Synthesis and preclinical evaluation of DOTAGA-conjugated PSMA ligands for functional imaging and endoradiotherapy of prostate cancer
© Weineisen et al.; licensee Springer. 2014
Received: 13 October 2014
Accepted: 6 November 2014
Published: 25 November 2014
Due to its high expression in prostate cancer, PSMA (prostate-specific membrane antigen) represents an ideal target for both diagnostic imaging and endoradiotherapeutic approaches. Based on a previously published highly specific PSMA ligand ([68Ga]DOTA-FFK(Sub-KuE)), we developed a corresponding metabolically stable 1,4,7,10-tetraazacyclododececane,1-(glutaric acid)-4,7,10-triacetic acid (DOTAGA) construct for theranostic treatment of prostate cancer.
All ligands were synthesized by a combined solid phase and solution phase synthesis strategy. The affinity of the natgallium and lutetium complexes to PSMA and the internalization efficiency of the radiotracers were determined on PSMA-expressing LNCaP cells. The 68Ga- and 177Lu-labelled ligands were further investigated for lipophilicity, binding specificity, metabolic stability, as well as biodistribution and μPET in LNCaP-tumour-bearing mice.
Radiochemical yields for 68Ga (3 nmol, 5.0 M NaCl/2.7 M HEPES (approximately 5/1), pH 3.5 to 4.5, 5 min, 95°C) and 177Lu labelling (0.7 nmol, 0.1 M NH4OAc, pH 5.5, 30 min, 95°C) were almost quantitative, resulting in specific activities of 250 to 300 GBq/μmol for the 68Ga analogues and 38 GBq/μmol for 177Lu complexes. Due to metabolic instability of l-amino acid spacers, d-amino acids were implemented resulting in a metabolically stable DOTAGA ligand. Compared to the DOTA ligand, the DOTAGA derivatives showed higher hydrophilicity (logP = -3.6 ± 0.1 and -3.9 ± 0.1 for 68Ga and 177Lu, respectively) and improved affinity to PSMA resulting in an about twofold increased specific internalization of the 68Ga- and 177Lu-labelled DOTAGA analogue. Especially, [68Ga]DOTAGA-ffk(Sub-KuE) exhibits favourable pharmacokinetics, low unspecific uptake and high tumour accumulation in LNCaP-tumour-bearing mice.
The pair of diagnostic/therapeutic PSMA-ligands [68Ga/177Lu]DOTAGA-ffk(Sub-KuE) possess remarkable potential for the management of prostate cancer.
KeywordsProstate cancer Theranostic Endoradiotherapy PET PSMA Prostate-specific membrane antigen PSMA ligand [68Ga]DOTAGA-ffk(Sub-KuE) [177Lu]DOTAGA-ffk(Sub-KuE)
Prostate-specific membrane antigen (PSMA, EC 18.104.22.168, synonym: glutamate carboxypeptidase II) is an extracellular hydrolase whose catalytic centre comprises two zinc(II) ions with a bridging hydroxido ligand . It is highly up-regulated in metastatic and hormone-refractory prostate carcinomas, but its endogenous expression has also been reported in kidneys, salivary glands, small intestine, brain and, to a low extent, also in healthy prostate tissue ,. In the intestine, PSMA facilitates absorption of folate by conversion of pteroylpoly-γ-glutamate to the pteroylglutamate (folate) . In the brain, it hydrolyses N -acetyl-l-aspartyl-l-glutamate (NAAG) to N-acetyl-l-aspartate and glutamate . The enzymatic function of PSMA in normal and diseased prostate has not been clarified yet . However, due to its overexpression on prostate cancer cells, PSMA represents an excellent target for molecular imaging and targeted radiotherapy of prostate cancer.
Recently, it has been demonstrated that the affinity of KuE-derived inhibitors is enhanced by extending the KuE-binding unit by an aromatic moiety, presumably due to an additional inhibitor-enzyme interaction via π/π-stacking of the additional aromatic residues with a remote arene-binding pocket . There is apparently an optimal distance between the KuE-inhibitor component and the additional arene moiety, leading to an enhancement of PSMA affinity (K D = 13.8 nM) by a factor of up to 5 .
Monoclonal anti-PSMA-antibodies labelled with therapeutic radionuclides such as 131I, 90Y and 177Lu, which are currently being evaluated in clinical trials (clinicaltrials.gov Identifier: NCT00006380, NCT00081172) already demonstrate promising tumour targeting, acceptable toxicity and therapeutic efficiency in humans -.
Amongst the currently available small-molecule PSMA inhibitors, only [131I]MIP-1375  and [131I]MIP-1466  (Figure 1) have been evaluated in therapy studies so far. In LNCaP-tumour-bearing nude mice, [131I]MIP-1375 inhibited tumour growth in a time- and dose-dependent manner. First, human applications of [131I]MIP-1095/1466 revealed promising therapy response with moderate side effects . The structurally corresponding diagnostics, 123I-labelled analogues [123I]MIP-1072 and [123I]MIP-1095, were shown to possess suitable characteristics for SPECT imaging. However, these compounds are comparably lipophilic (clogP = 0.20 and 1.97, respectively ), and their compact structure complicates further optimization of PSMA affinity and pharmacokinetics. Furthermore, PET imaging is only possible using 124I, a suboptimal PET-radionuclide with respect to resolution and dosimetry.
In contrast, radiometalated analogues based on the FFK(Sub-KuE)-scaffold represent a much more flexible and finely adjustable backbone for the development of KuE-based PSMA inhibitors, that allow labelling with both diagnostic and therapeutic radionuclides, e.g. 68Ga for PET imaging and 177Lu/90Y for therapy after conjugation of DOTA. However, in order to further facilitate the labelling procedure for 177Lu and 90Y, improve ligand pharmacokinetics, and potentially obtain radiometalated derivatives with higher affinity, we substituted DOTA in DOTA-FFK(Sub-KuE) by 1,4,7,10-tetraazacyclodocecane,1-(glutaric acid)-4,7,10-triacetic acid (DOTAGA) ,. Improved affinities, higher tumour uptake and faster kidney clearance have already been observed for the 68Ga-complex (one free carboxylate) compared to the 90Y-complex (all carboxylates coordinated) of DOTA-coupled somatostatin analogues before .
Consequently, we evaluated and compared the respective 68/natGa- and 177/natLu-DOTAGA analogues of FFK(Sub-KuE) and ffk(Sub-KuE) in terms of PSMA affinity, uptake in PSMA positive tumour cells, metabolic stability, in vivo biodistribution and μPET imaging. The previously described DOTA analogues  and [68Ga]HBED-CC-Ahx-KuE  were also included to allow direct comparability of our results with the data in the literature.
Fmoc-(9-fluorenylmethoxycarbonyl-) and all other protected amino acid analogues were purchased from Iris Biotech (Marktredwitz, Germany) or Bachem (Bubendorf, Switzerland). Tritylchloride polystyrene (TCP) resin was obtained from PepChem (Tübingen, Germany). The chelators DOTA-tris-t Bu-ester and DOTAGA-anhydride were purchased from Chematech (Dijon, France). Solvents and all other organic reagents were purchased from Sigma-Aldrich (Munich, Germany) or CLN (Freising, Germany). Solid phase peptide synthesis was carried out manually using an Intelli-Mixer syringe shaker (Neolab, Heidelberg, Germany). Analytical reversed-phase high performance liquid chromatography (RP-HPLC) was performed on a Nucleosil 100 C18 (5 μm, 125 × 4.0 mm) column (CS GmbH, Langerwehe, Germany) using a Sykam gradient HPLC System (Sykam GmbH, Eresing, Germany). The peptides were eluted applying different gradients of 0.1% (v/v) trifluoroacetic acid (TFA) in H2O (solvent A) and 0.1% TFA (v/v) in acetonitrile (solvent B) at a constant flow of 1 mL/min (specific gradients are cited in the text). UV detection was performed at 220 nm using a 206 PHD UV-Vis detector (Linear™ Instruments Corporation, Reno, USA). Both retention times t R as well as the capacity factors K' are cited in the text. Preparative RP-HPLC was performed on the same HPLC system using a Multospher 100 RP 18-5 (250 × 20 mm) column (CS GmbH, Langerwehe, Germany) at a constant flow of 9 mL/min. Radio-HPLC of the radioiodinated reference ligand was carried out using a Nucleosil 100 C18 (5 μm, 125 × 4.0 mm) column. For radioactivity detection, the outlet of the UV-photometer was connected to a NaI(Tl) well-type scintillation counter from EG&G Ortec (Munich, Germany). Analysis of 68Ga-labelled compounds was done as described previously . ESI-mass spectra were acquired on a Varian 500-MS IT mass spectrometer (Agilent Technologies, Santa Clara, USA).
Synthesis of carboxyl-protected Lys-urea-Glu-core (KuE)
(S)- di- tert- butyl 2-(1H- imidazole- 1- carboxamido)pentanedioate (1) was synthesized as described previously . HPLC (10% to 90% B in 15 min): t R = 12.2 min; K' = 5.78. Calculated monoisotopic mass for 1 (C17H27N3O5): 353.4; found: m/z = 376.0 [M + Na]+.
Cbz-(OtBu)KuE(OtBu)2 (2): A solution of 3.40 g (9.64 mmol, 1.0 eq) 1 in 45 mL 1,2-dichloroethane (DCE) was cooled to 0°C, and 2.69 mL (19.28 mmol, 2.0 eq) of triethylamine (TEA), and 3.59 g (9.64 mmol, 1.0 eq) of Cbz-Lys-OtBu • HCl was added under vigorous stirring. The reaction mixture was heated to 40°C overnight. The solvent was removed in vacuo, and the crude product was purified via silica gel flash-chromatography using an eluent mixture of ethyl acetate/hexane/TEA (500/500/0.8 (v/v/v)). Upon solvent evaporation, 4.80 g of 2 were obtained as a colourless, sticky oil (yield: 80% based on l-di-tert-butyl glutamate • HCl). HPLC (40% to 100% B in 15 min): t R = 14.3 min; K' = 8.53. Calculated monoisotopic mass for 2 (C32H51N3O9): 621.8; found: m/z = 622.2 [M + H]+, 644.3 [M + Na]+.
(OtBu)KuE(OtBu)2 (3): For Cbz deprotection, 6.037 g (9.71 mmol, 1.0 eq) of 2 was dissolved in 150 mL of ethanol (EtOH), and 0.6 g (1.0 mmol, 0.1 eq) of Palladium on activated charcoal (10%) was added. After purging the flask with H2, the solution was stirred overnight under light H2-pressure (balloon). The crude product was filtered through Celite, the solvent was evaporated in vacuo, and the desired product was obtained as a waxy solid (4.33 g, 91.5% yield). HPLC (10% to 90% B in 15 min): t R = 12.6 min; K′ = 6.41. Calculated monoisotopic mass for 3 (C24H45N3O7): 487.6; found: m/z = 488.3 [M + H]+, 510.3 [M + Na]+.
Synthesis of protected Sub-KuE conjugate
NHS- Sub-(OtBu)KuE(OtBu)2 (4): 3 (40 mg, 0.08 mmol, 1 eq) was dissolved in 500 μL N , N -dimethylformamide (DMF), and 57 μL (0.41 mmol, 5 eq) of TEA was added. This solution was added dropwise (within 30 min) to a solution of 33.2 mg (0.09 mmol, 1.1 eq) of disuccinimidyl suberate (DSS). After stirring for an additional 2 h at room temperature (RT), the reaction mixture was concentrated in vacuo, diluted with ethyl acetate and extracted with water (twice). The organic phase was dried over Na2SO4, filtered and evaporated to dryness. Due to sufficient purity of the crude 4, it was used for the following reaction step without further purification. HPLC (10% to 90% B in 15 min): t R = 16.9 min; K' = 8.39. Calculated monoisotopic mass for 4 (C36H60N4O12): 740.4; found: m/z = 741.2 [M + H]+, 763.4 [M + Na]+.
Synthesis of peptidic spacers
Fmoc- l - Phe- l - Phe- l - Lys(Boc) (Fmoc- FFK, 5) and Fmoc- d - Phe- d - Phe- d - Lys(Boc) (Fmoc- ffk, 6): Fmoc-Lys(Boc)-OH was coupled to TCP resin according to a previously published method . Briefly, Fmoc-Lys(Boc)-OH (1.5 eq) was dissolved in dry dichloromethane (DCM), and N,N-diisopropylethylamine (DIPEA) (1.25 eq) was added. Dry TCP resin was suspended and stirred at RT for 5 min. Another 2.5 eq of DIPEA was added, and stirring was continued for 90 min. Then, 1 mL methanol (MeOH) per gram resin was added to cap unreacted Tritylchloride groups. After 15 min, the resin was filtered off, washed twice with DCM, DMF and MeOH, respectively, and dried in vacuo. Final load of resin-bound Fmoc-Lys(Boc)-OH was calculated from the weight difference.
Assembly of the peptide sequence H2N-Phe-Phe- on resin-bound Lys(Boc) was performed according to a standard Fmoc-protocol using 1.5 eq of 1-hydroxybenzotriazole (HOBt) and O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium-tetrafluoroborate (TBTU) as coupling reagents and 4.5 eq DIPEA. After coupling of the last amino acid, the resin was washed, dried and stored in a desiccator until further functionalization.
Coupling of chelating moiety
Fmoc-Phe-Phe-Lys(Boc)-TCP resin was allowed to preswell in N-methyl-pyrrolidon (NMP) for 30 min. After cleavage of the N-terminal Fmoc-protecting group using 20% piperidine in DMF (v/v), the resin was washed eight times with NMP. The coupling of the respective chelators is described below. Cleavage from the resin (2 × 30 min) and concomitant t Bu-deprotection was performed using a mixture (v/v/v) of 95% TFA, 2.5% triisobutylsilane (TIBS) and 2.5% water. The combined product solutions were then concentrated, the crude peptide was precipitated using diethyl ether and was dried in vacuo. Due to sufficient purity of the crude products, they were used for the following reaction step without further purification.
DOTA- Phe- Phe- Lys (DOTA- FFK, 7) : For 38 μmol of resin-bound peptide, 33 mg of DOTA-tris-t Bu-ester (57 μmol, 1.5 eq), 108 mg of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU; 0.28 μmol, 5 eq) and 87 μL of DIPEA (570 μmol, 15 eq) in NMP were added to the resin. After 72 h of shaking, the resin was washed with NMP and DCM. HPLC (10% to 90% B in 15 min): t R = 8.2 min; K' = 4.13. Calculated monoisotopic mass for 7 (C40H58N8O11): 826.4; found: m/z = 827.3 [M + H]+, 849.3 [M + Na]+, 414.2 [M +2H]2+.
DOTAGA- Phe- Phe- Lys (DOTAGA- FFK, 8 and DOTAGA- ffk, 9): For 0.27 mmol peptide-bound resin, 190 mg DOTAGA-anhydride (0.42 mmol, 1.5 eq) and 470 μL DIPEA (2.7 mmol, 10 eq) in NMP were added to the resin. After 18 h of shaking, the resin was washed with NMP and DCM. HPLC (10% to 90% B in 15 min): t R = 10.6 min; K' = 5.63. Calculated monoisotopic mass for 8 and 9 (C43H62N8O13): 898.4; found: m/z = 899.4 [M + H]+, 921.4 [M + Na]+, 450.2 [M +2H]2+.
Condensation of the chelator-conjugated peptides and the PSMA binding motif
DOTA- FFK(Sub- KuE) (10) : To a solution of 7 (15 mg, 18 μmol, 1 eq) and TEA (13 μL, 90 μmol, 5 eq) dissolved in 600 μL of DMF was slowly added 13 mg of 4 (18 μmol, 1 eq) dissolved in 400 μL of DMF. After stirring for 2 h at RT, the reaction mixture was evaporated to dryness. Subsequent removal of t Bu-protecting groups was carried out by dissolving the crude product in TFA and stirring for 40 min. After precipitation in diethyl ether, the crude product was dissolved in water and purified using preparative RP-HPLC (25% to 40% B in 20 min). HPLC (10% to 90% B in 15 min): t R = 10.3 min; K' = 5.44. Calculated monoisotopic mass for 10 (C60H89N11O20): 1,283.6; found: m/z = 1,284.5 [M + H]+, 1,306.7 [M + Na]+, 642.8 [M +2H]2+.
DOTAGA- FFK( Sub- KuE) (11) and DOTAGA- ffk(Sub- KuE) (12): Either 8 or 9 (21μmg, 30μmol, 1 eq) was added to TEA (21 μL, 150 μmol, 5 eq) and 27 mg of 4 (30 μmol, 1 eq) as described for 10. HPLC (10% to 90% B in 15 min): t R = 9.7 min; K' = 4.11. Calculated monoisotopic mass for 11 and 12 (C63H93N11O22): 1,355.7; found: m/z = 1,356.2 [M + H]+, 1,378.2 [M + Na]+, 679.2 [M +2H]2+.
Synthesis of the radioiodination precursor (13)
Succinimidyl- 4- iodobenzoate (I- BA- NHS, 14): Under a nitrogen atmosphere, 500 mg (2.0 mmol, 1.0 eq) 4-iodobenzoic acid was dissolved in 10 mL DCM, and after addition of 278 mg (2.4 mmol, 1.2 eq) N-hydroxysuccinimide and 374 mg (1.81 mmol, 0.9 eq) dicyclohexyl carbodiimide, the suspension was stirred overnight. The precipitate was filtered off, and the filtrate was evaporated to dryness. The resulting solid was washed with a 1:1-mixture of DCM and hexane to yield the desired product (583.5 mg, 93%) as a white solid. Due to the limited detectability of the product in ESI-mass spectrometry (MS), a representative conjugate with H-Phe-OtBu (1 eq) was prepared in DMF in the presence of DIPEA (3 eq) and characterized via MS. HPLC (40% to 100% B in 15 min): t R = 10.6 min; K' = 5.63. Calculated monoisotopic mass for I-BA-Phe(OtBu) (C20H22INO3): 451.1; found: m/z = 396.1 [M + H-tBu]+.
Succinimidyl- 4- tributylstannyl- benzoate ( SnBu3- BA- NHS, 15): To a solution of 100 mg (0.29 mmol, 1.0 eq) 14 in 5 mL anhydrous toluene were added 234 μL (0.464 mmol, 1.6 eq) of hexabutylditin and 10.7 mg (9 μmol, 0.02 eq) of the catalyst tetrakis(triphenylphosphine)palladium under a nitrogen atmosphere. The mixture was heated under reflux until the solution turned black (overnight). After cooling, the toluene was removed in vacuo, and the resulting oil was purified using silica gel flash chromatography (ethyl acetate/hexane: 3/7 (v/v)) to yield 15 (78 mg, 53%) as a colourless oil. TLC (ethyl acetate/hexane: 3/7): R f = 0.46
(SnBu3- BA)(OtBu)KuE(OtBu)2 (13): To a solution of 19.0 mg (0.039 mmol, 1.0 eq) 3 in 2 mL DCM were added 26.3 μL (0.187 mmol, 4.8 eq) of TEA and 19.8 mg (0.039 mmol, 1.0 eq) of 15. The mixture was stirred at RT for 4 h and was then diluted with DCM. After washing with water, the organic phase was dried over Na2SO4, filtered and evaporated to dryness. 13 (30.8 mg, 89.7%) was obtained as a colourless oil. HPLC (10% to 90% B in 15 min): t R = 23.8 min; K' = 13.88. Calculated monoisotopic mass for 13 (C43H75N3O8Sn): 880.8; found: m/z = 902.2/903.3/904.3 [M + Na]+.
Synthesis of unlabelled reference compounds
( I- BA) KuE: To a solution of 15 mg (0.031 mmol, 1.0 eq) 3 in 2 mL DMF was added 11.4 mg (0.046 mmol, 1.5 eq) of 4-iodo-benzoic acid, 6.3 mg (0.046 mmol, 1.5 eq) of 1-Hydroxy-7-azabenzotriazole (HOAt), 7.2 μL (0.046 mmol, 1.5 eq) diisopropyl carbodiimide (DIC) and 23.7 μL (0.138 mmol, 4.5 eq) DIPEA. The yellow solution was stirred for 20 h, diluted with ethyl acetate and extracted with water. The organic layer was dried over Na2SO4, filtered and evaporated to dryness. For t Bu-deprotection, the crude product was dissolved in 200 μL TFA. After 30 min, the solvent was evaporated and the crude product was purified using preparative RP-HPLC (isocratic eluent: 18% B). HPLC (10% to 90% B in 15 min): t R = 10.5 min; K' = 5.56 calculated monoisotopic mass for (I-BA)KuE (C19H24IN3O8): 549.3; found: m/z = 550.0 [M + H]+, 571.9 [M + Na]+
nat Ga compounds: For the preparation of the natGa complexes, equal volumes of a 2 mM solution of Ga(NO3)3 in water and a 2 mM solution of the respective PSMA ligand in water were mixed and heated to 40°C for 30 min. After cooling, the natGaIII-chelate formation was confirmed using RP-HPLC and MS. The resulting 1 mM aqueous solutions of the respective natGa-complexes were then further diluted and used in the in vitro IC50 studies without further processing.
[natGa]DOTA-FFK(Sub-KuE) (natGa-10) HPLC (20% to 60% B in 15 min): t R = 11.6 min; K' = 6.3. Ccalculated monoisotopic mass (C60H86N11O20Ga): 1,349.5; found: m/z = 1,350.3 [M + H]+, 1,372.1 [M + Na]+, 675.7 [M +2H]2+.
[natGa]DOTAGA-FFK(Sub-KuE) (natGa-11) HPLC (25% to 45% B in 15 min): t R = 16.0 min; K' = 9.0. Calculated monoisotopic mass (C63H90N11O22Ga): 1,421.7, 1,423.7; found: m/z = 1,422.1/1,424.1 [M + H]+, 710.6/712.6 [M +2H]2+.
[natGa]DOTAGA-ffk(Sub-KuE) (natGa-12) HPLC (25% to 55% B in 15 min): t R = 12.1 min; K' = 7.6. Calculated monoisotopic mass (C63H90N11O22Ga): 1,421.7, 1,423.7; found: m/z = 1,422.6/1,424.5 [M + H]+, 1,444.4/1,446.4 [M + Na]+.
[natGa]HBED-CC-Ahx-KuE HPLC (25% to 43% B in 15 min): t R = 9.0 min; K' = 5.0. Calculated monoisotopic mass (C44H59N6O17Ga): 1,012.3, 1,014.3; found: m/z = 1,013.0/1,015.0 [M + H]+, 1,035.0/1,037.0 [M + Na]+.
nat Lu - compounds: The corresponding natLuIII complexes were prepared with a 2.5-molar excess of Lu3+, heated at 95°C for 30 min and evaluated similar to the natGaIII complexes.
[natLu]DOTA-FFK(Sub-KuE) (natLu-10) HPLC (25% to 45% B in 16 min): t R = 14.1 min; K' = 9.1. Calculated monoisotopic mass (C60H86N11O20Lu): 1,455.6; found: m/z = 1,456.4 [M + H]+, 1,478.5 [M + Na]+.
[natLu]DOTAGA-FFK(Sub-KuE) (natLu-11) HPLC (25% to 45% B in 16 min): t R = 14.4 min; K' = 9.3. Calculated monoisotopic mass (C63H90N11O22Lu): 1,527.6; found: m/z = 1,528.4 [M + H]+, 1,550.3 [M + Na]+, 764.2 [M +2H]2+.
[natLu]DOTAGA-ffk(Sub-KuE) (natLu-12) HPLC (25% to 55% B in 15 min): t R = 10.4 min; K' = 6.4. Calculated monoisotopic mass (C63H90N11O22Lu): 1,527.6; found: m/z = 1,528.1 [M + H]+, 764.5 [M +2H]2+.
([ 125 I]I - BA)KuE: The radioiodination was performed according to previously published methods ,,. Peracetic acid was prepared by mixing 130 μL of H2O2 (30%) and 50 μL of acetic acid. After a 2 h-incubation period, 20 μL peracetic acid solution and 5 μL (21.0 MBq) [125I]NaI (74 TBq/mmol, 3.1 GBq/mL 40 mM NaOH, Hartmann Analytic, Braunschweig, Germany) was added to a solution of approximately 0.1 mg 15 in 20 μL MeCN/acetic acid (1/1) and incubated at RT for 10 min. The product was diluted with 10 mL of water and loaded onto a C18 Sep Pak Plus cartridge, which had been preconditioned with 10 mL of MeOH and rinsed with 10 mL of water. The cartridge was then washed with 10 mL water, and the product was eluted in 300 to 500 μL fractions with a 1:1 mix (v/v) of EtOH/MeCN (2 mL). The radioactive fractions were evaporated to dryness, and the residue was dissolved in 200 μL TFA. After 30 min, the solvent was evaporated in vacuo. The crude product was isolated from unlabelled tributyltin precursor by RP-HPLC (20% to 40% B in 20 min) to afford the desired product (10.9 MBq). HPLC (20% to 40% B, 220 nm): t R = 13.0 min, K' = 6.22.
68 Ga - labelling: A 1.25 mL fraction of 68Ge/68Ga generator (iTHEMBA Labs, South Africa) eluate (1 M HCl) was loaded onto a self-filled cartridge containing 300 mg SCX material (Bond Elut-SCX, Varian). The cartridge was then washed with water (1.0 mL) and purged with air. The 68Ga3+ was eluted with aq. NaCl (5 M, 0.5 mL) and HEPES (2.7 M aq., 90 or 140 μL) was added (pH 3.5 or 4.5, respectively). That solution was mixed with the precursor (3 nmol in 10 μL) and heated at 95°C for 5 min. After cooling, labelling efficiency and radiochemical purity were determined using Radio-TLC and Radio-HPLC. Radiochemical purity of all 68Ga-labelled conjugates was ≥95%. Therefore, the tracers were diluted and used in vitro experiments without further purification. Tracers for in vivo use were prepared in GMP-compliant procedure using a fully automated synthesis module (Scintomics GmbH, Germany) similar to the procedure described previously . For animal studies, the EtOH for eluting the labelled tracer from a SPE cartridge was evaporated in vacuo.
177 Lu-labelling: A 0.1 mM aqueous solution of chelator-coupled peptide (0.66 nmol) was added to 25 MBq [177Lu]LuCl3 (170 GBq/μmol, 17 GBq/mL 0.05 M HCl, IDB Radiopharmacy bv). The pH was adjusted to pH 5 by the addition of approximately 150 μL 0.1 M NH4OAc solution. After 45 min at 95°C, the labelling efficiency was examined by TLC and RP-HPLC.
Determination of lipophilicity
To a solution of 0.5 to 1 MBq of radiolabelled peptide in 500 μl of phosphate-buffered saline (PBS, pH 7.4), 500 μl of n-octanol was added (n = 6). Vials were vortexed vigorously for 3 min. To achieve quantitative phase separation, the vials were centrifuged at 6,000 g for 5 min in a Biofuge 15 (Heraeus Sepatech, Osterode, Germany). The activity concentrations in 100 μl-samples of both the aqueous and the organic phase were measured in a γ-counter. Both the partition coefficient P(o/w), which is defined as the molar concentration ratio of a single species A between n-octanol and water at equilibrium, and logP(o/w), which is an important parameter used to characterize lipophilicity of a compound, were calculated.
177Lu-10 and 177Lu-11 were incubated in 80 μL of human serum (37°C) and 80 μL 0.4 M DTPA solution (RT), respectively and after 1, 24 and 48 h, the stability was determined by TLC analysis. The 68Ga-labelled tracers (40 to 45 MBq) were injected into the tail vein of CD-1 nu/nu mice. The animals were sacrificed after 30 min, and urine, blood and kidney samples were taken. The kidney was frozen in liquid nitrogen, homogenized with a ball mill and extracted with 0.2 to 1 mL PBS containing 200 nmol 2-(phosphonomethyl)pentane-1,5-dioic acid (PMPA). The suspension was first centrifuged (15,000 g), and after ultrafiltration, the extracts were analysed by RP-HPLC. The blood samples were centrifuged to separate the plasma from the blood cells. Plasma proteins were removed by precipitation with acetonitrile (10 min, 4°C) and subsequent centrifugation and ultrafiltration. The blood extract was also analysed by RP-HPLC.
In vitro assays
Cell culture: PSMA+ LNCaP cells (CLS: 300265) were grown in DMEM/Nutrition Mix F-12 with Glutamax-I (1:1) (Invitrogen, Life Technologies, Darmstadt, Germany) supplemented with 10% FCS. Cultures were maintained at 37°C in a 5% CO2/humidified air atmosphere. One day prior to the experiment, cells were harvested using Trypsin/EDTA (0.05% and 0.02%) in PBS, centrifuged and resuspended with culture medium. For cell counting, a Countesse automated cell counter (Invitrogen, Carlsbad, USA) was used. All in vitro binding and internalization studies were performed using live cells seeded 1 day prior to the experiment. For IC50 determination, 150,000 cells/mL were transferred to 24-well plates (1 mL/well), and for internalization studies, 125,000 cells/mL were transferred into PLL-coated 24-well plates.
Determination of IC 50 : The culture medium was removed, and the cells were washed once with 500 μL of HBSS (Hank's balanced salt solution, Biochrom, Berlin, Germany, containing 1% bovine serum albumin (BSA)), before being left to equilibrate in 200 μL of HBSS (1% BSA) on ice for 15 min. Then, 25 μL/well of either HBSS (1% BSA; control) or of solutions containing the respective unlabelled ligand of interest in increasing concentrations (10-10-10-4 M in HBSS (1% BSA)) was added, followed by the addition of 25 μL of ([125I]I-BA)KuE in HBSS (1% BSA). Experiments were carried out in triplicate for each concentration. The final radioligand concentration was 0.2 nM in all binding assays. Cells were incubated on ice for 60 min. Incubation was terminated by the removal of the incubation medium. Cells were thoroughly rinsed with 250 μL of HBSS. The wash medium was combined with the supernatant of the previous step. This fraction represents the amount of free radioligand. Cells were then lysed using 250 μL of 1 N NaOH, the lysate was transferred to vials and combined with 250 μL of HBSS used for rinsing the wells. Quantification of the amount of free and bound activity was performed in a γ-counter.
Internalization studies: The culture medium was removed, and the cells were washed once with 500 μL DMEM-F12 (5% BSA) before being left to equilibrate in 200 μL DMEM-F12 (5% BSA) at 37°C for a minimum of 15 min. Then, 25 μL (per well) of either DMEM-F12 (5% BSA) or of a 100-μM PMPA solution (blocking) was added, followed by the addition of 25 μL of 68Ga/177Lu-labelled PSMA ligand. The final radioligand concentration was 0.2/0.5 nM in all internalization assays. To determine internalization kinetics, cells were then incubated at 37°C for 5, 15, 30 and 60 min, respectively. Experiments were carried out in triplicate for each time point (control and blocking). Incubation was terminated by placing the plate on ice for approximately 1 min and by subsequent removal of the incubation medium. Cells were thoroughly rinsed with 250 μL of PBS. The wash medium was combined with the supernatant of the previous step. This fraction represents the amount of free radioligand. To remove receptor surface bound radioactivity, the cells were then incubated for 10 min with 250 μL of ice cold PMPA solution (10 μM in PBS). After removal of the supernatant, the cells were thoroughly rinsed with another 250 μL of ice cold PBS. Both fractions were combined. The internalized activity was released by incubation with 250 μL of 1 N NaOH, transferred to vials and combined with 250 μL of 1 N NaOH used for rinsing the wells. Quantification of the amount of free, PMPA-releasable and internalized activity was performed in a γ-counter.
In vivo experiments
All animal experiments were conducted in accordance with general animal welfare regulations in Germany (Deutsches Tierschutzgesetz, approval #55.2-1-54-2532-71-13). The prostate cancer cell line LNCaP was suspended 1/1 in serum-free medium and Matrigel (BD Biosciences, Germany) and approximately 107 cells in 200 μL were inoculated subcutaneously on the right shoulder of 6 to 8 weeks old CD-1 nu/nu mice (Charles River Laboratories). Tumours were grown for 2 to 4 weeks (males) and 4 to 6 weeks (females) to reach 4 to 8 mm in diameter.
Biodistribution: The 68Ga-labelled PSMA ligands (approximately 7 to 15 MBq, 0.1 to 0.2 nmol) were injected into the tail vein of isoflurane anesthetized animals. The organ distribution was examined 1 h p.i. and quantified in a γ-counter.
μPET imaging: Imaging studies were performed at a Siemens Inveon small animal PET, followed by data analysis using the Inveon Research Workplace software. The animals were anesthesized with isoflurane and injected via tail vein with 14 to 18 MBq (0.2 nmol) of tracer. Dynamic imaging was performed after on-bed injection for 1.5 h. Static images were recorded at 1 h p.i. with an acquisition time of 15 min. For the blockade image, animals were coinjected with 8 mg/kg of PMPA. Images were reconstructed using 3D ordered-subsets expectation maximum (OSEM3D) algorithm without scanner and attenuation correction.
The natGaIII and natLuIII complexes of compounds 10, 11 and 12 were prepared by incubation of 10, 11 and 12 with an equimolar amount of aq. Ga(NO3)3 at 40°C for 30 min and an 2.5-molar excess of LuCl3 at 95°C for 30 min, respectively. Quantitative metal complex formation was confirmed by RP-HPLC and MS.
([125I]I-BA)KuE, the reference radioligand in all PSMA binding assays carried out in this study, was synthesized in solution starting from 4-iodo-benzoic acid according to methods described in the literature ,,. The respective Bu3Sn-precursor 13 was destannylated with [125I]NaI using peracetic acid as the oxidizing agent within 10 min at RT. After cartridge purification subsequent t Bu-deprotection and RP-HPLC purification, the final product was obtained in a radiochemical yield of 44 ± 5% and radiochemical purity of >99%. The reference material for chromatographic analyses (cold) (I-BA)KuE was synthesized by coupling 4-iodo-benzoic acid to 3, subsequent t Bu-deprotection and RP-HPLC purification.
Preparation of 68Ga for labelling of 10, 11 and 12 was performed by combination of previously described methods , with minor modifications. The 68Ga3+ eluted with 1 M HCl from a 68Ge/68Ga generator (iThemba Labs, South Africa) was retained on a strong cation exchange cartridge followed by elution with 0.5 mL 5 M NaCl, resulting in highly concentrated 68Ga activity. Quantitative 68Ga-complexation was achieved using 3 nmol of the respective DOTA- or DOTAGA-conjugated PSMA inhibitors (95°C, 5 min, pH adjusted to 3.5 and 4.5 by addition of 2.7 M HEPES, respectively), allowing their use in in vitro studies without further purification. Specific activities of the 68Ga-labelled PSMA inhibitors were 250 to 300 GBq/μmol. The 68Ga-labelling for in vivo biodistribution and PET imaging studies were carried out using a fully automated GMP-compliant procedure using a GRP synthesizer (Scintomics GmbH, Germany) . In these cases, the obtained specific activities were 80 to 120 GBq/μmol.
To obtain RCY >95% for complexation of 10, 11 and 12 with [177Lu]LuCl3, 0.66 nmol of precursor were reacted with 25 MBq [177Lu]LuCl3 (AS = 170 GBq/μmol) at pH 5 (0.1 M NH4OAc, 95°C, 45 min) resulting in specific activities of AS ≥38 GBq/μmol.
PSMA binding affinity
PSMA affinities (IC 50 ) of the compounds investigated
5.7 ± 0.5
6.1 ± 0.8
13.1 ± 2.3
29.5 ± 6.6
54.7 ± 6.1
10.2 ± 1.5
12.1 ± 3.9
15.1 ± 1.5
13.9 ± 0.4
15.9 ± 0.5
13.1 ± 2.2
Internalization kinetics and specificity of cell binding
The cellular uptake and internalization kinetics of the different 68Ga- and 177Lu-labelled PSMA ligands was determined using PSMA-expressing LNCaP cells. In the case of the 68Ga-labelled compounds, the final peptide concentration in the assays was 0.2 nM. Since all 177Lu analogues were obtained in significantly lower specific activities than the 68Ga analogues, the ligand concentration had to be increased to 0.5 nM to obtain reasonable count rates for assay evaluation. All internalization studies were accompanied by reference studies using ([125I]I-BA)KuE (c = 0.2 nM). This experimental setup allows data normalization and eliminates the influence of cell count and cell viability on absolute cellular tracer uptake. To investigate the specificity of binding to PSMA, experiments were also carried out in the presence of 10 μM PMPA, a known high-affinity PSMA inhibitor (1.4 nM, ). Furthermore, to be able to discriminate between total cellular activity (sum of membrane associated and internalized activity) and internalized activity, all incubations were followed by a washing step with 10 μM PMPA at 4°C to remove specifically cell-surface bound radioligand by displacement.
Internalization of radiometalated ligands in percentage of external reference ([ 125 I]I-BA)KuE
91.1 ± 1.7
14.6 ± 0.8
19.3 ± 0.9
28.4 ± 0.7
36.1 ± 1.1
42.5 ± 1.7
44.4 ± 1.8
Determination of lipophilicity
Lipophilicity of the radiolabelled ligands (log P(o/w); distribution coefficient in n-octanol/PBS)
-4.1 ± 0.1
-3.1 ± 0.2
2.7 ± 0.02
-3.6 ± 0.1
-3.9 ± 0.1
The stability of 177Lu-10 and 177Lu-11 was investigated by incubation in human serum for 48 h at 37°C and by DTPA challenge (0.4 M, RT, 48 h) and subsequent TLC analysis. Release of 177LuIII from the complexes was not observed under the respective experimental conditions in the examined time frame.
Biodistribution and small-animal PET studies
Due to the consistent overexpression of PSMA in especially hormone-refractory and metastatic prostate cancer, this cell surface enzyme represents an excellent target for high-contrast PET imaging and also for therapeutic applications. The currently available PSMA-targeted radiopharmaceuticals are highly optimized either for imaging , or endoradiotherapeutic applications , but so far none of the compounds was evaluated for a theranostic concept. Our approach was therefore focused on the development of a PSMA ligand allowing for both, complexation with, e.g. 68Ga or 111In for PET or SPECT and with therapeutic M3+-radiometals, e.g. 177Lu, 90Y or 225Ac for endoradiotherapy. The most frequently used chelator for this purpose is DOTA, and recently, a first KuE-based DOTA-conjugated PSMA inhibitor (68Ga-10, ) has been described. In a comparative preclinical evaluation with [68Ga]HBED-CC-Ahx-KuE , 68Ga-10 showed significantly lower tumour uptake than [68Ga]HBED-CC-Ahx-KuE. However, since 68Ga-10 may be labelled with a broad palette of diagnostic and therapeutic radiometals, whereas [68Ga]HBED-CC-Ahx-KuE may not, we used 68Ga-10 as a starting point for the development of improved PSMA-targeted theranostics.
In a first attempt to improve the pharmacokinetics of the DOTA-coupled PSMA ligand 10, we increased the hydrophilicity of the ligand by substitution of DOTA by DOTAGA, resulting in 11. Although [68Ga]HBED-CC-Ahx-KuE contains the lipophilic HBED-CC chelator, which improves binding affinity , a logP of -4.1 ± 0.1 was determined, revealing a significantly higher hydrophilicity compared to logP = -3.1 ± 0.2 for 68Ga-10. The hydrophilicity of the 68Ga-labelled DOTAGA-coupled ligand 11 increased to log P = -3.6 ± 0.1. For 68Ga, as well as 177Lu labelling, the DOTA-conjugated ligand 10 and the DOTAGA-conjugated ligands 11 and 12 exhibited no differences in labelling efficiency.
To examine the metabolic stability of these new PSMA tracers in vivo, CD-1 nu/nu mice were injected with the 68Ga-labelled ligand 11. Due to its l-amino acid spacer, 68Ga-11 was rapidly metabolized. Furthermore, we demonstrated that fast metabolization of 68Ga-10 and 68Ga-11 resulted in discontinuous clearance kinetics and low tumour accumulation in vivo (Figure 7, 8, 9). This most likely explains the finding in the literature, that despite moderate affinity towards PSMA (IC50 = 29.5 ± 6.6 nM) the DOTA ligand 68Ga-10 exhibited unfavourable tumour targeting compared to [68Ga]HBED-CC-Ahx-KuE. Further, we observed significant inter-individual differences in metabolization kinetics for 68Ga-11 (with its l-amino acid spacer) in mice, which might explain inconsistent in vivo results obtained with PSMA ligands with l-amino acid spacers ,. To overcome the problem of rapid proteolytic cleavage of radiolabelled 11, we substituted the l-amino acid spacer (FFK) by its d-amino acids counterpart ffk (12) resulting in high metabolic stability in vivo.
In addition to the improvement in metabolic stability for 12, we found that the DOTAGA-conjugated ligands 11 and 12, as well as their gallium(III) and lutetium(III) complexes, showed a significantly increased affinity towards PSMA on LNCaP cells compared to the DOTA-coupled ligand 10/natGa-10/natLu-10. Especially, natLu-10 (IC50 = 54.7 ± 6.1 nM) was less affine compared to natLu-11 (15.1 ± 1.5 nM) or natLu-12 (13.1 ± 2.2 nM). This finding most likely is due to the increased charge of the DOTAGA-ligands. In our competitive binding assay, unlike reported for other assays in the literature , a significantly lower affinity for natGa-10 (IC50 of 29.5 ± 6.6 nM) was determined compared to [natGa]HBED-CC-Ahx-KuE (IC50 = 6.1 ± 0.8 nM).
As expected from their enhanced PSMA affinities, the DOTAGA ligands 68Ga/177Lu-11 and 68Ga/177Lu-12 showed up to twofold increased internalization compared to the corresponding DOTA ligands 68Ga/177Lu-10. Interestingly, 68Ga/177Lu-12 with the ffk spacer showed a higher internalization rate than ligand 11 with the FFK spacer. Blocking experiments with PMPA highlight the high PSMA-specificity of these tracers.
Accordingly, the pharmacokinetics and tumour uptake of the new ligands, i.e. 68Ga-12 in LNCaP-tumour bearing CD-1 nu/nu mice could be enhanced (Figure 7). The metabolically instable 68Ga-11 revealed higher activity levels in the blood pool and almost all organs, whereas the metabolically stable tracer 68Ga-12 displayed no significant uptake in the gastrointestinal tract (<0.5% ID/g) and high kidney accumulation of 96 ± 45% ID/g. The kidney uptake is PSMA-specific, as shown in the blocking experiment (Figure 8d'). Although [68Ga]HBED-CC-Ahx-KuE showed a higher affinity and increased internalization rate compared to 68Ga-12, the in vivo biodistribution (Figure 7) and μPET imaging studies (Figure 8) revealed favourable in vivo characteristics for 68Ga-12. The uptake of 68Ga-12 into the PSMA positive LNCaP tumour xenograft was somewhat increased compared to that of [68Ga]HBED-CC-Ahx-KuE (5.2 ± 1.2% vs. 4.7 ± 0.2% ID/g, respectively). Further, [68Ga]HBED-CC-Ahx-KuE showed enhanced tracer uptake into the lung (1.9 ± 0.4% vs. 1.1 ± 0.3% ID/g), liver (0.5 ± 0.1 vs. 0.2 ± 0.04% ID/g), spleen (14.7 ± 2.6 vs. 5.5 ± 0.2% ID/g) and pancreas (0.9 ± 0.2 vs. 0.4 0.1% ID/g). Therefore, the lower unspecific uptake of 68Ga-12 compared to [68Ga]HBED-CC-Ahx-KuE resulted in higher tumour-to-background ratios, i.e. tumour-to-blood (17.0 ± 0.5 vs. 12.5 ± 0.7) and tumour-to-skeletal muscle (19.8 ± 0.7 vs. 16.2 ± 0.1).
After the initial distribution phase of a radiopharmaceutical, straightforward clearance kinetics is typically characterized by a linear decrease in a semilogarithmic plot (two-compartment model). Interestingly, this kinetic profile (Figure 9) was only observed for 68Ga-12, whereas all other tracers (or their radiolabelled metabolites) exhibit unspecific retention or redistribution effects, resulting in non-linear kinetics (e.g. between 10 and 60 min) in the semilogarithmic plot. Considering the time-activity curves, it is worth noting that based on the kinetics of the reference compound [68Ga]HBED-CC-Ahx-KuE redistribution from tissues and compartments with unspecific uptake in the early phase seems likely and might explain the discontinuous clearance and increasing tumour uptake at later time points. The uptake of DOTA conjugate 68Ga-10 in the tumour is continuously increasing over time, but surprisingly without a corresponding decline of activity in muscle or blood (as measured over the heart). In contrast, a slow washout from the target and non-target tissues was observed for 68Ga-11. Most likely as a result of its metabolic instability, 68Ga-11 exhibited discontinuous and only moderate clearance kinetics. However, 68Ga-12 shows typical and nearly ideal in vivo pharmacokinetics, both for the target tissue (tumour and kidney) as well as the non-target tissue muscle and the blood. Equivalent to our DOTAGA ligand 12, the biodistribution of a naphthyl-containing [177Lu]DOTA-PSMA ligand was described recently , therefore, we assume that 177Lu-12 also reveals favourable properties in vivo.
Compared to the metabolically unstable DOTA conjugate 10 and DOTAGA-conjugate 11, 68Ga- and 177Lu-12 were found to be rapidly internalized into PSMA expressing cells, to have a favourable pharmacokinetic profile in vivo and only negligible unspecific uptake with no redistribution in vivo. Based on the DOTAGA chelator and the use of a d-amino acid spacer, compound 12 possesses remarkable potential for a PSMA based theranostics concept and is a suitable lead structure for the continuing development of PSMA ligands with further improved affinity.
The research leading to these results has received funding from the Deutsche Forschungsgemeinschaft (DFG) under Grant Agreement No. SFB 824 project Z1. We thank Simon Schmied for synthetic assistance, Sybille Reder, Markus Mittelhäuser and Marco Lehmann for μPET imaging and Johannes Notni for editing the figures.
- Mesters JR, Barinka C, Li W, Tsukamoto T, Majer P, Slusher BS, Konvalinka J, Hilgenfeld R: Structure of glutamate carboxypeptidase II, a drug target in neuronal damage and prostate cancer. EMBO J 2006, 25: 1375–1384. 10.1038/sj.emboj.7600969PubMed CentralView ArticlePubMedGoogle Scholar
- Ghosh A, Heston WD: Tumor target prostate specific membrane antigen (PSMA) and its regulation in prostate cancer. J Cell Biochem 2004, 91: 528–539. 10.1002/jcb.10661View ArticlePubMedGoogle Scholar
- Silver DA, Pellicer I, Fair WR, Heston WD, Cordon-Cardo C: Prostate-specific membrane antigen expression in normal and malignant human tissues. Clin Cancer Res 1997, 3: 81–85.PubMedGoogle Scholar
- Pinto JT, Suffoletto BP, Berzin TM, Qiao CH, Lin S, Tong WP, May F, Mukherjee B, Heston WDW: Prostate-specific membrane antigen: a novel folate hydrolase in human prostatic carcinoma cells. Clin Cancer Res 1996, 2: 1445–1451.PubMedGoogle Scholar
- Carter RE, Feldman AR, Coyle JT: Prostate-specific membrane antigen is a hydrolase with substrate and pharmacologic characteristics of a neuropeptidase. Proc Natl Acad Sci USA 1996, 93: 749–753. 10.1073/pnas.93.2.749PubMed CentralView ArticlePubMedGoogle Scholar
- Schulke N, Varlamova OA, Donovan GP, Ma D, Gardner JP, Morrissey DM, Arrigale RR, Zhan C, Chodera AJ, Surowitz KG, Maddon PJ, Heston WD, Olson WC: The homodimer of prostate-specific membrane antigen is a functional target for cancer therapy. Proc Natl Acad Sci USA 2003, 100: 12590–12595. 10.1073/pnas.1735443100PubMed CentralView ArticlePubMedGoogle Scholar
- Kozikowski AP, Nan F, Conti P, Zhang J, Ramadan E, Bzdega T, Wroblewska B, Neale JH, Pshenichkin S, Wroblewski JT: Design of remarkably simple, yet potent urea-based inhibitors of glutamate carboxypeptidase II (NAALADase). J Med Chem 2001, 44: 298–301. 10.1021/jm000406mView ArticlePubMedGoogle Scholar
- Pomper MG, Musachio JL, Zhang J, Scheffel U, Zhou Y, Hilton J, Maini A, Dannals RF, Wong DF, Kozikowski AP: 11C-MCG: synthesis, uptake selectivity, and primate PET of a probe for glutamate carboxypeptidase II (NAALADase). Mol Imaging 2002, 1: 96–101. 10.1162/153535002320162750View ArticlePubMedGoogle Scholar
- Foss CA, Mease RC, Fan H, Wang Y, Ravert HT, Dannals RF, Olszewski RT, Heston WD, Kozikowski AP, Pomper MG: Radiolabeled small-molecule ligands for prostate-specific membrane antigen: in vivo imaging in experimental models of prostate cancer. Clin Cancer Res 2005, 11: 4022–4028. 10.1158/1078-0432.CCR-04-2690View ArticlePubMedGoogle Scholar
- Chen Y, Foss CA, Byun Y, Nimmagadda S, Pullambhatla M, Fox JJ, Castanares M, Lupold SE, Babich JW, Mease RC, Pomper MG: Radiohalogenated prostate-specific membrane antigen (PSMA)-based ureas as imaging agents for prostate cancer. J Med Chem 2008, 51: 7933–7943. 10.1021/jm801055hPubMed CentralView ArticlePubMedGoogle Scholar
- Mease RC, Dusich CL, Foss CA, Ravert HT, Dannals RF, Seidel J, Prideaux A, Fox JJ, Sgouros G, Kozikowski AP, Pomper MG: N -[ N -[(S)-1,3-Dicarboxypropyl]carbamoyl]-4-[18F]fluorobenzyl- L-cysteine, [18F]DCFBC: a new imaging probe for prostate cancer. Clin Cancer Res 2008, 14: 3036–3043. 10.1158/1078-0432.CCR-07-1517PubMed CentralView ArticlePubMedGoogle Scholar
- Maresca KP, Hillier SM, Femia FJ, Keith D, Barone C, Joyal JL, Zimmerman CN, Kozikowski AP, Barrett JA, Eckelman WC, Babich JW: A series of halogenated heterodimeric inhibitors of prostate specific membrane antigen (PSMA) as radiolabeled probes for targeting prostate cancer. J Med Chem 2009, 52: 347–357. 10.1021/jm800994jView ArticlePubMedGoogle Scholar
- Hillier SM, Maresca KP, Femia FJ, Marquis JC, Foss CA, Nguyen N, Zimmerman CN, Barrett JA, Eckelman WC, Pomper MG, Joyal JL, Babich JW: Preclinical evaluation of novel glutamate-urea-lysine analogues that target prostate-specific membrane antigen as molecular imaging pharmaceuticals for prostate cancer. Cancer Res 2009, 69: 6932–6940. 10.1158/0008-5472.CAN-09-1682PubMed CentralView ArticlePubMedGoogle Scholar
- Banerjee SR, Foss CA, Castanares M, Mease RC, Byun Y, Fox JJ, Hilton J, Lupold SE, Kozikowski AP, Pomper MG: Synthesis and evaluation of 99mTc and Re-labeled inhibitors of the prostate-specific membrane antigen (PSMA). J Med Chem 2008, 51: 4504–4517. 10.1021/jm800111uPubMed CentralView ArticlePubMedGoogle Scholar
- Kularatne SA, Zhou Z, Yang J, Post CB, Low PS: Design, synthesis, and preclinical evaluation of prostate-specific membrane antigen targeted 99mTc-radioimaging agents. Mol Pharm 2009, 6: 790–800. 10.1021/mp9000712View ArticlePubMedGoogle Scholar
- Zhang AX, Murelli RP, Barinka C, Michel J, Cocleaza A, Jorgensen WL, Lubkowski J, Spiegel DA: A remote arene-binding site on prostate specific membrane antigen revealed by antibody-recruiting small molecules. J Am Chem Soc 2010, 132: 12711–12716. 10.1021/ja104591mPubMed CentralView ArticlePubMedGoogle Scholar
- Banerjee SR, Pullambhatla M, Byun Y, Nimmagadda S, Green G, Fox JJ, Horti A, Mease RC, Pomper MG: 68Ga-labeled inhibitors of prostate-specific membrane antigen (PSMA) for imaging prostate cancer. J Med Chem 2010, 53: 5333–5341. 10.1021/jm100623ePubMed CentralView ArticlePubMedGoogle Scholar
- Eder M, Schafer M, Bauder-Wust U, Hull WE, Wangler C, Mier W, Haberkorn U, Eisenhut M: 68Ga-complex lipophilicity and the targeting property of a urea-based PSMA inhibitor for PET imaging. Bioconj Chem 2012, 23: 688–697. 10.1021/bc200279bView ArticleGoogle Scholar
- Akhtar NH, Pail O, Saran A, Tyrell L, Tagawa ST: Prostate-specific membrane antigen-based therapeutics. Adv Urol 2012, 2012: 973820. 10.1155/2012/973820PubMed CentralView ArticlePubMedGoogle Scholar
- Milowsky MI, Nanus DM, Kostakoglu L, Sheehan CE, Vallabhajosula S, Goldsmith SJ, Ross JS, Bander NH: Vascular targeted therapy with anti-prostate-specific membrane antigen monoclonal antibody J591 in advanced solid tumors. J Clin Oncol 2007, 25: 540–547. 10.1200/JCO.2006.07.8097View ArticlePubMedGoogle Scholar
- Tagawa ST, Milowsky MI, Morris MJ, Vallabhajosula S, Christos PJ, Akhtar NH, Goldsmith SJ, Osborne J, Larson SM, Pandit-Taskar N, Scher HI, Bander NH, Nanus DM: Phase II study of Lutetium-177-labeled anti-prostate-specific membrane antigen (PSMA) monoclonal antibody J591 for metastatic castration-resistant prostate cancer. Clin Cancer Res 2013, 19: 5182–5191. 10.1158/1078-0432.CCR-13-0231PubMed CentralView ArticlePubMedGoogle Scholar
- Milowsky MI, Nanus DM, Kostakoglu L, Vallabhajosula S, Goldsmith SJ, Bander NH: Phase I trial of yttrium-90-labeled anti-prostate-specific membrane antigen monoclonal antibody J591 for androgen-independent prostate cancer. J Clin Oncol 2004, 22: 2522–2531. 10.1200/JCO.2004.09.154View ArticlePubMedGoogle Scholar
- Hillier S, Merkin R, Maresca K, Zimmerman C, Barrett J, Tesson M, Eckelman W, Mairs R, Joyal J, Babich J: [131I]MIP-1375, a small molecule prostate-specific membrane antigen (PSMA) inhibitor for targeted therapy of prostate cancer (PCa). J Nucl Med 2011, 52(Supplement 1):361.Google Scholar
- Hillier S, Rubino K, Maresca K, Marquis J, Tesson M, Zimmerman C, Eckelman W, Mairs R, Joyal J, Babich J: [131I]MIP-1466, a small molecule prostate-specific membrane antigen (PSMA) inhibitor for targeted radiotherapy of prostate cancer (PCa). J Nucl Med 2012, 53(Supplement 1):170.Google Scholar
- Zechmann CM, Afshar-Oromieh A, Armor T, Stubbs JB, Mier W, Hadaschik B, Joyal J, Kopka K, Debus J, Babich JW, Haberkorn U: Radiation dosimetry and first therapy results with a 124I/131I-labelled small molecule (MIP-1095) targeting PSMA for prostate cancer therapy. Eur J Nucl Med Mol Imaging 2014, 41: 1280–1292. 10.1007/s00259-014-2713-yPubMed CentralView ArticlePubMedGoogle Scholar
- Bernhard C, Moreau M, Lhenry D, Goze C, Boschetti F, Rousselin Y, Brunotte F, Denat F: DOTAGA-anhydride: a valuable building block for the preparation of DOTA-like chelating agents. Chemistry 2012, 18: 7834–7841. 10.1002/chem.201200132View ArticlePubMedGoogle Scholar
- Eisenwiener KP, Powell P, Macke HR: A convenient synthesis of novel bifunctional prochelators for coupling to bioactive peptides for radiometal labelling. Bioorg Med Chem Lett 2000, 10: 2133–2135. 10.1016/S0960-894X(00)00413-3View ArticlePubMedGoogle Scholar
- Heppeler A, Froidevaux S, Macke HR, Jermann E, Behe M, Powell P, Hennig M: Radiometal-labelled macrocyclic chelator-derivatized somatostatin analogue with superb tumour-targeting properties and potential for receptor-mediated internal radiotherapy. Chemistry 1999, 5: 1974–1981. 10.1002/(SICI)1521-3765(19990702)5:7<1974::AID-CHEM1974>3.0.CO;2-XView ArticleGoogle Scholar
- Simecek J, Zemek O, Hermann P, Wester HJ, Notni J: A monoreactive bifunctional triazacyclononane phosphinate chelator with high selectivity for 68Ga. Chem Med Chem 2012, 7: 1375–1378. 10.1002/cmdc.201200261View ArticlePubMedGoogle Scholar
- Schottelius M, Reubi JC, Eltschinger V, Schwaiger M, Wester HJ: N-terminal sugar conjugation and C-terminal Thr-for-Thr(ol) exchange in radioiodinated Tyr3-octreotide: effect on cellular ligand trafficking in vitro and tumor accumulation in vivo. J Med Chem 2005, 48: 2778–2789. 10.1021/jm040794iView ArticlePubMedGoogle Scholar
- Dekker B, Keen H, Shaw D, Disley L, Hastings D, Hadfield J, Reader A, Allan D, Julyan P, Watson A, Zweit J: Functional comparison of annexin V analogues labelled indirectly and directly with iodine-124. Nucl Med Biol 2005, 32: 403–413. 10.1016/j.nucmedbio.2005.02.002View ArticlePubMedGoogle Scholar
- Vaidyanathan G, Zalutsky MR: Preparation of N-succinimidyl 3-[*I]iodobenzoate: an agent for the indirect radioiodination of proteins. Nat Protoc 2006, 1: 707–713. 10.1038/nprot.2006.99View ArticlePubMedGoogle Scholar
- Martin R, Juttler S, Muller M, Wester HJ: Cationic eluate pretreatment for automated synthesis of [68Ga]CPCR4.2. Nucl Med Biol 2014, 41: 84–89. 10.1016/j.nucmedbio.2013.09.002View ArticlePubMedGoogle Scholar
- Mueller D, Klette I, Baum RP, Gottschaldt M, Schultz MK, Breeman WA: Simplified NaCl based 68Ga concentration and labeling procedure for rapid synthesis of 68Ga radiopharmaceuticals in high radiochemical purity. Bioconj Chem 2012, 23: 1712–1717. 10.1021/bc300103tView ArticleGoogle Scholar
- Notni J, Simecek J, Hermann P, Wester HJ: TRAP, a powerful and versatile framework for 68Ga radiopharmaceuticals. Chemistry 2011, 17: 14718–14722. 10.1002/chem.201103503View ArticlePubMedGoogle Scholar
- Afshar-Oromieh A, Malcher A, Eder M, Eisenhut M, Linhart HG, Hadaschik BA, Holland-Letz T, Giesel FL, Kratochwil C, Haufe S, Haberkorn U, Zechmann CM: PET imaging with a 68Ga-labelled PSMA ligand for the diagnosis of prostate cancer: biodistribution in humans and first evaluation of tumour lesions. Eur J Nucl Med Mol Imaging 2013, 40: 486–495. 10.1007/s00259-012-2298-2View ArticlePubMedGoogle Scholar
- Cho SY, Gage KL, Mease RC, Senthamizhchelvan S, Holt DP, Jeffrey-Kwanisai A, Endres CJ, Dannals RF, Sgouros G, Lodge M, Eisenberger MA, Rodriguez R, Carducci MA, Rojas C, Slusher BS, Kozikowski AP, Pomper MG: Biodistribution, tumor detection, and radiation dosimetry of 18F-DCFBC, a low-molecular-weight inhibitor of prostate-specific membrane antigen, in patients with metastatic prostate cancer. J Nucl Med 2012, 53(Supplement 1):1883–1891. 10.2967/jnumed.112.104661PubMed CentralView ArticlePubMedGoogle Scholar
- Baur B, Solbach C, Andreolli E, Winter G, Machulla HJ, Reske SN: Synthesis, radiolabelling and in vitro characterization of the 68Ga, 90Y and 177Lu-labelled PSMA ligand, CHX-A''-DTPA-DUPA-Pep. Pharmaceuticals (Basel) 2014, 7: 517–529. 10.3390/ph7050517View ArticleGoogle Scholar
- Winter G, Zlatopolskiy B, Kull T, Bertram J, Genze F, Cudek G, Machulla H-J, Reske S: 68Ga-DOTA-DUPA-Pep as a new peptide conjugate for molecular imaging of prostate carcinoma. J Nucl Med 2011, 52: 1597.Google Scholar
- Benesova M, Schaefer M, Bauder-Wuest U, Mier W, Haberkorn U, Eder M, Kopka K: Preclinical evaluation of a naphthyl-containing DOTA-conjugated PSMA inhibitor with improved targeting and pharmacokinetic properties. Eur J Nucl Med Mol Imaging 2014, 2(Supplement):438.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.