CA1286086C - Preparation of __ tc radiopharmaceuticals - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

Compounds of the formula I:

R+[99?TcNX4]- I

wherein R+ represents a cation and X represents a halo group, are prepared by reaction with an azide compound in the presence of a hydrohalic acid. The compounds are useful in production of 99?Tc-labelled-radiopharmaceuticals by reaction with ligands and monoclonal antibodies or antibody fractions.

Description

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This invention relates to the preparation of radio-pharmaceuticals, and in particular to the preparation of technetium-99m (9g~Tc) - labelled radiopharmaceuticals.

Radiopharmaceuticals are diagnostic or therapeutic agents by virtue of the physical properties o~ their constituent radionuclides. Thus, their utility is not based on any pharmacologic action. Most clinically used drugs of this class are diagnostic agents incorporating a gamma-emitting nuclide which, because of its physical or metabolic properties, localizes in a specific organ after intravenous injection. Images reflecting organ structure or function are then obtained by means of a scintillation camera that detects the distribution of ionizing radiation emitted by the radioactive drug. The principal isotope currently used in clinical diagnostic nuclear medicine is reactor-produced metastable technetium-99m.

Many methods have been described for the reduction of pertechnetate (99~TcV~04-) in the preparation of 998Tc-radiopharmaceuticals. Reducing agents which have been used include stannous ion, electrolysis, ferrous ion, ferrous ascorbate, formamidine sulphinic acid and sodium borohydride (Dsutsch et al, 1983). These labelIing procedures generally lead to the redu~tion of teshnetium to the Tc(IV) or TctV) oxidation state. In many cases the compound prepared contains the TcO moiety (Deutsch 1979). Because of problems experlenced with these reducing agents, the use of a substitution route for the production of 99~Tc-radiopharmaceuticals has been advocated (Deutsch & Barnett, 1980). The agents normally used for substitution reactions are TcOX52- and TcX6~- (X =
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Cl,Br) in which technetium is in the TcV and TcrV valency states respectively.

The present~inventors have investigated tho preparation of 99~Tc-radiopharmaceuticals containing the ,, : . .
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TcN moiety, and have discovered that the TcN moiety is extremely stable to hydrolysis and that the nitrido group remains firmly attached to the Tc atom throughout a number of substitution reactions.

According to the present invention, there is provided a novel group of compounds containing the TcN
moiety, as well as methods for the preparation thereof and methods for the preparation of 99nTc-radio- -pharmaceuticals utilising these compounds.

According to a first aspect of the present invention, there are provided compounds of the formula I:

R+[99mTcNX4]- I

wherein R' is a cation, preferably a soluble cation such as sodium or another alkali metal, or ammonium, and X
represents a halogen group, particularly a chloro or bromo group.

The compounds of this aspect of the invenkion are characterised by the presence of the nitridotetrahalo-technetium-99m anion in which Tc is in the TcV~ valency state, and which has been found to ha~e particular utility in the preparation of radiopharmaceuticals containing the TcN moiety.

In another aspect of this invention, there is provided a process for the preparation of compounds of the formula I as~described above, which comprises reaction of a compound containing the 99rTc-pertechnetate anion (R[99nT¢o~], wherein R represents a cation such as alkali metal, or ammonium), with an azide ion, such as sodium azide, in the presence of a hydrohalic acid, such as hydrochloric or hydrobromic acid.
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In another aspect, there is provided a method of producing a 99nTcN -labelled product, which comprises reacting a compound of the formula I with a ligand.
Suitable ligands include, for example, methylene diphosphonate (MDP), thiourea (TU), thiomalate (TMA), dimercaptosuccinate (DMSA), gluconate (GLUC), N-(2,6-diisopropylphenylcarbamoylmethyl)iminodiacetate (PIPIDA), N-(2,6-dimethylphenylcarbamoylmethyl)iminodiacetate (~IDA) ethane-1-hydroxy-1, 1-diphosphonate (EHDP), diethylenetriaminepentaacetate (DTPA), and cysteine (CYS). Other ligands which may be used in accordance with the present invention include thiouracil, diethyl-dithiocarbamate, mercaptopyridine, mercaptopyrimidine, thiooxine, acetylacetone, pyridoxal, oxine, tropolone and tetracycline. Monoclonal antibodies which may also be labelled in accordance with the present invention, and which have been shown to retain their specificity following labelling. This aspect of the invention is described in greater detail hereinafter.

In yet another aspect, there is provided a 99nTcN -labelled product which comprises the reaction product of a compound of formula I with a ligand.

The desirability of using à subst~itution route for the preparation of 99~Tc-radiopharmaceuticals has long been recognised. However the method has suffered because of the difficulty in obtaining 99~Tc in a suitable chemical form at the Tc concentrations used for radio-pharmaceutical production. The nitrido labelling technique descrlbed here is a comparatively simple method for the preparation of a wide range of radio-pharmaceuticals based on the TcN moiety. While Tc is initially present a= Tc ~VI) in TcNC1~-, it has been found that reduction usually takes place to the Tc(V) state if the ligand has the ability to act as a reducing agent. Ligand substitution then takes place around the " ' .
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Nitrido labelling has been found to be particularly suitable for the labelling of "soft" ligands The present invention has particular application in the coupling of 99~Tc to monoclonal antibodies (MAb) and the use of the resulting complexes, for example, in the specific detection of tumors in vivo, (see, for example, Rhodes, et al, 1982). At present a number of difficulties exist in the diagnostic radiolocalization of tumors, one of which is the choice of radionuclide. Many studies have used 131I, however this radionuclide has serious drawbacks including a poor quality image, significant radiation exposure due to its beta emissions, and short biological half-life.

Technetium -99m (~Tc) has an isotope for radio-localization offers several advantages: it has a reasonably short hal~-life; it is cheap, easy to produce, and is readily available. The isotope has an optimal gamma energy (140keV) for detection with currently available gamma scintigraphic instrumentation and produces very little radiation exposure to patients undergoing scanning procedures. However little use has been made of 99~Tc for labelling antibodies, presumably because most labelling methods used to date lead to loss of antibody activity, due to side reactions taking place and to transchelation reactions occurring in vl~o.

In accordance~with the present invention, compounds of the~formula I described above have been found to produce stable 99~Tc-labelled MAb by a substitution reaction in the same manner as other ligands described herein. Either whole monoclonal antibodies, or antibody fragments (such as~Fab fragments) which react with the ,, ~

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corresponding antigens may be labelled in accordance with this invention. Antibody specificity is maintained in the labelling process and the labelled product i.s table.
In addition, tests have shown tha~ when the labelled Mab is used in tumor detection, tumors may be visualised in as soon as two hours, and furthermore small tumors (about 0.4 cm) located near large vascular organs can be visualised.

Preferably, in labelling monoclonal antibody in accordance with this invention, the antibody is at least partially reduced for example by reaction with a reducing agent such as dithiothreitol, in order to convert disulfide linkages into sulfhydryl residues. Such a partial reduction procedure enables utilisation of the preference of the TcN moiety for sulfur atoms, and thus enables the production of more stable complexes in which the Mab ligand is bound through sulfur atoms to the TcN
moiety. It is also noted that these sulfhydryl groups would be removed from the sites responsible for antibody specificity, hence formation of the complexes is less likely to cause loss of specificity.
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In drawings illustrating the invention:

Figure 1 is an ultraviolet absorption spectrum showing the formation of TcNC14, complexes;
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Figure 2 is an ultraviolet absorption spectrum showing the formation of TcN-~DP complexes;

Figure 3 is an ultraviolet absorption spectrum showing the formation of PcN-DTPA compIexes: and Figure 4 shows the clearance rates of TcN-GLUC, TcN-HEDP, TcN-HIDA and TcN-PIPIDA complexes in bio-distribution studies.
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The following Examples illustrate the preparation of a [99nTcNX4]- compound as well as the biological behaviour of TcN radiopharmaceuticals containing various ligands.

A. Preparation of sodium tetrachloronitridotechnetate Na[99DTcNC1~] and 99nTcN-radiopharmaceuticals.

Unless otherwise stated all solvents and chemicals were of analytical grade. L(+)-Cysteine for biochemistry was obtained from E.Merck, Darmstadt, Methylenediphos-phonic acid (MDP) from Sigma Chemical Co., St. Louis and diethylenetriaminepentaacetic acid (DTPA) from Koch-Light Laboratories, Colnbrook. Sodium-2-gluconate was obtained from Fluka A.G. EHDP was prepared using the method described by Castronovo (1974).

HIDA and PIPIDA hydrochlorides were prepared by the reaction of the N-chloroacetanilides with iminodiacetic acid using a variation of the method of Callery et al (1976). A mixture of N-chloroacetanilide (0.05 mole), iminodiacetic acid (0.05 mole) and lOg anhydrous sodium carbonate was refluxed in 30mL of 75%`aqueous ethanol for 6 hours. On cooling the solution was acidified with concentrated hydrochloric acid and the pH adjusted to 1.5. The precipitate was filtered and recrystallised from 50% aqueous ethanol. Use of sodium carbonate resulted in improved yields (>60%). 99Tc in the form of ammonium pertechnetate in 0.lM ammonium hydroxide solution was obtained from Amersham International.

(i) A solution of 99~Tc-pertechnetate t50MCi~18 GBq for animal studies) was taken to dryness using a rotary evaporator. Sodium azide t~20mg) was added to the dry residue, followed by 10 ml of ,~

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concentrated hydrochloric acid (sg 1.18). The solution was refluxed for 5 minutes to complete the reduction and destroy excess azide before being taken to dryness in the rotary evaporator.
1 ml of ligand solution (PIPIDA 20mg/ml, pH 7, all others 5 mg/ml, pH 71 was added followed by 2 ml saline. If necessary, the pH was adjusted to 6-7 by the addition of 0.lM sodium hydroxide. After filtration through a 0.22 ~m membrane filter, the radiopharmaceutical was ready for use.
(ii) An alternative labelling procedure is to perform the ligand replacement in a non-aqueous solvent, such as in acetonitrile or ethanol solution. By way of example, 10 ml of acetonitrile is added to the dry residue after the --azide reduction followed by 100 ~1 of the ligand solution. ~fter heating on a water bath for 10 minutes, the acetonitrile is removed in a rotary evaporator and the dried extract dissolved in 1 ml o~ the ligand solution and 2 ml saline as before.

B. Animal;Distribution Studies 0.05-0.1 ml of the preparation (1-2mCij was injected into the tail vein of Swiss mice (20-30g) and the activity injected measurèd in an ionisation chamber.
Afker injection, mice were allowed free access to food and water. At each time interval skudied, three mice were sacrificed~by cervical dislocation and dissected.
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Organs were weighed and their activities measured in the ionisation~chamber. ~The original injected activity was 30 correc~ed~Por~the~activity found in the tail. Blood ;~
activity was calculated on~the assumption thak the overall blood volùme represents 7% of kotal body welght.

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C. Measurements of 99~Tc Labellina 2~L aliquots of each preparation were chromatographed on Whatman No. 1 paper in three solvent systems: saline, 70% methanol and methyl ethyl ketone.
After development all papers were dried and scanned in a radiochromatogram scanner (Packard Model 7220/21). Where appropriate, peaks were cut from the strips and counted for gg~rc activity in a gamma counter. All preparations contained less than 5% free pertechnetate.

D. W Spectral Studies Preparations were made using 300~g 99~Tc added as carrier for W spectral studies. UV spectra were recorded on a Beckman Acta CII spectrophotometer.

Pertechnetate was reduced using the following reduction systems:
(i) concentrated hydrochloric acid, (ii) concentrated hydrochloric acid/potassium iodide, (iii) concentrated hydrochloric acid/sodium azide, (iv) concentrated hydrochloric acid/stannous chloride, (v) concentrated hydrochloric acid/hypophosphorous acid, ~ ~
(vi) concentrated hydrochloric acid/hydroxylamine hydrochloride.

The spectra of all samples was measured in hydrochloric acid solution before and after heating on a hot plate.

W spectra~of the technetium complexes studied were obtained after taking the above solutions to dryness in a rotary evaporator and~dissolving the dry residue in 1 ml ligand soIution and~2 ml water.; The pH of the solution was adjusted to 7 using either O.lM or lM sodium hydroxide.

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E. RESULTS

The concentration of Tc in the solution used for uv absorption measurements was determined by beta counting.
lOmL scintillation fluid (Aquassure-NEN) were added to O.lmL aliquots of the solution for counting in a liquid scintillation spectrometer. Counting efficiency was determined by counting aliquots of a pertechnetate solution standardised by uv spectrophotometry. Quenching of the solution was checked by external standardisation.
An internal standard was added when a quench correction was necessary.

(i) W Spectral Studies (a) Hydrochloric Acid Studies Addition of potassium iodide, hydroxyl-amine hydrochloride, or hypophosphorous acid to pertechnetate in hydrochloric acid followed by heating all result in the formation of Tc(IV).
This is shown in Figure 1 by the characteristic W absorption spec~rum of TcC162- which has an-absorption maximum at 340nm and a minor peak at 305nm. Pertechnetate allowed to stand in concentrated hydrochloric ac~d in the cold, results in the production of TcOCl52- in which Tc is in the Tc(v) oxidation state. The W
spectrum however indicates that the TcOC15Z-produced also containa~Tc(~IV), the proportion of which is increased by heating. Heating of pertechnetate in the presence of azide results in the formation of TCNCl4- characterised by an absorption maximum at 395nm ( = 500m2 mol~l) and ~ free of contaminating Tc(IV).

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shown in Figure 2. When MDP was added to TCNC14- at pH 5.5, a pink colored compled (A~x-515nm) was produced. On heating the pink color disappeared producing a yellow complex with an adsorption maximum at 335nm. The spectrum of this complex was identical to that obtained by adding MDP to TcNC14- and adjusting the pH to 10 (A~=335nm, ~=21m2 mol~l).

(c) DTPA Complexes When prepared at room temperature, TcN-DTPA showed an absorption maximum at 505nm (~=150 m2mol~l) (Figure 3). On heating the peak disappeared producing a complex with no signi~icant absorption in the range 300-800nm.
TcIV-DTPA produced by adding DTPA to TcC162-showed no significant absorption maximum in the uv-vis region.

(d) Cysteine Complexes The~spectrum of TcN-CYS showed no significant absorption in the uv-visible region that could be attributed to the complex.
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(ii) Stability of dried T~oNCl~~ Preparation Samples of the~99nTcNCl4- preparation were taken to;dryness ln a rotary evaporator and stored for 24 hours~(a) in air at room temperature, (b) at 80 C, and (c~)~at room ~emperature and 100% humidity. They were then used to prepare 99~rcN-DTPA by addition of ligand as bafore. No significant difference in ~ chromatographic bahaviour as measured by high ; ~ perform~ance~liquid ohromatography (HPLC) was observed in any sample indicating that th~ dry 99~TcNCl4- reagent was stable for up to 24 hours.

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(iii) Animal ~istribution Studies (a) Results of biodistribution studies of 99'TcN-MDP, 99~TcN-DTPA and 99~TcN-CYS are given in Tables 1-3. 99~TcN-MDP was characterised by high blood pool activity and showed insignificant bone uptake. 9gnTcN-DTPA showed similar renal excre~ion behaviour to 99~Tc-DTPA(Sn) and was probably excreted by a similar mechanism. Blood pool activity however was higher than that observed for 99~Tc-DTPA (Sn).
99~Tc-cysteine showed rapid clearance with high renal localization. Urinary excretion rates for the three 99~TcN-complexes are given in Table 4. Excretion rates of 99~TcN-MDP and 99~TcN-DTPA are significantly less than that of the respective 99nTc(Sn) - complexes.

(b) Results of biodistribution studies of 99Yrc-GLUC, 99nTcN-HEDP, 99nTcN-HIDA and 9gnTcN--PIDIDA~are given in Tables 5-9. All preparations showed high blood pool activity with the 99nTcN-GLUC and 99nTcN-HEDP preparations cl aring slightly faster than 99nTcN-PIPIDA and 9g~TcN-HIDA. (Figure 4~. Overall, 99~TcN-GLUC~
99~TcN-HIDA and 99nTcN-HEDP showed comparable ;
patterns of biological behaviour. 99~TcN-PIPIDA
differed from the other agents in that it gave signlf~icantly higher actlvity in the intestines. With all preparations the clearance into the intestines~took place essentially in the first 30;minutes. The higher~ clearance of 99~TCN-PIPIDA could be due to gg~TcN-PIPIDA undergoing a slower rate of ;exchange~w1th serum proteins than 99~TCN-HIDA.

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(c) Results of biodistribution studies of 99~TcN-DMSA are given in Table 10.

(iv) Stability studies Stability studies of the dry 99~TcNC14-preparations have indicated that the agent is sufficiently stable to be prepared in a central laboratory or manufacturing site and distributed to users for use in the preparation of 9g~TcN-labelled radiopharmaceuticals. Preparation of the 99~TcN-radiopharmaceuticals in most cases takes place by simply dissolving the dry residue in a solution of the chelate.

99~rcNC14- may also be used to label ligands that are insoluble in water or which may be unstable in aqueous solutions. It is possible to extract the 9snTcN- activity from the dry salt residue into organic solvents such as acetonitrile. Labelling may be performed in the organic solvent~which may then be removed by evaporation. Labelling with-9g~TcNCl4~ takes place via a substitution mechanism and is expected~to be less susceptible to the hydrolytic type reactions which occur with many other~labelling proaedures. In addition, the presence of the nitrido group modifies~the chemistry of~the Tc atom in that~reactions with "soft" ligands are more favoured than when other reducing agents are~used~

A. ~Preparation~of 99'T~ ~belIed MAb (i) Tumor Cell Lines :
Two~tumor cell~lines were used: one, the E3 clonal variant of the thymoma ITT(1)75NS (Hogarth, , ..

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' et al, 1982), which was obtained by three successive rounds of cytofluorugraphic sorting of IGG(1)75NS
cells stained with monoclonal Ly-2 antibodles and selected for the most fluorescent 1% of cells. The murine cell line E(3) was maintained in vitro in DME
supplemented with 10% heat inactivated newborn calf serum (Flow Laboratories, Sydney, Australia), 2mM
glutamine (Commonwealth Serum Laboratories, CSL, Melbourne, Australia), 100 I.U./ml penicillin (CSL) and 100mg/ml streptomycin (Glaxo, Melb., Aust.). E3 cells were washed twice in DMI (without additives) and twice in DME containing 0.5% BSA and used in the in vitro binding assays. The E3 cell line was maintained in vivo by the passaging of cells from ascites fluid produced in (B6 x BALB/c)Fl mice or from solid tumors which grow after the subcutaneous injection of 106 or 107 cells. The second cell line used was a human colonic carcinoma, COLO 205 (Semple et al, lg78). It was maintained in culture with TPMI containing the same additives. Adherent COLO
205 cells were harvested with 0.125% trypsin (CSL) washed with RPMI and injected subcutaneously into-nude mice, where tumors appeared after the injection of 2 x 106 - 1 x I07 cells.
:, (ii) Monoclonal Antibodies ~MAb) Two monoclonal antibodies were used: (i) anti-Ly-2.1~(IgG2a), an antibody raised against the murine~alloantigen Ly-2.1 (Hogarth et al, 1982) and (ii) 250-30.6 (IgG2b), an antibody to human colonic secretory epi~helium (Thompson, et al, 1983). ~he monoclonal antibodies were isolated from ascitic fluid by preaipitation with 40% saturated ammonium sulphate, followed by dissolution in 0.01M Tris buffer pH 8.0 and extensive dialysis against the same buffer. The crude antibody preparations were further purified by affinity chromatography using :
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protein-A Sepharose (Pharmacia) and then purity determined by gel electrophoresis and antibody activity assayed by a rosetting test (Parish, et al, 1~78).

(iii) 99DTc-Labellinq of Antibodies 99~TcNC1~- was prepared as described above.
For labelling the MAb was first reduced with dithiothreitol (D~T) by adding 20~1 of DTT in PBS
(115mg/ml) to 200~g of MAb (lmg/ml in PBS~ and standing the mixture at room temperature for 30 minutes after which the reduced MAb was separated from DTT by gel chromatography using O.lM sodium acetate pH 4.0 as eluant on an 8cm x lcm column of Biogel P-6 (Biorad Laboratories, Richmond, USA).
The fractions (lml) containing the protein peak were added to the dried 99~rcNC1l salt residue and the mixture brought to pH 3.0 with 0.2M
hydrochloric acid. After 2 minutes at room temperature, 2 drops of O.lM sodium phosphate was added and the pH adjusted to 7 by the careful addition;of sodium hydroxide (lM or O.lM). - ~
Purification of the labelled MAb was then achieved by gel chromatography with a Sephadex G-25 disposable column (PD-10, Pharmacia) equilibrated in PBS, 500~1 aliquots were~collected and the radiolabelled protein~peak identified by gamma counting.
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B. RESULTS ~ ~
(i) In vitro stability and specificity of 99nTcN-MAb Three different specific.ity assays were performed. The first compared the activity of 99~Tc labelled~Ly-2.1 MAb to thymocytes from the mouse strains RF/~ (Ly-2.1 positive and C57~L/6 (Ly-2.1 negative)). A ten fold difference was observed in the binding of the labelled MAb to the specific '~

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cells (RF/J) when compared to the negative cell controls tC57BL/6). The procedure used to couple 99~TcNC14- to MAb produced a stable complex which was shown to be s~able and bind specifically antigen positive target cells ten times greater than antigen negative control cells.
In the second specificity assay two different MAb, one directed against colonic secretory epithelium (250-30.6) and the second the anti-Ly-2.1 MAb, were labelled with 99~TC under identical coupling conditions and the two complexes were tested for their ability to bind to the murine T
cell thymoma E3, which is posi-tive for the Ly-2.1 antigen but does not react with the anti-colon antibody. The anti-Ly-2.1 MAb bound ten times more efficiently than the 99~rcN-anti-colon complex. The stability of the 99'TcN-MAb was demonstrated here, where only the antibody reactive complex (99~TcN-anti-Ly-2.1) showed increased binding on E3 target cells. The non-reactive complex (99~TcN-anti-colon) exhibited a significantly reduced uptake of radioactivity on the same target cells.
The stabiIity of the TcN-MAb complexes was examined in a third assay. Aliquots of labelled MAb were stored at 4C overnight and~the stability of the label was determined by binding the stored material to thymocytes. The labelled MAb were shown to be s~able by their retention of binding ability.
RF/J thymocytes (Ly-2.1+) bound better than 10 fold more labelled Ly-2.1 MAb than C57BL/6 thymocytes (Ly-2.1~). In three diPPerent specificity assays the in vitro stabiIity of the 99~TcN-MAb complexes was established and were shown to be chemically stable, even when allowed to stand at 4C overnight, blnding only to antibody reactive target cells.
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(ii) Localization The in vivo localization and biodistribution of TcN-MAb complexes was performed in separate studies.
In the first, the mice were dissected normal organs, tumors and an aliquot of whole blood were counted in a gamma counter. The solid tissues were then weighed and the results were used to calculate the localization ratio derived as follows: tissue (cpm/g) / blood (cpm/g). Two groups of 16 (C57BL/6 x BALBjc)F1 mice bearing the E3 tumor (0.23 - l.llg) were injected i.v. via the tail vein, with one of two MAb (anti-Ly-2.1 or anti-colon carcinoma) labelled with 99~TcNCl4- under identical conditions.
(Each mouse received 115~Ci Tc and lO~g MAb). In the data in Table 11, 4 mice from each group were sacrificed at different time intervals after injection (20, 30.5, 35 hrs) and the distribution of the specific MAb (anti-Ly-2.1) was calculated for the individual tissues and compared to the observed distribution of the non-reactive MAb (anti-colon).
After 20 hrs the tumor localization was observed to be 3 times greater for the specific MAb than that observed for the non-specific MAb. This ratio increased to approximately 3.8 at 30.5 hrs, and 7.3 2~ at 35 hrs after injection. It is important to point out that the E3 tumor was observed to have the highest localization ratio (i.e. tissue (cpm/g) /
blood (cpm/g)) in the group injected with the specific MAb (anti-Ly-2.1) with the liver, spleen and kidney localization ratio being below or similar to the blood ratio. However with the non-specific antibody (250-30.6) the liver, spleen and kidney were observed to be higher than the blood ratio, with the liver biodistribution ratio being 5 times greater than the blood after 30.5 hrs.
In the second study, a specific MAb (anti-Ly- -2.1) was compared in two dif~erent tumors. Nude ~ ~ .

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38~3 mice bearing colo 205 xenografts were used as the non reactive tumor and the E3 as the positive tumor.
The data was obtained 20 hrs after the injection of the anti-Ly-2.1 labelled MAb (Table 12). The E3 thymoma (Ly-2.1') was observed to take up 3 times more radioactivity than the colo 205 xenografts (Ly-2.1-H). The two biodistribution studies illustrate a significant increased incorporation of radioactivity in the MAb reactive tumor when compared to the levels of radioactivity in the blood and that incorporated in the other normal tissues.

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TABLE 1 - Biological Distribution of 9~TcN-MDP in mice~
% Injected Dose/Organ 5Time after Injection 30 min 60 min 120 min -Heart 0.6(0) 0.4(0) 0.3(1) Lung 2.9(5) 3.0(14) 1.9(15) Liver 10.4(4) 9.4(8) 6.2(13) Spleen 0.4(1) 0.3(1) 0.4(1) Stomach 1.1(1) 0.8(2) 1.2(11) Kidneys 6.9(27) 3.3(5) 2.6(7) Intestines 4.3(4) 4.1(6) 6.1(13) Femurs 0.3(0) 0.3(0) 0.3(0) Blood 26.5(46) 18.8(18) 13.5(40) % Injected ~ose/Gram Organ Heart 4.2(2) 2.4(4) 2.0(4) Liver 5.7(3) 4.7(5) 3.8(1) Kidneys 13.0(54)+ 5.6(11) 5.8(18) Femurs 1.7(2) 1.4(2) 1.5(1) Blood 13~0(22) 8.1(3) ~. 6-7(19)+

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TABLE 2 - Biological Distribution of 99~TcN-DTPA in mice~
% Injected Dose/Organ Time after Injection 30 min 60 min 120 min -Heart 0.1(0) 0.1(0) 0.1(1) Lung 0.7(1) 0.7(2) 0.4(1) Liver 1.7(2) 1.5(3) 1.1(2) Spleen 0.1(0) 0~1(0) 0.1(0) Stomach 1.1(2) 1.3(4) 0-9(0) Kidneys 1.5(1) 1.3(2) 0.8(0) Intestines 2.1(4) 2.0(3) 2.1(2) Femurs 0.1(0) 0.1(0) 0.1(0) Blood 6.2(8) 4.0(5) 2.4(1) % Injected Dose/Gram Organ Heart 0.6(0) 0.5(1) 0.4(1) Liver 0.8(1) 0.7(1) 0.6(1) Kidneys 2.5(1) 2.2(4) 1.5(2) Femurs 0.5(1) 0.4(I) 0.4(1) Blood 2.8(5) 1.8(2) . 1.2(0) Standard deviation of last significant digit(s) in brackets.

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TABLE 3 - Biological Distribution of 99~TcN-CYSTEINE in mice~
% Injected Dose/Organ Time after Injection 30 min 60 min 120 min Heart 0.1(0) 0.1(0) 0.1(0) Lung 0.8(2) 0.5(1) o.~tl) Liver 2.7(5) 1.6(1) 1.3(1) Spleen 0.1(0) 0.1(0) 0.1(0) Stomach 0.4(1) 0.3(3) 0.3(1) Kidneys 4.4(5) 3.4(5) 2.8(2) Intestines 5.3(8) 3.4(1) 4.0(3) Femurs 0.2(0) 0.1(1) 0.1(0) Blood 4.9(7) 2.8(4) 1.8(1) -% Injected Dose/Gram organ .
Heart 0.7(1) 0.5(2) 0.4(2) Liver 1.3(2) 0.8(2) 0.6(1) Kidneys 7.4(11j 6.2(9) 4.8(5) Femurs ;0.8(1) 0.6(3) 0.2(1).
Blood 2.1(2) 1.3(1) ~- 0-8(1) : ' Standard deviatlon of last significant digit(s) in brackets.
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TABLE 4 - Urinary Clearance of g9~TcN-MDP, 99~TcN-DTPA
and 99~TcN-CYS~
~ Retained Activity 99~TcN-MDP 99~TCN-DTPA 99~TCN-CYS
30 min 71.9(43) 24.8(42) 35.8(43) 60 min 51.4(14) 21.4(31) 18.7(28) 120 min 44.7(57) 14.6(6) 15.7(15) Standard deviation of last significant digit(s) in brackets.
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TABLE 5 - Biological Distribution of 9g~TcN-GLUC in mice.
% Injected Dose/0rgan Time after Injection 30 min 60 min 120 min Heart 0.7(0.1) 0.7(0.1)0.4(0.1) Lung 2.2(0.6) 3.0(0.5)1.4(0.2) Liver 6.4(1.6) 6.2(1.7)7.1(2.1) Spleen 0.3(0.1) 0.3(0.1)0.2(0.1) Stomach 1.4(0.4) 1.5(0.4)1.6(0.7) Kidneys 4.7(0.6) 3.9(0.3)3.4(0-1) Intestines 5.4(0.7) 4.1(0-8)7.0(1.4) Femurs 0.3(0.0) 0.3(0.0)0.2(0.1) Blood 25.7(2.3) 21.7(3-6)12.7(1.9) % Injected Dose/Gram Organ Heart 4.5(0.7)~ 4.6(1.0) 2.6(0.6) Liver 3.3(0.8) 3.1(0.9) 3.3(0.8) Kidneys 8.4(1.0) 6.6(0.7) 5.6(0.6) Femurs 1.7(0.1) 1.3(0.1) 0.8(0.2) Blood 12.0(1.0) 9.5(1-8)5.2(0.3) , Standard deviation in brackets.
n = 3.

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, TABLE 6 - Biological Distribution of 9~TcN-HEDP in mice.
-% Injected Dose/Organ Time after Injection 30 min 60 min 120 min Heart 0,5(0,0) 0.3(0.0) 0.3(0.0) Lung 2.5(0.6) 1.8(0.3) 1.5(0.4) Liver 12.1(0.3) 9.0(1.0) 7.7(0.5) Spleen 0.6(0.1) 0.3(0.0) 0.3(0.1) Stomach 0.6(0.1) 0.7(0.1) 0.6(0.1) Kidneys 4.1(0.2) 3.2(0.2) 3.0(0.2) Intestines6.9(0.5) 7.0(0.2) 6.8(0.6) Femurs 0.5(0.0) 0.5(0.1) 0.4(0.1) Blood 28.0(5.0)$ 16.2(2.0) 13.2(1.0)~

% Injected Dose/Gram Organ -Heart 4,3(0,5) 2.4(0.3) 2.3(0.2) Liver 8.1(0.8) 5.5(0.7) 4.8(0.4) Kidneys 9.1(0.4) 6.5(0.4) 6.1(0.3) Femurs 3.4(0.5) 3.1(0.8) 2.7(0.6) Blood 16.2(1.0)$ 7.3(1.1) 7.7(0.5)~

Standard deviation in brackets.
n = 3 unless otherwise stated.
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TABLE 7 - Biological Distribution of 99~TcN-HIDA in mice.
% Injected Dose/Organ Time after Injection30 min 60 min 120 min Heart 0.4(0.1) 0.4(0.0) 0.2(0.0) Lung 2.3(0.3) 2.0(0.3) 1.2(0.3) Liver 7.2(1.4) 6.7(1.2) 3.6(0.2) Spleen 0.4(0.1) 0.3(0.0) 0.1(0.0]
Stomach 1.1(0.2) 2.0(0.4) 1.0(0.0) Kidneys 3.5(0.2) 3.4(0.2) 2.1(0.0) Intestines6.8(1.0)9.2(0.4) 6.9(0.2) Femurs 0.3(0.1) 0.4(0.0) 0.2(0.0) Blood 25.8(1.6)20.6(1.3) 16.3(2.2) ~ Injected Dose/Gram organ Heart 2.9(0.5) 2.7(0.4) 1.6(0.1) Liver 4.2(0.8) 3.9(0.6) 2.1(0.1) Kidneys 6.9(0.5) 6.7(0.4) 4.2(0.1) Femurs 2.0(0.4) 2.3(0.1) 1.0(0.1) Blood 13.6(0.6)10.8(0.6) 8.7(1.1) -Standard deviation in brackets.
n = 3.

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TABLE 8 - Biological Distribution of 99~TcN-PIPIDA in mice.
-% Injected Dose/Organ Time after Injection 30 min60 min~ 120 min Heart 0.4(0.0) 0.3(0.1)0.4(0.1) Lung 2.3(0.3) 1.8(0.6)1.8(0.2) Liver 14.6(1.0) 16.2(6.1)10.1(1.0) Spleen 0.2(0.1) Q.3(0.0)0.2(0.0) Stomach 0.7(0.1) 1.0(0.0)0.8(0.1) Kidneys 4.6(0.5) 7.6(5.6)3.3(0.1) Intestines12.8(1.5) 12.0(0.8)14.9(2.1) Femurs 0,3(0.0) 0.2(0.0)0.2(0.0) Blood 26.8(3.0) 23.2(0.4)20.0(3.7) ~ Injected Dose/Gram Organ Heart 3.4(0.2)2.1(0.8) 2.8(0.5) Liver 8.8(0.6)8.9(3.1) 5.8(0.8) Kidneys 9.4(0.6)14.1(7.2j 6.3(0.2) Femurs 2.0(0.3)1.1(0.1) 1.0(0.2) Blood 14.8(1.~9)11.5(0.7) ~ 10.2(1.5) Standard deviation in brackets.
n = 3.
n = 2 :

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TABLE 9 - Urinary Clearance of 99~TcN-GLUC, 99~TcN-HEDP, 99~TcN-HIDA and 99~TcN-PIPIDA
:
~ Retained Activity 99~TcN-GLUC 99~TcN-HEDP 99~TcN-HIDA 99~TcN-PIPIDA
30 min 72.6(4.2) 68.8(9.5~ 74.9(5.8) 79.5(1.2) 60 min 59.3(6.4) 52.2(1.2) 66.3(0.9) 73.2(2.7) 120 min 45.6(5.6) 47.2(4.5) 54.7(1.1) 62.0(2.5) Standard deviation in brackets.
n = 3.

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TABLE 10 - Biological Distribution o~ 99~TcN-DMSA
% Injected Dose/Organ Time after Injection30 min 60 min 120 min Heart 0.3(0,0) 0.3(0.0) 0.2(0.0) Lung 1.6(0.1) 1.1(0.2) 1.1(0.2) Liver 4.3(0.1) 3.9(0-3) 3.4(0.2) Spleen 0.4(0.1) 0.4(0.1) 0.3(0.0) Stomach 0.6(0.1) 0.7(0.2) 0.5(0.1) Kidneys 10.3(0.5)12.3(0.8) 17.1(0.7) Intestines5.3(0.7)5.1(0.4) 4.8(0.5) Femurs 0.5(0.1) 0.5(0.0) 0.4(0.1) Blood 18.0(1.7)13.5(0.9) 9.5(0.5) Urine 39.9(11.4)57.8(4.4) 65.3(4.3) % Injected Dose/Gram Organ Heart 1.8(0.1) 1.8(0.2) 1.2(0.0) Liver 2.0(0.2) 2.1(0.3) 1.7(0.2) Kidneys 16.4(0.7)22.2(1.4) 29.0(2.9) Femurs 2.0(0.5) 2.4(0.2) 1.9(0.4) Blood 7.1(0.8) 6.4(0.3) 4.1(0.2) .
5 Standard deviation in brackets.
n = 3.

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TABLE 11 - Biodistribution Ratio Tissue (cpm/g): Blood (cpm/g) Specific MAb (anti-Ly-2.1)/Non-specific Mab (anti-colon) Time after Injection Organ 20 hrs 30.5 hrs 35 hrs a2.1 30.6 a2.1 30.6 a2.1 30.6 Blood 1.00 1.00 1.00 1.00 1.00 1.00 Tumor 1.23 0.40 2.60 0.68 3.48 0.48 (0.23-l.llg) Stomach 0.08 0.12 0.12 0.17 0.07 0.12 Spleen 0.59 1.12 0.56 1.83 0.71 2.09 Kidney 0.77 1.48 0.94 2.14 0.92 2.50 Heart 0.33 0.39 0.19 0.17 0.33 0.19 Liver 0.84 0.27 1.02 3.26 1.20 5.19 Lung 0.38 0.22 0.86 0.66 0.36 0.60 Intestine 0.09 0.16 0.15 0.20 0.09 0.19 Tail 1.45 0.90 0.78 1.92 1.14 1.66 , T~BLE 12 - Biodistribution Ratio 20 Tissue (cpm/g): Blood (cpmjg)~
, ITT(l)E3 COLO 205 E3/COLO 205 Blood 1.0 1.0 1.0 Tumor 1.23 0.30 ~ 4.10 (.39 - l.llg) ~ (0.5 - 1.5g) Stomach 0.08 0.2 0.04 Spleen 0.59 0.60 0.98 Kidney 0.77 0.76 ~ 1.01 Heart 0.33 ~ 0.39 0.85 Liver 0.84 0.56 1.50 Lung 0.38 0.42 0.90 Intestlne~ o.09 ~ 0.15 0.60 :
~f '` : :

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2g~ 3~'i REFEREN~ S
~E .

1. Castronovo, F.P., J.Nucl.Med. 15, 127 (1974).

2. Callery, P.S., Faith, W.C., Loberg, M.D., Fields, A.T., Harvey, E.B. and Cooper, M.D. J.Med.Chem, 19, 962 (1976).

3. Deutsch, E., Libson, K., Jurisson, S. et al:
Technetium chemistry and technetium radiopharmaceuticals.
Prog.Inorq.Chem 30:75-139, 183.

4. Deutsch, E., Barnett, B.L.: Synthetic and structural aspects of technetium chemistry as related to nuclear medicine, in: Inorganic Chemistry in Biology and Medicine (ed Martel A.E.), ACS Symp.Series No. 150, Washington, Amer.Chem.Soc. 1980, pp.103-ll9.

5. Hogarth, P.M., Henning, M.M. and McKenzie, I.F.C.
The alloantigenic phenotype o~ radiation induced thymomas in the mouse. J.N.C.I. lg82; 69:619-626.

6. Hogarth, P.M., Edwards, J., McKenzie, I.F.C., Goding, J.W. and Liew, F.Y. Monoclonal antibodies to murine Ly-2.1 surface antigen. Immunoloay 1982; 46:135-144.
~' 7. Parish~ C.R. and McKenzie, I.F.C. A sensitive rosetting method for detecting subpopulations of Lymphocytes which react with alloantisera.
J.ImmunollMethods 1978, 20:173-183.

8. Rhodes,~B.A. and Burchiel, S.W. Radiolabelling of Antibodies with Technetium oggm. Radioimmunoimaging and Radioimmunotherapy. Editors Burchiel, S.W. and Rhodes, B.A. Elsevier Publishing Co. 1983, p. 207.
.
, .. : ~ . . . . .

, .

9. Semple, T.U., Quinn, L.A., Woods, L.F. and Moore, G.E. Tumor and Lymphoid cell line$ from a patient with carcinoma of the colon for a cytoxicity model. Cancer Res. 1978; 38:1345-1355.

10. Thompson, C.H., Jones, S.L., Pihl, E. and McKenzie, I.F.C. Monoclonal antibodies to human colon and colorectal carcinoma. Br.J.Cancer 1983; 47:595-605.

11. Tubis, M., Krishnamurthy, G.T., Endow, J.S., Blahd, W.H. tl975) - 9g~Tc-Penicillamine complexes:In:Subramanian G., Rhodes, B.A., Cooper, J.F., Sodd, V.J. (eds) "Radio-pharmaceuticalsi'. The Society of Nuclear Medicine Inc., New York, pp. 55-62.

~ : ~ ,. `' ~, :
,:
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Claims (12)

1. A compound of the formula I:

R+[99?TcNX4]- I

wherein R+ represents a cation and X represents a halo group.

2. A compound according to claim 1, wherein R+
represents a sodium or another alkali metal or ammonium cation, and X represents a chloro or bromo group.

3. A process for preparing a compound of the formula I
defined in claim 1, which comprises reaction of a compound of the formula II:

R+[99?TcO4]- II

wherein R+ is as defined in claim 1, with an azide compound in the presence of a hydrohalic acid.

4. A process according to claim 3, wherein R+
represents a sodium or another alkali metal or ammonium cation, the azide compound is sodium azide, and the hydrohalic acid is hydrochloric or hydrobromic acid.

5. A method of producing a 99?TcN-labelled product, which comprises reacting a compound of the formula I
defined in claim 1, with a ligand.

6. A method according to claim 5, wherein the ligand is selected from the group consisting of methylene diphosphonate (MDP), thiourea (TU), thiomalate (TMA), dimercaptosuccinate (DMSA), gluconate (GLUC), N-(2,6-diisopropylphenylcarbamoylmethyl)iminodiacetate (PIPIDA), N-(2,6-dimethylphenylcarbamoylmethyl)iminodiacetate (HIDA), ethane-1-hydroxy-1, 1-diphosphonate (EHDP), diethylenetriaminepentaacetate (DTPA), and cysteine (CYS).

7. A method according to claim 5, wherein the compound of the formula I defined in claim 1, is reacted with a monoclonal antibody, or an antibody fragment.

8. A method according to claim 7, wherein said monoclonal antibody or antibody fragment is first at least partially reduced to convert disulfide linkages into sulfhydryl residues.

9. A method according to claim 8, wherein said reduction step is performed by reaction of the said monoclonal antibody or antibody fragment with dithiothreitol or another reducing agent.

10. A 99?TcN-labelled radiopharmaceutical which comprises the reaction product of a compound of formula I defined in claim 1, with a ligand.

11. A 99?TcN-labelled product, which comprises the reaction product of a compound of formula I defined in claim 1, with a monoclonal antibody or antibody fragment.

12. A 99?TcN-labelled product, which comprises the reaction product of a compound of formula I defined in claim 1, with an at least partially reduced monoclonal antibody or antibody fragment.