The invention relates to a method of preparing a diagnostic agent for detecting and locating inflammations in a warm-blooded living being by attaching a detectable label to an immunospecific protein or proteinaceous substance. The invention further relates to a diagnostic agent obtained by using said method, to a pharmaceutical composition comprising said diagnostic agent, to the use of said composition and to a kit for preparing said pharmaceutical composition. Inflammations in the body of a warm-blooded living being cause many diseases and disorders, and may even turn out to be life-threatening. In order to be able to achieve a specific therapy, the detection and location of the inflammation site(s) in an early stage is of the utmost importance. A good diagnostic agent is also indispensable for supporting the therapy used. Various requirements have to be imposed on such a diagnostic agent, for example, non-toxic, no adverse influence on the host resistance and/or therapeutic treatment, well detectable and selective.

The required high selectivity means that the diagnostic agent, after having been introduced into the body, must accumulate selectively at the site of the inflammation to be detected. In order to be detectable from outside the body, the diagnostic agent should be labelled, preferably with a radionuclide or with a paramagnetic metal isotope. In the former case, the radioactive radiation can be detected by using a suitable detector (scanning). Modern techniques in this field use emission tomography; when gamma radiating isotopes are used, the so-called single photon emission computerized tomography (SPECT) may be applied. The use of paramagnetic diagnostic agents enables a detection by means of imaging by magnetic resonance (magnetic resonance imaging).

European Patent Application 241 106 describes the use of labelled immunoglobulins or fragments thereof for diagnosing inflammations. As is stated in said patent specification, immunoglobulins tend to accumulate more strongly in the inflamed sites than in the non-inflamed sites. As a result of this selectivity, i.e. a comparatively stronger concentration at the site of the inflammation, labelled immunoglobulins may be used for diagnostic imaging. From the examples of said patent specification a notable preference of radioactive-labelled immunoglobulins indeed appears for accumulation in inflamed sites over uptake in non-inflamed sites of the body.

However, the selectivity, i.e. the specific accumulation of the above-described labelled immunoglobulins in the inflamed sites, is still unsatisfactory in practice.

Selectivity is to be understood to mean the target-to-non- target ratio. This means that a comparatively large amount of radioactivity is found in the non-target tissues, e.g. in the blood, as a result of which the image of the inflammation to be examined is obscured. Moreover, when a considerable quantity of radioactivity remains circulating, the locating of inflammations is disturbed in case these are present in certain sites of the body. As a result of this a good imaging, in particular in an early stage of the inflammation, is disturbed so that a correct diagnosis is impeded. Therefore there exists a need for agents for diagnosing inflammations with a better selectivity than the above-described labelled immunoglobulins known for this purpose-

It has now been found surprisingly that said need can be met by using a protein or proteinaceous substance, which prior to the labelling procedure has been purified by using a system comprising an anion exchange separating substance. Consequently the present invention relates to a method of preparing a diagnostic agent as defined in the opening paragraph, and is characterized in that prior to the labelling procedure the protein or proteinaceous substance is purified by using a system comprising an anion exchange separating substance.

Suitable anion exchange separating substances which can be used for the method of the invention are, in particular, anion exchange resins. The use of anion exchange resins instead of bacterial proteins such as protein-A is advantageous in that no detrimental bacterial endotoxins or other pyrogens are applied by intravenous administration in a human being.

An example of a suitable system comprising an anion exchange resin is Mono- . According to a preferred procedure, the purification is carried out by subjecting a solution of the protein or proteinaceous substance to be purified, preferably in a suitable aqueous buffer solution, to anion exchange chromatography. In the chromatographic process said system, comprising an anion exchange resin, is used as the immobile phase and the solution of the protein or proteinaceous substance as the mobile phase. By eluting the immobile phase with a suitable eluent under appropriate conditions the purified protein or proteinaceous substance can be isolated and then be labelled at each desired instant.

After purification as described above, the protein or proteinaceous substance should be labelled by attaching a detectable label thereto. The detectable label may be a radionuclide, preferably selected from the group consisting of 1-123, 1-131 , Br-

75, Br-76, Tc-99m. Pb-203, Ga-67. Ga-68, As-72, ln-1 1 1. ln-1 13m. Ru-97, Cu-62, Cu-64, Cu-67, Fe-52, Mn-52m and Cr-51 , or a paramagnetic label, preferably

selected from the group consisting of F-19, D, Na-23, P-31 , Gd-157, Mn-55, Dy- 162, Cr-52 and Fe-56. The label may be attached to the purified protein or proteinaceous substance directly, if desired after a reductive treatment of said protein or proteinaceous substance, or through a suitable linker. Direct labelling of the protein with radioactive halogen can be carried out by reacting the protein with a suitable compound of the radionuclide in a preferably aqueous solution, if desired in the presence of an oxidant. Suitable radioactive halogen compounds are radioactive alkali halogenides or radioactive halo-substituted aromates, such as tyrosine. The direct labelling of the protein with a metal radionuclide has two disadvantages, via (i) that the biologically active site of the protein needed for a good selectivity can easily be blocked by said reaction so that the normal behaviour of the biologic macromolecule is disturbed, and (ii) that the affinity between metal isotope and macromolecule is often insufficient as a result of which the formed bond is insufficiently stable to remain intact under physiological conditions. Therefore in that case a reductive treatment of the protein, as suggested e.g. in European patent applications 237150 and 271806, is preferred. In such a pretreatment the disulphide bonds in the protein are reduced with a suitable agent, e.g. dithiothreitol, dithioerythritol, 2-mercaptoethanol or 2-mercaptoethγlamine, after which the reduced protein, now comprising free mercapto groups, may be reacted with a salt or chelate of the desired metal isotope. Stabilization of the reduced protein or the final complex may be desired. It is generally recognized, however, that the reductive pretreatment of the protein may exert an unfavourable influence on the biological behaviour of the protein. To avoid this disadvantage, it is proposed in the Netherlands patent application 9001 137 in the name of Academisch Ziekenhuis Leiden to use an effective amount of a borohydride in the labelling with technetium-99m, in order to obtain a Tc-99m labelled protein or proteinaceous substance which is excellently suitable for the purpose in view. Equally suitable is the use of a suitable linker or coupling agent for preparing a metal-radionuclide-Iabelled protein or proteinaceous substance, e.g. as described in the Intern, patent application (PCT) WO 89/07456 and the European patent application 78125, both in the name of Mallinckrodt Inc. The protein or proteinaceous substance to be labelled is then first modified by treating with a coupling agent, after which the resulting protein conjugate is brought into a complexing reaction with a salt or chelate of the desired metal isotopes. In literature various different coupling agents are described, via compounds which after coupling with the protein can complex the metal isotope by an N ₂ S ₂ -, N ₃ S- or N ₄ -

T/US93/03586

tetradentate ring structure, N-containing di- or polyacetic acids or their derivatives such as EDTA, DTPA, NTA, etc., amino-containing compounds such as the maleimide derivatives disclosed in the above EP 178125, peptide-derivatives, and compounds comprising chelating groups such as isocyanate, formyl, diazonium, isothϊocyanate, alkoxycarbimidoyl groups and the like. For the modification of the protein or proteinaceous substance, purified according to the invention, however, a coupling agent is preferred which is described in the above WO 89/07456 and which may generally be represented by the formula , - R - ^ ' ^\ -_ s- Y-'' wherein

R is a branched or non-branched, optionally substituted hydrocarbyl radical, which may be interrupted by one or more hetero-atoms selected from N, 0 and S and/or by one or more NH groups, and Y is a group which is capable of reacting with a functional group of the protein and which is preferably selected from the group consisting of carbonyl, carbimidoyl, N-CC,-C _β ) alkylcarbimidoyl, N-hydroxycarbimidoγl and

N-(C _t -C _β ) alkoxycarbimidoyl, or a water-soluble salt of this ring compound. Examples of suitable coupling agents described in said Intern, patent application are substituted or unsubstituted 2-iminothiolanes and 2-iminothiacγclohexanes.

The modification of the protein or proteinaceous substance, i.e. the reaction with the coupling agent resulting in the protein conjugate, can generally be carried out in a simple manner. In the subsequent complex forming reaction, the metal isotope is presented to the protein conjugate in the form of a salt or chelate. In the latter case relatively weak chelators are used, e.g. a phosphonate or polyphosphonate, an oxinate, a carboxylate, a hydroxycarboxylate, an aminocarboxylate or an enolate. Then the desired complex is formed by ligand exchange. The complex forming reactions can generally be carried out in a simple manner and under conditions which spare the protein or proteinaceous substance. The above-mentioned labelling with Tc-99m in the presence of a borohydride can equally be carried out in a simple manner and under very moderate conditions, e.g. at room temperature.

The method of the invention relates more in particular to the preparation of a diagnostic agent for detecting and locating inflammations, by labelling human immunoglobulin, a subclass of human im unoglobulin or a suitable fragment of human immunoglobulin, said immunoglobulin (fragment) being purified by using a system comprising an anion exchange separating substance.

S

The invention further relates to a diagnostic agent obtained by using the method as defined above and to a pharmaceutical composition which comprises, in addition to a pharmaceutically acceptable carrier, a diagnostic agent obtained as defined above. Such a pharmaceutical composition is intended for diagnostic application. If desired the composition so obtained can be brought into a form more suitable for intravenous or subcutaneous application, e.g. by adding a pharmaceutically acceptable liquid carrier material. For intravenous or subcutaneous application the solution should of course be in a sterile condition.

For performing a diagnostic examination the composition, as described above, if desired after dilution with a pharmaceutically acceptable liquid, preferably a physiological saline solution, can be administered to a warm-blooded living being in a quantity sufficient for externally imaging said being to detect and locate an inflammation in said being. In case a radioactive labelled protein or proteinaceous substance is used as a diagnostic agent, the radioactive material is generally administered to the living being in a quantity from 1 to 2000 MBq, preferably from

100 to 1200 Mbq, per 70 kg of body weight. Thereupon the being is subjected to external imaging to detect accumulated radioactivity and thus to determine the location thereof in the body of the being. Both sterile and asterile inflammations can be detected by using the above compositions. An example of the former is rheumascintigraphy; bacterial inflammations and abscesses are examples of asterile inflammations.

In case a radioactive labelled protein or proteinaceous substance is used as a diagnostic agent, it is frequently impossible to put the ready-for-use composition at the disposal of the user, in connection with the often poor shelf life of the radiolabelled compound and/or the short half-life of the radionuclide used. In such cases the user will carry out the labelling reaction with the radionuclide in the clinical hospital or laboratory. For this purpose the various reaction ingredients are then offered to the user in the form of a so-called "kit". It will be obvious that the manipulations necessary to perform the desired reaction should be as simple as possible to enable the user to prepare from the kit the radioactive labelled composition by using the facilities that are at his disposal. Therefore the invention also relates to a kit for preparing a radiopharmaceutical composition.

Such a kit according to the present invention may comprise (i) an immunospecific protein or proteinaceous substance purified as described above, to which substance, if desired, an inert pharmaceutically acceptable carrier and/or formulating agents and/or auxiliary substances is/are added, (ii) a solution of a compound of a radionuclide, and (iii) instructions for use with a prescription for

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reacting the ingredients present in the kit. If in such a kit the radionuclide is radioactive halogen, preferably a halogen compound as defined hereinbefore is used as an ingredient of the kit.

Alternatively, such a kit according to the present invention may comprise (i) a substance obtained by treating a human immuno-specific protein or proteinaceous substance purified as described above, with a suitable disulphide-reducing agent as defined hereinbefore, to which substance, if desired, an inert pharmaceutically acceptablecarrier and/or formulation agents and/or auxiliary substances is/are added, (ii) a solution of a salt or chelate of a metal-radionuclide, and (iii) instruction for use with a prescription for reacting the ingredients present in the kit. A suitable carrier is a physiological saline solution. Examples of auxiliary substances are stabilizers, antioxidants and filling agents. Suitable metal-radionuclides have been mentioned hereinbefore. As stated above, the desired radionuclide for this complex forming reaction prescribed may be presented to the reduced protein in the form of a chelate, bound to a comparatively weak chelator, in which the reaction may take place in a neutral environment, e.g. a buffered substantially aqueous solution. The kit to be supplied to the user may also comprise the ingredient(s) defined sub (i) above, together with instructions for use, whereas the solution of the salt or chelate of the radionuclide, defined sub (ii) above, which solution has a limited shelf life, may be put to the disposal of the user separately.

In case the kit serves to prepare a radiopharmaceutical composition labelled with technetium-99m, such a kit according to the present invention may comprise, in addition to the ingredient(s) defined sub (i) above, (ii) a pertechnetate-reducing agent and, if desired, a chelator, and (iii) instructions for use with a prescription for reacting the ingredients of the kit with technetium-99m in the form of a pertechnetate solution. If desired, the ingredients of the kit may be combined, provided they are compatible. The kit should comprise a reducing agent to reduce the pertechnetate, for example, a dithionite, a metallic reducing agent or a complex- stabilizing reducing agent, e.g. Sn(II)-tartrate,Sn(ll)-phosphonate or -pyrophosphate, or Sn(ll)-glucoheρtonate. The pertechnetate can simply be obtained by the user from a suitable generator. Examples of suitable chelators have been described hereinbefore.

In a preferred embodiment the kit according to the present invention and intended for the preparation of a Tc-99m labelled composition comprises in addition a borohydride in a quantity effective for the labelling procedure.

In an equally preferred embodiment the kit according to the present invention comprises, instead of a reduced protein or proteinaceous substance, a protein

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conjugate, obtained by modifying an immunospecific protein or proteinaceous substance, purified as described hereinbefore, by a treatment with a coupling agent. Suitable coupling agents have been described hereinbefore. The use of a compound of the general formula . _^ -R- - .

wherein the symbols have the meanings given hereinbefore, as a coupling agent is to be preferred.

When the radionuclide is present in the kit itself, the complex forming reaction with the protein conjugate can simply be produced by combining the components in a neutral medium and causing them to react. For that purpose the radionuclide is preferably presented to the protein conjugate in the form of a chelate bonded to a comparatively weak chelator. Examples of suitable chelators for the radionuclide are 8-hγdroxquinoline or derivatives hereof; dicarboxylic acids, polγcarboxγlic acids or hydroxycarboxylic acids, for example, oxalic acid, malonic acid, succinic acid, maleic acid, orthophtalic 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 disphosphόnate or hydroxymethylene diphosphonate; or enolates, for example, with a β-diketone, for example, acetyl acetone, furoyl acetone, thenoyl acetone, benzoyl acetone, dibenzoyl methane, tropolone or derivatives of these diketones. For this purpose are to be considered in particular 8-hydroxyquinoline, citric acid, tartaric acid, ascorbic acid, glucoheptonic acid or a derivative thereof, or acetyl acetone as chelators because it has been found that a chelate of a radionuclide, for example, indium- 1 1 1 or lead-203, with one of these chelators in a suitable medium, preferably a buffered aqueous solution, easily reacts at a physiological pH with a protein conjugate as defined hereinbefore, the desired radionuclide complex being formed in a high yield and purity by ligand exchange. A buffered aqueous indium-1 1 1- tropolonate solution which may be used for the desired complex formation and is suitable for this purpose is described in European patent application no.

131327. The supplied kit may also consist of the constituent defined sub (1 ) with instructions for use, while the solution mentioned sub (2), which is bound to an expiration date, can be placed at the user's disposal separately.

When the kit in addition comprises a borohydride, said borohydride is preferably NaBH ₄ or NaBH ₃ CN. A quantity of 0.01-1 ug of borohydride is generally sufficient for labelling 0.1 mg of the protein or proteinaceous substance. Preferably also a pertechnetate-reducing agent is present in such a kit, e.g. Sn(ll).

When the kit comprises a protein conjugate as defined hereinbefore and is intended for the preparation of a radiopharmaceutical composition, labelled with technetium-99m, the radionuclide will preferably be added separately in the form of a pertechnetate solution. In that case the kit will comprise a pertechnetate-reducing agent and, if desired, a chelator, the former to reduce the pertechnetate. As a pertechnetate-reducing agent may be used, for example, a dithionite or a metallic reducing agent. The ingredients may optionally be combined, provided they are compatible. Such a monocomponent kit, in which the combined ingredients are preferably freeze-dried, is excellently suitable for being reacted, by the user, with the radionuclide solution. As a reducing agent for the above-mentioned kits is preferably used a metallic reducing agent, for example, Sn(ll), Fe(ll), Cud), Ti(lll) or Sb(lll); Sn(ll) is excellently suitable. The protein constituent of the above-mentioned kits, r.e. preferably the protein conjugate, may be supplied as a solution, for example, in the form of a physiological saline solution, or in some buffer solution, but is preferably present in a dry state, for example, in the freeze-dried state. When used as a component for an injection liquid it should be sterile, in which, when the constituent is in the dry state, the user should preferably use a sterile physiological saline solution as a solvent. If desired, the above-mentioned constituent may be stabilized in the conventional manner with suitable stabilizers, for example, ascorbic acid, gentisic acid or salts of these acids, or it may comprise other auxiliary agents, for example, fillers, such as glucose, lactose, mannitol, an the like.

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

EXAMPLE

To show the results of purification of human immunoglobulin, by using Mono-Q® as the system comprising an anion exchange resin, the following experiments are carried out.

To be able to determine the results of the purification procedure, a protein is used, labelled with Tc-99m as described in the Intern, patent application WO

89/07456, mentioned hereinbefore. This product is prepared from a lyophilϊzed kit containing 1 mg 2-ϊminothiolane-modϊfϊed polyclonal human immunoglobulin and stannous tartrate, marketed by Mallinckrodt Medical B.V. under the registered :rade nameTechnescan HIG, and technetium-99m in the form of a pertechnetate solution, obtained from a molybdenum-technetium generator. ,

The above labelled human immunoglobulin (Ig) is purified by anion exchange chromatography by using a Mono C column. This column is regenerated with 100

E SHEET

ml 2M NaOH solution and then flushed with an elution buffer, viz. TRIS 20 mM.

1 mg of human immunoglobulin (Technescan HIGP) is labelled with 100 MBq of ^99m Tc-pertechnetate in 1 ml, at room temperature according to the product insert.

The labelled protein is brought on the regenerated Mono CP* column and the protein is fixed on the column with a 20 mM TRIS solution. The protein fractions are eluted from the column using mixtures of 25/75%, 50/50% and 0/100% of 20 mM TRIS and 1 M NaCI, resp.

The fractions obtained are incubated with 3.8x10 ⁷ CFU of Staphylococcus Aureus (St. Aureus) ATCC 25923 in 2 ml growth medium for 1.2 and 5 hours. The labelling of the bacterial pellet is measured in a well-type scintillation counter and reported as percentages of total activity bound. The following results are obtained: Table A.

Table A

- .

Separately, the fractions obtained are injected in mice, infected with 2x10 ⁷ CFU of St. Aureus 25923 in one thigh 18 hours before. After 15 minutes, 1 hour, 4 houri, and 24 hours, via a region of interest (ROD method, the ratios of injected-to- normal thigh are determined.

As a control, non-purified labelled HIG is used. The results are recorded in Table B below.

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