Synthesis and preclinical evaluation of DOTAGA-conjugated PSMA ligands for functional imaging and endoradiotherapy of prostate cancer

Abstract

Background: 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 ([(68)Ga]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.

Methods: All ligands were synthesized by a combined solid phase and solution phase synthesis strategy. The affinity of the (nat)gallium and lutetium complexes to PSMA and the internalization efficiency of the radiotracers were determined on PSMA-expressing LNCaP cells. The (68)Ga- and (177)Lu-labelled ligands were further investigated for lipophilicity, binding specificity, metabolic stability, as well as biodistribution and μPET in LNCaP-tumour-bearing mice.

Results: Radiochemical yields for (68)Ga (3 nmol, 5.0 M NaCl/2.7 M HEPES (approximately 5/1), pH 3.5 to 4.5, 5 min, 95°C) and (177)Lu 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 (68)Ga analogues and 38 GBq/μmol for (177)Lu 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 (68)Ga and (177)Lu, respectively) and improved affinity to PSMA resulting in an about twofold increased specific internalization of the (68)Ga- and (177)Lu-labelled DOTAGA analogue. Especially, [(68)Ga]DOTAGA-ffk(Sub-KuE) exhibits favourable pharmacokinetics, low unspecific uptake and high tumour accumulation in LNCaP-tumour-bearing mice.

Conclusions: The pair of diagnostic/therapeutic PSMA-ligands [(68)Ga/(177)Lu]DOTAGA-ffk(Sub-KuE) possess remarkable potential for the management of prostate cancer.

Figure 1

Structures of selected PSMA ligands. Depicted are the first ¹¹C-labelled and radiohalogenated PSMA ligands based on the XuE-scaffold (X = C, Y, K or E).

Figure 2

Previously reported PSMA ligands for ⁶⁸ Ga labelling [ 17,18 ]. Both compounds were included as references in this study.

Figure 3

Synthesis of the PSMA binding motif 3. (a) DCI, TEA, DMAP [DCM]; (b) H-Lys(Z)-OtBu, TEA [DCE]; (c) Pd/C (10%), H₂ [EtOH].

Figure 4

Modified solid-phase synthesis of the DOTA conjugate 10 [ 17 ] and DOTAGA conjugates 11 and 12. (a) 20% piperidine in NMP, Fmoc-Phe-OH, HOBt, TBTU, DIPEA, [NMP]; (b) 20% piperidine in NMP, DOTA-tris-tBu-ester, HATU, DIPEA [NMP]; (c) 95% TFA, 2.5% TIBS, 2.5% H₂O; (d) 20% piperidine in NMP, DOTAGA-anhydride, DIPEA [NMP]; (e) DSS, TEA [DMF]; (f) 7, TEA [DMF]; (g) TFA; (h) 8, TEA [DMF]; (i) 9, TEA [DMF].

Figure 5

Cell binding and internalization kinetics of ¹⁷⁷ Lu- 10 and ¹⁷⁷ Lu-11 in LNCaP cells. In PLL-coated plates, 125,000 cells/well were incubated with the respective radioligand (c = 0.5 nM) at 37°C in DMEM-F12 (5% BSA). The total cellular activity was corrected for non-specific binding (10 μM PMPA). All data are expressed as mean ± SD (n = 3).

Figure 6

Exemplary radio-HPLC analyses of extracts from homogenized organs and body fluids. HPLC-traces of CD-1 nu/nu mice (0.5 h p.i. of 40 to 45 MBq of ⁶⁸Ga labelled tracer, Chromolith column, flow rate 3 mL/min) for ⁶⁸Ga-11 (3% for 3 min, 3% to 95% in 6 min, 95% for 3 min) and ⁶⁸Ga-12 (3% to 95% in 6 min, 95% for 3 min).

Figure 7

Biodistribution data (in % ID/ g) at 1 h p.i.. The biodistribution of ⁶⁸Ga-12 in LNCaP tumour xenograft bearing CD-1 nu/nu mice (n =5) in comparison to ⁶⁸Ga-11 (n = 5) and [⁶⁸Ga]HBED-CC-Ahx-KuE (n = 4).

Figure 8

Maximum intensity projections (MIP) of μPET scans. MIP (1 h p.i. for 15 min, 0% to 6% ID/mL) of five LNCaP human prostate carcinoma xenograft bearing mice (right shoulder) after injection of approximately 15 MBq (a) [⁶⁸Ga]HBED-CC-Ahx-KuE, (b) ⁶⁸Ga-10, (c) ⁶⁸Ga-11, (d) ⁶⁸Ga-12, (d′) ⁶⁸Ga-12 + blocking (8 mg/kg PMPA).

Figure 9

Time -activity- curves (logarithmic plot) in % ID /mL for blood pool (heart), muscle, kidney and tumour. Graphs for (a) [⁶⁸Ga]HBED-CC-Ahx-KuE, (b) ⁶⁸Ga-10, (c) ⁶⁸Ga-11, (d) ⁶⁸Ga-12 are derived from dynamic small animal PET data.