WO1992010214A1

WO1992010214A1 - Method of radioactive labelling of blood cells and kit therefor

Info

Publication number: WO1992010214A1
Application number: PCT/US1991/009244
Authority: WO
Inventors: Wim Th. Goedemans
Original assignee: Mallinckrodt Medical Inc
Current assignee: Mallinckrodt Inc
Priority date: 1990-12-12
Filing date: 1991-12-11
Publication date: 1992-06-25
Anticipated expiration: 1993-06-12
Also published as: AU9172191A

Abstract

The invention relates to a method of radioactive labelling of blood cells by treating a suspension of blood cells with a composition, obtained by adding a Tc-99m pertechnetate solution to a mixture of (i) a metallic reducing agent, (ii) an exchange ligand, (iii) a suitable buffer, if desired, and (iv) a dithiocarbamate, or to an at least substantially aqueous solution of said mixture; and by then incubating the reaction mixture so obtained, preferably at room temperature. The invention further relates to a composition for the radioactive labelling of blood cells, and to a kit therefor.

Description

Method of radioactive labelling of blood cells and kit there for.

The invention relates to a method of radioactive labelling of blood cells and to a composition therefor. The invention further relates to a method of preparing said composition and to a kit, suitable for the radioactive labelling of blood cells.

Radioactive labelled blood cells such as leukocytes and erythrocytes form an important diagnostic tool, e.g. in abscess localization and blood pool studies. Up to the present, indium-111 labelled compounds such as indium-111 oxinate, indium-111 tropolonate and indium-111 pyrithionate have been described for this purpose, e.g. in European patent specifications 17355 and 131327. Technetium-99m labelled compounds, however, are to be preferred, because of the radiation characteristics of this radionuclide and the availability of technetium-99m in the form of a

pertechnetate solution. As a matter of fact, such a

pertechnetate solution can simply be obtained by the user from a molybdenum - technetium generator which is at his disposal.

It is the object of the invention to allow the use of Tc-99m labelled blood cells for the above purposes, e.g. inflammation scintigraphy and blood pool studies, and consequently to provide a method of preparing Tc-99m labelled blood cells.

This object can be achieved according to the present invention, in that a suspension of blood cells is treated with a composition, obtained by adding a Tc-99m

pertechnetate solution to a mixture of (i) a metallic reducing agent, (ii) an exchange ligand, (iii) a suitable buffer, if desired, and (iv) a dithiocarbamate of the general formula

wherein R is a hydrogen atom, an unsubstituted or

substituted C₁-C₈ alkyl group, a C₃-C₇

cycloalkyl group, or an unsubstituted or substituted benzyl group;

R₁ is an unsubstituted or substituted C₁-C₈ alkyl group, a C₃-C₇ cycloalkyl group, or an

unsubstituted or substituted benzyl group; or wherein R and R₁ together with the N atom to which they are connected constitute a five- or six- membered heterocyclic ring, which ring may be substituted and which ring may comprise a second hetero atom selected from N, O and S; and wherein further:

n is 1 - 5 ; and

Mⁿ⁺ is a metal cation with the indicated valency; or to an at least substantially aqueous solution of said mixture; and

by then incubating the reaction mixture so obtained, preferably at room temperature.

Surprisingly it has been found, that the composition obtained as described above is very suitable for the radioactive labelling of blood cells, such as leukocytes and erythrocytes. By this procedure the blood cells remain viable and consequently can be used successfully for the above purposes. This will become apparent from the examples.

Suitable substituents of the benzyl group and of the alkyl group are: hydroxy, amino, carboxy, C₁-C₄ - alkoxy- carbonyl, C₁-C₄ - alkylcarbonyl, C₁-C₄ - alkoxy, sulfo and C₁- C₄ - alkoxysulfonyl. Suitable metallic reducing agents to be used for the preparation of the above composition are Sn(II), Fe(II), Ce(III), Cu(I), Ti(III) and Sb(III). Of these is Sn(II) excellently suitable because of its reducing ability. It has been found, that an extremely small quantity of Sn(II) as the metallic reducing agent, viz. of approx. 0.1 to approx. 10 μg of metal per 0.2 mg of dithiocarbamate, is not only sufficient, but that the labelling efficiency as well as the stability of the composition obtained are considerably improved when such a small quantity of reducing agent is used.

The dithiocarbamate used for the preparation of the above composition is preferably characterized by the general formula

wherein R' and R'₁ are both methyl groups or ethyl groups if desired substituted by hydroxy,

or R' is a hydrogen atom and R'₁ is a benzyl group, or R' and R'₁ together with the N atom to which they are connected constitute an unsubstituted or substituted heterocyclic ring selected from pyrrolidine, morpholine, thiomorpholine,

piperidine, piperazine, imidazolidine and

pyrazolidine. Suitable examples of the above preferred dithiocarbamates are dimethyldithiocarbamate, diethyldithiocarbamates, benzyldithiocarmate, pyrrolidyldithiocarbamate,

piperazyldithiocarbamate, 4-benzylpiρerazyldithiocarbamate and 4-(1-piperidino)piperidyldithiocarbamate. Benzyl and piperidino are examples of substituents of the

heterocyclic ring; suitable substituents are in general C₁-C₄ alkyl, phenyl (C₁-C₄) alkyl, C₅-C₇ cycloalkyl, and a heterocyclic group, connected to the heterocyclic ring directly or through a C₁-C₄ alkyl spacing group. Examples of suitable heterocyclic groups are pyrrolidinyl,

morpholinyl, piperidino or piperidinyl, piperazinyl, imidazolinyl and pyrazolidinyl.

The invention further relates to a composition for the radioactive labelling of blood cells, obtained as described above, and a method of preparing said composition.

The preparation of technetium-99m dithiocarbamates is the subject of various publications. Sn(II), e.g. in the form of stannous chloride, is preferred to other reducing agents such as dithionite and formamidine sulfinic acid, because these latter reducing agents are not insuspicious qua purity and are therefore generally not used in

pharmaceutical compositions. The use of Sn(II) as a

reducing agent in the preparation of Tc-99m

dithiocarbamates, however, involves stability problems: see in this connection for example the publication bf Baldas et al in Eur. J. Nucl. Med. 7, 1982, 187-189. Ballinger et al (Nucl. Med. Biol. 16., 1989, 721-725), however, have

succeeded in labelling dithiocarbamate by using stannous chloride and glucoheptonate as an exchange ligand; so apparently the complex prepared was sufficiently stable at room temperature. The labelling procedure described by Ballinger et al is as follows: Tc-99m pertechnetate was added to glucoheptonate and stannous chloride in aqueous solution (pH8), followed by addition of the dithiocarbamate solution. It has been observed, however, that this known labelling procedure yields a composition which is less suitable for the purpose in view, viz. the labelling of blood cells. Apparently this is due to the different sequence of addition, viz. first Tc-99m pertechnetate and then dithiocarbamate.

As mentioned in the above publication of Ballinger et al, technetium-99m dithiocarbamates are potential brain imaging agents, e.g. for cerebral perfusion imaging with SPECT. It should be noted, that the composition, obtained as described above, in addition can be used for brain imaging.

In addition to the above-mentioned glucoheptonic acid, mentioned in the above publication by Ballinger et al, various other chelating agents can be used as exchange ligands. Examples of suitable chelating agents are 8-hydroxyquinoline or derivatives thereof; dicarboxylic acids, polycarboxylic acids or hydroxycarboxylic acids, for example, oxalic acid, malonic acid, succinic acid, maleic, acid, ortho-phthalic 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 ß-diketone, such as acetyl acetone, furoyl acetone, thenoyl acetone, benzoyl acetone, dibenzoyl methane, tropolone or

derivatives fo 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 for the desired ligand exchange. If desired, a suitable buffer can be used, for example, a phosphate buffer, a citrate buffer, a HEPES buffer, or an ammonium hydrogen carbonate buffer.

Since the above composition according to the invention can be prepared so easily and simply, said preparation can be performed particulary readily by the user himself. In connection with the relatively short half-life of the metal-radionuclide in question, viz. technetium-99m having a half-life of 6 hours, it is convenient to the user to perform the labelling reaction with the radionuclide by himself in the clinic or clinical laboratory. For this purpose the various non-radioactive reaction components are then offered to him in a so-called cold kit. It will be obvious that the operations to be performed must be as simple as possible, so without elaborate separation or purification, in order to enable the user to prepare the radioactive labelled composition with the means available from the supplied kit.

A kit according to the present invention and suitable for the radioactive labelling of blood cells comprises the following ingredients: a metallic reducing agent,

preferably Sn(II) in a minor quantity as defined

hereinbefore, an exchange ligand, a suitable buffer, if desired, and a dithiocarbamate, equally as defined

hereinbefore. Furthermore the usual formulation agents and/or auxiliary substances may be present in the kit. The kit further, comprises instructions for use with a

prescription for reacting the ingredients of the kit with technetium-99m in the form of a pertechnetate solution. The pertechnetate solution can simply be obtained by the user from a molybdenum-technetium generator available for this purpose.

The ingredients of the kit indicated above 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 condition, for example, in a lyophilized condition. If desired, the ingredients may be stabilised in a usual manner with suitable stabilizers like ascorbic acid, gentisic acid or salts of these acids, or inositol, or other auxiliary substances such as fillers, e.g. glucose, lactulose, mannitol, and the like, may be added. In a preferred embodiment of the kit of the present invention, the ingredients are combined in one vial. Such a monocomponent kit, in which the combined ingredients are preferably lyophilized, is excellently suitable for being reacted by the user with the Tc-99m pertechnetate solution in a simple manner.

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

EXAMPLE I

Preparation of a diethyldithiocarbamate kit (DPC kit)

A diethyldithiocarbamate solution is prepared by dissolving 2 mg sodium diethyldithiocarbamate, 20 mg disodiumtartrate .2H₂O and 20 μg Sn(II) chloride in 10 ml water under oxygen-free conditions (nitrogen cabinet). The solution is dispensed in vials (1 ml/vial) and lyophilized under exclusion of oxygen.

EXAMPLE II

Preparation of a pyrrolidine dithiocarbamate kit (PDC kit) A pyrrolidine dithiocarbamate solution is prepared by dissolving 1.5 mg ammonium pyrrolidine dithiocarbamate, 20 mg disodiumtartrate .2H₂O and 20 μg Sn(II) chloride in 10 ml water under oxygen-free conditions. The solution is dispensed in vials (1 ml/vial) and lyophilized under exclusion of oxygen.

EXAMPLE III

Labelling with Tc-99m

A vial with lyophilized DDC, prepared according to

Example I, is labelled with Tc-99m by adding 1 ml Tc-99m pertechnetate solution from a commercial Mo-Tc generator. After 25 min incubation at room temperature, the labelling yield is determined by dichloromethane extraction: 98.3% of all radioactivity is found in the organic phase; this means that the labelling yield is 98.3%. A paper chromatographic analysis (methyl ethyl ketone/physiological saline

solution) confirmed the high labelling yield: >90%.

In a corresponding manner lyophilized PDC, prepared according to Example II, is labelled with Tc-99m; labelling yield 96.0%.

EXAMPLE IV

Labelling of erythrocytes

Tc99m-DDC, obtained according to Example III, is added in a quantity of 222 MBq to a 2 ml concentrated bovine erythrocytes suspension in a physiological saline solution. After 20 min incubation at room temperature the labelling yield is determined by centrifugation and

measuring of the radioactivity of the cell-fraction and the supernatant. The labelling yield is 70.5%. In a second experiment a labelling yield of 92.0% is reached.

In a corresponding experiment bovine erythrocytes are labelled with Tc99m-PDC, obtained according to Example III, the labelling yield is 61.0%.

EXAMPLE V

Labelling of leukocytes Three so-called "mixed" leukocytes suspensions of human origin in physiological saline solutions are prepared. These solutions contain 8 × 10⁹, 4 × 10⁹ and 4 × 10⁹ cells per litre, respectively. These cell suspensions are labelled with Tc99m-DDC, obtianed according to Example III, by adding this labelling composition in a quantity of 370 MBq to 2 ml of the cell suspensions. Incubation one hour at room temperature. The labelling yields, determined as described in Example IV, are 79, 49 and 52%, respectively. The viability of the labelled cells is measured by the capacity of retaining the dye tryptophane blue. This capacity is undistrubed; conclusion: the cells remain viable.

In a corresponding way leukocytes suspensions are labelled with Tc99m-PDC, obtained according to Example III; the labelling yields are 70, 62 and 44%, respectively.

EXAMPLE VI

Preparation of a diethyldithiocarbonate kit (DDC kit) containing inositol.

A diethyldithiocarbamate solution is prepared by dissolving 2 mg sodium diethyldithiocarbamate, 20 mg disodium tartrate 2H₂O, 20 mg Sn(II)chloride and 100 mg inositol in 10 ml 15m NH₄HCO₃ solution under oxygen-free conditions (nitrogen cabinet). The solution is dispensed in vials (1 ml/vial) and lyophilized under exclusion of oxygen.

EXAMPLE VII

Stability testing of inositol containing diethyldithiocarbamate kits.

The stability of inositol containing DDC kits was tested by incubation of these kits at 4 C and -26 C for 12 months. The 99mTc labelling ability was tested at intervals. A comparison was done with DDC kits without inositol. At 4ºC all DDC kits are unstable if stored for longer than a month. At -26ºC the inositol containing kits are stable for 12 months. The kits without inositol are stable for only 2 months.

Claims

1. A method of radioactive labelling of blood cells, characterized in that a suspension of blood cells is treated with a composition, obtained by adding a Tc-99m pertechnetate solution to a mixture of (i) a metallic reducing agent, (ii) an exchange ligand, (iii) a suitable buffer, if desired, and (iv) a dithiocarbamate of the general formula

wherein R is a hydrogen atom, an unsubstituted or

substituted C₁-C₈ alkyl group, a C₃-C₇

cycloalkyl group, or an unsubstituted or substituted benzyl group;

R₁ is an unsubstituted or substituted C₁-C₈ alkyl group, a C₃-C₇ cycloalkyl group, or an unsubstituted or substituted benzyl group; or wherein R and R₁ together with the N atom to which they are connected constitute a five- or six- membered heterocyclic ring, which ring may be substituted and which ring may comprise a second hetero atom selected from N, 0 and S; and wherein further:

n is 1-5; and

Mⁿ⁺ is a metal cation with the indicated valency; or to an at least substantially aqueous solution of said mixture; and by then incubating the reaction mixture so obtained, preferably at room temperature.

2. A method as claimed in claim 1, characterized in that said composition is obtained by adding a Tc-99m pertechnetate solution to the mixture as defined in claim 1 or to said aqueous solution thereof, wherein the reducing agent is Sn(II) in a quantity of approx. 0.1 to approx. 10 μg of metal per 0.2 mg of dithiocarbamate.

3. A method as claimed in claim 1 or 2, characterized in that said composition is obtained by adding a Tc-99m pertechnetate solution to the mixture as defined in claim 1 or to said aqueous solution thereof, in which the

dithiocarbamate is an alkali metal salt, an alkaline earth metal salt, a magnesium salt of an ammonium salt of a dithiocarbamic acid of the general formula

wherein R' and R'₁ are both methyl groups or ethyl groups, if desired substituted by hydroxy,

piperidine, piperazine, imidazolidine and

pyrazolidine.

4. A composition for the radioactive labelling of blood cells, obtained by adding a Tc-99m pertechnetate solution to a mixture of a metallic reducing agent, an exchange ligand, a suitable buffer, if desired, and a dithiocarbamate of the general formula I, presented in claim 1, wherein the symbols have the meanings given in claim 1, or to an at least substantially aqueous solution of said mixture, and by then incubating the reaction mixture so obtained, preferably at room temperature.

5. A composition as claimed in claim 4, obtained by adding a Tc-99m pertechnetate solution to the mixture as defined in claim 1 or to said aqueous solution thereof, wherein the reducing agent is Sn(II) in a quantity of approx. 0.1 to approx. 10 μg of metal per 0.2 mg of

dithiocarbamate.

6. A composition as claimed in claim 4 or 5, obtained by adding a Tc-99m pertechnetate solution to the mixture as defined in claim 1 or to said aqueous solution thereof, in which the dithiocarbamate is an alkali metal salt, an alkaline earth metal salt, a magnesium salt of an ammonium salt of a dithiocarbamic acid of the general formula II, presented in claim 3, wherein the symbols have the meanings given in claim 3.

7. A method of preparing a composition as claimed in claim 4, characterized in that a Tc-99m pertechnetate solution is added to a mixture of a metallic reducing agent, an exchange ligand, a suitable buffer, if desired, and a dithiocarbamate of the general formula I, presented in claim 1, wherein the symbols have the meanings given in claim 1, or to an at least substantially aqueous solution of said mixture, after which the reaction mixture so obtained is incubated, preferably at room temperature.

8. A method as claimed in claim 7, characterized in that as a reducing agent Sn(II) is used in a quantity of approx. 0.1 to approx. 10 μg of metal per 0.2 mg of dithiocarbamate.

9. A method as claimed in claim 7 or 8, characterized in that as a dithiocarbamate is used an alkali metal salt, an alkaline earth metal salt, a magnesium salt or an ammonium salt of a dithiocarbamic acid of the general formula II, presented in claim 3, wherein the symbols have the meanings given in claim 3.

10. A kit for the radioactive labelling of blood cells, characterized in that the kit comprises a metallic reducing agent, an exchange ligand, a suitable buffer, if desired, and a dithiocarbamate of the general formula I, presented in claim 1, wherein the symbols have the meanings given in claim 1, optionally one or more formulation agents and/or auxiliary substances, and instructions for use with a prescription for performing the method as claimed in claim 7.

11. A kit as claimed in claim 10, comprising as a reducing agent Sn(II) in a quantity of approx. 0.1 to approx. 10 ug of metal per 0.2 mg of dithiocarbamate

12. A kit as claimed in claim 10 or 11, comprising as a dithiocarbamate an alkali metal salt, an alkaline earth metal salt, a magnesium salt or an ammonium salt of a dithiocarbamic acid of the general formula II, presented in claim 3, wherein the symbols have the meanings given in claim 3.