SEPTEMBER/OCTOBER 2007 Volume 18, Number 5 © Copyright 2007 by the American Chemical Society
COMMUNICATIONS Water-Soluble Contrast Agents Targeted at the Estrogen Receptor for Molecular Magnetic Resonance Imaging Chidambaram Gunanathan,†,‡ Adi Pais,‡ Edna Furman-Haran,‡ Dalia Seger,‡ Erez Eyal,‡ Sarbani Mukhopadhyay,† Yehoshoa Ben-David,† Gregory Leitus,§ Hagai Cohen,§ Ayelet Vilan,§ Hadassa Degani,*,‡ and David Milstein*,† Department of Organic Chemistry, Department of Biological Regulation, and Unit of Chemical Research Support, Weizmann Institute of Science, Rehovot 76100, Israel. Received June 21, 2007; Revised Manuscript Received August 1, 2007
Novel estrogen-conjugated pyridine-containing Gd(III) and Eu(III) contrast agents (EPTA-Gd/Eu) were designed and effectively synthesized. Convenient to administration and MRI experiments, both EPTA-Gd and EPTA-Eu are soluble in water. The EPTA-Gd selectively binds with a micromolar affinity to the estrogen receptor and induces proliferation of human breast cancer cells. The EPTA-Gd is not lethal and does not cause any adverse effects when administrated intravenously. It enhances T1 and T2 nuclear relaxation rates of water and serves as a selective contrast agent for localizing the estrogen receptor by MRI.
Targeting the delivery of hormones and drugs with a specific molecular recognition site and simultaneously monitoring their distribution and localization in the body is an important goal in pharmacology and medicinal chemistry, as well as in diagnostic molecular imaging. One such example (1) is targeting organs and tissues responsive to estrogen using a selective estrogen receptor modulator (SERM) that can be monitored in ViVo. Estrogens are steroids that function as the primary sex hormones in females, regulating an assortment of activity in the female reproductive organs and the development and function of the mammary gland. 17β-Estradiol (E2), estriol, and estrone are the three naturally occurring estrogens, with the former being * To whom the correspondence should be addressed. H.D.: E-mail
[email protected], Tel 972-8-9342017, Fax 972-89346154. D.M.: E-mail
[email protected], Tel 972-89342599, Fax 972-8-9344142. † Department of Organic Chemistry. ‡ Department of Biological Regulation. § Unit of Chemical Research Support.
the most potent one. Estrogens enter freely the cells and bind with a very high affinity to nuclear estrogen receptors (ERs), R or β, which act as potent transcription factors regulating many cellular functions (2, 3). The interaction of estrogen with its receptor also elicits ER degradation responsible for its cyclic activity (2). In breast cancer, specifically, evaluation of the ER level is a major and well-established tool for assessment of prognosis and for predicting response to endocrine therapy with antiestrogens such as tamoxifen (4, 5). The levels of ERR in human breast cancer biopsies span a broad range reaching 1000 fmol/mg cytosol protein (6). However, the current clinical methods for determining ERR are semiquantitative and suffer from technical difficulties and analytical variations which may lead to inconsistent results (7). Furthermore, these techniques measure the level in a small fraction of the tumor, which may not reflect the actual distribution in the entire tumor. Previous attempts to image ER in ViVo were based on using positron emission tomography or single-photon emission computed tomography of 18F- or 123I-labeled ER ligands, respectively
10.1021/bc700230m CCC: $37.00 © 2007 American Chemical Society Published on Web 09/05/2007
1362 Bioconjugate Chem., Vol. 18, No. 5, 2007 Scheme 1. Complexesa
Synthesis
of
EPTA
and
Gunanathan et al. EPTA-Lanthanide
Table 1. ERr Competitive Binding Affinities of EPTA and EPTA-Gd with 17β-Estradiol and Relaxivities (24) of EPTA-Gda compound
Ki, µM
r1 (mM‚s)-1
r2 (mM‚s)-1
EPTA (4) EPTA-Gd (5a)
0.40 ( 0.13 0.97 ( 0.07
6.8 ( 0.05
25 ( 1.0
a K values were calculated using a dissociation constant for 17R-estradiol i binding to ERR of 0.2 nM.
a Conditions: (a) trans-PdCl2(PPh3)2, CuI, dry iPr2NH/THF, rt, 60 h, 89%. (b) CF3CO2H, rt, 3 h, 92%; (c) (i) pH 5-7, GdCl3·3H2O, rt, 3 h, 86%; (ii) pH 5-7, EuCl3·6H2O, rt, 3 h, 77%.
(8-10). Although the high-spin gadolinium(III) ion is extremely toxic for living cells, when complexed with suitable chelates it provides useful agents, for magnetic resonance imaging (MRI) (11, 12). Potential target-directed contrast agents developed recently increase the scope of MRI experiments (13-15). We envision a novel method to detect ER in cancer patients using MRI by a new contrast agent derived from E2. As reported, upon introduction of bulky substituents at the 17R position of E2, a high affinity to ER was retained (16-18). Herein, we delineate the synthesis of a novel water-soluble, MRI contrast agent integrated SERM which binds to the estrogen receptor with high affinity and induces hormonal activity as well as contrast enhancement in human breast cancer cells. The Pd-catalyzed reaction of 17R-ethynyl estradiol (1) with compound 2 (19, 20) yielded E2-embedded pyridine tetracarboxylate 3, which on ester deprotection provided the desired ligand, [estradiol-17R-ylethynyl(pyridin-2,6-diyl)bis(methylenenitrilo)] tetrakis(aceticacid) EPTA) 4 (Scheme 1). Complexation of 4 with hydrated GdCl3 resulted in the formation of gadolinium chelate 5a, EPTA-Gd, in 86% yield. The structure of EPTA-Gd was confirmed by HR/MS, ES/MS, IR, magnetic moment, and XPS studies (21). EPTA-Gd displays paramagnetic properties without any hysteresis phenomenon. The Curie constant C and effective magnetic moment p (for instance, for
AC frequency f ) 74 Hz and temperature 2 K < T < 22 K, C ) 6.93(1) emu*K/mol and p ) 7.44 µB/molecule; for DC (2 K < T < 300 K) FC, C ) 6.640(4) emu/mol*K and p ) 7.29 µB/molecule) were well in accordance with the literature values (22). Complexation of 4 with EuCl3·6H2O provided EPTA-Eu 5b in 77% yield (21). Binding affinities of EPTA 4 and EPTA-Gd 5a to ER were determined by performing competitive radiometric binding assays using tritiated 17β-estradiol and human recombinant ER. Table 1 summarizes the values obtained for the competition constant, Ki (defined as the equilibrium concentration of the competing ligand that binds half of the ER binding sites) of both the metal-free ligand EPTA and the EPTA-Gd. Although the affinities of both EPTA and EPTA-Gd were about 3 orders of magnitude lower than that of 17β-estradiol, their effective concentration required for ER binding was within the micromolar range (8, 23). The effectiveness of EPTA-Gd as a nuclear relaxation agent was demonstrated by the measurement of water proton T1 and T2 relaxivities (r1 and r2, respectively) at 4.7 T (Table 1). The cellular hormonal-induced activities of both EPTA and EPTA-Gd were tested in ER+ MCF7 and T47D human breast cancer cells. Time- and dose-dependent growth curves of both cell lines in the presence of EPTA and EPTA-Gd proved a stimulating growth effect similar to that of E2 (Figure 1A). This stimulation was specific to the presence of ER in the cells, as neither EPTA nor EPTA-Gd exerted an effect on the growth of ER-negative MDA-MB-231 human breast cancer cells. In addition, EPTA and EPTA-Gd, like E2, induced ER degradation in the ER+ cells in a similar manner to that of E2, albeit at a slower rate, indicating their binding to ER (Figure 1B). The ability of EPTA-Gd to specifically bind to the nuclear ER in living cells and consequently enhance the MRI signal in a distinct manner was tested in MDA-MB-231 human breast cancer cells, engineered to express ERR under tetracycline. Cells cultivated with a medium containing tetracycline (tet on) expressed high levels of ERR (ER+), as determined by Western blots, and served to detect the enhancement due to the binding of EPTA-Gd with this receptor. The same cells treated with tetracycline-free medium (tet off) did not express ERR (ERcontrol cells), as determined by Western blots, and served to determine the effect of nonspecific binding of EPTA-Gd to the cells and of possible decomposition of the contrast agent inside the cells (25). To maintain their viability throughout the experiments, the cells were cultivated on Biosilon beads to a density of ∼40 million cells/mL beads (Figure 1C). Enhancement and T1 measurements by means of microscopic MRI of the cells on beads in the presence and absence of EPTA-Gd (6 µM) and after washout of EPTA-Gd indicated higher enhancement and difference in T1 relaxation rates, ∆R1, between EPTAGd-treated ER+ cells and the control ER+ cells as compared to the EPTA-Gd-treated ER- cells and the ER- control cells, specifically after removal of the free and nonbound EPTA-Gd from the cell system by washing with standard medium (Figure 1D). Macroscopic enhancement and T1 measurements of the same cell systems in wells, in the presence and after washout of EPTA-Gd (Figure 1E), confirmed the above results obtained by microscopic MRI (Figure 1F). Although one expects a null ∆R1 between EPTA-Gd-treated ER- cells and ER- control
Communications
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Figure 1. Biologic stimulation and MRI enhancement of EPTA-Gd in living human breast cancer cells. A. Growth curves of T47D cells in the presence of 17β-estradiol, 30 nM: b EPTA, 2 µM; O EPTA, 0.1 µM; 9 EPTA-Gd, 2 µM; 0 EPTA-Gd, 0.1 µM; [ Control, estrogen-free medium. B. Degradation of ER in MCF7 cells 6 h after treatment. C. High-resolution MR image of a tube containing tet-on MDA-MB-231 cells cultivated on Biosilon beads. D. The difference in T1 relaxation rate of water (∆R1): +EPTA-Gd designates ∆R1 between EPTA-Gd treated ER+ cells (incubated for 3 h with 6 µM EPTA-Gd) and ER+ control cells (incubated in standard medium) as compared to ∆R1 between EPTA-Gd treated ER- cells (incubated for 3 h with 6 µM EPTA-Gd) and ER- control cells (incubated in standard medium); after wash designates ∆R1 between EPTA-Gd treated ER+ cells (incubated for 3 h with 6 µM) after washout with standard medium and ER+ control cells as compared to ∆R1 between EPTA-Gd treated ER- cells (incubated for 3 h with 6 µM EPTA-Gd) after washout with standard medium and ER- control cells. E. Low-resolution images of ER+ and ER- MDA-MB-231 cells cultivated on Biosilon beads using standard medium, in the presence of 6 µM EPTA-Gd, and after washout of EPTA-Gd with standard medium (spin echo with TE/TR ) 15/300 ms). F. ∆R1 obtained from experiments demonstrated in E with ∆R1 defined as in D.
cells after washout, we found a small measurable value for this ∆R1 (Figure 1D,F) but significantly lower than the comparable ∆R1 for ER+ cells (Figure 1D,F) suggesting some nonspecific absorption of EPTA-Gd inside the cells. Thus, the difference between ∆R1 of the ER+ cells and that of the ER- cells reflects the change due to the specific EPTA-Gd binding to ER in the ER+ cells. Overall, targeting EPTA-Gd to ER in living breast cancer cells enabled us to detect differences in the intensity of T1-
weighted MR images, as well as in the T1 relaxation rates between ER+ and ER- breast cancer cells. In general, the amount of ER in cells is controlled by a balance between its synthesis and degradation and is influenced by the nature of the bound ligand. For example, as shown in Figure 1A,B, estrogen induction of ER proliferating activity is strongly associated with its proteasomal degradation, leading to a marked decrease in ER within few hours. Temporal changes in ER level can therefore reflect the functional state of ER as well as the
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presence or absence of SERMs. Hence, there is a strong scientific need to develop an in ViVo imaging method that will provide us with knowledge of the ER signaling function and monitor temporal changes of receptor down-regulation and desensitization in the different estrogen-responsive organs. The assessment of ER function is particularly important for the management of breast cancer. It has been well-established that the estrogen receptor has a pivotal role in prevention and treatment of breast cancer. Specifically, the ER level serves to select patients for adjuvant endocrine therapy, and together with the progesterone receptor, they serve as major prognostic factors predicting the likelihood of recurrence and survival. Assessment of the ER status in breast cancer patients by means of MRI is specifically advantageous, as it can be integrated with the unique diagnostic capabilities of MRI to record anatomical images, as well as produce parametric images that characterize tumor physiological and metabolic properties (26, 27). In order to extend the in Vitro measurements to in ViVo conditions, several key experimental criteria must be reached: nontoxicity; high-affinity for ER; reasonable pharmacodynamics; ability to penetrate vascular walls, interstitial spaces, and cell membrane; and sufficient MRI sensitivity. The above in Vitro results and preliminary in ViVo experiments in rodents (not shown) have indicated thatindeed EPTA-Gd meets these criteria, particularly, it is not lethal and does not cause any adverse effects when administered intravenously even at a high dose. However, in order to achieve MRI sensitivity it is necessary to design protocols that overcome the heterogeneity of tumors. Further studies of breast cancer xenografts implanted in mice are underway to find out whether utilization of EPTA-Gd provides sufficient MRI sensitivity for the detection of ER down-regulation in the same tumor. In conclusion, the first estrogen-conjugated Gd(III) contrast agent (EPTA-Gd) has been designed and synthesized. EPTA-Gd competes with E2 on binding to ERR, with a competition equilibrium constant Ki ) 0.97 µM, demonstrates like estrogens induced cell proliferation and down-regulation of ER level in ER+ breast cancer cells in Vitro, both requiring binding to ER. Free EPTA-Gd displays a ∼50% higher relaxivity than the common Gd-based contrast agents and increases the T1 relaxation rates of ER+ cells as compared to these rates in the same cells with null ER indicating a specific enhancement due to the binding to the ER. Structural modification studies aimed at enhancement of binding affinity are currently underway.
ACKNOWLEDGMENT This work was supported by DOA/BC 044499 and NIH/CA 42238. We thank Tamar Kreizman for technical assistance. C.G. is the recipient of Deans of Faculties Postdoctoral Fellowship. D.M. is the Israel Matz Professor of Organic Chemistry. H.D. holds the Fred and Andrea Fallek Chair for Breast Cancer Research. Supporting Information Available: Synthetic procedures, and spectral data of compounds 3, 4, and 5a,b. Magnetic moment and XPS analyses of 5a. ER competition binding assay, cell proliferation assay, assay for determination of ER protein level, and MRI experiments of living cells cultivated on beads. This material is available free of charge via the Internet at http:// pubs.acs.org.
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Communications (20) Takalo, H., Pasanen, P., and Kankare, J. (1988) Synthesis of 4-(phenylethynyl)-2,6[N,N-bis-(carboxymethyl)aminomethyl]pyridine. Acta Chem. Scand. B42, 373-377. (21) See Supporting Information for details. (22) Kittel, C. Introduction to Solid State Physics, 4th ed., John-Wiley & Sons, Inc., New York, 1971. (23) Similar affinities were demonstrated for other estrogen-derived steroidal diamagnetic metal complexes. See Jackson, A., Davis, J., Pither, R. J., Rodger, A., and Hannon, M. J. (2001) Estrogen-derived steroidal metal complexes: agents for cellular delivery of metal centers to estrogen receptor-positive cells. Inorg. Chem. 40, 39643973. (24) The T1 and T2 relaxivities of EPTA-Gd is higher than those of GdDTPA, which is the common MRI contrast agent.
(25) Cabella, C., Crich, S. G., Corpillo, D., Barge, A., Ghirelli, C., Bruno, E., Lorusso, V., Uggeri, F., and Aime, S. (2006) Cellular labeling with Gd(III) chelates: only high thermodynamic stabilities prevent the cells acting as ‘sponges’ of Gd3+ ions. Contrast Med. Mol. Imaging 1, 23-29. (26) Furman-Haran, E., and Degani, H. (2002) Parametric analysis of breast MRI. J. Comput. Assist. Tomogr. 26, 376-386. (27) Katz-Brull, R., Lavin, P. T., and Lenkinski, R. E. (2002) Clinical utility of proton magnetic resonance spectroscopy in characterizing breast lesions. J. Natl. Cancer Inst. 94, 1197-1203 and references cited therein. BC700230M
