99mTc-ethylenedicysteine-folate: a new tumor imaging agent. Synthesis, labeling and evaluation in animals

It is known that membrane folic acid receptors are responsible for cellular accumulation of folate and folate analogs such as methotrexate and overexpressed on various tumor cells. However, these receptors are highly restricted in normal differentiated tissues. Results of limited in vitro and in vivo animal studies suggest that folate receptors could be a potential target for tumor imaging. This study aimed to develop a 99mTc-labeled folic acid using ethylenedicysteine (EC) as a chelator and evaluate its labeling efficiency and potential use as a tumor seeking agent. Tissue distribution of 99mTc-EC-folate was determined in breast tumor-bearing rats at 20 min, 1, 2, and 4 h (n = 3/time interval, 370 KBq/rat, i.v.). Blocking study was employed to determine receptor-mediated process; 99mTc-EC-folate was co-administrated with 50 and 150 mumol/kg of cold folic acid to tumor-bearing rats. Planar imaging and whole-body autoradiograms were performed. The data was compared to that using 99mTc-EC (control). In animal studies, tumor/blood count density ratios at 20 min-4 h increased from 0.81 +/- 0.09 to 1.23 +/- 0.13 with 99mTc-EC-folate. Conversely, these values showed time-dependent decrease from 0.77 +/- 0.32 to 0.65 +/- 0.01 with 99mTc-EC in the same time period. Tumor/muscle and tumor/blood count density ratios significantly decreased with folic acid co-administrations. Planar images and autoradiograms confirmed that the tumors could be visualized clearly with 99mTc-EC-folate.     

3D radiobiological evaluation of combined radiotherapy and hyperthermia treatments

Purpose: Currently, clinical decisions regarding thermoradiotherapy treatments are based on clinical experience. Quantification of the radiosensitising effect of hyperthermia allows comparison of different treatment strategies, and can support clinical decision-making regarding the optimal treatment. The software presented here enables biological evaluation of thermoradiotherapy plans through calculation of equivalent 3D dose distributions.

Methods: Our in-house developed software (X-Term) uses an extended version of the linear-quadratic model to calculate equivalent radiation dose, i.e. the radiation dose yielding the same effect as the thermoradiotherapy treatment. Separate sets of model parameters can be assigned to each delineated structure, allowing tissue specific modelling of hyperthermic radiosensitisation. After calculation, the equivalent radiation dose can be evaluated according to conventional radiotherapy planning criteria. The procedure is illustrated using two realistic examples. First, for a previously irradiated patient, normal tissue dose for a radiotherapy and thermoradiotherapy plan (with equal predicted tumour control) is compared. Second, tumour control probability (TCP) is assessed for two (otherwise identical) thermoradiotherapy schedules with different time intervals between radiotherapy and hyperthermia.

Results: The examples demonstrate that our software can be used for individualised treatment decisions (first example) and treatment optimisation (second example) in thermoradiotherapy. In the first example, clinically acceptable doses to the bowel were exceeded for the conventional plan, and a substantial reduction of this excess was predicted for the thermoradiotherapy plan. In the second example, the thermoradiotherapy schedule with long time interval was shown to result in a substantially lower TCP.

Conclusions: Using biological modelling, our software can facilitate the evaluation of thermoradiotherapy plans and support individualised treatment decisions.     

Different biodistribution of 99mTc-labelled chimeric mouse-human monoclonal antibody between athymic mice model and human.

Biodistribution of chimeric mouse/human monoclonal antibody against non-specific cross-reacting antigen (chNCA Ab) was studied in athymic mice and patients with metastatic bone disease. 99mTc-chNCA Ab showed a high labelling efficiency, stability and also a high binding ratio to human granulocytes. Since NCA showed cross-reactivity with carcinoembryonic antigen (CEA), animal experiments showed that 99mTc-chNCA Ab was accumulated in the xenografted tumour which expressed CEA, suggesting the preserved immunoreactivity of labelled materials. In the clinical study, injected 99mTc-chNCA Ab formed a high molecular weight complex immediately after intravenous administration and was trapped mainly in liver. The first-phase plasma half-life was 6.4 +/- 1.1 min. None of the patients showed adverse reaction or human antimurine or anti-chimeric antibody in their serum. 99mTc-chNCA Ab demonstrated remarkably different biodistribution between patients and the animal model and showed different pharmacokinetics from other murine and chimeric Abs reported previously. For safety HPLC analysis should be performed before clinical radioimmunodetection or radioimmunotherapy by incubating radiolabelled MAb with human serum under strict conditions.     

Preclinical evaluation of a new bombesin analog for imaging of gastrin-releasing peptide receptors

Bombesin (BBN) is a peptide showing high affinity for the gastrin-releasing peptide receptor. Tumors such as prostate, small cell lung cancer, breast, gastric, and colon cancer are known to over express receptors to BBN and gastrin-releasing peptide (GRP). The goal of this study was to evaluate a new (67)Ga radiolabeled BBN analog based on the bifunctional chelating ligand DOTA (1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid), which could be used as a tool for diagnosis of GRP receptor-positive tumors. DOTA-GABA-BBN (7-14) NH(2) was synthesized using a standard Fmoc strategy. Labeling with (67)Ga was performed at 95°C for 30 minutes in ammonium acetate buffer (pH?=?4.8). Radiochemical analysis involved ITLC and HPLC methods. The stability of radiopeptide was examined in the presence of human serum at 37°C up to 24 hours. The receptor-bound internalization and externalization rates were studied in GRP receptor expressing PC-3 cells. Biodistribution of radiopeptide was studied in nude mice bearing PC-3 tumor. Labeling yield of >90% was obtained corresponding to a specific activity of approximatrly 2.6?MBq/nmol. Peptide conjugate showed good stability in the presence of human serum. The radioligand showed a good and specific internalization into PC-3 cells (16.13%?±?0.71% at 4 hours). After 4 hours, a considerable amount of activity (52.42%?±?1.86%) was externalized. In animal biodistribution studies, a receptor-specific uptake of radioactivity was observed in GRP-receptor-positive organs. After 4 hours, the uptake in mouse tumor and pancreas was 1.30%?±?0.18% ID/g (percentage of injected dose per gram of tissue) and 1.21%?±?0.13% ID/g, respectively. These data show that [(67)Ga]-DOTA-GABA-BBN (7-14) NH? is a specific radioligand for GRP receptor positive tumors and is a suitable candidate for clinical studies.     

99mTc-Tetraethylenepentamine-Folate--a new 99mTc-based folate derivative for the detection of folate receptor positive tumors: synthesis and biological evaluation

A new radiopharmaceutical, 99mTc-Tetraethylenepentamine(TEPA)-Folate has been synthesized introducing TEPA to the gamma-carboxyl group of folic acid. This binds with 99mTc high efficiency at ambient temperature. The resulting 99mTc-N5-Folate is stable under physiological conditions at least for 24 h after radiocomplexation. TEPA is a known open chain pentamine (N5) chelator, its four-nitrogen act as the binding site for 99mTc. The folate membrane receptor binding of the 99mTc-TEPA-Folate by established human tumor cell lines (KB, U-87MG and MDA-MB-468) showed Kd in microM range in normal DMEM (10% serum, 10 microM folic acid). The blood kinetic studies showed more than 70% clearance within five minutes from the circulation. The KB cell line tumors in mice were readily identifiable in the gamma images and revealed major accumulation of radiotracer in liver, kidneys and intestines. High tumor uptake was shown in the tumor bearing nude mice; tumorto-blood ratios reached 2.68 +/- 0.52 and 5.5 +/- 1.47 at 1 and 4 h after post injection respectively. Surviving fractions as obtained in clonogenic assay were 1.02 +/- 0.07 and 1.03 +/- 0.05 in U-87MG and MDA-MB-468 cell lines respectively. The 99mTc-N5-Folate conjugate have promising utility as a receptor specific radiopharmaceutical for imaging neoplastic tissues known to over express folate-binding protein.     

99mTc-labelled SM3 in the preoperative evaluation of axillary lymph nodes and primary breast cancer with change detection statistical processing as an aid to tumour detection.

The extent of primary surgery for breast cancer could be tailored to the patient if previous information on the presence or absence of lymph node involvement could be reliably determined. Prospective radioimmunoscintigraphy in 29 patients with primary breast cancer that was found on screening has been undertaken with 555 MBq (15 mCi) 99mTc SM3, an Imperial Cancer Research Fund (ICRF) murine monoclonal antibody, 0.5 mg with images at 10 min and 22 h, and analysis using a change detection algorithm. Sites of significant change between the early and later images were displayed as a map of probabilities. Image-positive and -negative axillary lymph nodes were compared by histology in the 28 evaluable patients. The correct identification of the presence or absence of node involvement, even if impalpable, has been shown in 24 out of 28 patients (29 lymph node groups). Sensitivity was 90% (nine out of ten), specificity 84% (16 out of 19) and accuracy 86%. These results encourage further assessment of this technique.     

99mTc-d-penicillamine-glucuronide: synthesis, radiolabeling, in vitro and in vivo evaluation

The current study was aimed at synthesizing a glucuronide derivative of D-penicillamine (D-PA) to be used for imaging purposes. First of all, D-PA-glucuronide (D-PA-Glu) was synthesized by experimental treatments starting with uridine 5'-diphospho-glucuronosyltransferase enzyme rich microsome preparate. Then, the synthesized compound was labeled with technetium ((99m)Tc) by using a reduction method with stannous chloride. Quality controls were performed by using high-performance liquid chromatography and thin-layer radio chromatography (TLRC). Radiolabeling yield of (99m)Tc-D-PA-Glu was more than 98% according to TLRC results. In vitro evaluations of radiolabeled complexes were investigated on PC-3 human prostate cancer cells. (99m)Tc-D-PA-Glu exhibited more accumulation on PC-3 cells versus (99m)Tc-D-PA at 240 minutes. In order to determine its radiopharmaceutical potential, biodistribution studies were carried out in male Albino Wistar rats. The biodistribution results of (99m)Tc-D-PA-Glu, showed the highest uptake in prostate at 120 minutes postinjection with the main excretion route being through kidneys and bladder. (99m)Tc-D-PA-Glu and (99m)Tc-D-PA have exhibited different biodistribution results.     

筛选化学过敏原的直接肽反应试验的建立

目的：建立筛选化学过敏原的体外替代法——直接肽反应试验。方法：赖氨酸/半胱氨酸肽与受试化学物反应24 h,经高效液相色谱检测肽损耗评价受试化学物的致敏性及其肽反应活性。结果：12种不同致敏级别化学物肽损耗率均值大于6.38%,为致敏阳性,反应活性不同;6种非致敏物肽损耗率均值小于6.38%,为致敏阴性,与LLNA致敏分类一致;致敏性未知的氯仿和苯肽损耗率均小于6.38%,为致敏阴性。结论：直接肽反应试验可较好地评价化学物的皮肤致敏性及致敏能力,该法简单、快速,适宜高通量筛选化学物致敏性,可推广应用于化学物安全性评价。     

Synthesis and biodistribution of peptide based 99mTc/186Re-MAGIPG-D612 monoclonal antibody in nude mice bearing colon cancer xenografts

A new bifunctional chelating agent MAGIPG was synthesized in good yield using Boc-p-nitro-phenylalanine as the starting material. 99mTc-labeled-MAGIPG-D612 conjugates reactive with human colon cancer were prepared in 3% to 15% yield with a radiochemical purity of 63% to 97%. 186Re-labeled MAGIPG-D612 conjugates were prepared with a 7% to 30% yield and a radiochemical purity of 82% to 98%. The biodistribution results demonstrate that these radioimmuno-conjugates selectively localized in LS174T human colon cancer xenografts.     

Synthesis of 99mTc-nitrido heterocomplex of piperidine and in vitro and in vivo evaluation of its affinity for sigma receptors

Sigma receptors are overexpressed in various types of cancer cells, making ligands that bind to these receptors attractive vectors for targeting radiation to specific sites for the imaging and therapy of oncologic disorders. In this paper, we report the synthesis of a dithiocarbamate derivative of 4-amino-N-benzylpiperidine and its radiolabeling with the [(99m)TcN(PNP)](2+) metal synthon. The radiolabeled tracer has been evaluated for sigma-receptor specificity. The radiochemical purity of the (99m)Tc-complex was >98%. The in vitro cell-binding and competition studies of the complex showed affinity and specificity toward fibrosarcoma and melanoma cells. In vivo studies carried out in mice bearing melanoma and fibrosarcoma tumors showed tumor uptakes of 1% and 1.9%, respectively, at 3 hours postinjection. In vivo blocking studies were carried out, using (+)-pentazocine, a sigma-receptor-specific agent where approximately 40% decrease in the tumor uptake was observed. The affinity of [(99m)TcN(PNP)Pip-DTC](+) complex for sigma-receptor sites ascertained through in vitro and in vivo studies makes it a potential agent for further investigation.     

Endocrinological and clinical evaluation of exemestane, a new steroidal aromatase inhibitor.

The androstenedione derivative, exemestane (FCE 24304), is a new orally active irreversible aromatase inhibitor. Fifty-six post-menopausal advanced breast cancer patients entered this study to evaluate the activity of four low exemestane doses in reducing oestrogen levels. The drug''s tolerability and clinical efficacy were also assessed. Exemestane was orally administered to four consecutive groups at daily doses of 25, 12.5, 5 and 2.5 mg, and the changes in oestrogen, gonadotrophins, sex-hormone binding globulin and dehydroepiandrosterone sulphate levels were evaluated. Drug selectivity was studied by measuring 17-hydroxycorticosteroid urinary levels. After 7 days of treatment, mean oestrone and oestradiol levels had decreased by respectively 64% and 65% (a decrease which was maintained over time); in the 2.5 mg group, oestrone sulphate levels also decreased by 74%. Gonadotrophin levels were significantly higher, whereas no changes in the other serum hormone levels or any interference with adrenal synthesis were detected. Treatment tolerability was satisfactory: nausea and dyspepsia were reported in 16% of patients. The overall objective response rate was 18%. In conclusion, exemestane is effective in reducing oestrogen levels at all of the tested doses and shows interesting clinical activity.     

Dosimetric and radiobiological evaluation of dose distribution perturbation due to head heterogeneities for Linac and Gamma Knife stereotactic radiotherapy

INTRODUCTION: In SRT/SRS, dedicated treatment planning systems are used for the calculation of the dose distribution. The majority of these systems utilize the standard TMR/OAR formalism for dose calculation as well as they usually neglect any perturbation due to head heterogeneities. The aim of this study is to examine the errors due to head heterogeneities for both absolute and relative dose distributions in stereotactic radiotherapy. MATERIALS AND METHODS: Dosimetric measurements in phantoms have been made for linac stereotactic irradiation. CT-based phantoms have been used for Monte Carlo simulations for both linac-based stereotactic system and Gamma Knife unit. Absolute and relative dose distributions have been compared between homogeneous and heterogeneous media. DVH and TCP results are presented for all cases. RESULTS: The maximum absolute dose difference at the isocenter was 2.2% and 6.9% for the linac and Gamma Knife respectively. The impact of heterogeneity in the target DVH was minor for the linac technique whereas considerable difference was observed for the Gamma Knife treatment. This was reflected also to the radiobiological evaluation, where the maximum TCP difference for the linac system was 2.7% and for the Gamma Knife was 4%. DISCUSSION AND CONCLUSIONS: The errors rising from the existence of head heterogeneities are not negligible especially for the Gamma Knife which uses lower energy beams. The errors of the absolute dose calculation could be easily eliminated by implementing a simple heterogeneity correction algorithm at the TPS. Nevertheless, the errors for not taking into account the lateral electron transport would require a more sophisticated approach and even direct Monte Carlo calculation.     

Synthesis and biological evaluation of new Rh (I) complexes with sulfonamide derivatives

New rhodium(I) complexes, belonging to the general structure [Rh(CO)2(L)], where L were sulfonamide derivatives, were synthesized and characterized by chemical analysis and IR determinations. These complexes were assayed as cytostatic and antitumour agents in vitro against KB cells and in vivo against P388, Ehrich ascites and advanced B16 melanoma. Assays against three Trypanosoma strains were also performed. Among the new compounds, the [Rh(CO)2-(sulfamethoxydiazine)] appeared to be active in all biological systems without showing evident nephrotoxicity. Relationships between biological activity and pi electronic charge localization on N atom of the ligand amidic group were also discussed.     

MDM2多肽抑制剂的发现与评价

目的:寻找MDM2的多肽抑制剂,从多角度探讨多肽药物在靶向肿瘤治疗中的潜在价值。方法:通过分子模拟对接技术对已有多肽库中的102条多肽进行结合测试;使用Alpha-lisa技术,分析多肽对MDM2与p53相互作用的影响;BIAcore技术研究多肽与MDM2的相互作用,及对MDM2和p53之间结合的影响;Western Blotting法检测多肽对MCF-7细胞中MDM2、p53及p21表达的影响。结果:Alpha-lisa对分子对接中得分较高的多肽进行验证,发现含苯环结构的Peptide 58:Ser-Tyr-Val-Trp及Peptide 95:Ala-Ser-Asp-Phe两条多肽对MDM2与p53结合破坏较大,分别在50.0和80.0 nmol/L即可产生抑制（Peptide 58:t=5.36,P<0.05,Peptide 95:t=31.26,P<0.05）;BIAcore实验发现,Peptide 58与Peptide 95可特异性的与MDM2结合,较p53相比。它们与MDM2的结合常数分别为(389.6±13.4)nmol/L和(1.6±0.2)μmol/L,而与p53的结合常数分别为(19.5±2.4)μmol/L（t=6.85,P<0.01）和(12.8±1.4)μmol/L（t=8.32,P<0.01）;Western Blotting结果表明,Peptide 58与Peptide 95能上调p53在MCF-7细胞内的表达水平,并引起p21表达的增加,相对p53基因,两肽对MDM2本身的表达水平影响较小。结论:Peptide 58与Peptide 95在体外对MDM2有较强的亲和抑制效应,但体内作用效果欠佳,原因可能是由于多肽进入细胞的比率较低或在细胞内由于蛋白酶的作用降解失效。     

99mTc] HYNIC-hEGF, a potential agent for imaging of EGF receptors in vivo: preparation and pre-clinical evaluation

Expression of epidermal growth factor receptors (EGFR) has prognostic and predictive value in many kinds of tumors. Imaging of expression of EGFR in vivo may give valuable diagnostic information. The epidermal growth factor (EGF), a natural ligand, is a possible candidate for the targeting of EGFR. The present study describes a method for preparation of (99m)Tc-EGF via the hydrazinopyridine-3-carboxylic acid (HYNIC) conjugation using tricine and ethylenediamine-N,N'-diacetic acid (EDDA) as co-ligands. Both conjugates bound EGFR expressing cells with nanomolar affinity, and demonstrated good intracellular retention. The complex with EDDA demonstrated much higher stability in blood serum and during cysteine challenge. Biodistribution of (99m)Tc-EDDA-HYNIC-EGF in normal mice demonstrated fast blood clearance of conjugate, and its ability to bind EGFR in vivo. (99m)Tc-EDDA-HYNIC-EGF is a promising candidate for visualization of EGFR expression in vivo.     

Synthesis and anti-cancer activity of covalent conjugates of artemisinin and a transferrin-receptor targeting peptide

Artemisinin, a natural product isolated from Artemisia annua L., shows a unique anti-cancer activity by an iron dependent mechanism. Artemisinin was covalently conjugated to a transferrin-receptor targeting peptide, HAIYPRH that binds to a cavity on the surface of transferrin receptor. This enables artemisinin to be co-internalized with receptor-bound transferrin. The iron released from transferrin can activate artemisinin to generate toxic radical species to kill cells. The artemisinin-peptide conjugates showed potent anti-cancer activity against Molt-4 leukemia cells with a significantly improved cancer/normal cells selectivity.