Double-labeled probe for molecular imaging and use thereof

Abstract

The present invention relates to a compound of a pharmaceutically acceptable salt thereof of formula (I) wherein (A) is at least one motif specifically binding to cell membranes of neoplastic cells; (B) at least one chelator moiety of radiometals; (C) a dye moiety; x.sub.1 is a spacer or a chemical single bond covalently connecting (A) to the rest of the molecule; x.sub.2 is a spacer or a chemical single bond covalently connecting (C) to the rest of the molecule. The invention further relates to compositions comprising said compounds as well as a method for detecting neoplastic cells in a sample in vitro with the aid of the compounds or composition. ##STR00001##

Claims

1. A compound or a pharmaceutically acceptable salt thereof of formula (I): ##STR00027## wherein (A) is at least one moiety specifically binding to cell membranes of neoplastic cells; (B) at least one chelator moiety of radiometals; (C) a dye moiety; x.sub.1 is a spacer or a chemical single bond covalently connecting (A) to the rest of the molecule; x.sub.2 is a spacer or a chemical single bond covalently connecting (C) to the rest of the molecule; wherein (C) has the formula ##STR00028## wherein X.sup.1 and X.sup.4 are independently selected from the group consisting of —N═, —N(R.sup.5)═, and —C(R.sup.6)═; X.sup.2 and X.sup.3 are independently selected from the group consisting of O, S, Se, N(R.sup.5), and C(R.sup.6R.sup.7); Y is a linker within (C) permitting electron delocalization within (C), wherein Y optionally comprises a group (L-).sub.cZ.sup.0; a and b are independently selected from the group consisting of 1, 2, and 3; each R.sup.1 and each R.sup.2 is independently (L-).sub.cZ, (L-).sub.cZ.sup.0 or H; and two adjacent R.sup.1 and/or two adjacent R.sup.2 can also form an aromatic ring, which is optionally substituted with one or more (L-).sub.cZ or (L-).sub.cZ.sup.0; R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.9 are independently selected from the group consisting of (L-).sub.cZ, (L-).sub.cZ.sup.0, and H; each c is independently 0, or 1; each L is independently T.sup.1, —OT.sup.1-, —ST.sup.1-, —C(O)T.sup.1-, —C(O)OT.sup.1-, —OC(O)T.sup.1-, —C(O)NHT.sup.1-, —NHC(O)T.sup.1, or a C.sub.1-10 alkylene group, which is optionally interrupted and/or terminated by one or more of —O—, —S—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)NH—, —NHC(O)O—, and T.sup.1; T.sup.1 is phenyl, naphthyl, indenyl, indanyl, tetralinyl, decalinyl, adamantyl, C.sub.3-7 cycloalkyl, 3 to 7 membered heterocyclyl, or 7 to 11 membered heterobicyclyl, wherein T.sup.1 is optionally substituted with one or more substituents selected from the group consisting of halogen, CN, C(O)R.sup.8, COOR.sup.8, OR.sup.8, C(O)N(R.sup.8R.sup.8a), S(O).sub.2N(R.sup.8R.sup.8a), S(O)N(R.sup.8R.sup.8a), S(O).sub.2R.sup.8, N(R.sup.8)S(O).sub.2N(R.sup.8aR.sup.8b), SR.sup.8, N(R.sup.8R.sup.8a), NO.sub.2; OC(O)R.sup.8, N(R.sup.8)C(O)R.sup.8a, N(R.sup.8)S(O).sub.2R.sup.8a, N(R.sup.8)S(O)R.sup.8a, N(R.sup.8)C(O)N(R.sup.8aR.sup.8b), N(R.sup.8)C(O)OR.sup.8a, OC(O)N(R.sup.8R.sup.8a), oxo (═O), wherein T.sup.1 is at least partially saturated, or C.sub.1-6 alkyl, wherein C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; each Z is independently H, halogen, CN, C(O)R.sup.8, C(O)OR.sup.8, C(O)O.sup.− OR.sup.8, C(O)N(R.sup.8R.sup.8a), S(O).sub.2OR.sub.8, S(O).sub.2O.sup.−, S(O).sub.2N(R.sup.8R.sup.8a), S(O)N(R.sup.8R.sup.8a), S(O).sub.2R.sup.8, S(O)R.sup.8, N(R.sup.8)S(O).sub.2N(R.sup.8aR.sup.8b), SR.sup.8, N(R.sup.8R.sup.8a), NO.sub.2; P(O)(OR.sup.8).sub.2, P(O)(OR.sup.8)O.sup.−, OC(O)R.sup.8, N(R.sup.8)C(O)R.sup.8a, N(R.sup.8)S(O).sub.2R.sup.8a, N(R.sup.8)S(O)R.sup.8a, N(R.sup.8)C(O)N(R.sup.8aR.sup.8b), N(R.sup.8)C(O)OR.sup.8a, or OC(O)N(R.sup.8R.sup.8a); R.sup.8, R.sup.8a, R.sup.8b are independently selected from the group consisting of H, or C.sub.1-6 alkyl, wherein C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; Z.sup.0 is a chemical bond connecting (C) to x.sub.2 or to the rest of the molecule in case x.sub.2 is a chemical single bond; provided that one of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.7, R.sup.9 is (L-).sub.cZ.sup.0 or that Y comprises (L-).sub.cZ.sup.0; wherein c is 1 and L is C.sub.3-7 alkylene; wherein any remaining positive or negative charge or charges are compensated by pharmaceutically acceptable negatively or positively charged counterion or counterions.

2. The compound or a pharmaceutically acceptable salt thereof of claim 1, wherein said compound has a molecular weight of not more than 10 kDa.

3. The compound or a pharmaceutically acceptable salt thereof of claim 1, wherein the moiety specifically binding to cell membranes of neoplastic cells (A) is a moiety specifically binding to cell membranes of cancerous cells.

4. The compound or a pharmaceutically acceptable salt thereof according to claim 3, wherein the moiety specifically binding to cell membranes of neoplastic cells (A) is a PSMA binding moiety having the following structure: ##STR00029## wherein Z.sup.1, Z.sup.2 and Z.sup.3 are each independently from another selected from the group consisting of —C(O)OR.sup.1a, —SO.sub.2R.sup.1a, —SO.sub.3R.sup.1a, —SO.sub.4R.sup.1a, —PO.sub.2R.sup.1a, —PO.sub.3R.sup.1a, and —PO.sub.4R.sup.1aR.sup.2a, wherein R.sup.1a and R.sup.2a are independently from another H or a C.sub.1-4-alkyl residue; wherein a′ represents a —[CH.sub.2].sub.o— residue, wherein o is an integer from 1 to 4, wherein b′ represents a residue selected from the group consisting of —NH—, —C(O)— and —O—; and wherein the wavy line indicates the conjugation site to the rest of the molecule.

5. The compound or a pharmaceutically acceptable salt thereof of claim 1, wherein the moiety specifically binding to cell membranes of neoplastic cells (A) is a PSMA binding moiety having the following structure: ##STR00030## wherein the wavy line indicates the conjugation site to the rest of the molecule.

6. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the x.sub.1 is a chemical single bond.

7. The compound or a pharmaceutically acceptable salt thereof of claim 1, wherein, in formula (I), (A) and x.sub.1 are selected to give the following structure: ##STR00031##

8. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein x.sub.2 a chemical single bond.

9. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the chelator moiety of radiometals (B) is derived from a chelator selected from: ##STR00032##

10. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein, in formula (I), (A), x.sub.1 and (B) are selected to give the following structure: ##STR00033##

11. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein in formula (C) X.sup.1 and X.sup.4 are the same.

12. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein in formula (C) X.sup.2 and X.sup.3 are the same.

13. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein in formula (C) Y does not comprise (L-).sub.cZ.sup.0.

14. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein in formula (C) a and b are the same.

15. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein in formula (C) a and b are the same and 2.

16. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein in formula (C) one of R.sup.3 and R.sup.4 is (L-).sub.cZ.sup.0 and the other is (L-).sub.cZ with L-=C.sub.1-10 alkylene and Z═H or SO.sub.3—.

17. The compound or a pharmaceutically acceptable salt thereof of claim 1, wherein (L-).sub.cZ.sup.0 is C.sub.5 alkylene connecting (C) to x.sub.2 or to the rest of the molecule in case x.sub.2 is a chemical single bond.

18. The compound or a pharmaceutically acceptable salt thereof of claim 1, wherein the dye moiety (C) is selected from the group consisting of the following structures: ##STR00034## ##STR00035## wherein X.sup.− is a pharmaceutically acceptable negatively charged counterion; wherein Y.sup.− is a pharmaceutically acceptable positively charged counterion; and wherein the wavy line indicates the conjugation site to the rest of the compound.

19. The compound of claim 1, wherein in the dye moiety (C) is a fluorescent dye moiety having an emission maximum in the range from 400 nm to 1000 nm.

20. The compound or a pharmaceutically acceptable salt thereof of claim 1, wherein said compound has the following chemical structure: ##STR00036##

21. A composition comprising: (a) the compound or a pharmaceutically acceptable salt thereof of claim 1; and (b) a radiometal and optionally (c) one or more pharmaceutically acceptable carriers.

22. A composition according to claim 21 for use as a diagnostic and therapeutic agent.

23. A method for detecting neoplastic cells in a sample in vitro, comprising the following steps: (i) providing cells which are neoplastic or at risk of being neoplastic; (ii) administering the compound or a pharmaceutically acceptable salt thereof according to claim 1 to said cells; (iii) detecting the fluorescence and/or radioactive signal of said cells.

24. The compound or a pharmaceutically acceptable salt thereof of claim 2, wherein said compound has a molecular weight of not more than 5 kDa.

25. The compound or a pharmaceutically acceptable salt thereof of claim 3, wherein said moiety comprises a prostate-specific membrane antigen (PSMA) binding moiety.

26. The compound or a pharmaceutically acceptable salt thereof according to claim 4, wherein o is 3 or 4 and/or wherein b′ is —NH—.

27. The compound or a pharmaceutically acceptable salt thereof according to claim 26, wherein o is 4.

28. The compound or a pharmaceutically acceptable salt thereof according to claim 11, wherein in formula (C) X.sup.1 and X.sup.4 are C(R.sup.6).

29. The compound or a pharmaceutically acceptable salt thereof according to claim 11, wherein in formula (C) X.sup.1 and X.sup.4 are CH.

30. The compound or a pharmaceutically acceptable salt thereof according to claim 12, wherein in formula (C) X.sup.2 and X.sup.3 are C(R.sup.6R.sup.7).

31. The compound or a pharmaceutically acceptable salt thereof according to claim 30, wherein R.sup.6 and R.sup.7 are the same.

32. The compound or a pharmaceutically acceptable salt thereof according to claim 31, wherein R.sup.6 and R.sup.7 are L-Z with L=C.sub.1-10 alkylene and Z═H.

33. The compound or a pharmaceutically acceptable salt thereof according to claim 32, wherein L=CH.sub.2.

34. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein in formula (C) Y is ##STR00037## wherein g is 1, 2, 3, or 4 and each R.sup.9a is (L-).sub.cZ, or H; and two R.sup.9a can also form a carbocyclic ring having 5, 6, or 7 carbon atoms or a 4 to 7 membered heterocyclic ring; each c is independently 0, or 1; each L is independently T.sup.1, —OT.sup.1-, —ST.sup.1-, —C(O)T.sup.1-, —C(O)OT.sup.1-, —OC(O)T.sup.1-, —C(O)NHT.sup.1-, —NHC(O)T.sup.1, or a C.sub.1-10 alkylene group, which is optionally interrupted and/or terminated by one or more of —O—, —S—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)NH—, —NHC(O)O—, and T.sup.1; T.sup.1 is phenyl, naphthyl, indenyl, indanyl, tetralinyl, decalinyl, adamantyl, C.sub.3-7 cycloalkyl, 4 to 7 membered heterocyclyl, or 7 to 11 membered heterobicyclyl, wherein T.sup.1 is optionally substituted with one or more substituents selected from the group consisting of halogen, CN, C(O)R.sup.8, COOR.sup.8, OR.sup.8, C(O)N(R.sup.8R.sup.8a), S(O).sub.2N(R.sup.8R.sup.8a), S(O)N(R.sup.8R.sup.8a), S(O).sub.2R.sup.8, N(R.sup.8)S(O).sub.2N(R.sup.8aR.sup.8b), SR.sup.8, N(R.sup.8R.sup.8a), NO.sub.2; OC(O)R.sup.8, N(R.sup.8)C(O)R.sup.8a, N(R.sup.8)S(O).sub.2R.sup.8a, N(R.sup.8)S(O)R.sup.8a, N(R.sup.8)C(O)N(R.sup.8aR.sup.8b), N(R.sup.8)C(O)OR.sup.8a, OC(O)N(R.sup.8R.sup.8a), oxo (═O), wherein T.sup.1 is at least partially saturated, or C.sub.1-6 alkyl, wherein C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; R.sup.8, R.sup.8a, R.sup.8b are independently selected from the group consisting of H, or C.sub.1-6 alkyl, wherein C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; and each Z is independently H, halogen, CN, C(O)R.sup.8, C(O)OR.sup.8, C(O)O.sup.− OR.sup.8, C(O)N(R.sup.8R.sup.8a), S(O).sub.2OR.sup.8, S(O).sub.2O.sup.−, S(O).sub.2N(R.sup.8R.sup.8a), S(O)N(R.sup.8R.sup.8a), S(O).sub.2R.sup.8, S(O)R.sup.8, N(R.sup.8)S(O).sub.2N(R.sup.8aR.sup.8b), SR.sup.8, N(R.sup.8R.sup.8a), NO.sub.2; P(O)(OR.sup.8).sub.2, P(O)(OR.sup.8)O.sup.−, OC(O)R.sup.8, N(R.sup.8)C(O)R.sup.8a, N(R.sup.8)S(O).sub.2R.sup.8a, N(R.sup.8)S(O)R.sup.8a, N(R.sup.8)C(O)N(R.sup.8aR.sup.8b), N(R.sup.8)C(O)OR.sup.8a, or OC(O)N(R.sup.8R.sup.8a).

35. The compound or a pharmaceutically acceptable salt thereof according to claim 29, wherein in formula (C) Y is selected from: ##STR00038## with g=2 and each R.sup.9a═H.

36. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein in formula (C) a and b are 1; and/or wherein R.sup.1 and R.sup.2═SO.sub.3—.

37. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein in formula (C) two adjacent R.sup.1 and two adjacent R.sup.2 form a phenyl ring.

38. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein in formula (C) one of R.sup.3 and R.sup.4 is (L-).sub.cZ.sup.0 and the other is (L-).sub.cZ with L-=alkylene and Z═H or SO.sub.3— and c=1.

39. The composition of claim 21, wherein the radiometal is selected from the group consisting of .sup.89Zr, .sup.44Sc, .sup.111In, .sup.90Y, .sup.67Ga, .sup.68Ga, .sup.177Lu, .sup.99mTc, .sup.82Rb, .sup.64Cu, .sup.67Cu, .sup.153Gd, .sup.155Gd, .sup.157Gd, .sup.213Bi, .sup.225Ac and .sup.59Fe.

40. The composition of claim 21, wherein the radiometal is .sup.68Ga.

41. The method of claim 23, wherein the cells which are neoplastic or at risk of being neoplastic are cancerous or at risk of being cancerous.

Description

EXAMPLES

Abbreviations

(1) Glu-urea-Lys-2-Nal-Chx-Lys(IRDye800CW)-DOTA

(2) ##STR00026##

Experimental Procedures

(3) All commercially available chemicals were of analytical grade and used without further purification. .sup.68Ga (half-life 68 min; β.sup.+89%; E.sub.β+ max. 1.9 MeV) was obtained from a .sup.68Ge/.sup.68Ga generator based on a pyrogallol resin support (1). 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 (0.1% aqueous TFA (A) to 100% B (0.1% TFA in CH.sub.3CN)) in 10 min at 2 mL/min. The system L6200 A (Merck-Hitachi, Darmstadt, Germany) was equipped with a variable UV and a gamma detector (Bioscan; Washington, USA).

(4) For preparative HPLC the system LaPrep P110 (VWR, Darmstadt, Germany) was supplied with a variable UV detector (P314, VWR, Darmstadt, Germany). Analytical HPLC runs were performed using the system Agilent 1100 series (Agilent Technologies, Santa Clara, Calif., USA). UV absorbance was measured at 214 and 254 nm, respectively. For mass spectrometry a MALDI-MS (Daltonics Microflex, Bruker Daltonics, Bremen, Germany) was used.

Synthesis of Glu-urea-Lys-2-Nal-Chx-Lys(IRDye800CW)-DOTA

(5) The synthesis of the pharmacophore Glu-urea-Lys was performed as described previously (3). Briefly, the synthesis started with the formation of the isocyanate of the glutamyl moiety using triphosgene. A resin-immobilized (2-chloro-tritylresin, Merck, Darmstadt) ε-allyloxycarbonyl protected lysine was added and reacted for 16 h with gentle agitation. The resin was filtered off and the allyloxy-protecting group was removed by reacting twice with Pd(PPh.sub.3).sub.4 (0.3 eq.) and morpholine (15 eq.) under ambient conditions (1 h, RT). Subsequently, the linkers were introduced by standard Fmoc solid phase protocols.

(6) For all compounds in a first step Fmoc-2-Nal-OH and N-Fmoc-tranexamic acid (4 eq. each) with HBTU (4 eq.) and DIPEA (4 eq.) were coupled in DMF. Afterwards the resin was split.

(7) For synthesis of Glu-urea-Lys-2-Nal-Chx-Lys(Boc)-DOTA (i=0-2), Fmoc-Lys(Boc)-OH was coupled and subsequently tris(tBu)DOTA (tris(tBu)-ester of 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetic acid) (4 eq. each) with HBTU (4 eq.) and DIPEA (4 eq.) in DMF.

(8) The products were cleaved from the resin for 3 hours at RT using TFA/TIPS/H.sub.2O (95/2.5/2.5, v/v/v). All products were purified using RP-HPLC and identified with mass spectrometry. Purification was done using a NUCLEODUR® Sphinx RP column (VP250/21, 5 μm 250×21 mm; Macherey-Nagel, Duren, Germany) with a 20 min gradient starting at 10% B, raised to 100% B within 20 min. Solvent A consisted of 0.1% aqueous TFA and solvent B was 0.1% TFA in CH.sub.3CN. The flow rate was 20 mL/min.

(9) IRDye800CW-NHS ester (1 eq.) (LI-COR Biosciences) was conjugated to Glu-urea-Lys-2-Nal-Chx-Lys-DOTA in PBS-buffer (pH 8.5) for 24 h at RT.

(10) The products were isolated via semipreparative HPLC using a Chromolith RP-18e column (100×10 mm; Merck, Darmstadt, Germany) (0-100% B in 10 min, flow 5 ml/min) and identified with mass spectrometry.

(11) TABLE-US-00002 TABLE 1 Analytical data of the final compound. Mass spectrometry (MALDI-MS) was performed with the metal-free substances. m/z m/z (g/mol, M.sub.calc.) (M.sub.found.) Glu-urea-Lys-2-Nal-Chx-Lys 2156.5 2157.8 (IRDye800CW)-DOTA

(12) Reference compound (Compound 1 in Banerjee S et al. Angew Chem Int Ed Engl. 2011 Sep. 19; 50(39):9167-70) was prepared as described by Banerjee S et al.

(13) .sup.68Ga-Labeling

(14) The precursor peptides [1 nmol in HEPES buffer (580 mg/ml), 40 μL] were added to 40 μL [.sup.68Ga]Ga.sup.3+ eluate (˜40 MBq). The pH was adjusted to 3.8 using 30% NaOH and 10% NaOH, respectively. The reaction mixture was incubated at 98° C. for 10 minutes. The radiochemical yield (RCY) was determined by HPLC.

(15) Cell Culture

(16) PSMA.sup.+ LNCaP cells (ATCC CRL-1740) were cultured in RPMI medium supplemented with 10% fetal calf serum and 2 mmol/L L-glutamine (all from PAA). Cells were grown at 37° C. in humidified air with 5% CO.sub.2 and were harvested using trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA; 0.25% trypsin, 0.02% EDTA, Invitrogen).

(17) Cell Binding and Internalization

(18) The competitive cell binding assay and internalization experiments were performed as described previously (4). Briefly, the cells (10.sup.5 per well) were incubated with a 0.75 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 (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).

(19) For internalization experiments, 10.sup.5 cells per well were seeded in poly-L-lysine coated 24-well cell culture plates 24 h before incubation. After washing, the cells were incubated with 30 nM of the radiolabeled compound for 45 min at 37° C. and at 4° C., respectively. Cellular uptake was terminated by washing 3 times with 1 mL of ice-cold PBS. To remove surface-bound radioactivity, cells were incubated twice with 0.5 mL glycine-HCl in PBS (50 mM, pH=2.8) for 5 min. The cells were washed with 1 mL of ice-cold PBS and lysed using 0.3 N NaOH (0.5 mL). The surface-bound and the internalized fractions were measured in a gamma counter. The cell uptake was calculated as percent of the initially added radioactivity bound to 10.sup.5 cells [% ID/10.sup.5 cells].

(20) Biodistribution

(21) For the experimental tumor models 5×10.sup.6 cells of LNCaP (in 50% Matrigel; Becton Dickinson, Heidelberg, Germany) were subcutaneously inoculated into the right trunk of 7- to 8-week-old male BALB/c nu/nu mice (Charles River). The tumors were allowed to grow until approximately 1 cm.sup.3 in size. The .sup.68Ga-labeled compounds were injected into a tail vein (1-2 MBq; 60 pmol). At 1 h after injection the animals were sacrificed. Organs of interest were dissected, blotted dry, and weighed. The radioactivity was measured using a gamma counter and calculated as % ID/g. All animal experiments complied with the current laws of the Federal Republic of Germany.

(22) Statistical Aspects

(23) All experiments were performed at least in triplicate and repeated at least for three times. Quantitative data were expressed as mean±SD. If applicable, means were compared using Student's t test. P values<0.05 were considered statistically significant.

(24) Results

(25) The internalization efficiency and the PSMA binding affinity of the compounds were determined in order to investigate the influence of the linker and its position on the binding properties. The results are summarized in Table 2.

(26) TABLE-US-00003 TABLE 2 Cell binding and internalization data of the investigated compounds [radioligand: [.sup.68Ga]Ga -PSMA-10 (K.sub.d = 2.1 ± 1.4 nM(3), C.sub.radioligand = 0.75 nM)] Specific cell Specific IC.sub.50[nM] surface bound internalized metal-free ligand [% ID/10.sup.5 cells] [% ID/10.sup.5 cells] Glu-urea-Lys-2-Nal-Chx-Lys 15.87 ± 5.46 25.51 ± 9.73 27.64 ± 12.80 (IRDye800CW)-DOTA

(27) The IRDye800CW structure exhibits a high binding affinity and specific internalization.

(28) A high binding affinity and specific internalization was conserved after conjugation of IRDye800CW.

(29) Organ distribution was performed in comparison to the reference (Compound 1 in Baneriee S et al. Angew Chem Int Ed Engl. 2011 Sep. 19; 50(39):9167-70).

(30) TABLE-US-00004 TABLE 3 Organ distribution of .sup.68Ga-labeled Glu-urea-Lys-2-Nal-Chx- Lys(IRDye800CW)-DOTA in comparison to the reference in LNCaP-tumor bearing balb/c nu/nu mice 1 h p.i. (n = 3). .sup.68Ga-labeled .sup.68Ga- Glu-urea-Lys- reference compound 1 2-Nal-Chx-Lys synthesized according to (IRDye800CW)-DOTA Banerjee et al. (2) Mean SD Mean SD Blood 1.14 0.14 0.69 0.91 Heart 0.51 0.10 0.68 0.67 Lung 1.47 0.14 1.35 1.24 Spleen 2.82 1.19 10.13 5.02 Liver 0.95 0.15 0.83 0.58 Kidney 65.64 6.60 166.68 43.06 Muscle 0.34 0.03 0.83 0.63 Intestine 0.43 0.12 0.54 0.54 Brain 0.07 0.01 0.13 0.1 Tumor 4.13 0.15 5.74 2.0

(31) In comparison to the reference, the synthesized compound .sup.68Ga-Glu-urea-Lys-2-Nal-Chx-Lys(IRDye800CW)-DOTA exhibits a significantly reduced kidney uptake while the tumor uptake is comparable. Furthermore, the uptake of this compound in all other analyzed background organs was similar to the reference as there were no significant differences detected.

REFERENCES

(32) 1. Schuhmacher J, Maier-Borst W. A new Ge-68/Ga-68 radioisotope generator system for production of Ga-68 in dilute HCl. Int J Appl Radiat Isot. 1981; 32:31-36. 2. Banerjee S R, Pullambhatla M, Byun Y, et al. Sequential SPECT and optical imaging of experimental models of prostate cancer with a dual modality inhibitor of the prostate-specific membrane antigen. Angew Chem Int Ed Engl. 2011; 50:9167-9170. 3. Schafer M, Bauder-Wust U, Leotta K, et al. A dimerized urea-based inhibitor of the prostate-specific membrane antigen for 68Ga-PET imaging of prostate cancer. EJNMMI Res. 2012; 2:23. 4. Eder M, Schafer M, Bauder-Wust U, et al. (68) Ga-Complex lipophilicity and the targeting property of a urea-based PSMA inhibitor for PET imaging. Bioconjug Chem. 2012; 23:688-697.