Double-labeled probe for molecular imaging and use thereof

Abstract

The present invention relates to a compound or a pharmaceutically acceptable salt thereof having a chemical structure comprising: (A) at least one motif specifically binding to cell membranes of neoplastic cells; (B) at least one chelator moiety of radiometals; and (C) at least one dye moiety; wherein said compound has a molecular weight of not more than 5 kDa. Further, the invention refers to a method for producing such compound and to the in vivo and in vitro uses thereof.

Claims

1. A method for diagnosing a neoplasm in a patient suffering therefrom or being at risk thereof, comprising the following steps: (i) administering to said patient sufficient amounts of a composition comprising: (a) a compound or a pharmaceutically acceptable salt thereof having a chemical structure comprising: (A) a prostate-specific membrane antigen (PSMA) binding motif having the structure: ##STR00025## (B) a chelator moiety N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N-diacetic acid (HBED-CC) of radiometals; and (C) a fluorescent dye moiety; wherein the wavy line indicates the conjugation site to the chelator moiety; wherein said compound has a molecular weight of not more than 5 kDa; and wherein the compound has the following molecular structure: (A)-(B)-(C), wherein (A) and (B) are linked with each other via a spacer moiety of not more than 2 nm in length or via a direct bond, and wherein (B) and (C) are linked with each other via a spacer moiety containing polyethylene glycol; wherein the radiometal comprises .sup.68Ga; and optionally (b) one or more pharmaceutically acceptable carriers; (ii) imaging the patient's body or a part thereof, wherein imaging is selected from the group consisting of positron emission tomography (PET), fluorescence imaging, and a combination of PET and fluorescence imaging; and (iii) diagnosing the neoplasm based on the imaging of step (ii).

2. The method according to claim 1, wherein the neoplasia is cancer.

3. The method according to claim 1, wherein step (ii) comprises imaging the patient's body or a part thereof to detect the radioactive signal of the radiometal.

4. The method according to claim 3, step (ii) comprises imaging the patient's body or a part thereof to detect the fluorescent dye moiety (C).

5. The method according to claim 1, wherein step (ii) comprises imaging the patient's body or a part thereof to detect the fluorescent dye moiety (C) during surgery, wherein cancerous tissue is at least partly laid open.

6. The method according to claim 1, wherein step (ii) comprises the following steps: (iia) imaging the patient patient's body or a part thereof by means of positron emission tomography (PET); and subsequently (iib) imaging patient's tissue of interest by means of fluorescence imaging, wherein the tissue of interest is laid open during a surgery.

7. The method according to claim 6, wherein step (iia) is conducted prior to step (iib).

Description

FIGURES

(1) FIG. 1A shows one embodiment of the compound disclosed herein in its non-complexed form. The motif specifically binding to cell membranes of neoplastic cells (A) enables binding to the cellular target structure on neoplastic surfaces (black on the left-hand side) and the dye moiety (C) emits fluorescent light upon excitation.

(2) FIG. 1B shows one embodiment of the compound disclosed herein that is complexed with a radiometal. Here, additionally, positrons are emitted upon disintegration of the radiometal. When these hit on an electron in the sample, photons are emitted in two traverse directions, which can be detected.

(3) FIG. 2A shows one embodiment of the compound disclosed herein in its non-complexed form.

(4) FIG. 2B shows one embodiment of the compound disclosed herein that is complexed with the radiometal .sup.68Ga. The amino acid moieties (i.e., the glutamate moiety and the lysine moiety) preferably bear the L-configuration.

(5) FIG. 3 exemplifies the synthesis of Glu-urea-Lys-HBED-CC-PEG.sub.2-fluorescein. Herein, a indicates triphosgene, DIPEA, CH.sub.2Cl.sub.2, 0° C.; b indicates H-Lys(Alloc)-2CT-Resin, CH.sub.2Cl.sub.2; c indicates Pd[P(C.sub.6H.sub.5).sub.3].sub.4, morpholine, CH.sub.2Cl.sub.2; d indicates hexafluoroisopropanol/CH.sub.2Cl.sub.2; e indicates (HBED-CC)TFP.sub.2, DIPEA, DMF; f indicates 1,8-Diamino-3,6-Dioxaoctane; g indicates fluorescein isothiocyanate (isomer I), DIPEA, DMF; and h indicates TFA.

(6) FIG. 4 shows the comparison of Glu-urea-Lys-HBED-CC-Fluorescein with the references Glu-urea-Lys-HBED-CC, both labeled with .sup.68Ga, in terms of their specific cell surface binding and internalization properties on LNCaP cells. Specific cell uptake was determined by blocking with 500 μM 2-PMPA. Values are expressed as % of applied radioactivity bound to 10.sup.6 cells. Data are expressed as mean±SD (n=3).

(7) FIG. 5 depicts the organ distribution at one hour post injection of 0.06 nmol of the PSMA inhibitor Glu-urea-Lys either with [.sup.68Ga]HBED-CC or [.sup.68Ga]HBED-CC-fluorescein. Data are expressed as mean % ID/g tissue±SD (n=3).

(8) FIG. 6 shows the synthesis of Glu-urea-Lys-HBED-CC-PEG.sub.2-NH.sub.2 as precursor for the conjugation of various fluorescent dyes. Herein a. indicates triphosgene, DIPEA, CH.sub.2Cl.sub.2, 0° C.; b. indicates H-Lys(Alloc)-2CT-Resin, CH.sub.2Cl.sub.2; c. indicates Pd[P(C.sub.6H.sub.5).sub.3].sub.4, morpholine, CH.sub.2Cl.sub.2; d. indicates hexafluoroisopropanol/CH.sub.2Cl.sub.2; e. indicates (HBED-CC)TFP.sub.2, DIPEA, DMF; and f. indicates 1,8-Diamino-3,6-Dioxaoctane.

(9) FIG. 7 shows the synthesis of Glu-urea-Lys-HBED-CC-PEG.sub.2-Fluorescein. Herein, g. indicates fluorescein isothiocyanate (isomer I), DIPEA, DMF; and h. indicates TFA.

(10) FIG. 8 shows the comparison of Glu-urea-Lys-HBED-CC-Fluorescein with the references Glu-urea-Lys-HBED-CC, both labeled with .sup.68Ga, in terms of their specific cell surface binding and internalization properties on LNCaP cells. Specific cell uptake was determined by blocking with 500 μM 2-PMPA. Values are expressed as % of applied radioactivity bound to 10.sup.6 cells. Data are expressed as mean±SD (n=3).

(11) FIG. 9 shows the organ distribution at one hour post injection of 0.06 nmol of the PSMA inhibitor Glu-urea-Lys either with [.sup.68Ga]HBED-CC or [.sup.68Ga]HBED-CC-fluorescein. Data are expressed as mean % ID/g tissue±SD (n=3).

(12) FIG. 10 shows PET imaging experiments in LNCaP tumor bearing nude mice 1 hour post injection of 10 MBq of the shown .sup.68Ga labeled compounds. If aromatic groups in the linker part of the molecule are completely missing, the tumor cannot be visualized. The arrow and the circle in the center of the dashed white cross indicate the tumor. In the HBED-CC structure, the tumor is particularly well visible.

(13) FIG. 11 shows comparative PET-imaging of .sup.68Ga-labeled PSMA-HBED-CC conjugates. The PET-imaging shows comparable distribution of radioactivity and tumor uptake for PSMA-Ahx-HBED-CC, PSMA-HBED-CC-FITC, and PSMA-Ahx-HBED-CC-cyanine 5.5 1 hour post injection in LNCaP tumor-bearing nude mice.

(14) FIG. 12 shows the comparative organ distribution at 1 hour post injection of a reference compound published by Banerjee et al. (Banerjee et al., 2011, Angew Chem Int Ed Engl. 50(39):9167-9170, page 6, scheme 1, final product) and the corresponding compound without the fluorescent dye IRDye800CW bearing a free amino group of the lysyl moiety the dye IRDye800CW can be bound to and PSMA-Ahx-HBED-CC-FITC. As can be seen, the tumor uptake was significantly improved using PSMA-Ahx-HBED-CC-FITC.

EXAMPLES

Example 1

(15) Materials and Methods

(16) Analysis of the synthesized molecules was performed using reversed-phase high performance liquid chromatography (RP-HPLC; Chromolith RP-18e, 100×4.6 mm; Merck, Darmstadt, Germany) with a linear A-B gradient (0% B to 100% B in 6 min) at a flow rate of 4 mL/min (analysis) or 6 mL/min (purification). Solvent A consisted of 0.1% aqueous TFA and solvent B was 0.1% TFA in CH.sub.3CN. The HPLC system (L6200 A; Merck-Hitachi, Darmstadt, Germany) was equipped with a UV and a gamma detector (Bioscan; Washington, USA). UV absorbance was measured at 214 nm, respectively. Mass spectrometry was performed with a MALDI-MS Daltonics Microflex system (Bruker Daltonics, Bremen, Germany). .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 pyrogallol resin support (Schuhmacher et al. 1981).

(17) Synthesis

(18) An exemplarily synthesis protocol for producing a double-labeled probe for molecular imaging is exemplified in FIG. 3.

(19) To synthesize the pharmacophore Glu-urea-Lys, the isocyanate of the glutamyl moiety (indicated as 1 in FIG. 3) was generated in situ by adding a mixture of 3 mmol of bis(tert-butyl) L-glutamate hydrochloride (Bachem, Switzerland) and 1.5 mL of N-ethyldiisopropylamine (DIPEA) in 200 mL of dry CH.sub.2Cl.sub.2 to a solution of 1 mmol triphosgene in 10 mL of dry CH.sub.2Cl.sub.2 at 0° C. over 4 h. After agitation of the reaction mixture for one further hour at 25° C., 0.5 mmol of a resin-immobilized (2-chloro-tritylresin, Merck, Darmstadt) ε-allyloxycarbonyl protected lysine was added in 4 mL DCM and reacted for 16 h with gentle agitation leading to compound indicated as 3 in FIG. 3. The resin was filtered off and the allyloxy-protecting group was removed using 100 mg tetrakis(triphenyl)palladium(0) (Sigma-Aldrich, Germany) and 400 μL morpholine in 4 mL CH.sub.2Cl.sub.2 for 3 hours resulting in the compound indicated as 4 in FIG. 3. Compound 4 was cleaved from the resin by reacting with 4 mL of a 30% 1,1,1-3,3,3-hexafluoroisopropanole (HFIP) in CH.sub.2Cl.sub.2 for two hours at ambient temperature resulting in the tert-butyl protected crude product 5 which was purified via RP-HPLC.

(20) The bis-activated ester (HBED-CC)TFP.sub.2 was synthesized as previously described (Schafer et al. 2012). The precursor for the conjugation of the Dye was synthesized by reacting 66 mg (0.08 mmol) of the bis-activated ester (HBED-CC)TFP.sub.2 with 39 mg (0.072 mmol) of the TFA salt of bis(tert.butyl)Glu-urea-Lys (5) in 1 ml of dry DMF and 25 μl of DIPEA at room temperature. After 4 hours 75 μL of 1,8-Diamino-3,6-Dioxaoctane (0.52 mmol) were added and the reaction was carried out at room temperature for 16 hours. After evaporation of the solvent, the crude product indicated as 6 in FIG. 3 was purified via RP-HPLC (Gradient: 10% CH.sub.3CN to 40% CH.sub.3CN in 10.5 min, Flow 6 ml/min; Detection at 214 nm). (yield: 32 mg; 34%). (Calc. 1076.24; Found: 1077.2 (M+H.sup.+)).

(21) Conjugation of fluorescein was performed by reacting 6 mg of the HBED-CC conjugate indicated as 6 in FIG. 3 (0.005 mmol) with 2.3 mg (0.006 mmol) Fluorescein isothiocyanate(isomer I) in 1 mL of dry DMF supplemented with 15 μL DIPEA at room temperature for 16 hours. After evaporation of the solvent, the product 7 was isolated via RP-HPLC (Gradient: 15% CH.sub.3CN to 51% CH.sub.3CN in 9.2 min, Flow 6 mL/min; Detection at 214 nm). (yield: 4.2 mg; 57%): (Calc. 1465.62; Found: 1466.4 (M+H.sup.+)). The cleavage of the remaining protecting groups was done by using TFA. (Calc. 1353.4; Found: 1354.3 (M+H.sup.+)).

(22) .sup.68Ga-Labelling

(23) The conjugates (0.1-1 nmol in 0.1 M HEPES buffer, pH=7.5, 100 μL) were added to a mixture of 10 μL HEPES solution (2.1 M) and 40 μL [.sup.68Ga]Ga.sup.3+ eluate (25-60 MBq). The pH of the labelling solution was adjusted to 4.2 using 30% NaOH. The reaction mixture was incubated at 80° C. for 2 minutes. The radiochemical yield (RCY) was determined via analytical RP-HPLC.

(24) Cell Culture

(25) For binding studies and in vivo experiments LNCaP cells (metastatic lesion of human prostatic adenocarcinoma, ATCC CRL-1740) were cultured in RPMI medium supplemented with 10% fetal calf serum and Glutamax (PAA, Austria). During cell culture, cells were grown at 37° C. in an incubator with humidified air, equilibrated with 5% CO.sub.2. The cells were harvested using trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA; 0.25% trypsin, 0.02% EDTA, all from PAA, Austria) and washed with PBS.

(26) Cell Binding and Internalization

(27) The competitive cell binding assay and internalization experiments were performed as described previously (Eder et al. 2012). Briefly, the respective cells (10.sup.5 per well) were incubated with the radiometal (.sup.68Ga-labeled [Glu-urea-Lys(Ahx)].sub.2-HBED-CC (Schafer et al. 2012)) in the presence of 12 different concentrations of analyte (0-5000 nM, 100 μL/well). After incubation, washing was carried out using a multiscreen vacuum manifold (Millipore, Billerica, Mass.). Cell-bound radioactivity was measured using a gamma counter (Packard Cobra II, GMI, Minnesota, USA). The 50% inhibitory concentration (IC50) was calculated by fitting the data using a nonlinear regression algorithm (GraphPad Software). Experiments were performed three times.

(28) To determine the specific cell uptake and internalization, 10.sup.5 cells were seeded in poly-L-lysine coated 24-well cell culture plates 24 h before incubation. After washing, the cells were incubated with 25 nM of the radiolabeled compounds for 45 min at 37° C. and at 4° C., respectively. Specific cellular uptake was determined by competitive blocking with 2-(phosphonomethyl)pentanedioic acid (500 μM final concentration, PMPA, Axxora, Loerrach, Germany). Cellular uptake was terminated by washing 4 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 remove the surface-bound fraction. 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 per cent of the initially added radioactivity bound to 10.sup.6 cells [% ID/10.sup.6 cells].

(29) Biodistribution

(30) 7- to 8-week-old male BALB/c nu/nu mice (Charles River Laboratories) were subcutaneously inoculated into the right trunk with 5×10.sup.6 cells of LNCaP (in 50% Matrigel; Becton Dickinson, Heidelberg, Germany). The tumors were allowed to grow until approximately 1 cm.sup.3 in size. The radiolabeled compounds were injected into the tail vein (approx. 1 MBq per mouse; 0.06 nmol). 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.

(31) Results

(32) In Vitro Cell Binding Properties

(33) An in vitro competitive cell binding assay was performed in order to determine the binding potential expressed as IC.sub.50-values of Glu-urea-Lys-HBED-CC-Fluorescein in comparison to the reference Glu-urea-Lys(Ahx)-HBED-CC. The IC.sub.50 values of Glu-urea-Lys-HBED-CC-Fluorescein and Glu-urea-Lys(Ahx)-HBED-CC were 11.14±1.16 nM and 9.82±1.26 nM, respectively, indicating that the PSMA specificity was not affected by the conjugation of fluorescein.

(34) The functionality of the dual-imaging agent Glu-urea-Lys-HBED-CC-fluorescein was additionally investigated on cellular basis by analyzing the internalization and cell surface binding properties (FIG. 4). Glu-urea-Lys-HBED-CC-fluorescein was specifically internalized by LNCaP cells shown by competitive blocking with the PSMA inihibor 2-PMPA (P<0.001). The cell uptake and internalization profile was not considerably changed in the course of the chemical combination of Glu-urea-Lys-HBED-CC and Fluorescein, indicating that the dye on this position has no influence on the cell binding properties of the PSMA-binding molecule.

(35) Organ Distribution

(36) In order to demonstrate the functionality of the molecule in vivo, organ distribution studies with tumor bearing xenografts were performed. FIG. 5 and Table I show that the tumor uptake of the dye-conjugate Glu-urea-Lys-HBED-CC-fluorescein in PSMA positive LNCaP tumors (10.86±0.94% ID/g) was higher compared to the non-conjugated reference Glu-urea-Lys(Ahx)-HBED-CC (4.89±1.34% ID/g). Furthermore, the distribution profiles of both compounds, Glu-urea-Lys-HBED-CC-fluorescein and Glu-urea-Lys(Ahx)-HBED-CC, in healthy organs were comparable indicating similar background activity of both compounds in vivo. Thus, the in vivo tumor-targeting properties of Glu-urea-Lys-HBED-CC-fluorescein are at least comparable to the reference Glu-urea-Lys(Ahx)-HBED-CC.

(37) TABLE-US-00001 TABLE I Organ distribution of the labeled probes at 1 h post injection. Glu-urea-Lys- Glu-urea-Lys- HBED-CC HBED-CC-dye Mean SD N Mean SD N Blood 0.53 0.04 3 1.34 0.40 3 Heart 0.83 0.08 3 2.22 0.68 3 Lung 2.36 0.27 3 3.09 1.07 3 Spleen 17.88 2.87 3 45.24 13.48 3 Liver 1.43 0.19 3 1.71 0.54 3 Kidney 139.44 21.40 3 138.18 39.08 3 Muscle 1.00 0.24 3 1.84 1.06 3 Intestine 1.14 0.46 3 0.81 0.23 3 Brain 0.40 0.19 3 0.31 0.22 3 Tumor 4.89 1.34 3 10.86 0.94 3

(38) Discussion

(39) As the cell binding properties were not affected by the conjugation of the dye, Glu-urea-Lys-HBED-CC-dye conjugates as exemplified might represent a tool to follow the intracellular distribution of .sup.68Ga-labeled Glu-urea-Lys(Ahx)-HBED-CC. An organ distribution study showed that the absolute tumor uptake and the tumor-to-background ratios were at least comparable to non-conjugated Glu-urea-Lys(Ahx)-HBED-CC which has recently provided promising results as a novel clinical PET-tracer for the diagnosis of recurrent prostate cancer (Afshar-Oromieh et al. 2013, Afshar-Oromieh et al. 2014). Consequently, conjugated to clinical relevant dyes this tracer may serve as a multimodal imaging agent offering staging by PET imaging on the one hand and fluorescence signals such as, e.g., intraoperative fluorescence signals, on the other hand which might help to distinguish between neoplasia and healthy tissue during surgery.

Example 2

(40) Syntheses of the Preferred Precursor

(41) To synthesize the pharmacophore Glu-urea-Lys, the isocyanate of the glutamyl moiety 1 was generated in situ by adding a mixture of 3 mmol of bis(tert-butyl) L-glutamate hydrochloride (Bachem, Switzerland) and 1.5 mL of N-ethyldiisopropylamine (DIPEA) in 200 mL of dry CH.sub.2Cl.sub.2 to a solution of 1 mmol triphosgene in 10 mL of dry CH.sub.2Cl.sub.2 at 0° C. over 4 h. After agitation of the reaction mixture for one further hour at 25° C., 0.5 mmol of a resin-immobilized (2-chloro-tritylresin, Merck, Darmstadt) ε-allyloxycarbonyl protected lysine was added in 4 mL DCM and reacted for 16 h with gentle agitation leading to compound 3. The resin was filtered off and the allyloxy-protecting group was removed using 100 mg tetrakis(triphenyl)palladium(0) (Sigma-Aldrich, Germany) and 400 μL morpholine in 4 mL CH.sub.2Cl.sub.2 for 3 hours resulting in 4. Compound 4 was cleaved from the resin by reacting with 4 mL of a 30% 1,1,1-3,3,3-hexafluoroisopropanole (HFIP) in CH.sub.2Cl.sub.2 for two hours at ambient temperature resulting in the tert-butyl protected crude product 5 which was purified via RP-HPLC.

(42) The bis-activated ester (HBED-CC)TFP.sub.2 was synthesized as previously described (Schäfer et al., 2012). The precursor for the conjugation of the Dye was synthesized by reacting 66 mg (0.08 mmol) of the bis-activated ester (HBED-CC)TFP.sub.2 with 39 mg (0.072 mmol) of the TFA salt of bis(tert.butyl)Glu-urea-Lys (5) in 1 ml of dry DMF and 25 μl of DIPEA at room temperature. After 4 hours 75 μL of 1,8-Diamino-3,6-Dioxaoctane (0.52 mmol) were added and the reaction was carried out at room temperature for 16 hours. After evaporation of the solvent, the crude product 6 was purified via RP-HPLC (Gradient: 10% CH.sub.3CN to 40% CH.sub.3CN in 10.5 min, Flow 6 ml/min; Detection at 214 nm). (yield: 32 mg; 34%). (Calc. 1076.24; Found: 1077.2 (M+H.sup.+))

(43) This procedure is further depicted in FIG. 6.

(44) In order to introduce the most preferred aminohexanoic acid spacer between the binding motif and the chelator HBED-CC compound 4 is reacted with 2 mmol of the Fmoc-protected 6-amino-hexanoic acid (Sigma-Aldrich, Germany), 1.96 mmol of HBTU (Merck, Darmstadt, Germany), and 2 mmol of N-ethyl-diisopropylamine in a final volume of 4 mL DMF.

(45) Example PSMA-HBED-CC-FITC

(46) Conjugation of fluorescein was performed by reacting 6 mg of the HBED-CC conjugate 6 (0.005 mmol) with 2.3 mg (0.006 mmol) Fluorescein isothiocyanate (isomer I) in 1 mL of dry DMF supplemented with 15 μL DIPEA at room temperature for 16 hours. After evaporation of the solvent, the product 7 was isolated via RP-HPLC (Gradient: 15% CH.sub.3CN to 51% CH.sub.3CN in 9.2 min, Flow 6 mL/min; Detection at 214 nm). (yield: 4.2 mg; 57%): (Calc. 1465.62 Found: 1466.4 (M+H.sup.+)). The cleavage of the remaining protecting groups was done by using TFA. (Calc. 1353.4; Found: 1354.3 (M+H.sup.+)). This is further depicted in FIG. 7.

(47) Analogously, other fluorescent dyes such as, e.g., Alexa488, Cy5.5, sulfoCy5, ATTO647N, ICG and IRdye800CW were conjugated. Then, a corresponding activated form of the respective fluorescent dye is used instead of FITC. Analogously also rhodamine type dyes such as those shown by Kolmakov et al. (cf., Kolamkov et al., 2012; Kolmakov et al., 2014), such as e.g., KK114 or Abberior Star 635P shown therein, are conjugated.

(48) Examples for the structures obtainable thereby are the following:

(49) ##STR00023## ##STR00024##

(50) wherein X.sup.− is a pharmaceutically acceptable negatively charged counterion; and

(51) wherein Y.sup.+ is a pharmaceutically acceptable positively charged counterion.

(52) Herein the respective counterion depends on the used surrounding liquids such as those comprised in the buffer the compound is dissolved in and the body fluids after injection in vivo. In vivo, extracellularly, one of the main, but not sole positively charged counterions is Na.sup.+ and one of the main, but not sole negatively charged counterions is Cl.sup.−.

(53) .sup.68Ga-Labelling

(54) The conjugates (0.1-1 nmol in 0.1 M HEPES buffer, pH=7.5, 100 μL) were added to a mixture of 10 μL HEPES solution (2.1 M) and 40 μL [.sup.68Ga]Ga.sup.3+ eluate (25-60 MBq). The pH of the labelling solution was adjusted to 4.2 using 30% NaOH. The reaction mixture was incubated at 80° C. for 2 minutes. The radiochemical yield (RCY) was determined via analytical RP-HPLC.

(55) Cell Culture

(56) For binding studies and in vivo experiments LNCaP cells (metastatic lesion of human prostatic adenocarcinoma, ATCC CRL-1740) were cultured in RPMI medium supplemented with 10% fetal calf serum and Glutamax (PAA, Austria). During cell culture, cells were grown at 37° C. in an incubator with humidified air, equilibrated with 5% CO.sub.2. The cells were harvested using trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA; 0.25% trypsin, 0.02% EDTA, all from PAA, Austria) and washed with PBS.

(57) Cell Binding and Internalization

(58) The competitive cell binding assay and internalization experiments were performed as described previously (Eder et al., 2012). Briefly, the respective cells (10.sup.5 per well) were incubated with the radioligand (.sup.68Ga-labeled [Glu-urea-Lys(Ahx)].sub.2-HBED-CC (Schäfer et al., 2012)) in the presence of 12 different concentrations of analyte (0-5000 nM, 100 μL/well). After incubation, washing was carried out using a multiscreen vacuum manifold (Millipore, Billerica, Mass.). Cell-bound radioactivity was measured using a gamma counter (Packard Cobra II, GMI, Minnesota, USA). The 50% inhibitory concentration (IC50) was calculated by fitting the data using a nonlinear regression algorithm (GraphPad Software). Experiments were performed three times.

(59) To determine the specific cell uptake and internalization, 10.sup.5 cells were seeded in poly-L-lysine coated 24-well cell culture plates 24 h before incubation. After washing, the cells were incubated with 25 nM of the radiolabeled compounds for 45 min at 37° C. and at 4° C., respectively. Specific cellular uptake was determined by competitive blocking with 2-(phosphonomethyl)pentanedioic acid (500 μM final concentration, PMPA, Axxora, Loerrach, Germany). Cellular uptake was terminated by washing 4 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 remove the surface-bound fraction. 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 per cent of the initially added radioactivity bound to 10.sup.6 cells [% ID/10.sup.6 cells].

(60) Biodistribution

(61) 7- to 8-week-old male BALB/c nu/nu mice (Charles River Laboratories) were subcutaneously inoculated into the right trunk with 5×10.sup.6 cells of LNCaP (in 50% Matrigel; Becton Dickinson, Heidelberg, Germany). The tumors were allowed to grow until approximately 1 cm.sup.3 in size. The radiolabeled compounds were injected into the tail vein (approx. 1 MBq per mouse; 0.06 nmol). 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.

(62) Results

(63) PSMA-HBED-CC-FITC

(64) In Vitro Cell Binding Properties

(65) An in vitro competitive cell binding assay was performed in order to determine the binding potential expressed as IC.sub.50-values of Glu-urea-Lys-HBED-CC-Fluorescein in comparison to the reference Glu-urea-Lys(Ahx)-HBED-CC. The IC.sub.50 values of Glu-urea-Lys-HBED-CC-Fluorescein and Glu-urea-Lys(Ahx)-HBED-CC were 11.14±1.16 nM and 9.82±1.26 nM, respectively, indicating that the PSMA specificity was not affected by the conjugation of fluorescein.

(66) The functionality of the dual-imaging agent Glu-urea-Lys-HBED-CC-fluorescein was additionally investigated on cellular basis by analyzing the internalization and cell surface binding properties (FIG. 8). Glu-urea-Lys-HBED-CC-fluorescein was specifically internalized by LNCaP cells shown by competitive blocking with the PSMA inihibor 2-PMPA (P<0.001). The cell uptake and internalization profile was not considerably changed in the course of the chemical combination of Glu-urea-Lys-HBED-CC and Fluorescein, indicating that the dye on this position has no influence on the cell binding properties of the PSMA-binding molecule.

(67) Organ Distribution

(68) In order to demonstrate the functionality of the molecule in vivo, organ distribution studies with tumor bearing xenografts were performed. FIG. 9 and Table II show that the tumor uptake of the dye-conjugate Glu-urea-Lys-HBED-CC-fluorescein in PSMA positive LNCaP tumors (10.86±0.94% ID/g) was higher compared to the non-conjugated reference Glu-urea-Lys(Ahx)-HBED-CC (4.89±1.34% ID/g). Furthermore, the distribution profiles of both compounds, Glu-urea-Lys-HBED-CC-fluorescein and Glu-urea-Lys(Ahx)-HBED-CC, in healthy organs were comparable indicating similar background activity of both compounds in vivo. Thus, the in vivo tumor-targeting properties of Glu-urea-Lys-HBED-CC-fluorescein are at least comparable to the reference Glu-urea-Lys(Ahx)-HBED-CC.

(69) TABLE-US-00002 TABLE II Organ distribution data 1 h post injection Glu-urea-Lys- Glu-urea-Lys- HBED-CC HBED-CC-dye Mean SD N Mean SD N Blood 0.53 0.04 3 1.34 0.40 3 Heart 0.83 0.08 3 2.22 0.68 3 Lung 2.36 0.27 3 3.09 1.07 3 Spleen 17.88 2.87 3 45.24 13.48 3 Liver 1.43 0.19 3 1.71 0.54 3 Kidney 139.44 21.40 3 138.18 39.08 3 Muscle 1.00 0.24 3 1.84 1.06 3 Intestine 1.14 0.46 3 0.81 0.23 3 Brain 0.40 0.19 3 0.31 0.22 3 Tumor 4.89 1.34 3 10.86 0.94 3

(70) Discussion

(71) As the cell binding properties were not affected by the conjugation of the fluorescent dye, Glu-urea-Lys-HBED-CC-fluorescein might represent a tool to follow the intracellular distribution of .sup.68Ga-labeled Glu-urea-Lys(Ahx)-HBED-CC. An organ distribution study showed that the absolute tumor uptake and the tumor-to-background ratios were at least comparable to non-conjugated Glu-urea-Lys(Ahx)-HBED-CC which has recently shown promising results as a novel clinical PET-tracer for the diagnosis of recurrent prostate cancer (Afshar-Oromieh et al., 2013, Afshar-Oromieh et al., 2014). Consequently, conjugated to clinical relevant dyes this tracer might serve as a multimodal imaging agent offering staging by PET imaging on the one hand and intraoperative fluorescence signals on the other hand which might help to distinguish between prostate cancer and healthy tissue during surgery.

Example 3

(72) It was found that the chelator HBED-CC (N,N′-bis-[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N′-diacetic acid), represents an acyclic complexing agent especially allowing efficient radiolabeling with .sup.68Ga even at ambient temperature (Eder et al., 2010; Eder et al., 2008). It was found that combining HBED-CC with the PSMA inhibitor Glu-urea-Lys, a favorable aromatic part is introduced into the radiotracer which was found to be beneficial for a sustainable interaction with the PSMA receptor, putatively with the accessory hydrophobic pocket of the PSMA binding site (Eder et al., 2012; Kularatne et al., 2009; Liu et al., 2008). Indeed, it has been shown in a preclinical study that the replacement of HBED-CC by DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid) resulted in a molecule not able to image the tumor at all (Eder et al., 2012). This is depicted in FIG. 10, wherein the tumor (in an LNCaP tumor model in balb/c nu/nu nude mice) is indicated by the arrow. Therefore, HBED-CC conjugates are particularly beneficial.

Example 4

(73) The .sup.68Ga-labeled compounds PSMA-HBED-CC-FITC (the compound depicted above), PSMA-Ahx-HBED-CC-cyanine 5.5 (the compound depicted above, of which the mass was determined by mass spectrometry as being M(calculated)=1642.98; M(found)=1642.7) and PSMA-Ahx-HBED-CC (the corresponding compound without cyanine 5.5) and were injected into an LNCaP tumor-bearing nude mice. One hour post injection, μPET imaging was performed.

(74) It could be shown that all three compounds showed comparable distribution of radioactivity and tumor uptake (cf., FIG. 11). Therefore, it could be demonstrated that the conjugation of the fluorescent dyes and insertion of a spacer do not significantly influence the distribution of radioactivity and tumor uptake of PSMA-HBED-CC-conjugates.

Example 5

(75) PSMA-Ahx-HBED-CC-FITC (as shown above) was compared with a reference compound published by Banerjee et al. (Banerjee et al., 2011, Angew Chem Int Ed Engl. 50(39):9167-9170, page 6, scheme 1, final product) and the corresponding compound without the fluorescent dye IRDye800CW bearing a free amino group of the lysyl moiety the dye IRDye800CW can be bound to. These compounds were injected into LNCaP tumor-bearing nude mice.

(76) It could be shown that the tumor uptake was significantly improved using PSMA-Ahx-HBED-CC-FITC (cf., FIG. 12). The absence of the fluorescent dye of the reference compound published by Banerjee et al. resulted in a significantly reduced kidney, spleen (both organs express PSMA), and tumor uptake of 0.71±0.03% ID/g (cf., FIG. 12).

(77) Thus, this organ distribution study confirms that using branched compounds bears significant disadvantages such as diminished binding to the target structure unless these compounds such as the reference compound published by Banerjee et al. are not combined with the particular fluorophore structures such as IRDye800CW. Therefore, the structures known from Banerjee et al. are not usable in a modular manner. In particular, the dyes conjugated therewith are not freely selectable and several fluorophors regularly and preferably used in the art are not usable with the branched compounds as those shown by Banerjee et al. (Banerjee et al. (2011) and WO 2010/108125, in particularly not when the chelator is DOTA.