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The vitamin folic acid was covalently linked to the chelating agent deferoxamine (DF) via an amide bond using a simple carbodiimide coupling reaction. A mixture of two isomers, DF--folate(alpha) and DF--folate(gamma), was produced involving the alpha- and gamma-carboxyl group of folic acid, respectively. These two isomers were separated by anion-exchange chromatography using a NH4HCO3 gradient. Competitive binding studies revealed that only the DF-folate(gamma) is recognized by the folate receptor on KB cells, interacting with an affinity comparable to unconjugated folic acid. The DF--folate conjugates were radiolabeled with the gamma-emitting radionuclide 67Ga3+ and tested for uptake by cultured KB cells overexpressing the folate receptor. The cellular accumulation of 67Ga-DF-folate(gamma) tracer exhibited rapid uptake kinetics in cell culture with a t1/2 of approximately 3 min. The KB cell association of 67Ga-DF--folate(gamma) was competitively blocked by free folic acid, indicating that uptake of the 67Ga-DF--folate(gamma) was specifically mediated by the folate receptor. Since the folate receptor is overexpressed on the surfaces of many neoplastic cells, these results suggest that 67Ga-DF--folate(gamma) complex might be useful as a diagnostic agent for noninvasive imaging of folate receptor-positing tumors.
Folate-conjugated metal chelates have been proposed as potential imaging agents for cancers that overexpress folate receptors. In a previous study, folic acid was linked through its gamma-carboxyl group to deferoxamine (DF), and the 67Ga-labeled complex ([67Ga]DF-folate) was examined for in vivo tumor targeting efficiency in athymic mice with a human tumor cell implant. Although superb tumor-to-background contrast was obtained, slow hepatobiliary clearance would compromise imaging of abdominal tumors such as ovarian cancer. In the present study, folic acid was conjugated to an alternative chelator, diethylenetriaminepentaacetic acid (DTPA), via an ethylenediamine spacer. The desired DTPA-folate (gamma) regioisomer was synthesized by two different approaches, purified by reversed phase column chromatography, and characterized mainly by analytical HPLC, mass spectroscopy, and NMR. In cultured tumor cells, uptake of [111In]DTPA-folate (gamma) was found to be specific for folate receptor-bearing cells, and the kinetics of uptake were similar to those of free folate and other folate-conjugated molecules. In the normal rat, intravenously administered [111In]DTPA-folate (gamma) was found to be rapidly excreted into the urine, giving intestinal levels of radiotracer 10-fold lower than those observed with [67Ga]DF-folate (gamma) at 4 h. In a preliminary mouse imaging study, a folate receptor-positive KB cell tumor was readily visualized by gamma scintigraphy 1 h following intravenous administration of [111In]DTPA-folate (gamma).
We have prepared three analogues of 16 alpha-fluoroestradiol (FES) substituted either with an 11 beta-methoxy group (1, 11 beta-MeO-FES), an 11 beta-ethyl group (2, 11 beta-Et-FES), or a 17 alpha-ethynyl group (3, 17 alpha-ethynyl-FES). These substituents all lower the binding of FES to the serum proteins alphafetoprotein and sex steroid binding protein, but their effect on estrogen receptor binding varies: Receptor binding is increased by the 11 beta-ethyl and 17 alpha-ethynyl groups, but decreased by the 11 beta-methoxy group. These substituents also have a parallel effect on the lipophilicity, and hence the nonspecific binding estimated for these compounds. All three compounds were prepared in fluorine-18 labeled form, at effective specific activities of 90-1600 Ci/mmol, by fluoride ion displacement reactions as done previously with FES. Tissue distribution studies in immature rats show high uptake selectivity by target tissue (uterus) and effective competition by an excess of unlabeled estradiol. Percent injected dose per gram values (% ID/g) at 1 h are 6% for 11 beta-MeO-FES and 11-13% for 11 beta-Et-FES and 17 alpha-ethynyl-FES (FES itself has a % ID/g of 9%). Uptake selectivity in terms of uterus to blood or muscle ratios at 1 h is highest for 11 beta-MeO-FES and 17 alpha-ethynyl-FES (43-149). Metabolic consumption studies show that most activity in uterus is unmetabolized and in blood is rapidly and nearly completely metabolized. In muscle, FES and the substituted estrogens show intermediate levels of metabolic consumption; in some cases activity in muscle extracts is nearly unmetabolized. Thus, the substituents on FES cause major alterations in receptor and nonreceptor binding affinity, uptake efficiency and selectivity, and extent of metabolism. It is not readily clear, however, whether the alterations in uptake efficiency and selectivity are the result of differences in receptor or nonreceptor binding or lipophilicity, or altered patterns of metabolism. Nevertheless, these compounds should be useful in providing a spectrum of uptake properties that could be used for imaging different estrogen-receptor-containing structures.
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