99mTc glucarate high-resolution imaging of drug sensitive and drug resistant human breast cancer xenografts in SCID mice

BACKGROUND AND AIM: Previous studies have showed that 99mTc labelled glucarate (GLA) might be an agent for non-invasive detection of breast tumours. In xenografted BT20 breast tumours, GLA was found to have higher uptake than 99mTc sestamibi (MIBI). It is unclear whether GLA can localize in all cell line breast cancer xenografts, as well as breast tumours with multidrug resistance (MDR). The present study aimed to investigate the properties of GLA in detecting drug sensitive and drug resistant MCF7 breast cancer xenografts in mice by using dynamic single photon emission computed tomography (SPECT) imaging. METHODS: MCF7/S cells are drug sensitive breast carcinoma cells. MCF7/D40 cells are 40-fold more resistant to doxorubicin compared to MCF7/S. Subcutaneous tumours were grown in SCID mice for 10-14 days after injection of 1 x 10(6) cells into the right thigh. Anaesthetized mice with MCF7/S (MIBI, n=9; GLA, n=8) and MCF7/D40 (MIBI, n=6; GLA, n=5) tumours were imaged using a high-resolution SPECT system called FASTSPECT. Dynamic images were acquired for 2 h after intravenous injection of GLA or MIBI. Expression of MDR P-glycoprotein (Pgp) in the tumours was demonstrated in the MCF7/D40 tumours by western blotting, not in the MCF7/S tumours. RESULTS: The xenografted tumours were visualized unequivocally within 10-30 min in GLA images and remained detectable for at least 2 h after injection. Drug resistant tumours, from which MIBI was rapidly expelled, retained GLA as readily as did drug sensitive tumours. The biodistribution data of GLA demonstrated significantly higher accumulation (%ID/g) compared to MIBI. CONCLUSION: MCF7 tumour xenografts can be detected by 99mTc glucarate imaging. More importantly, 99mTc glucarate can potentially localize drug resistant breast tumours.     

Development of 99mTc labelled Lipiodol: biodistribution following injection into the hepatic artery of the healthy pig

BACKGROUND: We develop a method for the radiolabelling of Lipiodol with Tc, using a lipophilic complex, [99mTc-(S2CPh)(S3Ph)2], dissolved in Lipiodol (99mTc-SSS Lipiodol). RESULTS: The labelling yield is high (96 +/- 0.8%), and the radiochemical purity satisfactory (92 +/- 2.6%). This labelling is reproducible and stable for up to 24 h in vitro. Studies carried out after injection into the hepatic artery of the healthy pig show that the biodistribution of 99mTc-SSS Lipiodol is comparable with that observed for 188Re Lipiodol. MATERIALS AND METHODS: The 99mTc-SSS lipiodol was obtained after dissolving a chelating agent, previously labelled with 99mTc, in cold lipiodol. The radiochemical purity (RCP) of the labelling was checked immediately and at 24 h. The 99mTc-SSS lipiodol was injected into the hepatic artery of four healthy pigs for an ex-vivo biodistribution study. An autoradiographic study was performed in two cases. CONCLUSIONS: Apart from the specific interest of a Lipiodol-bearing technetiated agent for carrying out dosimetric studies, the labelling of Lipiodol with 99mTc is a preliminary step towards the use of radiolabelling with the 188Re analogue.     

Influence of different chelators (HYNIC, MAG3 and DTPA) on tumor cell accumulation and mouse biodistribution of technetium-99m labeled to antisense DNA

We have shown recently that cell accumulation in culture of antisense DNA is strongly influenced by the presence of a 99mTc-MAG3 group for radiolabeling. We have now compared the in vitro and mouse in vivo behavior of 99mTc when radiolabeled to one antisense phosphorothioate DNA by three different methods. The 18-mer antisense DNA against the RIalpha subunit of PKA was conjugated via a primary amine on the 5'-end with the NHS esters of HYNIC and MAG3 and by the cyclic anhydride of DTPA. Surface plasmon resonance measurements revealed that the association rate constant for hybridization was unchanged for all three chelators as compared with that of the native DNA. Size exclusion HPLC showed rapid and quantitative protein binding for all three chelators upon incubation of labeled DNAs in 37 degrees C serum and cell culture medium. However, in each case, radiolabeled and intact oligonucleotide was still detectable after 24 h. Cellular uptake was tested in an RIalpha mRNA-positive cancer cell line. The order of cellular accumulation of 99mTc was DTPA>HYNIC(tricine) >MAG3, with the differences increasing with time between 4 and 24 h. The rate of 99mTc egress from cells was found to be MAG3>HYNIC>DTPA, which may explain the order of cellular accumulation. The biodistribution in normal mice was heavily influenced by the labeling method and followed a pattern similar to that seen previously by us for peptides labeled with the same chelators. In conclusion, although these studies concerned only one antisense DNA in one cell line, the results suggest that the success of antisense imaging may depend, in part, on the method of radiolabeling.     

Molecular-size fractionation of pentastarch, radiolabelling with 99mTc, and evaluation of biological behaviour in mice

BACKGROUND: Pentastarch is used clinically as a plasma volume expander for the management of substantial blood loss. 99mTc labelled pentastarch may be useful as a diagnostic agent in place of 99mTc labelled red blood cells. METHODS: Commercial pentastarch (PS; molecular weight (MW) 240 kDa) was separated according to molecular size by using chromatography, and the fractions were pooled as small (MW 128 kDa), medium (MW 277 kDa) and large (MW 510 kDa) pentastarch. We studied the effect of various physicochemical parameters on the efficiency of radiolabelling with 99mTc and on the stability of the products, and evaluated the biological properties of the 99mTc labelled preparations. RESULTS: We developed an optimised kit formulation containing 3.25 mg pentastarch and 0.13 mg gentisic acid that can be reliably labelled with 99mTc at pH 6.6-8.2 with good stability. In mice, the 99mTc labelled medium pentastarch showed the more favourable blood retention properties (56% of initial blood activity is retained after 3 h) with lower liver levels.     

The labelling of human serum transferrin with 99mTc and a study concerning uptake of the complex by tumour cells

The direct labelling of serum transferrin (sTf) with 99mTc on high-affinity binding sites, producing a complex of excellent stability, is described. The high-affinity binding sites were prepared by pre-treating sTf with 2-mercaptoethanol. For the radiolabelling step, thiourea was used as an exchange ligand and a filtration procedure used to remove 99mTc that had not complexed with the protein. RT112 bladder tumour cells incubated in the presence of labelled sTf showed a rapid initial uptake of 99mTc, reaching a plateau after about 20 min. Radiolabelling was also carried out without a pre-reduction step in an attempt to form a co-ordination complex between 99mTc and the Fe3+-binding site of sTf, analogous to that formed by Fe3+. The tumour cell uptake of sTf labelled without pre-reduction was then examined. In contrast to 59Fe3+ and other radio-metals co-ordinated with the Fe3+-binding site which show a continuous increase in incorporation with time, the uptake of 99mTc rapidly reached a plateau.     

Development and validation of hydroxy ethyl starch kits for instant use in gastroesophageal reflux and gastric motility studies

A reduction method based on stannous chloride is described to prepare hydroxy ethyl starch kits for gastrointestinal reflux and gastric motility studies. Following verification of the consistency of radiolabelling, in vitro experiments were carried out to validate 99mTc hydroxy ethyl starch as a liquid phase and solid phase gastric motility imaging radiotracer. Gastroesophageal reflux, liquid phase and solid phase studies were then conducted in 13 adult volunteers to examine the in vivo stability of the radiotracer. High labelling efficiency (>95% when prepared at neutral pH) was consistently achieved, which remained stable in conditions simulating gastric environment. Twelve of the 13 volunteers did not show absorption of any radioactivity from the gastro-intestinal tract. 99mTc hydroxy ethyl starch is a new agent suitable for gastroesophageal reflux and gastric motility studies. It is available in kit form and is a more 'physiological' agent than 99mTc sulphur colloid for preparing a solid radioactive meal. 99mTc hydroxy ethyl starch represents a true carbohydrate meal, and unlike 99mTc sulphur colloid, is easy to prepare and can easily be standardized to produce a standard vegetarian meal.     

Labeling of phosphorothioate antisense oligonucleotides with yttrium-90

Novel yttrium-90 (90Y)-labeled phosphorothioate antisense oligonucleotides were designed as a potential targeted radionuclide therapeutic agent for malignant tumors. A 15-mer phosphorothioate antisense oligonucleotide, which was complementary to the translation start region of the N-myc oncogene mRNA, was conjugated with isothiocyanobenzyl ethylenediamine tetraacetic acid (SCN-Bn-EDTA), via a C-5-substituted deoxyuridine that had replaced a thymine in the oligonucleotide, and was then labeled with 90Y-acetate. Following purification, the radiochemical purity of the 90-Y-Bn-EDTA-phosphorothioate antisense oligonucleotides was estimated by 2.0% agarose gel electrophoresis, and the specific hybridization of 90Y-Bn-EDTA-phosphorothioate antisense oligonucleotide to a phosphorodiester sense oligonucleotide was investigated by 20% polyacrylamide gel electrophoresis in a cell-free system. Radiochemical purity was 98.7 +/- 0.4% at 72 h after labeling and 90.3 +/- 0.9% after 72-h incubation with human normal serum. The 90Y-Bn-EDTA-phosphorothioate antisense oligonucleotide hybridized specifically to a complementary phosphorodiester sense oligonucleotide. In conclusion, phosphorothioate antisense oligonucleotides can be labeled stably with 90Y using SCN-Bn-EDTA without loss of hybridization properties.     

Radioguided sentinel node detection in breast cancer patients: comparison of 99mTc phytate and 99mTc rhenium colloid efficacy

BACKGROUND: Radioguided sentinel node biopsy (SNB) of breast cancer patients has become a standard method for detecting early stage breast cancer. However, no standard radiopharmaceutical exists. METHODS: 99mTc rhenium colloid or 99mTc phytate SNB was used to aid detection in breast cancer patients. For each radiopharmaceutical, 100 patients were examined. The following points were compared: (1) scintigraphic detection rate of axillary sentinel nodes (detectability and number when detectable) and internal mammary sentinel nodes; (2) the number of nodes detected scintigraphically and the number detected during surgery; (3) sensitivity, specificity, accuracy, negative predictive value, and positive predictive value for axillary sentinel nodes. RESULTS: Axillary sentinel nodes of patients were biopsied using either 99mTc rhenium or 99mTc phytate. The number of axillary nodes surgically removed from patients given 99mTc rhenium was 2.28+/-1.08 (mean+/-SD), and the number of axillary nodes surgically removed from patients given 99mTc phytate was 1.68+/-0.82. Some patients given 99mTc rhenium showed a spill-over of radioactivity from sentinel nodes. Concordance of scintigraphically detected nodes and surgical removed nodes was superior for 99mTc phytate compared to that with 99mTc rhenium, with a statistically significant difference. The sensitivity and negative predictive value was superior with 99mTc phytate compared to that with 99mTc rhenium, even though no statistical difference was detectable. However, visualization of internal mammary nodes was superior with 99mTc rhenium. CONCLUSION: In breast cancer patients, 99mTc phytate is a better choice for the detection of axillary SNB than 99mTc rhenium colloid. However, 99mTc rhenium colloid is a better choice for the detection of internal mammary nodes.     

Initial observations of 99mTc labelled locked nucleic acids for antisense targeting

BACKGROUND AND OBJECTIVE: The most recent DNA analogues to become commercially available are known as locked nucleic acids (LNAs). The aim of this study was to evaluate the properties of LNAs for antisense targeting. METHODS: A 15 mer LNA antisense to RIalpha mRNA was studied in cell culture. The antisense LNA (5'-amine linker-TGCCTCCTCACTGGC) was purchased along with its sense control LNA. Surface plasmon resonance was used to compare affinity constants with uniform 18 mer phosphorothioate (PS) DNA and uniform 18 mer phosphodiamidate morpholinos (MORFs, another DNA analogue). After radiolabelling with 99mTc via MAG3, the antisense and sense LNAs were added at 5 nM to wells containing ACHN cells in culture and accumulations measured over 24 h. Subcellular partition was determined after 16 h of incubation by separating membrane bound, cytoplasmic and nuclear fractions. The cell studies were conducted both with naked LNAs and with liposomes (oligofectamine) as carrier. RESULTS: Radiochemical purity was about 95% after purification on a P4 column and each LNA was radiolabelled at about 20 GBq.micromol(-1) (100.microCi.microg(-1)). The surface plasmon resonance results showed a more favourable dissociation constant for the duplex with DNA of the 15 mer LNA (0.55 x 10(-10).M(-1)) compared to the duplex with 18 mer DNA and 18 mer MORFS (2.05 and 1.06 x 10(-10).M(-1), respectively). Because of lower dissociation constants, the hybridization affinities of LNAs are therefore higher than those of uniform and identical PS DNAs or MORFs. The cellular accumulations suggested an antisense effect in that the antisense LNA accumulation was higher than sense both when added naked (1.8% vs. 0.4% at 24 h) and with liposome carrier (3.8% vs. 1.0% at 24 h). Thus while absolute cellular uptake was lower than that observed by this laboratory with other oligomers, the antisense/sense differential was higher. The number of antisense LNAs accumulating per cell specifically (i.e., antisense minus sense) was about 45,000 naked and about 100,000 with carrier. Subcellular partition showed that both LNAs were partitioned to each fraction with antisense accumulations greater than sense and carrier accumulations greater than naked as before. That as much as 2.9% of the antisense LNA (with carrier) was in the cytoplasmic or nuclear factions demonstrates that the LNA was internalized. CONCLUSIONS: LNAs appear to be attractive oligomers for antisense targeting and other radiopharmaceutical applications.     

Direct labelling of octreotide with 99mTc: effect of different concentration of reducing agents and amount of sodium pertechnetate on radiolabelling efficiency.

Octreotide, a synthetic analog of natural hormone somatostatin, was labelled with 99mTc. Labelling was accomplished by reduction of the cysteine bridge, which provided sulfhydryl groups for chelating with 99mTc. Sodium ascorbate and sodium dithionite in different concentrations were used as reducing agents. Different amounts of sodium pertechnetate were used for labelling of peptide. When the mass ratio of peptide and sodium ascorbate was 1:100 and the final concentration of dithionite in the labelling vial was 0.2-0.4 microg/microl with 0.18-1.48 GBq sodium pertechnetate more than 80% radiolabelling efficiency was confirmed by RP-HPLC, ITLC-SG and C18 Cartridge analysis. The stability of the 99mTc-peptide bond was evaluated by human serum challenge and that showed the stability was 90% after 4h.     

99Tcm标记反义肽核酸探针的制备及其荷瘤裸鼠体内分布

目的 探讨99Tcm标记反义肽核酸(PNA)探针的新方法及其在生物体内的分布.方法 合成12mer且5'端含有四肽G-(D)-A-G-G的c-myc mRNA反义、无义PNA片段,利用G-(D)-A-G-G形成的N4结构为螯合基团进行99Tcm标记,用聚酰胺薄膜层析法和高效液相色谱仪法(HPLC)测定其标记率和标记物的稳定性,并行人结肠癌荷瘤裸鼠体内分布[每克组织百分注射剂量率(%ID/g)]及显像研究.采用SAS 6.22软件对数据进行分析.结果 反义、无义PNA片段合成物的纯度>95%.99Tcm标记c-myc mRNA反义、无义PNA的标记率>95%,标记物室温放置18 h测定其标记率仍可达95%以上.c-myc mRNA反义、无义PNA片段4～8℃下放置3个月标记率仍>95%.HPLC测定标记物呈单峰.99Tcm标记c-myc mRNA反义PNA主要分布在荷瘤鼠肾、脾、肿瘤、肠道、肝组织中,99Tcm标记c-myc mRNA无义PNA在荷瘤鼠血液、脾、肾、肝及肺组织中分布较多;注射后4 h两者在荷瘤鼠肿瘤组织中的分布差异有统计学意义[(1.11±0.12)%ID/g和(0.14±0.02)%ID/g;t=14.75,P<0.01].99Tcm标记c-mycmRNA反义PNA在小鼠体内肿瘤/肌肉、肿瘤/肺的摄取比值较高,肿瘤显像明显.结论 用99Tcm标记c-myc mRNA反义、无义PNA的方法简单,标记率高,标记物稳定,前者有望成为一种新型肿瘤显像剂.     

HER-2反义寡核苷酸对靶基因抑制作用的时相变化与微观分布研究

目的:研究靶向HER-2基因、具有不同活性的反义硫代寡核苷酸(S-ODNs)在SK-BR-3细胞内的微观分布差异,以及与靶基因转录和翻译抑制作用的关系.方法:运用实时定量PCR和Western印迹方法检测反义寡核苷酸对细胞靶基因转录和翻译抑制的时相性变化,用激光扫描共聚焦显微镜(LSM)观察6-羧基荧光素标记S-ODNs在靶细胞内的分布.结果:反义S-ODNs能够在mRNA与蛋白水平抑制HER-2的表达.不同序列的抑制活性各不相同,但最大抑制强度均出现于转染后48～72 h之间,此后靶基因表达逐渐恢复.高活性序列主要均匀分布于细胞核,且持续时间长,低活性序列呈点状分布于胞质,少量分布于细胞核且持续时间短.结论:活性不同的S-ODNs亚细胞分布有显著区别,且分布变化与对靶基因的抑制效应具有对应关系.     

99mTc-labelled human serum transferrin for tumour imaging: an in vitro and in vivo study of the complex

This pilot study reports the uptake of 99mTc-transferrin by MCF7 cells and the biodistribution of the complex in mice bearing MCF7 tumours. Human serum holo-transferrin was labelled with 99mTc using pre-reduction with 2-mercaptoethanol. The uptake of the complex by MCF7 breast tumour cells, which was found to be competitively inhibited by the presence of unlabelled transferrin, reached a plateau within 30 min. Two groups of xenografted mice with small and large tumours, respectively, were injected with the complex, and the uptake was followed for up to 24 h post-administration using a dedicated gamma camera. The mean tumour uptake values, determined after dissection, in the two groups of mice were 6% (small tumours) and 1.7% (large tumours) of the injected dose per gram of tissue, with mean tumour/blood ratios of 2.7 and 1.7, respectively.     

Preparation and evaluation of 99mTc-EDDA/HYNIC-[Lys 3]-bombesin for imaging gastrin-releasing peptide receptor-positive tumours

BACKGROUND: Bombesin is a peptide that was initially isolated from frog skin and which belongs to a large group of neuropeptides with many biological functions. The human equivalent is gastrin-releasing peptide (GRP), whose receptors are over-expressed in a variety of malignant tumours. AIM: To prepare a HYNIC-[Lys 3]-bombesin analogue that could be easily labelled with 99mTc from lyophilized kit formulations and to evaluate its potential as an imaging agent for GRP receptor-positive tumours. METHODS: HYNIC was conjugated to the epsilon-amino group of Lys 3 residue at the N-terminal region of bombesin via succinimidyl-N-Boc-HYNIC at pH 9.0. 99mTc labelling was performed by addition of sodium pertechnetate solution and 0.2 M phosphate buffer pH 7.0 to a lyophilized formulation. Stability studies were carried out by reversed phase HPLC and ITLC-SG analyses in serum and cysteine solutions. In-vitro internalization was tested using human prostate cancer PC-3 cells with blocked and non-blocked receptors. Biodistribution and tumour uptake were determined in PC-3 tumour-bearing nude mice. RESULTS: 99mTc-EDDA/HYNIC-[Lys 3]-bombesin was obtained with radiochemical purities >93% and high specific activity ( approximately 0.1 GBq.nmol). Results of in-vitro studies demonstrated a high stability in serum and cysteine solutions, specific cell receptor binding and rapid internalization. Biodistribution data showed a rapid blood clearance, with predominantly renal excretion and specific binding towards GRP receptor-positive tissues such as pancreas and PC-3 tumours. CONCLUSION: 99mTc-EDDA/HYNIC-[Lys 3]-bombesin obtained from lyophilized kit formulations has promising characteristics for the diagnosis of malignant tumours that over-express the GRP receptor.     

Induction of unstable and stable chromosomal aberrations by 99mTc: in-vitro and in-vivo studies

BACKGROUND: Biological dosimetry, which determines the dose of acquired radiation by measuring radiation-induced variation of biological parameters, can help assess radiation damage in an individual. Evaluation of radiation exposure requires setting up reference curves for each type of radiation. AIM: To evaluate the potential induction of chromosome aberrations by a clinical diagnostic dose of 99mTc. METHODS: Dicentrics, rings, excess fragments, complete reciprocal translocations and incomplete reciprocal translocations were scored in peripheral blood lymphocytes from patients exposed to a 99mTc bone scintigraphy. A specific relationship between the radiation dose delivered by 99mTc and the frequency of stable and unstable chromosomal aberrations was established in vitro to estimate whole-body dose. Chromosome analysis using fluorescence plus Giemsa and fluorescence in-situ hybridization was undertaken on six patients before and after a 99mTc bone scintigraphy. Dicentrics, rings, excess fragments, and translocations were scored in blood lymphocytes after in vitro 99mTc external irradiation in order to construct dose calibration curves. RESULTS: Analysis of the in-vitro data shows that the number of both unstable and stable aberrations has a quadratic linear relationship to the dose. Our in-vivo irradiation studies showed that activities of 99mTc-hexamethylene diphosphonate (99mTc-HDP) used for bone investigations do not induce any additional unstable chromosome aberrations and translocations. The frequencies obtained did not differ significantly from background values. CONCLUSIONS: 99mTc can produce unstable and stable chromosomal aberrations in vitro. 99mTc-HDP administration does not induce supplementary chromosomal aberrations. The dose-response curves will allow a more accurate evaluation of the risk related to in-vivo administration of 99mTc labelled radiopharmaceuticals, and they can be used to assess the safe upper limit of injected activity in humans.