Angelo Lubag

A Concentration-Independent Method to Measure Exchange Rates in PARACEST Agents

The efficiency of chemical exchange dependent saturation transfer (CEST) agents is largely determined by their water or proton exchange kinetics, yet methods to measure such exchange rates are variable and many are not applicable to in vivo measurements. In this work, the water exchange kinetics of two prototype paramagnetic agents (PARACEST) are compared by using data from classic NMR line‐width measurements, by fitting CEST spectra to the Bloch equations modified for chemical exchange, and by a method where CEST intensity is measured as a function of applied amplitude of radiofrequency field. A relationship is derived that provides the water exchange rate from the X‐intercept of a plot of steady‐state CEST intensity divided by reduction in signal caused by CEST irradiation versus 1/ω12, referred to here as an omega plot. Furthermore, it is shown that this relationship is independent of agent concentration. Exchange rates derived from omega plots using either high‐resolution CEST NMR data or CEST data obtained by imaging agree favorably with exchange rates measured by the more commonly used Bloch fitting and line‐width methods. Thus, this new method potentially allows access to a direct measure of exchange rates in vivo, where the agent concentration is typically unknown. Magn Reson Med 63:625–632, 2010.

A New Gadolinium-Based MRI Zinc Sensor

The properties of a novel Gd(3+)-based MRI zinc sensor are reported. Unlike previously reported Gd(3+)-based MRI contrast agents, this agent (GdL) differs in that the agent alone binds only weakly with human serum albumin (HSA), while the 1:2 GdL:Zn(2+) ternary complex binds strongly to HSA resulting in a substantial, 3-fold increase in water proton relaxivity. The GdL complex is shown to have a relatively strong binding affinity for Zn(2+) (K(D) = 33.6 nM), similar to the affinity of the Zn(2+) ion with HSA alone. The agent detects as little as 30 microM Zn(2+) in the presence of HSA by MRI in vitro, a value slightly more than the total Zn(2+) concentration in blood (approximately 20 microM). This combination of binding affinity constants and the high relaxivity of the agent when bound to HSA suggests that this new agent may be useful for detection of free Zn(2+) ions in vivo without disrupting other important biological processes involving Zn(2+).

Noninvasive MRI of β-cell function using a Zn2+-responsive contrast agent.

Elevation of postprandial glucose stimulates release of insulin from granules stored in pancreatic islet β-cells. We demonstrate here that divalent zinc ions coreleased with insulin from β-cells in response to high glucose are readily detected by MRI using the Zn2+-responsive T1 agent, GdDOTA-diBPEN. Image contrast was significantly enhanced in the mouse pancreas after injection of a bolus of glucose followed by a low dose of the Zn2+ sensor. Images of the pancreas were not enhanced by the agent in mice without addition of glucose to stimulate insulin release, nor were images enhanced in streptozotocin-treated mice with or without added glucose. These observations are consistent with MRI detection of Zn2+ released from β-cells only during glucose-stimulated insulin secretion. Images of mice fed a high-fat (60%) diet over a 12-wk period and subjected to this same imaging protocol showed a larger volume of contrast-enhanced pancreatic tissue, consistent with the expansion of pancreatic β-cell mass during fat accumulation and progression to type 2 diabetes. This MRI sensor offers the exciting potential for deep-tissue monitoring of β-cell function in vivo during development of type 2 diabetes or after implantation of islets in type I diabetic patients.

MRI detection of paramagnetic chemical exchange effects in mice kidneys in vivo

In this report, the On resonance PARamagnetic CHemical Exchange Effects (OPARACHEE) method was implemented in vivo using WALTZ‐16* as a preparation pulse with a standard spin echo sequence to detect the accumulation and clearance of the TmDOTA‐4AmC− in mouse kidney. The performance of the technique in vivo is described in terms of the magnitude of the contrast effect versus the bolus agent concentration and signal‐to‐noise ratio (SNR) levels. The lowest injected concentration of TmDOTA‐4AmC−, 200 μL of a 2‐mM stock solution (corresponds to ∼0.2 mM agent in plasma), reduced the total water signal in the kidney papilla by 45% 3 min after the a bolus injection. The results show that the OPARACHEE methodology employing low‐amplitude RF trains can detect paramagnetic exchanging agents in vivo. Magn Reson Med 58:650–655, 2007.

The detection limit of a Gd3+-based T1 agent is substantially reduced when targeted to a protein microdomain

Simple low molecular weight (MW) chelates of Gd(3+) such as those currently used in clinical MRI are considered too insensitive for most molecular imaging applications. Here, we evaluated the detection limit (DL) of a molecularly targeted low MW Gd(3+)-based T(1) agent in a model where the receptor concentration was precisely known. The data demonstrate that receptors clustered together to form a microdomain of high local concentration can be imaged successfully even when the bulk concentration of the receptor is quite low. A GdDO3A-peptide identified by phage display to target the anti-FLAG antibody was synthesized, purified and characterized. T(1-)weighted MR images were compared with the agent bound to antibody in bulk solution and with the agent bound to the antibody localized on agarose beads. Fluorescence competition binding assays show that the agent has a high binding affinity (K(D)=150 nM) for the antibody, while the fully bound relaxivity of the GdDO3A-peptide/anti-FLAG antibody in solution was a relatively modest 17 mM(-1) s(-1). The agent/antibody complex was MR silent at concentrations below approximately 9 microM but was detectable down to 4 microM bulk concentrations when presented to antibody clustered together on the surface of agarose beads. These results provided an estimate of the DLs for other T(1)-based agents with higher fully bound relaxivities or multimeric structures bound to clustered receptor molecules. The results demonstrate that the sensitivity of molecularly targeted contrast agents depends on the local microdomain concentration of the target protein and the molecular relaxivity of the bound complex. A model is presented, which predicts that for a molecularly targeted agent consisting of a single Gd(3+) complex with bound relaxivity of 100 mM(-1) s(-1) or, more reasonably, four tethered Gd(3+) complexes each having a bound relaxivity of 25 mM(-1) s(-1), the DL of a protein microdomain is approximately 690 nM at 9.4 T. These experimental and extrapolated DLs are both well below current literature estimates and suggests that detection of low MW molecularly targeted T(1) agents is not an unrealistic goal.

MRI Detection of VEGFR2 in Vivo Using a Low Molecular Weight Peptoid−(Gd)8-Dendron for Targeting

The synthesis of a polylysine dendron containing eight GdDOTA units conjugated to a peptoid dimer known to have a high affinity for the vascular endothelial growth factor receptor 2 (VEGFR2) is described. This simple low molecular weight system with a molecular r(1) relaxivity of ∼48 mM(-1) s(-1) is shown to enhance MR images of tumors grown in mice in vivo.

Silencing of phosphonate-gadolinium magnetic resonance imaging contrast by hydroxyapatite binding

Rationale and Objectives:GdDOTP5− is a highly charged, bone-seeking paramagnetic complex that could potentially detect bone lesions by magnetic resonance imaging (MRI). To date, its pharmacokinetics, effects on organ relaxivity, and interaction with hydroxyapatite (HA) has not been described. Methods:Liver, kidney, and bone MRI images were obtained on male white rabbits after the administration of GdDOTP5− or a gold standard MRI contrast agent, GdDTPA2−. Parallel in vitro experiments quantified the effect of HA binding on GdDOTP5− -induced changes in relaxivity. Results:The 2 compounds showed similar MRI enhancements in visceral tissues, but no enhancement of bone was evident with GdDOTP5− despite confirmation of bone and HA binding of the radioactive 153SmDOTP5− and 111InDOTP5− derivatives. In vitro experiments demonstrated that GdDOTP5−-induced changes in relaxivity were silenced upon HA binding but could be recovered by acid elution of the complex. Conclusions:HA binding assays revealed that GdDOTP5− is essentially MR silent when bound to bone, likely because of the exclusion of all outer sphere water molecules from the surface of the complex. These data suggest a novel strategy for creating highly sensitive, switchable MRI contrast agents.

Advances in molecular imaging of pancreatic beta cells.

The development of non-invasive imaging methods for early diagnosis of beta cell associated metabolic diseases, including type 1 and type 2 diabetes (T1D and T2D), has recently drawn interest from the molecular imaging community and clinical investigators. Due to the challenges imposed by the location of the pancreas, the sparsely dispersed beta cell population within the pancreas, and the poor understanding of the pathogenesis of the diseases, clinical diagnosis of beta cell abnormalities is still limited. Current diagnostic methods are invasive, often inaccurate, and usually performed post-onset of the disease. Advances in imaging techniques for probing beta cell mass and function are needed to address this critical health care problem. A variety of imaging techniques have been tested for the assessment of pancreatic beta cell islets. Here we discuss current advances in magnetic resonance imaging (MRI), bioluminescence imaging (BLI), and nuclear imaging for the study of beta cell diseases. Spurred by early successes in nuclear imaging techniques for beta cells, especially positron emission tomography (PET), the need for beta cell specific ligands has expanded. Progress for obtaining such ligands is presented. We report our preliminary efforts of developing such a peptidic ligand for PET imaging of pancreatic beta cells.

Zinc-sensitive MRI contrast agent detects differential release of Zn(II) ions from the healthy vs. malignant mouse prostate.

Significance The normal prostate gland contains the most Zn(II) of all mammalian tissues, and there are marked differences in Zn(II) content between the healthy, malignant, and benign hyperplastic prostate. Given that multiparametric MRI does not always reliably distinguish between these tissue conditions, the release of Zn(II) ions from the prostate in response to an external stimulus may prove valuable as a specific biomarker of prostate cancer progression. In this work, we show that glucose stimulates the release of Zn(II) from intracellular stores in healthy prostate tissue and that Zn(II) secretion is reduced in a transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Many secretory tissues release Zn(II) ions along with other molecules in response to external stimuli. Here we demonstrate that secretion of Zn(II) ions from normal, healthy prostate tissue is stimulated by glucose in fasted mice and that release of Zn(II) can be monitored by MRI. An ∼50% increase in water proton signal enhancement is observed in T1-weighted images of the healthy mouse prostate after infusion of a Gd-based Zn(II) sensor and an i.p. bolus of glucose. Release of Zn(II) from intracellular stores was validated in human epithelial prostate cells in vitro and in surgically exposed prostate tissue in vivo using a Zn(II)-sensitive fluorescent probe known to bind to the extracellular surface of cells. Given the known differences in intracellular Zn(II) stores in healthy versus malignant prostate tissues, the Zn(II) sensor was then evaluated in a transgenic adenocarcinoma of the mouse prostate (TRAMP) model in vivo. The agent proved successful in detecting small malignant lesions as early as 11 wk of age, making this noninvasive MR imaging method potentially useful for identifying prostate cancer in situations where it may be difficult to detect using current multiparametric MRI protocols.

A second generation MRI contrast agent for imaging zinc ions in vivo

A Zn2+ specific GdDOTA derivative containing two bis-(3-pyrazolyl) units was prepared and characterized. Unlike a previously reported Zn2+ binding agent, the new agent binds to human albumin both in the presence and absence of Zn2+.