An important tool in clinical diagnosis of disease is the use of the magnetic resonance imaging (MRI) contrast agents. In MRI, images are acquired by employing radio frequency pulses to excite nuclear spins of a specimen. The observed signal is from the protons of water molecules in the specimen. MRI can capture three-dimensional images without the need for invasive procedures. By imposing one or more orthogonal magnetic field gradients onto a target, an MR image can be obtained using radio frequency pulses to excite nuclear spins as in NMR. Images are based upon the NMR signal from the protons of water molecules, where the signal intensity in a given volume element is a function of the water concentration and relaxation times (T1 and T2).
MRI has several advantages over other imaging modalities. MRI can image in three dimensions with high spatial and temporal resolution. Unlike while light microscopy and fluorescent microscopy, MRI is not limited by the distance of scattered light onto the cells of interest or dye intensity. It avoids the harmful ionizing radiation of X-ray and CT. Finally, while positron emission tomography (PET) has higher sensitivity, the resolution is much lower than for MRI. MRI can visualize opaque organisms in three dimensions and can follow organisms over time, making it an ideal biological imaging tool.
Sensitivity and intrinsic contrast can be enhanced by using paramagnetic contrast agents, such as the commonly used paramagnetic Gd(III) ion, that decreases the local T1 relaxation of nearby water protons (Caravan et al. Chem Rev 1999, 99, 2293-352., herein incorporated by reference in its entirety). Images derived from changes in T1 regions that are associated with a Gd(III) ion have a higher signal intensity (Aime et al. Curr Pharm Biotechnol 2004, 5, 509-18., herein incorporated by reference in its entirety). The ability of a contrast agent to decrease T1 and therefore increase signal intensity at a given concentration is relaxivity (mM−1 s−1). High relaxivity agents result in area of increased signal.
Free Gd(III) ions are toxic to biological systems, and a suitable ligand or chelate must bind the lanthanide to form a nontoxic complex. For many years, Gd(III) contrast agents have been used in a chelated form to eliminate toxicity in humans. Several factors influence the stability of chelate complexes including enthalpy and entropy effects (e.g., number, charge and basicity of coordinating groups, ligand field, and conformational effects). Recently, there has been concern associated with the use of Gd(III) agents due to an apparent link to a disabling condition called NSF (Kurtkoti & Hiremagalur. Nephrology 2008, 13, 235-41., Kay Ann Rheum Dis 2008, 67 Suppl 3, iii66-9., herein incorporated by reference in their entirety). Many patients with this condition experience a thickening of the skin that inhibits joint movement. Clinical data on known cases of NSF have revealed that the condition is only present in patients with renal failure (low pH) and only when certain classes of contrast agent are used. Three clinically approved contrast agents have been associated with the onset of NSF: OMNISCAN, MAGNEVIST and OPTIMARK. These are all linear Gd(III) chelates based on the structure of DTPA.
Macrocyclic chelates have higher thermodynamic stability constants and have not been associated with NSF. The reduced thermodynamic stability constants (and the presence of an amide) in linear chelates is thought to be the cause of NSF when Gd(III) is released from the chelate and displaced by other naturally occurring metals. The medical community has studied the risks of using gadolinium in patients with renal failure and has published guidelines to minimize the risk (Shellock & Spinazzi, AJR Am J Roentgenol 2008, 191, 1129-39., herein incorporated by reference in its entirety).
Gd(III) ions are toxic to living tissues, presumably due to binding to calcium channels and therefore, it must be chelated to reduce the bioavailability. These chelates are synthetically versatile and provide the means to attach targeting moieties (Allen & Meade. Met Ions Biol Syst 2004, 42, 1-38., herein incorporated by reference in its entirety).
Mammary epithelial cells express the progesterone receptor (PR) and estrogen receptors (ER) (Ismail et al. Steroids 2003, 68, 779-87., herein incorporated by reference in its entirety). The PR is present in two distinct isoforms both derived from the same gene, PRA and PRB. Each subtype is critical to mammary gland lobuloalveolar development and epithelial differentiation (Lanari & Molinolo Breast Cancer Res 2002, 4, 240-3., herein incorporated by reference in its entirety). The receptor consists of several regions that serve as functional units such as the DNA binding domain (DBD), the ligand binding domain (LBD), and transcriptional activation domains (AFs). The expression of these receptors is a critical parameter typically examined using immunohistochemistry in biopsies of human breast cancers (Jacobsen et al. J Mammary Gland Biol Neoplasia 2003, 8, 257-68., herein incorporated by reference in its entirety).
The presence of both receptors correlates significantly with the survival rate of breast cancer patients (Hopp et al. Clin Cancer Res 2004, 10, 2751-60., herein incorporated by reference in its entirety). The PR is an estrogen-regulated gene that becomes activated and expressed in the presence of estradiol and ER. Therefore, it is not surprising that treatment with tamoxifen (an anti-estrogenic therapy) reduces PR. Decreased PR correlates with tamoxifen resistance, although the mechanism of resistance is still being debated (Arpino et al. J Natl Cancer Inst 2005, 97, 1254-61., herein incorporated by reference in its entirety). Tumors that are ER+/PR− are considered more metastatic and aggressive than PR+ tumors and correlate with a lower survival rate (Cui et al. J Clin Oncol 2005, 23, 7721-35., herein incorporated by reference in its entirety).
An important prognostic marker is the presence of ER+/PR− tumors because these cancers respond much better to aromatase inhibitors than ER+/PR+ tumors that can be effectively treated with tamoxifen (Fuqua et al. J Clin Oncol 2005, 23, 931-2; author reply 932-3., Osborne et al. Breast 2005, 14, 458-65., herein incorporated by reference in their entireties). In addition, expression of PR may also reflect activation of the growth factor pathway Her2/neu. Since monoclonal antibodies directed against the Her2/neu receptor are being developed for breast cancer, knowing the PR status might help determine if a patient will respond to these treatments (Montemurro & Aglietta, Clin Breast Cancer 2005, 6, 77-80., herein incorporated by reference in its entirety). Therefore, non-invasively delineating whether or not a mammary cancer expresses the PR may be crucial to determining the best chemotherapeutic agent for the patient and ultimately improve survival.
The progression of endometrial cancer resembles that of breast cancer in regards to the expression of progesterone receptors. For example, the loss of both PRA and PRB is associated with a poor prognosis and inversely correlated with disease free survival (Boruban et al. Eur J Cancer Prey 2008, 17, 133-8., Uharcek. Obstet Gynaecol Res 2008, 34, 776-83., herein incorporated by reference in their entireties). When each isoforms of the receptor is analyzed separately, the correlation to disease progression is less clear (Arnett-Mansfield et al. Cancer Res 2001, 61, 4576-82., herein incorporated by reference in its entirety); however, the contrast agent would bind to all available progesterone receptors and therefore the overall loss of PR is more critical for imaging purposes. In cell lines, a reduction in the level of PRs is associated with increases in genes that regulate invasion (Miyamoto et al. J Steroid Biochem Mol Biol 2004, 92, 111-8., Saito et al. Cancer Sci 2006, 97, 1308-14., herein incorporated by reference in their entireties). Further, in tissue samples PR is inversely correlated to Ki67, a marker of cellular proliferation. Progesterone is also a therapeutic agent for endometrial cancers with many patients receiving progesterone to slow the growth of their cancer (Ito et al. Endocr J 2007, 54, 667-79., herein incorporated by reference in its entirety).
Recent reports have addressed the use of MR imaging for analyzing breast tumors (Lehman et al. N Engl J Med 2007, 356, 1295-303., Tozaki. Breast Cancer 2008, 15, 205-11., Lee et al. Radiology 2008, 246, 763-71., herein incorporated by reference in their entireties). Although the majority of physicians conclude that traditional mammography is the best for routine screening of the general population, MR imaging is increasingly used in tumor imaging. For patients with familial risk of breast cancer lesions tend to form quickly and have varying appearance using mammography. When a patient has a positive mammography and biopsy, MR imaging is a second line technique to discover other lesions and identify lesions in the contralateral breast. MR can be helpful for guiding biopsies so that the needle is inserted directly into the cancerous area for accurate results.
MR imaging is valuable for determining if a patient is responding to therapy. Response to therapy is one of the critical areas that a targeted steroid-based contrast agent can be used because many drugs for breast cancers down regulate estrogen inducible genes, such as the progesterone receptor. For uterine cancers in particular, progesterone is often given to the patient as part of treatment and in the case of the PR-imaging agent, the technology would possibly be both therapeutic and diagnostic (theranostic).
Progesterone agents have been developed for positron emission topography (PET) imaging with success in targeting breast cancer cells and tissues in rat models (Zhou et al. J Med Chem 2006, 49, 4737-44., Vijaykumar et al. A. J Org Chem 2002, 67, 4904-10., Pomper et al. J Med Chem 1988, 31, 1360-3., herein incorporated by reference in their entireties). Metabolic conversion of reported progestin based PET agents prevented the application of these probes in humans (Dehdashti et al. J Nucl Med 1991, 32, 1532-7., herein incorporated by reference in its entirety).
In vivo imaging agents could provide a tool for basic scientific investigations into the etiology of disease by providing size and molecular profiles of tumors without the need to euthanize the animal. Mouse models of cancer and uterine tumors are an essential component of understanding how to prevent and treat disease. Many models could be improved by applying imaging techniques such that tumors could develop and differentiate without the need to remove the tumor mass directly.
For example, models of ductal carcinoma in situ (DCIS) are available, but understanding when the lesion forms, where, and whether it is steroid responsive is difficult to accomplish without sacrificing the animal and performing a dissection. However, if contrast agents were applied, small lesions could be identified very early, allowing one to determine if they form invasive cancers, and then categorizing the cancer as steroid responsive or unresponsive. Progesterone receptor positive breast and uterine cancer cells can be subcutaneously injected into nude mice and produce solid tumors monitored with magnetic resonance imaging (Zong et al. Magn Reson Med 2005, 53, 835-42., Preda et al. J Magn Reson Imaging 2004, 20, 865-73., herein incorporated by reference in their entireties). The breast cancer cell lines typically used for such tumors include T47D and MCF7 cell lines, both of which express estrogen receptor (ER) and progesterone receptor (PR) (Hoffmann et al. J Natl Cancer Inst 2004, 96, 210-8., herein incorporated by reference in its entirety). For uterine cancers, the Ishikawa cell line lacks receptors and stable clones of the cell line with the PR gene integrated allow the investigator to analyze both receptor positive and negative tumors. Xenografted tumors visibly protrude from the mouse but may be analyzed earlier and with more ease using MR imaging (Bhujwalla et al. Neoplasia 2001, 3, 143-53., herein incorporated by reference in its entirety). These nude mouse tumor models will be used to study the targeting ability of progesterone based contrast agents to image PR+ receptor positive tumors and for quantitative imaging to determine tumor response to drug therapies.