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.     

(99m)Tc-HYNIC-derivatized ternary ligand complexes for (99m)Tc-labeled polypeptides with low in vivo protein binding

6-Hydrazinopyridine-3-carboxylic acid (HYNIC) is a representative agent used to prepare technetium-99m ((99m)Tc)-labeled polypeptides with tricine as a coligand. However, (99m)Tc-HYNIC-labeled polypeptides show delayed elimination rates of the radioactivity not only from the blood but also from nontarget tissues such as the liver and kidney. In this study, a preformed chelate of tetrafluorophenol (TFP) active ester of [(99m)Tc](HYNIC)(tricine)(benzoylpyridine: BP) ternary complex was synthesized to prepare (99m)Tc-labeled polypeptides with higher stability against exchange reactions with proteins in plasma and lysosomes using the Fab fragment of a monoclonal antibody and galactosyl-neoglycoalbumin (NGA) as model polypeptides. When incubated in plasma, [(99m)Tc](HYNIC-Fab)(tricine)(BP) showed significant reduction of the radioactivity in high molecular weight fractions compared with [(99m)Tc](HYNIC-Fab)(tricine)(2.) When injected into mice, [(99m)Tc](HYNIC-NGA)(tricine)(BP) was metabolized to [(99m)Tc](HYNIC-lysine)(tricine)(BP) in the liver with no radioactivity detected in protein-bound fractions in contrast to the observations with [(99m)Tc](HYNIC-NGA)(tricine)(2.) In addition, [(99m)Tc](HYNIC-NGA)(tricine)(BP) showed significantly faster elimination rates of the radioactivity from the liver as compared with [(99m)Tc](HYNIC-NGA)(tricine)(2.) Similar results were observed with (99m)Tc-labeled Fab fragments where [(99m)Tc](HYNIC-Fab)(tricine)(BP) exhibited significantly faster elimination rates of the radioactivity not only from the blood but also from the kidney. These findings indicated that conjugation of [(99m)Tc](HYNIC)(tricine)(BP) ternary ligand complex to polypeptides accelerated elimination rates of the radioactivity from the blood and nontarget tissues due to low binding of the [(99m)Tc](HYNIC)(tricine)(BP) complex with proteins in the blood and in the lysosomes. Such characteristics would render the TFP active ester of [(99m)Tc](HYNIC)(tricine)(BP) complex attractive as a radiolabeling reagent for targeted imaging.     

生长抑素受体显像剂 99Tcm-HTOC的制备

目的　研制99Tcm 标记的生长抑素受体 (SSTR)阳性肿瘤显像剂99Tcm 6 肼基烟酰基 [Tyr3] octreotide (HTOC)。方法　通过四步化学反应合成 ,HPLC纯化 ,得到HTOC ;用乙二胺 N ,N′ 二乙酸 (EDDA)作为配体进行99Tcm 标记 ,测定标记率与标记物稳定性。结果　所合成HTOC质谱的分子离子峰与HTOC相对分子质量一致 ,99Tcm HTOC标记率 >95 % ,标记后 6h放化纯仍 >95 %。结论该法研制的99Tcm HTOC标记率高 ,体外稳定性好。     

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.     

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.     

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.     

Confirmation of hydrazone formation in HYNIC-peptide conjugate preparation, and its hydrolysis during labeling with (99m)Tc.

Because of its monodenticity, 6-hydrazinopyridine-3-carboxylic acid (HYNIC) is of interest as a bifunctional chelator for labeling peptide with (99m)Tc. Here, we confirm the formation of hydrazone in HYNIC-conjugated peptide. The preparative HPLC was used to purify the HYNIC conjugated somatostatin-based peptide and the result showed two peaks, even after two consecutive purifications. Analysis of these peaks by mass spectrometry indicated the presence of hydrazone, produced during preparation conjugate. Further, we have shown that presence of hydrazone really does not matter because under (99m)Tc-labeling conditions, hydrazone is hydrolyzed back to HYNIC that then chelates (99m)Tc. A HYNIC-peptide conjugate freeze-dried kit was also prepared in a mildly acidic or neutral condition with a final pH of 6-7. The kit was then labeled by (99m)Tc and incubated in 100 degrees C for 10min, and a labeling yield of >95% was obtained.     

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.     

Radiolabeled bombesin analogs for prostate cancer diagnosis: preclinical studies

INTRODUCTION: Radionuclide imaging can be a useful tool for the diagnosis of prostate cancer. Bombesin (BBN) is a molecule with high affinity for gastrin releasing peptide (GRP) receptors which are over-expressed in that tumor. This report compares (99m)Tc-HYNIC-betaAla-BBN(7-14)NH2 [(99m)Tc-HYNIC-BBN] and (99m)Tc identical withN(PNP6)-Cys-betaAla-BBN(7-14)NH2 [(99m)TcN(PNP6)-Cys-BBN] with regard to labeling procedures as well as in vitro and in vivo evaluation (biodistribution and scintigraphic imaging). METHODS: Peptide synthesis was performed in an automated peptide synthesizer. HYNIC-BBN was radiolabeled with pertechnetate using tricine and ethylenediamine diacetic acid (EDDA) as coligands. Cys- BBN was radiolabeled in a two-step procedure with the preparation of the precursor (99m)Tc-Nitrido first and then introducing diphosphine (PNP6). Radiochemical evaluation of conjugates, as well as studies of stability, transchelation toward cysteine, and partition coefficient were done. Biological studies included internalization, biodistribution in healthy animals and in animals bearing PC3 cancer cells with acquisition of images from the tumor-bearing animals. RESULTS: Both complexes showed a high radiochemical yield along with good stability. Biodistribution studies pointed out strong renal excretion for the former complex due to its hydrophilic profile and marked hepatobiliary excretion for the latter, corresponding to observed lipophilicity. Tumor uptake was higher for (99m)Tc-HYNIC-BBN and the same occurred with internalization findings, which exceeded those of (99m)TcN(PNP6)-BBN. Blocking studies in mice bearing PC-3 tumor cells revealed significantly reduced pancreas and tumor uptake, demonstrating receptor specificity of the conjugates. CONCLUSION: The best radiotracer was (99m)Tc-HYNIC-BBN on the basis of high radiochemical yield, fast radiolabeling procedure without need for a purification step, and more consistent tumor uptake.     

Optimization of the preparation of 99mTc-labeled Hynic-derivatized Annexin V for human use

Hydrazino nicotinate (Hynic) is one of the most attractive bifunctional agents designed for the labeling of proteins with (99m)Tc. Recently, a (99m)Tc-labeled Hynic-Annexin V derivative has been described and successfully evaluated in animal models of apoptosis. Prior to a phase I human study, the preparation of (99m)Tc-Hynic-Annexin V has been optimized. The influence of the Hynic-load of Annexin V, amount of protein, nature and amount of reducing agent, activity and co-ligand on the labeling yield were evaluated using ITLC and size-exclusion FPLC. Optimal labeling yields were obtained when 60-90 microgram Hynic-Annexin V was labeled with up to 1.11 GBq (30 mCi) (99m)TcO(4)-using 10-20 microgram SnCl(2).2H(2)O as reducing agent and 1.5 mg tricine as the co-ligand. Biodistribution in normal mice was comparable to literature data.     

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。     

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.     

Breast tumor targeting with (99m)Tc-HYNIC-PR81 complex as a new biologic radiopharmaceutical

Human epithelial mucin, MUC1, is commonly overexpressed in adenocarcinoma that includes more than 80% of breast cancers. The PR81 is a murine anti-MUC1 monoclonal antibody (MAb) that was prepared against the human breast cancer. We developed an indirect method for labeling of this antibody with (99m)Tc in order to use the new preparation in immunoscintigraphy studies of BALB/c mice bearing breast tumors. The (99m)Tc-PR81 complex was prepared using the HYNIC as a chelator and tricine as a coligand. The labeling efficiency determined by instant thin-layer chromatography (ITLC) was 89.2%+/-4.7%, and radiocolloides measured by cellulose nitrate electrophoresis were 3.4%+/-0.9%. The in vitro stability of labeled product was determined at room temperature by ITLC and in human serum by gel filtration chromatography - 88.3%+/-4.6% and 79.8%+/-5.7% over 24 h, respectively. The integrity of labeled MAb was checked by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis, and no significant fragmentation was seen. The results of cell binding studies showed that both labeled and unlabeled PR81 were able to compete for binding to MCF 7 cells. Biodistribution studies performed in female BALB/c mice with breast tumor xenografts at 4, 16 and 24 h after the (99m)Tc-HYNIC-PR81 injection demonstrated a specific localization of the compound at the site of tumors and minimum accumulation in non target organs. The tumor imaging was performed in BALB/c mice with breast xenograft tumors at 4, 8, 12, 16, 20, 24, 28, 32 and 36 h after the complex injection. The tumors were visualized with high sensitivity after 8 h. The findings showed that the new radiopharmaceutical is a promising candidate for radioimmunoscintigraphy of the human breast cancer.     

99mTc-labeled annexin V fragments: a potential SPECT radiopharmaceutical for imaging cell death

INTRODUCTION: Annexin V is a protein that binds to phosphatidylserine exposed on dying cells. The phosphatidylserine-specific sequence is attributed to a chain on the N-terminal of annexin consisting of 13 amino acid sequence. Radiolabeled annexin V is used for imaging apoptosis. METHODS: With an aim to synthesize a probe that can detect cell death akin to annexin V but smaller in size, annexin-13 fragments were derivatized to contain cysteine, cysteine-cysteine and histidine in their sequence at N terminal and were labeled with (99m)Tc via nitrido and carbonyl precursors. The (99m)Tc-labeled annexin-13 derivatives were characterized by HPLC and studied for their stability. In vitro and in vivo studies were carried out in apoptotic HL-60 cells and fibrosarcoma tumor-bearing Swiss mice, respectively. RESULTS: The (99m)Tc complexes were formed in high yields and were found to be stable. HPLC pattern of (99m)Tc nitrido complex of cysteine-cysteine-annexine 13 (CC-Anx13) and (99m)Tc carbonyl complex of histdine-annexin 13 (H-Anx13) revealed the formation of single species. In vitro cell uptake studies with (99m)Tc nitrido complex of cysteine-cysteine-annexin 13 fragment showed 6.5% uptake in apoptotic HL-60 cells. The uptake was found to be specific on testing with apoptotic HL-60 cells. Biodistribution studies of (99m)Tc nitrido complex with CC-Anx13 in fibrosarcoma tumor-bearing Swiss mice revealed optimum tumor uptake of 0.52 (0.17) %ID/g at 1 h pi. CONCLUSION: (99m)Tc(N)-CC-anx13 showed specific uptake in apoptotic tumor cells and warrants further evaluation.     

Synthesis of 99mTc(CO)3-deoxyuridine derivatives as potential HSV1-tk gene expression imaging agents.

In this study, we synthesized (99m)Tc(CO)(3)-2'-aminomethylpyridyl-2'-deoxyuridine ((99m)Tc(CO)(3)-AMPDU) and (99m)Tc(CO)(3)-aminoethylpyridyl-2'-deoxyuridine ((99m)Tc(CO)(3)-AEPDU) as potential agents for imaging the expression of the non-invasive herpes simplex virus type-1 thymidine kinase. AMPDU and AEPDU were synthesized from uridine in five chemical steps and then labeled with [(99m)Tc(CO)(3)(H(2)O)(3)](+) (370MBq/0.5 mL) at 100 degrees C for 10 min. Under optimal conditions (0.5 and 1.0mg for AMPDU and AEPDU and heating for 10 min), the labeling efficiency was 95.3+/-2.8% for AMPDU and 94.2+/-5.1% for AEPDU. To validate the chemical structure of (99m)Tc(CO)(3)-labeled compounds, we also synthesized ReBr(CO)(3)-AMPDU and ReBr(CO)(3)-AEPDU by reacting [Et(4)N][ReBr(3)(CO)(3)] and AMPDU or AEPDU in methanol at 25 degrees C for 6h. (99m)Tc(CO)(3)-AMPDU and (99m)Tc(CO)(3)-AEPDU had the same retention time on HPLC analysis as ReBr(CO)(3)-AMPDU and ReBr(CO)(3)-AEPDU. (99m)Tc(CO)(3)-AMPDU and (99m)Tc(CO)(3)-AEPDU had high radiochemical stabilities of 98.1+/-1.5% and 98.0+/-1.7% for 6h, respectively.     

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.     

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.     

Radiolabeling morpholinos with 90Y, 111In, 188Re and 99mTc

This laboratory is investigating morpholinos (MORF), a DNA analogue, for radiopharmaceutical applications. While we routinely radiolabel with (99m)Tc, we have now labeled MORFs with (111)In, (188)Re and (90)Y in anticipation of therapeutic studies. METHODS: A 25 mer MORF with a primary amine on the 3' equivalent end attached via a 10 member linker was conjugated with an isothiocyanate backbone derivative of DOTA (for labeling with (111)In and (90)Y) and with NHS-MAG(3) (for labeling with (188)Re and (99m)Tc). The in vitro stability of labeled MORFs were investigated and biodistribution was carried out in normal mice. RESULTS: As evident by size exclusion HPLC, ITLC and Sep-Pak analysis, all four radiolabeled MORFs were successfully radiolabeled. In each case, the labeled MORFs showed one sharp peak in HPLC that shifted completely to earlier retention times following addition of a polymer conjugated with the complementary MORF. In saline at room temperature and in 37 degrees C serum, the radioactivity profile of (111)In, (188)Re and (99m)Tc was unchanged over 48 h while over the same period, the (90)Y profile showed a pronounced lower molecular weight peak which did not shift and was shown to be most probably due to (90)Y-DOTA resulting from radiolysis. In addition, the recovery of (188)Re on HPLC decreased as samples aged probably due to oxidation to perrhenate which was retained by the HPLC column. The biodistributions at 1, 3 and 6 h in normal mice showed no important differences among all four labels with the exception that levels of radioactivity in stomach and thyroid were higher in the case of (188)Re due to in vivo oxidation of the radiolabel to perrhenate. CONCLUSIONS: When radiolabeled with DOTA, (90)Y-labeled MORF showed increased instabilities relative to that of (111)In and when radiolabeled with MAG(3), (188)Re showed in vitro and in vivo instabilities compared to (99m)Tc, but all labels were still largely intact after 48 h in saline or serum. Possibly because of the rapid clearance of MORFs, no important differences in biodistribution among (90)Y, (111)In and (99m)Tc labels were evident in normal mice. These strategies for labeling MORF with (90)Y and (188)Re therefore appear to be suitable for therapeutic applications although both show some evidence of instabilities.     

Preparation and characterization of 99mTc(CO)3-BPy-RGD complex as alphav beta3 integrin receptor-targeted imaging agent.

The aim of this study is to develop a novel arginine-glycine-aspartic acid (RGD) peptide-containing ligand for (99m)Tc labeling as alpha(v)beta(3) integrin receptor-targeted imaging agent. BPy-RGD conjugate was successfully synthesized by coupling of 5-carboxylate-2,2'-bipyridine and c(RGDyK) peptide through EDC/SNHS in aqueous solution and was characterized by MADLI-TOF-MS (m/z=802.72, C(38)H(48)N(11)O(9)). (99m)Tc(CO)(3)-BPy-RGD was prepared by exchange reaction between [(99m)Tc(H(2)O)(3)(CO)(3)](+) and BPy-RGD. Final product was purified by HPLC and tested for octanol/water partition coefficient. Cell-binding assays of BPy-RGD and unmodified c(RGDyK) were tested in MDA-MB-435 cells ((125)I-echistatin as radioligand). Preliminary biodistribution of the (99m)Tc(I)-labeled radiotracer in orthotopic MDA-MB-435 breast tumor xenograft model was also evaluated. The BPy-RGD conjugate had good integrin-binding affinity (50% inhibitory concentration (IC(50))=92.51+/-22.69 nM), slightly lower than unmodified c(RGDyK) (IC(50)=59.07+/-11.03 nM). The hydrophilic radiotracer also had receptor-mediated activity accumulation in MDA-MB-435 tumor (1.45+/-0.25 percentage of injected dose per gram (%ID/g) at 1.5h postinjection (p.i.)), which is known to be integrin positive. After blocking with c(RGDyK), the tumor uptake was reduced from 0.71+/-0.01%ID/g to 0.33+/-0.18%ID/g at 4h p.i. (99m)Tc(I) tricarbonyl complex of cyclic RGD peptide is a promising strategy for integrin targeting. Further modification of the bipyridine-conjugated RGD peptide by using more potent RGD peptides and fine tuning of the tether group between the RGD moiety and (99m)Tc(CO)(3)(+) core to improve the tumor targeting efficacy and in vivo kinetic profiles is currently in progress.