AU671465B2 - Metal complexes of hydroxyaryl containing aminocarboxylic acid chelating agents - Google Patents

Description

-1 I P/00/011 Regulation 3.2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT o r r r r Invention Title: Metal Complexes of Hydroxyaryl Containing Aminocarboxylic Acid Chelating Agents

D

r r The following statement is a full description of this invention, including the best method of performing it known to us: GH&CO REF: P13160-KH:AMP 2 The present invention relates to radiolabelled derivatives of ethylenediaminetetraacetic acid (EDTA) suitable for use as radiopharmaceutical agents. In particular, the present invention relates to EDTA derivatives, which when complexed with radionuclides may be used as imaging agents for example to assess hepatobiliary function or in radiolabelling of monoclonal antibodies, proteins, peptides, oligonucleotides and the like for in vivo imaging or therapy.

US patent 3,833,590 describes a class of EDTA derivatives which are useful for the control of metal-deficiency phenomena in biological systems. We have found that these derivatives are useful as radiopharmaceutical agents for example, for studying organ function or in 15 radiolabelling of monoclonal antibodies and the disclosure of US patent 3,833,590 is incorporated herein by reference.

Thus, according to one aspect, the present invention S: provides radiolabelled compounds of general formula (I) 20 X-NH-C-CH 2

[(CH

2

,-CH

2

-C-NH-Y

II I II I 0 CH 2

CH

2 0 COOH COOH where k is an integer from 2 to 1 is an integer from 1 to X and Y are independently selected from phenyl, naphthyl, pyridine or quinoline radical, each having at position 2 one of -OH, -SH, -NH 2 or -COOH and each of X and Y can be optionally substituted by one or more amino, halogen, hydroxy, mercapto, nitro, cyano, thiocyano, alkyl, alkoxy, halogenoalkyl, acyl, acylamino, acyloxy, carboxyl, alkoxycarbonyl, carbamoyl, pyridoylamino, N-carboxyalkyl-carbamoyl, Ssulpho, sulphamoyl, mono- or dialkylated or fT 3 3 phenylated sulphamoyl which can also carry one or S:13160KH/25.06.96 3 more substituents alkylsulphonyl, alkoxysulphonyl, or by an optionally hydroxycontaining phenylsulphonyl or phenoxysulphonyl; where R' is as defined for X and Y; or pharmaceutically acceptable salts thereof.

Preferably, X and Y are the same. The group -OH, -SH,

-NH

2 or -COOH is a donor functional group which is ortho to the position of attachment of the ring to the -NH of formula Compounds of formula and salts thereof, are labelled by nuclides such as technetium-99m 99 mTc), copper- 64( 64 Cu) or copper-67( 67 Cu) and subsequently administered intravenously into a subject for evaluating organ function, for example, the hepatobiliary system and for the purpose of therapy. The specificity for various organs may be achieved by the substitution of hydrophobic or hydrophilic groups on the ring structure at positions X and Y. Preferably, X and Y are each phenyl. For example, specificity for the liver may be achieved by the 20 substitution of hydrophobic groups such as a chloride or a tertiary butyl group on the phenyl ring. The technetium complexes of some of the compounds of formula have shown some specificity, high uptake and prompt excretion into the urine in rats and mice. The compound 25 of formula and salts thereof can also be labelled with radionuclides, such as 67Ga, 111 In, and other radionuclides from metals such as Y, Co (ie. 57 Co, 55 Co) Re, Fe, Au, Ag, Pb, Bi, Rh, Hg, Ti and Lanthanides such as Sm, Ho (ie. 1 66 Ho) or Gd.

The radiolabelling of compounds of formula and salts thereof can be accomplished by using procedures recognised in the art. The radiolabelling of the Schelator with copper-67 can be achieved by adding copper in an aqueous acetate solution to a compound of formula S:13160KH/25.06.96 4 in an aqueous solution and incubating for 15 minutes at room temperature.

Alternatively, the radiolabelling of a chelator of formula with technetium, for example, may be achieved by adding a reducing agent such as stannous chloride, to an aqueous solution of a compound of formula followed by reaction with aqueous sodium pertechnetate solution (Na 9 9mTcO 4 The order of mixing these three components is believed not to be critical. However, 10 preferably the reducing agent is added to the chelator of formula The non-radioactive composition can be supplied to the radio-chemists, technicians, radiopharmacists, doctors and the like for radiolabelling prior to use.

15 Technetium-99m in the form of an aqueous solution of sodium pertechnetate is readily obtainable from commercially available molybdenum-99/technetium-99m generators or alternatively, instant 99 mTc may be used.

64 Cu is commercially available from Australian Nuclear Science Technology Organisation and 67 Cu from the US Department of Energy, Brookhaven, USA.

The compounds of formula are obtainable by the reaction of the anhydride of EDTA with one or two equivalents, as desired, of the corresponding amine. The reaction is performed in an inert-gas atmosphere and in the presence of solvent and diluents preferably inert to the reactants. Suitable solvent are aliphatic, aromatic or halogenated hydrocarbons such as benzene, toluene, xylenes, chlorobenzene, chloroform, methylene chloride, ethylene chloride; ethers and ethereal compounds such as dialkyl ether, ethylene glycol mono or -dialkyl ether, THF, dioxane; alkanols such as methanol, ethanol, npropanol, isopropanol; ketones such as acetone, methyl ethyl ketone; nitriles such as acetonitrile or 2methoxypropionitrile; N,N-dialkylated amides such as S:13160KH/27.07.93

C

5 dimethylformamide; dimethylsulphoxide, tetramethylurea; as well as mixtures of these solvents with each other.

If the amine or a salt thereof is soluble in water, then the reaction medium may be water at low temperature.

The compounds of formula can be converted to pharmaceutically acceptable salts using art-recognised procedures.

In a second aspect, the present invention provides a diagnostic formulation suitable for labelling with 9 9mTc, 10 188 Re or 1 8 6 Re comprising a compound of formula or a pharmaceutically acceptable salt thereof and a reducing agent in a pharmaceutically acceptable carrier.

In a third aspect, the present invention provides a Sformulation comprising a radiolabelled compound of S 15 formula or a pharmaceutically acceptable salt thereof in a pharmaceutically acceptable carrier.

In a fourth aspect, the present invention provides a method of diagnosis or therapy comprising administering to a subject a radiolabelled compound of formula or a S 20 pharmaceutically acceptable salt thereof.

We have further found that the nitro substituents on the X and Y groups of compounds of formula may be further modified to form new compounds of formula The synthesis of these new compounds has provided an opening to a new array of compounds, which have been found to be suitable for radiolabelling of monoclonal antibodies, receptor specific proteins, peptides, or oligonucleotides.

Radiolabelled antibodies are especially useful in medicine, for example, in locating specific tissue types and in the treatment of cell disorders. The labelled antibodies can be used to target metal ions to a specific S:13160KH/27.07.93 6 tissue type, both in vitro and in vivo.

Thus, in a further aspect, the present invention provides novel compounds of formula pharmaceutically acceptable salts and/or radiolabelled complexes thereof

R-NH-C-CH

2

-CH-C-NH-R

II I I II 0 CH 2 C H2 O o o COOH COOH where k is an integer from 2 to 10 1 is an integer from 1 to R is independently selected from i R Ar OH wherein Ar is an aryl or heteroaryl group;

R

1 is -NR 2

R

3 where R 2 and R 3 are independently selected from hydrogen, (CH) -NH; -(CH 2 )p-Ar- 15 (CH 2 )m-NH 2

-(CH

2 )p-COH; (C 2 )p-Ar-CO 2 H;

(CH

2 CH20)n-CH 2

CH

2

NH

2

-(CH

2 )p-NCS, -(CH 2 )p-Ar-NCS; or

-(CH

2 NHCOR", -COR"; or

R

2 and R 3 together with the nitrogen atom to which they are attached form an optionally substituted 20 saturated or partially unsaturated ring optionally containing one or more heteroatoms 0, S or N; or

R

1 is -NCS, -N=N or -C(=NH)-OCH 3 n and m are independently an integer from 0 to 4 p is an integer from 1 to 4; R" is alkyl-L where L is halogen or other leaving group; 2,5-diketo-pyrrolinyl, -Het-CH=CH 2 wherein Het is an optionally substituted 5 or 6 membered heterocyclic ring containing one or more heteroatoms 0, N or S.

Preferably, aryl is phenyl, naphthyl, anthracenyl or the like and heteroaryl is preferably pyridine, quinoline, imidazolyl or the like. Het is preferably pyridine and Het-CH=CH 2 is preferably, 2- or 4-vinylpyridine.

S:13160KH/27.07.93 7 In yet a further aspect, the present invention provides a process of preparing compounds of formula which comprises treating the corresponding nitro compound with a catalyst such as palladium/C in the presence of a reducing agent such as sodium borohydride and as required, further derivatising the amino group following procedures known in the art.

Compounds of formula (II) where R 1 is mono- or disubstituted amino and where the substituent is 10 substituted alkyl, are readily prepared by treating the amino compound with the appropriate halo-substituted alkyl. For example, compound of formula (II) where R 1 is

-NH-CH

2

-CH

2

-NH

2 can be prepared by treating a compound of formula (II) where R 1 is NH 2 with BrCH 2

CH

2

NH

2 in the presence of NaHCO 3 or the like.

V a Compounds of formula (II) where R 1 is -NCS is prepared by reacting the amino compound with thiophosgene (see W087/12631), Kozak et al., Cancer Res. 49, 2639 (1989).

Substituted acid halide compounds are produced by reacting a compound of formula (II) where R 1 is NH 2 with BrCH 2 COBr at 4 0 C according to the procedure of C J SMathias et al., Bioconjugate Chem., 1, 204 (1990).

Compounds with an electrophilic moiety can also be prepared by methods known in the art, such as in ACC Chem. Res. 17 202-209 (1984). Compounds with active esters R-C(O)-X can be formed by the procedures of Bodanszky M, The Peptide. Analysis, Synthesis, Biology, Ed. E. Gross and J Meienhofer, Vol 1. pp105-196, Academic Press, Inc., Orlando, FL. (1979) and Bodanszky M, Principles of Peptide Synthesis, pp9-58, Springerverlag, New York, (1984). 2,5-diketo-pyrrolinyl, -Het-CH=CH 2 or 2,4-vinylpyridine compounds can be prepared according to the procedure of Morphy et al., J. Chem. Soc., Chem.

Commun., 156, (1988). Compounds containing maleimides and succinimide may be prepared by procedures outlined in Y Arano et al., Bioconjugate Chem., 2, 71, (1991) and S S:13160KH/27.07.93 8 Garg et al., Bioconjugate Chem., 2, 50, (1991). Other standard procedures are to be found in Modern Synthetic Reactions, H 0 House, 2nd Edition, Benjamin, Inc.

Phillippines, 1972.

The R 1 group may be termed the reactive group which provides the point of attachment for a protein such as a monoclonal antibody either directly, ie where R 1 is NH 2 or through a linker, ie other R 1 groups defined above.

The isothiocyanate and the halogen functionalised 10 derivatives may be directly linked to a protein (eg.

antibody) thiol or amino group. In a structural comparison, it can be seen that R 1 is equivalent to a 0:* substituent on the X or Y group of compounds of formula 15 Preferably, the -OH group is ortho to the position of attachment of the phenyl ring to the -NH of formula (II).

R

1 can be on any other position on the ring.

Compounds of formula (II) provide a method of attachment of radionuclide metal ions such as In(III), Gd(III), 20 Ga(III), Fe(III), Tc0 4 3 Cu(II), Ti(IV) and other radionuclides from the Lanthanides, Rhenium, Samarium, Holmium, Yttrium and the like to monoclonal antibodies, receptor specific proteins, peptides or oligonucleotides for in vivo imaging and therapy.

Preferred use of the compounds would be to radiolabel monoclonal antibodies specific for colon, ovarian, lymphoma, breast and/or bladder cancer, with beta emitter radionuclides of metals such as Sm, Ho, Re, Sc, Cu, Pd, Pb, Rh and Y for therapy of above mentioned cancer. A further preferred use is in the radiolabelling of a monoclonal antibody specific for metastasis of colon cancer for diagnosis and therapy.

It is generally preferable to couple the compounds of S:I3160KH/27.07.93 I a_ 9 formula (II) to other molecules such as proteins, peptides, antibodies or carbohydrates to form conjugate compounds for use as radiopharmaceutical agents or cytotoxic agents.

Compounds of formula (II) may be coupled through any thiol, amino, carboxyl, hydroxyl, aldehyde, aromatic or heteroaromatic group present in the protein, peptide, carbohydrate or oligonucleotide.

In particular, the present invention provides a conjugate compound which comprises a compound of formula a radiolabelled complex and/or salt thereof, coupled to an antibody, protein, peptide, carbohydrate or oligonucleotide.

The conjugate compound according to the invention may S 15 contain more than one molecule of a compound of formula (II) coupled to any one protein, peptide or carbohydrate molecule. The antibody in the conjugates may be a e S. complete antibody molecule or a fragment thereof or an analogue of either of these, provided that the antibody 20 comprises a specific binding region. The antibody may be humanized monoclonal or a fragment thereof. The antibody may also be a recombinant antibody. The antibody may be specific for any number of antigenic determinants, but is preferably specific for one antigenic determinant.

In another aspect, the present invention provides a pharmaceutical formulation comprising a compound of formula a radiolabelled complex and/or salt thereof in a pharmaceutically acceptable carrier.

In a further aspect, the present invention provides a method of diagnosis or therapy in a subject comprising administering to the subject a compound of formula (II) or a radiolabelled complex and/or salt thereof.

S:13160KH/27.07.93 10 Radiolabelling of proteinaceous materials using bifunctional chelators of the present invention can be conducted in two ways, namely: prelabelling of chelator followed by conjugation of resultant radiocomplex to proteinaceous material, or conjugating chelator to proteinaceous material for subsequent radiolabelling.

Other suitable applications are: o* 10 PET imaging *9 When chelators of formula or (II) are labelled with 64 Cu (Positron 0.278 MeV; half-life 12.70 hours), or 62 Cu (Positron 0.93 MeV; half-life 9.8 minutes), such radiocomplexes may be useful for quantitative dynamic 15 flow measurements of blood flow and bile flow in the hepatobiliary system. Alternatively the chelators may be labelled with Ti-45, a positron emitter (1.04 MeV) with a half life of 3.08 hours.

Radiolabelling monoclonal antibodies with 67 Cu, (Beta 20 and Gamma emitters) for combined radioimmunoscintography (RIS) and radioimmunotherapy (RIT) Auger emitting agents where a chelator is coupled to an intercalator and labelled with auger emitting isotope such as Fe-59 two step pretargeted radioimmunotherapy A monoclonal antibody with one or more biotin molecules or marker molecules attached is injected into a patient.

Once the antibody has cleared from the system and localized to the tumour, a second injection is administered. This second injection involves the radiolabelled chelator attached to a smaller molecule S:13160KH/27.07.93 11 such as avidin or streptavidin which recognises the biotin or marker on the targeted antibody.

Alternatively, the second injection may be avidin or streptavidin and when cleared from the system, the radiolabelled chelator attached to biotin is administered. Both procedures provide amplification of the target site and reduce exposure to normal tissue.

Another two step procedure involves the administration of an antibody-DNA conjugate or antibody-oligonucleotide S' 10 conjugate followed by targeting with a radiolabelled complementary DNA or complementary oligonucleotide. This procedure also provides amplification of the target site and reduces exposure to normal tissue.

Radiotherapy of Liver metastasis The chelators compound 2 and compound 4 specifically traverse the hepatobiliary system (see data in Tables 2 and 4, Biodistribution of 99 mTc complex of compound 2 and 4 respectively), and empty into the gastrointestinal 0. tract. When chelators, such as compound 2 and compound 4, are labelled with a therapeutic radionuclide such as 67 Cu, they would be useful for treatment of metastasis of the liver.

Magnetic Resonance Imaging (MRI) agents We envisage the use of these compounds as MRI agents where complexes formed with any paramagnetic metal ion such as Fe (III) may be used as a contrast agent to enhance images. Also complexes such as these may be attached to a pharmaceutically acceptable carrier for the same purpose. We also envisage the industrial use of these compounds for attachment to solid surfaces such as polymers, electrode surfaces etc. for use in the concentration of metal ions, purification of water or the S:13160KH/27.07.93 12 like.

The present invention also encompasses mixed combinations of radioactive labels on a given monoclonal antibody or a mixture of monoclonals and metal radionuclides attached to one or more different ligands. These would be useful for cancer therapy.

Specific embodiments of the present invention are illustrated by the following examples. It will be understood, however, that the invention is not confined 10 to specific limitations set forth in the individual examples.

Experimental A series of derivatives incorporating carboxyl and .phenolate donor groups were synthesised by 15 literature method and their radiolabelling characteristic and biological behaviour evaluated. Initial work involved an investigation of radiochemical properties of 0 the 9 9mTc complexes df the ligands and their biodistribution in rats and mice (see Tables 1-6).

a.

Compound No. 3 To a solution of 4-nitro-2-amino-phenol (0.18g) in acetonitrile (100ml) was added EDTA anhydride (5.05g).

The reaction mixture was stirred vigorously with reflux under N 2 gas overnight. The solid which formed was filtered off while the solution was still warm. The yellow solid was then washed with copious amount of methanol and finally acetone. Yield: 8.92, 81%.

All compounds listed below were prepared in a similar manner. Yields and all spectral data for some compounds are presented in Table A.

S:13160KH/27.07.93 13 Compound No. X and Y Formula 0

CLQ

a a a a.

*0 a a a a. a. a.

a a a a

N~H,

0 S:131 6OKH/27.07.93 f16 0 a 0 4 TABLE A Physical Data for Selected Compounds Cpd Ms, Mptb FTIRc NMRd (yield%) (OC) (cm'1) (ppm) No. 1 475 204-205 NCCO, 1708.2 8.02,s,2H,OH;6.91-6.72,m,6H;Ar;3.46,3.38,s,4H,4H, COOH, 1635.8 CH 2 CONH, CH 2 COOH;2.86,s,4H,NCHCH; OH, 3314.1 No. 2 543 222-224 NHCO, 1716.9 9.63,s,2H,OH;8. 17,6.01,6.81 ,d,6H,Ar;0.45,3.39,s,41, COOH, 1659.0 4H,CH 2

,CONH,CH

2 COOH;2.83,s,4H,NCH, 2

CH

1 OH, 3331.6 No. 3 564 222-226 NHCO, 1728.4 9.88,s,2H,OH;9.06,7.89,6.94,d,2H,2H,Ar;3i.5-76,3J.52,s, COOH, 1693.9 4H,4A-,CH 2 CONH,CH, COOH;2.83,s,4H,NCHCH 2 OH, 3299.3 No. 4 587 0 NHCO, 1708.9 9.59,s,2H,OH;8.1 1,6.90,6.715,d,6H,Ar;3.42,3.38,s,4H, COOH, 1658.8 4H,CH 2

CONH,CH

2 COOH;2.83,s,4H,NCH,; 1.2 1,s, OH, 3318.9 18H,tBu; No. 5 509 'NHCO, 1740.1 9.33,s,2H,OH;8.41I-8.28,m,6H,Ar;5.42,5.35,s,4H,4H, Method A COOH, 1683.3 CH 2

CONH,CH

2 COOII;3.80,s,4H,NCHCH 2 OH, 3300 br No. 5 504 NHCO, 1734.0 07.52,s,2H,6.90-6.81l,m,4H,Ar;3.92,3.76,s,4H,4H, Method B COGH, 1684.0 CH 2

CONH,CH

2 COOH;3.28,s,4H,NCHCH,; OH, 2998 br 'Positive FAB mass spectra were determined of Jeol DX-300 mass spectrometer. MS:parent Ion (FAB):M/C, LH'. bAll melting points are uncorrected. 'Diffuse reflectance FT Infrared spectra were recorded on B ioRad Giglab FTS-60 in 1% Kbr. dThe 'H NMR spectra were determined in d 6 DMSO0 at 298'K, on a Jeol Gx-400MHz spectrometer. 'The 1'H NMR spectra were determined in D, 2 0 at 298'K, on a Jeol Gx- 400MHz spectrometer. 0 Compound hygroscopic. 'Decomposes.

15 Preparation of New Compound No. Method A To Palladium/C catalyst (50mg) in water (10ml) was added sodium borohydride (0.24g) in water (10ml) under nitrogen gas. To this mixture was then added compound 3 (l.0g) in ethanol/sodium hydroxide solution (10ml/lml, 8% NaOH) slowly. The mixture was left to stir at room temperature for a further 20 minutes or till the solution becomes clear. To this mixture is added concentrated hydrochloric acid dropwise till all excess sodium borohydride is quenched. Palladium catalyst is filtered off and the filtrate is reduced under vacuum. The pale pink powder is dissolved in a minimum of methanol and any salt remaining filtered off. Yield: 0.85g; Method B To palladium on activated charcoal (50 mg) in distilled water (2 cm 3 was added sodium borohydride (0.16 g) under nitrogen. Ethanol 8 cm 3 was added to the reaction mixture, then ligand (1.0 g) in concentrated ammonia (10 cm 3 :28% ammonia). The mixture was left to stir at room temperature for a further 30 minutes or until the solution became clear in colour. Concentrated hydrochloric acid was added dropwise to the mixture to destroy any unreacted sodium borohydride or until gases ceased to evolve. The final pH of the solution was The palladium catalyst was removed by filtration and the filtrate reduced under vacuum. Water was azeotroped off using copious amounts of ethanol and acetone. The purple/brown residue was suspended in a minimum amount of ethanol 20 cm 3 and any undissolved salt removed by filtration. Yield 0.85 g; Final product was characterised by 1 H NMR, FT-IR and electrospray massspectrometry and melting point. Data are tabulated in Table A.

S:13160KH/25.06.96 Radiolabelling of Chelators with 99 mTc A typical method of radiolabelling of the chelator was achieved by dissolving the chelator in a basic solution of saline. The pH was then adjusted to approx. 6 with HC1. Excess stannous chloride was added to the resultant solution followed by one equivalent volume of sodium 99 mTc04 (10-50 mCi/ml). Final concentration of the chelator was approx. Img/ml. The efficacy of labelling was determined by ITLC-SG. For each product only one main radiochemical species was observed. Each complex was diluted to 15 pCi/100l with saline. In vitro saline stability studies showed the complexes were stable for up to 6 hours. Serum stability studies indicated that no more than 15 per cent of the technetium is lost from the :15 chelator during the first hour at 37 0 C. (See Figure S 9 S:13160KH/25.06.96 :16 Serum stability of Technetium complex Complex of (Time-0) 04 *4 *q 4 4 9*44 6844 4 .rC.

4* *4 A 4*44

S

4* 66 @5 4 4 4 a 12 16 20 24 Volume (ml) Serum stability of Technetium complex Complex of (tim-i hr) 0 4 Figure 1I a 12 16 20 24 28 Volume (ml) Serum stability of Technetium complex of (1) S:131 6OKH/27.07.93 17 Biodistribution studies in rats show that the 99 mTc complexes are extracted rapidly and efficiently from the blood by the kidney and the liver.

Selected data from biodistribution results of technetium- 99m complexes of No. 1, No. 2 and No 4 in the liver, kidney and blood at 3 minutes is tabulated below.

Compound Liver Kidney Blood No. 1 1.54 4.12 1.16 No. 2 5.93 8.90 1.32 No. 4 7.89 4.54 1.71 The structural variation in this selection of derivatives is the substitution of a chloride atom in No. 2 for a hydrogen atom on both aromatic rings. For No. 3, the substitution of a tertiary butyl group on each aromatic S 15 ring. The substitution of these groups make the parent S: compound more hydrophobic hence there becomes a preference for the liver uptake as opposed to the kidney.

Effect of pH on Complexdaion of 99Tc with ligand Radiolabelling with 99 mTc was investigated over a range of pHs (3.0 10.0) (described in Table To ligand (1.0 ml 1 mg/ml) dissolved in appropriate buffer was added an excess of stannous chloride (20 Al 2 mg/ml) in 0.1 N HC1. To this mixture is added an appropriate activity of 99 mTc0 4 (20 il) eluted from a technetium 25 generator.

All reactions were carried out at room temperature.

Percent complexation was monitored by ITLC-SG using two solvent systems. System 1 cyclohexane/acetone 1:2 showed free 99 mTc0 4 migrated with the solvent front and any colloid or complex formed remained at the origin.

System 2 saline showed complex and free 99 mTc0 4 migrate with the solvent front and colloid remained at the origin. Results are illustrated in Table C.

S:13160KH/25.06.96 17A S~o 0500 **0

*S

S S 500)s

S

5

S.

Table B. Buffers and range of pH used in 99mTc and 64/67Cu radiolabelling of pH range Buffer (0.5 M) 4.0 acetate 5.0 7.0 phosphate 9.0 bicarbonate 10.0 carbonate Table C. Percent 99 mTc complexed by ligand verses reaction time Reaction Time pH 1 min 3 min 5 min 10min 15 min 30 min 1h 2h 3 89 93 90 91 90 92 92 94 4 94 97 92 94 96 91 100 100 84 83 91 83 81 71 85 88 6 80 80 83 80 82 90 85 91 7 73 83 81 74 87 80 83 52 8 76 88 81 83 87 85 77 81 9 90 100 94 86 100 83 J85 74 10 15 16 11 13 9 ND J8 8 This study shows that complexation for is rapid and optimum at pH 3.0 4.0. The final product formed is stable for upto 2 hours throughout pH range 3.0 However complexation is poor at pH 10.0 and what product formed is unstable.

15 Radiolabelling of Chelators with 64 Cu and 67 Cu A typical procedure involved the dissolving Cu-67 20pl of original stock in an acetate buffer (0.2 M; pH 4.5) to a total volume of 500pl. To this solution was added 500l of the ligand (20mg/ml) solution in acetate buffer. The mixture was then left to react at 37 0 C for 20 minutes.

ITLC confirmed the labelling was greater than Serum Stability Studies of Copper-67 Complexes The serum stability was determined for selected derivatives and typical procedure is as follows.

The ligand was labelled with Cu-67 at a ligand:metal ratio of 4:1. ITLC showed the efficiency of labelling to be 95%. Serum stability studies demonstrated a loss of 4% over the initial 4 hours and no further loss after 24 hours. See Figure 2.

S:13160KH/25.06.96 a a a a Figure 2: Serum stability of Cu-67 complex of ligand Serum StabTI I ty TTme 0 0 2 4 6 9 10 12 14 16 19 VOW-m Cml) Figure 2: continued.

TTrno lhr a 2 4 6 2 10 12 14 16 163 Volumo CmID Cl)

C)

50 e S 0 Figure 2: continued.

Time 4hr 0 2 4 6 e 10 12 14 is 19 VolUMQ CMID a S a..

a. .aa Figure 2: continued.

Time 24hr 0 2 4 6 e 10 VOlum Cml) 12 14 i6 12 23 Biodistribution of Copper-67 Complex of The biodistribution of the copper-67 complex of ligand was evaluated in balb/c mice (5 animals per time point) at 1, 3, 5, 10, 30 minute time intervals. The data are presented in Table 7.

The results show that the Cu-67 complex of clears from the kidneys, blood and urine over the 30 minute period. However there appears to be an active uptake of the complex in the liver over the 30 minute period.

Effect of pH on radiolabellinq ligand with 64/ 6 7 Cu Radiolabelling with 64 67 Cu was investigated with ligand over a range of pHs (see Table Conditions employed are similar to that reported above for where the 64 67 Cu in appropriate buffer was added directly to a buffered solution of ligand Rate and percent of complexation was monitored over time by ITLC-SG. Mobile phase employed was methanol water ammonia trace Samounts). The copper complex moves with the solvent front while the free copper is retained at the origin.

20 Complexation was complete within the first minute for all S. pHs (pH 3.0 1 0.0) and maintained for upto 2 hours.

Radiolabelling of Protein Radiolabelling of protein may be achieved by two methods First the chelator may be radiolabelled by a procedure outlined previously. The radiolabelling of the proteinaceous material may be accomplished using a crossconjugation linker such as EDC (CH 3

CH

2

(CH

3

(CH

3 2 -HCl or the like. For example, to IgG (such as B72.3) in phosphate buffered saline is added the radiolabelled ligand at a molar ratio of 20:1 and EDC in SR a 1000:1 molar ratio. The mixture is then left to react -3 at 37 0 C for 11 hour. The unreacted reagents are removed S: 13160KH/25.06.96 24 by size exclusion ultra filtration membrane.

Radiolabelling of Protein with 67 Cu The radiolabelling of human serum albumin (HSA, Sigma Chemicals) and streptavidin (Sigma Chemicals) and IgG was achieved at values ranging from 0.12-0.16LCi/pg. The purified radiolabelled IgG was analyzed on HPLC sizeexclusion column and fractions collected. Greater than was associated with the main protein peak. No evidence of crosslinking was observed under the labelling conditions used, ie similarly, the chelators can be labelled with a range of radionuclides such as 62Cu, 1 66 Ho, 153 Sm, 188 Re, 186 Re and 45 Ti.

Typical reaction for radiolabellin B72.3 antibody with 64 6 7 Cu To ligand dissolved in 0.2 M sodium acetate buffer (pH 7.0) was added 64 67 Cu in acetate buffer. Greater than >99% complexation was confirmed by ITLC-SG. Antibody 15 mg/ml) was then reacted with the solution of complex L at a 20:1 ligand to antibody ratio in the presence of 1000:1 EDC at 37 0 C. A range of antibody concentrations were investigated to determine the effect on crosslinking of protein. Each product was purified by sizeexclusion chromatography and then analysed for crosslinking using HPLC Bio-Sil SEC-250 column (eluent PBS:0.7 ml/min). The results are illustrated in Figure 3. The data shows that as the protein concentration was increased the amount of cross-linking also increased.

S:1 3160KH/25.06.96 *9 9 9 9* 99 9* a 9999 9 W *9 *9 a *9 a 9*eS 9 a 9 15 mg/mi mg/mi -starting material 10 15 20 Time (min) Effect of protein concentration on cross-linking when radiolabelling with Figure 3.

~1_ 26 The second method involves the conjugation of the chelator to the antibody or proteinaceous material as described but using the unlabelled ligand and prepared in a form suitable for subsequent labelling with a suitable radionuclide. This is the preferred form for preparation of the proteinaceous material in a kit form, suitable for manufacturing.

Ga-67 citrate (t 1 /2 78.25 h, y 184.6 keV is commonly used in the diagnosis of lymphoma. While Ga-67 citrate shows relatively fast and selective uptake at the disease sites, it clears slowly from the blood, through the liver, accumulating in the bowel. Its long biological half-life necessitates repeated scanning at delayed intervals (upto 72 h postinjection), resulting in 15 repeated visits of the patient and increased demands on physician time.

In an attempt to address these short comings, 67 Gacomplexes of a series of hydroxyaryl EDTA derivatives (1) have been synthesized and their mode complexation 20 and biological behaviour investigated.

Radiolabelling with 6 7 Ga The series of derivatives to were radiolabelled with 67Ga. The kinetics of radiolabelling were monitored at various pHs and temperatures. The rate of complexation was found to be temperature, pH and concentration dependent. The optimum pH for complexation was -12. The data are represented in Figures 4 18.

A typical method for radiolabelling with 6 7 Ga Dissolve the ligand [10mg] in 1M NaOH (200ul) and then adjust the volume to 1 mL with 0.2 M NaOAc (pH The final pH of solution should be 13. Dissolve the I 6 7 Ga citrate (purchased from Australian Radioisotopes, Australia) with 0.2 M NaOAc (pH 7.0) buffer. Mix equal S:13160KH/25.06.96 27 volumes of the ligand and 67 Ga NaOac solutions and monitor complexation by ITLC-SG (mobile phase ethanol:water 9:1).

Rf for 67Ga complex migrate with solvent front and any colloid or uncomplexed 67Ga remained at the origin.

Typical preparation of 67 Ga complex for serum stability studies and biodistributions.

To ligand (50 AL, 10 mg/mL, 0.2 M NaOAc, pH 12) was added 67 Ga citrate (18.5 MBq) and the resultant mixture diluted to 1 mL (0.2 M NaOAc, pH The pH was adjusted to 12 with 1 M NaOH (20 AL) and heated at 40 0 C for 40 min. The final volume was adjusted (to 5 mL) with saline and sterilised by filtering through a 0.22 pm filter.

Each animal was injected via tail vein with 67Ga complex (100 pL, ~3.7x10 5 Bq). Animals were sacrificed at 15 various time intervals (5 animals per time point/6 time points). Data are illustrated in Figure 26 30 and in Tables 13 17.

Serum stability of the selected 6Ga-complexes.

S- The 67 Ga complexes were prepared as described, then diluted in human plasma The mixtures were incubated at 37 0 C fo.: upto six days.

Aliquots were sampled at regular intervals and the protein separated from the complexes usin4 size-exclusion chromatography (sephadex-G25; eluent: phosphate buffered saline). Fractions (1.5 mL) were collected and monitored for UV absorbance and radioactivity. Profiles show no radioactivity associated with the protein indicating 67 Ga complexes are stable under conditions described. See Figure 19 Biodistribution studies of 67 Ga complexes Each 67 Ga-ligand complex has been evaluated in balb/c R mice. Studies show that the complex route of excretion x may be directed by the substituents on the phenolic S:13160KH/25.06.96 28 groups of the ligand. (see Figure 26 The present study shows that complexation of the hydroxyaryl ligands is rapid (at pH >12) and the 67 Ga complexes are stable. Data show that substituents on the phenolic rings of these ligands can effect the mode of excretion of the 67 Ga complexes of the hydroxyarvl-EDTA derivatives. High yields in synthesis of the hydroxyaryl-EDTA derivatives and rapid comDlexation make these ligands attractive for "kit formulation" and indicate that they may have a role as imaging agents in nuclear medicine.

Complexation of these ligands have also been investigated wi th 5Ni, 5Co and 55 Co.

Typical preparation involves dissolving the ligand 15 mg) in 0.1 N NaOH and then adjusting the pH to 6.5. The final mixture is then diluted with acetate buffer (0.2 N e NaAc; pH The radioisotope dissolved in acetate buffer is then added to the mixture. Complexation is J confirmed by ITLC-SG using methanol: water: ammonia (9:l:trace). In each case the complexes move with the solvent front while free radioisotope remains at the origin. Under these conditions complexation was complete in less than 1 h.

Complexation and stability of with 16 6 Dy and 166Ho Radiolabelling with of with 166 Dy and 166 Ho and the serum stability of the complex formed has been investigated. A typical procedure involves dissolving (2.6 10"l in 1.0 M NaOH (150 p1) and then adding saline (669 1l). Then equal volume of ligand mixture and the 1 66 Dy/ 166 Ho in 0.2 M NaAc (pH to 4.5) are mixed. The complexation is monitored by ITLC-SG. Mobile phase SRAL, acetonitrile: water:4:1 shows 99% complexation is complete within 40 mins at RT.

S:13160KH/25.06.96 C I_ 29 The radiolabelling is then incubated in human serum at 37 0 C. At various time intervals the aliquots are separated and monitored for UV absorbance and radioactivity. The data illustrated in Figure 31 show the complex is stable for 46 hrs.

Formulations of the radiolabelled compounds of formula or (II) are prepared and administered in according to standard techniques. A pharmaceutical formulation of the present invention comprises the active compound together with one or more pharmaceutically acceptable carriers and optionally any other therapeutic ingredient. The formulation may conveniently be prepared in unit dosage form and may be prepared according to conventional pharmaceutical techniques. Additionally, the formulation 15 may comprise one or more accessory ingredients such as

S

e diluents or buffers. The effective amount of the active compound will vary both with the route of administration, the condition under treatment and host undergoing treatment.

o S:13160KH/25.06.96 TABLE 1 BIODISTRIBUTION OF 99 m TC COMPLEX OF NO. 1a MEAN SD MEAN SD MEAN SD MEAN SD MEAN SD SEC 1 MIN 3 MIN 5 MIN 10 MI!N LIVER 1.73 0.21 2.01 0.15 1.54 0.10 1.34 0.15 0.76 0.15 SPLEEN 0.53 0.04 0.42 0.04 0.26 0.03 0.20 0.03 0.15 0.01 KIDNEYS 6.73 0.45 6.55 0.50 4.12 0.62 2.87 0.21 2.72 0.34 MUSCLE 0.28 0.02 0.27 0.03 0.26 0.07 0.27 0.13 0.15 0.01 SKIN 0.37 0.03 0.43 0.05 0.49 0.04 0.53 0.06 0.40 0.10 BONE 0.31 -0.03 0.32 0.03 0.26 0.02 0.25 0.07 0.19 0.02 LUNGS 1.15 0.10 1.13 0.11 0.80 0.05 0.54 0.03 0.46 0.03 HEART 0.75 0.04 0.67 0.09 0.46 0.06 0.33 0.03 0.27 0.03 BLOOD 1.93 0.09 1.71 0.14 1.16 0.14 0.83 0.05 0.71 0.04 STOMACH 0.14 0.08 0.28 0.14 0.32 0.28 0.11 0.01 0.12 0.03 TOT GIT 0.27 0.02 0.34 0.03 0.59 0.19 0.79 0.08 1.22 0.16 TAIL 0.68 0.03 0.56 0.05 0.80 0.32 0.73 0.04 0.83 0.10 BRAIN 0.14 0.30 0.09 0.03 0.06 0.02 0.05 0.01 0.04 0.01 THYROID 1.42 0.14 1.15 0.24 1.17 1.12 0.59 0.09 0.40 0.07 EYES 0.19 0.02 0.17 0.01 0.18 0.02 0.17 0.01 0.13 0.02 PANCREAS 0.49 0.08 0.51 0.11 0.44 0.19 0.44 0.18 0.30 0.02 THYMUS 0.38 0.04 0.35 0.04 0.27 0.03 0.19 0.03 0.14 0.01 aAustralian Albino Wistar Rats, Injected dose per gram (X SD). Average of 5 animals per time point.

IJ

0 23 at. a 0 0 0* a a 0 0* a *0 a TABLE 2 BIODISTRIBUTION OF 99 TC COMPLEX OF NO. 2a MEAN SD MEAN SD MEAN SD MEAN SID MEAN SID 3SEC 1iMIN 3 MIN 5 MIN LIVER 4.61 0.69 6.28 1.07 5.93 0.73 3.93 0.66 1.89 0.36 SPLEEN 0.62 0.04 0.58 0.031 0.33 0.03 0.25 0.04 0.16 0.03 KIDNEYS 6.42 0.45 8.37 0.63 8.90 0.73 7.26 2.45 3.35 0.54 MUSCLE 0.22 0.03 0.24 0.01 0.20 0.03 0.16 0.02 0.11 0.02 SKIN 0.29 0.02 0.45 0.07 0.42 0.02 0.35 0.02 0.33 0.05 BONE 0.34 0.03 0.37 0.03 0.25 0.03 0.19 0.021 0.15 0.03 LUNGS 1.20 0.09 1.17 0.14 0.79 0.06 0.59 0.02 0.48 0.09 HEART 0.87 0.11 0.93 0.07 0.46 0.02 0.36 0.03 0.28 0.05 BLOOD 2.79 0.33 2.77 0.20 1.32 0.09 0.94 0.08 0.77 0.16 STOMACH 0.41 0.10 0.68 0.46 2.08 3.10 0.98 0.94 0.90 1.51 TOT G IT 0.48 0.12 0.58 0.08 1.56 0.25 2.27 0.19 3.18 0.71 TAIL 0.81 0.22 0.66 0.09 0.64 0.07 1.16 0.78 0.55 0.08 BRAIN 0.14 0.01 0.09 0.01 0.06 0.01 0.04 0.01 0.03 0.01 THYROID 1.41 0.36 1.71 0.66 0.81 0.18 0.58 0.14 0.44 0.12 EYES 0.11, 0.01 0.15. 0.03. 0.12, 0.02 0.10 0.02, 0.10 0.02 ~PANCREAS 0.49 0.06 0.66 0.16 0.44 0.17 0.52 0.34 0.27 0.04 [THYMUS 0.39 0.0 15 0.37 0.08 0.271 0.04 0.17 0.02 0.15 0.03 aAustralian Albino Wistar Rats, Injected dose per gram (X SID). Average of 5 animals per time point.

TABLE 3 BIODISTRIBUTION OF 99 mTC COMPLEX OF NO. 3" MEAN SD MEAN SD MEAN SD MEAN SD MEAN SD SEC 1 MIN 3 MIN 5 MIN 10 MIN LIVER 1.43 0.03 1.61 0.18 1.61 0.19 1.24 0.20 0.92 0.16 SPLEEN 0.49 .0.06 0.42 0.05 0.29 0.03 0.23 0.03 0.17 0.01 KIDNEYS 3.92 0.22 4.63 0.64 4.27 1.12 3.00 0.31 2.10 0.21 MUSCLE 0.26 0.02 0.24 0.03 0.23 0.04 0.21 0.02 0.22 0.06 SKIN 0.49 0.06 0.43 0.07 0.58 0.11 0.64 0.08 0.63 0.04 BONE 0.30 0.03 0.29 0.04 0.30 0.11 0.29 0.08 0.22 0.04 LUNGS 1.24 0.16 1.04 0.12 0.87 0.04 0.72 0.06 0.57 0.07 HEART 0.78 0.11 0.65 0.05 0.55 0.06 0.45 0.05 0.34 0.02 BLOOD 2.10 0.10 1.83 0.17 1.37 0.14 1.17 C.11 0.86 0.05 TAIL 0.85 0.30 2.43 3.83 0.63 0.10 0.84 0.14 0.84 0.07 BRAIN 0.18 0.04 0.14 0.03 0.09 0.02 0.08 0.01 0.05 0.01 THYROID 1.11 0.22 1.14 0.36 0.92 0.10 0.70 0.24 0.63 0.04 EYES 0.20 0.03 0.23 0.05 0.23 0.01 0.17 0.03 0.18 0.05 PANCREAS 0.47 0.07 0.41 0.09 0.38 0.07 0.42 0.13 0.21 0.01 THYMUS 0.38 0.03 0.34 0.03 0.32 0.06 0.25 0.04 0.20 0.06 aAustralian Albino Wistar Rats, Injected dose per gram (X SD). Average of 5 animals per time point.

I

TABLE 4 BIODISTRIBUTION OF 99m TC COMPLEX OF NO. 4a MEAN SD rMEAN SD MEAN SD MEAN SD MEAN SD 1 MIN 3 MIN 5 MIN LIVER 6.83 0.51 8.03 0.95 7.89 0.57 7.39 1.20 4.90 1.06 SPLEEN 0.93 0.12 0.80 0.051 0.42 0.05 0.32 0.051 0.27 0.05 KIDNEYS 5.57 0.88 4.86 0.78 4.54 0.68 3.89 0.26 2.33 0.50 MUSCLE 0.361 0.05 0.29 0.04 0.22 0.05 0.18 0.05 0.13 0.04 SKIN 0.53 0.09 0.51 0.07 0.46 0.06 0.37 0.05 0.36 0.04 BONE 0.61 0.10 0.54 0.05, 0.32 0.06 0.26 0.051 0.19 0.04 LUNGS 2.37 0.46 1.67 0.13 1.00 0.21 0.66 0.131 0.53 0.09 HEART 1.78 0.32 1.26 0.07 0.65 0.07 0.48 0.09 0.36 0.10 BLOOD 5.84 1.01 3.96 0.32 1.71 0.83 1.33 0.33 0.99 0.22 URINE I- 0.08 0.05 STOMACH 1.04 0.41 0.95 0.30 3.09 1.27 2.71 4.081 3.80 6.73 TOT G IT 0.61 0.08 0.74 0.26 1.59 0.63 2.62 0.70 5.16 1.86 TAIL 1.03 1.02 0.53 0.23 0.39 0.07 1.23 2.00 0.32 0.10 BRAIN 0.43 0.04 0.26 0.051 0.12 0.01 0.08 0.02, 0.06 0.06 THYROID 3.49 0.83 2.28 0.25 1.11 0.63 0.89 0.24 0.79 0.18 EYES 0.22 0.03 0.19 0.05 0.15 0.04 0.13 0.08 0.16 0.04 PANCREAS 0.90 0.13 0.69 0.20 0.55 0.34 0.32 0.07 0.39 0.22 THYMUS 0.66 0.08 0.49 0.08 0.31 0.02 0.22 0.03 0.191 0.63 aAustralian Albino Wistar Rats, Injected dose per gram (X Average of 5 animals per time point.

cc r t O TABLE BIODISTRIBUTION OF 99m TC COMPLEX OF NO. MEAN SD MEAN SD MEAN SD MEAN SD MEAN SD SEC 1 MIN 3 MIN 5 MIN 10 MIN LIVER 1.11 0.16 0.97 0.16 0.83 0.13 0.79 0.07 0.69 0.07 SPLEEN 0.83 0.12 0.98 0.10 0.83 0.08 0.83 0.05 0.72 0.03 KIDNEYS 4.35 0.55 5.70 0.45 4.70 0.10 6.04 1.92 3.61 0.36 MUSCLE 0.25 0.03 0.30 0.01 0.27 0.04 0.25 0.01 0.23 0.04 SKIN 0.31 0.03 0.38 0.06 0.43 0.04 0.41 0.0 0.46 0.04 BONE 0.37 0.02 0.45 0.02 0.42 0.03 0.37 0.08 0.36 0.03 LUNGS 1.66 0.19 1.69 0.20 1.31 0.09 1.25 0.15 1.17 0.10 HEART 1.81 0.22 1.62 0.18 1.24 0.09 1.15 0.11 1.06 0.17 BLOOD 4.92 0.67 4.22 0.34 3.26 0.29 3.13 0.17 2.59 0.19 TAIL 0.22 0.11 0.14 0.01 0.17 0.12 0.15 0.03 0.19 0.05 BRAIN 0.20 0.02 0.22 0.04 0.16 0.03 0.17 0.02 0.14 0.03 THYROID 1.85 0.31 2.43 0.67 1.59 0.46 1.73 0.45 1.71 0.29 EYES 0.17 0.01 0.23 0.03 0.19 0.03 0.18 0.02 0.18 0.02 PANCREAS 0.80 0.10 0.73 0.09 0.55 0.07 0.56 0.06 0.43 0.06 THYMUS 0.63 0.12 0.56 0.08 0.45 0.03 0.54 0.13 0.39 0.05 STOMACH 0.47 0.10 0.42 0.12 0.36 0.11 0.37 0.1 0.24 0.06 TOT GIT 0.35 0.04 0.34 0.04 0.31 0.04 0.29 0.06 0.27 0.02 aAustralian Albino Wistar Rats, Injected dose per gram (X SD). Average of 5 animals per time point.

35 TABLE 6 COMPOUND NO. 6" p 4o* p MEAN SD MEAN SD 3 MIN 5 MIN LIVER 6.52 0.62 9.39 1.04 SPLEEN 2.24 0.42 1.78 0.15 KIDNEYS 24.05 3.93 18.35 0.89 MUSCLE 1.81 0.33 1.26 0.08 SKIN 3.96 0.41 3.47 0.39 BONE 1.91 0.02 1.39 0.11 LUNGS 6.38 i.04 4.66 0.39 HEART 3.84 0.62 2.80 0.38 BLOOD 10.65 0.87 7.83 0.54 URINE 1.30 0.88 0.95 0.43 STOMACH 1.78 0.38 6.85 6.36 TOT GIT 1.03 0.05 2.12 0.38 TAIL 8.94 3.42 6.18 2.50 BRAIN 0.74 0.21 0.35 0.11 THYROID 5.76 1.26 3.73 0.74 PANCREAS 1.86 0.18 1,41 0.22 THYMUS 3.67 1.03 2.50 0.41 aBalb/c mice, Injected dose per gram (X SD).

Average of 5 animals per time point.

S:13160KH/27.07.93 6 h-i r't.

.t a Table 7 Biodistribution of Copper-67 complex of Biodistribution of Ligand (5)-67Cu Times Animals 25 Balb/c mice.

Results: Injected dose.

Inj 1 min S.D. 3 min S.D 5 min S.D. 10 S.D. 30 S.D.

Dose min min Liver 5.71 2.27 8.48 0.89 9.04 1.94 11.79 0.80 17.10 1.02 Spleen 0.18 0.11 0.19 0.03 0.16 0.04 0.16 0.02 0.19 0.03 Kidney 8.35 3.82 5.65 0.81 4 99 0.99 4.48 0.42 3.38 0.35 Muscle 13.69 9.74 18.00 3.97 11.95 2.94 8.38 1.81 7.45 4.44 Skin 9.61 3.87 19.21 1.31 16.23 3.68 16.46 2.03 14.14 3.19 Bone 5.46 1.97 3.35 0.50 2.74 0.57 3.21 0.80 2.05 0.16 Lung 0.85 0.38 1.20 0.27 1.10 0.21 1.20 0.22 1.29 0.41 Heart 0.27 0.09 0.28 0.05 0.19 0.03 0.18 0.03 0.20 0.04 Blood 5.86 3.44 4.76 2.07 4.05 0.63 2.33 0.49 2.12 0.26 Urine 0.35 0.47 1.98 1.33 3.72 3.97 4.81 3.87 1.33 1.13 Stomach 0.52 0.22 0.93 0.15 0.87 0.26 0.80 0.41 1.22 0.26 Tail 8.27 9.95 5.19 4.72 5.36 2.39 3.45 2.23 1.51 0.47 Pancreas 0.20 0.13 0.11 0.03 0.12 0.04 0.34 0.22 0.34 0.05 Brain 0.23 0.12 0.22 0.02 0.23 0.09 0.19 0.15 0.54 0.88 S. Int. 0.50 0.23 1.10 0.23 1.08 0.24 0.90 0.48 0.71 0.32 L. Int. 0.35 0.14 0.75 0.26 0.54 0.15 0.79 0.25 0.31 0.15 .5.

'0 t o S S S 55 S S SS 55.55.50 5

S

S 55 MEAN S.D. MEAN S.D. MEAN S.D. MEAN S.D. MEAN S.D.

ORGAN 1 min 3 min 5 min 10 min 30 min Liver 12.07 0.43 12.83 0.86 11.82 0.77 5.96 3.53 3.09 1.1 Spleen 0.17 0.01 0.1 0.01 0.07 0.02 0.06 0.01 0.02 0.01 Kidney 11.78 2.27 11.23 1.4 9.17 2.29 5.58 1.43 3.19 0.28 Muscle 23.48 4.91 13.18 4.19 9.81 2.24 16.44 13.81 4.98 3.91 Skin 19.66 3.67 19.28 1.67 19.68 3.48 15.68 2.13 7.31 5.21 Bone 5.2~t 0.42 3.1 0.7 2.88 0.4 5.06 3.58 1.12 0.41 Lungs 1.54 0.48 0.98 0.31 0.77 0.13 0.48 0.13 0.2 0.06 Heart 0.57 0.1 0.23 0.05 0.21 0.03 0.12 0.02 0.05 0 Blood 27.09 3.45 12.02 1.57 9.77 0.73 5.34 0.74 1.82 0.3 Urine 0 0 5.62 1.01 4.51 5.04 11.64 5.79 8.84 5.77 Bladder 0.12 0.02 0.63 0.42 0.68 0.21 1.96 1.71 1.23 2.01 Stomach 0.5 0.09 0.98 0.21 1.02 0.26 1.16 0.69 0.49 0.31 GIT 3.82 0.88 6.06 0.59 4.77 2.1 12.35 1.63 20 0.9 Tail 2.42 1.4 1.58 0.68 3.76 2.12 2.07 0.69 1.53 1.1 Balb/c mice, Injected dose (X SD), Average of 5 animals per time point.

Table 8. Biodistribution of 99mTc complex of compound 1.

d a a (4 J( e a. Oa a a a a a. a MEAN S.D. MEAN S.D. MEAN S.D. MEAN S.D. MEAN S.D.

ORGAN 1 min 3 min 5 min 10 min 30 min Liver 14.68 0.57 14.52 2.24 7.75 4.13 7.95 1.15 5.71 1.53 Spleen 0.19 0.02 0.16 0.03 0.07 0.04 0.06 0.01 0.05 0.01 Kidney 8.04 1.53 10.44 1.83 5.45 3.53 5.14 0.73 4 Muscle 19.67 5.83 14.89 4.78 6.73 3.57 8.73 3.93 5.48 2.78 Skin 13.37 0.85 13.1 0.89 7.96 4.6 11.02 3.76 5.58 1.27 Bone 7.88 1.45 6.29 0.87 2.99 1.42 2.76 0.4 2.27 0.53 Lungs 1.36 0.28 1.19 0.27 0.72 0.3 0.53 0.07 0.43 0.15 Heart 0.49 0.07 0.34 0.08 0.22 0.09 0.15 0.01 0.09 0.01 Blood 23.14 3.9 14.96 2.56 7.4 3.51 7.15 0.55 3.58 0.24 Urine 0.09 0.03 2.78 3.55 1.06 0.95 2.67 2.31 3.84 1.08 Bladder Stomach 0.37 0.09 0.98 0.07 0.6 0.34 1.3 0.26 2.06 0.56 GIT 3.74 0.52 4.86 0.8 3.38 2.46 8.11 4.14 16.24 5.2 Tail 4.05 3.97 6.89 5.52 30.12 14.89 18.31 8.6 7.67 4.69 Balb/c mice, Injected dose (X SD), Average of 5 animals per time point.

Table 9. Biodistribution of 99mTc complex of compound 2.

0* S*S S C 5 S a r. a SC SC S 5 S sSSSSC**

S.

S S S abcs 0* S S MEAN S.D. MEAN S.D. MEAN S.D. MEAN S.D. MEAN S.D.

ORGAN 1 min 3 mir 5 min 10 min 30 min Liver 31.82 3.89 40.23 4.84 30.24 3.75 21.19 3.76 12.48 2.01 Spleen 0.21 0.03 0.12 0.03 0.1 0.02 0.07 0.03 0.06 0.02 Kidney 7.83 1.04 6.27 0.57 4.18 0.73 3.57 1.09 3.67 0.36 Muscle 11.85 5.14 9.92 0.68 7.77 2.09 4.95 1.66 3.35 1.39 Skin 8.94 1.18 9.02 0.74 8.67 0.53 7.3 0.55 4 0.33 Bone 6.4 1.53 2.5 0.5 2.84 1.71 2.08 0.74 1.67 0.98 Lungs 1.29 0.85 0.56 0.16 0.68 0.25 0.52 0.16 0.26 0.11 Heart 0.6 0.13 0.22 0.02 0.16 0.04 0.13 0.03 0.05 0.01 Blood 34.01 4.04 12.06 1.89 7.87 0.38 5.04 0.26 3.88 1.01 Urine 0 0 0.84 0.47 1.55 0.73 4.27 2.46 3.21 3.26 Bladder 0.06 0.04 0.09 0.07 0.04 0.03 0.17 0.09 0.08 0.1 Stomach 0.52 0.07 0.61 0.25 3.96 2.59 0.7 0.52 4.84 4.82 GIT 3.96 1.76 8.34 2.99 13.7 5.33 33.63 3.55 39.97 10.4 Tail 4.62 1.56 2.02 0.74 4.63 4.59 3.58 3.36 1.91 0.74 Balb/c mice, Injected dose (X SD), Average of 5 animals per time point.

Table 10. Biodistribution of 9 9mTc complex of compound 4.

I--

0 A. U I

S

C

MEAN S.D. MEAN S.D. MEAN S.D. MEAN S.D. MEAN S.D.

ORGAN 1 min 3 mir: 5 min 10 min 30 min Liver 5.72 0.55 5.04 0.7 4.34 0.84 5.34 0.82 2.86 0.37 Spleen 0.14 0.04 0.16 0.02 0.12 0.02 0.11 0.01 0.06 0.01 Kidney 9.08 2.1 8.71 1.52 6.71 1.87 5.97 0.71 3.75 0.66 Muscle 23.78 3.73 15.39 1.79 11.74 2.25 9.67 1.93 23.84 33.4 Skin 18.09 4.65 19.66 4.38 19.83 6.59 23.93 4.64 17.37 6.21 Bone 6.02 1.8 4 0.29 2.79 0.32 2.98 0.54 1.86 0.82 Lungs 1.64 0.29 1.65 0.7 1.28 0.32 1.1 0.36 0.46 0.1 Heart 0.61 0.1 0.43 0.03 0.26 0.06 0.23 0.05 0.12 0.05 Blood 26.59 1.56 14.84 1.03 10.06 1.33 12.87 8.12 3.98 0.56 Urine 0 0 2.97 1.49 6.52 4.77 10.66 8.48 5.91 3.45 Bladder 0.09 0.05 1.16 0.43 2.15 1.62 1.04 0.97 0.83 0.99 Stomach 0.65 0.11 0.56 0.09 0.4 0.08 0.63 0.08 0.49 0.35 GIT 3.89 0.86 3.22 0.08 2.42 0.53 3.94 0.75 4.61 0.75 Tail 2.65 0.84 4.28 4.1 11.55 12.68 6.98 6.75 2.01 0.6 Balb/c mice, Injected dose (X SD), Average of 5 animals per time point.

Table 11. Biodistribution of 9 9 mTc complex of compound II I -r I I I_ Lj S °o y MEAN S.D. MEAN S.D. MEAN S.D. MEAN S.D. MEAN S.D.

ORGAN 1 min 3 min 5 min 10 min 30 min Liver 22.44 3.45 35.66 2.09 30.85 6 16.04 1.79 14.05 2.58 Spleen 0.2 0.04 0.09 0.03 0.14 0.05 0.07 0.02 0.06 0.03 Kidney 7.57 1.71 5.68 0.79 4.53 0.57 2.44 0.34 1.9 0.19 Muscle 13.95 2.12 7.87 2.49 7.24 1.79 5.27 2.52 6.92 8.06 Skin 8.13 1.14 8.31 1.33 9.28 3.33 5.17 1.92 4.23 2.25 Bone 7.37 0.86 3.5 1.17 3.86 0.52 2.76 1.63 2.54 1.37 Lungs 1.71 0.72 0.65 0.33 0.69 0.19 0.45 0.13 0.39 0.09 Heart 0.46 0.04 0.2 0.04 0.16 0.02 0.08 0.04 0.11 0.09 Blood 41.63 3.94 15.59 1.22 10.31 0.77 6.34 1.8 9.66 14.1 Urine 0.07 0.15 0.81 0.52 0.59 0.49 1.21 0.59 1.64 0.74 Bladder 0.06 0.01 0.08 0.05 0.07 0.01 0.23 0.19 0.2 0.14 Stomach 0.4 0.06 0.43 0.21 5.42 5.84 3.42 4.01 1.73 1.39 GIT 3.58 1.37 11.98 7.7 27.85 7.9 45.71 6.16 51.18 3.62 Tail 8.46 7.08 7.37 4.71 7.64 5.59 4.77 2.15 2.41 3.11 Balb/c mice, Injected dose (X SD), Average of 5 animals per time point.

Table 12. Biodistribution of 99 mTc complex of compound 6.

I -r II r 1 1 II S

S

S S

*.S

S S S S. S S 100 0 mg 0 20 40 60 80 100 120 140 lime (min) Figure 4. Complexation Profile of 67 Ga at T=21 0

C

I i 1 60 180

S

a

S

a. S S *aSaSSa*S a S S 55

*S

100 10 Mg rng 1 rng 30 40 50 60 time (min) Figure 5. Complexation Profile of 67 Ga at T 30 0

C

S

a *ae*.sa..

S a C C C C a a **CS C S S a a 100 a 10 mg 5 mg Mg 0 10 20 30 40 50 60 time (min) Figure 6. Complexation Profile of 67 Ga at T 36 'C 100 A,-.-iMg 0 I I 9 S 50 75 100 125 150 175 200 time (min) Figure 7. Complexation Profile of 67 Ga at T=21 0

C

o, 100 o o S o 0 S S 5 5 4 5 S 5 OS S S 0 1- mg -1 Mg 40 60 80 100 time (min) Figure 8. Complexation Profile of 67 Ga at T 30 0

C

er.. ~a 0 0.

0 0 0 C 0 0 a 100 0 -u0- Mg mg -1 M g 40 60 80 100 120 140 16E time (min) Figure 9. Complexation Profile of 67 Ga at T 37 'C *.bC C CC CC C. CC CCC. C CC C C C CC C C C C C C C C C CCCC S C C C C CC C C 1 00 mg/mL mg/mL -1 mglmL 0 30 40 50 60 70 time (min) Figure 10. Fir 1.Complexation Profile of 67 Ga at T 21 0

C

a a.

a a a a a a a a a a a a o a *a a a 100 ep, -u10 mg/mL A -5 mg/mL 1mg/mL 30 40 50 time (min) Figure 11. Complexation Profile of 1 7 Ga at T 25 0

C

S. enS 55 @5 StSI.

55 00 S S **55 S S S *S S S 55 @5 S C S Sc S tO S S 55555555 S C S5SS 6 5 S a S ~S S C 00 0 1 mg/mL A -5 mg/mL mg/mL 40 60 80 100 120 140 160 time (min) Figure 12. Fir 1.Complexation Profile of 67 Ga at T 30 'C ~j~jd. 0t C C* C C C C C S C 00 0 -a-10 mg/mL A &5 mrg/mL 1 mg/mL 40 60 80 100 120 140 160 180 time (min) Figure 13. Fir 1.Complexation Profile of 67 Ga at T 37 0

C

Ea*S 0.

S

S

S.

S

0 0 5 0 55 5 S 5 mg/mL A -1 mg/mL 5 mg/mL I I 100 1 20 140 160 time (min) Figure 14. Fir 1.Complexation Profile of 67 Ga at T 21 0

C

C C

C.

S f* 5 04 4 mg/mL 1 mg/mL mg/mL i 100 1 time (min) Figure 15. Fir 1.Complexation profile of 67 Ga at T 35 0

C

S

*.SS

I.

*5 A~'TS S 55*5

S

*5S* 5' .55.

S S S S 00 0 Mg 1 Mg 40 60 80 100 120 140 time (min) Figure 16. Fir 1.Complexation Profile of 67 Ga at T 21 0

C

0 .e 0b 0000 0 0 0* 4 0 t0 o 0 0. p 0* 0 0 0 0 0 CO 0 100 10 mg/mL 5 mg/mL -1 mg/mL 20 30 time (min) Figure 17. Fir 1.Complexation Profile of 67 Ga at T 27 'C o 0 0* *0* 0 0 0 0* 0 100 6 0 0- 0 A Mg 1 mg I I. I 50 time (min) 100 Figure 18. Fir 1.Complexation Profile of 67 Ga at T 32 0

C

a se,.

*0as a a a a. a a *a.a a a a a. 6.0E+5 1 0.0E+0 WM 0.00 120 1 00 0o 40 activity UV (abs) [0 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 vol(mL) Figure 19. Serum Stabi lity 67 Ga at t=O S. *e*e it S S S S CS S S 55555555 S *S*5 S S S S S *5 S S 2.5E+5 1.I5E+5 co 5.0E+4 0.OE+0 -120 -100 .2 40 activity UV abs lo 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 vol (mL) Figure 20. Serum Stablify 67 Ga t=6 days LIII o S C .OSS .0 C S *5 S 15 oa-m 0.00 UV abs *U activity 2.5E+5 2.0E+5

E

1.5E+5

Z

5.OE+4 0.0 E+0 10.00 20.00 30.00 40.00 50.00 vol(rnL) 60.00 70.00 Figure 21. Serum Stabil ity 67 Ga S ijdS~ S a 0 S S S S Sa 5 9.OE+4 8.OE+4 7.OE+4 6.OE+4 5.0E+4 4.OE+4 3.OE+4 E+4 1.OE+4 0.0E+0 140 120 100 -4-activity -E-absorbance 0 40 50 vol (mL) Serum Stability 67 Ga t 6 days Figure 22.

.0*0 .0 0 00 S 0 00000000 0 **00 0 0 0 04 0 3.5E+5 100 2.5E+5 t w~1.5E4-5

OU

(I)

C

counts A*-UV-abs E+4 0.OE+0 -11 0 i i i i 0 70 80 90 100 10 20 30 40 50 e vol (mL) Figure 23. Serum Stability of 67 Ga t=O efl. A S a. a 0 a a a.

a S a S S S 2.5E+5 E 1.5E+5 5.OE+4 0.OE+0 100 90 100 activity UV-abs 0 10 20 30 Figure 24.

40 50 60 70 80 volume (ml-) Serum Stability 67 Ga t=22h t a Fe a. a.

a 4. a a a a a a a a a. 2.5E+5 1.5E,5 4

O.OE+O

C

4~_ activity UV-abs 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 volume (ml-) Figure 25. Serum Stabi lity 6 7 Ga t=46 h .4 4.0* 4 0 04 a 4 4 0 0 i Em1 030 E0120

BONE

LIVER KIDNEY

BLOOD

ORGAN

Figure 26. BIODISTRIBUTION OF 67 GA (1)

AN

I.

~Ym S V S. *S S S S S 5 5 0550 5 5 5 5 S S fU Ml MIN 03 MIN 010 MIN *30 MIN Ill120 MIN 2 5 1-- 0~ 150 0

KIDNEY

BONE

BLOOD

LIVER

organ Figure 27. BIODISTRIBUTION OF 1 7 GA (2) C C 0 0 Liver Kidney Bone

ORGAN

Figure 28. BIODISTRIBUTION OF 7 GA (4) 2030 .4 12 12 min- 18 16 14 12 0 LIVER KIDNEY BONE

BLOOD

ORGAN

Figure 29. BIODISTRIBUTION OF 67 GA 35.00 Ell MIN 30.00 S3 MIN

MIN

25.00 010 MIN 030 MIN C~ 20.00 E nRI 15.00 LIVER KIDNEY BONE BLOOD organ Figure 30. BIODISTRIBUTION OF 6 7 GA (6) a a.

a.

a a 18 16 14 :12 6 4 2 0 -1% Radioactivity

UV

0.9 0.8 0.7 C

C*

0.6 En 0

CI

0

CO

0.4N 0.

0.2 0.1 0.0 0 5 10 Volume Eluted (ml) Figure 31. Serum Stability Elution Profile of 166 D y/' 66 Ho(1) at 46 hours.

b e C*b a D a C CC C be C C. C 1 MIN 3MIN 5 MIN 10 MIN 30 MIN 120 MIN ORGAN MEAN S.D. MEAN S.D. MEAN S.D. MEAN S.D. MEAN S.D. MEAN S.D.

LIVER 15.45 1.65 24.21 1.93 25.79 1.7 22.77 1.74 10.11 1.15 0.88 0.56 SPLEEN 0.29 0.05 0.14 0.02 0.1 0.01 0.06 0.02 0.01 0.01 0.01 0 2 i. .0 0 6 KIDNEY 9.28 1.50 5.26 2.06 4.37 1.97 1.91 0.33 0.57 0.12 0.05 0.01 MUSCLE 22.75 2.64 12.70 1.58 12.14 6.29 26.13 39.89 1.80 1.44 0.48 0.26 .5.07 2. 3O. 0.7 2 2 5 0 22 SKIN 11.72 0.62 13..76 1.63 13.02 1.63 .8.47. 0.99 1.56 0.52 0.24 0.09 BONE 5.07 0.42 3.30 0.57 2.83 0.71 2.20 1.04 0.50 0.22 0.42 0.22 LUNGS 1.28 0.42 0.83 0.17 1.11 0.49 0.46 0.12 0.12 0.06 0.04 0.02 HEART 0.41 0.07 0.19 0.02 0.15 0.03 0.08 0.01 0.01 0.00 0.01 0.00 BLOOD 16.86 1.74 9.81 0.34 7.82 1.59 3.52 0.55 0.78 0.41 0.35 0.21 BLADDER 0.08 0.02 0.11 0.03 0.09 0.06 0.09 0.07 0.03 0.03 0.01 0.01 L, 2 3 Q.9 0 6 9..0 9 0..07 3 1 STOMACH SQ 0.08 0.03 0.08 0.02 0.11 0.0 0.10 0.08 0.02 0.01 0.02 0.02 STOMACH GL 0.36 0.05 0.19 0.10 0.21 0.08 0.18 0.12 0.04 0.04 0.03 0.03 DUODENUM 0.12 0.05 0.09 0.04 0.17 0.13 0.24 0.13 0.06 0.05 0.02 0.01 JEJUNUM 1.27 0.22 0.69 0.16 0.73 0.28 1.73 0.50 2.99 1.09 0.85 0.47 o ILEUM 0.25 0.12 0.13 0.06 0.11 0.07 0.18 0.18 0.11 0.13 0.66 0.65 CAECUM 0.45 0.18 0.26 0.06 0.22 0.06 0.17 0.13 0.06 0.04 0.88 0.53 COLON 0.53 0.13 0.43 0.13 0.37 0.10 0.21 0.10 0.08 0.04 0.24 0.14 TAIL 1.13 0.31 1.50 0.32 1.95 0.76 1.48 0.28 1.06 0.46 0.10 0.08 PANCREAS 0.35 0.11 0.16 0.05 0.11 0.01 0.07 0.03 0.02 0.01 0.01 0.00 SALIVARY GL 0.16 0.04 0.11 0.04 0.10 0.05 0.07 0.03 0.01 0.01 0.01 0.01 OVARIES 0.07 0.03 0.06 0.02 0.07 0.02 0.03 0.01 0.01 0.00 0.00 0.00 a Balb/c mice, %ID (X Average of 5 animals per timepoint.

TABLE 13 BIODISTRIBUTION OF GA-67 (1) A! c e c a A U* A @Oeee A A *A ORGAN 1 MIN 3MIN 5 MIN 10 MIN 30 MIN 120 MIN MEAN SD MEAN SD MEAN SD MEAN SD MEAN SD MEAN SD ULIVER 24.9 5.47 35.52 3.34 33.09 3.19 23.34 4.55 3.7 1.22 0.92 0.86 SPLEEN 0.29 0.1 0.1 0.063 0.07 0.01 0.02 0.01 0.02 0.02 0 0 KIDNEY 9.54 0.93 4.62 0.93 3.52 0.81 1.00 0.17 0.21 0.05 0.01 0.01 MUSCLE 20.55 5.85 8.63 1.46 5.50 1.60 2.00 0.68 2.00 3.54 0.31 0.38 SKIN 11.55 1.74 9.27 1.08 8.04 1.05 3.80 1.07 0.88 0.64 0.17 0.17 BONE 6.16 1.15 2.82 0.42 2.13 0.36 0.74 0.12 0.50 0.54 0.38 0.61 LUNGS 1.79 0.69 0.56 0.12 0.41 0.11 0.12 0.06 0.06 0.06 0.01 0.01 HEART 0.42 0.09 0.14 0.03 0.09 0.03 0.03 0.00 0.01 0.01 0.00 0.00 BLOOD 17.45 4.46 6.02 0.86 3.31 0.70 2.16 2.31 3.20 4.40 0.07 0.13 BLADDER 0.08 0.02 0.06 0.01 0.09 0.03 0.03 0.01 0.01 0.01 0.00 0.00 STOMACHSQ. 0.07 0.03 0.08 0.03 0.18 0.28 0.25 0.40 0.03 0.05 0.03 0.03 ST OMACHGL 0.26 0.12 0.20 0.08 0.53 0.72 0.28 0.28 0.11 0.21 0.03 0.04 DUODENUM 0.13 0.04 0.34 0.31 0.67 0.26 1.22 1.24 0.31 0.15 0.04 0.06 JEUUNEM 1.42 0.42 1.67 0.82 3.68 1.80 5.99 5.13 6.64 4.73 0.99 1.06 ILEUM 0.15 0.07 0.11 0.09 0.21 0.20 0.03 0.01 0.78 1.49 0.28 0.26 CAcUM 0.42 0.14 0.23 0.10 0.15 0.06 0.04 0.01 0.20 0.24 7.06 7.34 COLON 0.48 0.17 0.28 0.07 0.25 0.11 0.10 0.02 0.16 0.08 2.03 2.01 PANCREAS 0.34 0.14 0.11 0.03 0.10 0.06 0.03 0.01 0.08 0.14 0.00 0.00 SALIVARYGL. 0.11 0.06 0.05 0.01 0.02 0.01 0.01 0.00 0.00 0.00 0.00 0.00

OVARIES

TAIL

C 06 J.00 0.01 0.00 0.05 0.01 0.04 0.00 0.00 0.00 0.01 0.01 0.00 0.01 0.02 0.00 0.00 0.00 0.00 0.00 0.56 0.13 0.12 0.06 a Balb/c mice, %ID (X Average of 5 animals per timepoint.

TABLE 14 BIODISTRIBUTION OF GA-67 (2) p~8a~ I,--cl P ~b L d.

S S sc

J

6: S *eS.**S*S

S.

S

S S 1 MIN 3 MIN 5 MIN 20 MIN 60 MIN 120 MIN ORGAN MEAN S.D. MEAN S.D. MEAN S.D. MEAN S.D. MEAN S.D. MEAN S.D.

LIVER 39.06 1.27 43.23 1.17 39.61 2.10 8.29 2.00 06 0.9 6 0.29 0..18 SPLEEN 0.15 0.03 0.04 0.02 0.03 0.00 0.00 0.00 0.00 0.00 0.01 0.00 S 0. 3. 0 0. KIDNEY 6.91 0.97 2.12 0.26 1.59 0.15 0.43 0.0b 0.17 0.09 0.07 0.01 LE. 1.34 .3 6 8 8 PT P P 0..09 MUSCLE 11.34 3.66 5.08 1.98 4.29 0.65 0.65 0.38 0.32 0.28 0.21 0.09 8 8. .5 2.

SKIN 11.94 0.78 8.12 0.66 8.22 1.18 2.92 3.85 0.18 0.12 0.15 0.02 BONE 3.20 0.57 1.31 0.09 0.98 0.20 0.20 0.12 0.13 0.04 0.15 0.05 O 0 5 0. 0.O LUNGS 0.64 0.21 0.26 0.05 0.18 0.07 0.03 0.00 0.01 0.00 0.01 0.00 BLOOD 9.95 2.46 3.06 0.41 2.28 0.50 0.27 0.04 0.13 0.03 0.10 0.02 6 6 6 1 T .1 BLADDER 14 0.13 0.28 0. 24 0.14 0.09 0.41 0.57 0.00 0.01 0.00 0.00 STOMACH SQ. 0.06 0.03 0.03 0.01 0.07 0.07 0.04 0.08 0.01 0.02 0.21 0.42 STOMACH GL 0.14 0.03 0.06 0.02 0.47 0.48 0.19 0.35 0.02 0.01 0.02 0.02 DUODENUM 0.09 0.03 0.25 0.27 0.44 0.32 0.36 0.37 0.53 1.16 0.04 0.05 SDUDN i 09 .063 25T 2.7 0..32. .6 7 0...56 1.16 0T.0: 6 805 JEJUNEM 1.16 0.28 1.17 0.32 1.19 0.59 5.43 1.56 0.61 0.38 0.20 0.18 ILEUM 0.20 0.07 0.08 0.03 0.05 0.02 0.06 0.04 1.52 1.97 0.12 0.09 CAECUM 0.19 0.10 0.15 0.05 0.06 0.01 0.09 0.03 0.49 0.12 1.19 0.33 COLON 0.32 0.09 0.11 0.03 0.14 0.07 0.06 0.04 0.28 0.25 0.56 0.34 TAIL 0.00 0.00 0.88 0.17 0.73 0.25 0.57 0.11 0.14 0.04 .11 0.06 I .o 2 0 3 .Q 0 0 Q 0 PANCREAS 17 0.05 0.02 0.10 0.0..3 0.04 0.00 0.00 0.01 0.01 SALIVARY GL 0.12 0.08 0.06 0.03 0.03 0.02 0.01 0.02 0.00 0.00 0.00 0.01 OVARIES 0.13 0.03 0.12 0.06 0.10 0.03 0.02 0.01 0.00 0.00 0.00 0.00 a Balb/c mice, %ID (X Average of 5 animals per timepoint.

TABLE 15 BIODISTRIBUTION OF GA-67 (4) I I a e k «o o oo oo 3 MIN 5 MIN 10 MIN 30 MIN 60 MIN 120 MIN ORGAN MEAN S.D. MEAN S.D MEAN S.D MEAN S.D MEAN S.D MEAN S.D LVER 3.08 0.29 3.02 0.41 2.04 0.33 1.22 0.15 1.33 0.32 0.86 0.12 2 4 o. 3 6 SPLEEN 0.18 0.03 0.15 0.01 0.10 0.02 0.05 0.02 0.03 0.01 0.02 0.00 KIDNEY 8.39 2.08 5.78 0.54 3.17 0.17 2.39 0.31 1.72 0.26 1.60 0.16 MUSCLE 23.50 9.80 19.47 7.65 20.11 19.15 4.58 2.05 2.65 1.01 1.44 0.77 SrN 20.79 1.42 21.98 1.64 18.72 1.68 10.65 1.90 5.12 0.75 2.82 0.28 BONE 4.85 0.31 5.90 2.14 4.51 1.59 2.13 0.65 1.69 1. 72 0.80 0.45 LUNGS 1.15 0.22 0.89 0.10 0.76 0.22 0.22 0.03 0.16 0.08 0.10 0.03 HEART 0.35 0.03 0.27 0.03 0.19 0.02 0.07 0.01 0.04 0.01 0.03 0.01 BLOOD 17.48 0.91 12.82 0.86 8.06 0.54 2.86 0.19 1.16 0.19 0.65 0.07 URIEE 6.30 3.49 8.94 5.16 15.34 3.12 14.23 4.14 7.76 2.85 3.55 2.07 BLADDER 0.48 0.54 0.62 1.01 0.47 0.43 0.10 0.04 1.04 1.90 0.32 0.57 STOMACH SQ 0.08 0.02 0.10 0.01 0.10 0.07 0.03 0.01 0.02 0.01 0.01 0.00 STOMACH GL 0.27 0.05 0.17 0.04 0.11 0.03 0.04 0.01 0.03 0.01 0.02 0.01 DUODENUM 0.12 0.02 0.10 0.01 0.05 0.02 0.03 0.02 0.02 0.01 0.01 0.00 JEJUNUM 0.81 0.35 0.61 0.17 0.46 0.11 0.29 0.06 0.18 0.03 0.09 0.02 ILEUM 0.32 0.12 0.20 0.10 0.18 0.07 0.08 0.07 0.07 0.02 0.06 0.03 CAECUM 0.36 0.08 0.26 0.06 0.31 0.35 0.09 0.06 0.04 0.01 0.03 0.02 COLON 0.59 0.15 0.53 0.09 0.37 0.12 0.11 0.04 0.05 0.01 0.05 0.01 TAIL.. 0.86 0.19 64 17 0_-Q .1 PANCREAS 0.36 0.20 0.19 0.04 0.16 0.03 0.05 0.01 0.05 0.04 0.01 0.00 SALIV 0.29 0.06 0.28 0.04 0.16 0.05 0.06 0.01 0.04 0.01 0.03 0.02 TESTIES 0.25 0.05 0.25 0.07 0.32 0.20 0.08 0.01 0.10 0.05 0.05 0.03 a Balb/c mice, %ID (X Average of 5 animals per timepoint.

TABLE 16 BIODISTRIBUTION OF GA-67 0. IuffI~ 0 t*0* 00 0* 0 0 .0 *0 0* 0 .000 S 0 0 S *0 0 ORGAN 1 MIN WMIN 5 MIN 10 MIN 30 MIN 120 MIN MEAN S.D. MEAN S.D. MEAN S.D. MEAN S.D. MEAN S.D. MEAN S.D.

LIVER 26.45 1.01 34.06 1.45 30.21 2.02 18.58 2.52 3.15 1.92 1.33 1.18 SPLEEN 0.22~ 0.02 0.08 0.03 0.04 0.02 0.03 0.01 0.01 0.00 0.00 0.00 KIDNEY 9.95 0.5? 5.24 1.09.3.36 0.50 1.98 0.16..0.79 0.09 0.06 0.01 MUSCLE 17.93 3.63 10.67 1.25 6.35 1.58 4.37 1.3? 7.73.11.64 1.85 3.85 SKIN 13.91 1.44.12.80.1.65 10.62.1.41 5.76 0.93 0.86, 0.24 0.10 0.05 BONE 4.33 1.43.2.41 0.34 1.67 0.31 0.96 0.62 0.34 0..1.41 0Q06 0.04 LUNGS 0.78 0.17 0.64 0.37 0.25 0.06 0.17 0.08 0.19 0. .101 0.01 HEA 0.29 0.02 0.13 0.02 0.09* 0.03 0.04 0.01 0.01 0.00 0.00 0.00 BOOD 13.61 2.51 6.08 0.-9'4 3 .'02 0.'60 1.44 0.14 033 0..25. 1.79 -391 BLDDER 03 0.1 0.62 0.45 0.94 1.13 0.37 0.59..0.27 0.45 0.62 1.39 STOMACHS6. 0.08 0.06 0.07 0.03 0.04 0.02 0.32 0.53 0.03.0.02 0.01 0.00 STOMACH GL 0.20 0.1? 0.22 0.11 0.20 0.21 1.61, 1.28 0.35 0.43 0.01 0.01 DUODENUM 0.08 0.02 0.43 0.64 0.41, 0.22 1.75 0.63 0.27---0.26 0.02 0.02 JEJUNBVI 1.19 0.26 1.25 0.7? 2.25 1.48 5 3 4 3 .57..41 1.70 ILEUM 0.21 0.07 0.15 0.16.0.08 0.02 0.1 ,3 0.17 0.06 0.06 0.10 0.06 CAECUM 0.27 0.09 0.20 0.06 0.13 0.02 0.17 0.07 0.15 0.20 1.70 0.74 COLON 0.49 0.11 0.28 0.09 0.18 0.05 0.27 0.31 -0.27 .7J2 0.35.

TAIL 073 0.00 0.84 1_0.10 0.55 0.31 0.1 0.0 .ACE 25 0.11 0.11 0.2 006 0.03 0.12 0 17* 008 0.14 0.00 0.O0 SALVAYGL 0.8 016 0.2 005 0.0 004 0.02 0.01 00 0.01 0.00 0.00 TESTES 0.17 0.03 0.17 0.041 0. 12 0.031 0.07 0.02 0.07 0.05 .2 00 8 Balb/c mice, %ID (X Average of 5 animals per timepoint.

TABLE 17 BIODISTRIBUTION OF GA-67 (6)

Claims (12)

1. A physiologically acceptable, radiolabelled compound of general formula consisting of a compound of the formula X-NH-C-CH 2 [(CH 2 -NI -CH 2 -C-NH-Y II I I 1I 0 CH 2 CH 2 O (I) I I COOH COOH where k is an integer from 2 to 1 is an integer from 1 to X and Y are independently selected from phenyl, naphthyl, pyridine or quinoline radical, each having at position 2 one of -OH, -SH, -NH 2 or COOH and each of X and Y can be optionally 15 substituted by one or more amino, halogen, hydroxy, mercapto, nitro, cyano, thiocyano, alkyl, alkoxy, halogenoalkyl, acyl, acylamino, acyloxy, carboxyl, alkoxycarbonyl, carbamoyl, pyridoylamino, N-carboxyalkyl-carbamoyl, sulpho, sulphamoyl, mono- or dialkylated or phenylated sulphamoyl which can also carry one or more substituents alkylsulphonyl, alkoxysuli honyl, or by an optionally hydroxy-containing phenylsulphonyl or phenoxysulphonyl; where R' is as defined for X and Y; or pharmaceutically acceptable salts thereof.

2. A diagnostic formulation suitable for labelling with 99 mTc, 188 Re or 1 86 Re comprising a compound of formula or a pharmaceutically acceptable salt thereof and a reducing agent in a pharmaceutically acceptable carrier.

3. A formulatioi comprising a radiolabelled compound Aof formula as defined in claim 1 or a pharmaceutically acceptable salt thereof in a pharmaceutically acceptable carrier. S:13160KH/25.06.96 76

4. A method of diagnosis or therapy in a subject comprising administering to the subject a radiolabelled compound of formula as defined in claim 1 or a pharmaceutically acceptable salt thereof. A compound of formula pharmaceutically acceptable salts and/or radiolabelled complexes thereof R-NH--CCH 2 [(CH 2 k-N-CH2-C-NH-R SI II 0 CH 2 CH 2 0 COOH COOH where k is an integer from 2 to 1. is an integer from 1 to 15 R is independently selected from R I Ar OH wherein Ar is an aryl or heteroaryl group; R 1 is -NR 2 R 3 where R 2 and R 3 are independently selected from hydrogen, (CH 2 p NH 2 -(CH 2 )p-Ar- 20 (CH 2 )m-NH 2 (CH 2 )p-CO 2 H; (CH 2 )p-Ar-CO 2 H; (CH 2 CH 2 0) n-CCH 2 CHNH 2 (CH 2 )p-NCS, (CH 2 pAr-NCS; or (CH 2 )pNHCOR", -COR"; or R 2 and R 3 together with the nitrogen atom to which they are attached form an optionally substituted saturated or partially unsaturated ring optionally containing one or more heteroatoms 0, S or N; or R 1 is -NCS, -N=N or -C(=NH)-OCH 3 n and m are independently an integer from 0 to 4 p is an integer from 1 to 4; R" is alkyl-L where L is halogen or other leaving group; 2,5-diketo-pyrrolinyl, -Het-CH=CH 2 wherein A ~Het is an optionally substituted 5 or 6 membered p heterocyclic ring containing one or more ,i heteroatoms 0, N or S. S:13160KH/25.06.96 I I 77

6. A process of preparing a compound of formula (II) as defined in claim 5 which comprises treating the corresponding nitro compound with a catalyst in the presence of a reducing agent to provide the amino compound and as required, further derivatising the amino group following procedures known in the art.

7. A conjugate compound which comprises a compound of formula (II) or a radiolabelled complex and/or salt thereof coupled to an antibody, protein, peptide, carbohydrate or oligonucleotide

8. A pharmaceutical formulation comprising a compound of formula (II) as defined in claim 5, a radiolabelled complex and/or salt thereof in a 15 pharmaceutically acceptable carrier. A method of diagnosis or therapy in a subject comprising administering to the subject a compound of formula (II) as defined in claim 5 or a radiolabelled complex and/or salt thereof. S 20 10. A method of diagnosis or therapy in a subject o"i comprising administering to the subject a conjugate compound of formula (II) as defined in claim 7.

11. A compound of formula (II) according to claim where each R is 2-hydroxy-5-aminophenyl, 1=1 and k=2.

12. A process according to claim 6 for preparing a compound of formula (II) where each R is 2- 1=1 and k=2. S:13160KH/25.06.96 I 78

13. A pharmaceutical formulation according to claim 8 wherein in the compound of formula (II) each R is 1=1 and k=2.

14. A radiolabelled compound of formula (II) according to claim 5 where each R is aminophenyl, 1=1 and k=2. A method according to claim 9 wherein in the compound of formula (II) each R is aminophenyl, 1=1 and k=2.

16. A compound of formula (II) or a radiolabelled compound of formula substantially as herein described with reference to the examples. Dated this 25th June 1996 AUSTRALIAN NUCLEAR SCIENCE TECHNOLOGY ORGANISATION 15 By their Patent Attorney Griffith Hack Co o i S:13160KH/25.06.96 I