A dimerized urea-based inhibitor of the prostate-specific membrane antigen for 68Ga-PET imaging of prostate cancer

Abstract

Background: Alternative positron-emission tomography (PET) probes like labeled inhibitors of the prostate-specific membrane antigen (PSMA) are of emerging clinical impact as they show the ability to image small lesions of recurrent prostate cancer. Here, the dimerization of the pharmacophore Glu-ureido-Lys via the 68Ga chelator N,N'-bis[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N'-diacetic acid (HBED-CC) was investigated to further improve the binding characteristics and pharmacokinetics.

Methods: The peptidomimetic structures were synthesized by solid-phase chemistry, and the resulting products were coupled with the respective 2,3,5,6-tetrafluorophenol esters of HBED-CC to form the monomeric reference and the dimeric Glu-ureido-Lys derivative. The binding properties were analyzed in competitive binding, internalization, and cell surface retention experiments. PET images and biodistribution data were obtained 1 h after injection in BALB/c nu/nu mice bearing LNCaP tumor xenografts.

Results: Cell binding data revealed significant better binding properties of the dimer (IC50 = 3.9 ± 1.8 nM; IC50 (monomer) = 12.1 ± 2.1 nM). The inhibition potency investigated by the enzyme-based NAALADase assay confirmed these results. Specific internalization in LNCaP cells was demonstrated for both, the monomer and dimer. As shown by efflux measurements, the dimeric compound was more effectively retained on the cell surface, resulting in advanced in vivo properties (T/BMonomer = 9.2; T/BDimer = 26.5).

Conclusions: The dimeric [68Ga]7 is a promising imaging agent for PSMA-expressing tumors as it shows higher tumor uptake while observing more favorable background clearance. As compared to the respective monomer, the higher affinity and prolonged tumor retention additionally represent promising features and warrant further evaluation regarding 68Ga-PET imaging of PSMA expression.

Figure 1

Syntheses of Glu‐ureido‐Lys(Ahx)-HBED-CC (6) and [Glu‐ureido‐Lys(Ahx)]₂-HBED-CC (7). (a) Triphosgene, DIPEA, CH₂Cl₂, 0°C; (b) H-Lys(Alloc)-2CT-Resin, CH₂Cl₂; (c) Pd[P(C₆H₅)₃]₄, morpholine, CH₂Cl₂; (d) Fmoc-6-Ahx-OH, HBTU, DIPEA, DMF; (e) 20% piperidine, DMF; (f) hexafluoroisopropanol/CH₂Cl₂; (g) HBED-CC-TFP ester, DIPEA, DMF; (h) TFA; (i) (HBED-CC)TFP₂ diester, DIPEA, DMF; (j) TFA.

Figure 2

Cell binding and internalization of [⁶⁸Ga]6 and [⁶⁸Ga]7 (A). Specific cellular uptake was evaluated by blockage using 100 μM 2-PMPA. (B) The graph shows the release of radioactivity from cells in percentage of initially bound compound. (C) The release of radioactivity related to the initial cellular uptake derived from A. Values in A and C are expressed as percentage of applied radioactivity bound to 10⁶ cells. Data are expressed as mean ± SD (n = 3).

Figure 3

Organ distribution expressed as % ID/g tissue 1 h post-injection. (A) Comparison of the monomer [⁶⁸Ga]6 and the dimer [⁶⁸Ga]7. (B) PSMA blocking by co-administration of 2 mg of 2-PMPA/kg body weight. Data are expressed as mean ± SD (n = 5).

Figure 4

Whole-body coronal microPET image of an athymic male nude mice bearing LNCaP tumor xenograft. The monomer [⁶⁸Ga]6 (A) and the dimer [⁶⁸Ga]7 (B) were evaluated by a dynamic microPET scan followed by a static scan. The static scans 1 h post-injection of [⁶⁸Ga]6 and [⁶⁸Ga]7 are shown in (A) and (B), respectively. Approximately 15 MBq/mouse was injected. (C) The graph shows the respective time-activity curves in the muscle and tumor for both tracers. (D) The graph demonstrates the elimination of [⁶⁸Ga]7 from other organs in PET.

Figure 5

Time-activity curves of [⁶⁸Ga]7 and [⁶⁸Ga]d-7 taken from dynamic PET measurements and expressed as % ID/g.