EP0163701A1 - PREPARATION OF RADIOPHARMACEUTICALS BASED ON TECHNETIUM-99m/ - Google Patents

Abstract

A method of reducing a radioactive material includes depositing a reducing agent on an internal surface of a reaction vessel and contacting a solution of the radioactive material with the surface so as to obtain a radio solution -active reduced substantially free of reducing agent. Technetium (99mTc) in the form of a solution of pertechnetate ions is reduced by stannous chloride which is deposited as a coating layer on the inside of a reaction vessel to obtain a stannous chloride-free technetium solution in excess. A kit for preparing technetium-labeled binders used as a radiopharmaceutical is also described.

Description

PREPARATION OF RADIOPHAMACEUTIOALS BASED ON TECHNETIUM-99m/

This invention relates in general to nuclear medicine, and in particular it relates to radiopharmaceuticals, and to a novel method for their production. Even more particularly, this invention relates to a novel process of producing radiopharmaceuticals comprising suitable ligands "labelled" or "tagged" with radioactive technetium ( ^99m Tc).

Nuclear medicine is a specialised field of medicine practised predominantly in large hospitals which have considerable resources and equipment. Usually, patients present themselves at the hospital with a problem of a diagnostic nature and receive injections of a low dosage radioactive chemical which is known as a radiopharmaceutical. The radiopharmaceutical consists of a radioactive portion which emits radioactivity that can be detected by a suitable detector, and a ligand portion which is non-radioactive and which allows the compound to enter into chemical reactions within the patient's body. The ligand portion is selected from a range of suitable ligands, the choice being dependent upon the presumed diagnosis of the disease being investigated since it is the chemical properties of the ligand which determine the chemical reactions the radiopharmaceutical will participate in, and hence where in the body it will be distributed. After the injection into a patient of the radioactive chemical, the emitted radiation may be detected by the detector in order to follow the metabolic or other distribution pathways of the administered radioactive chemical. Thus the distribution of the radioactive chemical within the patient's body and throughout the various organs by pathological and physiological processes may be monitored and studied.

The process of attaching the radioactive portion to the ligand portion of the radiopharmaceutical is known as "labelling", and in effect results in the ligand being "tagged". One commonly used radioactive element is technetium 99m (^99mTc) (usually available in the form of the pertechnetate ion (TcO₄-)) which is metastable and has a half life of around 6 hours, decaying by means of gamma emissions which can be detected by a gamma camera. Thus, when a patient is injected with ^99mTc attached to a selected ligand, the distribution of the ligand within the patient's body may be monitored by obtaining an image from the gamma camera of the distribution of the ^99mTc in the patient. It is to be noted that throughout this specification, the terms technetium, pertechnetate ion and ^99mTc are used to denote the radioactive portion of the radiopharmaceutical or its precursor.

Technetium (^99mTc) is usually obtained from a molybdenum 99 generator in the form of a saline solution of pertechnetate ion, TcO₄- , in which the technetium is in the +7 oxidation state. Technetium in this form is not suitable for reaction with the various ligands usually used to form radiopharmaceuticals, and in general it must be reduced to the technetium cation in either the +3 or +4 oxidation state prior to the reaction with the selected ligand. The reduction of technetium is usually achieved by reacting the pertechnetate ion with a stannous chloride (SnCl₂) solution. It has been found, however, that the stannous chloride solution must be freshly prepared since it readily undergoes atmospheric oxidation, particularly in the presence of saline.

Most ^99mTc compounds used for in-vivo studies obtained by reduction using stannous chloride are prepared from commercially available kits which involve the direct reaction between the stannous ions, the technetium in the form of the pertechnetate ion, and the selected ligand, with the stannous ions being in excess to ensure that all of the pertechnetate ion is converted to a lower oxidation state. A typical commercially available kit comprises a sealed, sterile container containing a powdered mixture of stannous chloride and the selected ligand. Immediately prior to use, the seal on a sterile container is broken and a saline solution of pertechnetate ion is added to the container. Reduction of the pertechnetate ions occurs simultaneously with reaction of the reduced technetium cation with the ligand. The container is agitated to ensure complete reaction and a sample of the reaction liquid is withdrawn into a syringe which is then injected into the patient. As the injection solution is taken straight from the container, any unreacted ligand and unreacted excess stannous chloride is injected into the patient along with the desired radiopharmaceutical. Furthermore, excess stannous ions may react with the ligand itself rather than with the pertechnetate ions and therefore prevent, or hinder, the desired reaction between the reduced technetium cation and the ligand. The free stannous chloride injected into the patient as discussed above can enter into unwanted metabolic reactions and alterations during and after the studies on the patient such as, for example, following a bone scan using technetium-labelled methylendiphosphonate. Furthermore, stannous chloride present in the general body circulation makes it difficult to perform some other scan studies on the same patient within a reasonably short time.

Therefore, it is an aim of the present invention to at least alleviate some of the problems associated with the prior art methods of making radiopharmaceuticals, particularly those which consist of radioactive technetium-labelled ligands. United States Patent Specification No. 4,272,503, issued June 9, 1981, discloses a reductant composition for technetium 99m and a method for making technetium 99m labelled ligands. In particular, this specification discloses in one embodiment a reductant for reducing technetium comprising a substrate having a reducing complex attached thereto, the reducing complex preferably comprising a reducing agent and a chelating ligand for binding the reducing agent to the substrate. The chelating ligand may be any known chelating ligand for the reducing agent and may be bound to the substrate either directly or through a linking group. Alternatively, substrates having chelating ligand already bound thereto, for example, those commercially available as functionalized glass beads or functionalized polysaccharide beads, may be used. It is an object of the present invention to provide an alternative method of making radioactive-labelled ligands which is simpler and more economical than the method described in the above mentioned patent specification and yet which provides an effective means for producing the desired radiopharmaceuticals.

In general terms, the present invention provides a method of overcoming the unwanted effects of excess stannous chloride by depositing stannous chloride on the surface of a reaction vessel, for example, as a thin layer, coating or the like, and then reducing the pertechnetate ion in this reaction vessel so that there is no excess stannous chloride in the solution injected into the patient's body. Further advantages of this method are that unlike the established methods, ligands which are unstable in dilute acids or which precipitate with tin ions or which are insoluble in saline can still be combined with technetium to form radiopharmaceuticals.

According to one aspect of the present invention there is provided a method of reducing a radioactive material, particularly reducing pertechnetate ion to Tc(III) or Tc(IV), which method comprises the steps of depositing a suitable reducing agent onto a surface, for example the internal wall of a reaction vessel, and reacting the radioactive material with the reducing agent by contacting a solution containing the radioactive material with the surface thereby reducing the radioactive material and providing a solution containing reduced radioactive material substantially free of reducing agent.

In contrast to the method disclosed in U.S. Patent Specification No. 4,272,503, in the present method the reducing agent is deposited directly onto the substrate surface, and accordingly the need to prepare a "reducing complex" between the reducing agent and a chelating ligand therefor, or to use a special functionalized substrate, is avoided.

Preferably the reducing agent is a source of stannous ions, particularly stannous chloride. Other known reducing agents may, however, be used, including for example, ferrous ions, cuprous ions, ferric-ascorbate complexes, and reduced zirconium. Furthermore, salts other than the chloride salts, such as pyrophosphate salts, may also be used. The surface onto which the reducing agent is deposited may be either a glass surface or a plastics surface such as polyethylene. In a particularly preferred embodiment the surface is the inner wall of a reaction vessel, such as a syringe or a length of tubing.

According to another aspect of the present invention there is provided a method of preparing a radioactive labelled ligand substantially free of reducing agent, particularly a technetium-labelled ligand substantially free of reducing agent, comprising the steps of:

(a) depositing a reducing agent onto the internal surface of the vessel;

(b) admitting a radioactive material to the vessel;

(c) admitting a ligand to be labelled by the radioactive material to the vessel:

(d) reacting the radioactive material, ligand to be labelled and reducing agent to reduce the radioactive material and form a radioactive labelled ligand whilst substantially retaining the reducing agent on the internal surface of the reaction vessel.

If desired, a silicon-containing material such as dimethyl-siloxane may be applied to the internal surface of the reaction vessel before deposition of the reducing agent, however this is not essential.

According to another aspect of the present invention there is provided a kit for producing a radioactive labelled ligand suitable for use as an injectable radiopharmaceutical comprising a reaction vessel having a reducing agent deposited on an inner surface thereof, means for admitting a radioactive material to the reaction vessel and means for admitting a ligand to be labelled by the radioactive material to the reaction vessel, whereby the radioactive material is reduced and ligand is labelled by the reduced radioactive material within the reaction vessel, and means for discharging the radioactive-labelled ligand substantially free of the reducing agent from the reaction vessel.

According to another aspect of the present invention there is provided a radioactive labelled ligand, particularly a radioactive technetium-labelled ligand, whenever prepared by a method of the present invention.

Any ligand capable of being labelled, for example with technetium 99m, can be labelled in accordance with this invention. Particularly useful ligands are polyhydroxy polycarboxylic acids, aminocarboxylic acids, phosphonates, phosphates and mercaptans, etc. Examples of such ligands include, for instance, plasma proteins such as human serum albumin (HSA), ethylhydroxydiphosphonate (EHDP), methylenediphosphonate (MDP), pyrophosphate, ethylenediaminεtetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), dimercaptosuccinic acid (DSMA), gluconate, glucoheptonate, N-(2,6-dimethylphenylcarbamoylmethyl) iminodiacetic acid (HIDA), analogs of HIDA such as N-(2,6-diisopropylphenylcarbamoylmethyl) iminodiacetic acid (PRIDA), N-(4-butylphenylcarbamoylmethyl)- iminodiacetic acid (BIDA), clotting factors such as fibrinogen, gamma globulins, antibodies and their fractions, phytate, and the like.

The present invention will now be described by way of example with reference to the accompanying drawings in which:

FIGURE 1 is a bone scan of rabbit using 9^9mTc-labelled methylene diphosphonate (MDP) prepared in accordance with the present invention shown in three different intensities;

FIGURE 2 is a bone scan similar to Figure 1 using ^99mTc-labelled methylene diphosphonate prepared by commercially available kits also shown in three different intensities;

FIGURE 3 is a kidney scan of rabbit using 9^9mTc-labelled diethylenetriamine penta-acetic acid (DTPA) prepared in accordance with the present invention shown in three different intensities.

Technetium in the form of pertechnetate ion ( ^99mTcO₄-) in a saline solution is obtained by elution from a sterile ⁹⁹Mo-^99mTc generator, such as for example, a Mallinckrodt Aiishi generator. A solution having typically 50 to 100 mCi ^99mTcO₄- in saline solution is used in the subsequent labelling reaction.

Preparation of Vessel with thin coating of stannous chloride

Any suitable material such as glass or plastic may form a suitable reaction vessel. However, in the described embodiment a sterile non-pyrogenic polyethylene catheter pipette was used, the internal surface of the pipette was washed with dimethyl-siloxane in order to make the internal bore of the catheter smooth by depositing silicon on it. (In alternative embodiments, a polyethylene syringe may be used as the reaction vessel in place of the catheter pipette.) Typically, a solution of Sigmacote which is a commercially available silicone solution from Sigma Inc. may be used. It should be noted that siliconizing the surface of the reaction vessel is an optional step, although siliconised vessels have been found to assist in some instances in prevention of microcolloid formation. The vessel is then dried at room temperature and afterwards rinsed with a normal saline solution. The vessel, whether pretreated with silicon or not, is coated with stannous chloride by using a plastic syringe to flush the stannous chloride solution which has been previously freshly prepared backwards and forwards through the vessel before returning the solution back into the vial containing the solution. Thus, stannous ions are directly deposited on the inside of the vessel by means of a combination of physical and chemical mechanisms. It is thought that stannous and chloride ions are held inside the vessel by the combined effect of the ions lodging in minute surface troughs in the internal bore of the vessel and the chemical attraction between the ions and the surface. As previously mentioned, it has been discovered that a siliconised plastic surface or smooth glass surface in some instances inhibits and may even eliminate, microcolloid formation which is caused by unlabelled free TcO₄- and reduced uncomplexed technetium.

In order to prevent residual excess free stannous chloride from remaining inside the vessel the vessel is rinsed with typically 5 ml of normal saline solution (0.9% NaCl). After thus depositing the stannous chloride in the vessel, the vessel is quickly dried at room temperature (to stop autooxidation) in preparation for its use in the next step. Typically, it may be dried with a purge of nitrogen or other conventional means.

Formation of Technetium attached to a ligand i.e. labelling

The process of attaching the radioactive portion to the ligand is known as labelling. A mixture is prepared by mixing an aqueous solution of the ligand and the previously eluted pertechnetate ion saline solution. No reaction between these two reactants takes place since the technetium is in the +7 oxidation state and accordingly it will not readily react with the ligand. The mixture is then passed through the previously prepared vessel by means of a plastic syringe which is used to flush the mixed solution backwards and forwards repeatedly through the vessel. The simultaneous reduction of Tc and combination of the ligand and Tc takes place in the presence of the SnCl₂ on the walls of the vessel. The SnCl₂ stays substantially on the vessel walls at all times and does not enter the saline solution of Tc and ligand being flushed through the vessel. Therefore, no SnCl₂ can enter into the solution to be injected into the patient.

By this means the pertechnetate ion is reduced and the ligand complex is formed i.e. the ligand is labelled by the technetium.

Verification of the Invention

The analytic method employed to verify that a solution substantially free of SnCl₂ is obtained is thin-layer chromatography using saline and methyl ethyl ketone as solvents.

After the reduction of the pertechnetate ion by the stannous chloride there exist in solution the following species (i) free unreacted pertechnetate ion TcO₄-, (ii) reduced but uncomplexed technetium in the form of ^99mTc(III) or ^99mTc(IV), and (iii) technetium labelled ligand. Saline solution is used to separate the reduced uncomplexed Tc from the unreacted pertechnetate ion and the labelled ligand. Methyl ethyl ketone is used to separate the free unreduced pertechnetate ion from the technetium labelled ligand and the reduced uncomplexed technetium.

Generally, the stationary phase used in the chromatographic process is prepared from pure cellulose sheets of chromatographic quality such as

Watman No. 1 which are cut into strips typically about 7 mm wide by 57 mm long. These strips which are used with the saline medium are marked transversely with a graphite pencil about 10 mm from one end. Similarly, the strips which are used with the methyl ethyl ketone are marked about 10 mm from the same one end.

For each labelling two strips are used. One drop of the labelled compound under test is placed on strip A and one drop of the same compound is placed on strip B. Without drying, strip A is inserted vertically into a flat-bottomed vial containing saline solution to a depth of about 2 to 3 mm and strip B is inserted into a flat bottom vial containing methyl ethyl ketone to a depth of about 2 to 3 mm. Just before the solvent front reaches the top of each chromatographic strip (2-3 min), the strips are removed from the vial. They are then cut at the pencil marks and the two components are placed in counting tubes for measurement in a well counter or a dose calibrator. These sections are designated as A₁ and A₂ for strip A and B₁ and B₂ for strip B.

The percentages of reduced uncomplexed ^99mTC, free pertechnetate ion and ^99mTC labelled ligand are calculated from the counts obtained, using the following formulas: ^99mTc Labelled = 100 - (R + F)

All of components are tested and measured by this method (Table 1).

As illustrated in Table 1 the technique of the present invention employed 14 different chemical compounds which are usable in nuclear medicine for diagnosis. Some of the ligands used in the method of the present invention include citrate which is used in the study of tumours; pyrophosphate which is used in the study of bones; ethylenediaminetetraacetic acid which is used in the study of kidneys; diethylenetriamine penta-acetic acid which is used in the study of kidneys also generally ligands labelled with technetium are useful in the diagnosis of cancer, particularly metastas and in the dynamic study of kidneys, particularly kidney failure.

As mentioned below, the effects of pH and Stannous chloride concentration which are two important aspects of the reaction of the present invention were investigated.

Effect of pH

Solutions containing technetium and the various ligands were made at various pHs and checked more than five times. (Table 1 shows the mean value of pH for each compound), then mixed with 0.5 - 1mCi/ml (18.2 -37.0 MBq) of TcO₄. The ligands are usually soluble in normal saline and stirred for a few minutes. pH is adjusted by using alkaline solution, 0.1 M NaOH or acid solution, HCl I M. The solution of the ligand to be labelled is filtered through a 0.22 μm Millipore filter. The highest efficiency for complex formation is obtained at pH 5 - 7. In this procedure the formation of free unreacted ^99mTcO₄- and uncomplexed 9^9mTcO₂ was very low for certain compounds and negligible for most of them. Reference may be made to Table I in this regard where the percentages of each of (i) free pertechnetate ion, (ii) reduced uncomplexed technetium, and (iii) labelled technetium are given in the appropriately headed columns at the right hand side of the table.

Effect of the concentration of Stannous chloride

Various concentrations of aqueous stannous chloride solutions are made and filtered through 0.22 um Millipore filter material. After a few experiments, the concentration of 1mM is used for all compounds apart from red blood and platelet studies where a concentration of less than 1 was used.

Use of other reducing salts

As previously described, reducing salts other than stannous chloride can be used for preparation of certain labelled ligands. By way of example, Tc-red blood cell labels prepared by the method of the present invention using stannous chloride as the reducing agent were found to be unstable in vivo. However, where stannous pyrophosphate (10μg/ml) was deposited as the reducing agent on the internal surface of a syringe then suitably labelled red blood cells were obtained in good yield (76%) with excellent in vivo stability. Other stannous salts including Sn-oxime and Sn-EDTA may also be used.

Stability of deposited reducing agent

Polyethylene syringes having Sn Cl₂ deposited on the internal surface thereof in accordance with this invention were stored in air at -10°C for periods of three and six months. Following this storage period, these syringes were used in the preparation of labelled ligands by the method of this invention, using sodium pertechnate and DTPA, MDP and PYP as ligands. Labelling yields (calculated as previously described) are set out in Table 2.

Study of Rabbits

Solutions obtained by the method of the present invention were injected into rabbits which were used for in vivo studies. The results of the scans are shown in Figures 1 to 3. The Gamma camera used for obtaining the images of the scans was a Toshiba GLA402. For example, the radiopharmaceutical Tc-labelled DTPA (Figure 3) was intravenously injected into the rabbit's ear in an amount of 250 μCi (9 MBq). Following the intravenous injection into the rabbit, ^99mTc-DTPA is rapidly cleared from the blood circulation by excretion into urine. Kidney uptake was clear as shown in Figure 3. There is no accumulation or retention in the thyroid gland or salivary glands as illustrated. The bladder only was visualised in the scan.

As a further example, and as a comparison, a commercially prepared as well as a surface labelled Tc MDP complexes were injected intravenously into two rabbits. A bone scan was performed two hours after the injection. Figures 1 and 2 show the relative quality of each of the scans.

The present invention provides a method producing a simpler way of obtaining technetium-labelled compounds, and of obtaining many more technetium compounds than has hitherto previously been obtainable by conventional commercial methods. This method is of commercial value in nuclear medicine where on a world-wide basis millions of dollars worth of compounds which are used for labelling with technetium are sold annually.

One of the advantages of the present invention is that there is none or at worst a negligible amount (about < 4 μg) of stannous chloride injected into the biological system of the patient receiving the invention. Accordingly, there is less chance of there being adverse side reactions in the patient and interference with subsequent treatments and investigations.

The method of the present invention provides a simple and reliable method which offers considerable promise in combining various compounds (ligands) with technetium using the stannous ions that hithertofore have not been possible or entirely successful due to the ligands precipitating out of solution or due to pH incompatibility. Therefore, although the present invention has been described for some ligands with technetium, many other ligand reactions are possible and it is applicable to other radioactive materials other than technetium.

These and other modifications may be made without departing from the spirit and scope of the invention which includes every novel feature and combination of novel features herein disclosed.

Claims

1. A method of reducing a radioactive material, which method comprises the steps of depositing a suitable reducing agent onto a surface, and reacting the radioactive material with the reducing agent by contacting a solution containing the radioactive material with the surface thereby reducing the radioactive material and providing a solution containing reduced radioactive material substantially free of reducing agent.

2. A method according to claim 1, wherein the surface onto which the reducing agent is deposited is the internal wall of a reaction vessel.

3. A method according to claim 1, wherein the radioactive material comprises pertechnetate ions, which are reduced to Tc(III) or Tc (IV).

4. A method according to claim 1, wherein the reducing agent comprises stannous ions.

5. A method of preparing a radioactive labelled ligand substantially free of reducing agent, comprising the steps of:

(a) depositing a reducing agent onto the internal surface of the vessel;

(b) admitting a radioactive material to the vessel; (c) admitting a ligand to be labelled by the radioactive material to the vessel;

(d) reacting the radioactive material, ligand to be labelled and reducing agent to reduce the radioactive material and form a radioactive labelled ligand whilst substantially retaining the reducing agent on the internal surface of the reaction vessel.

6. A method according to claim 5, wherein the radioactive material comprises pertechnetate ions, which are reduced to Tc(III) or Tc(IV).

7. A method according to claim 5, wherein the reducing agent comprises stannous ions.

8. A method according to claim 5, wherein the vessel comprises a glass or plastics syringe or length of tubing.

9. A kit for producing a radioactive labelled ligand suitable for use as an injectable radiopharmaceutical comprising a reaction vessel having a reducing agent deposited on an inner surface thereof, means for admitting a radioactive material to the reaction vessel and means for admitting a ligand to be labelled by the radioactive material to the reaction vessel, whereby the radioactive material is reduced and ligand is labelled by the reduced radioactive material within the reaction vessel, and means for discharging the radioactive-labelled ligand substantially free of the reducing agent from the reaction vessel.

10. A radioactive labelled ligand, prepared by the method according to claims 5 to 8, or by use of the kit according to claim 9.