Preclinical Evaluation of Minigastrin Analogs and Proof-of-Concept [68Ga]Ga-DOTA-CCK-66 PET/CT in 2 Patients with Medullary Thyroid Cancer

Abstract

Because of the need for radiolabeled theranostics for the detection and treatment of medullary thyroid cancer (MTC), and the yet unresolved stability issues of minigastrin analogs targeting the cholecystokinin-2 receptor (CCK-2R), our aim was to address in vivo stability, our motivation being to develop and evaluate DOTA-CCK-66 (DOTA-γ-glu-PEG₃-Trp-(N-Me)Nle-Asp-1-Nal-NH₂, PEG: polyethylene glycol) and DOTA-CCK-66.2 (DOTA-glu-PEG₃-Trp-(N-Me)Nle-Asp-1-Nal-NH₂), both derived from DOTA-MGS5 (DOTA-glu-Ala-Tyr-Gly-Trp-(N-Me)Nle-Asp-1-Nal-NH₂), and clinically translate [⁶⁸Ga]Ga-DOTA-CCK-66. Methods: ⁶⁴Cu and ⁶⁷Ga labeling of DOTA-CCK-66, DOTA-CCK-66.2, and DOTA-MGS5 was performed at 90°C within 15 min (1.0 M NaOAc buffer, pH 5.5, and 2.5 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, respectively). ¹⁷⁷Lu labeling of these 3 compounds was performed at 90°C within 15 min (1.0 M NaOAc buffer, pH 5.5, 0.1 M sodium ascorbate). CCK-2R affinity of ^natGa/^natCu/^natLu-labeled DOTA-CCK-66, DOTA-CCK-66.2, and DOTA-MGS5 was examined on AR42J cells. The in vivo stability of ¹⁷⁷Lu-labeled DOTA-CCK-66 and DOTA-MGS5 was examined at 30 min after injection in CB17-SCID mice. Biodistribution studies at 1 h ([⁶⁷Ga]Ga-DOTA-CCK-66) and 24 h ([¹⁷⁷Lu]Lu-DOTA-CCK-66/DOTA-MGS5) after injection were performed on AR42J tumor-bearing CB17-SCID mice. In a translation to the human setting, [⁶⁸Ga]Ga-DOTA-CCK-66 was administered and whole-body PET/CT was acquired at 120 min after injection in 2 MTC patients. Results: Irrespective of the metal or radiometal used (copper, gallium, lutetium), high CCK-2R affinity (half-maximal inhibitory concentration, 3.6-6.0 nM) and favorable lipophilicity were determined. In vivo, increased numbers of intact peptide were found for [¹⁷⁷Lu]Lu-DOTA-CCK-66 compared with [¹⁷⁷Lu]Lu-DOTA-MGS5 in murine urine (23.7% ± 9.2% vs. 77.8% ± 2.3%). Overall tumor-to-background ratios were similar for both ¹⁷⁷Lu-labeled analogs. [⁶⁷Ga]Ga-DOTA-CCK-66 exhibited accumulation (percentage injected dose per gram) that was high in tumor (19.4 ± 3.5) and low in off-target areas (blood, 0.61 ± 0.07; liver, 0.31 ± 0.02; pancreas, 0.23 ± 0.07; stomach, 1.81 ± 0.19; kidney, 2.51 ± 0.49) at 1 h after injection. PET/CT examination in 2 MTC patients applying [⁶⁸Ga]Ga-DOTA-CCK-66 confirmed multiple metastases. Conclusion: Because of the high in vivo stability and favorable overall preclinical performance of [^nat/67Ga]Ga-/[^nat/177Lu]Lu-DOTA-CCK-66, a proof-of-concept clinical investigation of [⁶⁸Ga]Ga-DOTA-CCK-66 was completed. As several lesions could be identified and excellent biodistribution patterns were observed, further patient studies applying [⁶⁸Ga]Ga- and [¹⁷⁷Lu]Lu-DOTA-CCK-66 are warranted.

Keywords: CCK-2R; DOTA-CCK-66; clinical translation; medullary thyroid cancer.

Graphical abstract

FIGURE 1.

Chemical structures of compounds evaluated. All comprise same C-terminal tetrapeptide binding motif and N-terminal DOTA chelator but differ in linker section. Orange = Ala-Tyr-Gly sequence; green = PEG₃ moiety; blue = α-bridged d-glutamic acid moiety; violet = γ-bridged d-glutamic acid moiety.

FIGURE 2.

In vitro data of ^nat/64Cu-, ^nat/67Ga-, and ^nat/177Lu-labeled CCK-2R ligands. Data are expressed as mean ± SD. (A) Affinity data (n = 3) on AR42J cells (2.0 × 10⁵ cells per well) using [¹⁷⁷Lu]Lu-DOTA-PP-F11N (0.3 pmol/well) as radiolabeled reference (3 h, 37°C, RPMI 1640, 5 mM l-Gln, 5 mL nonessential amino acids [×100], 10% fetal calf serum + 5% bovine serum albumin [v/v]). (B) logD_7.4 (n = 6). (C) Human serum albumin binding as determined by high-performance affinity chromatography. n.s. = not significant. *P < 0.05. **P < 0.01. ***P < 0.0001.

FIGURE 3.

In vivo stability of CCK-2R ligands: amount of intact compound at 30 min after injection (3 each) in murine serum (red) and urine (blue) for [¹⁷⁷Lu]Lu-DOTA-MGS5 (A) and [¹⁷⁷Lu]Lu-DOTA-CCK-66 (B) (quality control in gray).

FIGURE 4.

(A) Biodistribution of [⁶⁷Ga]Ga-DOTA-CCK-66 (1 h after injection), as well as [¹⁷⁷Lu]Lu-DOTA-CCK-66 and [¹⁷⁷Lu]Lu-DOTA-MGS5 (24 h after injection), in selected organs in AR42J tumor–bearing CB17-SCID mice (100 pmol each). Data are %ID/g (mean ± SD, 4 each). (B) Maximum-intensity projection of AR42J tumor (arrows)–bearing CB17-SCID mice (1 each) injected with [⁶⁷Ga]Ga-DOTA-CCK-66, as well as [¹⁷⁷Lu]Lu-DOTA-CCK-66 and [¹⁷⁷Lu]Lu-DOTA-MGS5 (100 pmol each). Images were acquired at either 1 or 24 h after injection. n.s. = not significant; p.i. = after injection. *P < 0.05. **P < 0.01.

FIGURE 5.

Maximum-intensity projection (left) and fused transaxial slices (right) of 64-y-old male MTC patient undergoing PET/CT at 2 h after intravenous injection of 151 MBq of [⁶⁸Ga]Ga-DOTA-CCK-66. CCK-2R–expressing lymph node metastases could be detected in left retroclavicular region and in upper mediastinum (arrows).

FIGURE 6.

Maximum-intensity projections (left) and fused transaxial sections (right) of 46-y-old female MTC patient undergoing PET/CT at 2 h after intravenous injection of 193 MBq of [⁶⁸Ga]Ga-DOTA-CCK-66. Several lymph node (e.g., right retroclavicular), liver, and bone metastases (e.g., right femur and right ischium) could be detected (arrows).