated with 50 ml, 100 ml and 300 ml, respectively, 1% hydroxylaraine solution (0.5 rag, 1.0 mg and 3.0 mg) as a reducing- agent : fractions "B", "C", and "D" , respectively. The labelling yields are determined after 20 * an 80 min by means of gel chromatography . The results are recorded in the table below. Table: labelling yield fraction after 20 min after 80 min A 94.7% 96.6% B 57.2% 73.4% C 44.4% 65.9% D 59.9% 58.0% It will be obvious from the above results that the labelling yield in the absence of a reducing agent is significantly higher than in the presence thereof. So the formed protein conjugate, i.e. , the IgG treated with SATA, yields the desired technetium-99m-labelled protein directl in a very high labelling efficiency and in a very short period of time. SUBSTITUTE SHEET

Claims: C LA I M S : 1. A method of preparing a metal - radionucl ide labelled material which is intended for diagnostic or therapeutic application, by reacting a protein or a proteina ceous material with a bifunctional agent for coupling the radionuclide to the protein or proteinaceous material, a protein conjugate being formed by reaction between bifuncti onal agent and free amino groups or mercapto groups in the protein or the proteinaceous material, and by by then complexing the radionuclide with the conjugate thus formed t a radionuclide complex, characerised in that, the coupling reaction is carried out with a bifunctional agent consisting at least sunstantially of a thio compound of the general formula: Y - R - S - X (I) wherein X is a halogenated or non-halogenated alkanoyl group having 2-5 carbon atoms or a substituted or non- substituted benzoyl group; R is an optionally substituted hydrocarbon radical having 1-10 carbon atoms; and Y is at least one terminal reactive group which is capable of reacting with a free amino group or mercapto group in the protein or the proteinace- ous material ; and that the formed protein conjugate is then reacted with the radionuclide. 2. A method as claimed in Claim 1, characterised in that the coupling reacton is carried out with a bifunctional agent consisting at least substantially of a thio compound o the general formula Y' - R - S - X (II) wherein X and R have the meanings given in Claim 1; and Y' is an isocyanato group, a formyl group, a diazonium group, an iso hiocyanate group, an epoxyethylene group, a trichloro-s - triazinyl group, an ethylene imino group, a halocarbonyl group, a halosulphonyl group, a maleimido group, sulphonated or non-sulphonated alkylcarbonyl- oxycarbonyl group, a sulphonated or non-sulphona¬ ted alkylcarbonyliminocarbonyl group, a 2,4- dinitrophenoxycarbonyl group, or a sulphonated or non-sulphonated, nitrogen-containing heterocyclic five- or six-membered ring which is bound to R with the ring nitrogen via a carbonyl group or oxycarbonyl group and which is substituted in the ortho position with an oxo function or thioxo function, or wherein R and Y' together form an epoxyethylene group, a sulphonated or non-sulphonated- alkylcarbonyl- oxycarbonyl group, or a sulphonated or non- sulphonated alkylcarbonyliminocarbonyl group. 3. A method as claimed in Claim 1, characterised in that the coupling reaction is carried out with a bifunctional agent consisting at least substantially of a thio compound of the general formula Y * ' - R - S - X' (III) wherein X' is an acetyl group, a trifluoroacetyl group, an unsubstituted benzoyl group or a benzoyl group substituted with nitro, halogen or sulpho ; R has the meaning given in Claim 1; and Y * ' is a 2 , -dinitrophenoxycarbonyl group, or a sulphonated or non-sulphonated nitrogen-containi heterocyclic five- or six-membered ring which is bound to R with the ring-nitrogen via a carbonyl group or an oxycarbonyl group and which is substituted in the ortho position with an oxo function or a thioxo function. 4. A method as claimed in Claim 1, characterised in that the coupling reacton is carried out with a bifunctiona agent of the general formula ύ II I! γ « i ■ — c — R * — S — C — CZ 3 (IV) wherein Z is a hydrogen atom or a fluorine atom, R' is a methylene group or an ethylene group; and Y 1 ' ' is a sulphonated or non-sulphonated succinimido- oxy group or 2 - hioxo- hiazolidin- 3 -yl group. 5. A metal- radionuclide- labelled protein or protein ceous material obtained by using the method as claimed in a of the preceding claims 1-4. 6. A .radiopharmaceutical composition which comprises in addition to a pharmaceutically acceptable liquid carrier material, a metal-radionuclide - labelled protein or proteina¬ ceous material, characterised in that the composition comprises a protein or a proteinaceous material labelled wit a metal-radionuclide , obtained by using the method as claime in any of the Claims 1-4. 7. A method of performing a radiodiagnostic examina¬ tion, charaterised in that a composition as claimed in claimed in Claim 6, if desired after dilution with a pharmaceutically acceptable liquid, is administered to a warm-blooded living being in a quantity from 100 Ci to 30 mCi, preferably from 0.5 to 10 mCu , per 70 kg of bodyweight, and the radioactive radiation emitted by the being is then recorded. 8. A method of subjecting a warm-blooded living being to a radiotherapeutic treatment, characterised in that the composition as claimed in Claim 6, if desired after dilution with a pharmaceutically acceptable liquid, is administered to said being in a quantity effective for combating or controlling tumours. 9. A kit for preparing a radiopharmaceutical composition, comprising, (1) in an optionally dry condition a composition of a protein conjugate formed by reaction of protein or proteinaceous material with a bifunctional agent consisting at least substantially of a thio compound of the general formula I, wherein the symbols have the meanings given in Claim 1, (2) a solution of a salt or chelate of* a metal-radionuclide , and (3) instructions for use with a prescription for reacting the ingredients present in the ki 10. A kit for preparing a radiopharmaceutical composition, comprising, (1) in an optionally dry condition a composition of a protein conjugate formed by reaction of protein or proteinaceous material with a bifunctional agent consisting at least substantially of a thio compound of the general formula I, wherein the symbols have the meanings given in Claim I, (2) a chelator and a reducing agent, ingredients (1) and (2) optionally being combined, and (3) instructons for use with a prescription for reacting the ingredients of the kit with technetium- 99 , in the form of a pertechnetate solution or with rhenium-186 or rhenium-188 i the form of a perrhenate solution. 11. A kit for preparing a radiopharmaceutical composition comprising, (1) in an optionally dry condition, a bifunctional agent consisting at least substantially of a thio compound of the general formula I, wherein the symbols have the meanings given in Claim 1, as well as a chelator a a reducing agent, and (2) instructions for use with a prescription for reacting the ingredients mentioned sub (1) with a protein or proteinaceous material and then with technetium- 9m in the form of a pertechnetate solution or with rhenium-186 or rhenium-188 in the form of a perrhenate solution. 12. A kit for preparing a radiopharmaceutical composition comprising, (1) in an optionally dry condition, a bifunctional agent, consisting at least substantially of a thio compound of the general formula I, wherein the symbols have the meanings given in Claim 1, (2) a solution of a salt or chelate of a metal-radionuclide, and (3) instructions for use with a prescriptio'n for reacting the ingredient mentione sub (1) with a protein or proteinaceous material and then with the ingredients mentioned sub (2) . 13. A kit as claimed in any of the claims 9-12, comprising a bifunctional agent or a protein conjugate, formed by reaction of a protein or proteinaceous material with a bifunctional agent, respectively, said bifunctional agent consisting at least substantially of a thio compound of the general formula II, wherein X and R have the meanings given in Claim 1 and Y' has the meaning given in Claim 2. 14. A kit as claimed in any of the Claims 9-12, comprising a bifunctional agent or a protein conjugate, formed by reaction of a protein or proteinaceous material with a bifunctional agent, respectively, said bifunctional agent consisting at least substantially of a thio compound of the general formula III, wherein R has the meaning given in Claim 1 and X' and Y' ' have the meanings given in Claim 3. 15. A kit as claimed in any of the Claims 9-12, comprising a bifunctional agent or a protein conjugate, formed by reaction of a protein or proteinaceous material with a bifunctional agent, respectively, said bifunctional agent having the general formula IV, wherein Z, R 1 and Y' ' 1 have the meanings given in Claim 4.

Inventors: GOEDEMANS WILHELMUS THEODORUS, GOEDEMANS, WILHELMUS, THEODORUS
Applicants: MALLINCKRODT MEDICAL INC, MALLINCKRODT MEDICAL, INC.
Application year: 1990
Main classification: A61K49
IPC codes: A61K51