MXPA94003278A - Method for preparing a metal-radionuclide-labelled protein. - Google Patents

Abstract

The invention relates to 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 proteinaceous material, and then complexing the radionuclide with the conjugate thus formed to a radionuclide complex. The invention further relates to the labelled protein or proteinaceous material thus obtained, and to a kit for preparing a radiopharmaceutical composition, which kit comprises the above-mentioned polyfunctional agent or a protein conjugate formed by reaction of a protein or proteinaceous material with said polyfunctional agent.

Description

METHOD FOR PREPARING A PRQTEIN MARKED WITH METAL RADIDNUCLIDQ Inventor (s): ALFONS M. VERBRUGBE, Belgian domiciled in Herestraat 49, B-300G, Leuven, Belgium.

Cause holder: MALLINCKRODT MEDICAL a North American corporation organized and existing in accordance with the laws of the State of Missouri, domiciled at 675 McDonell Blvd, St. Louis Missouri 63134 ·, E.U.A.

SUMMARY The invention relates to a method for preparing a pro-na or a proteinaseous material, labeled with a metal radionuclide, which is intended for application in diagnosis or a therapeutic application, by reacting a protein or a proteinase material with a bi-functional agent. for coupling the radionuclide to the protein or proteinase material; forming a protein conjugate by reaction between the bifunctional agent and the free amine or free mercapto groups in the protein or proteinaseous material, and then complexing the radionuclide with the conjugate thus formed to a radionuclide complex In this reaction, a poly i Fune i onal general formula Y - R - (SX) wherein: X is a halogenated or non-halogenated alkanoyl group having 2 to 5 carbon atoms, or a substituted or unsubstituted benzoyl group; R is a saturated high aliphatic hydrocarbyl radical, having 2 to 20 carbon atoms and wherein the main chain, if desired, may be interrupted by a nitrogen atom and / or a carbon atom; And it is at least one terminal reactive group, which is capable of reacting with a free amino group or a free mercapto group in the protein or proteinase material; and n is from 2 to 6; and then, if necessary after deprotection of the mercapto protected groups, react the protein conjugate formed with the solid radionuclide. The invention relates additionally to the protein or protein-labeled material thus obtained, and to a kit for preparing a radiopharmaceutical composition, a kit comprising the polyfunctional agent or the protein conjugate mentioned above, formed by reaction of a protein. or a proteinaseous material with the polyfunctional agent.

DESCRIPTIVE MEMORY The invention relates to a method for preparing a protein or a proteinaseo material marked with metallic radionuclide, which is intended for diagnostic or therapeutic application. Radionuclide-labeled compounds can be used for diagnostic examination, for example, in deviations of form and function of internal organs, and in the presence and location of pathological processes in the body. For this purpose, a composition is present in which the radioactive compound is administered to the patient, for example, in the form of an injectable liquid. By means of a suitable detection apparatus, for example, a gamma camera, images are taken, for example, of the organ or the pathological process in which the radioactive compound has been incorporated, recording the radiation emitted ("scanning"). Biological materials labeled with radioactive elements, in particular proteins and proteinase materials, for example, blood cells, serum albumin, immunoglobulins, glycoproteins, monoclonal antibodies similar to antimyosin and monocytes. lonal against tumor antigens, peptides, hormones containing amino functions, such as somatostat ina and ACTH and other proteins suitable for this purpose, such as plasmin and plasmin derivatives, for example, miniplasmin and tissue plasminogen activator, present interesting perspectives for application of dignostics. Some proteins have a very large specificity for the target organ, they can react very selectively with the biological macromolecules present in it; A good example of them is the selective reaction of antibodies or antibody fragments with antigens present in the body. A number of metal radionuclides can be used satiially as long as they are attached to selective biological macromolecules for a tumor., such as the glycopeptide bleomycin, to control tumors, and in such a way, form a powerful tool in radiotherapy. The macromolecules used thus serve as carriers for the transport of the desired radiation dose, i.e., the metal radionuclide to the tumor to be exposed to the radiation. Direct labeling of a protein or proteinase material with a metal radionuclide has two disadvantages. Firstly, the biologically active site of the protein necessary for a good specificity or selectivity towards the target organ can be easily blocked by this reaction, so that the normal behavior of the biological macromolecule is not altered. In addition, the affinity between the metal radionuclide and the macromolecule is often insufficient, as a result of which the bond formed is not stable enough to remain intact under physiological conditions. The material administered then is no longer useful even as a diagnosis (the behavior of the protein in the body can no longer be followed) or as a therapeutic material (the dose of radiation is no longer transported to the desired site, but causes a load of undesirable radiation somewhere else). In order to mitigate these disadvantages, it is suggested in European patent application No. 237150 and in the PTC patent application W0 &The proteinaseas substances comprising disulfide ligands are treated with a sulfide reducing agent, for example, dit iotrei tol, before reacting the reduced proteinasease substance, which now comprises free mercapto groups, specifically with the radionuclide species. , for example, with Tc-99m tartrate or Tc-99m glucoheptonate. The disadvantage of this method is the reductive treatment of the protein in which the protein is "unfolded" by breaking the disulphide bonds to or the desired mercapto groups. It can then easily damage the pratein molecules. In the last few years, a large number of patent ions have appeared, and biological macromolecules, usually proteins or proteinaceous substances, which comprise chelating groups for a binding to the desired radionuclide have been described therein. Recent patent publications, in this field, are U.S. Patent Nos. 4479930; 4511550; 4652440; 4652519 and 4676667, European patent applications Nos. £ 3.129? 173,629 and 166,256, the Netherlands patent application No. 6,204,106 and the PCT patent application WO 65/03231 and WO 66/03010. Of course, the biological behavior of the original macromolecule must be maintained as well as possible by this modification. This means that the chelator or the bifunctional agent with which the metal radionuclide is bound to the protein may not be too bulky; certainly not when it is used for relatively small protein molecules. Additionally, the protein or proteinase material, which is habitually extremely sensitive, should be exposed as little as possible to conditions of damage during coupling with the chelator or bifunctional agent, which may adversely include the properties of the macromolecule. Long-term incubations, treatments at elevated temperatures, the presence of organic solvents or organic acid conditions that differ from physiological pH, reactions in the presence of oxidizing or reducing agents, all these treatments should be avoided as much as possible. As already noted above, the bifunctional agent selected must secure one. strong binding between protein to proteinase material, on the one hand, and metallic radionuclide, on the other. In the event that the junction does not remain intact under radiological conditions, ie, the radionuclide is released into the bloodstream and can be transported to undesirable sites in the body by other particles in the blood, the radioactive material can cause a load of undesirable radiation to the tissue at those sites and can even seriously damage the tissue, if present in therapeutically effective amounts. In addition, with respect to the storage life, often deficient, of the marked macromolecule and / or the short half-life of the metal radionuclide used, it is often not possible to place the protein or protein material marked, ready to be used, available to the user . In such cases, the user himself must perform the marker reaction with the radionuclide in the clinic or in the clinical laboratory, for which purpose the various components of the reaction are offered in terms of denominates a "case". It will be obvious that the operations that have to be carried out and that must be carried out under aseptic conditions, should be as simple as possible (so that a minimum number of reaction steps is carried out and a minimum number of reaction components is present, without separation or laborious purification), in order to allow the user to prepare the protein or proteinase material labeled with the radioactive material, with the auxiliary medium that is supplied from the kit. The efficiency or performance of marking also plays an important role. In addition to the loss of valuable material, the unconverted raw material must be removed from the resulting product, in the case of incomplete labeling, as a result of which a laborious purification of a product can usually be carried out by the user. radioactive, under aseptic conditions. The chelators or binary agents described in the patent publications mentioned above are not satisfactory with regard to one or more of the requirements mentioned hitherto. For example, a comparatively voluminous chelator is used in US 4, 479,930, US 4, 511,550, US 4,652,519, US 4,676,667 and EP 156,256. In order to couple the chelator with the protein, conditions that are damaging to the protein during the coupling reaction are applied in the methods described in US 4,652,440, EP 177,629, O 65/03231 and WO 66/03010. The binding between the protein and the metallic radionuclide is not strong enough in the proteins labeled according to US 4,479.93 and WO 65/03231. In many cases, the labeling method is laborious and a further purification of the labeled protein is necessary: US 4,652,440, EP 63,129, EL 166,256, NL 6,204,106, W0 66/03010. Sometimes, the marking is also incomplete to a degree such that, as a result, an additional processing step is necessary: US 4,479,930, US 4,652,440, W0 65/03231 and W0 66/03010. It is suggested in WO 67/04164 the use of anhydride S-acet and lmercapto succinic (SAMSA) as a bifunctional agent to prepare a conjugate of antibody labeled with technetium, by reaction with Tc (Hal) ^. however, in this method, an extra reduction step is necessary to form a -SH group, which is capable of reacting with the technetium compound. A few years ago, a patent application was published, namely W0 69/07456, in which 2-iminothiolane (and related compounds) is used as a bi-functional agent for coupling metal radionuclides with proteins. This agent, in fact, has considerable advantages over the known agents, because it can be reacted very easily with the protein, in which the protein conjugate formed can react with the radionuclide in a single step of reaction. This latter reaction directly provides the desired conjugate of labeled protein, without byproducts that are disruptors. However, it has been discovered that by labeling certain proteins with the method described in W0 & amp;9 / 07l + 5o, sometimes polymerization occurs, even under the conditions that are usual for this reaction, which are deficient in oxygen. Recently, a method has been described for preparing a metal radionuclide labeled protein, where IM-succinimidyl S-acet i lt-ioacetate (SATA, suitably S-acet and N-suc in imidylmercaptoacetate) and related compounds are used , as bifunctional coupling agents: WO 91/07991. It appears that the polymerization of the above ion of the protein conjugate during the preparation could be avoided and that an additional reduction step was not necessary. By using this method, the protein conjugate formed allows a direct labeling with the desired metal radionuclide, which is particularly advantageous for a case formulation. However, it has been observed that even the use of SATA with a coupling agent is not completely satisfactory, in particular as regards the in vivo stability of the labeled protein, as is apparent from the retention in the blood of the protein, determined in a living being. Apparently, the radionuclide label is not tightly unit tightly in the SATA-modified protein. It is the object of the present invention to provide a method for preparing a protein or a metallic radionuclide-labeled proteinase material, which is intended for diagnostic or therapeutic application, by reacting a protein or proteinase material with an agent for coupling the radionuclide to the protein or proteinaseous material; forming a protein conjugate by reaction between the agent and the amino groups or free mercapto groups in the protein or proteinase material, and then complexing the radionuclide with the conjugate thus formed, to a radionuclide complex, in which, for a In part, the above advantages of the method described in WD 91/07991 are maintained, but on the other hand, the radionuclide labeled protein, formed, does not show the disadvantages mentioned above for SATA: This objective can be achieved by effecting a coupling reaction with a pol i fune ional agent of the general formula Y - R - (SX) < I) wherein: X is a halogenated or non-halogenated alkanoyl group having 2 to S carbon atoms, or a substituted or unsubstituted benzoyl group; R is a saturated aliphatic hydrocarbyl radical, mu.lt ivalent, having from 2 to 20 carbon atoms and wherein, if desired, the main chain may be interrupted by a nitrogen atom and / or a carbonyl group; And it is at least one terminal reactive group, which is capable of reacting with a free amino group or free mercapto group present in the protein or proteinase material; and n is from 2 to 6; and then, if necessary after the deprotection of the protected mercapto groups, react the conjugate of protein formed with the radium on the left. The metal radionuclides suitable for use in the method according to the invention are Tc-99m, Re-1 & , Re-lña, G? -111, Ga-¿> 7, As-72 and As- 77. Of these radionuclides, Tc-99m, In-111, Ga-67 and As-72 can be used for diagnostic purposes; the other radionuclides are particularly useful as therapeutically active compositions. The above term "multivalent hydrocarbyl radical" means a hydrocarbyl group having at least three free valencies, such as a trivalent radical, etc.

The formation of derivatives of the protein or proteinase material, ie the reaction with the polyfunctional agent, can be carried out in a very simple manner (pH between 6.5 and &) and at room temperature. Subsequent deprotection of the protected mercapto groups, i.e., cleavage of the protective groups X in the formula I, can be readily effected immediately after the coupling reaction, preferably by a simple treatment with hydroxyl and lamina. In the complex formation reaction, the desired radionuclide for the protein conjugate is present in the form of a salt or, preferably, in the form of a chelate attached to comparatively weak chelators, for example, a pyrophosphate, a phosphate or a polyphosphonate, an oxinate, a carboxylate, a hydroxycarboxylate, an aminocarboxylate, a phenolate or a mixture thereof; likewise, in a neutral medium. In the latter case, the complex is formed by means of the principle of ligand exchange, wherein the sulfur atom of the compound t forms a strong chelate linkage with the metal radionuclide. The use of a mercapto protecting group X is very important because the polymerization of the protein conjugate during preparation is avoided. Examples of suitable protecting groups X for the mercapto group are: acetyl, halogenated acetyl and substituted or unsubstituted benzoyl, wherein particular electron withdrawing groups, such as nitro, halogen and sulfo, should be considered as suitable substituents. A suitable protein to be labeled, using the method of the present invention, is albumin, for example, human serum albumin CASH). It has been found that ASH labeled with Tc-99m, by means of the method of the present invention, ie using the poly-functional coupling agent of formula I, is very suitable for certain diagnostic tests, specifically for ventr iculograf í with rad u onúcl ido. As will be evident from the appended examples, a high retention in the vascular compartment of a human being is obtained using a radiolabelled albumin prepared by the method of the present invention. The present invention also relates to the conjugate of pratein complexed to the radionuclide, as mentioned above, and to the protein conjugate per se, prepared as described above, by reacting a protein or a proteinase agent with the agent pol i fune i onal coupler of the formula I. For the method according to the invention, a polymeric agent of the formula II Y '- R (SX) (II) can be used in particular. where X, n and R have the meanings given previously; and Y 'is an isocyanate group, a formyl group, a diazonium group, a sotiocyanate group, a wood epoxy group, a tri- lor-s-tr i az ini lo group, a group et i len imi no , a halogen-carbonyl group, a halogen-1-onyl group, a maleimide group, an alkylenecarbonyl locarbonyl group, sulphonated or unsulfonated, an alkylcarbonyliminocarbonyl group, sulphonated or non-sulfonated, group 2, 4-din i rof enox icarbon i lo or a 5 or 6 membered heterocyclic ring, containing nitrogen, sulphonated or unsulfonated, which is attached to R with the ring nitrogen by means of a carbonyl group uo icarbon i lo, and that is substituted in the ortho position with an oxo function or a thioxo function. Suitable examples of alkylcarbon and loxcarbon groups and of the alkylcarbon and loiminocarbon groups are the radicals derived from succinic anhydride and succinimide, respectively. As noted above, examples of suitable protecting groups X are: acetyl, halogenated acetyl, such as trichloroacetyl and benzoyl, whether substituted or not, with a nitro group, alkyl of 1 to 1 + atoms of carbon, alkoxy of 1 to 4 carbon atoms, halogen or sulfo. The following groups for Y 'are preferred: a group,? - d in i t rof enox i carbon i or a heterocyclic ring 5 or 6 > members, which contains nitrogen, sulphonated or unsulfonated, which is attached to R with the ring nitrogen, by means of a carbonyl group or an oxycarbonyl group and which is substituted in the ortho position with an oxo function or a thioxo function. A pol i fune i onal agent of the general formula has proved to be particularly suitable: ? II II Y "- C - R - (SCCZ3) n (IV) wherein Z is a hydrogen atom or a fluorine atom; R has the meaning given above; and Y "is a succinyl group-do-ox, sulphonated or non-sulphonated, or a 2-tioxo-thiazole id in-3-i.l group A succinimido-or i group, also referred to as a N-succinimidyl group The suitable examples of the above multivalent radicals -R-CO- are: (1) - CHa - CH - CO - (V) I (2) CH - Re CH CO - (VI) (3) N - Ra - CD - (VII) / < > | + > - RH \ - CH - - CH - (VIII) / - R s? k wherein: RL is hydrogen or alkyl of 1 to M- carbon atoms; a is alkylene of O to 3 carbon atoms; 3 is alkylene of 1 to 5 carbon atoms; Ru ", Rs and RA, are each, individually, alkylene from 1 to & carbon atoms, straight or branched chain; A and ± are hydrogen or (CH ^) -. - CO -, where m = 0-5; provided that A = H if Aj. is (CHK) m - CO -; and that At = H if A is (¾! ra - CO - .The specific examples of the multivalent radicals preferred in (3) and C- +) are: 1 & The preferred agents mentioned at the end are excellently suitable for use in the preparation of proteins or labeled proteinase materials according to the present invention. It has been found that the abovementioned polynuclear agents can react with the amine functions of the protein, selectively and without damage to the protein molecule. The specific examples of functional agents included in the general formula IV mentioned at the end, under < i), can be represented by the following formulas: wherein Z has the meaning given above, and Aa is hydrogen or an alkaline sulfonate group < or ammonium). The invention also relates to a protein marked with metallic radionuclide or a proteinase material obtained using the method described above, and to a radiopharmaceutical composition comprising, in addition to the pharmaceutically acceptable liquid carrier material, a protein or proteinaseous material labeled with metallic radionuclide. The resulting solution of the labeled protein or 1 < ? Protein labeled material can be used directly as a radioactive composition. If necessary, the solution can be brought to a form that is suitable for intravenous or subcutaneous administration, for example, by the addition of a pharmaceutically acceptable liquid carrier material, preferably a physiological saline solution. Of course, it must be ensured that the protein is not damaged during treatment and that the solution must be sterile for intravenous or subcutaneous administration. To carry out a radiodiagnosis examination, the composition described above can be administered, optionally after dilution with a pharmaceutically acceptable liquid, preferably a physiological saline solution, to a warm-blooded animal, in an amount of 100. , uCi at 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 radiotherapeutic treatment, a metal radionuclide suitable for the marker reaction should be selected, as indicated above. In use, the composition, optionally after dilution with a pharmaceutically acceptable liquid, is administered to the warm-blooded animal in an amount effective to fight or control tumors. Since the radiopharmaceutical composition according to the invention can be prepared easily and simply, said preparation can be carried out particularly easily by the user himself. Therefore, the invention also relates to what is referred to as a "kit", comprising: (1) in an optionally dry condition, a composition of a protein conjugate that is formed by reaction of a protein or a material proteinase with a poly functional agent, as described here above; (2) a solution of a salt or chelate of a metallic solid radionuc; and (3) the instructions for use with a prescription to react the ingredients present in the kit. A protein, hereinafter, should be understood to mean a protein that includes a proteinase material. As noted above, for this complex-forming reaction, the desired radionuclide is preferably present in the protein conjugate in the form of a chelate attached to comparatively weak chelators, for example, a pyrophosphate, a phosphonate or a polyphosphonate, a oxinate, a carboxylate, a hydroxycarbonate, an aminocarbonate, an enolate or a mixture thereof; wherein the reaction can be carried out in a neutral medium. Examples of suitable chelating agents for the radionuclide are 6-hydroxyquinoline or its 2.1 derivatives? dicarboxylic acids, carboxylic acids, or hydrocarboxylic acids, for example, oxalic acid, malonic acid, succinic acid, maleic acid, orthophthalic acid, melic acid, lactic acid, tartaric acid, citric acid, ascorbic acid, salicylic acid or derivatives of these acids; phosphonates, phosphonates or polyphosphonates, for example, methylene phosphonate, hydrogen diphosphonate or diphosphonates of hydroxides and methyles; or enolates, for example, with ß-diketone such as acetone, chloramphenol, lacetum, tenoi laceton, bensoi lacetona, d iben zolemethane, tropolane or derivatives of these diketones. They should be considered as particularly suitable as chelators, iodine, citric acid, tartaric acid, ascorbic acid, glucoheptonic acid or a derivative thereof, or acetyl lacetone; because it has been found that a chelate of a radionuclide with these chelators in a suitable medium, preferably in a regulated aqueous solution, readily reacts at a physiological pH with a protein conjugate, as defined hereinabove, forming the desired radionuclide complex by exchange of ligand in a high yield and with high purity. The supplied kit can also contain the constituents mentioned under (1) with instructions for use, while the solution of the metal radionuclide defined under (2), which has a limited shelf life, can be supplied to the user separately. In another similar, extremely favorable embodiment, the kit according to the invention is equipped to comprise the following ingredients: (1) in an optionally dry condition, a composition of a protein conjugate that is formed by reaction of a protein with a pol i functional agent, as previously defined; (2) a chelator, as described hereinabove, and a reducing agent; and (3) instructions for use with a prescription to react 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, dithionite or stannous ions. Said case is intended for the preparation of a pharmaceutical composition labeled with Tc-99m. The pertechnetate solution can simply be obtained by the user of an ibdeno-technec generator, or made available to it. A similar kit can be used for the preparation of a pharmaceutical preparation labeled with Re-166 or Re-lfi, in which the perrhenate solution can also be reduced with a suitable reducing agent, for example, a dithionite or stannous ions. . If desired, the ingredients defined above under (1) and (2) can be combined, as long as they are compatible. Said case, in which the combined ingredients are preferably injected, is extremely suitable to be reacted by the user with the solution of the irradiated solution in a simple manner. In another excellently suitable embodiment, the case according to the invention is equipped to comprise: (1) in an optionally dry condition, a poly functional agent, as defined above, as well as a chelator, such as described here before, and a reducing agent? and (2) instructions for use with a prescription to react the ingredients mentioned under (1), which are preferably accommodated in a vial, with a protein that is supplied separately to the user, and then, with tcnecía-99m in the form of a pertechnetate solution or with rhenium-166 or rhenium-ififi, in the form of a per-renato solution. By means of what is called a "multi-purpose case", the user can mark any desired protein that is made available with the radioactive technetium or rhenium, wherein the protein conjugate required for this is formed in an intermediary manner. In a modality related to the case mentioned at the end, the case according to the invention comprises: (1) a polyfunctional agent, as defined 2 · - + here before; (2) a solution of a salt or chelate of a metal radionuclide; and (3) instructions for use with a prescription to react the ingredient indicated in (1) with a protein, and then with the ingredient mentioned in (2). A metal reducing agent, for example, Sn (II), Fe (II), Cu < I), Ti (III) or Sb (III), is preferably used as a reducing agent for the aforementioned kits; Sn (II) is excellently adequate. The constituent of the kits mentioned above, mentioned under (1), can be supplied as a solution, for example, in the form of a physiological saline solution, or in some regulatory solution or otherwise, but is preferably present in a dry condition, for example, in a condition linked. When used as a component for an injection liquid, it must be sterile, where, if the constituent is present in a dry condition, the user should use a sterile physiological saline solution, as a solvent. If desired, the above-mentioned constituent can be stabilized in the usual manner with suitable stabilizers, or it can comprise other auxiliary means, such as fillers, glucose, lactose, mannitol and the like. The invention will now be described in more detail with reference to the specific examples that follow.

EXAMPLES EXAMPLE I SYNTHESIS OF 2.3- (DI-S-ACETYLMERCAPTQ) N-SUCCINIMIDYL PR0CIQNAT (SATP) The reactions described in Examples I to III are presented in the attached reaction scheme.

REACTION SCHEME The albumin label with "¡" r ^ Tc after the derivation with SATP Albumin - (NHj).

Albumin- NH-CO-CH-S-CO-CH, CH S-CO-CH3 Deprotection- (HjOH) Albumin- NH-CO-CH-SH CHj-SH Albumin- O-CH-S- CH, -S- 2,3- (Di-S-acet lmercapto &propyonic acid is prepared by reaction of 2,3-dibromopropionic acid and mercaptoacetic acid, from according to Ondetti and co-inventors (German Patent Application No. 2,752,720) To a solution of 5.05 g, 22.7 mmoles of 2, 3- (di-S-acet i lmercapto) prop in ico and 2.61 g, 22.7 mmoles, of IM-h idrox i succ in imi da 45 ml of CHSC13, add 4.66 g of 1,3-dicyclohexyl-1-carboxyidimide (22.7 mmoles) The mixture is stirred at room temperature for 1 hour. the mixture is filtered and the filtrate is evaporated, the residue is dissolved in a minimum amount of dichloromethane and refiltered to remove the traces of 1, 3-dihydrohexylurea, and the filtrate is concentrated to give 5.2 g of the title compound. title, as a light yellow oil NMR with lH (DMSO): or 2.3fi (s, 3H), 2.46 (s, 3H), 2.63 (s, 4H), 3.46 (d, 2H), 4.69 (t, 1H ).

EXAMPLE II COUPLING ALBUMIN WITH SATP To 1 ml of a 2% solution m / v of albumin (0.3 jumol, Merius) in 0.05 M phosphate buffer pH 7.5 containing 1 mM EDTA, add 10 joules of a solution containing variable amounts (3-30). jumóles) of SATP in DMSO. 2. 7 After incubating for 2 hours at room temperature, the unreacted ligand is removed by chromatography on a Sephadex column; puffing in saline solution and in elution with 0.05 M phosphate regulator pH 7.5 containing, 1 mM EDTA. The fractions containing the albumin (native and formed to derivatives) are combined. The deacetylation of the mercapto groups protected with S-acetyl is carried out, incubating for 2 hours after the addition of 0.2 ml of a mixture of deacetyl ion (50 mM sodium phosphate, 25 mM EDTA, 0.5 m of hydroxyl and lamina, pH 7.5). Further purification can be carried out by size exclusion HPLC to remove hydroxylamine, using a Biosil® SEC 250 column; Elution with phosphate buffer, pH 7. The peak with a retention time similar to that of the native serum albumin is collected and used for labeling.

EXAMPLE III MARKING OF MODIFIED ALBUMIN WITH SATP WITH TECNECI0-99M The direct labeling is carried out with "^" "Tc by adding 10 g of SnCl5a-2HaO dissolved in 5 μ? Of HC1 0.05 N and 1 ml of eluate from a Technetium generator 26 commercially containing 0.55 GBq of * 5"5'mTc- in the form of sodium pertechnetate, to the fraction purified by HPLC, containing the modified and deacetylated albumin After incubation for a few minutes, the mixture is analyzed. of reaction that is labeled and purified by previous SEC-HPLC The effluent of the column is monitored both for UV absorbance at 260 nm and for radioactivity The peak with a retention time similar to that of albumin is isolated. The exchange labeling is carried out by mixing 2 ml of the deacetylated and modified albumin solution with 0.5 ml of a gluconate solution of <5"5" T, Tc, and incubation for 20 minutes at 37 ° C. the gluconate of "Te, dissolve 20 ml of sodium gluconate in 1 ml of 0.5 M phosphate regulator pH 7, and add 100 pg of SnCl1a-2HaO in 25 JJI of HC1 0.05 N, and then 1 ml of a pertechnetate solution of ^ "" Tc containing 0.55 GBq of * 5> * 5, '"Tc . The analysis and purification are carried out as for the direct labeling reaction mixture. The product is indicated in the following examples as 99mTc-DMP-HSA.

EXAMPLE VI RETENTION IN THE 5ANBRE IN A RABBIT Radi-blocked preparations are diluted with saline at a concentration of 3.7 HBq / ml and 12 IS-albumin is added to a concentration of 0.37 MBq / ml. An albino male rabbit (2.5-3.1 kq) is sedated and 0.5 ml of the diluted tracer solution is injected through a vein in the ear. At fixed time intervals (5, 10, 15, 20, 30, 40, 50, 60, 75, 90, 105 and 120 minutes, p.i.), a blood sample of 2 ml is taken from the artery of the contralateral ear. The activity of iSBSI and "mTc, as described above, is measured and compared to the activity of a common solution of the injected preparation.The percentage of activity injected in .100 g of blood is calculated at each interval. of time, and also the ratio between the percentage of activity of the injected "" that remains in the plasma and the corresponding percentage of activity of 1 5I injected, at the same time interval. "The results are shown in figure 1. For comparison HSA is used, modified with SATA, marked with "* '* mTc. In the ordinate, the proportion between the percentage of activity of, mTc injected, remaining in the blood, and the residual percentage of iSaaI-HSA, co-injected, at the same time interval is presented. In this way, the graphs show a direct comparison between the retention in the blood of agents marked with < 5 " 5 > mTc, and the reference compound I A = aeI-lbumina- From Figure 1 it is evident that both albumin preparations modified with "I" "" '' T show a relatively higher retention in the vascular compartment that the preparation of "" "" "Tc-HSA tested. 99mTc-DMP-albumin, especially, mimics very similarly to SSI-HSA. Immediately after the injection, its retention in the blood is 96% of the 1¾SI-HSA and the diffusion from the vascular compartment is almost identical. The comparatively tested 99mTc-MA- albumin, prepared using SATA known as coupling agent, is retained in the blood at a significantly lower level, immediately after injection, and diffuses more rapidly to the extra-vascular space. For reasons of comparison, the curve obtained in a similar manner with a conventional preparation of "" T -HSA (Technescan HSA), is included in the figure.

EXAMPLE V RETENTION IN THE BLOOD IN A HUMAN BEING "'" ^ "' Tc-DMP-albumin is compared, as well as an HSA preparation labeled with < ? s', T, Tc (TechnescanRHSA), with a one-week time interval, in a healthy male volunteer. The radiolabeled preparations are diluted with a saline solution at a concentration of 3.7 MBq / ml and 0.5 ml is injected through a vein in the arm. At fixed intervals (see example IV), a blood sample of 2 ml is taken from the contralateral breasts and the activity of "5" is measured., nr, Tc as described above. The data are related to the activity in the blood at 5 minutes after the injection, which allows to compare the disappearance in the plasma of the tested preparations. The volunteer was asked to urinate at 30 minutes, 60 minutes and 120 minutes after the injection, and the percentage of injected dose excreted at those time intervals, without correction for residual urine after emptying of the bladder. The disappearance curves in the blood, obtained, are shown in figure 2. The activity in the blood at 5 ml after the invention, that is, after sufficiently mixing the substances injected with the blood is equal to 100. %, and the activities at the other time intervals are related to this value. With 99mTc-DMP-albumin (albumin derived from SATP in a 1:25 ratio), there is a small decrease in activity in the blood during the first 30 minutes after injection, but subsequently, the retention time is fairly constant. On the other hand, the blood activity of Tc-HSA decreases continuously during the two hours of the investigation.After two hours, only 60% of the initial activity is retained in the blood. The cumulative activity of "*, mTc, after administration of the prepared ions is shown in figure 3. Two hours after the injection, the percentage of the injected dose excreted in the urine is only 2% in the case of 99mTc-albumin. However, when "" ^ "" Tc-HSA is used, ..5% of the injected dose is already excreted during the first 30 minutes, and 17% after two hours.

Claims (1)

1. NOVELTY OF THE INVENTION CLAIMS A method for preparing a radionuclide labeled material that is intended for diagnostic application or by reacting a protein a proteinase material with an agent for coupling the radionuclide to the protein or material forming a protein conjugate by reaction between the agent and the free groups or free groups present in the protein or material and then complexing the radionuclide with the conjugate thus formed to a complex characterized in that method because the coupling reaction with a polynucleotide agent is carried out. Onal of the formula YR in X is a halogenated or non-halogenated alkanoyl group having 2 to 5 atoms or a substituted group or is not an aliphatic hydrocarbon radical i valent having 2 carbon atoms and wherein the chain if it can be interrupted by a nitrogen atom a group Y is at least a reactive group that is able to react with a group libr eo a mercapto group in the protein or material and n is 2 a and if necessary after the deprotection of the mercapto groups to react the protein conjugate formed with the A method according to the claim further characterized in that the coupling reaction is takes place with a political agent of the general formula R where n and have the meanings in the and is a group a group a group a group i a group i a group tr ii a group a group a group a group a alkoxycarbonyl group and whether or not an alkylated carbonyl group is sulphonated or non-sulfonated or a non-sulfonated or heterocyclic ring group or group that is attached to R with the middle ring nitrogen of a group carbonyl u and substituted in the position with a function or a function A method according to claim also in that the coupling reaction is carried out with a polyalkyl agent of the general formula 0 II C wherein Z is a hydrogen atom or an atom of R has the meaning given in and is a succ-sulfonated or non-sulfonated group or an iazole group in accordance with the method further characterized in that the radical i-valent is is selected from the group of CH IR CH CH CO I in is hydrogen or alkyl of 1 to atoms of alkylene of O to 3 atoms of is alkylene of 1 to 5 atoms and are each alkylene of 1 to atoms of straight chain o and A and they are hydrogen or CO wherein ma condition that AH is CO and that H if A is CQ A protein or a proteinaseo material marked with radionuclide characterized because they are obtained using the method according to any of the claimed ions 1 to A conjugate of preparation by reacting a protein or a proteinase agent with a poly coupling agent as described in any of claims 1 to 7 A composition that in addition to a pharmaceutically liquid carrier material a protein om radionuclide labeled proteinase labeled with said composition because it comprises a protein or a proteinase material labeled with a metal radionuclide obtained using the method according to any of claims 1 to A method for performing a radiodiaxin test characterized in that a composition according to with the claim if after dilution with a liquid pharmaceutically to a living being of blood in an amount of 100 to 30 preferably mCi to 10 by 70 weight of the radioactive radiation emitted by the being is a method of the claim in particular for ventriculograf with a composition having the active ingredient labeled technet albumin obtained using the method according to any of claims 1 to A method for subjecting a warm-blooded living being to a treatment characterized in that is administered The composition according to the claim is desired after dilution with a liquid pharmaceutically said in an effective amount to combat or control A kit for preparing a composition because in optional condition a composition of a protein conjugate that is formed by reaction of a protein or a proteinase material with a polymeric agent. of the formula wherein the symbols have the meanings given in the claim to a solution of a salt or chelate of a radionuclide and instructs them for use with a prescription for reacting the ingredients present in the kit to prepare a composition ica characteristic because in a condition optionally a composition of a protein conjugate that is formed by reaction of a protein or a proteinase material with a poly-agent of the formula wherein the symbols have the meanings given in the claim, a chelator and a reducing agent being combined the ingredients and and instructions for use with a prescription n to react the ingredients of the kit with in the form of a pertechnetate solution or with or in the form of a kit prepare a composition because in one condition an agent i of the general formula wherein the symbols have the meaning given in as well as a chelator and an agent and of use with a prescription to react the ingredients in with a pratein or a proteinaseo material and after the con in the form of a pertechnetate solution or with or in the form of a solution of A kit prepares a composition characterized in that in a condition optionally a polyational agent of the general formula wherein the symbols have the meanings given in the solution of a salt or chelate of a radionuclide and instructions for use with a prescription to make react the ingredient mentioned in with a protein or a proteinaseo material after with the ingredients mentioned in a box of The composition according to any one of claims 11 is further characterized in that it comprises a polya tional agent of the general formula II or a conjugate of formed by reaction of a protein or a material with the agent wherein n and R have the meanings given in the text. It has the meaning given in the A kit according to any of claims 11 a further characterized in that it comprises a pol i agent of the general formula IV or a conjugate formed by reaction of a protein or a material with the ionic agent where y n they have the meanings given in the claim and Z have the meanings given in the claim In testimony of which I sign the above in this city of the 3 days of the month of May of ODT MEDICAL insufficientOCRQuality