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 compound or a pharmaceutically acceptable salt thereof of formula (I): (A)-x.sub.1-(B)-x.sub.2-(C), 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 covalently connecting (A) and (B); x.sub.2 is a spacer or a chemical single bond connecting (B) and (C); wherein (C) has the formula

##STR00001##

wherein R.sup.1 to R.sup.4, R.sup.9, a, b, Y and X.sup.1 to X.sup.4 have the meaning as indicated in the claims and description. 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.

Claims

1.-29. (canceled)

30. A method for diagnosing a neoplasm in a patient suffering therefrom or being at risk thereof, comprising administering sufficient amounts of a composition, the composition comprising: (a) a compound or a pharmaceutically acceptable salt thereof; (b) a radiometal; and optionally (c) one or more pharmaceutically acceptable carriers, wherein the compound or a pharmaceutically acceptable salt thereof is represented by formula (I):
(A)-x.sub.1-(B)-x.sub.2-(C)  (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 covalently connecting (A) and (B); X.sub.2 is a spacer or a chemical single bond connecting (B) and (C); wherein the dye moiety (C) has the formula ##STR00031## 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 connecting the two moieties of (C) and permitting electron delocalization between said moieties, 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), where the ring 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 (B) 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 x.sub.1 comprises a group -AA-, wherein AA is an amino acid sequence of 4 to 8 naturally occurring amino acids and wherein at least two amino acids are histidine and wherein any remaining positive or negative charge or charges are compensated by pharmaceutically acceptable negatively or positively charged counterion or counterions.

31. The method of claim 30, wherein said compound has a molecular weight of not more than 10 kDa.

32. The method of claim 30, wherein the motif specifically binding to cell membranes of neoplastic cells (A) is a motif specifically binding to cell membranes of cancerous cells.

33. The method according to claim 32, wherein the motif specifically binding to cell membranes of neoplastic cells (A) is a PSMA binding motif having the following structure: ##STR00032## 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 chelator moiety of radiometals (B), conjugated via a spacer molecule x.sub.1.

34. The method of claim 30, wherein the motif specifically binding to cell membranes of neoplastic cells (A) is a PSMA binding motif having the following structure: ##STR00033## wherein the wavy line indicates the conjugation site to the chelator moiety of radiometals (B), conjugated via a spacer molecule x.sub.1.

35. The method of claim 30, wherein AA comprises three histidine amino acids.

36. The method of claim 30, wherein AA consists of histidine and glutamic acid.

37. The method of claim 30, wherein AA is represented by the formula -His-Glu-His-Glu-His-Glu-.

38. The method of claim 30, wherein the C-terminus or the N-terminus of AA forms an amide bond with (A).

39. The method according to claim 30, wherein the spacer x.sub.1 bears the following structure:
-AA-[b″-e-b′″].sub.n-b″″-d.sup.1-, wherein b″ is —C(O)— or —N(H)—; wherein e represents a residue selected from the group consisting of an C.sub.1-8-alkylene wherein one or more —CH.sub.2— moieties may optionally be replaced by one or more —O—, —S—, —C(O)NH— —C(O)N(C.sub.1-6 alkyl), —C(O)O—, succinimide, triazole; wherein b′″ is selected from the group consisting of —NH—, and —C(O)—; wherein b ″″ is selected from the group consisting of —C(O)—, and —NH—; and wherein b′″ and b″″ or a terminus of AA- and b″″ together form an amide group; wherein d.sup.1 is —[CH.sub.2].sub.p—, wherein p is 1 or 2; and wherein n is 0 or 1.

40. The method of claim 30, wherein (A)-x.sub.1- is represented by the following structure: ##STR00034##

41. The method according to claim 30, wherein the chelator moiety of radiometals (B) is selected from the group consisting of: ##STR00035## ##STR00036## wherein in each of the chelator moieties one of the wavy lines indicates the conjugation site to the at least one motif specifically binding to cell membranes of neoplastic cells (A) via a spacer x.sub.1, and the other wavy line indicates the conjugation site to the at least one dye moiety (C).

42. The method according to claim 30, wherein (A)-x.sub.1-(B)-x.sub.2- is represented by the following structure: ##STR00037##

43. The method of claim 30, wherein the dye moiety (C) is selected from the group consisting of the following structures: ##STR00038## ##STR00039## 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.

44. The method of claim 30, wherein in the dye moiety (C) is a fluorescent dye moiety having an emission maximum in the range from 400 nm to 1000 nm.

45. The method of claim 30, wherein said compound has the following chemical structure, wherein the positive charge is compensated by a pharmaceutically acceptable negatively charged counterion: ##STR00040##

46. The method of claim 30, 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.

47. The method of claim 30, wherein the radiometal is .sup.68Ga.

48. The method of claim 30, wherein the cells which are neoplastic or at risk of being neoplastic are cancerous or at risk of being cancerous.

49. The method of claim 30, wherein the diagnosing includes a readout of a radioactive signal occurring from the radiometal and/or a fluorescence signal occurring from the dye moiety, both signals may enable the detection of the localizations and/or size of a neoplasm.

50. The method of claim 30, comprising the steps of (i) administering said composition to a patient; (ii) detecting a radioactive signal of the radiometal.

51. The method of claim 30, further including the step of admixing the composition before the step of administering.

52. The method of claim 30, further comprising: (iii) detecting the dye moiety (C).

Description

EXAMPLES

Abbreviations

PSMA-(HE)3-HBED-CC-IRdye800CW (Synonym: Glu-urea-Lys-(HE).SUB.3.-HBED-CC-PEG.SUB.2.-IRDye800CW)

[0348] ##STR00025##

PSMA-(HE)1-HBED-CC-IRdye800CW (Synonym: Glu-urea-Lys-(HE).SUB.1.-HBED-CC-PEG.SUB.2.-IRDye800CW) (Comparative Example)

[0349] ##STR00026##

PSMA-(WE)1-HBED-CC-IRdye800CW (Synonym: Glu-urea-Lys-(WE).SUB.1.-HBED-CC-PEG.SUB.2.-IRDye800CW) (Comparative Example)

[0350] ##STR00027##

PSMA-HBED-CC-(HE)3-IRDye800CW (Synonym: Glu-urea-Lys-HBED-CC-(HE).SUB.3.-PEG.SUB.2.-IRDye800CW) (Comparative Example)

[0351] ##STR00028##

PSMA-HBED-CC-IRDye800CW (Synonym: Glu-urea-Lys-HBED-CC-PEG.SUB.2.-IRDye800CW) (Comparative Example)

[0352] ##STR00029##

PSMA-HBED-CC (Synonym: PSMA-11, Glu-urea-Lys-(Ahx)-HBED-CC) (Comparative Example)

[0353] ##STR00030##

[0354] Experimental Procedures

[0355] 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).

[0356] 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.

[0357] Glu-urea-Lys-(Ahx)-HBED-CC (PSMA-11) was purchased from ABX, Radeberg, Germany. PSMA-HBED-PEG.sub.2-IRDye800CW was synthesized according to US Patent (Publication number: 20150110715) Double-Labeled Probe for Molecular Imaging and Use Thereof.

Synthesis of Glu-urea-Lys-(HE).SUB.3.-HBED-CC-PEG.SUB.2.-IRDye800CW

Synthesis of Glu-urea-Lys-(HE).SUB.3.-CO(CH.SUB.2.).SUB.4.—N.SUB.3

[0358] The synthesis of the pharmacophore Glu-urea-Lys was performed as described previously (2). 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). The resin was split and the (HE).sub.3 linker was synthesized by standard Fmoc solid phase protocols. For (HE).sub.3-containing molecules the coupling of Fmoc-His(Trt)-OH, Fmoc-Glu(otBu)-OH and 5-azidopentanoic acid (4 eq.) was performed using HBTU (4 eq.) and DIPEA (4 eq.) in DMF. In order to form (HE).sub.3 the coupling of Fmoc-His(Trt)-OH and Fmoc-Glu(otBu)-OH was repeated, respectively. 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) resulting in the azido-functionalized intermediates. All products were purified using RP-HPLC and identified with mass spectrometry. Purification of Glu-urea-Lys-(HE).sub.3-CO—(CH.sub.2).sub.4—N.sub.3 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.

Synthesis of Glu-urea-Lys-(HE).SUB.3.-HBED-CC-PEG.SUB.2

[0359] The HBED-CC(TFP).sub.2 was synthesized as previously described by protection using Fe.sup.3+ and the formation of [Fe(HBED-CC)].sup.− (3). The bis-TFP ester was isolated with preparative HPLC using a NUCLEODUR® Sphinx RP column with a 20 min gradient starting at 10% B, raised to 100% B in 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. The product was identified with mass spectrometry (MW: 828.7).

[0360] [Fe (HBED-CC)] TFP.sub.2 and 0.95 eq of Propargylamine were solved in DMF in the presence of DIPEA. After 4 h at room temperature an excess of 2,2′-(ethylenedioxy)bis(ethylamine) (100 μl) was added and stirred for 16 h at RT. The alkenyl-functionalized chelator was purified via preparative HPLC using a NUCLEODUR® Sphinx RP column with a 20 min gradient starting at 10% B, raised to 100% B. 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 (MW: 699.8).

[0361] Subsequently, PEG.sub.2-[Fe (HBED-CC)]-propargylamine (1 eq.) was reacted with Glu-urea-Lys-(HE).sub.3-CO(CH.sub.2).sub.4—N.sub.3 (1 eq.) via CuAAC, CuSO.sub.4 (1 eq.), Na-Ascorbate (1 eq.), in 3 mL THF/H.sub.2O (1:1, v/v) for 16 h at RT. The Fe-protected product was isolated via preparative HPLC using a NUCLEODUR® Sphinx RP column (0-100% B in 20 min, flow 20 ml/min) and identified with mass spectrometry (MW: 1943.1).

Glu-urea-Lys-(HE).SUB.3.-HBED-CC-PEG.SUB.2.-IRDye800CW

[0362] IRDye800CW-NHS ester (1 eq.) (LI-COR Biosciences) was conjugated to Glu-urea-Lys-(HE).sub.3-[Fe(HBED-CC)]-PEG.sub.2 in PBS-buffer (pH 8.5) for 24 h at RT. The Fe-protected product was isolated via semipreparative HPLC using a Chromolith RP-18e column

[0363] (100×10 mm; Merck, Darmstadt, Germany) (0-100% B in 10 min, flow 5 ml/min) and identified with mass spectrometry (MW: 2929.2).

[0364] Complexed Fe.sup.3+ was removed as described previously (3). Briefly, the Fe-containing product was trapped on a C18 cartridge (Waters SepPak-Classic C18; Waters Corp., Milford, Mass., USA) and was subsequently flushed with 10 mL 1 M HCl and washed with 5 mL H.sub.2O. The remaining product was eluted with 2 mL H.sub.20/CH.sub.3CN (3:1) and evaporated to dryness.

[0365] .sup.68Ga—Labeling

[0366] The precursor peptides [1 nmol in HEPES buffer (580 mg/ml), 90 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.

[0367] Cell Culture

[0368] 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).

[0369] Cell Binding and Internalization

[0370] 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 0.2 nM solution of .sup.68Ga-labeled radioligand [Glu-urea-Lys(Ahx)]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, Minnesota, USA). The 50% inhibitory concentration (IC50) values were calculated by fitting the data using a nonlinear regression algorithm (GraphPad Software).

[0371] 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].

[0372] Biodistribution

[0373] 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.

[0374] Statistical Aspects

[0375] 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.

[0376] Results

[0377] The internalization efficiency and the PSMA binding affinity of the conjugates were determined in order to investigate the influence of the linkers on the binding properties. The results are summarized in Table 1. The binding affinity (Ki) determined on LNCaP cells was slightly reduced from 9.82±1.26 nM PSMA-HBED-CC (PSMA-11) to 17.53±4.98 nM for PSMA-HBED-CC-PEG.sub.2-IRDye800CW and 36.70±9.77 nM for PSMA-(HE).sub.3-HBED-CC-PEG.sub.2-IRDye800CW.

TABLE-US-00002 TABLE 1 Cell binding and internalization data of the investigated conjugates PSMA-HBED-CC-PEG.sub.2-IRDye800CW and PSMA-(HE).sub.3-HBED-CC-PEG.sub.2-IRDye800CW compared to the reference PSMA-HBED-CC (PSMA-11). Specific cell Specific surface bound internalized K.sub.i [nM] [% ID/10.sup.5 cells] [% ID/10.sup.5 cells] PSMA-HBED-CC  9.82 ± 1.26 2.31 ± 0.61 3.57 ± 0.51 (PSMA-11) PSMA-HBED-CC- 17.53 ± 4.98 13.62 ± 4.79  18.70 ± 7.03  PEG.sub.2-IRDye8000W PSMA-(HE).sub.3- 36.70 ± 9.77 8.30 ± 3.93 6.45 ± 3.36 HBED-CC-PEG.sub.2- IRDye800CW

[0378] The specific cell surface binding and specific internalization of PSMA-HBED-CC-PEG.sub.2-IRDye800CW and PSMA-(HE).sub.3-HBED-CC-PEG.sub.2-IRDye800CW were improved as compared to the reference structure PSMA-11.

[0379] The higher PSMA-specific internalization of the conjugates observed in these assays resulted in a significant higher tumor accumulation of 13.66±3.73% ID/g (PSMA-HBED-CC-PEG.sub.2-IRDye800CW) and 7.59±0.95% ID/g (PSMA-(HE).sub.3-HBED-CC-PEG.sub.2-IRDye800CW) as compared to the reference (4.89±1.34% ID/g (PSMA-11); P<0.05) shown by organ distribution using LNCaP-tumor bearing nude mice (Table 2). PSMA-(HE).sub.3-HBED-CC-PEG.sub.2-IRDye800CW showed a lower uptake in all background organs as compared to PSMA-HBED-CC-PEG.sub.2-IRDye800CW. Through the introduction of (HE).sub.3 the spleen uptake was extremely reduced from 38.12±14.62% ID/g (PSMA-HBED-CC-PEG.sub.2-IRDye800CW) to 3.47±1.39% ID/g (PSMA-(HE).sub.3-HBED-CC-PEG.sub.2-IRDye800CW).

TABLE-US-00003 TABLE 2 Organ distribution of .sup.68Ga labeled PSMA-11, PSMA-HBED-CC-PEG.sub.2- IRDye800CW and PSMA-(HE).sub.3-HBED-CC-PEG.sub.2-IRDye800CW in LNCaP-tumor bearing balb/c nu/nu mice 1 h p.i. (n = 3). PSMA-(HE).sub.3-HBED- PSMA-HBED-CC-PEG.sub.2- CC-PEG.sub.2- PSMA-11 IRDye800CW IRDye800CW Mean SD Mean SD Mean SD Blood 0.53 0.04 3.04 1.36 1.12 0.64 Heart 0.83 0.08 2.78 0.83 1.36 0.37 Lung 2.36 0.27 5.60 1.79 2.34 0.37 Spleen 17.88 2.87 38.12 14.62 3.47 1.39 Liver 1.43 0.19 2.76 0.51 1.25 0.18 Kidney 139.44 21.40 204.98 56.24 109.27 10.33 Muscle 1.00 0.24 2.86 0.88 1.18 0.15 Intestine 1.14 0.46 2.41 0.72 1.31 0.00 Brain 0.40 0.19 0.43 0.14 0.18 0.02 Tumor 4.89 1.34 13.66 3.73 7.59 0.95

TABLE-US-00004 TABLE 3 Tumor-to-Organ ratio of .sup.68Ga labeled PSMA-11, PSMA-HBED-CC-PEG.sub.2-IRDye800CW and PSMA-(HE).sub.3-HBED-CC-PEG.sub.2-IRDye800CW in LNCaP-tumor bearing balb/c nu/nu mice 1 h p.i. (n = 3). PSMA-HBED- PSMA-(HE).sub.3- CC-PEG.sub.2- HBED-CC-PEG.sub.2- PSMA-11 IRDye8000W IRDye8000W T/Blood  9.23  4.49  6.80 T/Heart  5.89  4.92  5.57 T/Lung  2.07  2.44  3.24 T/Spleen  0.27  0.36  2.19 T/Liver  3.42  4.94  6.08 T/Kidney  0.04  0.07  0.07 T/Muscle  4.89  4.78  6.45 T/Intestine  4.29  5.66  5.80 T/Brain 12.23 31.39 42.63

[0380] In order to evaluate the influence of the (HE).sub.3 linker introduction on the uptake in background organs, tumor-to-organ ratios were calculated and summarized in Table 3. Compared to PSMA-HBED-CC-PEG.sub.2-IRDye800CW, the linker bearing compound PSMA-(HE).sub.3-HBED-CC-PEG.sub.2-IRDye800CW showed a higher T/O-ratio for all organs except kidney, clearly indicating an improved pharmacokinetic profile.

[0381] Noticeably, localizing the (HE).sub.3 as linker between the binding motif and the chelator resulted in a clear reduction of the spleen signal.

[0382] To compare the influence of the (HE).sub.3 linker introduction to other amino acid linkers at different positions, their uptake in background organs were determined and the tumor-to-organ ratios calculated. The data is summarized in and Table 4.

TABLE-US-00005 TABLE 4a Tablea 4a and 4b. Organ distribution of .sup.68Ga labeled PSMA-11, PSMA-HBED-CC- PEG.sub.2-IRDye800CW, PSMA-(HE).sub.3-HBED-CC-PEG.sub.2-IRDye800CW; PSMA-HBED-CC- (HE).sub.3-PEG.sub.2-IRDye8000CW, PSMA-(HE).sub.1-HBED-CC-PEG.sub.2-IRDyeS800CW and PSMA- (WE).sub.1-HBED-CC-PEG.sub.2-IRDye8800CW in LNCaP-tumor bearing balb/c nu/nu mice 1 h p.i. (n = 3). PSMA-HBED- PSMA-(HE).sub.3- CC-PEG.sub.2- HBED-CC- PSMA-11 IRDye8000W PEG.sub.2-IRDye8000W Mean SD Mean SD Mean SD Blood 0.53 0.04 3.04 1.36 1.12 0.64 Heart 0.83 0.08 2.78 0.83 1.36 0.37 Lung 2.36 0.27 5.60 1.79 2.34 0.37 Spleen 17.88 2.87 38.12 14.62 3.47 1.39 Liver 1.43 0.19 2.76 0.51 1.25 0.18 Kidney 139.44 21.40 204.98 56.24 109.27 10.33 Muscle 1.00 0.24 2.86 0.88 1.18 0.15 Intestine 1.14 0.46 2.41 0.72 1.31 0.00 Brain 0.40 0.19 0.43 0.14 0.18 0.02 Tumor 4.89 1.34 13.66 3.73 7.59 0.95

TABLE-US-00006 TABLE 4b PSMA-(HE).sub.3- PSMA-HBED-CC- PSMA-(HE).sub.1- PSMA-(WE).sub.1- HBED-CC-PEG.sub.2- (HE).sub.3-PEG.sub.2- HBED-CC-PEG.sub.2- HBED-CC-PEG.sub.2- IRDye800CW IRDye800CW IRDye800CW IRDye800CW Mean SD Mean SD Mean SD Mean SD Blood 1.12 0.64 0.80 0.31 1.52 0.64 1.61 0.55 Heart 1.36 0.37 0.57 0.26 0.94 0.14 1.49 0.51 Lung 2.34 0.37 1.78 1.19 2.46 0.46 2.57 1.01 Spleen 3.47 1.39 8.88 2.93 8.17 0.23 12.94 2.00 Liver 1.25 0.18 0.72 0.05 1.19 0.30 1.73 0.61 Kidney 109.27 10.33 92.54 21.07 84.34 2.13 104.58 20.79 Muscle 1.18 0.15 0.97 0.38 1.03 0.34 1.00 0.37 Intestine 1.31 0.00 0.65 0.21 1.29 0.36 0.96 0.37 Brain 0.18 0.02 0.18 0.06 0.30 0.10 0.15 0.04 Tumor 7.59 0.95 3.32 1.51 3.92 0.31 3.85 1.10

[0383] PSMA-(HE).sub.3-HBED-CC-PEG.sub.2-IRDye800CW (7.59±0.95% ID/g) showed a significant higher tumor uptake compared to other linker bearing compounds (PSMA-HBED-CC-(HE).sub.3-PEG.sub.2-IRDye800CW: 3.32±1.51% ID/g, PSMA-(HE).sub.1-HBED-CC-PEG.sub.2-IRDye800CW: 3.92±0.31% ID/g, PSMA-(WE).sub.1-HBED-CC-PEG.sub.2-IRDye800CW: 3.85+1.10% ID/g). The spleen uptake of PSMA-(HE).sub.3-HBED-CC-PEG.sub.2-IRDye800CW is extremely reduced as compared to all other compounds represented in Table 4.

[0384] The tumor-to-organ ratios (Table 5) of for all background organs are the highest for PSMA-(HE).sub.3-HBED-CC-PEG.sub.2-IRDye800CW (except T/heart: 5.57 vs PSMA-HBED-CC-(HE).sub.3-PEG.sub.2-IRDye800CW T/heart: 5.80).

TABLE-US-00007 TABLE 5 Tumor-to-Organ ratio of .sup.68Ga labeled PSMA-11, PSMA-HBED-CC-PEG.sub.2- IRDye800CW, PSMA-(HE).sub.3-HBED-CC-PEG.sub.2-IRDye800CW; PSMA-HBED-CC- (HE).sub.3-PEG.sub.2-IRDye800CW, PSMA-(HE).sub.1-HBED-CC-PEG.sub.2-IRDye800CW and PSMA-(WE).sub.1-HBED-CC-PEG.sub.2-IRDye800CW in LNCaP-tumor bearing balb/c nu/nu mice 1 h p.i. (n = 3). PSMA-(HE).sub.3- PSMA-HBED- PSMA-(HE).sub.1- PSMA-(WE).sub.1- PSMA-HBED- HBED-CC- CC-(HE).sub.3- HBED-CC- HBED-CC- CC-PEG.sub.2- PEG.sub.2- PEG.sub.2- PEG.sub.2- PEG.sub.2- PSMA-11 IRDye800CW IRDye800CW IRDye800CW IRDye800CW IRDye800CW T/Blood 9.23 4.49 6.80 4.12 2.58 2.39 T/Heart 5.89 4.92 5.57 5.80 4.19 2.59 T/Lung 2.07 2.44 3.24 1.86 1.60 1.50 T/Spleen 0.27 0.36 2.19 0.37 0.48 0.30 T/Liver 3.42 4.94 6.08 4.60 3.30 2.23 T/Kidney 0.04 0.07 0.07 0.04 0.05 0.04 T/Muscle 4.89 4.78 6.45 3.43 3.81 3.86 T/Intestine 4.29 5.66 5.80 5.13 3.05 4.01 T/Brain 12.23 31.39 42.63 18.15 13.01 25.56

[0385] To further analyze the pharmacokinetic influence of the (HE).sub.3 linker, additional organ distribution studies at 2 h p.i. with the favourable .sup.68Ga-labeled compound PSMA-(HE).sub.3-HBED-CC-PEG.sub.2-IRDye800CW in comparison to .sup.68Ga labeled PSMA-11 PSMA-HBED-CC-PEG.sub.2-IRDye800CW were performed (Table 6).

TABLE-US-00008 TABLE 6 Organ distribution of .sup.68Ga labeled PSMA-11, PSMA-HBED-CC-PEG.sub.2- IRDye800CVV, PSMA-(HE).sub.3-HBED-CC-PEG.sub.2-IRDye800CW in LNCaP-tumor bearing balb/c nu/nu mice 2 h p.i. (n = 3). PSMA-HBED-CC-PEG.sub.2- PSMA-(HE).sub.3-HBED-CC- PSMA-11 IRDye8000W PEG.sub.2-IRDye8000W Mean SD Mean SD Mean SD Blood 0.32 0.14 0.76 0.23 0.65 0.01 Heart 0.31 0.01 1.00 0.11 0.53 0.08 Lung 1.19 0.11 2.53 0.27 0.89 0.05 Spleen 12.41 0.7 13.73 1.63 0.76 0.16 Liver 0.34 0.05 1.21 0.05 0.76 0.11 Kidney 129.73 13.77 119.56 5.13 55.81 9.76 Muscle 2.06 0.36 1.96 0.57 0.47 0.11 Intestine 1.19 0.35 1.37 0.27 0.67 0.17 Brain 0.23 0.05 0.22 0.01 0.23 0.15 Tumor 6.09 0.71 7.94 0.19 3.10 1.17

[0386] Introduction of (HE).sub.3 between PSMA-binding motif and chelator resulted in rapid clearance from background organs. Especially, kidney and spleen uptake was reduced compared to the other compounds.

REFERENCES

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