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 compound having the following chemical structure: ##STR00035## or a pharmaceutically acceptable salt thereof, wherein the residue (C) is a fluorescent dye moiety.

2. The compound or a pharmaceutically acceptable salt thereof according to claim 1, having the following chemical structure: ##STR00036##

3. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein said fluorescent dye moiety (C) is an infrared fluorescence dye.

4. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein said fluorescent dye moiety (C) is IRDye 800CW.

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

6. The composition according to claim 5, wherein the radiometal or a salt thereof is 68Ga or a salt thereof.

7. The composition according to claim 5, wherein the composition is useful as a diagnostic.

8. The composition according to claim 5, wherein the composition is usable in a method for diagnosing a neoplasm in a patient suffering therefrom.

9. The composition for use according to claim 5, wherein the neoplasia is cancer.

10. A kit comprising: (a) the compound or a pharmaceutically acceptable salt thereof according to claim 1; and (b) a user manual.

11. A kit comprising: (a) the composition according to claim 5; and (b) a user manual.

Description

FIGURES

(1) FIG. 1 depicts the basic principle of the compound according to the present invention. FIG. 1A shows the compound 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. FIG. 1B shows the compound complexing 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.

(2) FIG. 2 exemplarily shows a compound according to the present invention. FIG. 2A shows the compound in its non-complexed form. FIG. 2B shows the compound complexing the radiometal .sup.68Ga. The amino acid moieties (i.e., the glutamate moiety and the lysine moiety) preferably bear the L-configuration.

(3) 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.

(4) 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 M 2-PMPA. Values are expressed as % of applied radioactivity bound to 10.sup.6 cells. Data are expressed as meanSD (n=3).

(5) 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 tissueSD (n=3).

(6) 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.

(7) 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.

(8) 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 M 2-PMPA. Values are expressed as % of applied radioactivity bound to 10.sup.6 cells. Data are expressed as meanSD (n=3).

(9) 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 tissueSD (n=3).

(10) 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.

(11) 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.

(12) 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

Materials and Methods

(13) Analysis of the synthesized molecules was performed using reversed-phase high performance liquid chromatography (RP-HPLC; Chromolith RP-18e, 1004.6 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; .sup.+ 89%; E.sub.+ max. 1.9 MeV) was obtained from a .sup.68Ge/.sup.68Ga generator based on pyrogallol resin support (Schuhmacher et al. 1981).

(14) Synthesis

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

(16) 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) -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 L 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.

(17) 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 l of DIPEA at room temperature. After 4 hours 75 L 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+W)).

(18) 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 L 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+W)). The cleavage of the remaining protecting groups was done by using TFA. (Calc. 1353.4; Found: 1354.3 (M+H.sup.+)).

(19) .sup.68Ga-Labelling

(20) The conjugates (0.1-1 nmol in 0.1 M HEPES buffer, pH=7.5, 100 L) were added to a mixture of 10 L HEPES solution (2.1 M) and 40 L [.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.

(21) Cell Culture

(22) 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).

(23) 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.

(24) Cell Binding and Internalization

(25) 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 L/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.

(26) 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 M 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 percent of the initially added radioactivity bound to 10.sup.6 cells [% ID/10.sup.6 cells].

(27) Biodistribution

(28) 7- to 8-week-old male BALB/c nu/nu mice (Charles River Laboratories) were subcutaneously inoculated into the right trunk with 510.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.

(29) Results

(30) In Vitro Cell Binding Properties

(31) 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 11.141.16 nM and 9.821.26 nM, respectively, indicating that the PSMA specificity was not affected by the conjugation of fluorescein.

(32) 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 inhibitor 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.

(33) Organ Distribution

(34) 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 (10.860.94% ID/g) was higher compared to the non-conjugated reference Glu-urea-Lys(Ahx)-HBED-CC (4.891.34% 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.

(35) 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
Discussion

(36) 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

Syntheses of the Preferred Precursor

(37) 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) -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 L 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.

(38) The bis-activated ester (HBED-CC)TFP.sub.2 was synthesized as previously described (Schfer 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 l of DIPEA at room temperature. After 4 hours 75 L 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.+)).

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

(40) 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.

(41) Example PSMA-HBED-CC-FITC

(42) 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 L 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.

(43) 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.

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

(45) PSMA-Ahx-HBED-CC-FITC:

(46) ##STR00028##
PSMA-Ahx-HBED-CC-Alexa488:

(47) ##STR00029##
PSMA-Ahx-HBED-CC-cyanine 5.5:

(48) ##STR00030##
PSMA-Ahx-HBED-CC-sulfoCy5:

(49) ##STR00031##
PSMA-Ahx-HBED-CC-ATT0647N:

(50) ##STR00032##
PSMA-Ahx-HBED-CC-ICG:

(51) ##STR00033##
PSMA-Ahx-HBED-CC-IRdye800CW:

(52) ##STR00034##
wherein X.sup. is a pharmaceutically acceptable negatively charged counterion; and
wherein Y.sup.+ is a pharmaceutically acceptable positively charged counterion.

(53) 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 Cr.

(54) .sup.68Ga-Labelling

(55) The conjugates (0.1-1 nmol in 0.1 M HEPES buffer, pH=7.5, 100 L) were added to a mixture of 10 L HEPES solution (2.1 M) and 40 L [.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.

(56) Cell Culture

(57) 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 PM, Austria) and washed with PBS.

(58) Cell Binding and Internalization

(59) 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 (Schfer et al., 2012)) in the presence of 12 different concentrations of analyte (0-5000 nM, 100 L/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.

(60) 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 M 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 percent of the initially added radioactivity bound to 10.sup.6 cells [% ID/10.sup.6 cells].

(61) Biodistribution

(62) 7- to 8-week-old male BALB/c nu/nu mice (Charles River Laboratories) were subcutaneously inoculated into the right trunk with 510.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.

(63) Results

(64) PSMA-HBED-CC-FITC

(65) In Vitro Cell Binding Properties

(66) 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 11.141.16 nM and 9.821.26 nM, respectively, indicating that the PSMA specificity was not affected by the conjugation of fluorescein.

(67) 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.

(68) Organ Distribution

(69) 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 (10.860.94% ID/g) was higher compared to the non-conjugated reference Glu-urea-Lys(Ahx)-HBED-CC (4.891.34% 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.

(70) 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
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, WET 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 0.710.03% 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.

REFERENCES

(78) WO 2010/108125 Afshar-Oromieh A, Malcher A, Eder M, Eisenhut M, Linhart H G, Hadaschik B A, Holland-Letz T, Giesel F L, Kratochwil C, Haufe S, Haberkorn U and Zechmann C M (2013a); PET imaging with a [68Ga]gallium-labelled PSMA ligand for the diagnosis of prostate cancer: biodistribution in humans and first evaluation of tumour lesions; Eur J Nuci Med Mol Imaging; 40(4):486-495. Afshar-Oromieh A, Zechmann C M, Malcher A, Eder M, Eisenhut M, Linhart H G, Holland-Letz T, Hadacschik B A, Giesel F L, Debus J, Haberkorn U (2014); Comparison of PET imaging with a Ga-labelled PSMA ligand and F-choline-based PET/CT for the diagnosis of recurrent prostate cancer. Eur J Nucl Med Mol Imaging 41:11-20. Banerjee S R, Pullambhatla M, Byun Y, Nimmagadda S, Foss C, Green G, Fox J J, Lupoid S E, Mease R, Pomper M G (2011); 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. 50(39):9167-9170. Eder M, Wngler B, Knackmuss S, LeGall F, Little M, Haberkorn U, Mier W, Eisenhut M (2008); Tetrafluorophenolate of HBED-CC: a versatile conjugation agent for (68)Ga-labeled small recombinant antibodies. Eur J Nucl Med Mol Imaging 35(10):1878-1886. Eder M, Krivoshein A V, Backer M, Backer J M, Haberkorn U, Eisenhut M (2010); ScVEGF-PEG-HBED-CC and scVEGF-PEG-NOTA conjugates: comparison of easy-to-label recombinant proteins for [68Ga]PET imaging of VEGF receptors in angiogenic vasculature. Nucl Med Biol, 37(4):405-412. Eder M, Schfer M, Bauder-Wst U, Hull W E, Wngler C, Mier W, Haberkorn U and Eisenhut M (2012); .sup.68Ga-complex lipophilicity and the targeting property of a urea-based PSMA inhibitor for PET imaging; Bioconjug Chem. 23(4):688-697. Kolmakov K, Wurm C A, Meineke D N, Gttfert F, Boyarskiy V P, Belov V N, Hell S W (2014); Polar red-emitting rhodamine dyes with reactive groups: synthesis, photophysical properties, and two-color STED nanoscopy applications. Chem. Eur. J. 20:146-157. Kolmakov K, Wurm C A, Hennig R, Rapp E, Jakobs S, Belov V N and Hell S W (2012); Red-emitting rhodamines with hydroxylated, sulfonated, and phosphorylated dye residues and their use in fluorescence nanoscopy. Chem. Eur. J. 18:12986-12998. Kularatne, S A (2009); Design, synthesis, and preclinical evaluation of prostate-specific membrane antigen targeted (99m)Tc-radioimaging agents. Mol Pharm. 6(3):790-800. Liu T, Toriyabe Y, Kazak M, Berkman C E (2008); Pseudoirreversible inhibition of prostate-specific membrane antigen by phosphoramidate peptidomimetics. Biochemistry, 47(48):12658-12660. Schfer M, Bauder-Wst U, Leotta K, Zoller F, Mier W, Haberkorn U, Eisenhut M and Eder M (2012); A dimerized urea-based inhibitor of the prostate-specific membrane antigen for 68Ga-PET imaging of prostate cancer; EJNMMI Res. 2(1):23. Schuhmacher J and Maier-Borst W (1981); A new .sup.68Ge/.sup.68Ga radioisotope generator system for production of .sup.68Ga in dilute HCl; Int J Appl Radiat Isot 32:31-36. Seibold U, Wngler B, Schirrmacher R and Wngler C (2014); Bimodal imaging probes for combined PET and OI: recent developments and future directions for hybrid agent development. Biomed Res Int. 2014:153741. doi: 10.1155/2014/153741.