Alun Hughes

Bromine-80m-labeled estrogens: Auger electron-emitting, estrogen receptor-directed ligands with potential for therapy of estrogen receptor-positive cancers

To assess their possible use for estrogen receptor (ER)-directed radiotherapy of estrogen receptor-containing cancers, two estrogens were synthesized with the Auger electron-emitting nuclide bromine-80m and administered to immature female rats. Both the triphenylethylene-based estrogen, [80mBr]-2-bromo-1,1-bis(4-hydroxyphenyl)phenylethylene (Br-BHPE) and the steroidal estrogen [80mBr]17 alpha-bromovinylestradiol, showed substantial diethylstilbestrol-inhibitable localization only in the estrogen target tissues, the uterus, pituitary, ovaries, and vagina and, except for the liver and intestines, generally lower concentrations in all other tissues at both 0.5 and 2 h. The [80mBr]Br-BHPE (specific activity, 8700 Ci/mmol), was shown to bind specifically to the low salt extractable ER of the rat uterus. Comparing i.p., i.v., and s.c. administration of [80mBr]BHPE the i.p. route was found to be particularly advantageous to effect maximum, DES-inhibitable concentrations of radiobromine in the ER-rich target organs in the peritoneal cavity. When the tissue distribution of the [80mBr]Br-BHPE was compared with that of sodium bromide-80m, it was apparent that no substantial amounts of radiobromine were released from the bromoestrogen prior to its target tissue localization. The substantial concentration of these bromine-80m-labeled estrogens in ER-rich tissues, combined with previously reported evidence for the effective radiotoxicity of Auger electron-emitting nuclides within cell nuclei suggest a good potential for such ligands for therapy of ER positive cancers.

Relative biological effectiveness of accumulated 125IdU and 125I-estrogen decays in estrogen receptor-expressing MCF-7 human breast cancer cells

Abstract Yasui, L. S., Hughes, A. and DeSombre, E. R. Relative Biological Effectiveness of Accumulated 125IdU and 125I-Estrogen Decays in Estrogen Receptor-Expressing MCF-7 Human Breast Cancer Cells. The therapeutic potential for delivering a cytotoxic dose of radiation (using the decay of Auger-electron emitters) to the cell nucleus of cancer cells that express estrogen receptors (ERs) by radiolabeled estrogen was investigated in the ER-expressing human breast cancer cell line, MCF-7. The radiolabeled estrogen/ER complex irradiates the cell nucleus by binding specific DNA sequences called estrogen response elements (EREs). Cell clonogenicity and induction of DNA double-strand breaks (DSBs) by γ radiation or accumulation of 125I-iododeoxyuridine (125IdU) or E-17α[125I]iodovinyl-11βmethoxyestradiol (125IVME2) decays were determined. MCF-7 cells were efficiently killed by accumulation of 125IdU (D0 = 30 decays per cell) and 125IVME2 decays (D0 = 28 decays per cell). DNA DSBs were induced by the accumulation of 125IdU (approximately 3750 decays per cell required to reduce the mean value of the elution profile to 50%) or 125IVME2 decays (approximately 465 decays per cell required to reduce the mean value to 50%). For survival of MCF-7 cells after γ irradiation, the D0 was 1 Gy, and approximately 65 Gy was required to reduce the mean value to 50% for induction of DSBs. The RBE values for cell killing and induction of DSBs by 125IVME2 relative to γ radiation were 4.8 and 18.8, respectively. The RBE values for cell killing and induction of DSBs by 125IdU relative to γ radiation were 4.5 and 2.3, respectively. Cell killing in a manner similar to that induced by high-LET radiation and the high RBE for induction of DSBs by 125IVME2 in the ER-expressing MCF-7 cells provide a biological rationale for the use of Auger electron-emitting radionuclides covalently bound to estrogen to deliver a cytotoxic dose of radiation to ER-positive cancers.

Therapy of estrogen receptor-positive micrometastases in the peritoneal cavity with Auger electron-emitting estrogens--theoretical and practical considerations.

Previous studies have demonstrated that Auger electron-emitting estrogens, when associated with the estrogen receptor (ER), can effect breaks in DNA and ER-dependent radiotoxicity. To evaluate the potential of [123I]-iodoestrogens, ([123I]-IE) to treat ER-positive human cancer cells, we have studied the effect of incubation of [123I]-IE with ER-positive MCF-7 breast cancer cells on cell survival in vitro and found that subnanomolar concentrations of [123I]-IE effectively reduce survival, with a mean lethal dose of about 800 decays per cell. MCF-7 cells incubated 30 min with 2 nM [123I]-IE (13 MBq/ml) showed a 2 log reduction in the ability to form tumors in immunodeficient mice. Evaluation of a mathematical model for [123I]-IE therapy for intraperitoneal micrometases in vivo in the mouse, based on variables related to the (a) specific activity of [123I]-IE; (b) its affinity for ER; (c) the characteristics of the uptake and retention of [123I]-IE by the target cells; (d) the concentration of ER in the tumor cells and (e) the tumor weight suggest that such therapy may be feasible.

Evaluation of 17α-E-(trifluoromethylphenyl)vinyl estradiols as novel estrogen receptor ligands

As part of our program to develop novel ligands for the estrogen receptor, we synthesized the series of isomeric 17alpha-(trifluoromethyl)phenylvinyl estradiols using our solid-phase organic synthesis methodology. The compounds were evaluated for their relative binding affinity (RBA) using the ERalpha-LBD and in vivo potency using the immature rat uterotrophic growth assay. The ortho-isomer had the highest RBA values, 48-223, and the highest estrogenicity in vivo. The other isomers had significantly lower affinities and were weaker agonists in the uterotrophic assay. The results suggest that introduction of substituents at the 17alpha-position of estradiol is tolerated by the ER-LBD and permit agonist responses in the intact animal, however, the effect is sensitive to the position of groups on the phenyl ring. This study demonstrates that the 17alpha-position of estradiol is a reasonable site for modification but the position and physicochemical properties of such modifications may significantly affect the affinity and efficacy of the ligand.

CELLULAR AND SUBCELLULAR STUDIES OF THE RADIATION EFFECTS OF AUGER ELECTRON-EMITTING ESTROGENS

We studied the effect of 123I-labeled estrogen (123I-E) in estrogen receptor (ER)-rich cells in culture and in cell free model systems in vitro to elucidate the nature of the radiotoxicity for ER + cells of estrogens containing nuclides which emit Auger electrons. In cells the 123I-E caused a dose-dependent, unlabeled estrogen-inhibitable induction of chromosome aberrations. A dose of about 1000 decays per cell, which is approximately the mean lethal dose for these cells, resulted in an average of 1 chromosome break per cell. This supports the hypothesis that the lethal lesion induced by 123I-E is a chromosome break. Incubation of 123I-E/ER complex, but not 123I-E alone, with 27-mer duplex estrogen response element (ERE) DNA produced a dose-dependent cleavage of the ERE. However, we were unable to detect any fragmentation of either the 66 kDa full length ER in cell extracts or a purified 31 kDa hormone binding domain when incubated with excess 123I-E. Thus it appears that 123I-E effects its radiotoxicity by binding to ER, associating with ERE DNA and, by directing high LET radiation to DNA, inducing lethal chromosome breaks.

Cytotoxicity of 125 I-oestrogen decay in non-oestrogen receptor-expressing human breast cancer cells, MDA-231 and oestrogen receptor-expressing MCF-7 cells

Purpose : To compare the cytotoxicity of 125 I-oestrogen (E-17 α [ 125 I]iodovinyl-11 β methoxyoestradiol or 125 IVME2) decay accumulation in human breast adenocarcinoma cells that do not express oestrogen receptor (ER) (MDA-231 cells) with human breast adenocarcinoma cells that do express ER (MCF-7 cells). Materials and methods : MDA-231 cells were labelled with 125 IVME2 or [ 125 I]iododeoxyuridine (125 IdU), frozen for decay accumulation, thawed and then plated for colony formation. γ-irradiation survival was also determined. A whole-cell 3 H-oestrogen-binding assay and a specific-binding assay were used to detect ER. Results : No MDA-231 cell killing by accumulated 125 IVME2 decays (up to 440 dpc) was observed but ER-positive MCF-7 cells were killed by 125 IVME2 (D o =28 dpc). MDA-231 cells were not significantly more radioresistant to γ-rays (D o =1.7Gy for MDA-231 cells; 1 Gy for MCF-7 cells) or to 125 IdU decays (D o = 44dpc for MDA-231 cells; 30 dpc for MCF-7 cells). No ER were detected in MDA-231 cells. Conclusions : ER-negative cells, MDA-231, are not killed by 125 IVME2 decay accumulation. It is speculated that without ER (required to translocate the 125 IVME2 to its nuclear target), formation of the 125 IVME2-ER-DNA oestrogen-response element (ERE) complex and subsequent specific irradiation of the DNA at the ERE cannot occur. These results support the hypothesis that the nuclear genome is a critical target for radiation-induced cell death.

Radiotoxicity of Auger electron-emitting estrogens in MCF-7 spheroids: a potential treatment for estrogen receptor-positive tumors.

To approach treatment of micrometastases of steroid receptor-rich cancers using estrogen receptor-directed therapy with Auger electrons, multicellular spheroids of the estrogen receptor-rich human breast cancer cell line, MCF-7, were prepared and exposed to a range of concentrations of an Auger electron-emitting estrogen, E-17alpha-[123I]-iodovinyl-11beta-methoxyestradiol, [123I]IVME2, in vitro. After washing, the treated spheroids were dissociated to single cells and plated for assay of colony survival, whereby we observed a dose-dependent reduction in survival that was inhibited by inclusion of an excess of unlabeled estradiol in the initial incubation with [123I]IVME2. Spheroids of a range of sizes from 40 to 280 microm showed similar sensitivity to the Auger electron-emitting estrogen. The mean lethal dose was approximately 700 decays per cell and corresponded to an initial [123I]IVME2 concentration of less than 0.5 nM. If the control and treated spheroids were not trypsinized but rather were allowed to grow intact, there was not only a significant reduction in the growth of the treated spheroids, but in 18 days nearly half became necrotic, while few control spheroids were necrotic. Considering the low concentrations of Auger electron-emitting estrogen required for a significant reduction in survival, we believe this approach has merit to pursue in vivo, especially in cases where it may be possible to target the steroid receptor-rich micrometastases directly, such as ovarian cancer.

Uptake and interconversion of the Z and E isomers of 17α-iodovinyl-11β-methoxyestradiol in the immature female rat

Abstract This study compares the specific uptake and retention of the Z and E isomers of 17α-iodovinyl-11β-methoxyestradiol (IVME2) in estrogen target tissues in immature female rats following intraperitoneal injection. Estrogen receptor binding studies in vitro showed that the Z-IVME2 had greater affinity than the E-IVME2, but our initial in vivo data, comparing 125 I-labeled E- or Z-IVME2 in separate studies showed no difference between the two isomers in either uptake or retention. These results were confirmed when the two isomers, labeled either with 123 I or 125 I, were injected simultaneously, allowing direct comparison of uptake and retention patterns in the same animal. Analysis of the nature of the radioiodinated estrogens in estrogen target tissues showed that at all time points, the estrogen target tissues contained mainly the E isomer, even at short times after injection of Z isomer. Although the Z-IVME2 was reasonably stable in the injectant, analysis of a peritoneal lavage soon after intraperitoneal injection of Z-[ 125 I]-IVME2 showed that mainly the E isomer was present, suggesting that the conversion occurred prior to uptake by the tissues. In vitro studies with intraperitoneal fluid and serum showed that denaturing by heating at 65°C substantially reduced their ability to affect the conversion of Z to E isomer, suggesting a possible enzymatic conversion.

DNA and Chromosome Breaks Induced by iodine-123-Labeled Estrogen in Chinese Hamster Ovary Cells

The effects of the Auger electron-emitting isotope 123I, covalently bound to estrogen, on DNA single- and double-strand breakage and on chromosome breakage was determined in estrogen receptor-positive Chinese hamster ovary (CHO-ER) cells. Exposure to the 123I-labeled estrogen induced both single- and double-strand breaks with a ratio of single- to double-strand breaks of 2.8. The corresponding ratio with 60Co gamma rays was 15.6. The dose response was biphasic, suggesting either that receptor sites are saturated at high doses, or that there is a nonrandom distribution of breaks induced by the 123I-labeled estrogen. The 123I-labeled estrogen treatment induced chromosome aberrations with an efficiency of about 1 aberration for each 1000 disintegrations per cell. This corresponds to the mean lethal dose of 123I-labeled estrogen for these cells, suggesting that the lethal event induced by the Auger electron emitter bound to estrogen is a chromosome aberration. Most of the chromosome-type aberrations were dicentrics and rings, suggesting that 123I-labeled estrogen-induced chromosome breaks are rejoined. The F ratio, the ratio of dicentrics to centric rings, was 5.8 +/- 1.7, which is similar to that seen with high-LET radiations. Our results suggest that 123I bound to estrogen is an efficient clastogenic agent, the cytotoxic damage produced by 123I bound to estrogen is very like damage induced by high-LET radiation, and the 123I in the estrogen receptor-DNA complex is probably in proximity to the sugar-phosphate backbone of the DNA.

Dna Damage Induction by 125I-Estrogen

DNA damage induced by the radioactive decay of 125I-estrogen (125I-VME2) in an estrogen receptor expressing CHO cell line, CHO-ER, was measured. 125I-VME2 targeted 125I atoms proximal to DNA estrogen response elements (EREs). 125I decays were accumulated at -135 degrees C, and thereafter assayed by alkaline and neutral filter elution techniques to measure DNA single strand break (ssb) and double strand break (dsb) induction respectively. Increasing DNA damage (both ssbs and dsbs) was detected after exposure of cells to increasing concentrations of 125I-VME2. DNA ssb and dsb dose-response curves for 125I-VME2 were multiphasic. The rates of DNA damage induction by the decay of 125I-VME2 was determined by comparing slopes of all data or by comparing initial slopes. DNA ssb induction per 125I-VME2 decay was approximately 2 times greater compared with DNA dsb induction. 125I-VME2 decay induced approximately 4-8 times more DNA dsbs than 125IUdR decay.