Preclinical evaluation of new GnRH-I receptor radionuclide therapy with 177 Lu-peptide tracer.

The purpose of this study was to develop preclinical evaluation of a novel radiolabeled gonadotropin-releasing hormone (GnRH) receptor targeting peptide for prostate cancer therapy. The new antiproliferative agent of GnRH-I analogue was developed on the basis of the D-Trp6 -GnRH-I scaffold, and in vivo pharmacokinetics and receptor binding affinity were enhanced by the substitution of Gly-NHNH2 for Gly-NH2 at position 10 in D-Trp6 -GnRH-I. To evaluate 177 Lu-DOTA-triptorelin-hydrazide as radionuclide therapy of tumor, the quality control tests and preclinical stage assessment were carried out. Solid-phase method was used to synthesize new peptide. Characterization and purity of peptide were done by mass spectroscopy and high-performance liquid chromatography (HPLC). In order to be utilized in targeted therapy, the new GnRH-I agonist was coupled with pSCN-Bn-DOTA. The precipitate crude of DOTA-triptorelin-hydrazide was then purified via preparative HPLC. At optimal conditions of time, temperature, ligand amount, and lutetium content, DOTA-triptorelin-hydrazide was labeled with 177 Lu (specific activity not less than 925 GBq/mg). Investigation of the in vivo biodistribution and in vitro studies for 177 Lu-DOTA-TRPHYD was performed in three different ways, and the binding of radiopeptide to GnRH receptors was expressed on the human cell lines using 125 I-labeled D-TRP6 GnRH-I as a tracer, respectively. Synthesized novel GnRH-I was obtained with purity greater than 98%. Paper chromatography was found to be the most suitable with Rf of the complex and observed radiochemical purity of RTLC and HPLC greater than 97%. For in vivo studies, 177 Lu-DOTA-triptorelin-hydrazide showed promising results with fast clearance from the blood and resulted in good T/NT ratios at 1, 4, and 24 hours postinjection and satisfactory biodistribution with no significant activity seen in normal tissue. The values of internalization efficiency and receptor affinity of new radiopeptide binding were IC50 = 0.47 ± 0.06 vs 0.13 ± 0.01 nM for triptorelin and cellular uptake: 3.4 ± 0.7% at 1 hour and 6.8 ± 1.17% at 4 hours of the internal reference. The results showed a good stability and radiochemical purity of the obtained radioconjugate. For in vivo and in vitro studies, new radiopeptide showed a high uptake of 177 Lu conjugate in tumor and rapid clearance from the blood stream almost entirely via the renal/urinary pathway and binding to the GnRH receptors with high specificity and affinity, respectively.

Preparation of a radiolabeled GnRH-I analogue derivative with 111 In as a new anti-proliferative agent.

The new GnRH-Ιanalogue developed in this paper was based on the D-Trp6 -GnRH-Ι-scaffold, and its potency was increased by the replacement Gly-NH2 by NH-NH2 binding to the Gly at position 10. Triptorelin-Hydrazide analogue was synthesized using solid phase. For 111 In labeling, synthesized peptide was followed by conjugation with DOTA using pSCN-Bn-DOTA. The conjugated Triptorelin-Hydrazide was labeled with 500-550 MBq of 111 In-chloride (in 0.2 M HCl). At optimized conditions after labeling, radio-chromatography showed radiochemical purity of approximately equal to 98% (RTLC) and greater than 95% (HPLC). The serum stability of the tracer was determined up to 24 hr. Binding affinities of Triptorelin-Hydrazide analogue were determined in a binding assay for both human and rat GnRH receptors. For in vivo studies, 111 In-peptide was injected intravenously via the tail vein into rats and significant ovaries uptake consist with reported GnRH receptor mappings. In vitro radioligand binding assays performed with GnRHR-expressing human cell lines using 125 I-Triptorelin as the standard radioligand. The quantities of internalization efficiency and receptor affinity of the new radioligand were IC50  = 0.20 ± 0.04 nM vs 0.13 ± 0.08 nM for Triptorelin and internalization: 3.5 ± 0.9% at 1 hr and 12.8 ± 1.8% at 4 hr of the internal reference.

Development of a (68)Ga-peptide tracer for PET GnRH1-imaging.

OBJECTIVE: Total synthesis, quality control and preclinical evaluation of [(68)Ga]-DOTA-triptorelin ([(68)Ga]-DOTA-TRP) is reported as a possible PET radiotracer for GnRH receptor imaging. METHODS: DOTA-TRP was totally synthesized in two steps and after characterization went through radiolabelling optimization studies followed by tracer stability. The biodistribution of the tracer in normal male rats and 4T1 tumour-bearing mice was performed in 120 min after i.v. injection. RESULTS: The peptide and the conjugates were synthesized with >95 % chemical purity. [(68)Ga]-DOTA-TRP complex was prepared in high radiochemical purity (>99 %, ITLC, HPLC) and specific activity of 1400-2100 MBq/nM at 95 °C using 40-60 µg of the peptide in 5-7 min followed by solid phase purification. The IC50 [nM] DOTA-TRP was comparable to the intact peptide, 0.11 ± 0.01 and 0.22 ± 0.05, respectively. The biodistribution of the tracer demonstrated kidney, stomach, and testes significant uptake, all in accordance with GnRH receptor ligands. Significant tumour uptake was observed in 4T1 tumour-bearing female mice 30-120 min post-injection with tumour:blood and tumour:muscle ratios of 28 and >50 in 60 min, respectively. Kidney is rapidly washed from the tracer. [(68)Ga]-DOTA-TRP can be proposed as a possible tracer for GnRH-R imaging studies.