[en] Summary of Progress The specific aims of this project can be summarized as follows: Aim 1: Prepare and evaluate radiolabeled ligands for the peroxisome proliferator-activated receptor γ (PPARγ), a new nuclear hormone receptor target for tumor imaging and hormone therapy. Aim 2: Prepare steroids labeled with a cyclopentadienyl tricarbonyl technetium or rhenium unit. Aim 3: Prepare and evaluate other organometallic systems of novel design as ligand mimics and halogenated ligands for nuclear hormone receptor-based tumor imaging. As is described in detail in the report, we made excellent progress on all three of these aims; the highlights of our progress are the following: (1) we have prepared the first fluorine-18 labeled analogs of ligands for the PPARγ receptor and used these in tissue distribution studies in rats; (2) we have developed three new methods for the synthesis of cyclopentadienyltricarbonyl rhenium and technetium (CpRe(CO)3 and CpTc(CO)3) systems and we have adapted these to the synthesis of steroids labeled with these metals, as well as ligands for other receptor systems; (3) we have prepared a number of fluorine-18 labeled steroidal and non-steroidal androgens and measured their tissue distribution in rats; (4) we have prepared iodine and bromine-labeled progestins with high progesterone receptor binding affinity; and (5) we have prepared inorganic metal tricarbonyl complexes and steroid receptor ligands in which the metal tricarbonyl unit is an integral part off the ligand core

[en] The development of technetium-99m-labeled small-molecule radiopharmaceuticals directed at specific high-affinity binding sites, as are found in receptors for hormones and neurotransmitters, transport systems, and certain enzymes, is a natural outgrowth from the successful development of technetium radiopharmaceuticals for imaging flow and metabolism. Although many receptor-specific radiopharmaceuticals labeled with PET and other SPECT isotopes already exist, the low cost and widespread availability of technetium-99m would make their ^99mTc-labeled counterparts much more accessible to the medical community. This review has four goals: (a) To survey and analyze critically the results of a flurry of research activity in this area in recent years, which has led to the preparation of a number of novel technetium-labeled radiopharmaceuticals targeted at high-affinity sites, a few of which appear to be very promising; (b) to provide a conceptual analysis of how these agents are being designed; (c) to provide a context in terms of binding and uptake behavior by which these agents should be judged; and (d) to highlight emerging knowledge on the structure of receptors and related high-affinity binding biomolecules and their distribution, which may serve as reference points for understanding the results that have been obtained so far, and may be useful guides for future design

[en] We have prepared two estrogens labeled with carbon-11, 17α-[¹¹C]methylestradiol and 11β-ethyl-17α-[¹¹C]methylestradiol, at a specific activity of 300-1000 Ci/mmol (11.1-37 TBq/mmol), and we have determined their in vivo biodistribution in immature female rats. Both compounds accumulated selectively in two target tissues, the uterus and ovaries, reaching levels of 3.5-4.9 %ID/g at 20 min and 4.6-6.6 %ID/g at 40 min; uterus-to-blood ratios reached 12-23. Uterine uptake showed a saturation dependence with the amount of injected mass, and was displaced by unlabeled estradiol, indicating that this uptake was receptor mediated. These results suggest that these compounds may be useful in estrogen receptor-based imaging of breast tumors

[en] Doisynolic acids, D-ring seco-steroids derived from alkaline fusion of estrones, are hormonal curiosities: Their binding affinity for the estrogen receptor is low (ca. 1-2% that of estradiol), but their in vivo potency is high and they have a long duration of action. To study the in vivo behavior of the doisynolic acids, we prepared fluorine-substituted analogs of both trans-doisynolic acid (with the natural 14α-hydrogen configuration, trans-FDA) and the more active cis-doisynolic acid (with the unnatural 14β-hydrogen configuration, cis-FDA) from estrone and 14β-estrone, respectively. Modification of the D-ring haloform cleavage approach of Meyers allowed us to introduce fluorine (or fluorine-18) on the carbon atom derived from C-16 in the estrones. Fluorine substitution had little effect on the estrogen receptor binding affinity of the doisynolic acids. Tissue distribution of the fluorodoisynolic acids (trans-[¹⁸F]FDA and cis-[¹⁸F]FDA) was unusual and very different from that of typical, high-affinity ligands for the estrogen receptor. At 1-3 h in immature female rats, trans-[¹⁸F]FDA shows low and rather nonselective uptake in the principal estrogen target tissue (uterus) and slow clearance. By contrast, cis-[¹⁸F]FDA shows high uptake in nearly all tissues, with significant uterine uptake that continues to increase over the 1-6-h period. The uterine uptake of this isomer was blocked at the later times by a sufficiently high dose of unlabeled cis-FDA. After administration of the trans-[¹⁸F]FDA, a more polar metabolite slowly accumulates in the blood. The cis-[¹⁸F]FDA, however, showed no apparent metabolism, with 84% of the blood activity at 5 h assigned as the unmetabolized radioligand. After 5 h, only limited clearance from blood, liver, and kidneys has occurred. No metabolite from this isomer accumulates in the uterus. Although fluorodoisynolic acids will not be useful breast-tumor imaging agents, their behavior was found to be interesting as it deviates from that of other F-18 estrogens. Further long-term studies of cis-doisynolic acid, labeled with tritium, may be needed to explicate fully its unusual distribution properties and high in vivo activity

[en] We describe the synthesis, in vitro metabolism and biodistribution of [17α-²H]16α-[¹⁸F]fluoroestradiol ([¹⁸F]DFES). The clinically useful breast cancer imaging agent, 16α-[¹⁸F]fluoroestradiol-17β ([¹⁸F]FES), was deuterated at the C-17α position to lower the rate of C-17 alcohol oxidation. Metabolism studies in immature female rat and mature female baboon isolated hepatocytes showed [¹⁸F]DFES being consumed ca. 2.5 times slower than [¹⁸F]FES. Biodistribution studies and time-activity curve measurements in female rats showed [¹⁸F]DFES to have superior uptake characteristics compared to [¹⁸F]FES for imaging estrogen-receptor rich targets

[en] We have evaluated 6α-[¹⁸F]fluoroprogesterone as a potential imaging agent for progesterone receptor (PgR-ppositive breast cancer. 6α-Fluoroprogesterone (1) was obtained via halofluorination of the C-5 double bond in pregnenolone, followed by oxidation of the 3β-OH group, elimination of HBr from C-4,5, and epimerization at the C-6 center. The relative binding affinity (RBA) of 6α-fluoroprogesterone (1) to PgR is 11 (R5020 = 100), and its binding selectivity index (BSI, i.e. the ratio of the RBA to the non-specific binding, NSB) is 14.4; these values are similar to those of progesterone. 17α-Acetoxy-6α-fluoroprogesterone (2) was also prepared by the same method, but was not used for fluorine-18 labeling studies because its binding affinity for PgR is very low (0.9). The synthesis of 1 was adapted to fluorine-18 labeling and although the overall radiochemical yield was low (decay-corrected, 0.3%), progestin [¹⁸F]1 was obtained in moderately high effective specific activity (147 Ci/mmol). In vivo distribution studies using estrogen-primed immature female rats show that 6α-fluoroprogesterone ([¹⁸F]1) has low uterine uptake, low target tissue selectivity, and high fat uptake, presumably due to its low RBA and BSI. High uptake in bone, which indicates extensive metabolic defluorination, suggests that the C-6 position of steroids may not be a good site for fluorine-18 labeling

[en] We have observed that intraperitoneal administration of [¹⁸F]fluoroestradiol (FES), a radiolabeled estrogen receptor ligand, results in higher abdominal organ uptake and slower blood clearance than intravenous administration in female mice. In SCID mice bearing MCF-7 human tumors SC, IP administration resulted in tumor uptake that was only about one third that obtained with IV administration. Thus, the route of administration of a radiopharmaceutical for imaging or radiotherapy of a tumor in the abdomen, an ovarian tumor, for example, could have a profound effect on the efficiency and selectivity of delivery of the agent to the tumor

[en] The two estrogen receptor subtypes, ERα and ERβ, play important roles in breast cancer. To develop an ERα imaging agent, we synthesized fluoropropyl pyrazole triol (FPPT, 2), an analog of our ERα-selective ligand PPT. FPPT retains the high ERα binding selectivity of its parent PPT. We prepared [¹⁸F]FPPT (¹⁸F-2) in high specific activity, but estrogen target tissue uptake in female rats was minimal and was not displaceable by unlabeled estradiol, probably because of the lipophilicity and triphenolic nature of FPPT

[en] Octreotide was labeled at its N-terminus with Tc-99m-cyclopentadienyltricarbonyltechnetium, and the biodistribution of the labeled conjugate was studied in adult female Sprague-Dawley rats. The synthesis began with the preparation of Tc-99m-(methoxycarbonylcyclopentadienyl)tricarbonyltechnetium from Tc-99m-pertechnetate using a novel double ligand transfer reaction; it was completed in a total of five steps, with an 8% overall radiochemical yield (15% decay-corrected). The ^99mTc-cyclopentadienyltricarbonyltechnetium-labeled octreotide conjugate (^99mTc-CpTT-octreotide) showed receptor-mediated uptake in the pancreas and adrenals, which was blocked (80% and 93%, respectively) by excess unlabeled octreotide. These studies illustrate a new method for labeling a peptide with Tc-99m through an organometallic linkage that is stable, nonpolar, and low molecular weight; it complements labeling approaches that utilize inorganic metal complexes

[en] 16β-[¹⁸F]Fluoromoxestrol ([¹⁸F]βFMOX) is an analog of 16α-[¹⁸F]fluoroestradiol-17β ([¹⁸F]FES), a radiopharmaceutical known to be an effective positron emission tomography (PET) imaging agent for estrogen receptor-positive (ER+) human breast tumors. Based on comparisons of target tissue uptake efficiency and selectivity in a rat model, [¹⁸F]βFMOX was predicted to be as effective an imaging agent as [¹⁸F]FES. However, in a preliminary PET imaging study with [¹⁸F]βFMOX of 12 patients, 3 of whom had ER+ breast cancer, no tumor localization of [¹⁸F]βFMOX was observed. In search for an explanation for the unsuccessful [¹⁸F]βFMOX clinical trial, we have examined the rate of metabolism of [¹⁸F]βFMOX and [¹⁸F]FES in isolated rat, baboon, and human hepatocytes. We have also studied the effect of the serum protein sex hormone-binding globulin (SHBG), which binds [¹⁸F]FES better than [¹⁸F]βFMOX, on these rates of metabolism. Immature rat hepatocytes were found to metabolize [¹⁸F]FES 31 times faster than [¹⁸F]βFMOX, whereas mature rat cells metabolized [¹⁸F]FES only 3 times faster, and baboon and human hepatocytes only 2 times faster than [¹⁸F]βFMOX. In the presence of SHBG, the metabolic consumption rate for [¹⁸F]FES in mature rat hepatocytes decreased by 26%. Thus, the very favorable target tissue uptake characteristics of [¹⁸F]βFMOX determined in the rat probably result from its comparative resistance to metabolism (vis-a-vis [¹⁸F]FES) in this species, an advantage that is strongly reflected in comparative metabolism rates in rat hepatocytes. In the baboon and human, [¹⁸F]FES is extensively protein bound and protected from metabolism, an effect that may be reflected to a degree as a decrease in the rate of metabolism of this compound in baboon and human hepatocytes relative to [¹⁸F]βFMOX. Thus in primates, SHBG may potentiate the ER-mediated uptake of [¹⁸F]FES in ER+ tumors by selectively protecting this ligand from metabolism and ensuring its delivery to receptor-containing cells. In addition to current screening methods for ¹⁸F-estrogens that involve evaluating in vivo ER-mediated uptake in the immature female rat, studies comparing the metabolism of the new receptor ligands in isolated hepatocytes, especially those from primates or humans, may assist in predicting the potential of these ligands for human PET imaging