Fluorine-substituted ligands for the peroxisome proliferator-activated receptor gamma (PPARgamma): potential imaging agents for metastatic tumors.

The peroxisome proliferator-activated receptor gamma (PPARgamma), a primary regulator of lipid metabolism, is present in many tumor cell lines and animal tumor systems and, in some cases, can mediate effective antitumor therapy with potent synthetic ligands. In an approach to image tumors with positron-emission tomography (PET) based on their content of PPARgamma, we have synthesized two fluorine-substituted analogues of a high affinity ligand from the phenylpropanoic acid class. The analogue having the highest affinity for PPARgamma was labeled with the positron-emitting radionuclide fluorine-18. In tissue distribution studies in normal rats and in SCID mice bearing human breast tumor xenografts, this compound did not show evidence of receptor-mediated uptake. The prospects for using PPARgamma as a target for imaging tumors may be limited by the low receptor concentrations in tumors and by the pharmacokinetic behavior of this class of ligands, which appears to be more favorable for therapy than for imaging.

Comparative breast tumor imaging and comparative in vitro metabolism of 16alpha-[18F]fluoroestradiol-17beta and 16beta-[18F]fluoromoxestrol in isolated hepatocytes.

16beta-[18F]Fluoromoxestrol ([18]betaFMOX) is an analog of 16alpha-[18F]fluoroestradiol-17beta ([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]betaFMOX was predicted to be as effective an imaging agent as [18F]FES. However, in a preliminary PET imaging study with [18F]FMOX of 12 patients, 3 of whom had ER+ breast cancer, no tumor localization of [18F]betaFMOX was observed. In search for an explanation for the unsuccessful [18F]betaFMOX clinical trial, we have examined the rate of metabolism of [18F]FMOX 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]betaFMOX, on these rates of metabolism. Immature rat hepatocytes were found to metabolize [18F]FES 31 times faster than [18F]betaFMOX, whereas mature rat cells metabolized [18F]FES only 3 times faster, and baboon and human hepatocytes only 2 times faster than [18F]betaFMOX. 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]betaFMOX 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]betaFMOX. 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.

Synthesis, biological evaluation, and baboon PET imaging of the potential adrenal imaging agent cholesteryl-p-[18F]fluorobenzoate.

Cholesteryl-p-[18F]fluorobenzoate ([18F]CFB) was investigated as a potential adrenal positron emission tomography (PET) imaging agent for the diagnostic imaging of adrenal disorders. We describe the synthesis, biodistribution, adrenal autoradiography, and baboon PET imaging of [18F]CFB. The synthesis of [18F]CFB was facilitated by the use of a specially designed microwave cavity that was instrumental in effecting 70-83% incorporation of fluorine-18 in 60 s via [18F]fluoro-for-nitro exchange. Tissue distribution studies in mature female Sprague-Dawley rats showed good accumulation of [18F]CFB in the steroid-secreting tissues, adrenals and ovaries, at 1 h postinjection. The effectiveness of [18F]CFB to accumulate in diseased adrenals was shown through biodistribution studies in hypolipidemic rats, which showed a greater than threefold increase in adrenal uptake at 1 h and increased adrenal/liver and adrenal/kidney ratios. Analysis of the metabolites at 1 h in the blood, adrenals, spleen, and ovaries of hypolipidemic and control rats showed the intact tracer representing greater than 86%, 93%, 92%, and 82% of the accumulated activity, respectively. [18F]CFB was confirmed to selectively accumulate in the adrenal cortex versus the adrenal medulla by autoradiography. Normal baboon PET imaging with [18F]CFB effectively showed adrenal localization as early as 15 min after injection of the tracer, with enhanced adrenal contrast seen at 60-70 min. These results suggest that [18F]CFB may be useful as an adrenal PET imaging agent for assessing adrenal disorders.

Methyl hypofluorite in the synthesis of 16-methoxyestradiol stereoisomers.

The usual chemistry of methyl hypofluorite provides a previously unexplored route for functionalizing the 16-position of estradiol. Three isomers of 16-methoxyestradiol were prepared via two synthetic routes, each using methyl hypofluorite. The estrogen receptor binding affinity of these compounds was determined to evaluate their potential as positron emission tomographic (PET) imaging agents targeting estrogen receptor-positive breast cancer. Radiolabeled methyl hypofluorite ([11C]CH3OF) would allow the rapid preparation of novel carbon-11 PET imaging agents. The 17-trimethylsilyl enol ethers of 3-benzyloxy and 3-trifloxyestrone were prepared as substrates to react with methyl hypofluorite. Conditions for the reaction of methyl hypofluorite with simple substrates were optimized to provide reasonable reaction yields with the steroidal substrates. Following introduction of the methoxy substituent at the 16-position, reduction and deprotection conditions were manipulated to yield the various methoxyestradiol isomers. Two-dimensional NMR techniques (HMQC and HMQC-TOCSY) were instrumental in the characterization of the methoxyestradiol isomers. NOESY experiments confirmed the stereochemistry of the 16- and 17-positions. 16 alpha-Methoxyestradiol-17 beta and 16 beta-methoxyestradiol-17 beta each with the preferred beta orientation for the 17-alcohol, were determined to have relative binding affinities of 1.5% and 2.3%, respectively. The stereoisomer with the unfavored alpha orientation at the 17-position, 16 alpha-methoxyestradiol-17 alpha, exhibited only a 0.5% relative binding affinity for the estrogen receptor. The biological evaluation of these compounds was not pursued further because of their low binding affinities.

PET imaging of breast cancer with fluorine-18 radiolabeled estrogens and progestins.

Through the use of fluorine-18 radiolabeled estrogen receptor ligands and Positron Emission Tomography (PET), imaging of estrogen receptor-positive (ER+) breast lesions has been accomplished. Targeting the estrogen and progesterone receptors found in receptor-positive breast cancer provides a means of diagnosing the disease non-invasively. The structure-activity relationship of evaluated fluorine-18 ligands are summarized and design considerations for construction of novel target ligands discussed. The role of the serum protein sex hormone-binding globulin (SHBG) in transport and metabolism of estrogens is related to target tissue uptake. A historical review of fluorine-18 radiolabeled estrogens includes the clinical study of 16 alpha-[18F]fluoroestradiol-17 beta (18FES) for imaging ER+ breast lesions. The success of 18FES in the clinical setting has shown the significance of PET in imaging primary and metastatic breast cancer by allowing for assessment of tumor response to tamoxifen therapy after as little as 7 days of treatment. Advantages of visualizing the tumor through targeting the progesterone receptor (PR) include PET imaging to follow the progress of tamoxifen therapy while the estrogen receptors are blocked. Clinical studies with the PR ligand 21-[18F]fluoro-16 alpha-ethyl-19-norprogesterone (18FENP) were not successful due to high hepatic uptake and poor correlation of tumor uptake with receptor content. Second generation PR ligands with decreased non-specific binding are predicted to be effective imaging agents for human PR+ breast cancer from studies in the immature rat and are ready for clinical evaluation.

Synthesis, estrogen receptor binding, and tissue distribution of [18F]fluorodoisynolic acids.

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 14 alpha-hydrogen configuration, trans-FDA) and the more active cis-doisynolic acid (with the unnatural 14 beta-hydrogen configuration, cis-FDA) from estrone and 14 beta-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.

Retardation of 17-oxidation of 16 alpha-[18F]fluoroestradiol-17 beta by substitution of deuterium for hydrogen in the 17 alpha position(6).

We describe the synthesis, in vitro metabolism and biodistribution of [17 alpha-2H]16 alpha-[18F]fluoroestradiol ([18F]DFES). The clinically useful breast cancer imaging agent, 16 alpha-[18F]fluoroestradiol-17 beta ([18F]FES), was deuterated at the C-17 alpha 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.