Method for the preparation of technetium or rhenium complex for radiopharmaceuticals

The present invention relates to a method of preparing technetium or rhenium complex for radiopharmaceuticals, through reaction of pertechnetate or perrhenate with a ligand in the presence of a reducing agent, wherein the reducing agent is a borohydride exchange resin (BER). Also, the present invention is concerned with a kit for radiolabelling with technetium or rhenium.

It is known that technetium(Tc)-99m ( 99m Tc), as a radionuclide, has desirable nuclear properties, for example, a short half-life of 6 hours and 140 keV gamma ray emission energy suitable for obtaining gamma image, as well as a low price and a general utility, thus is commonly applied in nuclear medicine as radiopharmaceuticals for diagnosis and therapy. Tc-99m forms a stable complex with a compound having an unshared electron, such as isocyanate, amine, carboxy or thiol groups, and is thus used as an imaging agent or a radiolabelling agent for organs including lungs, liver and brain, or their tissues.

As isotopes of rhenium(Re), which is an analogous atom to Tc-99m, rhenium(Re)-186 ( 186 Re) and rhenium-188 ( 188 Re) are well known. The rhenium isotopes emit both beta ray suitable for therapy and gamma ray capable of imaging organs or living tissues. In practice, rhenium-186 and rhenium-188 are applied for alleviating pain in bone caused by secondary transition of various cancers, including prostate gland cancer, lung cancer and breast cancer, to bone. In addition, since Re-186 and Re-188 have similar chemical properties to technetium-99m, they can be applied for the radiolabelling methods using rhenium through improvement of the radiolabeling methods using Tc-99m (Lin, W. et al., Eur. J. Nucl. Med . 1997, 24, 590-595; Lewington, V. J. et al., Eur. J. Nucl. Med . 1993, 20, 66-74; Lewington, V. J. et al., Phys. Med. Biol . 1996, 41, 2027-2042; Hashimoto, K. et al., Appl. Radiat. Isot . 1996, 47, 195-199).

Using the metals technetium or rhenium, a target compound can be labeled through a process of forming a complex of a ligand with technetium or rhenium, and then performing a substitution reaction with another ligand. For example, when a lyophilized glucoheptonate is reacted with 99m Tc-sodium pertechnetate, 99m Tc-glucoheptonate complex is formed, and an active site of the complex is demonstrated to be [Tc v =O] 3+ (Owunwanne, A. et al., The Handbook of Radiopharmaceuticals , Chapman & Hall Medical, London, UK, p. 94-95)

Based on this finding, production of technetium-labeled complex and its structure can be identified by performing a transchelation reaction with another ligand having an affinity higher than the bound ligand, glucoheptonate, and then by confirming a peak at a homologous position to [Tc v =O] 3+ through thin layer chromatography (TLC) or reverse-phase high performance liquid chromatograph (HPLC).

Also, there was another attempt to form 99m TcNCl 4 − precursor, where pertechnetate or perrhenate is refluxed with sodium azide (NaN 3 ) in the presence of strong HCl to form 99m TcNCl 4 − complex, and [ 99m Tc v ≡N] 2+ complex may be formed via a ligand-exchange reaction from the complex 99m TcNCl 4 − (John Baldas and John Bonnyman Int. J. Appl. Radiot. Isot ., 1985, 36, 133-139; Florian Demaimay, Leontine Dazord, Alain Roucoux, Nicolas Noiret, Herri Patin and Annick Moisan, Nuclear Medicine & Biology , 1997, 24, 701-705).

In addition, Alberto, et al. disclose that 99m Tc-tricarbonyl complex of the low oxidation state(+1), a precursor which may be used in radiolabeling biomaterials, can be formed (Alberto R. et al., J. Am. Chem. Soc ., 1998, 120, 7987-7988; Egli A. et al., J. Nucl. Med ., 1999, 40(11), 1913-1917; Alberto R. et al., Radiochimica Acta ., 1997, 79, 99-103; Alberto R. et al., J. Organometallic Chem ., 1995, 493, 119-127; Reisgys M. et al., Bioorganic & Medicinal Chemistry Letters , 1997, 7(17), 2243-2246).

As described above, technetium or rhenium should be reduced prior to reaction of pertechnetate or perrhenate with a ligand to form complexes.

The reduction of Tc or Re may be achieved by electrolysis, or mostly in the presence of a reducing agent, including SnCl 2 .2H 2 O, ferrous ion, ferrous-ascorbate, formamidinesulphinic acid and sodium borohydride. The most commonly used reducing agent is tin chloride dihydrate (SnCl 2 .2H 2 O)

However, the reducing agent described above, tin chloride dihydrate (SnCl 2 .2H 2 O) is stable in an acidic condition, but it induces formation of precipitates in an alkaline condition. In contrast, sodium borohydride is stable at high pH, while being unstable at low pH. In addition, the reducing agents described above are typically utilized in a liquid state, when they are used excessively, impurities such as colloids can be generated, in addition to a danger of potentially residual toxicity, thereby limiting the amount of used reducing agents. These problems can be overcome through employment of a borohydride exchange resin to which borohydride ion (BH 4 − ) is bound, with which the metal Tc or Re is reduced in a solid phase, and after that, borohydride ion can be removed by filtration, regardless of its used amount.

Because the reduction of Tc or Re has been achieved in stringent conditions as described above, there is a need for developing methods capable of reducing pertechnetate or perrhenate in a wide range of pH 2 to 14.

Leading to the present invention, the intensive and thorough research into a reducing agent for preparation of technetium or rhenium complex for radiopharmarceuticals, with the aim of solving the problems of the conventional reducing agents, resulted in the finding that technetium or rhenium complex with high radiochemical purity and excellent labeling efficiency is prepared by reacting pertechnetate or perrhenate with a ligand in the presence of borohydride exchange resin as a reducing agent.

It is therefore an object of the present invention to provide a method of preparing technetium or rhenium complex for radiopharmarceuticals.

It is another object of the present invention to provide a method of preparing technetium or rhenium complex through reduction of pertechnetate or perrhenate in the presence of borohydride exchange resin.

It is a further object of the present invention to provide a kit for radiorabelling with technetium or rhenium, comprising a ligand and a borohydride exchange resin.

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

FIGS. 1 a and 1 b are results of thin-layer chromatography (TLC) of 99m Tc-glucoheptonate prepared in Step 1 of Example 1;

FIGS. 1 c and 1 d are results of TLC of 99m Tc-cysteine prepared in Example 1;

FIG. 1 e is a result of high performance liquid chromatography (HPLC) of 99m Tc-cysteine prepared in Example 1;

FIGS. 2 a and 2 b are results of TLC of 99m Tc-diamine disulfide prepared in Example 2;

FIG. 2 c is a result of HPLC of 99m Tc-diamine disulfide prepared in Example 2;

FIG. 3 a is a result of HPLC of 99m Tc-tricarbonyl precursor prepared in Example 3;

FIG. 3 b is a result of HPLC of 99m Tc-(H 2 O) 3 (CO) 3 L-cysteine prepared in Example 3;

FIGS. 4 a and 4 b are results of TLC of 99m Tc-dimercaptosuccinic acid prepared in Example 4;

FIG. 4 c is a result of HPLC of 99m Tc-dimercaptosuccinic acid prepared in Example 4;

FIGS. 5 a and 5 b are results of TLC of 99m Tc-methylene diphosphate prepared in Example 5;

FIG. 6 is a result of TLC of 99m Tc-MAMA prepared in Example 6;

FIG. 7 is a result of TLC of 99m Tc-human serum albumin prepared in Example 7;

FIG. 8 a is a result of TLC of 99m Tc-Nitrido prepared in Example 8;

FIG. 8 b is a result of TLC of 99m TcN-DADS prepared in Example 8;

FIG. 8 c is a result of HPLC of 99m TcN-DADS prepared in Example 8;

FIGS. 9 a and 9 b are results of TLC of 99m Tc-1-thio-β-D-glucose prepared in Example 9;

FIG. 9 c is a result of HPLC of 99m Tc-1-thio-β-D-glucose prepared in Example 9;

FIG. 10 is a result of TLC of 99m Tc-glycylglycylglycine prepared in Example 10;

FIG. 11 is a result of TLC of 99m Tc-human IgG prepared in Example 11;

FIG. 12 a is a result of TLC of 188 Re-diamine disulfide prepared in Example 12;

FIG. 12 b is a result of HPLC of 188 Re-diamine disulfide prepared in Example 12; and

FIG. 13 is a result of TLC of 188 Re-Nitrido prepared in Example 13.

The present invention is directed to a method of preparing technetium or rhenium complex for radiopharmarceuticals.

In more detail, the preparation of technetium or rhenium complex is achieved by reacting pertechnetate (TcO 4 − ) or perrhenate (ReO 4 − ) with a ligand in the presence of a borohydride exchange resin (hereinafter referred to as “BER”), where the BER functions to reduce pertechnetate or perrhenate, thus being capable of directly combining technetium or rhenium to the ligand to form a complex.

Therefore, the BER of the present invention, as a novel reducing agent with ability to reduce pertechnetate and perrhenate, can be applied in all fields where the conventional reducing agents are used.

The BER carries borohydride ion (BH 4 − ) bound to cation supported on the resin. The preferred cation is a quaternary ammonium functionality. The BER is preferably used in an amount sufficient for complete reduction of pertechnetate or perrhenate.

The borohydride ion (BH 4 − ) can be immobilized on any of commercially available anion exchange resins containing quaternary ammonium group. Examples of the resins useful in the present invention include polystyrene, high-density polyethylene, and amberlite.

The BER has advantages of being stable in a wide range of pH, including strong acidic and strong alkaline conditions, and thus being applicable to biomolecules, in addition the borohydride ion of the BER is easily removable by filtration when the biomolecules are applied to the BER.

The ligand useful in the present invention is any one selected from those capable of forming complex with technetium or rhenium, preferably, monodentate, and more preferably, bidentate or tetradentate, which serve to stereochemically stabilize the metal when complex is formed.

The ligand has a functional group including amine, carboxy group, thiolate, nitrido, isocyanate, alcohol, ester, halogen atom, alkoxy group, sulfonic acid, nitro group, amide group, nitrile group, and isonitrile group. In detail, the ligand is preferably selected from the group consisting of nitrido, glucoheptonate, L-cysteine, L-cysteine.HCl.H 2 O, diamine disulfide, dimercaptosuccinic acid, thio-β-D-glucose, methylene diphosphate, diethylenetriamine pentaacetic acid, and N-[2-((2-triphenyl methyl)thio)ethyl] acethyl]-S-(triphenyl methyl)-2-aminoethanethiol.

In addition, according to the present invention, because the BER is stable in a wide range of pH including extreme acidic and alkaline pH, it is directly applied to the biomolecules, thus they can be used as ligands. The preferred biomolecules are human serum albumin, peptide, and human immunoglobulin.

In accordance with the present invention, technetium (that is, Tc-99m or 99m Tc) or rhenium (that is, Re-188 or 188 Re), supplied in a form of pertechnetate or perrhenate, respectively, is reduced in the presence of the BER, thus being converted to a lower oxidation state capable of forming complex. The reduced technetium or rhenium is then reacted with a ligand to form a complex, where the reaction is performed in situ under inert gas atmosphere, for example, nitrogen atmosphere.

The method of the present invention does not have a limitation in an order of adding the ligand and pertechnetate or perrhenate.

The ligand and pertechnetate or perrhenate may be added together to the borohydride exchange resin, or according to a predetermined order. Also, pertechnetate or perrhenate can be added to a mixture of a lyophilized ligand and the BER.

The reduction of pertechnetate or perrhenate is achieved at room temperature in a proper medium depending on the used chemical reagents. Then, the reactant, which may contain a radiolabelled complex of interest, is filtered with a 0.2 μm membrane filter, yielding a product with high radiochemical purity and high labeling efficiency. In case of being suitable for use as radiopharmaceuticals, immediately after the filter-purification step, the complexes may be applied for medical imaging. When technetium is employed, technetium is typically reduced using only the BER, in which the reaction is completed in about 10 to about 30 minutes at room temperature, depending on the used chemical reagents and conditions. In contrast, since rhenium has electron density higher than technetium and thus forms stronger bonds, its reduction is preferably carried out in combination with other reducing agents used in the art, and completed in about 10 to about 30 minutes at 80 to 100° C. In accordance with embodiments of the present invention, technetium or rhenium-labeled complexes, prepared according to the method of the present invention, have a radiochemical purity of 99% and a labeling efficiency of over 95% (see, FIGS. 1 a to 13 ).

Additionally, the present invention is directed to a kit for radiolabelling with technetium or rhenium, comprising a ligand and a borohydride exchange resin. Before the kit is applied to patients, pertechnetate or perrhenate should be added thereto to form radiolabelled complex. The kit is preferably lyophilized or dried at room temperature to guarantee its stability during storage. When being impossible to be lyophilized, the kit can be stored in a frozen state. The ligand and borohydride exchange resin are preferably stored in a tightly sealed sterile vessel which contains no exoergic materials, and a reaction vessel is preferably filled with inert gas, such as nitrogen. To make sure of its stability during storage, the kit can additionally contain an additive commonly used in the art (for example, sodium citrate and manitol), use of which is well known in the art.

In accordance with the present invention, using the method of preparing technetium or rhenium radiopharmarceuticals and the kit for providing the radiopharmarceuticals, with which a complex of a ligand and Tc-99m or Re-188 is formed after reduction of pertechnetate or perrhenate metal by the borohydride exchange resin, the radiolabelled complex is produced under conditions milder than those required for the conventional reducing agents, as well as having high radiochemical purity and high labeling efficiency. Especially, when technetium is used, radiolabelled complexes with high radiochemical purity and high labeling efficiency are prepared using only the BER as a reducing agent. Thus, the conventional reducing agents can be replaced by the BER of the present invention.

In embodiments of the present invention, all of radiolabelled compounds prepared using the BER or tin chloride dihydrate (SnCl 2 .2H 2 O) as a reducing agent are distributed in bone. Those are variably distributed in vivo, depending on reducing agent. In addition, radiolabelled complexes formed using tin chloride dihydrate are predominantly distributed in brain, while those formed using the BER are dominantly distributed in liver, thus allowing selection of a reducing agent depending on organs or tissues to which they will be applied when preparing radiopharmaceuticals.

The present invention will be explained in more detail with reference to the following examples in conjunction with the accompanying drawings. However, the following examples are provided only to illustrate the present invention, and the present invention is not limited to them.

Preparation of 99m Tc-L-cysteine

Lyophilized glucoheptonate was first added to a vial containing 0.5 ml of a liquid solution of Na 99m TcO 4 (25 mCi) The reaction mixture was well mixed for about 20 sec at room temperature under nitrogen atmosphere, and the reaction was carried out until that the glucoheptonate powder was completely dissolved, thus forming 99m Tc-glucoheptonate.

A liquid solution of L-cysteine-HCl.H 2 O, prepared by dissolving 1.0 mg of L-cysteine.HCl.H 2 O in 0.1 ml distilled water, and 0.1 ml of 99m Tc-glucoheptonate (5 mCi) prepared in the Step 1 were added simultaneously to a vacuum vial containing 5.0 mg BER, and transchelation reaction was then performed. The reaction mixture was well mixed for 30 minutes at room temperature under nitrogen atmosphere. After mixing, the reaction mixture was filtered using a 0.2 μm membrane filter, thus giving 99m Tc-L-cysteine, as a technetium-labeled compound of interest.

Preparation of 99m Tc-DADS

To a vacuum vial containing 5.0 mg of BER were simultaneously added 99m Tc-glucoheptonate prepared in Step 1 of Example 1, and a liquid solution of 3,3,10,10-tetramethyl-1,2-dithia-5,8-diazacyclodecane (diamine disulfide; DADS) as a ligand, prepared by dissolving 0.1 mg thereof in 0.1 ml distilled water, and transchelation was performed according to the same method as in Example 1, resulting in formation of 99m Tc-diamine disulfide ( 99m Tc-DADS), as a technetium-labelled compound of interest.

Preparation of 99m Tc(H 2 O) 3 (CO) 3 L-cysteine

STEP 1: Preparation of a precursor [ 99m Tc(H 2 O) 3 (CO) 3 ] +

8 mg of sodium bicarbonate and 15 mg of BER were put into a 10 ml vial, and the vial was sealed with a rubber cover and held at room temperature for 30 minutes under CO gas. The vial was then supplemented with 6 ml of Tc-99m sodium pertechnetate (Na 99m TcO 4 ) (60 mCi) dissolved in physiological saline, and the reaction mixture was heated at 75° C. for 30 minutes under CO gas. After being cooled to room temperature in a chamber containing ice, and neutralized with 0.6 ml phosphate buffer (1M, pH 7.4), the reaction mixture was then filtered with a 0.22 μm membrane filter, giving a 99m Tc-tricarbonyl precursor [ 99m Tc (H 2 O) 3 (CO) 3 ] + .

0.1 ml of 10 mM L-cysteine.HCl.H 2 O was mixed with 0.9 ml of the 99m Tc-tricarbonyl precursor [ 99m Tc (H 2 O) 3 (CO) 3 ] + , prepared in the above Step 1, and the mixture was heated at 75° C. for 30 minutes. After being cooled to room temperature in a chamber containing ice, the reaction mixture was filtered with a 0.22 μm membrane filter, giving 99m Tc(H 2 O) 3 (CO) 3 L-cysteine as a technetium-labeled compound of interest.

The same procedure as in Example 4 was carried out, except that a liquid solution of sodium azide (NaN 3 ) prepared by dissolving 1.0 mg thereof in 0.1 ml distilled water, and 0.1 ml of a liquid solution of Tc-99m sodium pertechnetate (5 mCi) were simultaneously added to a vacuum vial containing 5.0 mg of BER, resulting in the formation of 99m TcNCl 4 − . 99m Tc-Nitrido was formed by substituting ligand of 99m TcNCl 4 − .

The same procedure as in Example 4 was carried out, except that 99m Tc-Nitrido prepared in Step 1 of Example 8 and a liquid solution of 3,3,10,10-tetramethyl-1,2-dithia-5,8-diazacyclodecane (diamine disulfide or DADS) as a ligand prepared by dissolving 1.0 mg thereof in 0.1 ml distilled water were simultaneously added to a vacuum vial containing 5.0 mg of BER, resulting in the formation of 99m Tc-diamine disulfide ( 99m Tc-DADS) as a technetium-labelled compound of interest.

As described hereinbefore, according to the present invention, employing the borohydride exchange resin (BER) as a novel reducing agent, technetium or rhenium complexes having high radiochemical purity as well as high labeling efficiency can be prepared by reacting pertechnetate or perrhenate with a ligand. The BER is advantageous in terms of being stable in most pH ranges including extreme acidic and alkaline pH conditions and applicable to biological materials, as well as being easily removable through filtration when being administrated, thus providing the potential to economically and effectively produce technetium or rhenium radiopharmarceuticals by replacing the conventional reducing agent requiring very stringent condition for preparation thereof.

The present invention has been described in an illustrative manner, and it is to be understood that the terminology used is intended to be in the nature of description rather than of limitation. Many modifications and variations of the present invention are possible in light of the above teachings. Therefore, it is to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.