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 GnRE-I (pGlu(1)-HiS(2) -Trp(3)-Sef(4)-Tyr(5)-Gly(6) -Leu(7) -Arg(8)-Pro(9)-Gly(10)-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 F-18- and Ga-68-labeled GnRH analogues have been developed. In vitro radioligand binding assays performed with various GnRHR-expressing human cell lines using [I-125]Triptorelin (D-Trp(6)-GnRH-1) as the standard radioligand revealed a very low level of GnRH receptor expression on the cell surface. Generally, total cellular activity was very low (similar to 3% of the applied activity), and only a small fraction (max. 40%) of cell-associated activity could be attributed to receptor-specific radioligand binding/intemalization. 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-Lys(6)-GnRH-I-scaffold. For Ga-68-labeling, the latter was coupled with DOTA at D-LyS(6). To allow F-18-labeling via chemoselective oxime formation, D-LyS(6)-GnRH-I was also conjugated with Ahx (ammohexanoic acid) or beta-Ala, which in turn was coupled with Boc-aminooxyacetic acid. F-18-labeling via oxime formation with 4-[F-18]fluorobenzaldehyde was performed using the Boc-protected precursors. Receptor affinities of [Ga-68]DOTA-GnRH-I, D-LyS(6)-AhX([F-18]FBOA)GnRH-I, and D-Lys(6) -beta Ala([F-18]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-Lys(6)-Ahx([F-18]FBOA)-GnRH-1 (IC50 = 0.50 +/- 0.08 nM vs 0.13 +/- 0.08 nM for Triptorelin; internalization: 86 +/- 16% of the internal reference [I-125]Triptorelin), already substantially reduced in the case of the -fiAla([F-18]FBOA)-derivative (IC50 = 0.86 +/- 0.13 nM; internalization: 42 +/- 3% of [I-125]Triptorelin), while the [Ga-68]DOTA-analogue showed almost complete loss of binding affinity and ligand internalization (IC50 = 13.3 +/- 1.0 nM; internalization: 2.6 +/- 1.0% of [I-125]Triptorelin). Generally, the lipophilic residue [F-18]FBOA- is much better tolerated as a modification of the D-Lys(6)-side chain, with receptor affinity of the respective analogues strongly depending upon spacer length between the D-Lys(6)-side chain and the [F-18]FBOA-moiety. In summary, D-LyS6 (Ahx-[F-18]FBOA)-GnRH-1 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 thusvery 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 [I-125]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-Lys(6) (Ahx-[(8) F]FBOA)-GnRH-I.