Plasma protein binding of (99m)Tc-labeled hydrazino nicotinamide derivatized polypeptides and peptides

6-Hydrazinopyridine-3-carboxylic acid (HYNIC) constitutes one of the most attractive reagents to prepare (99m)Tc-labeled polypeptides and peptides of various molecular weights in combination with two tricine molecules as coligands. Indeed, (99m)Tc-HYNIC-conjugated IgG showed biodistribution of radioactivity similar to that of (111)In-DTPA-conjugated IgG. However, recent studies indicated significant plasma protein binding when the (99m)Tc labeling procedure was expanded to low molecular weight peptides. In this study, pharmacokinetics of (99m)Tc-HYNIC-conjugated IgG, Fab and RC160 using tricine were compared with their radioiodinated counterparts to evaluate this (99m)Tc-labeling method. In mice, [(99m)Tc](HYNIC-IgG)(tricine)(2) and [(99m)Tc](HYNIC-Fab)(tricine)(2) showed persistent localization of radioactivity in tissues when compared with their (125)I-labeled counterparts. [(99m)Tc](HYNIC-IgG)(tricine)(2) eliminated from the blood at a rate similar to that of (125)I-labeled IgG, while [(99m)Tc](HYNIC-Fab)(tricine)(2) showed significantly slower clearance of the radioactivity than (125)I-labeled Fab. On size-exclusion HPLC analyses, little changes were observed in radiochromatograms after incubation of [(99m)Tc](HYNIC-IgG)(tricine)(2) in murine plasma. However, [(99m)Tc](HYNIC-Fab)(tricine)(2) and [(99m)Tc](HYNIC-RC160)(tricine)(2) demonstrated significant increases in the radioactivity in higher molecular weight fractions in plasma. Formation of higher molecular weight species was reduced when [(99m)Tc](HYNIC-RC160)(tricine)(2) was stabilized with nicotinic acid (NIC) to generate [(99m)Tc](HYNIC-RC160)(tricine)(NIC). [(99m)Tc](HYNIC-RC160)(tricine)(NIC) also demonstrated significantly faster clearance of the radioactivity from the blood than [(99m)Tc](HYNIC-RC160)(tricine)(2). These findings suggested that one of the tricine coligands in (99m)Tc-HYNIC-labeled (poly)peptides would be replaced with plasma proteins to generate higher molecular weight species that exhibit slow blood clearance. In addition, the molecular sizes of parental peptides played an important role in the progression of the exchange reaction of one of the tricine coligands with plasma proteins.     

Coligand effects on the solution stability, biodistribution and metabolism of the (99m)Tc-labeled cyclic RGDfK tetramer

In this study, we present the evaluation of two new ternary ligand (99m)Tc complexes [(99m)Tc(HYNIC tetramer)(tricine)(L)] [L=isonicotinic acid (ISONIC) and 2,5-pyridinedicarboxylic acid (PDA)] as potential radiotracers for tumor imaging. Athymic nude mice bearing MDA-MB-435 human breast cancer xenografts were used to evaluate their biodistribution and metabolic properties. Solution stability data showed that [(99m)Tc(HYNIC tetramer)(tricine)(L)] (L=ISONIC and PDA) had significant decomposition (14% and 35%, respectively) at 6 h in the absence of excess ISONIC or PDA coligand. Biodistribution data clearly showed that [(99m)Tc(HYNIC tetramer)(tricine)(PDA)] had a much lower uptake in most organs of interest than [(99m)Tc(HYNIC tetramer)(tricine)(ISONIC)] during the 2-h study period. Results from metabolism studies revealed that approximately 50% of [(99m)Tc(HYNIC tetramer)(tricine)(ISONIC)] remained intact in fecal samples at 120 min postinjection, whereas only 10% of [(99m)Tc(HYNIC tetramer)(tricine)(PDA)] remained intact in fecal samples. The extent of metabolism correlated well with radiotracer solution stability. The results from this and our previous studies clearly demonstrated that coligands [trisodium triphenylphosphine-3,3',3'-trisulfonate (TPPTS), ISONIC and PDA] have a significant impact on the tumor uptake, excretion kinetics and metabolism of the (99m)Tc-labeled cyclic RGDfK tetramer. Among the three radiotracers evaluated in this tumor-bearing animal model, [(99m)Tc(HYNIC tetramer)(tricine)(TPPTS)] remained the best with respect to blood clearance, tumor uptake and target/background ratios.     

99mTc]HYNIC-RGD for imaging integrin alphavbeta3 expression

There has been increasing interest in peptides containing the Arg-Gly-Asp (RGD) sequence for targeting of alpha(v)beta(3) integrins to image angiogenesis. [(18)F]Galacto-RGD has been successfully used for positron emission tomography applications in patients. Here we report on the preclinical characterization of a (99m)Tc-labeled derivative for single-photon emission computed tomography. c(RGDyK) was derivatized with HYNIC at the amino group of the lysine [c(RGDyK(HYNIC)) or HYNIC-RGD]. (99m)Tc labeling was performed using coligands (tricine and EDDA), as well as (99m)Tc(CO)(3)(H(2)O)(3). Radiolabeled peptides were characterized with regard to lipophilicity, protein binding and stability in buffer, serum and tissue homogenates. Integrin receptor activity was determined in internalization assays using alpha(v)beta(3)-receptor-positive M21 and alpha(v)beta(3)-receptor-negative M21L melanoma cells. Biodistribution was evaluated in normal and nude mice bearing M21, M21L and small cell lung tumors. HYNIC-RGD could be labeled at high specific activities using tricine, tricine-trisodium triphenylphosphine 3,3',3'-trisulfonate (TPPTS), tricine-nicotinic acid (NA) or EDDA as coligands. [(99m)Tc]EDDA/HYNIC-RGD, [(99m)Tc]tricine-TPPTS/HYNIC-RGD and [(99m)Tc]tricine-NA/HYNIC-RGD showed protein binding (<5%) considerably lower than [(99m)Tc](CO)(3)/HYNIC-RGD and [(99m)Tc]tricine/HYNIC-RGD. [(99m)Tc]EDDA/HYNIC-RGD revealed high in vitro stability accompanied by low lipophilicity with a log P value of -3.56, comparable to that of [(18)F]Galacto-RGD. In M21 cells for this compound, the highest level of specific and rapid cell uptake (1.25% mg protein(-1)) was determined. In vivo, rapid renal excretion, low blood retention, low liver and muscle uptakes and low intestinal excretion 4 h postinjection were observed. Tumor uptake values were 2.73% ID/g in M21 alpha(v)beta(3)-receptor-positive tumors versus 0.85% ID/g in receptor-negative tumors 1 h postinjection. Small cell lung tumors could be visualized using gamma camera imaging. [(99m)Tc]EDDA/HYNIC-RGD shows encouraging properties to target alpha(v)beta(3) receptors in vivo with high stability and favorable pharmacokinetics. Tumor uptake studies showed specific targeting of alpha(v)beta(3)-receptor-positive tumors with tumor-to-organ ratios comparable to those of [(18)F]Galacto-RGD.     

99mTc-labeled mannosyl-neoglycoalbumin for sentinel lymph node identification

99mTc-labeled mannosyl-neoglycoalbumin (NMA) was prepared and evaluated as a radiopharmaceutical for sentinel lymph node (SLN) identification, since 99mTc-labeled human serum albumin (HSA) rapidly cleared from injection sites. NMA was conjugated with 6-hydrazinopyridine-3-carboxylic acid (HYNIC) and reacted with [99mTc](tricine)2 to prepare [99mTc](HYNIC-NMA)(tricine)2. After subcutaneous injection of [99mTc](HYNIC-NMA)(tricine)2 from murine foot pad, radioactivity levels in the popliteal and lumbar lymph nodes, the injection site and other tissues were compared with those of [99mTc](HYNIC-HSA)(tricine)2 and 99mTc-labeled colloidal rhenium sulfate ([99mTc]colloid). [99mTc](HYNIC-NMA)(tricine)2 demonstrated significantly higher radioactivity levels in the popliteal lymph node, the SLN in this model, than did [99mTc](HYNIC-HSA)(tricine)2 and [99mTc]colloid at 0.5, 1, and 6 h post-injection. [99mTc](HYNIC-NMA)(tricine)2 showed a dose-dependent decrease in the popliteal accumulation while the radioactivity levels in the blood, liver and spleen increased with an increase in the molar dose of NMA. [99mTc]colloid registered a decrease in the radioactivity levels in the popliteal lymph node, blood, liver, and spleen with dilution. However, the radioactivity levels at the injection site increased with dilution of [99mTc] colloid. Both [99mTc](HYNIC-NMA)(tricine)2 and [99mTc](HYNIC-HSA)(tricine)2 showed the radioactivity levels at the injection site similar each other. These findings indicated that an addition of a macrophage binding function to 99mTc-labeled HSA provided high and selective accumulation of the radioactivity in the SLN without affecting the elimination rate from the injection site. Such characteristics render [99mTc](HYNIC-NMA)(tricine)2 attractive as a radiopharmaceutical for SLN identification. This study also demonstrated that the number of non-radiolabeled colloidal particles and the molar dose of mannosylated compounds play a crucial role in the SLN accumulation.     

Imaging of HER2/neu expression in BT-474 human breast cancer xenografts in athymic mice using [(99m)Tc]-HYNIC-trastuzumab (Herceptin) Fab fragments

OBJECTIVE: To evaluate the ability of trastuzumab (Herceptin) Fab, labelled with (99m)Tc through introduced hydrazinenicotinamide (HYNIC) functionalities, to image HER2/neu-overexpressing human breast cancer xenografts in athymic mice. METHODS: Fab fragments were produced by immobilized papain digestion of trastuzumab immunoglobulin G (IgG), followed by purification by ultrafiltration. The immunoreactivity of trastuzumab Fab was evaluated by receptor-binding assays against HER2/neu-positive SK-BR-3 human breast cancer cells. Trastuzumab Fab fragments were labelled with (99m)Tc following modification with HYNIC N-hydroxysuccinimide ester. Biodistribution and tumour imaging studies were performed in athymic mice bearing subcutaneous HER2/neu-overexpressing BT-474 human breast cancer xenografts following intravenous injection of 1.1 or 25 MBq of [(99m)Tc]-trastuzumab Fab (30 microg), respectively. The specificity of tumour uptake was assessed by comparison with that of [(99m)Tc]-labelled irrelevant anti-CD33 HuM195 Fab. RESULTS: Trastuzumab Fab was pure and exhibited preserved immunoreactivity towards SK-BR-3 cells (K(d) = 1.6 x 10(-8) M). Modification with HYNIC diminished its receptor-binding affinity fourfold. [(99m)Tc]-trastuzumab Fab localized avidly and specifically in BT-474 xenografts, achieving a tumour uptake of 10.7% of the injected dose (ID) per gram and a tumour to blood (T/B) ratio of 3 : 1 at 24 h. The tumour uptake and T/B ratio for [(99m)Tc]-trastuzumab Fab were significantly higher than those for control [(99m)Tc]-HuM195 Fab (2.6% ID x g(-1) and 0.9 : 1, respectively; P<0.05). Tumours were imaged as early as 2 h post-injection of [(99m)Tc]-trastuzumab Fab, but were more clearly visualized at 6 and 24 h post-injection. CONCLUSIONS: [(99m)Tc]-HYNIC-trastuzumab Fab localized specifically in HER2/neu-overexpressing human breast cancer xenografts in athymic mice, allowing imaging of the tumours within the useful lifetime of the radionuclide.     

Radiolabeling of annexin A5 with (99m)Tc: comparison of HYNIC-Tc vs. iminothiolane-Tc-tricarbonyl conjugates

In the perspective of expanding the use of annexin A5 (anx A5) as radioactive tracer of cell death in vivo, we recently described its radiolabeling with (99m)Tc-tricarbonyl [(99m)Tc(H(2)O)(3)(CO)(3)](+) via the mercaptobutyrimidyl group (anx A5-SH). The aim of the present article was to compare this new method with the HYNIC strategy (anx A5-HYNIC), recognized at present as the reference for the radiolabeling of proteins with (99m)Tc. Similar radiolabeling yields and better chemical stability were obtained with the [anx A5-SH-(99m)Tc-tricarbonyl] complex. Since the [anx A5-HYNIC-(99m)Tc(tricine)(2)] conjugate shows isomeric forms which can affect the biological properties whereas [anx A5-SH-(99m)Tc-tricarbonyl] is less or not prone to such drawback, the latter seems superior to the former. Furthermore, (anx A5-SH) is readily obtained via commercial sources of Traut's reagent whereas (anx A5-HYNIC) is not. The results provide encouraging evidence in the development of anx A5-labeled reagent for apoptose imaging.     

Nitriles form mixed-coligand complexes with (99m)Tc-HYNIC-peptide.

Using a 12-amino acid peptide conjugated with HYNIC as a model, we investigated nitriles as possible coligands for labeling with (99m)Tc. After the preparation of the (99m)Tc labeled HYNIC-peptide using tricine as coligand, the addition of acetonitile was found by reverse phase HPLC to block further coligand exchange with ethylenediamine diacetic acid (EDDA) at room temperature. The addition of this nitrile changed the pharmacokinetics of the (99m)Tc labeled peptide in normal mice towards faster clearance and significant differences in accumulation in most tissues sampled. By replacing acetonitrile with cyanoacetate, a nitrile not present in the HPLC eluant, it was possible to show the existence of a new, more hydrophilic, species by reverse phase HPLC. We conclude that nitriles can act as coligands for HYNIC-conjugated peptides labeled with (99m)Tc and tricine. Furthermore, the presence of acetonitrile during Sep-Pak or HPLC purification may inadvertently generate a mixed tricine/acetonitile coligand (99m)Tc-HYNIC-peptide complex.     

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.     

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.     

Surfactant protein B labelled with [(99m)Tc(CO)3(H20)3](+) retains biological activity in vitro.

Labelling of the hydrophobic surfactant protein B (SP-B) under non-reducing conditions was achieved with [(99m)Tc(CO)(3)(H2O)(3)](+) prepared according to Alberto et al. (JACS, 1998). The binding of radioactivity was protein-specific, with an overall radiochemical yield of 50%. Gel electrophoresis and Westernblot analyses showed no structural changes of SP-B. Spreading properties and surface activity of (99m)Tc-labelled SP-B in an air/water interface coincided with those of unlabelled SP-B. (99m)Tc-SP-B seems to be a promising agent to observe surfactant spreading under clinical conditions. BACKGROUND: Therapeutic results for surfactant instillation in clinical trials are conflicting. The (99m)Tc-labelling of surfactant would allow to observe its spreading in the lung under clinical conditions. METHODS: [(99m)Tc(CO)(3)(H2O)(3)](+) was prepared as described by Alberto et al. (JACS, 1998). This carbonyl complex was used for the direct labelling of surfactant protein B (SP-B) under non-reductive conditions by direct incubation with SP-B at elevated temperature followed by extraction into CHCl(3)/MeOH. RESULTS: The hydrophobic protein SP-B was labelled with [(99m)Tc(CO)(3)(H2O)(3)](+). An overall radiochemical yield of about 50% was achieved. HPLC-analysis revealed a single radiolabelled species according to UV elution profile of SP-B, supported by paper and size exclusion chromatography. Gel electrophoresis confirmed that the dimer structure of SP-B was preserved. Spreading properties of (99m)Tc-labelled SP-B in an air/water interface coincided with those of unlabelled SP-B. Spreading of radioactivity observed in a glass trough of 26 cm x 27 cm with a gamma camera was completed during the first 7-9 sec after application of (99m)Tc-labelled SP-B. The corresponding decrease of surface tension to 45 mN/m at the peripheral surface tension sensors took 7 sec +/- 2 sec (MEAN +/- STD; n = 3). CONCLUSIONS: Direct and specific (99m)Tc-labelling of the hydrophobic surfactant protein B was achieved using the [(99m)Tc(CO)(3)(H2O)(3)](+) precursor. This procedure can easily be used to prepare specifically labelled surfactant mixtures with spreading properties that coincide with those of unlabelled surfactant.     

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 N₃S system is described and a comparison made with [Tyr³]-octreotide (TOC). Conjugates of both peptides with HYNIC, and of RC160 with benzoyl-MAG₃ and an N₃S-adipate derivative were prepared and radiolabelling performed at high specific activities using tricine, tricine/nicotinic acid and ethylenediamine-N,N’-diacetic adic (EDDA) as co-ligands for HYNIC conjugates. All conjugates and ^99mTc-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 ^99mTc-RC160 derivatives compared with ^99mTc-EDDA/HYNIC-[Tyr³]-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 ^99mTc to achieve complexes with favourable biodistribution. Despite the relatively low tumour uptake compared with ^99mTc-EDDA/HYNIC-[Tyr³]-octreotide, ^99mTc-RC160 could play a role in imaging tumours that do not bind octreotide derivatives. Received 26 January and in revised form 16 April 1999     

A comparison in monkeys of (99m)Tc labeled to a peptide by 4 methods.

Although a number of different strategies for labeling peptides with (99m)Tc have been developed, only a few studies have compared the in vivo properties of (99m)Tc when attached to different chelators. Furthermore, these comparisons are usually in mice, whereas results obtained in nonhuman primates may be expected to be more relevant to the clinical situation. METHODS: We evaluated the influence of 4 common chelators on the biodistribution in monkeys of (99m)Tc-labeled HNE-2, a 6.7-kDa peptide being investigated as an inflammation/infection imaging agent. The peptide was conjugated with the N-hydroxysuccinimide ester of mercaptoacetyltriglycine (MAG3), mercaptoacetyltriserine (MAS3), hydrazinonicotinamide (HYNIC), and the cyclic anhydride of diethylenetriaminepentaacetic acid (DTPA). After radiolabeling, each peptide was administered intravenously to rhesus monkeys with a Staphylococcus aureus-induced focal inflammation/infection. RESULTS: Quantification of radioactivity accumulation by regions of interest over 3 h after administration in monkeys showed important differences among labeling methods: For example, at 3 h, kidney accumulation varied in percentage injected dose per organ (%ID per organ) from 31 %ID per organ (HYNIC) to 18 %ID per organ (MAG3), whereas liver varied from 7.8 %ID per organ (MAG3) to 2.8 %ID per organ (MAS3). Radioactivity accumulation in the lesion was independent of labeling method. These organ accumulations were compared with that obtained earlier in mice by sacrifice and dissection also at 3 h and at the same administered dosage. In the rodent, kidney levels varied from 45 %ID per organ (HYNIC) to 12 %ID per organ (MAS3) and liver levels varied from 6.5 %ID per organ (DTPA) to 2.0 %ID per organ (MAS3). CONCLUSION: In agreement with previous work from this laboratory and elsewhere, the method of radiolabeling had an important effect on the biodistribution of (99m)Tc. Furthermore, although biodistribution results in mice should be used with caution to predict biodistributions in primates, in major organs, these results in mice and monkeys were similar.     

Direct 99m Tc labeling of Herceptin (trastuzumab) by 99m Tc(I) tricarbonyl ion.

By simply incubating Herceptin (trastuzumab) with [99m Tc(CO)3(OH2)3]+ ion in saline, a significant yield of 99m Tc-labeled trastuzumab was found to be achievable. The effective labeling may be based on that trastuzumab is inherent with endogenous histidine group to which 99m Tc(I) tricarbonyl ion can be strongly bound. For practical 99m Tc labeling processing, trastuzumab was purified beforehand from the commercial product, Herceptin (Genentech) via size exclusion chromatography to remove the excipient, alpha-histidine and a high-labeled yield could be obtained by incubating the purified trastuzumab with [99m Tc(CO)3(OH2)3]+. Retention of bioactivity of the 99m Tc(I)-labeled trastuzumab was validated using a cell binding test.     

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。     

The effect of molecular weight on nonspecific accumulation of (99m)T-labeled proteins in inflammatory foci

Although several proteins have been proposed and tested for scintigraphic detection of infection, the most optimal characteristics of a protein for this application have not yet been determined. Molecular weight (MW) of the protein, its charge, shape, carbohydrate content, characteristics of the radionuclide and receptor interactions are factors that could affect the in vivo behavior of the infection imaging agent. The effect of molecular weight on nonspecific accumulation of (99m)Tc-labeled proteins in inflammatory foci was studied in a rat model.Methods: Eleven proteins whose MWs ranged from 2.5 kDa up to 800 kDa were labeled with (99m)Tc using the hydrazinonicotinamide (HYNIC) chelator. Rats with S. aureus infection were injected i.v. with 15 MBq (99m)Tc-labeled protein. Gamma camera images were acquired and biodistribution of the radiolabel was determined ex vivo.Results: From biodistribution data no significant correlation was found between abscess uptake and molecular size of the (99m)Tc-labeled proteins that were studied. Fast blood clearance with predominant uptake in liver and spleen was found for the largest proteins (MW 669 kDa-800 kDA). For proteins of intermediate size (MW 66 kDa -206 kDa) we found relatively slow blood clearance with relatively moderate uptake in liver and spleen. For smaller proteins (MW 2.5 kDa -29 kDa) rapid blood clearance with predominant kidney uptake was observed. The abscess uptake of the (99m)Tc-labeled proteins (%ID/g, 24 h p.i.) was highest for serum proteins IgG and BSA. Abscess uptake correlated well with blood levels: r = 0.95 and 0.84 at 4 and 24 h respectively (P < 0.005). The abscess-to-muscle ratios varied from 2.1 to 17.8 at 24 h p.i. with highest values for alpha-2 macroglobulin (MW 725 kDa) and the intermediate sized proteins (MW 66-206 kDa). Gamma camera imaging showed localization of all radiotracers at the site of infection with abscess-to-background ratios (A/B) ranging from 1.4 to 7.0 (IgG) at 20 h p.i. The serum proteins IgG and BSA showed highest blood levels and best infection imaging characteristics.Conclusion: Not molecular weight but blood residence time is the principal factor that determines localization of a nonspecific tracer protein in infectious foci. The ideal nonspecific infection imaging agent is a protein with a long circulatory half-life. From the proteins tested here IgG and albumin showed the best characteristics for an infection imaging agent.     

First evaluation of a 99mTc-tricarbonyl complex, 99mTc(CO)3(LAN), as a new renal radiopharmaceutical in humans.

(99m)Tc-Mercaptoacetyltriglycine ((99m)Tc-MAG3), (99m)Tc-dd- and ll-ethylene-dicysteine ((99m)Tc-EC), and (99m)Tc-mercaptoacetamide-ethylene-cysteine ((99m)Tc-MAEC) contain N(3)S or N(2)S(2) ligands designed to accommodate the 4 ligating sites of the ((99m)TcO)(3+) core; they are all excellent renal imaging agents but have renal clearances lower than that of (131)I-orthoiodohippurate ((131)I-OIH). To explore the potential of the newly accessible but less polar [(99m)Tc(CO)(3)](+) core with 3 ligating sites, we decided to build on the success of (99m)Tc-EC, with its N(2)S(2) ligand and 2 dangling carboxylate groups; we chose an N(2)S ligand that also has 2 dangling carboxylate groups, lanthionine, to form (99m)Tc(CO)(3)(LAN), a new renal radiopharmaceutical. METHODS: Biodistribution studies were performed on Sprague-Dawley rats with (99m)Tc(CO)(3)(LAN) isomers, meso-LAN and dd,ll-LAN (an enantiomeric mixture), coinjected with (131)I-OIH. Human studies also were performed by coinjecting each (99m)Tc-labeled product ( approximately 74 MBq [ approximately 2 mCi]) and (131)I-OIH ( approximately 7.4 MBq [ approximately 0.2 mCi]) into 3 healthy volunteers and then performing dual-isotope imaging by use of a camera system fitted with a high-energy collimator. Blood samples were obtained from 3 to 90 min after injection, and urine samples were obtained at 30, 90, and 180 min. RESULTS: Biodistribution studies in rats revealed rapid blood clearance as well as rapid renal extraction for both preparations, with the dose in urine at 60 min averaging 88% that of (131)I-OIH. In humans, both agents provided excellent renal images, with the plasma clearance averaging 228 mL/min for (99m)Tc(CO)(3)(meso-LAN) and 176 mL/min for (99m)Tc(CO)(3)(dd,ll-LAN). At 3 h, both (99m)Tc(CO)(3)(meso-LAN) and (99m)Tc(CO)(3)(dd,ll-LAN) showed good renal excretion, averaging 85% and 77% that of (131)I-OIH, respectively. Plasma protein binding was minimal (10% and 2%, respectively), and erythrocyte uptake was similar (24% and 21%, respectively) for (99m)Tc(CO)(3)(meso-LAN) and (99m)Tc(CO)(3)(dd,ll-LAN). CONCLUSION: Although the plasma clearance and the rate of renal excretion of the (99m)Tc(CO)(3)(LAN) complexes were still lower than those of (131)I-OIH, the results of this first application of a (99m)Tc-tricarbonyl complex as a renal radiopharmaceutical in humans demonstrate that (99m)Tc(CO)(3)(LAN) complexes are excellent renal imaging agents and support continued renal radiopharmaceutical development based on the (99m)Tc-tricarbonyl core.     

Development of a novel 99mTc-chelate-conjugated bisphosphonate with high affinity for bone as a bone scintigraphic agent.

In bone scintigraphy using (99m)Tc with methylenediphosphonate ((99m)Tc-MDP) and hydroxymethylenediphosphonate ((99m)Tc-HMDP), it takes 2-6 h after an injection before imaging can start. This interval could be shortened with a new radiopharmaceutical with higher affinity for bone. Here, based on the concept of bifunctional radiopharmaceuticals, we designed a (99m)Tc-mercaptoacetylglycylglycylglycine (MAG3)-conjugated hydroxy-bisphosphonate (HBP) ((99m)Tc-MAG3-HBP) and a (99m)Tc-6-hydrazinopyridine-3-carboxylic acid (HYNIC)-conjugated hydroxy-bisphosphonate ((99m)Tc-HYNIC-HBP). METHODS: (99m)Tc-MAG3-HBP was prepared by complexation of MAG3-HBP with (99m)Tc using SnCl(2) as a reductant. The precursor of (99m)Tc-HYNIC-HBP, HYNIC-HBP, was obtained by deprotection of the Boc group after the coupling of Boc-HYNIC to a bisphosphonate derivative. (99m)Tc-HYNIC-HBP was prepared by a 1-pot reaction of HYNIC-HBP with (99m)TcO(4)(-), tricine, and 3-acetylpyridine in the presence of SnCl(2). Affinity for bone was evaluated in vitro by hydroxyapatite-binding assays for (99m)Tc-HMDP, (99m)Tc-MAG3-HBP, and (99m)Tc-HYNIC-HBP. Biodistribution experiments for the 3 (99m)Tc-labeled compounds were performed on normal rats. RESULTS: (99m)Tc-MAG3-HBP and (99m)Tc-HYNIC-HBP were each prepared with a radiochemical purity of >95%. In the in vitro binding assay, (99m)Tc-MAG3-HBP and (99m)Tc-HYNIC-HBP had greater affinity for hydroxyapatite than (99m)Tc-HMDP. In the biodistribution experiments, (99m)Tc-MAG3-HBP and (99m)Tc-HYNIC-HBP had higher levels of radioactivity in bone than (99m)Tc-HMDP. (99m)Tc-MAG3-HBP was cleared from the blood slower than (99m)Tc-HMDP, whereas there was no significant difference in clearance between (99m)Tc-HYNIC-HBP and (99m)Tc-HMDP. Consequently, (99m)Tc-HYNIC-HBP showed a higher bone-to-blood ratio than (99m)Tc-HMDP. CONCLUSION: We developed a novel (99m)Tc-chelate-conjugated bisphosphonate with high affinity for bone and rapid clearance from blood, based on the concept of bifunctional radiopharmaceuticals. The present findings indicate that (99m)Tc-HYNIC-HBP holds great potential for bone scintigraphy.     

Synthesis and biodistribution of a new (99m)Tc nitrido complex for cerebral imaging

The bis(N-isopentyl dithiocarbamato) nitrido technetium-99m complex [(99m)TcN(IPEDTC)(2)] (IPEDTC: N-isopentyl dithiocarbamato) has been synthesized by the reduction of (99m)TcO(4)(-) into [(99m)TcN](2+) with stannous chloride in the presence of succinic dihydrazide and propylenediamine tetraacetic acid, followed by the addition of the sodium salt of N-isopentyl dithiocarbamate. The radiochemical purity of the complex was over 90% as measured by thin layer chromatography. In vitro studies showed that the complex possessed good stability under physiological conditions. Its partition coefficient indicated that it was a lipophilic complex. The electrophoresis results showed the complex was neutral. Biodistribution in mice showed that the complex accumulated in brain with high uptake and good retention. The brain uptake (ID%/g) was 2.22, 2.06 and 2.45 and the brain/blood ratio was 1.01, 2.34 and 3.22 at 5, 30 and 60 min post-injection, respectively. These results for the complex suggested that it could be a potential brain perfusion imaging agent.     

Evaluation of [(99m)Tc] liposomes as lymphoscintigraphic agents: comparison with [(99m)Tc] sulfur colloid and [(99m)Tc] human serum albumin

This study investigates the use of [(99m)Tc] liposomes for the detection of sentinel lymph nodes. A variety of [(99m)Tc] liposome formulations were compared with common lymphoscintigraphic agents including [(99m)Tc] regular-sulfur colloid (SC), [(99m)Tc] 0.22 microfiltered-SC, [(99m)Tc] reduced heating time 0.22 microfiltered-SC, and [(99m)Tc] human serum albumin (HSA) in rabbits. Images were acquired for the first 60 minutes and at 24 hours, followed by tissue biodistribution study. All agents except [(99m)Tc] regular SC demonstrated good migration from the injection site. Agents were retained in the popliteal node at 24 hours to varying degrees as follows: both [(99m)Tc] filtered SC preparations > [(99m)Tc] regular SC > [(99m)Tc] liposomes > [(99m)Tc] HSA. [(99m)Tc] liposome imaging can be used to develop novel liposome compositions with improved lymph node diagnostic and drug delivery characteristics.     

Picolylamine-methylphosphonic acid esters as tridentate ligands for the labeling of alcohols with the fac-[M(CO)3]+ core (M = 99mTc, Re): synthesis and biodistribution of model compounds and of a 99mTc-labeled cobinamide

[(Methyl-pyridin-2-ylmethyl-amino)-methyl]-phosphonic acid is a new bifunctional chelator for the fac-[(99m)Tc(CO(3))](+) core which can be linked to biomolecules via formation of phosphonic acid esters. Its synthesis and the coupling to model alcohols and to a bioactive molecule (cobinamide) are described. The rhenium complexes [Re(CO)(3)L] of the esters have been prepared and characterized, one of them by X-ray crystallography. The model esters could be labeled with [(99m)Tc(OH(2))(3)(CO)(3)](+) under mild conditions and relatively low ligand concentration with >97% yield and only one isomer formed. The (99m)Tc-labeled cobinamide analog was a mixture of four isomers. It bound strongly to transcobalamin I (TC I, haptocorrin) but only slightly to transcobalamin II (TC II) and intrinsic factor (IF), reflecting the binding abilities of cobinamide. Biodistribution studies in mice with B(16) melanoma exhibited fast clearance with no specific tissue binding.