99mTc-glutamate peptide 3-aminoethyl estradiol


Chopra A.

Publication Details



In vitro Rodents



A favorable prognosis of estrogen therapy for breast cancer can often be predicted on the basis of response to the hormone because the estrogen receptor (ER) is a valuable marker to determine treatment outcome (1). The ER status of the tumors determines the likelihood of a favorable response to the treatment, and patients with ER-positive tumors have a better chance of recovery compared to those with ER-negative tumors. The presence of ER in the tissue is measured in a biopsy sample or after resection of a tumor. The random biopsy or tumor sampling may yield a false negative result because primary tumors usually have a heterogeneous ER distribution (2). In this regard a radioscintigraphic procedure could be more useful to determine the ER status of tumors because agents used for scintigraphy have a binding specificity for their targets.

Tamoxifen is a selective ER modulator that blocks estrogen binding to the receptor and is often used to treat breast cancer. Using radioactive halogen ([18F] fluorine and [131I] iodine) derivatives of tamoxifen in conjunction with positron emission tomography (PET), some investigators have investigated and monitored the effects of this drug at the cellular and clinical levels for the treatment of cancer (3-6). Although the results obtained from these studies are encouraging, the expenses, availability, and water solubility of these derivatives are limiting factors for clinical use. As an alternative, Takahashi et al. decided to develop a meta-stable, technetium (99mTc)-labeled derivative of estradiol (EDL) to possibly investigate and monitor ER status in cancer patients because the isotope is inexpensive and easily available, and the derivative is water-soluble (7); this estradiol was conjugated to a poly-glutamate peptide (GAP) that could chelate the 99mTc isotope to obtain 99mTc-GAP-EDL for imaging and radiotherapeutic purposes. The investigators also explored the possibility of using 99mTc-GAP-EDL to detect endometriosis, an ER-associated condition, using a rabbit model (7-9).



The synthesis of 99mTc -GAP-EDL was performed as described by Takahashi et al. (7, 9). To start, estrone was dissolved in anhydrous ethanol. Sodium ethoxide and bromoacetonitrile were added to the solution and the mixture was heated under reflux for 3 h. The ethanol was allowed to evaporate completely and the residue was dissolved in ethyl acetate. The mixture was washed with water and the organic layer was filtered after drying over magnesium sulphate. The ethyl actetate was evaporated under reduced pressure and the remaining solid product, 3-acetonitrile estradiol, was washed with ether. The yield of this reaction was 75%. The solid product was dissolved in tetrahydrofuran, and lithium aluminum hydride was added to the mixture and stirred for 4 h. The solvent was evaporated to yield a solid that was dissolved in ethyl acetate. This solution was washed with water, dried over magnesium sulphate, and filtered. The solvent was evaporated to obtain 3-cyanomethyl estrone (EDL) with a yield of 92%. The structure of EDL was confirmed by nuclear magnetic resonance.

For the synthesis of GAP-EDL, the sodium salt of GAP was converted to the acid form by the addition of 2 N hydrochloric acid (HCl). This solution was dialyzed to remove all the HCl and freeze-dried to obtain the acidic form of GAP. The GAP acid was dissolved in dimethylformamide (DMF), and dicyclohexyl carbodiimide and 4-N,N-dimethyl aminopyridine were added to it. This mixture was stirred at room temperature for 2 days. The DMF was evaporated under reduced pressure; 1 N sodium bicarbonate was added to it and extracted with chloroform. The aqueous solution was dialyzed, filtered, and freeze-dried to obtain GAP-EDL. The percent yield of this reaction was not provided in the publication. The GAP-EDL complex contained 15% (weight/weight) EDL as determined by ultraviolet spectroscopy.

For the synthesis of 99mTc-GAP-EDL, 99mTc was added to a vial containing lyophilized GAP-EDL and tin (II) chloride. Using a radio thin-layer chromatography scanner, the radiochemical purity of the product was determined to be 97%. The Rf value and specific activity of 99mTc-GAP-EDL were not provided in the publication (7). The synthesis and specific activities of 99mTc-GAP and 99mTc-labeled diethylenetriamene pentaacetate (DTPA), used as controls in the studies, were not presented in the publications (7, 9).

In Vitro Studies: Testing in Cells and Tissues


The uptakes of 99mTc-GAP and 99mTc-GAP-EDL were investigated in ER-positive cell lines (ie, MCF-7 (of human breast adenocarcinoma origin), T-47D (of human breast carcinoma origin), and 13762 MAT B III (of rat mammary adenocarcinoma origin) (7). An ER-negative cell line, RAB-9 (of rabbit skin origin), was used as a control in the studies (9). The uptake of 99mTc-GAP-EDL was significantly higher than 99mTc-GAP in the MCF-7, T-47D, and 13762 MAT B III cells. Prior exposure of these cells to diethylstilbestrol (DES), a synthetic estrogen, resulted in a 70% reduction of 99mTc-GAP-EDL uptake (7). In the same study, MCF-7 cells treated with tamoxifen showed a 10% decrease in uptake of the radiochemical. The rate of uptake and accumulation of 99mTc-GAP-EDL was ~4-fold higher in the MCF-7 versus the RAB cells (9). On the basis of these observations, the investigators concluded that the in vitro uptake of 99mTc-GAP-RDL was an ER-mediated process.

Animal Studies



Tissue distribution studies of 99mTc-GAP-EDL were performed in rats bearing 13762 MAT B III cell tumors (7). The animals (n = 3 per time point) were injected with either 99mTc-GAP-EDL or 99mTc-GAP and euthanized 0.5–4 h later. Selected tissues from the animals were excised, collected, and counted for incorporated radioactivity. Radioactivity levels, given as % injected dose/gram (%ID/g) for tumor/muscle increased as a function of time and were significantly higher (P<0.05) 4 h after administration for animals injected with 99mTc-GAP-EDL (7.92 ± 0.56% ID/g) compared to those injected with 99mTc-GAP (6.01 ± 0.05% ID/g). Similar observations were also made for the uterus/muscle and uterus/blood count ratios.

Planar scintigraphy with 99mTc-GAP-EDL and 99mTc-DTPA was also performed on rats bearing 13762 MAT B III cell tumors (7). In blocking studies, the rats were treated with DES 1 h before administration of 99mTc-GAP-EDL. Region-of-interest analysis of the planar images 0.5 and 4 h after administration of the labels showed that the tumor/muscle ratios were 1.67–2.95 and 1.26–1.75 for 99mTc-GAP-EDL and 99mTc-DTPA, respectively. In the blocking study, the ratios were 1.98–2.39 and 1.21–1.63 for 99mTc-GAP-EDL and the DES pre-treated animals, respectively.

Other Non-Primate Mammals


The use of 99mTc-GAP-EDL for the detection of endometriosis was evaluated in a rabbit model (7, 9). An established procedure was used to induce endometriosis in the animals (10). Eight weeks after surgery, 99mTc-GAP-EDL was intravenously administered to the animals, and scintigraphic images were obtained 0.5–2 h after the injection. The rabbits were then euthanized, and the grafts were excised for histological examination. In comparison to the surrounding tissue, the uterus, ovaries, and the implants showed an increased uptake of 99mTc-GAP-EDL. The radiotracer was also detected in the microinvasive endometrial tissue. A microscopic examination of the tissue supported the observations made after imaging. From these observations the investigators suggest that 99mTc-GAP-EDL was taken up by the tissue through an ER-mediated process. No blocking studies for 99mTc-GAP-EDL uptake were reported.

Non-Human Primates


No publications are currently available.

Human Studies


No publications are currently available.


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