WO1994025488A1

WO1994025488A1 - Method for preparing a metal-radionuclide-labelled protein

Info

Publication number: WO1994025488A1
Application number: PCT/US1994/005022
Authority: WO
Inventors: Alfons M. Verbruggen
Original assignee: Mallinckrodt Medical, Inc.
Priority date: 1993-05-03
Filing date: 1994-05-02
Publication date: 1994-11-10
Also published as: EP0654043A4; CA2141739A1; EP0654043A1; AU6670894A; MXPA94003278A

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 metal-radionuclide-labelled protein

The invention relates to a method of preparing a metal-radionuclide-labelled protein or proteinaceous material 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 radioactive 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, serum albumin, immunoglobulins, glycopeptides, monoclonal antibodies like antimyosin and monoclonals against tumour antigens, peptides, aminofunctions-containing hormones like somatostatin and ACTH, and other proteins suitable for this purpose, such as plasmin and plasmin derivatives, e.g., miniplas in 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 macro-molecules present therein; a good example thereof is the selective reaction of antibodies or antibody fragments with antigens present in the body. 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 proteinaceous material with a metal-radionuclide has two disadvantages. First, the biologically active site of the protein necessary for a good target organ specificity or selectivity, may easily be blocked by this reaction, so that the normal behaviour of the biological macromolecule is disturbed. In addition, the affinity between metal-radionuclide and macromolecule often is insufficient, as a result of which the formed bond is not sufficiently stable to remain intact under 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 WO87/04164 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 radionuclide species, for example, with Tc-99m-tartrate or -gluccheptonate. The disadvantage of this method is the reductive 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 macromolecules, usually proteins or proteinaceous substances, have been described which comprise chelating groups for a bond with the 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 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 to be too bulky, certainly not when used for comparatively small protein molecules. Moreover, the usually extremely sensitive protein or proteinaceous material 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 macromolecule. 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 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 the 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 conditions 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 used in US 4479930, US 4511550, US 4652519, US 4678667 and EP 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-acetylmercapto succinic anhydride (SAMSA) as bifunctional agent to prepare a technetium-labelled antibody conjugate by reaction with TcN(Hal)₄. 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. Some years ago a Patent Application was published, namely WO 89/07456, in which 2-iminothiolane (and related compounds) is 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. Recently a method of preparing a metal-radionuclide- labelled protein has been described, wherein N-succinimidyl-S-acetylthioacetate (SATA; properly: N-succinimidyl-S-acetylmercapto-acetate) and related compounds are used as bifunctional coupling agents: WO 91/07991. It has appeared, that the above polymerisation of the protein conjugate during preparation could be avoided and that an additional reduction step was not needed. By using this method, the formed protein conjugate permits a direct labelling with the desired metal-radionuclide, which is of particular advantage for a kit formulation.

It has been observed, however, that even the use of SATA as a coupling agent is not completely satisfactory, in particular with respect to the in vivo stability of the labelled protein, as appears from the blood retention of said protein determined in a living being. Apparently the radionuclide label is not perfectly firmly bound in the SATA-modified protein.

It is the objective 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 an agent for coupling the radionuclide to the protein or proteinaceous material, a protein conjugate being formed by reaction between said 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 above advantages of the method described in WO 91/07991 are maintained, but on the other hand the formed radionuclide-labelled protein does not show the disadvantage mentioned hereinbefore for SATA. This objective can be achieved by performing the coupling reaction with a polyfunctional agent of the general formula

Y - R -(SX)_n (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 a multivalent saturated aliphatic hydrocarbyl radical, having 2-20 carbon atoms and wherein the main chain, if desired, may be interrupted by a nitrogen atom; 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 proteinaceous material; and π is 2-6; and by then, if needed after deprotection of the protected mercapto groups, reacting the formed protein conjugate with the radionuclide.

Metal-radionuclides suitable for use in the method according to the invention are Tc-99m, Re-186, Re-188, In-Ill, Ga-67, As-72 and As-77. Of these radionuclides Tc-99m, In-Ill, Ga-67 and As-72 may be used for diagnostic purposes, the other radionuclides are paricularly useful in therapeutically active compositions.

The above term multivalent hydrocarbyl radical means a hydrocarbyl group having at least three free valencies, such as a trivalent, etc. radical.

The derivatisation of the protein or the proteinaceous material, i.e. the reaction with the polyfunctional agent, may be carried out in a very simple manner in a neutral medium (pH between 6.5 and 8) and at room temperature. The subsequent deprotection of the protected mercapto groups, i.e. the cleavage of protecting groups X in the above formula I, can easily be carried out immediately following the coupling reaction, preferably by a simple treatment with hydroxylamine. In the complex formation reaction the desired radionuclide is presented to the protein conjugate in the form of a salt or preferably in the form of a chelate bound to comparatively weak chelators, for example, a pyrophosphate, a 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-radionuclide.

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. Examples of suitable protecting groups X for the mercapto group are: acetyl, halogenated acetyl and substituted or non-substituted benzoyl, in which in particular electrons-withdrawing groups, such as nitro, halogen and sulpho are to be considered as suitable substituents. A suitable protein to be labelled by using the method of the present invention is albumin, e.g. human serum albumin (HSA) . It has been found, that HSA labelled with Tc-99m via the method of the invention, i.e. by using the polyfunctional coupling agent of formula I, is very suitable for certain diagnostic examinations, in particular for radionuclide ventriculography. As will become apparent from the accompanying Examples, a high retention in the vascular compartment of a human being is obtained by using a radiolabelled albumin prepared according to the method of the present invention.

The present invention also relates to the protein conjugate complexed with the radionuclide, as mentioned above, and to the protein conjugate per se, prepared as described above by reacting a protein or proteinaceous material with the polyfunctional coupling agent of formula I.

For the method according to the invention may be used more in particular a polyfunctional agent of the general formula

Y'- R -(SX)_n (II) wherein X, n and R have the meanings given hereinbefore; and

Y' is an isocyanate group, a for yl group, a diazonium group, an isothiocyanato group, an epoxyethylene group, a trichloro-s-triazinyl group, an ethylene i ino group, a halocarbonyl group, a halosulphonyl group, a malei ido group, a sulphonated or non-sulphonated alkylcarbonyloxycarbonyl group, a sulphonated or non-sulphonated 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 a thioxo function.

Suitable examples of alkylcarbonyloxycarbonyl groups and of alkylcarbonyliminocarbonyl groups are radicals derived from succinic anhydride and succinimide, respectively.

As stated herinbefore, examples of suitable protecting groups X are: acetyl, halogenated acetyl such as trifluoroacetyl, and benzoyl whether or not substituted with nitro, (C₁-C₄)alkyl, (C₁-C₄) alkoxy, halogen or sulpho. The following groups for Y' are 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 polyfunctional agent of the general formula

Y" - 8C - R -(S BCCZ₃)_n (IV) wherein Z is a hydrogen atom or a fluorine atom;

R has the meaning given above; and

Y" is a sulphonated or non-sulphonated succinimido-oxy group or a 2-thioxo-thiazolidin-3-yl group. A succinimido-oxy group is also called a

N-succinimidyl group.

Suitable examples of the above multivalent radicals

-R-CO- are:

(1) -CH₂-CH-CO- (V)

(2) R,-CH-R,-CH-R,-CO- (VI)

R- is hydrogen or Cχ-C.₄ alkyl;

R₂ is C₀-C₃ alkylene;

R₃ is C_!-C₅ alkylene; R₄, R₅ and R₆ are each individually straight or branched Cx-Cβ alkylene;

A and Ai are hydrogen or (CH₂)_B-CO-, wherein m=

0-5, with the proviso, that A = H if A-. is (CH₂)_m-CO-, and that A_x = H if A is (CH₂)_m-CO-.

Specific examples of the preferred multivalent radicals defined sub (3) and (4) above are:

co

CI — CI ci — CE ClI CE } — C_E,— co and ^ \

CE- CE CE- CE ι ι i t

The last-mentioned preferred agents are excellently suitable for use in the preparation of labelled proteins or proteinaceous materials according to the present invention. It has been found in addition that the last-mentioned polyfunctional agents can react with the amino functions of the protein selectively and without damage to the protein molecule. Specific examples of trifunctional agents included in the last-mentioned general formula IV, sub (1), may be presented by the following formulas:

wherein Z has the meaning given hereinbefore, and A₂ is hydrogen or an alkali- (or ammonium-)sulphonate group.

The invention also relates to a metal-radionuclide-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 proteinaceous material may be used directly as a radiopharmaceutical composition. If necessary, the solution may be brought into a form which is better 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 should be ensured that the protein is not damaged during the said treatment and that 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, preferably a physiological saline solution, may be administered to a warmblooded living being in a quantity from 100 μCi 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 radiotherapeutic treatment, a suitable metal-radionuclide should be selected for the labelling reaction, as indicated hereinbefore. 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", 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 polyfunctional 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 ingredients present in the kit. A protein hereafter is to be understood to mean a protein including a proteineous material. As stated 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 chelators, for example, a pyrophosphate, and phosphonate or polyphosphonate, an oxinate, a carboxylate, a hydroxycarboxylate, an aminocarboxylate, an enolate or a mixture thereof, 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 polyphosphonates, for example, methylene diphosphonate, hydroxyethylene diphosphonate or hydroxymethylene diphosphonate; or enolates, for example with a B-diketone such as acetyl acetone, furoyl acetone, thenoyl acetone, benzoyl acetone, dibenzoyl methane, tropolone or derivatives of these diketones. 8-Hydroxyquinoline, citric acid, tartaric acid, ascorbic acid, glucoheptonic acid or a derivative thereof, or acetyl acetone, are to 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 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 instructions for use, whereas the solution of the metal- radionuclide defined sub (2), having a limited shelf life, may be supplied to the user separately.

In another likewise extremely favourable embodiment 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 polyfunctional agent as defined hereinbefore; (2) a chelator as described hereinbefore and a reducing agent; and (3) instructions for use with a prescription for reacting the ingredients of the kit with technetium-9 m 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 molybdenum-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 extremely 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 polyfunctional agent 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 accomodated 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-called "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 polyfunctional 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 ingredient 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.

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 dry condition, for example, in a lyophilized condition. When used as a component for an injection liquid it should be sterile, in which, 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 may be stabilised in the usual manner with suitable stabilisers, or may comprise other auxiliary means like fillers, e.g, glucose, lactose, mannitol, and the like.

The invention will now be described in greater detail with reference to the ensuing specific examples.

EXAMPLES

Example I

Synthesis of N-succinimidyl-2,3- (di-S-acetylmercapto) propionate (SATP) The reactions described in Examples I - III are presented in the Reaction Scheme enclosed.

2,3- (di-S-acetylmercapto)propionic acid is prepared by reaction of 2,3-dibromopropionic acid and ercaptoacetic acid according to Ondetti et al. (German pat. appln. 2,752,720). To a solution of 2,3-(di-

S-acetylmercapto)-propionic acid (5.05 g, 22.7 mol) and N-hydroxysuccinimide (2.61 g, 22.7 mmol) in 45 ml of CH₂C1₂ is added 4.68 g of 1,3-dicyclohexyl-carbodiimide (22.7 mmol). The mixture is stirred at room temp, for 18 h. The mixture is filtered and the filtrate is evaporated. The residue is dissolved in a minimal amount of dichloromethane and filtered again to remove traces of 1,3-dicyclohexylurea. The filtrate is concentrated to give 5.2 g of the title compound as a clear yellow oil. Η-NMR (DMSO) :δ 2.38 (s, 3H) , 2.48 (s, 3H) , 2.83 (s, 4H) , 3 .46 (d, 2H) , 4. 69 ( t , 1H) .

Example II Coupling of albumin with SATP To 1 ml of a 2% m/V solution of albumin (0.3 μmol; Merieux) in 0.05M phosphate buffer pH 7.5 containing 1 mM EDTA is added 10 μl of a solution containing varying amounts (3-30 umol) of SATP in DMSO. After incubation for 2 h. at room temp., the unreacted ligand is removed by chromatography over a Sephadex G25M column; swelling in saline and elution with a 0.05 M phosphate buffer pH 7.5 containing ImM EDTA. The fractions containing the albumin (native and derivatized) are combined. Deacetylation of the S-acetyl protected mercapto groups is carried out by incubation for 2 h. after addition of 0.2 ml of a deacetylation mixture (50 mM sodium phosphate, 25 mM EDTA, 0.5 M hydroxylamine, pH 7.5).

An additional 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 similar retention time as native serum albumin is collected and used for labelling.

Example III Labelling of SATP-modified albumin with technetium-99m Direct labelling with ^99πTc is performed by addition of 10 μg of SnCl₂.2H₂0 dissolved in 5 μl HCL 0.05 N and 1 ml eluate of a commercial Tc-generator containing 0.55 GBq ^99mTc in the form of sodium pertechnetate to the HPLC-purified fraction containing the deacylated modified albumin. After incubation for a few min, the labelling reaction mixture is analyzed and purified by the above SEC-HPLC. the column effluent is monitored for both UV absorbance at 280 nm and radioactivity. The peak with a retention time similar to that of albumin is isolated. Exchange labelling is performed by mixing 2 ml of the deacylated modified albumin solution with 0.5 ml of a ^99mTc-gluconate solution and incubation for 20 min at 37°C. To prepare ^99mTc-gluconate, 20 mg of sodium gluconate is dissolved in 1 ml of 0.5 M phosphate buffer pH 7 and 100 μg SnCl₂.2H₂0 in 25 μl HC1 O.05N is added, followed by 1 ml of ^99mTc-pertechnetate solution containing 0.55 GBq ^99mTc. Analysis and purification are performed as for the direct labelling reaction mixture. The product is indicated in the following Examples as 99mTc-DMP-HSA.

Example IV

Blood retention in a rabbit

The radiolabelled preparations are diluted with saline to a concentration of 3.7 MBq/ml and ¹²⁵I-albumin is added to a concentration of 0.37 MBq/ml. A male albino rabbit (2.5-3.1 kg) is sedated and 0.5 ml of the diluted tracer solution is injected via an ear vein. At fixed time intervals (5, 10, 15, 20, 30, 40, 50, 60, 75, 90, 105 and 120 min p.i.) a 2 ml blood sample is taken from the artery of the contralateral ear. The ¹²⁵I- and ^99mTc-activity are measured as described above and compared with the activity of a standard solution of the injected preparation. The percentage of injected activity in 100 g of blood is calculated at each time interval and also the ratio between the percentage of injected ^99mTc-activity remaining in the plasma and the corresponding percentage of injected ¹²⁵I-activity at the same time interval.

The results are shown in Figure 1. For comparison ^99mTc-labelled SATA-modified HSA is used. In the ordinate is presented the ratio between the percentage of the injected ^99mTc-activity remaining in the blood and the residual percentage of co-injected ¹⁵I-HSA activity at the same time interval. In this way the graphs show a direct comparison between the blood retention of the ^99mTc-labelled agents and the reference compound ¹²⁵I-albumin.

From figure 1 it is evident that both ^99mTc-modified-albumin preparations show a remarkably higher retention in the vascular compartment than the tested conventional ^99mTc-HSA preparation. Especially 99mTc-DMP-albumin mimics ¹²⁵I-HSA very closely. Immediately after injection, its blood retention is 96% of that of ¹⁵I-HSA and the diffusion out of the vascular compartment is almost identical. The comparatively tested 99mTc-MA-albumin, prepared by using the known SATA as the coupling agent, is retained in the blood to a significantly lower extent immediately after injection and diffuses faster to the extravascular space. For reasons of comparison, the curve obtained in a similar way with a conventional ^99mTc-HSA-preparation (Technescan® HSA) is included in the figure.

Example V

Blood retention in a human being

^99mTc-DMP-albumin as well as a common ^99mTc-labelled HSA preparation (Technescan® HSA) are compared with a one week time interval in a healthy male volunteer. The radiolabelled preparations are diluted with saline to a concentration of 3.7 MBq/ml and 0.5 ml is injected via an arm vein. At fixed intervals (see Example IV) a 2-ml blood sample is taken from the contralateral arm and the ^99mTc-activity is measured as described above. Data are related to the activity in the blood at 5 min p.i., which allows to compare the plasma disappearance of the preparations tested.

The volunteer is asked to void at 30 min, 60 min and 120 min a.i., and the percentage of injected dose excreted at these time intervals is determined without correction for the postvoid residual urine.

The blood disappearance curves obtained are shown in figure 2. The activity in the blood at 5 min p.i., i.e. after sufficient mixing of the injected substances with the blood, is equalled to 100% and the activities at the other time intervals are related to this value. With

99mTc-DMP-albumin (albumin deivatized with SATP in a 1:25 ratio) there is a small decrease in blood activity during the first 30 min a.i., but afterwards the blood retention is quite constant. On the other hand, the blood activity of ^99mTc-HSA decreases continuously during the 2 hours of the investigation. After 2 hours, only 60% of the initial activity is retained within the blood.

The cumulative urinary excretion of the ^99mTc-activity after administration of the preparations is shown in figure 3. Two hours a.i., the percentage of the injected dose excreted in the urine is only 2% in the case of 99mTc-DMP-albumine. However, when ^99πιTc-HSA is used, 4.5% of the injected dose is already excreted during the first 30 min and 17% after 2 hours.

Claims

CLAIMS :

1. A method of preparing a metal-radionuclide labelled material which is intended for diagnostic or therapeutic application, by reacting a protein or a proteinaceous material with an agent for coupling the radionuclide to the protein or proteinaceous material, a protein conjugate being formed by reaction between said 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 to a radionuclide complex, characterised in that the coupling reaction is carried out with a polyfunctional agent of the general formula: Y-R-(SX)_n (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 a multivalent saturated aliphatic hydrocarbyl radical, having 2-20 carbon atoms and wherein the main chain, if desired, may be interrupted by a nitrogen atom or/and a carbonyl group; 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 proteinaceous material; and π is 2-6; and by then, if needed after deprotection of the protected mercapto groups, reacting the formed protein conjugate with the radionuclide.

2. A method as claimed in Claim 1, characterised in that the coupling reacton is carried out with a polyfunctional agent of the general formula

Y'-R-(SX)_n (II) wherein X, n and R have the meanings given in Claim 1; and Y' is an isocyanate group, a foππyl 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, a sulphonated or non-sulphonated alkylcarbonyloxycarbonyl group, a sulphonated or non-sulphonated 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 a thioxo function.

3. A method as claimed in Claim 1, characterised in that the coupling reacton is carried out with a polyfunctional agent of the general formula

0 0 II ⁽I y"-C-R-(SCCZ₃)_n (IV)

wherein Z is a hydrogen atom or a fluorine atom; R has the meaning given in Claim 1; and Y" is a sulphonated or non-sulphonated succinimido-oxy group or a 2-thioxo-thiazolidin-3-yl group.

4. A method as claimed in Claim 3, wherein the multivalent radical -R-CO- is selected from the group of:

(1) -CH₂-CH-CO- (V)

(2) Rj-CH-Rj-CH-Ra-CO- (VI)

wherein:

R_x is hydrogen or Ci-C₄ alkyl;

R₂ is C₀-C₃ alkylene;

R₃ is Ci-Cs alkylene; R₄, R₅ and R₆ are each individually straight or branched Ci-Cβ alkylene;

A and Ai are hydrogen or (CH₂)_m-CO-, wherein m= 0-5, with the proviso, that A = H if A_x is (CH₂)_m-CO-, and that A₁ = H if A is (CH₂)_B-CO-.

5. A metal-radionuclide-labelled protein or proteinaceous material obtained by using the method as claimed in any of the preceding claims 1-4.

6. A protein conjugate, prepared by reacting a protein or proteinaceous material with a polyfunctional coupling agent as defined in any of Claims 1-4.

7. A radiopharmaceutical composition which comprises, in addition to a pharmaceutically acceptable liquid carrier material, a metal radionuclide-labelled protein or proteinaceous material, characterised in that the composition comprises a protein or a proteinaceous material labelled with a metal-radionuclide, obtained by using the method as claimed in any of the Claims 1-4.

8. A method of performing a radiodiagnostic examination, characterised in that a composition as claimed in Claim 7, if desired after dilution with a pharmaceutically acceptable liquid, is administered to a warm-blooded living being in a quantity from 100 μCi 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.

9. A method as claimed in Claim 8, in particular for radionuclide ventriculography, characterized in that a composition is administered, containing as the active ingredient albumin labelled with technetium-99m, obtained by using the method as claimed in any of Claims 1-4.

10. A method of subjecting a warm-blooded living being to a radiotherapeutic treatment, characterised in that the composition as claimed in Claim 7, if desired after dilution with a pharmaceutically acceptable liquid, is administered to said being in a quantity effective for combating or controlling tumours.

11. A kit for preparing a radiopharmaceutical composition, comprising, (1) in an optionally dry condition a composition of a protein conjugate formed by reaction of a protein or proteinaceous material with a polyfunctional agent 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 kit.

12. A kit for preparing a radiopharmaceutical composition, comprising, (1) in an optionally dry condition, a composition of a protein conjugate formed by reaction of a protein or proteinaceous material with a polyfunctional agent of the general formula I, wherein the symbols have the meanings given in Claim 1, (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-99m in the form of a pertechnetate solution, or with rhenium-186 or rhenium-188 in the form of a perrhenate solution.

13. A kit for preparing a radiopharmaceutical composition comprising, (1) in an optionally dry condition, a polyfunctional agent of the general formula I, wherein the symbols have the meanings given in Claim 1, as well as a chelator and 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, after deprotection, with technetium-99m in the form of a pertechnetate solution or with rhenium-186 or rhenium-188 in the form of a perrhenate solution.

14. A kit for preparing a radiopharmaceutical composition comprising, (1) in an optionally dry condition, a polyfunctional agent 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 ingredient mentioned sub (1) with a protein or proteinaceous material and then, after deprotection, with the ingredients mentioned sub (2).

15. A kit as claimed in any of the claims 11-14, comprising a polyfunctional agent of the general formula II or a protein conjugate, formed by reaction of a protein or proteinaceous material with said polyfunctional agent, respectively, wherein X, n and R have the meanings given in Claim 1 and Y' has the meaning given in Claim 2.

16. A kit as claimed in any of the Claims 11-14, comprising a polyfunctional agent of the general formula IV or a protein conjugate, formed by reaction of a protein or proteinaceous material with said polyfunctional agent, respectively, wherein R and n have the meanings given in Claim 1 and Y" and Z have the meanings given in Claim 3.