Development of Novel 68Ga- and 18F-Labeled GnRH-I Analogues with High GnRHR-Targeting Efficiency
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- 作者：Margret Schottelius ; Sebastian Berger ; Thorsten Poethko ; Markus Schwaiger ; Hans-Jü ; rgen Wester
- 刊名：Bioconjugate Chemistry
- 出版年：2008
- 出版时间：June 2008
- 年：2008
- 卷：19
- 期：6
- 页码：1256 - 1268
- 全文大小：282K
- 年卷期：v.19,no.6(June 2008)
- ISSN：1520-4812
文摘
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-Tyr5-Gly6-Leu7-Arg8-Pro9-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-Lys6-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.
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.