Folic acid (FA; folate) belongs to the B group of vitamins, and its activity is mediated through the membrane-bound folate receptor (FR; also known as the folate-binding protein), which has two isoforms (α and β). A third constitutively secreted form of the receptor (γ) has also been identified in some hematopoietic cells (1). The α and β isoforms of FR show a high affinity for FA and have a restricted expression in certain tissues. FR-α is found in the lungs, glandular tissue, and choroid plexus of the brain. A functional β form is present only in activated monocytes and macrophages, although it is also borne by mature neutrophils and CD34+ cells (as an inactive form) (1). Low amounts of FR-α are expressed in normal tissue, but it has been shown to be overexpressed in several cancerous tumors such as those of the breast, colon, head and neck, kidneys, and lungs (2). In addition, overexpression of the FR in tumors indicates a poor prognosis for the patient (3, 4). Therefore, the FR has been targeted in clinical trials for the detection, diagnosis, and treatment of cancers. Several folate-based imaging agents that use techniques such as magnetic resonance imaging, optical imaging, computed tomography, positron emission tomography, and single-photon emission tomography (SPECT) have been developed and evaluated for the noninvasive detection of malignant tumors that overexpress the FR (2). However, except for SPECT-based agents, all imaging agents had limitations and were determined to be unsuitable for the detection of tumors overexpressing the FR as detailed by Sega et al. (2).
An indium (111In)-labeled folate-based SPECT agent with a good tumor specificity was developed earlier and evaluated in clinical trials, but the nuclide is expensive to produce, has a high γ energy emission (171 and 245 keV) and a long half-life (~68 h) (2), and the detection of recurrent cancer with this agent is difficult (5). To overcome the problems associated with the use of 111In, some investigators developed and evaluated the use of technetium (99mTc)-labeled FR imaging agents because this nuclide has a low energy emission (140 keV), has a short half-life (~6 h), is inexpensive to produce and, compared with 111In, produces high-quality images (6-10). Among the 99mTc-labeled FR imaging agents, only the peptide-based agent 99mTc-EC20 has been evaluated preclinically (10) and more recently in the clinics (11). In an effort to develop an alternate FR imaging agent, Panwar et al. synthesized and evaluated 99mTc-tetraethylenepentamine-folate (99mTc-TEPA-folate) for scintigraphy in nude mice bearing FR-overexpressing tumors (12). The biodistribution of this radiolabeled compound was also studied in these animals.
Synthesis of the TEPA-folate conjugate was described by Panwar et al. (12). The final product was purified with high-performance liquid chromatography, and the chemical yield of the conjugate was reported to be 78%. Characterization of the conjugate was performed with mass spectroscopy and nuclear magnetic resonance.
The radiolabeling of TEPA-folate with 99mTc was performed in the presence of stannous chloride (pH 7.0) for 15 min at room temperature (12). The yield of the labeling reaction was reported to be >95% as determined with instant thin-layer chromatography–silica gel. The specific activity of 99mTc-TEPA-folate was calculated to be 74 kBq/μg (2 μCi/μg).
99mTc-Labeled folate (99mTc-folate) was also synthesized as described above for use in one of the studies (12). The yield and specific activity of 99mTc-folate was not reported.
The stability of 99mTc-TEPA-folate in 0.9% saline after exposure for 24 h was reported to be >95% (12). The stability of 99mTc-TEPA-folate in human serum under physiological conditions was determined to be >95% at 6 h (12). For comparison, 99mTc-folate was exposed to human serum under the same conditions, with a reported stability of <20% after 6 h.
In Vitro Studies: Testing in Cells and Tissues
Receptor binding assays were performed with 99mTc-TEPA-folate using three FR-overexpressing cell lines: KB (originally believed to be derived from human carcinoma of the mouth, but is believed to be contaminated with HeLa cells), U-87MG (a human glioblastoma cell line), and MDA-MB-468 (a human breast adenocarcinoma cell line), as described by Panwar et al. (12). The affinity constants of 99mTc-TEPA-folate for the FR on the three cell lines were reported to be 5.0 ± 0.06, 27.46 ± 0.01, and 25.85 ± 0.005 μM, respectively. By comparison the Ka of folic acid for the folate receptor was reported to be 5 X 10-10 M by Ke et. al. (5) and the uptake of 99mTc-6-Hydrazinopyridine-3-carboxylic acid (HYMIC)-folate by KB and 24JK-FBP cells (a methylcholan threne-induced mouse sarcoma cell line transfected with the human folate receptor gene) was reported to be 10 and 3.4 pmol/mg cellular protein (8). In another study 19F click folate was reported to have a Ki of 9.76 ± 3.13 nM with FR expressing KB tumor cells (13).
The biodistribution of 99mTc-TEPA-folate was investigated in nude mice bearing KB cell tumors (12). The animals (n = 3 mice/time point) were injected with the radiolabeled compound through the tail vein and euthanized at 1, 4, and 24 h after treatment. The major organs, including tumors, were removed to count accumulated radioactivity (presented as percent injected dose/gram tissue (% ID/g)) and to determine the tumor/blood, tumor/liver, and tumor/kidney ratios. At 1 h after injection, high levels of radioactivity were detected in the liver (20.5 ± 3.4% ID/g) and kidneys (13.0 ± 1.2% ID/g), followed by the spleen (4.30 ± 0.7% ID/g) and the intestines (3.77 ± 1.1% ID/g). By 24 h after treatment, the radioactivity in these organs was reduced to 0.38 ± 0.01, 1.25 ± 0.7, 0.26 ± 0.06 and 0.14 ± 0.0% ID/g, respectively. During the same period, the amount of label in the tumor was 4.06 ± 0.9% ID/g at 1 h, 4.26 ± 1.3% ID/g at 4 h, and 3.1 ± 1.2% ID/g at 24 h. The tumor/blood ratio increased from 2.68 ± 0.52 at 1 h to 13.9 ± 1.8 at 24 h. A similar trend, though less pronounced, was noted for the tumor/liver and tumor/kidney ratios (for details see Table 1 in Panwar et al. (12)).
Whole-body scintigraphy was performed on mice bearing KB cell tumors on the right thigh at 30 min, 1 h, and 4 h after an intravenous injection of 99mTc-TEPA-folate through the tail vein (12). The number of animals used for this study was not reported. An accumulation of label in the tumor was evident at 30 min after the injection, and the tumor was clearly visible at 1 h for up to 4 h after treatment.
From these studies, the investigators concluded that 99mTc-TEPA-folate could be used for the detection of FR-overexpressing tumors in animals and could probably be used as a diagnostic and therapeutic agent for humans after coupling with other suitable nuclides.
Other Non-Primate Mammals
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- Leamon C.P. Folate-targeted drug strategies for the treatment of cancer. Curr Opin Investig Drugs. 2008;9(12):1277–86. [PubMed: 19037834]
- Sega E.I., Low P.S. Tumor detection using folate receptor-targeted imaging agents. Cancer Metastasis Rev. 2008;27(4):655–64. [PubMed: 18523731]
- Hartmann L.C., Keeney G.L., Lingle W.L., Christianson T.J., Varghese B., Hillman D., Oberg A.L., Low P.S. Folate receptor overexpression is associated with poor outcome in breast cancer. Int J Cancer. 2007;121(5):938–42. [PubMed: 17487842]
- Allard J.E., Risinger J.I., Morrison C., Young G., Rose G.S., Fowler J., Berchuck A., Maxwell G.L. Overexpression of folate binding protein is associated with shortened progression-free survival in uterine adenocarcinomas. Gynecol Oncol. 2007;107(1):52–7. [PubMed: 17582475]
- Ke C.Y., Mathias C.J., Green M.A. The folate receptor as a molecular target for tumor-selective radionuclide delivery. Nucl Med Biol. 2003;30(8):811–7. [PubMed: 14698784]
- Mathias C.J., Hubers D., Low P.S., Green M.A. Synthesis of [(99m)Tc]DTPA-folate and its evaluation as a folate-receptor-targeted radiopharmaceutical. Bioconjug Chem. 2000;11(2):253–7. [PubMed: 10725102]
- Trump D.P., Mathias C.J., Yang Z., Low P.S., Marmion M., Green M.A. Synthesis and evaluation of 99mTc(CO)(3)-DTPA-folate as a folate-receptor-targeted radiopharmaceutical. Nucl Med Biol. 2002;29(5):569–73. [PubMed: 12088727]
- Guo W., Hinkle G.H., Lee R.J. 99mTc-HYNIC-folate: a novel receptor-based targeted radiopharmaceutical for tumor imaging. J Nucl Med. 1999;40(9):1563–9. [PubMed: 10492380]
- Ilgan S., Yang D.J., Higuchi T., Zareneyrizi F., Bayhan H., Yu D., Kim E.E., Podoloff D.A. 99mTc-ethylenedicysteine-folate: a new tumor imaging agent. Synthesis, labeling and evaluation in animals. Cancer Biother Radiopharm. 1998;13(6):427–35. [PubMed: 10851435]
- Leamon C.P., Parker M.A., Vlahov I.R., Xu L.C., Reddy J.A., Vetzel M., Douglas N. Synthesis and biological evaluation of EC20: a new folate-derived, (99m)Tc-based radiopharmaceutical. Bioconjug Chem. 2002;13(6):1200–10. [PubMed: 12440854]
- Fisher R.E., Siegel B.A., Edell S.L., Oyesiku N.M., Morgenstern D.E., Messmann R.A., Amato R.J. Exploratory study of 99mTc-EC20 imaging for identifying patients with folate receptor-positive solid tumors. J Nucl Med. 2008;49(6):899–906. [PubMed: 18483093]
- Panwar P., Shrivastava V., Tandon V., Mishra P., Chuttani K., Sharma R.K., Chandra R., Mishra A.K. 99mTc-Tetraethylenepentamine-Folate--a new 99mTc-based folate derivative for the detection of folate receptor positive tumors: synthesis and biological evaluation. Cancer Biol Ther. 2004;3(10):995–1001. [PubMed: 15467429]
- Ross T.L., Honer M., Lam P.Y., Mindt T.L., Groehn V., Schibli R., Schubiger P.A., Ametamey S.M. Fluorine-18 click radiosynthesis and preclinical evaluation of a new 18F-labeled folic acid derivative. Bioconjug Chem. 2008;19(12):2462–70. [PubMed: 19053298]
This MICAD chapter is not included in the Open Access Subset, because it was authored / co-authored by one or more investigators who was not a member of the MICAD staff.
Created: August 25, 2009; Last Update: October 1, 2009.
National Center for Biotechnology Information (US), Bethesda (MD)
Panwar P, Srivastava V, Tandon V, et al. 99mTc-Labeled tetraethylenepentamine-folate. 2009 Aug 25 [Updated 2009 Oct 1]. In: Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.