Wayne Tansey

In vivo and in vitro measurement of apoptosis in breast cancer cells using 99mTc-EC-annexin V.

OBJECTIVE The purpose of this study was to develop an imaging technique to measure and monitor tumor cells undergoing programmed death caused by radiation and chemotherapy using 99mTc-EC-annexin V. Annexin V has been used to measure programmed cell death both in vitro and in vivo. Assessment of apoptosis would be useful to evaluate the efficacy and mechanisms of therapy and disease progression or regression. METHODS Ethylenedicysteine (EC) was conjugated to annexin V using sulfo-N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCl as coupling agents. The yield of EC-annexin V was 100%. In vitro cellular uptake, pre- and post-radiation (10-30 Gy) and paclitaxel treatment, was quantified using 99mTc-EC-annexin V. Tissue distribution and planar imaging of 99mTc-EC-annexin V were determined in breast tumor-bearing rats at 0.5, 2, and 4 hrs. To demonstrate in vivo cell apoptosis that occurred during chemotherapy, a group of rats was treated with paclitaxel and planar imaging studies were conducted at 0.5-4 hrs. Computer outlined region of interest (ROI) was used to quantify tumor uptake on day 3 and day 5 post-treatment. RESULTS In vitro cellular uptake showed that there was significantly increased uptake of 99mTc-EC-annexin V after irradiation (10-30 Gy) and paclitaxel treatment. In vivo biodistribution of 99mTc-EC-annexin in breast tumor-bearing rats showed increased tumor-to-blood, tumor-to-lung and tumor-to-muscle count density ratios as a function of time. Conversely, tumor-to-blood count density ratios showed a time-dependent decrease with 99mTc-EC in the same time period. Planar images confirmed that the tumors could be visualized clearly with 99mTc-EC-annexin. There was a significant difference of ROI ratios between pre- and post-paclitaxel treatment groups at 2 and 4 hrs post injection. CONCLUSION The results indicate that apoptosis can be quantified using 99mTc-EC-annexin and that it is feasible to use 99mTc-EC-annexin to image tumor apoptosis.

Synthesis of [99mTc]ethylenedicysteine-colchicine for evaluation of antiangiogenic effect.

Angiogenesis is in part responsible for tumor growth and the development of metastasis. Radiolabeled angiongenesis inhibitors would be useful to assess tumor microvasculature density. Colchicine (COL), a potent antiangiogenic agent, is known to inhibit microtubule polymerization and cell arrest at metaphase. This study aimed to develop 99mTc-labeled COL (EC-COL) using ethylenedicysteine (EC) as a chelator to assess tumor microvascular density. EC was conjugated to trimethylcolchicinic acid using N-hydroxysuccinimide and 1-ethyl-3-dimethylaminopropyl carbodiimide as coupling agents with a yield of 50-60%. In vivo stability was analyzed in rabbit serum at 0.5-4 h. Tissue distribution and planar imaging studies of [99mTc]EC-COL were evaluated in breast tumor-bearing rats at 0.5, 2 and 4 h. The data was compared to that using [99mTc]EC (control). The radiochemical yield of [99mTc]EC-COL was greater than 95%. [99mTc]EC-COL was stable in rabbit serum. In vivo biodistribution of [99mTc]EC-COL in breast tumor-bearing rats showed increased tumor-to-blood (0.52+/-0.12 to 0.72+/-0.07) and tumor-to-muscle (3.47+/-0.40 to 7.97+/-0.93) ratios as a function of time. Conversely, tumor-to-blood values showed a time-dependent decrease with [99mTc]EC over the same time period. Planar images confirmed that the tumors could be visualized clearly with [99mTc]EC-COL from 0.5 to 4 h. [99mTc]EC-COL may be useful to assess antiangiogenic and therapeutic effects during chemotherapy.

Synthesis of [18F]Fluoroalanine and [18F]Fluorotamoxifen for Imaging Breast Tumors

To develop ligands for imaging breast tumors, [18F]fluoro analogue of tamoxifen and [18F]fluoroalanine were radiosynthesized. In vivo biodistribution studies were performed in mammary tumor-bearing rats. In studies on the biodistribution of an [18F]fluoro analogue of tamoxifen, tumor uptake decreased when rats were pretreated with diethylstilbestrol (DES), suggesting that tracer uptake in tumors was receptor-mediated. An estrogen receptor assay indicated that tumors have a receptor density of 7.5 fmol/mg protein. Studies of the distribution of [18F]fluoroalanine in tissue showed that the tumor-to-tissue ratio increases as a function of time. Positron emission tomography (PET) images of tumor-bearing rats demonstrated that tumors can be visualized 1 h after rats are injected with an [18F]fluoro analogue of tamoxifen. PET imaging of pigs after injection of 10 mCi of [18F]fluoro analogue of tamoxifen showed uterine uptake that could be blocked by DES (50 mg). The findings suggest that both radiotracers are useful for imaging breast tumors.

Formation and Characterization of Cisplatin-Loaded Poly(benzyl 1-glutamate) Microspheres for Chemoembolization

Chemoembolization using microspheres of 100- to 200-µm is a useful way to treat primary and secondary hepatic tumors. In a search for a better embolic material, we described in detail the preparation and characterization of a poly(benzyl 1-glutamate) (PBLG) microspheres containing cisplatin (CDDP). We determined the optimal experimental conditions to produce spherical free-flowing microspheres that were able to release drug content (44% [w/w] CDDP) in a sustained manner. We found that solvent viscosity played a key role in determining the resulting microsphere characteristics. Microscopic studies showed that increasing the polymer concentration (to 10% [w/v]) and the viscosity of the organic phase produced microspheres with uniform drug distribution. Increasing polymer concentration also markedly improved drug incorporation efficiency. In vitro release studies revealed that the release of CDDP was a function of drug loading; microspheres with a higher amount of entrapped CDDP had a slower release rate. This observation and the fact that CDDP/ PBLG microspheres did not show “burst effect” at higher loading is ascribed to the formation of uniformly distributed drug crystal networks within the polymer matrix. The favorable properties of the CDDP/PBLG system warrants its further evaluation on experimental animal models for the treatment of hepatic tumors.

Biodistribution of Cyclic Carbonate of Ioxilan: A Radiopaque Particulate Macrophage Imaging Agent

RATIONALE AND OBJECTIVES A biodegradable radiopaque particulate contrast agent formulated from cyclic carbonate of ioxilan (IXC), which is a prodrug of nonionic water-solubel contrast ioxilan, recently has been developed. This contrast agent enhances liver attenuation and is cleared from the body as ioxilan. In the current study, we tested whether the biodistribution of IXC particles would be affected by the characteristics of particles. METHODS IXC nanoparticles (average diameter = 290 nm) and IXC microparticles (average diameter = 1.7 mm) were prepared, characterized, and injected intravenously (i.v.; 50 mg I/kg body weight) into rats. Two sensitive, reproducible analytic methods--inductively coupled plasma-mass spectrometry (ICP-MS) and high-performance liquid chromatography (HPLC)- were used to quantify tissue iodine and ioxilan concentrations. RESULTS Both IXC nanoparticles and microparticles were taken up in the liver and spleen. The IXC nanoparticles remained in the liver at high concentrations for 6 hr and were slowly cleared. They also gave a high blood iodine concentration in the first 5 min after i.v. injection, suggesting their potential use as a blood-pool imaging agent. Unlike the nanoparticles, the microparticles had a significantly lower uptake by the kidney. CONCLUSION Because of reduced renal uptake, microparticles are a preferred macrophage imaging agent. Biodegradable radiopaque particles may be used either as blood-pool imaging agents or as macrophage imaging agents depending on their size and distribution characteristics. The ICP-MS and HPLC methods are useful for biodistribution studies of iodinated contrast agents.

Biodistribution and imaging of polyethyleneimine - A gene delivery agent

Polyethyleneimine (PEI) has been described as a potentially effective agent for gene delivery. To track the delivery of this gene vector, the biodistribution and imaging of PEI labeled with 111Indium (111In) was studied in Fischer 344 rats. PEI was conjugated with diethylenetriamine pentaacetic acid dianhydride (DTPA), dialyzed, and chelated with 111In. Breast adenocarcinoma 13762 tumor cells were inoculated into the thighs of the rats. The first group of rats (n = 3) were injected intravenously with 300 mu g of the Indiumlabeled DTPA-polyethlyeimine (111In-DTPA-PEI) (50 mu Ci per rat) or 111In-DTPA. These animals were imaged with a gamma camera with a medium energy parallel hole collimator at 5 min, 2 hr, and 24 hr postinjection. The percentage of uptake in tumor (region of interest) was quantitated by a computer image analyzer and expressed as a percentage of injected dose (%ID) per pixel. To further characterize the tissue distribution of 111In-DTPA-PEI (300 mu g, 10 mu Ci per rat) and 111In-DTPA (10 m...

N,N-Diethylfluoromethyltamoxifen: Synthesis, assignment of 1H and 13C spectra and receptor assay

Abstract (cis) and (trans)N,N-diethylfluoromethyltamoxifen (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-5-fluoro-1-pentene) were synthesized from clomiphene in a three-step procedure and both isomers were differentiated by the concerted application of two-dimensional NMR techniques. In in vitro estrogen receptor assay from pig uterus using [3H]-estradiol (5 nM), the IC50 values were: N,N-diethylfluorotamoxifen cis 5 μM, trans 1 μM; tamoxifen 30 μM. In human MCF7 cell growth assay, the IC50 values were cis 4.5 μM, trans 11.8 μM, tamoxifen 11.0 μM. The data suggest that the fluoro analog of tamoxifen has potential for imaging estrogen receptors by positron emission tomography (PET) and may be used to predict the efficacy of breast tumor therapy.

Evaluation of [131I]iodoerythronitroimidazole as a predictor for the radiosensitizing effect

The aim of this study was to evaluate whether radiolabeled iodoerythronitroimidazole (IETNIM) could predict the radiosensitization effect on tumors. Tumor-bearing mice were irradiated at a dose of 25, 31 and 37 Gy after the injection of IETNIM. They were also exposed to 37 Gy radiation at 35, 70, 140 and 240 min after the i.p. injection of IETNIM. After the irradiation, tumor growth assays were conducted and the effect of IETNIM as a radiosensitizer was estimated as enhancement factor (EF). Tumor uptake was measured at 35, 70, 140 and 240 min after i.p. injection of [131I]IETNIM, which were the same intervals used in the radiosensitization study. EF of IETNIM in mice treated with 25, 30 and 37 Gy irradiation was 0.72, 0.98 and 1.28, respectively. EF of IETNIM in mice irradiated at 35, 70, 140 and 240 min after the injection was 1.50, 1.69, 1.46 and 1.08, which corresponded to the tumor uptake and blood clearance of [131I]IETNIM. [131I]IETNIM may be a suitable radiopharmaceutical to predict the radiosensitization effect of misonidazole analogs on tumors.

Radiolabeled Tamoxifen Analogues for Imaging Breast Cancer with Spect

This study is aimed at developing SPECT ligands to image breast tumors. 99mTc-labeled tamoxifen (TX) analogue was prepared by reacting hydroxyethylthio TX analogue with reduced Tc-IV. The yield was 50–60% (purity >99%). 131I-TX was prepared by treating tosyl-TX with Na131I. The yield was 20–25% (>99% purity). Biodistribution studies of both analogues were performed in DMBA-induced mammary tumor-bearing rats (10 μCi/rat, i.v., n=3/timeinterval). Biodistribution 99mTc-TX at 1, 2, 4, 6 and 18 hrs showed a tumor uptake value (% injected dose/gram tissue) of 0.37±0.058, 0.38±0.066, 0.27±0.041, 0.28±0.124 and 0.10±0.013. Tumor/blood ratio ranged from 0.11 to 0.07. Tumor/muscle ratio ranged from 5.68 to 7.38. Biodistribution of 131I-TX at 1, 3, 6 and 24 hrs showed a tumor uptake value 0.18±0.062, 0.23±0.152, 0.26±0.166 and 0.27±0.016. When rats primed with estradiol (60 μg/rat, 3 days, s.c), the value changed to 0.30±0.033, 0.42± to 0.039, 0.48±0.107, and 0.40±0.123. Tumor/blood ratio ranged from 1.95 to 11.0. When rats pretreated with DES (1.2 mg/rat, iv), the tumor uptake value changed to 0.32±0.058 (99mTc-TX, 2 h) and 0.22±0.059 (131I-TX, 6 h). In rats pretreated with estradiol, a significant increase in tumor uptake value was observed after 131I-TX postinjection. 99mTc-TX uptake in tumor could not be blocked by DES, suggesting the uptake was not due to a receptor-mediated process. 131I-TX may be useful in differentiating functioning ER(+) breast tumors.

Evaluation of intratumoral injection of poly(d,l-lactide) cisplatin microspheres in rats with breast tumors using [131I]iodomisonidazole (IMISO)

AbstractThis study utilized [131I]iodomisonidazole (IMISO) to examine changes in tumor hypoxia after therapy of breast cancer with poly(d,l-lactide) cisplatin microspheres (PLA-CDDP MSs) by an intratumoral injection technique. PLA-CDDP MSs were prepared by a solvent evaporation process. Breast tumor cells were inoculated into the thighs of rats. After therapy with CDDP or PLA-CDDP MSs (6 mg/kg, subcutaneously, single injection), the tumor volume and blood chemistry of breast tumor-bearing rats were measured and compared daily with those of a control group given saline alone for 16 days. A group of rats were administered [131I]IMISO (50 μCi per rat, intravenously, n = 3) on day 5 and planar scintigraphy was then acquired at 2 h after injection. The percentage of tumor uptake (region of interest) was quantitated by a computer image analyzer and expressed as percentage of injected dose (ID) per pixel. PLA-CDDP microspheres (50-100 (Jim) contained 40.04% (w/w) cisplatin and produced sustained-release properti...