Development of novel ⁶⁸Ga- and ¹⁸F-labeled GnRH-I analogues with high GnRHR-targeting efficiency

A large majority of tumors of the reproductive system express the gonadotropin releasing hormone receptor (GnRHR). Blockade and activation of this receptor with various antagonistic and agonistic analogues of native GnRH-I (pGlu¹-His²-Trp³-Ser⁴-Tyr ⁵-Gly⁶-Leu⁷-Arg⁸-Pro ⁹-Gly¹⁰-NH₂), respectively, has shown efficient suppression of tumor growth. In this study, the GnRH-receptor system has been evaluated with respect to its suitability as a target for in vivo peptide receptor targeting using radiolabeled GnRH-analogues, and in parallel, new ¹⁸F- and ^68Ga-labeled GnRH analogues have been developed. In vitro radioligand binding assays performed with various GnRHR-expressing human cell lines using [¹²⁵I]Triptorelin (D-Trp⁶-GnRH-I) as the standard radioligand revealed a very low level of GnRH receptor expression on the cell surface. Generally, total cellular activity was very low (∼3% of the applied activity), and only a small fraction (max. 40%) of cell-associated activity could be attributed to receptor-specific radioligand binding/internalization. However, substitution of fetal calf serum by NU serum in the culture medium led to increased and stable GnRHR-expression, especially in the ovarian cancer cell line EFO-27, thus allowing for a stable experimental setup for the evaluation of the new radiolabeled GnRH-I analogues. The new radiolabeled GnRH-I analogues developed in this study were all based on the D-Lys⁶-GnRH-I-scaffold. For ⁶⁸Ga-labeling, the latter was coupled with DOTA at D-Lys⁶. To allow ¹⁸F-labeling via chemoselective oxime formation, D-Lys⁶-GnRH-I was also conjugated with Ahx (aminohexanoic acid) or β-Ala, which in turn was coupled with Boc-aminooxyacetic acid. ¹⁸F-labeling via oxime formation with 4-[¹⁸F]fluorobenzaldehyde was performed using the Boc-protected precursors. Receptor affinities of [⁶⁸Ga]DOTA-GnRH-I, D-Lys ⁶-Ahx([¹⁸F]FBOA)-GnRH-I, and D-Lys⁶- βAla([¹⁸F]FBOA)-GnRH-I (FBOA = fluorobenzyloxime acetyl) were determined using GnRHR-membrane preparations, and internalization efficiency of the new radioligands was determined in EFO-27 cells. Both quantities were highest for D-Lys⁶-Ahx([¹⁸F]FBOA)-GnRH-I (IC₅₀ = 0.50 ± 0.08 nM vs 0.13 ± 0.08 nM for Triptorelin; internalization: 86 ± 16% of the internal reference [¹²⁵I] Triptorelin), already substantially reduced in the case of the -βAla([ ¹⁸F]FBOA)-derivative (IC₅₀ = 0.86 ± 0.13 nM; internalization: 42 ± 3% of [¹²⁵I]Triptorelin), while the [⁶⁸Ga]DOTA-analogue showed almost complete loss of binding affinity and ligand internalization (IC₅₀ = 13.3 ± 1.0 nM; internalization: 2.6 ± 1.0% of [¹²⁵I]Triptorelin). Generally, the lipophilic residue [¹⁸F]FBOA is much better tolerated as a modification of the D-Lys⁶-side chain, with receptor affinity of the respective analogues strongly depending upon spacer length between the D-Lys⁶-side chain and the [¹⁸F]FBOA-moiety. In summary, D-Lys⁶(Ahx-[¹⁸F]FBOA)-GnRH-I shows the highest potential for efficient GnRHR-targeting in vivo of the compounds investigated. Unfortunately, however, the very low cell surface expression of GnRH-receptors and thus very low radioligand uptake by GnRHR-positive tumor cells found in vitro was also confirmed by a preliminary biodistribution study in OVCAR-3 xenografted nude mice using the standard GnRHR radioligand [¹²⁵I] Triptorelin. Tumor uptake was lower than blood activity concentration at 1 h p.i. (0.49 ± 0.05 vs 0.96 ± 0.13 for tumor and blood, respectively). These data seriously challenge the suitability of the GnRHR-system as a suitable target for in vivo peptide receptor imaging using radiolabeled GnRH-I derivatives, despite the availability of high-affinity radiolabeled receptor-ligands such as D-Lys⁶(Ahx-[ ¹⁸F]FBOA)-GnRH-I.