Technetium-99m somatostatin analogues: effect of labelling methods and peptide sequence.

In this paper the preclinical evaluation of the somatostatin analogue RC160 labelled with technetium-99m using bifunctional chelators (BFCs) based on the hydrazinonicotinamide (HYNIC) and N(3)S system is described and a comparison made with [Tyr(3)]-octreotide (TOC). Conjugates of both peptides with HYNIC, and of RC160 with benzoyl-MAG(3) and an N(3)S-adipate derivative were prepared and radiolabelling performed at high specific activities using tricine, tricine/nicotinic acid and ethylenediamine-N,N'-diacetic acid (EDDA) as co-ligands for HYNIC conjugates. All conjugates and (99m)Tc-labelled peptides showed preserved binding affinity for the somatostatin receptor (IC50, Kd<5 nM). The biodistribution was markedly dependent on the BFC and co-ligand used, with the amidothiol ligands showing a greater degree of hepatobiliary clearance, the HYNIC/tricine complex higher blood levels and the HYNIC/EDDA complex the highest level of renal excretion and lowest blood levels. All peptide conjugates showed receptor-mediated uptake in tumour xenografts, but tumour uptake was significantly lower for the (99m)Tc-RC160 derivatives compared with (99m)Tc-EDDA/HYNIC-[Tyr(3)]-octreotide (0.2%-3.5%ID/g vs 9.7%ID/g) and correlated well with the reduced internalisation rate for RC160 derivatives. Our results show that the selection of the labelling approach as well as the right choice of the peptide structure are crucial for labelling peptides with (99m)Tc to achieve complexes with favourable biodistribution. Despite the relatively low tumour uptake compared with (99m)Tc-EDDA/HYNIC-[Tyr(3)]-octreotide, (99m)Tc-RC160 could play a role in imaging tumours that do not bind octreotide derivatives.     

99mTc-HYNIC-[Tyr3]-octreotide for imaging somatostatin-receptor-positive tumors: preclinical evaluation and comparison with 111In-octreotide.

In this paper we describe the preclinical evaluation of 99mTc-hydrazinonicotinyl-Tyr3-octreotide (HYNIC-TOC) using different coligands for radiolabeling and a comparison of their in vitro and in vivo properties with 111In-diethylenetriaminepentaacetic acid (DTPA)-octreotide. METHODS: HYNIC-TOC was radiolabeled at high specific activities using tricine, ethylenediaminediacetic acid (EDDA), and tricine-nicotinic acid as coligand systems. Receptor binding was tested using AR42J rat pancreatic tumor cell membranes. Internalization and protein binding studies were performed, and biodistribution and tumor uptake were determined in AR42J tumor-bearing nude mice. RESULTS: All 99mTc-labeled HYNIC peptides showed retained somatostatin-receptor binding affinities (Kd < 2.65 nM). Protein binding and internalization rates were dependent on the coligand used. Specific tumor uptake between 5.8 and 9.6 percentage injected dose per gram (%ID/g) was found for the 99mTc-labeled peptides, compared with 4.3 %ID/g for 111In-DTPA-octreotide. Tricine as coligand showed higher activity levels in muscle, blood, and liver, whereas tricine-nicotinic acid produced significant levels of activity in the gastrointestinal tract. EDDA showed the most promising overall biodistribution profile, with tumor-to-liver and tumor-to-gastrointestinal tract ratios similar to those obtained with 111In-DTPA-octreotide, lower ratios in blood and muscle, but considerably higher tumor-to-kidney ratios. CONCLUSION: TOC can be radiolabeled to high specific activities using HYNIC as a bifunctional chelator. The high specific tumor uptake, rapid blood clearance, and predominantly renal excretion make 99mTc-EDDA-HYNIC-TOC a promising candidate for an alternative to 111In-DTPA-octreotide for tumor imaging.     

Investigation of 99mTc-labelling of recombinant human interleukin-2 via hydrazinonicotinamide

IntroductionInterleukin-2 (IL-2) when radiolabelled with ^99mTc has been proved useful in imaging the side of lymphocytic infiltration in patients with autoimmune disorders and plays a significant role as a T-cell imaging agent. However, the labelling procedures used so far appeared to be rather complex and laborious. The aim of present study was to develop an efficient procedure of ^99mTc-labelling of recombinant human interleukin-2 (rhIL-2) via hydrazinonicotinamide (HYNIC) to develop a dry kit formulation.MethodsVarious molar ratios of rhIL-2/HYNIC (from 1:2 to 1:12) were used at the conjugation step. The conjugates were purified on a PD-10 column to remove the excess of unbound HYNIC, as well as of any aggregates. The final peptide concentration was quantified by the BCA method, and the number of HYNIC molecules incorporated into a rhIL-2 molecule was determined based on the reaction with 2-sulfobenzaldehyde. The ^99mTc-labelling was optimized using various amounts of HYNIC–rhIL-2, ^99mTc, SnCl₂, tricine and nicotinic acid (NA). Quality control included GF-HPLC, ITLC, SDS-PAGE and biological assay. Biodistribution studies were performed in Swiss mice and Wistar rats.ResultsGenerally, the highest radiolabelling yields were achieved when the HYNIC–rhIL-2 conjugates of ca. 2–4 HYNIC molecule substitution ratios were used. The optimal pH of the reaction medium was found to be in the range of 6.5 to 7.0. GF-HPLC analysis indicated that monomer and aggregates of ^99mTc-HYNIC–rhIL-2 are formed during radiolabelling. At optimized conditions of wet radiolabelling, the ^99mTc-HYNIC–rhIL-2 monomer was obtained with radiochemical purity >99%, specific activity of ca. 4 GBq/mg rhIL-2 and overall yield of ca. 65%. The two-vial freeze-dried kit was prepared: the first vial contained 30 μg HYNIC–rhIL-2, co-ligands, buffer and antioxidant; the second vial contained tricine and SnCl₂. The monomer of ^99mTc-HYNIC–rhIL-2 was obtained by gel chromatography on a PD-10 column. No differences between labelled and unlabelled IL2 in terms of biological activity were observed.ConclusionsOur study shows that rhIL-2 can be efficiently radiolabelled with ^99mTc via HYNIC, with tricine and NA as co-ligands using a two-vial freeze-dried kit. This enables the preparation of sterile and ready-to-use ^99mTc-HYNIC(tricine,NA)-rhIL-2 within 1 h.     

99Tcm-（HYNIC-[Lys3]-BBS）（tricine）（tricine）的制备及对胰腺癌荷瘤鼠显像研究

目的制备99Tcm-（联肼尼克酰胺-蛙皮素类似肽）（N-三羟甲基甘氨酸）（三苯基膦三间磺酸钠盐）[（HYNIC-[Lys3]-BBS）（tricine）（TPPTS）]三重配位化合物,评价其在正常小鼠及胰腺癌荷瘤裸小鼠的生物分布。方法双功能螯合剂HYNIC与[Lys3]-BBS偶联（pH值9．0）,以SnCl2为还原剂,tricine和TPPTS为协同配体,进行99Tcm-标记,合成三重配位化合物99Tcm-（HYNIC-[Lys。]-BBS）（tricine）（TPPTS）。用Sep-PakC18cartridge和HPLC对其纯化和分析,测定其标记率和放化纯,研究其在人血清中的稳定性,并进行正常小鼠体内的生物分布研究以及胰腺癌荷瘤裸小鼠活体显像。结果99Tcm-（HYNIC_[Lys3]-BBS）（tricine）（TPPTS）标记率为（90±2）％,放化纯〉95％,在人血清中放置4h其放化纯仍大于85％。正常小鼠体内分布结果表明,99Tcm-（HYNIC-[Lys3]-BBS）（tricine）（TPPTS）血液清除迅速,2h血液中放射性为（0．07±0．01）％ID／g,主要经。肾排泄,肝、胃肠道摄取较少,2h时肝放射性为（0．27±0．03）％ID／g,胃为（0．06±0．03）％ID／g,肠为（0．04±0．00）％ID／g。胰腺癌荷瘤裸小鼠吖显像可见肿瘤部位有放射性浓聚影,2h后肿瘤与对侧正常肌肉的T／NT比值最高达3．71±0．57。结论99Tcm-（HYNIC-[Lys3]-BBS）（tricine）（TPPTS）三重配位化合物制备成功,所用标记方法可行,标记物稳定性较好,标记率和放化纯较高,生物分布特性良好,有望用于胰腺癌的显像研究。     

Preclinical evaluation of [99mTc/EDDA/tricine/HYNIC0, 1-Nal3, Thr8]-octreotide as a new analogue in the detection of somatostatin-receptor-positive tumors

PURPOSE: Radiolabeled somatostatin analogues are important tools for the in vivo localization and targeted radionuclide therapy of somatostatin-receptor-positive tumors. The aim of this study was to evaluate a new somatostatin analogue designed for the labeling with (99m)Tc: [6-hydrazinopyridine-3-carboxylic acid (HYNIC(0)), 1-Nal(3), Thr(8)]-octreotide ([HYNIC]-NATE), using ethylenediamine-N,N'-diacetic acid (EDDA) and tricine as coligands. METHODS: Synthesis was preformed on a solid phase using a standard Fmoc strategy. Labeling with (99m)Tc was performed at 100 degrees C for 10 min using SnCl(2) as a reductant. Radiochemical analysis involved ITLC and high-performance liquid chromatography methods. Peptide conjugate affinity was determined in AR4-2J cell membranes. The internalization and externalization rates were studied in sstr(2)-expressing AR4-2J cells. Biodistribution of radiopeptide was studied in rats bearing the AR4-2J tumor. RESULTS: Radiolabeling was performed at high specific activities, and radiochemical purity was >95%. Peptide conjugate showed high affinity binding for sstr(2). The radioligand showed a moderate and specific internalization into AR4-2J cells (14.13+/-0.61% at 4 h). In animal biodistribution studies, a receptor-specific uptake of radioactivity was observed in somatostatin-receptor-positive organs. After 4 h, uptake in the AR4-2J tumor was 1.33+/-0.23%ID/g (percentage of injected dose per gram of tissue). CONCLUSION: These data show that [(99m)Tc/EDDA/tricine/HYNIC]-NATE is a specific radioligand for the somatostatin-receptor-positive tumors and is a suitable candidate for clinical studies.     

99m-Technetium-labelled peptide-HYNIC conjugates: effects of lipophilicity and stability on biodistribution.

The aim of this study was to explore the effects of lipophilicity and stability on the biodistribution of 99mTc labelled peptides through the use of different co-ligands. 6-Hydrazinopyridine-3-carboxylic acid (HYNIC) was coupled to the somatostatin analogue RC160 and radiolabelled using a range of ethylendiaminediacetic acid (EDDA) and ethylenediaminetetraacetic acid (EDTA) derivatives as well as tricine and pyridine/tricine as co-ligands. After labelling with technetium-99m, chromatographic, stability, protein-binding, and rat biodistribution studies were performed. For most co-ligands, biodistribution correlated well with in vitro properties. Lipophilic substitution on EDDA resulted in higher protein binding, increased liver uptake, and intestinal excretion. Stabilisation of tricine with pyridines reduced blood levels and lowered liver uptake. EDTA derivatives showed high instability in vitro and in vivo.     

Radio-guided surgery with the use of [<Superscript>99m</Superscript>Tc-EDDA/HYNIC]octreotate in intra-operative detection of neuroendocrine tumours of the gastrointestinal tract

Purpose Radio-guided surgery (RGS) is an intra-operative localising technique which enables identification of tissue “marked” by a specific radiotracer injected before surgery. It is mainly used for sentinel node mapping and for detection of parathyroid adenomas and other tumours, including neuroendocrine tumours of the gastrointestinal tract (GEP-NET). The aim of this study was to determine whether intra-operative radio-detection with the use of [^99mTc-EDDA/HYNIC]octreotate, a new somatostatin analogue, is able to reveal an unknown primary and secondary sites, thereby improving surgical treatment and the final outcome of GEP-NET. Methods The study group included nine patients with suspected GEP-NET (four carcinoids, five pancreatic NET) localised with somatostatin receptor scintigraphy (with [^99mTc-EDDA/HYNIC]octreotate), who had negative results on other pre-operative imaging tests. At surgery, suspected tumours were measured in situ and ex vivo and precise exploration of the abdominal cavity was performed with the intra-operative scintillation detector (Navigator). Results Intra-operative gamma counting localised three carcinoids. In one patient SRS was false positive (owing to inflammatory infiltration). Compared with SRS, RGS revealed additional lymph node metastases in one case. RGS resulted in successful localisation of all pancreatic NET (the smallest lesion was 8 mm in diameter). Conclusion [^99mTc-EDDA/HYNIC]octreotate SRS followed by RGS is a promising technique to improve the rate of detection and efficacy of treatment of GEP-NET, especially in the presence of occult endocrine tumours. The imaging properties of [^99mTc-EDDA/HYNIC]octreotate and the 1-day imaging protocol offer opportunities for more widespread application of this tracer followed by RGS in oncology.     

Lys and Arg in UBI: a specific site for a stable Tc-99m complex?

The aim of this study was to help establish if ubiquicidin peptide 29-41 fragment (UBI) contains a specific site for 99mTc labeling by a new direct method under alkaline conditions. Since this peptide does not have cysteine residues, it is possible that neighboring arginine and lysine in the peptide amino acid sequence (Thr-Gly-Arg-Ala-Lys-Arg-Arg-Met-Gln-Tyr-Asn-Arg-Arg) could be a specific coordination site to form a stable 99mTc-UBI complex. Following direct labeling, the in vitro stability of 99mTc-UBI was compared to UBI radiolabeled by one indirect method using HYNIC/tricine and HYNIC/tricine/EDDA. Radiochemical purity of 99mTc-UBI averaged 97% compared to 88% for 99mTc-HYNIC-UBI/tricine and 98% for 99mTc-HYNIC-UBI/tricine/EDDA. Both 99mTc-HYNIC-UBI (tricine or EDDA) and 99mTc-UBI showed stability in human serum and solutions of cysteine. 99mTc-UBI radiochemical purity 24 h after dilution in 0.9% NaCl was greater than 90% at pH 9 and greater than 95% at pH 6.5. Under one set of experimental conditions, in vitro binding to bacteria of 99mTc-UBI was 35% and identical to that of 99mTc-HYNIC-UBI/tricine and 99mTc-HYNIC-UBI/tricine/EDDA at 32% and 31% respectively. The biodistribution of 99mTc-UBI in mice showed a rapid renal clearance. To help identify the site(s) of 99mTc binding following direct labeling, molecular mechanics and quantum-mechanical calculations were performed which showed that the amine groups of Arg(7) and Lys are the most probable site. The calculations show that these groups can form a square pyramid with two water molecules for the Tc cation (dxysp(3)). It will be necessary to isolate and characterize the 99Tc(V)(O)-UBI.(H2O)n complex to confirm these results.     

Affinity profiles for human somatostatin receptor subtypes SST1–SST5 of somatostatin radiotracers selected for scintigraphic and radiotherapeutic use

In vivo somatostatin receptor scintigraphy using Octreoscan is a valuable method for the visualisation of human endocrine tumours and their metastases. Recently, several new, alternative somatostatin radioligands have been synthesised for diagnostic and radiotherapeutic use in vivo. Since human tumours are known to express various somatostatin receptor subtypes, it is mandatory to assess the receptor subtype affinity profile of such somatostatin radiotracers. Using cell lines transfected with somatostatin receptor subtypes sst1, sst2, sst3, sst4 and sst5, we have evaluated the in vitro binding characteristics of labelled (indium, yttrium, gallium) and unlabelled DOTA-[Tyr³]-octreotide, DOTA-octreotide, DOTA-lanreotide, DOTA-vapreotide, DTPA-[Tyr³]-octreotate and DOTA-[Tyr³]-octreotate. Small structural modifications, chelator substitution or metal replacement were shown to considerably affect the binding affinity. A marked improvement of sst2 affinity was found for Ga-DOTA-[Tyr³]-octreotide (IC₅₀ 2.5 nM) compared with the Y-labelled compound and Octreoscan. An excellent binding affinity for sst2 in the same range was also found for In-DTPA-[Tyr³]-octreotate (IC₅₀ 1.3 nM) and for Y-DOTA-[Tyr³]-octreotate (IC₅₀ 1.6 nM). Remarkably, Ga-DOTA-[Tyr³]-octreotate bound at sst2 with a considerably higher affinity (IC₅₀ 0.2 nM). An up to 30-fold improvement in sst3 affinity was observed for unlabelled or Y-labelled DOTA-octreotide compared with their Tyr³-containing analogue, suggesting that replacement of Tyr³ by Phe is crucial for high sst3 affinity. Substitution in the octreotide molecule of the DTPA by DOTA improved the sst3 binding affinity 14-fold. Whereas Y-DOTA-lanreotide had only low affinity for sst3 and sst4, it had the highest affinity for sst5 among the tested compounds (IC₅₀ 16 nM). Increased binding affinity for sst3 and sst5 was observed for DOTA-[Tyr³]-octreotide, DOTA-lanreotide and DOTA-vapreotide when they were labelled with yttrium. These marked changes in subtype affinity profiles are due not only to the different chemical structures but also to the different charges and hydrophilicity of these compounds. Interestingly, even the coordination geometry of the radiometal complex remote from the pharmacophoric amino acids has a significant influence on affinity profiles as shown with Y-DOTA versus Ga-DOTA in either [Tyr³]-octreotide or [Tyr³]-octreotate. Such changes in sst affinity profiles must be identified in newly designed radiotracers used for somatostatin receptor scintigraphy in order to correctly interpret in vivo scintigraphic data. These observations may represent basic principles relevant to the development of other peptide radioligands. Received 8 August and in revised form 26 October 1999     

[99mTc]Demotate 2 in the detection of sst2-positive tumours: a preclinical comparison with [111In]DOTA-tate

Purpose The aim of this study was to evaluate [^99mTc]Demotate 2 ([^99mTc-N₄ ^0-1,Asp⁰,Tyr³]octreotate) as a candidate for in vivo imaging of sst₂-positive tumours and to compare it with [¹¹¹In]DOTA-tate ([¹¹¹In-DOTA⁰,Tyr³]octreotate). Methods Labelling of Demotate 2 with ^99mTc was performed at room temperature using SnCl₂ as reductant in the presence of citrate at alkaline pH. Radiochemical analysis involved ITLC and HPLC methods. Peptide conjugate affinities for sst₂ were determined by receptor autoradiography on rat brain cortex sections using [DOTA⁰,¹²⁵I-Tyr³]octreotate as the radioligand. The affinity profile of Demotate 2 for human sst₁–sst₅ was studied by receptor autoradiography in cell preparations using the universal somatostatin radioligand [¹²⁵I][Leu⁸,(D)Trp²²,Tyr²⁵]somatostatin-28. The internalisation rates of [^99mTc]Demotate 2 and [¹¹¹In]DOTA-tate were compared in sst₂-positive and -negative control cell lines. Biodistribution of radiopeptides was studied in male Lewis rats bearing CA20948 tumours. Results Peptide conjugates showed selectivity and a high affinity binding for sst₂ (Demotate 2 IC₅₀=3.2 nM and DOTA-tate IC₅₀=5.4 nM). [^99mTc]Demotate 2, like [¹¹¹In]DOTA-tate, internalised rapidly in all sst₂-positive cells tested, but not in sst₂-negative control cells. After injection in CA20948 tumour-bearing rats both radiopeptides showed high and specific uptake in the sst₂-positive organs and in the implanted tumour and rapid excretion from non-target tissues via the kidneys. Conclusion [^99mTc]Demotate 2, similarly to the known sst₂-targeting agent [¹¹¹In]DOTA-tate, showed promising biological qualities for application in the scintigraphy of sst₂-positive tumours.     

In vitro cell studies of technetium-99m labeled RGD-HYNIC peptide,a comparison of tricine and EDDA as co-ligands

The level of alpha(V)beta(3) integrins on endothelial cells is elevated in angiogenesis. The high binding specificity to alpha(V)beta(3) integrins of peptides containing Arg-Gly-Asp (RGD) residues suggests that the radiolabeled RGD peptides may be useful as tumor specific imaging agents. In this research, cyclised peptides containing Arg-Gly-Asp (RGD) and Arg-Gly-Glu (RGE, as control) residues were conjugated with HYNIC and labeled with (99m)Tc. OBJECTIVE: The goal was to evaluate the influence of co-ligand, either tricine or ethylenediamine-N,N'-diacetic acid (EDDA) on protein and integrin binding and on cellular uptake in culture. METHODS: The n-octanol/water partition coefficient, binding to bovine serum albumin (BSA) and human umbilical vein endothelial (HUVE) cells, and cell lysate distributions of the radiolabeled peptides were evaluated. RESULTS: The co-ligands had a significant effect on the labeling efficiency of the HYNIC conjugates and on certain properties of the (99m)Tc complexes. The labeling efficiency with tricine was 10 fold higher and BSA binding was over 8 fold greater compared to EDDA. Both RGD labels showed higher (6 to 28 fold) binding to HUVE cells than that of the RGE labels, indicating binding specificity. After cell-lysis, only a small percentage of the total RGD label that accumulated in the cells was found bound to cellular proteins (9% of RGD/tricine and 5% of RGD/EDDA), implying that over 90% of the radiolabeled peptides were internalized for both radiolabeled RGDs. The number of the RGD molecules bound to proteins was estimated to be approximately three per cell, suggesting that only a small number of alpha(V)beta(3) integrin proteins are expressed on the cells. CONCLUSIONS: Apart from the differences in radiolabeling, the only important effect of substituting EDDA for tricine as co-ligand on the HYNIC-peptides was the lower degree of serum protein binding. In spite of the lower serum protein binding potential, in vivo tumor accumulation of the RGD/EDDA may not be improved compared to RGD/tricine since quantitation of the cell binding results suggests that the number of alpha(V)beta(3) integrin proteins per cell might be limited.     

Technetium-99m labelled hydrazinonicotinamido human non-specific polyclonal immunoglobulin G for detection of infectious foci: a comparison with two other technetium-labelled immunoglobulin preparations

Recently a new linker — hydrazinonicotinate (HYNIC) — was introduced for labelling of proteins and peptides with technetium-99m. HYNIC and other linkers have been used for labelling of human non-specific polyclonal immunoglobulin G (hIgG) with^99mTc for the detection of infections. In this study we compared the tissue distribution of three different^99mTc-hIgG preparations in groups of five Wistar rats with a focal intramuscular infection withStaphylococcus aureus. We compared^99mTc-HYNIC-hIgG with^99mTc-hIgG labelled via the so-called Schwarz method (reduction of disulphide bonds) and with the^99mTc-labelled commercially available Technescan-HIG. Unlike the HYNIC linker, in the two other labelling methods free sulph-hydryl groups are involved in the binding of^99mTc. High-performance liquid chromatography analysis of the labelled preparations and of plasma samples revealed aggregate or polymer formation in all three agents; this was least pronounced in the product labelled by means of the Schwarz method. The tested preparations did not show signs of degradation in vitro. The difference in linker chemistry was reflected in the tissue distribution. Thus the biodistribution of^99mTc-HYNIC-hIgG was significantly different from the distribution of the two other preparations: abscess (1.4%±0.2%ID/g), muscle, liver, spleen, plasma, lung, bone marrow, and small intestine concentrations were higher at 24 h p.i.; kidney uptake (1.19%±0.08%ID/g) was significantly lower. The abscess-to-plasma and the abscess-to-muscle ratios (0.5 and 11, respectively), however, were in the same range for the three preparations tested. Quantitative analysis of the scintigraphs revealed that the total body clearance of^99mTc-HYNIC-hIgG was significantly slower than for the other agents. The abscess uptake of^99mTc-HYNIC-hIgG as a percentage of the remaining body activity was significantly higher. Based on its high abscess uptake, its low uptake in the kidneys and the high percentage of its abscess uptake in relation to the remaining body activity, we conclude that^99mTc-HYNIC-hIgG seems superior to the two other preparations tested for the detection of infections.     

<Superscript>99m</Superscript>Tc-labelled HYNIC-minigastrin with reduced kidney uptake for targeting of CCK-2 receptor-positive tumours

Purpose Different attempts have been made to develop a suitable radioligand for targeting CCK-2 receptors in vivo, for staging of medullary thyroid carcinoma (MTC) and other receptor-expressing tumours. After initial successful clinical studies with [DTPA⁰,DGlu¹]minigastrin (DTPA-MG0) radiolabelled with ¹¹¹In and ⁹⁰Y, our group developed a ^99mTc-labelled radioligand, based on HYNIC-MG0. A major drawback observed with these derivatives is their high uptake by the kidneys. In this study we describe the preclinical evaluation of the optimised shortened peptide analogue, [HYNIC⁰,DGlu¹,desGlu^2–6]minigastrin (HYNIC-MG11). Methods ^99mTc labelling of HYNIC-MG11 was performed using tricine and EDDA as coligands. Stability experiments were carried out by reversed phase HPLC analysis in PBS, PBS/cysteine and plasma as well as rat liver and kidney homogenates. Receptor binding and cell uptake experiments were performed using AR4-2J rat pancreatic tumour cells. Animal biodistribution was studied in AR4-2J tumour-bearing nude mice. Results Radiolabelling was performed at high specific activities and radiochemical purity was >90%. ^99mTc-EDDA-HYNIC-MG11 showed high affinity for the CCK-2 receptor and cell internalisation comparable to that of ^99mTc-EDDA-HYNIC-MG0. Despite high stability in solution, a low metabolic stability in rat tissue homogenates was found. In a nude mouse tumour model, very low unspecific retention in most organs, rapid renal excretion with reduced renal retention and high tumour uptake were observed. Conclusion ^99mTc-EDDA-HYNIC-MG11 shows advantages over ^99mTc-EDDA-HYNIC-MG0 in terms of lower kidney retention with unchanged uptake in tumours and CCK-2 receptor-positive tissue. However, the lower metabolic stability and impurities formed in the labelling process still leave room for further improvement. Parts of this study were presented at the Annual Congress of the European Association of Nuclear Medicine, Istanbul, Turkey, October 2005 and at the 7th International Symposium on Technetium in Chemistry and Nuclear Medicine, Bressanone, Italy, September 2006.     

In vivo tumor angiogenesis imaging with site-specific labeled 99mTc-HYNIC-VEGF

Purpose We recently developed a cysteine-containing peptide tag (C-tag) that allows for site-specific modification of C-tag-containing fusion proteins with a bifunctional chelator, HYNIC (hydrazine nicotinamide)-maleimide. We then constructed and expressed C-tagged vascular endothelial growth factor (VEGF) and labeled it with HYNIC. We wished to test ^99mTc-HYNIC-C-tagged VEGF (^99mTc-HYNIC-VEGF) for the imaging of tumor vasculature before and after antiangiogenic (low continuous dosing, metronomic) and tumoricidal (high-dose) cyclophosphamide treatment. Methods HYNIC-maleimide was reacted with the two thiol groups of C-tagged VEGF without any effect on biologic activity in vitro. ^99mTc-HYNIC-VEGF was prepared using tin/tricine as an exchange reagent, and injected via the tail vein (200–300 μCi, 1–2 μg protein) followed by microSPECT imaging 1 h later. Results Sequencing analysis of HYNIC-containing peptides obtained after digestion confirmed the site-specific labeling of the two accessible thiol groups of C-tagged VEGF. Tumor vascularity was easily visualized with ^99mTc/VEGF in Balb/c mice with 4T1 murine mammary carcinoma 10 days after implantation into the left axillary fat pad in controls (12.3±5.0 tumor/bkg, n=27) along with its decrease following treatment with high (150 mg/kg q.o.d. ×4; 1.14±0.48 tumor/bkg, n=9) or low (25 mg/kg q.d. ×7; 1.03±0.18 tumor/bkg, n=9) dose cyclophosphamide. Binding specificity was confirmed by observing a 75% decrease in tumor uptake of ^99mTc/biotin-inactivated VEGF, as compared with ^99mTc-HYNIC-VEGF. Conclusion ^99mTc can be loaded onto C-tagged VEGF in a site-specific fashion without reducing its bioactivity. ^99mTc-HYNIC-VEGF can be rapidly prepared for the imaging of tumor vasculature and its response to different types of chemotherapy.     

[<Superscript>99m</Superscript>Tc]Demotensin 5 and 6 in the NTS1-R-targeted imaging of tumours: synthesis and preclinical results

Purpose The aim of this study was to evaluate the applicability of [^99mTc]Demotensin 5 and 6 in the targeted diagnostic imaging of neurotensin subtype 1 receptor (NTS1-R)-expressing tumours. Methods Labelling of Demotensin 5 and 6 with ^99mTc was conducted by brief incubation with ^99mTcO₄ ⁻, SnCl₂ and citrate anions in alkaline medium at ambient temperature. Affinities of conjugates for the NTS1-R were determined by competition binding experiments in WiDr cell membranes using [¹²⁵I-Tyr³]NT as the radioligand. Saturation binding assays were conducted for [^99mTc/^99gTc]Demotensin 6 in WiDr cell membranes. Internalisation of [^99mTc]Demotensin 5 and 6 was studied at 37°C in WiDr cells. Biodistribution of [^99mTc]Demotensin 5 and 6 was performed in female Swiss nu/nu mice bearing human WiDr xenografts. Results Unlabelled conjugates showed a high affinity for the human NTS1-R (Demotensin 5 IC₅₀=0.03±0.01 nM; Demotensin 6 IC₅₀=0.08±0.02 nM), while high affinity was also exhibited by (radio)metallated [^99mTc/^99gTc]Demotensin 6 (K _d=0.13±0.01 nM). [^99mTc]Demotensin 5 and 6 internalised rapidly and specifically in WiDr cells. After injection in WiDr tumour-bearing mice, radiopeptides, and especially the doubly stabilised [^99mTc]Demotensin 6, showed NTS1-R-mediated uptake in the intestines and in the implanted tumour (4.30±0.45%ID/g at 1 h post injection) and rapid renal excretion from non-target tissues into the urine. Conclusion [^99mTc]Demotensin 6 shows a favourable preclinical profile and further testing in patients is warranted to monitor its eventual applicability as a radiotracer in the diagnostic imaging of NTS1-R-positive tumours.     

Evaluation of [99mTc/EDDA/HYNIC0]octreotide derivatives compared with [111In-DOTA0,Tyr3, Thr8]octreotide and [111In-DTPA0]octreotide: does tumor or pancreas uptake correlate with the rate of internalization?

Radiolabeled somatostatin analogs are important tools for the in vivo localization and targeted radionuclide therapy of somatostatin receptor-positive tumors. The aim of this study was to compare 3 somatostatin analogs designed for the labeling with (99m)Tc (where HYNIC is 6-hydrazinopyridine-3-carboxylic acid): 6-hydrazinopyridine-3-carboxylic acid(0)-octreotide (HYNIC-OC/(99m)Tc-(1)), [HYNIC(0),Tyr(3)]octreotide (HYNIC-TOC/(99m)Tc-(2)), and [HYNIC(0),Tyr(3),Thr(8)]octreotide (HYNIC-TATE/(99m)Tc-(3)), using ethylenediamine-N,N'-diacetic acid (EDDA) as a coligand. In addition, we compared the (99m)Tc-labeled peptides [(111)In-diethylenetriaminepentaacetic acid(0)]octreotide ([(111)In-DTPA]-OC) and [(111)In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid(0),Tyr(3),Thr(8)]octreotide ([(111)In-DOTA]-TATE) with regard to the rate of internalization and the biodistribution in AR4-2J (expressing the somatostatin receptor subtype 2) tumor-bearing rats. The main attention was directed toward a potential correlation between the rate of internalization and the tumor or pancreas uptake. METHODS: Synthesis was performed on solid phase using a standard Fmoc strategy. Internalization was studied in cell culture (AR4-2J) and biodistribution was studied using a Lewis rat tumor model (AR4-2J). RESULTS: The 5 radiopeptides showed a specific internalization into AR4-2J cells in culture (as shown by blocking experiments). The rate of internalization of the 5 radiopeptides differed significantly according to the following order: (99m)Tc-(1) approximately = [(111)In-DTPA]-OC < (99m)Tc-(2) < (99m)Tc-(3) approximately = [(111)In-DOTA]-TATE. All radiopeptides displayed a rapid blood clearance and a fast clearance from all somatostatin receptor-negative tissues predominantly via the kidneys. A receptor-specific uptake of radioactivity was observed for all compounds in somatostatin receptor-positive organs such as the pancreas, the adrenals, and the stomach. After 4 h, the uptake in the AR4-2J tumor was comparable for (99m)Tc-(2) (3.85 +/- 1.0 injected dose per gram tissue (%ID/g)), (99m)Tc-(3) (3.99 +/- 0.58%ID/g), and [(111)In-DOTA]-TATE (4.12 +/- 0.74%ID/g) but much lower for [(111)In-DTPA]-OC (0.99 +/- 0.08%ID/g) and (99m)Tc-(1) (0.70 +/- 0.13%ID/g). The specificity was determined by blocking experiments using a large excess of [Tyr(3)]octreotide. (99m)Tc-(3) displayed the highest tumor-to-kidney ratio (2.5:1), followed by (99m)Tc(2) (1.9:1) and [(111)In-DOTA]-TATE (1.7:1). CONCLUSION: These data show that the 5 radiopeptides are specific radioligands for the somatostatin receptor subtype 2. The rate of internalization correlates with the uptake in the tumor (R(2) = 0.75; P = 0.026) and pancreas (R(2) = 0.98; P = 7.4.10(-5)). [Tyr(3),Thr(8)]octreotide derivatives show superiority over the corresponding octreotide and [Tyr(3)]octreotide derivatives, indicating that [(111)In-DOTA]-TATE and [(99m)Tc/EDDA/HYNIC]-TATE are suitable candidates for clinical studies.     

Click chemistry for [99mTc(CO)3] labeling of Lys3-bombesin

^99mTc-HYNIC labeled Lys³-bombesin has shown specific binding to gastrin-releasing peptide receptors (GRP-r) over-expressed in cancer cells. Click chemistry offers an innovative functionalization strategy for biomolecules such as bombesin. The aim of this research was to apply a click chemistry approach for [^99mTc(CO)₃] labeling of Lys³-bombesin and to compare the in vitro MCF7 breast cancer cell uptake and biodistribution profile in mice with that of ^99mTc-EDDA/HYNIC-Lys³-bombesin. The results suggest a higher lipophilicity for ^99mTc(CO)₃-triazole-Lys³-bombesin which explains its higher in vivo hepatobiliary elimination. Pancreas-to-blood ratio for ^99mTc(CO)₃-triazole-Lys³-bombesin was 4.46 at 3 h and both bombesin radiopharmaceuticals showed specific recognition for GRP receptors in MCF7 cancer cells. Click chemistry is a reliable approach for [^99mTc(CO)₃] labeling of Lys³-bombesin.     

Glucagon-like peptide-1 receptor imaging with [Lys40(Ahx-HYNIC-99mTc/EDDA)NH2]-exendin-4 for the detection of insulinoma

Purpose

The objective of this article is to present a new method for the diagnosis of insulinoma with the use of [Lys⁴⁰(Ahx-HYNIC-^99mTc/EDDA)NH₂]-exendin-4.

Methods

Studies were performed in 11 patients with negative results of all available non-isotopic diagnostic methods (8 with symptoms of insulinoma, 2 with malignant insulinoma and 1 with nesidioblastosis). In all patients glucagon-like peptide-1 (GLP-1) receptor imaging (whole-body and single photon emission computed tomography/CT examinations) after the injection of 740 MBq of the tracer was performed.

Results

Both sensitivity and specificity of GLP-1 receptor imaging were assessed to be 100 % in patients with benign insulinoma. In all eight cases with suspicion of insulinoma a focal uptake in the pancreas was found. In six patients surgical excision of the tumour was performed (type G1 tumours were confirmed histopathologically). In one patient surgical treatment is planned. One patient was disqualified from surgery. In one case with malignant insulinoma pathological accumulation of the tracer was found only in the region of local recurrence. The GLP-1 study was negative in the other malignant insulinoma patient. In one case with suspicion of nesidioblastosis, a focal accumulation of the tracer was observed and histopathology revealed coexistence of insulinoma and nesidioblastosis.

Conclusion

[Lys⁴⁰(Ahx-HYNIC-^99mTc/EDDA)NH₂]-exendin-4 seems to be a promising diagnostic tool in the localization of small insulinoma tumours, but requires verification in a larger series of patients.     

[99mTc(CO)3]+-(HE)3-ZIGF1R:4551, a new Affibody conjugate for visualization of insulin-like growth factor-1 receptor expression in malignant tumours

Purpose

Radionuclide imaging of insulin-like growth factor type 1 receptor (IGF-1R) expression in tumours might be used for selection of patients who would benefit from IGF-1R-targeted therapy. We have previously shown the feasibility of IGF-1R imaging using the Affibody molecule ¹¹¹In-DOTA-His₆-Z_IGF1R:4551. The use of ^99mTc instead of ¹¹¹In should improve sensitivity and resolution of imaging, and reduce the dose burden to patients. We hypothesized that inclusion of a HEHEHE tag instead of a His₆ tag in Z_IGF1R:4551 would permit its convenient purification using IMAC, enable labelling with [^99mTc(CO)₃]⁺, and improve its biodistribution.

Methods

Z_IGF1R:4551 was expressed with a HEHEHE tag in the N terminus. The resulting (HE)₃-Z_IGF1R:4551 construct was labelled with [^99mTc(CO)₃]⁺. Targeting of IGF-1R-expressing cells using [^99mTc(CO)₃]⁺-(HE)₃-Z_IGF1R:4551 was evaluated in vitro and in vivo.

Results

(HE)₃-Z_IGF1R:4551 was stably labelled with ^99mTc with preserved specific binding to IGF-1R-expressing DU-145 prostate cancer cells in vitro. In mice, [^99mTc(CO)₃]⁺-(HE)₃-Z_IGF1R:4551 accumulated in IGF-1R-expressing organs (pancreas, stomach, lung and salivary gland). [^99mTc(CO)₃]⁺-(HE)₃-Z_IGF1R:4551 demonstrated 3.6-fold lower accumulation in the liver and spleen than ¹¹¹In-DOTA-Z_IGF1R:4551. In NMRI nu/nu mice with DU-145 prostate cancer xenografts, the tumour uptake was 1.32?±?0.11 %ID/g and the tumour-to-blood ratio was 4.4?±?0.3 at 8 h after injection. The xenografts were visualized using a gamma camera 6 h after injection.

Conclusion

^99mTc(CO)₃]⁺-(HE)₃-Z_IGF1R:4551 is a promising candidate for visualization of IGF-1R expression in malignant tumours.     

Somatostatin receptor scintigraphy using <Superscript>99m</Superscript>Tc-EDDA/HYNIC-TOC in patients with medullary thyroid carcinoma

Purpose Several new somatostatin analogues have been developed for the diagnosis and therapy of different tumours. Since somatostatin receptors are often over-expressed in medullary thyroid carcinoma (MTC), the aim of our study was to evaluate the utility of scintigraphy with the somatostatin analogue ^99mTc-EDDA/HYNIC-TOC in MTC in comparison with other diagnostic techniques. Methods Forty-five patients with MTC, aged 14–83 years, were investigated. Scintigraphy using ^99mTc-EDDA/HYNIC-TOC (Tektrotyd) was performed 2 and 4 h post injection of 740 MBq (20 mCi) of the tracer. Other imaging techniques were also applied and analysed in individual cases (ultrasonography, computed tomography, ^99mTc(V)-DMSA, ¹³¹I-MIBG, ^99mTc-MDP, ¹¹¹In-DTPA-octreotide and ¹⁸F-FDG-PET) and compared with ^99mTc-EDDA/HYNIC-TOC. Results In group 1 (eight patients before thyroidectomy), uptake of the tracer was found in the primary tumours. In group 2 (six patients with remission), a false positive result was found in one patient; in the remaining five patients, no pathological foci were visualised. In group 3 (31 patients with post-surgical hypercalcitoninaemia), scintigraphy was true positive in 23 patients (74.2%): uptake in the thyroid bed was found in five patients, in the lymph nodes in 18 and in bone metastases in four. Using ^99mTc-EDDA/HYNIC-TOC scintigraphy, the overall sensitivity was 79.5%, specificity 83.3%, accuracy 80.0%, positive predictive value 96.9% and negative predictive value 38.5%. Conclusion ^99mTc-EDDA/HYNIC-TOC is clinically useful for scintigraphy in the follow-up of patients with MTC. It can be used in clinical practice for preoperative evaluation, for localisation of local recurrence or distant metastases and particularly for therapy decision making.