PROSTATE SPECIFIC MEMBRANE ANTIGEN (PSMA) LIGANDS COMPRISING AN AMYLASE CLEAVABLE LINKER

Abstract

In particular, the present invention relates to a PSMA binding ligand comprising an oligosaccharide building block which comprises a bond being cleavable by alpha-amylase.

Typically, this PSMA binding ligand further comprises a PSMA binding motif Q and a chelator residue A, wherein the PSMA binding motif Q and the chelator residue A are preferably linked via at least one linker L.sup.AQ comprising the oligosaccharide building block, the PSMA binding ligand thus preferably having the structure (I)

A-L.sup.AQ-Q

or a pharmaceutically acceptable salt or solvate thereof.

Claims

1. PSMA binding ligand comprising an oligosaccharide building block which comprises a bond being cleavable by alpha-amylase.

2. PSMA binding ligand according to claim 1, further comprising a PSMA binding motif Q and a chelator residue A, wherein the PSMA binding motif Q and the chelator residue A are preferably linked via at least one linker L.sup.AQ comprising the oligosaccharide building block, the PSMA binding ligand preferably having the structure (I)
A-L.sup.AQ-Q   (I), or a pharmaceutically acceptable salt or solvate thereof.

3. PSMA binding ligand according to claim 1, having the structure (Ia) ##STR00113## or a pharmaceutically acceptable salt or solvate thereof, wherein Q is the PSMA binding motif, A is the chelator residue, AS.sup.a and AS.sup.b are amino acid building blocks and q is an integer of from 0-4 and p is an integer of from 0 to 3.

4. PSMA binding ligand according to claim 1, wherein the oligosaccharide building block comprises of from 2 to 10, preferably of from 3 to 10, more preferably of from 3 to 6 monosaccharide units, most preferably 3 monosaccharide units.

5. PSMA binding ligand according to claim 1, comprising a PSMA binding motif Q and a chelator residue A, wherein the chelator residue A is derived from a chelator selected from the group consisting of 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (=DOTA), N,N″-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N″-diacetic acid, 1,4,7-triazacyclononane -1,4,7-triacetic acid (=NOTA), 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentanedioic acid, (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid (DOTAGA), 1,4,7-riazacyclononane phosphinic acid (TRAP), 1,4,7-triazacyclononane phosphinic acid (TRAP), 1,4,7-triazacyclononane-1-[methyl(2-carboxyethyl)phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid] (NOPO), 3,6,9,15-tetraazabicyclo[9.3.1.]pentadeca -1(15),11,13-triene-3,6,9-triacetic acid (=PCTA), N′-{5-[Acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-arninopentyl)(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO), Diethylenetriaminepentaacetic acid (DTPA), trans-cyclohexyl-diethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid (oxo-Do3A) p-isothiocyanatobenzyl-DTPA (SCN-Bz-DTPA), 1-(p-isothiocyanatobenzyl)-3-methyl-DTPA (1 B3M), 2-(p-isothiocyanatobenzyl)-4-methyl-DTPA (1 M3B) and 1-(2)-methyl-4-isocyanatobenzyl -DTPA (MX-DTPA).

6. PSMA binding ligand according to claim 5, wherein A is a chelator residue having a structure selected from the group consisting of ##STR00114##

7. PSMA binding ligand according to claim 1, comprising a PSMA binding motif Q having the structure ##STR00115## wherein R.sup.1 is H or —CH.sub.3, preferably H, wherein R.sup.2, R.sup.3 and R.sup.4 are independently of each other, selected from the group consisting of —CO.sub.2H, —SO.sub.2H, —SO.sub.3H, —OSO.sub.3H, —PO.sub.2H, —PO.sub.3H and —OPO.sub.3H.sub.2.

8. PSMA binding ligand according to formula (Ia) ##STR00116## or a pharmaceutically acceptable salt or solvate thereof, wherein Q is a PSMA binding motif, A is a chelator residue, AS.sup.a and AS.sup.b are amino acid building blocks and q is an integer of from 0 to 4, wherein p is an integer of from 1 to 3.

9. PSMA binding ligand according to claim 8, wherein AS.sup.a has the structure ##STR00117## wherein Q.sup.1 is selected from the group consisting of alkylaryl, arylalkyl, aryl, alkylheteroaryl, heteroarylalkyl and heteroaryl.

10. PSMA binding ligand according to claim 8, wherein AS.sup.b has the structure (b) ##STR00118## wherein Q.sup.2 is selected from the group consisting of aryl, alkylaryl, arylalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl, and wherein q is an integer of from 0-4, preferably wherein Q.sup.2 is ##STR00119## preferably ##STR00120##

11. PSMA binding ligand according to claim 8, wherein A is a chelator residue derived from a chelator selected from the group consisting of 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″′-tetraacetic acid (=DOTA), N,N″-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N″-diacetic acid, 1,4,7-triazacyclononane-1,4,7-triacetic acid (=NOTA), 2-(4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl)pentanedioic acid, (NODAGA), 2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid (DOTAGA), 1,4,7-riazacyclononane phosphinic acid (TRAP), 1,4,7-triazacyclononane phosphinic acid (TRAP), 1,4,7-triazacyclononane-1-[methyl(2-carboxyethyl)phosphinic acid]-4,7-bis[methyl(2-hydroxymethyl)phosphinic acid] (NOPO), 3,6,9,15-tetraazabicyclo[9.3.1.]pentadeca -1(15),11,13-triene-3,6,9-triacetic acid (=PCTA), N′-{5-[Acetyl(hydroxy)amino]pentyl}-N-[5-({4-[(5-arninopentyl)(hydroxy)amino]-4-oxobutanoyl}amino)pentyl]-N-hydroxysuccinamide (DFO), Diethylenetriaminepentaacetic acid (DTPA), Trans-cyclohexyl-diethylenetriaminepentaacetic acid (CHX-DTPA), 1-oxa-4,7,10-triazacyclododecane-4,7,10-triacetic acid (oxo-Do3A) p-isothiocyanatobenzyl-DTPA (SCN-Bz-DTPA), 1-(p-isothiocyanatobenzyl)-3-methyl-DTPA (1 B3M), 2-(p -isothiocyanatobenzyl)-4-methyl-DTPA (1 M3B) and 1-(2)-methyl-4-isocyanatobenzyl -DTPA (MX-DTPA).

12. Complex comprising (a) a radionuclide, and (b) the PSMA binding ligand according to claim 1 or a pharmaceutically acceptable salt or solvate thereof.

13. The complex of claim 12, wherein, the radionuclide is selected from the group consisting .sup.89Zr, .sup.44.sub.Sc, .sup.111ln, .sup.90Y, .sup.66Ga, .sup.67Ga, .sup.68Ga, .sup.177Lu, .sup.99mTc, .sup.60Cu, .sup.61Cu, .sup.62Cu, .sup.64Cu, .sup.66Cu, .sup.67Cu, 149Tb, .sup.152Tb, .sup.155Tb, .sup.153Sm, .sup.161Tb, .sup.153Gd, .sup.155Gd, .sup.157Gd, .sup.213Bi, .sup.225Ac, .sup.230U, .sup.223Ra, .sup.165Er, .sup.52Fe, .sup.59Fe, and radionuclides of Pb (such as .sup.203Pb and .sup.212Pb, .sup.211Pb, .sup.213Pb, 214Pb, .sup.209Pb, .sup.198Pb, .sup.197Pb).

14. A pharmaceutical composition comprising the PSMA binding ligand of claim 1.

15. A method for treating and/or preventing prostate cancer and/or metastases thereof with the PSMA binding ligand of claim 1.

16. A method for diagnosing cancer, preferably prostate cancer and/or metastases thereof, with a PSMA binding ligand of claim 1.

17. PSMA binding ligand according to claim 9, wherein AS.sup.b has the structure (b) ##STR00121## wherein Q.sup.2 is selected from the group consisting of aryl, alkylaryl, arylalkyl, cycloalkyl, heterocycloalkyl, heteroaryl, heteroarylalkyl and alkylheteroaryl, and wherein q is an integer of from 0-4, preferably wherein Q.sup.2 is ##STR00122## preferably ##STR00123##

Description

FIGURES

[0238] FIG. 1. The time-dependent cleavability of the compound PSMA-MT by α-amylase is shown. .sup.177Lu-PSMA-MT was incubated with an α-amylase (isolated from the human salivary gland) and enzymatic cleavage was monitored at different times by analytical HPLC.

[0239] FIG. 2: μPET imaging with .sup.Ga-labeled PSMA-MT.

[0240] FIG. 3: Internalization of PSMA-MT in comparison to PSMA-617

[0241] FIG. 4: Organ distribution of PSMA-MT in comparison to PSMA-617 (.sup.177Lu-labeled)

[0242] FIG. 5: Chemical Formula of PSMA-MT with potential alpha-amylase cleavage sites A and B indicated, as well as cleavage products

EXAMPLES

[0243] All commercially available chemicals were of analytical grade and used without further purification. [.sup.177Lu]LuCl.sub.3 was obtained from ITG. The compounds were analyzed using reversed-phase high performance liquid chromatography (RP-HPLC; Chromolith RP-18e, 100×4.6 mm; Merck, Darmstadt, Germany). Analytical HPLC runs were performed using a linear gradient 5% (A) (0.1% aqueous TFA) to 50% B (0.1% TFA in CH.sub.3CN)) in 24 min at 1 mL/min.

[0244] Analytical HPLC runs were performed using the system Agilent 1200 series (Agilent Technologies, Santa Clara, Calif., USA). UV absorbance was measured at 220 and 280 nm, respectively. For mass spectrometry a LC-MS SQ300 (Perkin Elmer, Waltham, Mass., USA) was used.

[0245] The precursor PSMA-617 (2-[3-(1-Carboxy-5-{3-naphthalen-2-yl-2-[(4-{[2-(4,7,10-tris-carboxymethyl-1,4,7,10-tetraaza-cyclododec-1-yl)-acetylamino]-methyl}-cyclohexanecarbonyl) -amino]-propionylamino}-pentyl)-ureido]-pentanedioic acid) was purchased from ABX, Radeberg, Germany.

Example 1: Synthesis of “PSMA-MT”

1-Fmoc-4-(3-(diethoxymethyl)phenyl)piperazine

[0246] ##STR00107##

[0247] A suspension of 648 mg (2.50 mmol) 1-bromo-3-(diethoxymethyl)benzene, 646 mg (7.50 mmol) Piperazine, 280 mg (2.50 mmol) potassium tert-butoxide, 18.7 mg (30.0 μmol) rac-BINAP and 11.5 mg (12.5 μmol) Tris(dibenzylideneacetone)dipalladium(0) in 5 mL dry dioxane is heated to 60° C. over 16 h under N.sub.2. After cooling to room temperature, the red suspension is filtered over a short pad of SiO.sub.2 (ca. 5 g) and eluted with 100 mL of ethyl acetate containing 1% triethylamine. 843 mg (2.50 mmol) Fmoc N-hydroxysuccinimide ester are directly added to the filtrate and left stirring at room temperature for 2 h. After completion the solvent is removed, the residue taken up in dichloromethane and purified by column chromatography (50 g SiO.sub.2; hexanes/ethyl acetate 4:1). After evaporation, 778 mg (1.60 mmol; 64%) of the desired product are obtained.

[0248] R.sub.f(hexanes/ethyl acetate 2:1)=0.35

1-(4-(3-allyloxy-3-oxopropyl)phenyl)decaacetylmaltotriose

[0249] ##STR00108##

[0250] 500 mg (517 μmol) undecaacetylmaltotriose and 200 mg (1.04 mmol) ally 3-(4-hydroxyphenyl)propanoate[1] are dissolved in 20 mL dichloromethane. The solution is cooled to 0° C. and 185 μL (213 mg; 1.50 mmol) boron trifluoride diethyl etherate is added dropwise under an inert atmosphere. The reaction is allowed to reach room temperature and stirred under reflux for 24 h. The reaction mixture is poured to 50 g ice and stirred for 30 min. The layers are separated and the water phase is extracted with dichloromethane (3×15 mL). The organic phases are washed with sat. sodium bicarbonate and brine and dried over magnesium sulfate. After evaporation the residue is filtrated over SiO.sub.2 (10 g; 120 mL hexanes/ethyl acetate 1:1) and purified by RP-HPLC (50-70% acetonitrile over 25 min). After freeze drying 305 mg (274 μmol; 53%) of the desired trisaccharide are obtained.

1-(4-(3-carboxypropyl)phenyl)maltotriose

[0251] ##STR00109##

[0252] 305 mg (274 μmol) of lyophilized 1-(4-(3-allyloxy-3-oxopropyl)phenyl)decaacetylmaltotriose are dissolved in 10 mL tetrahydrofuran/water 6:4 and stirred with 100 mg lithium hydroxide overnight. After completion Tetrahydrofuran is removed and the resulting solution purified by RP-HPLC (5-20% acetonitrile in 25 min). After freeze drying 135 mg (207 μmol; 75%) of the desired product are obtained.

1-(4-(3-carboxypropyl)phenyl)-4″,6″-(3-(4-Fmoc-piperazine)benzyliclene)maltotriose

[0253] ##STR00110##

[0254] 9.60 mg (14.7 μmol) 1-(4-(3-carboxypropyl)phenyl)maltotriose, 27.8 mg (57.1 μmol) and 3.51 mg (18.4 μmol) p-toluenesulfonic acid monohydrate are dissolved in 100 μL dimethylformamide and heated to 60° C. for 1 h. The rection mixture is directly purified by RP-HPLC (20-50% acetonitrile in 25 min) yielding 6.76 mg (6.46 μmol; 44%) of the desired compound after freeze drying.

1-(4-(3-oxo-3-PSMA-propyl)phenyl)-4″,6″-(3-(4-Fmoc-piperazine)benzyliclene)maltotriose

[0255] ##STR00111##

[0256] 1.94 mg (6.43 mmol) N,N,N′,N′-Tetramethyl-O-(N-succinimidyl)uronium tetrafluoroborate (TSTU) are added to 5.61 mg (5.36 μmol) 1-(4-(3-carboxypropyl)phenyl)-4″,6″-(3-(4-Fmoc -piperazine)benzylidene)maltotriose and 1.23 mg (10.7 μmol) N-hydroxysuccinimide in 95 dimethylformamide and 5 μL N,N-diisopropylethylamine. The solution is left for 15 min until 5.27 mg (8.03 μmol) of the PSMA building block (((1R)-5-(2-(4-(aminomethyl)cyclohexane-1-carboxamido)-3-(naphthalene-2-yl)propanamido)-1-carboxypentyl)carbamoyl)-L-glutamic acid, (obtained following Benesova et al. For this purpose the resin bound compound (8) in doi: 10.2967/jnumed.114.147413 was cleaved from the resin: After Fmoc-deprotection of the trans-1,4-aminomethylcyclohexylcarboxamide residue, the compound was cleaved with 95% TFA (2.5% TIS, 2.5% water) and purified by HPLC followed by freeze drying.) are added. Another 95 dimethylformamide and 5 μL N,N-diisopropylethylamine are added to the turbid solution, which is then heated to 45° C. until the solution clears up. The reaction mixture was quenched with 500 acetonitrile/water 1:1 and purified by RP-HPLC (20-50% acetonitrile in 25 min). The fraction containing the desired product was evaporated to dryness and directly used for the following step.

1-(4-(3-oxo-3-PSMA-propyl)phenyl)-4″,6″-(3-(4-DOTA-piperazine)benzylidene)maltotriose (PSMA-MT)

[0257] ##STR00112##

[0258] The residue obtained in the previous step (1-(4-(3-oxo-3-PSMA-propyl)phenyl)-4″,6″-(3-(4-Fmoc-piperazine)benzylidene)maltotriose) is taken up in 4 mL of dimethylformamide/piperidine 4:1 and left standing for 5 min. After thorough evaporation of the solvent 10.0 mg (19.0 μmol) DOTA-p-nitrophenylester, 4 mL dimethylformamide and 40 μL N,N-diisopropylethylamine are added. After 2 h, the solvent is removed and the residue taken up in 2 mL acetonitrile/water 1:1 before purification via RP-HPLC (10-30% acetonitrile in 25 min). 3.31 mg (1.79 μmol; 33%) of the final product are obtained after freeze drying. [0259] 1. M.-K. Lee, Y. B. Park, S.-S. Moon, S. H. Bok, D.-J. Kim, T.-Y. Ha, T.-S. Jeong, K.-S. Jeong, M.-S. Choi, Chemico-Biological Interactions 2007, 170, 9-19.

Example 1A: Radiolabeling with .SUP.177.Lu

[0260] The γ- and β-emitter [.sup.177Lu]LuCl.sub.3 (ITG, Munich), with a t.sub.1/2 of 6.7 days was used. Radiolabeling was performed by adding 13 μL [.sup.177Lu]LuCl.sub.3 (˜30-37 MBq) in 0.4 mM HCl, 7 μL of diluted compound (0.1 mM solution of PSMA-MT in nanopure H.sub.2O) to 230 μL ammonium-acetate buffer (0.5 M, pH 5.4). The reaction mixture was incubated at 95° C. for 30 min to yield .sup.177Lu-PSMA-MT. The radiochemical yield (RCY) was determined using high performance liquid chromatography (RP-HPLC; Chromolith RP-18e, 100×4.6 mm; Merck, Darmstadt, Germany). Analytical HPLC runs were performed using an Agilent 1200 series (Agilent Technologies, Santa Clara, Calif., USA) equipped with a γ-detector. HPLC runs were performed using a linear gradient of A (0.1% trifluoroacetic acid (TFA) in water) to B (0.1% TFA in acetonitrile) (gradient: 5% B to 80% B in 15 min) at a flow rate of 2 mL/min.

Example 1B: Radiolabeling with .SUP.68.Ga

[0261] .sup.68Ga (half-life 68 min; β.sup.+ 89%; E.sub.β+ max. 1.9 MeV) was obtained from an in house .sup.68Ge/.sup.68Ga generator (DKFZ Heidelberg, Germany) based on pyrogallol resin support. Approximately 0.5-1 GBq .sup.68Ga was eluted using 5.5 M HCl. The activity was trapped on a small anion-exchanger cartridge (AG1×8, Biorad, Richmond, Calif., USA) as [.sup.68Ga]GaCl.sub.4.sup.−. The radiogallium was eluted from the cartridge in a final volume of 300 μL ultrapure water (Merck, Darmstadt, Germany) as [.sup.68Ga]GaCl.sub.3. The precursor peptides (1 nmol in 2.4 M HEPES buffer, 90 μL) were added to 40 μL [.sup.68Ga]Ga.sup.3+ eluate (˜40 MBq). The pH was adjusted to 4.2 using 30% NaOH and 10% NaOH. The reaction mixture was incubated at 98° C. for 10 minutes. The radiochemical yield (RCY) was determined by HPLC.

Example 2: Cleavability of the Linker

[0262] The basic cleavability of the linker by α-amylases was demonstrated after radioactive labeling of PSMA-MT in vitro according to example 1A (FIG. 1). The radiolabelled compound was incubated with α-amylase (isolated from the human salivary gland, Sigma-Aldrich) according to instructions of the manufacturer and enzymatic cleavage was monitored at different times by analytical HPLC (see FIG. 1).

Example 3: μPET-Imaging

[0263] For μPET imaging, mice bearing a PSMA positive tumor were anaesthetized (2% sevoflurane, Abbott), placed into a small animal PET scanner (Inveon PET, Siemens) and injected with .sup.68Ga-labeled PSMA-MT (see example 1B). A 20 min transmission scan, a 50 min dynamic scan and a static scan from 100 to 120 min p.i. were performed. Images were reconstructed iteratively using the space alternating generalized expectation maximization method (SAGE, 16 subsets, 4 iterations) applying median root prior correction and were converted to standardized uptake value (SUV) shown in maximum intensity projection (MIP) images. Quantitation was done using a ROI (region of interest) technique and expressed as SUVmean (see FIG. 2).

Example 4: Competitive Cell Binding

[0264] For the determination of competitive cell binding the cells (10.sup.5 per well) were incubated with a 0.8 nM solution of .sup.68Ga-labeled radioligand [Glu-urea-Lys(Ahx)].sub.2-HBED-CC (PSMA-10, precursor ordered from ABX, Radeberg, Germany) in the presence of 12 different concentrations of analyte (non-labeled compounds, 0-5000 nM, 100 μL/well). After incubation, the mixture was removed and the wells were washed 3 times with PBS using a multiscreen vacuum manifold (Millipore, Billerica, Mass.). Cell-bound radioactivity was measured using a gamma counter (Packard Cobra II, GMI, Minn., USA). The 50% inhibitory concentration (IC50) values were calculated by fitting the data using a nonlinear regression algorithm (GraphPad Software).

[0265] In first preliminary experiments for PSMA-MT an IC.sub.50 of around 39 nM was determined, which is in the same range as the IC.sub.50 of PSMA-617 (ABX, Radeberg, Germany) which was detected to be around 20 nM.

Example 5: Internalization of PSMA-MT in Comparison to PSMA-617

[0266] 10.sup.5 LNCaP cells were seeded in poly-L-lysine coated 24-well cell culture plates. 24 h later, wells were washed and the cells incubated with a 30 nM solution of .sup.177Lu radiolabeled PSMA-ligand (PSMA-617 or PSMA-MT) in 250 μL medium, for 45 min at 37° C. In the blocking experiments, incubation was performed in the presence of excess of 2-PMPA to assess competition for PSMA. Following incubation, PSMA-ligand containing medium was removed by washing 3 times with 1 mL of ice-cold PBS. Cells were subsequently incubated twice with 0.5 mL glycine-HCl in PBS (50 mM, pH=2.8) for 5 min to extract the cell membrane associated fraction. The cells were then washed with 1 mL of ice-cold PBS, and lysed with 0.5 mL 0.3 M NaOH. The membrane bound and lysed (internalized) fractions were assessed for radioactivity levels using a gamma counter (Packard Cobra II, GMI, Minn., USA). Cell uptake was calculated as percent of the initially added radioactivity detected in 10.sup.5 cells [% IA/10.sup.5 cells].

[0267] The results are given in the following table (see also FIG. 3):

TABLE-US-00001 37° C. Mean Standard Deviation 177Lu-PSMA-MT Specifically internalized 2,14459836 0,24994955 Specifically cell surface bound 4,86794251 0,56488708 177Lu-PSMA-617 Specifically internalized 2,63598315 0,51667575 Specifically cell surface bound 4,09228993 1,14873804

Example 6: Organ Distribution of PSMA-MT in Comparison to PSMA-617 (.SUP.177.Lu-labeled)

[0268] 5×10.sup.6 LNCaP cells were subcutaneously inoculated into the right flank of male 6-week-old BALB/c nu/nu mice (Charles River Laboratories). Tumors were grown for ˜3 weeks, to ˜200 mm.sup.3. 60 pmoles of .sup.177Lu labelled PSMA-ligands were dissolved in 100 μL 0.9% NaCl and injected per mouse. The solution was injected via the tail vein, followed by sacrifice at various time points. Organs of interest (blood, heart, lung, spleen, liver, kidney, muscle, small intestine, brain, tumor, and femur) were dissected, blotted dry, and weighed. The radioactivity was measured using a gamma counter and calculated as % ID/g, and corrected for .sup.177Lu decay.

[0269] The results are given in the following tables (mean+standard deviation; per time point n=3 animals) (see also FIG. 4):

TABLE-US-00002 .sup.177Lu-PSMA-617: 1 h Small Blood Heart Lung Spleen Liver Kidney Muscle Intestine Brain Tumor Kidney Tail 0.31 0.17 0.61 5.86 4.73 143.55 0.07 0.18 0.02 19.20 146.77 3.46 0.10 0.05 0.12 2.21 0.90 56.58 0.03 0.07 0.00 3.01 57.40 2.25

TABLE-US-00003 .sup.177Lu-PSMA-617: 4 h Small Blood Heart Lung Spleen Liver Kidney Muscle Intestine Brain Tumor Kidney Tail 0.03 0.03 0.08 1.20 1.57 5.52 0.01 0.04 0.01 14.77 5.50 0.78 0.01 0.01 0.01 0.34 0.31 1.00 0.00 0.00 0.00 0.98 0.82 0.57

TABLE-US-00004 .sup.177Lu-PSMA-MT: 1 h Small Blood Heart Lung Spleen Liver Kidney Muscle Intestine Brain Tumor Kidney Tail 0.35 0.16 0.56 3.83 2.07 144.27 0.08 0.15 0.02 12.83 142.04 4.61 0.24 0.07 0.14 1.36 1.90 23.66 0.04 0.06 0.01 1.30 26.70 5.36

TABLE-US-00005 .sup.177Lu-PSMA-MT: 4 h Small Blood Heart Lung Spleen Liver Kidney Muscle Intestine Brain Tumor Kidney Tail 0.04 0.04 0.10 3.94 4.58 20.59 0.02 0.04 0.01 9.18 19.45 0.50 0.02 0.01 0.03 0.25 0.30 12.68 0.01 0.02 0.00 1.58 10.01 0.12

Example 7: Identification of the Alpha-Amylase Cleavage Site in PSMA-MT

[0270] 10 μl of a solution of PSMA-MT in ultrapure water with a concentration of 1 mM were analyzed by analytic RP-HPLC and MALDI-TOF before the digestion. For digestion 10 μl of α-Amylase (1 U/μl in H2O ) were added to 10 μl of a solution of PSMA-MT in ultrapure water with a concentration of 1 mM and incubated at RT for 1 h. The solution was afterwards analyzed with analytic RP-HPLC and MALDI-TOF to evaluate the reaction.

[0271] In MALDI-TOF analysis a peak of the original mass of 1848.97 g/mol was detected, which disappeared after 1 h of digestion with α-Amylase, indicating a complete turnover of PSMA-MT.

[0272] The potential cleavage sites of alpha-amylase in PSMA-MT are shown in FIG. 5, as are products of cleavage at cleavage position A.

[0273] To investigate the exact digest position of PSMA-MT the different fragments found in the MALDI-TOF after the digest were compared. Two main peaks occurred at 902 g/mol and at 989 g/mol. The first fragment with the DOTA-chelator C.sub.35H.sub.60N.sub.6O.sub.18+H.sup.+ (901+1 g/mol) and the second fragment with the PSMA-binding-motive C.sub.46H.sub.63N.sub.5O.sub.16+Na.sup.+ (966+23 g/mol) predicted for cleavage site A were found in MALDI-TOF. Fragments corresponding to cleavage at cleavage site B could not be detected in the MALDI-TOF.