Somatostatin Analogues ~ (99m) Tc-edda/hynic-lys ~ 0-toca Preparation And Glycosylation Derivatives Synthesis Research

The naturally occurring somatostatin is a cyclic neuropeptide of 14-amino acid residues and possess high affinity to all somatostatin receptors sst1-sst5. Somatostatin-14 has a very short biological half-life of several minutes, because of rapid in vivo enzymatic degradation, and this disadvantage precludes its use in vivo. To circumvent this problem, the eight-amino acid somatostatin analogue "Octreotide(OC)", with enhanced resistance to in vivo enzymatic degradation and preservation of the biological activity of the original somatostatin peptide, has been developed by molecular modifications to the orginal somatostatin peptide. Tyr³-Octreotide (TOC) was developed by replacing the Phe³ in octreotide with a Tyr residue, and ¹²³I-Tyr³-octreotide became the first somatostatin analogue used for in vivo imaging of somatostatin receptor-expressing tumors.¹¹¹In-DTPA-D-Phe¹-octreotide was developed later, by modifying at the N-terminus of TOC with a DTPA moiety(for labelling with ¹¹¹In). And it became the first peptide-based radiopharmaceutical to obtain regulatory approval in Europe and the USA for neuroendocrine tumors. Tyr³-Thr(OH)⁸-Octreotide (TOCA) is one of another octreotide analuges, which developed from TOC by replaced the Thr(ol)⁸ in TOC with Thr(OH). In contrast with TOC, TOCA significantly reduced the lipophilicity, and shows increased sst2 binding affinity. Daniel S. et al found that, the higher hydrophilicity and higher tumor-to-liver ratio of ^99mTc-EDDA/HYNIC-TOCA compared with ^99mTc-EDDA/HYNIC-TOC may consequently lead to an improved sensitivity in the detection of liver metastases.There are several approaches to get farther improvement of the in vivo physicochemical behavior of small radiolabled peptide pharmaceuticals. And carbohydration seems to be an appropriate method. Studies on the improvement of the in vivo behavior of glycosylated TOC and TOCA analogues had been done by Wester H. J. et al. The improved in vivo behavior of these analogues, such as blood and whole-body clearance, clearance pathways, reduction of liver and renal uptake, enhancement of urinary excretion, increased tumor uptake, and reduced unspectific binding was observed. These advantages make the radiolabelled glycosylated somatostatin analogues seem to have the potential use as radiopharmaceuticals for tumor imaging and therapy.[Objective]①The new somatostatin analogue ^99mTc-EDDA/HYNIC-Lys⁰-TOCA was designed and synthesized, and biological studies were carried out in normal and tumor-bearing nude mice. The impact on in vivo pharmacokinetics of ^99mTc-EDDA/HYNIC-Lys⁰-TOCA, in which lysine residue is introduced to the N-terminal of TOCA, was investigated by compared with ^99mTc-EDDA/HYNIC-TOCA, and its potential application was also discussed as an imaging agent for somatostatin receptor positive tumor.②Following H. J Wester's method, synthesis of glycosylated analogue of TOCA, N^α-Mtr, N^ε-HYNIC-Lys⁰-TOCA, was performed through Maillard-Amadori reaction[Methods]①N^α-Fmoc, N^ε-HYNIC(BOC)-Lys was synthesized, and N^ε-HYNIC-Lys⁰-TOCA was obtained by conjugation of N^α-Fmoc, N^ε-HYNIC(BOC)-Lys with TOCA(Lys⁵-BOC) followed by deprotection of Fmoc and BOC.②^99mTc labelling of N^ε-HYNIC-Lys⁰-TOCA was carried out with two-step approach using tricine/EDDA as coligands, and the radiolabelling conditions (such as the amount of SnCl₂. 2H₂O, pH, reaction time and reaction volume) were investigated.③The lipophilicity of ^99mTc-EDDA/HYNIC-Lys⁰-TOCA and its in vivo and in vitro stability were evaluated.④The biodistribution of ^99mTc-EDDA/HYNIC-Lys⁰-TOCA was exercised in normal mice.⑤The biodistribution and scintigraphy imaging of ^99mTc-EDDA/HYNIC-Lys⁰-TOCA were also done in tumor-bearing nude mice⑥N^α-Mtr, N^ε-HYNIC(BOC)-Lys⁰-TOCA(Lys⁵-BOC) was synthesized through Maillard-Amadori reaction, and followed by deprotection with TFA/TIPS/H₂O.[Results]①N^ε-HYNIC-Lys⁰-TOCA was synthesized, and characterized by ¹H-NMR and ESI-TOF mass spectra.②The labelling efficiency of ^99mTc-EDDA/HYNIC-Lys⁰-TOCA was approximately 96% under the optimized conditions, and more than 99% could be reached after Sep-pak column purification.③The radiolabelled complex showed satisfactory in vitro stability.④^99mTc-EDDA/HYNIC-Lys⁰-TOCA displayed a rapid blood clearance ( about 20min of half-life) and the predominant excretion via the kidney and urinary system in normal mice. Meanwhile. the substantial uptake of radioactivity in stomach, lung and adrenals was observed.⑤The biodistribution in tumor-bearing nude mice of ^99mTc-EDDA/HYNIC-Lys⁰-TOCA suggested that there was high radioactivity uptake in tumor and rapid blood clearance, and thus high T/NT ratios of tumor to heart, blood and muscle were obtained 1h post injection. The radio-tracer remained almost stable in urine as assayed by HPLC. Planar gamma imaging allowed contrasting visualisation of tumours at 1h and 2h post injection⑥Using modified approach, N^α-Mtr, N^ε-HYNIC(BOC)-Lys⁰-TOCA(Lys⁵-BOC) was synthesized in much higher yield (＞60%, 14h in this work; and＜10%, 20h in the literature). The resulted compound was characterized by ESI-TOF mass spectra. Unfortunately, the desired product was not obtained in the subsequent deprotection, in which O- and N-glycosidic bonds were probably cleaved in aqueous TFA. and the reaction became complicated as monitored by ESI-TOF mass spectra.[Conclusion]①^99mTc-EDDA/HYNIC-Lys⁰-TOCA with high labelling yield was obtained though two-step radiolabelling approach using Tricine/EDDA as coligand. The radio-compound has high in vitro and in vivo stability and low lipophilicity. The preliminary animal studies showed that ^99mTc-EDDA/HYNIC-Lys⁰-TOCA had rapid blood clearance and ideal excretion pathway. High uptake in tumor allowed contrasting visualisation of tumors in tumor-bearing nude mice. All these results indicated that ^99mTc-EDDA/HYNIC-Lys⁰-TOCA is a novel promising candidate for somatostatin receptor-positive tumor imaging.②N^α-Mtr, N^ε-HYNIC(BOC)-Lys⁰-TOCA(Lys⁵-BOC) was synthesized successfully by using two-step approach, and a yield of more than 60% was achieved. The expertise from this work will be helpful in the future pursuing peptide based radiophamerceuticals...