Peter Caravan

Strategies for increasing the sensitivity of gadolinium based MRI contrast agents

Gadolinium(III) complexes are often used in clinical MRI to increase contrast by selectively relaxing the water molecules near the complex. There is a desire to improve the sensitivity (relaxivity) of these contrast agents in order to detect molecular targets. This tutorial review describes the molecular factors that contribute to relaxivity and illustrates with recent examples how these can be optimized. It may be of interest to senior undergraduates and more advanced researchers interested in lanthanide chemistry, biophysics, and/or molecular imaging.

Protein-Targeted Gadolinium-Based Magnetic Resonance Imaging (MRI) Contrast Agents: Design and Mechanism of Action

Magnetic resonance imaging (MRI) is a powerful medical diagnostic technique: it can penetrate deep into tissue, provide excellent soft tissue contrast with sub-millimeter resolution, and does not employ ionizing radiation. Targeted contrast agents provide an additional layer of molecular specificity to the wealth of anatomical and functional information already attainable by MRI. However, the major challenge for molecular MR imaging is sensitivity: micromolar concentrations of Gd(III) are required to cause a detectable signal change, which makes detecting proteins by MRI a challenge. Protein-targeted MRI contrast agents are bifunctional molecules comprising a protein-targeting moiety and typically one or more gadolinium chelates for detection by MRI. The ability of the contrast agent to enhance the MR image is termed relaxivity, and it depends upon many molecular factors, including protein binding itself. As in other imaging modalities, protein binding provides the pharmacokinetic effect of concentrating the agent at the region of interest. Unique to MRI, protein binding provides the pharmacodynamic effect of increasing the relaxivity of the contrast agent, thereby increasing the MR signal. In designing new agents, optimization of both the targeting function and the relaxivity is critical. In this Account, we focus on optimization of the relaxivity of targeted agents. Relaxivity depends upon speciation, chemical structure, and dynamic processes, such as water exchange kinetics and rotational tumbling rates. We describe mechanistic studies that relate these factors to the observed relaxivities and use these findings as the basis of rational design of improved agents. In addition to traditional biochemical methods to characterize ligand-protein interactions, the presence of the metal ion enables more obscure biophysical techniques, such as relaxometry and electron nuclear double resonance, to be used to elucidate the mechanism of relaxivity differences. As a case study, we explore the mechanism of action of the serum-albumin-targeted angiography agent MS-325 and closely related compounds and show how small changes in the metal chelate can impact relaxivity. We found that, while protein binding generally improves relaxivity by slowing the tumbling rate of the complex, in some cases, the protein itself can also negatively affect hydration of the metal complex and/or inner-sphere water exchange. Drawing on these findings, we designed next-generation agents targeting albumin, fibrin, or collagen and incorporating up to four gadolinium chelates. Through judicious molecular design, we show that high-relaxivity complexes with high target affinity can be realized.

Influence of molecular parameters and increasing magnetic field strength on relaxivity of gadolinium- and manganese-based T1 contrast agents.

Simulations were performed to understand the relative contributions of molecular parameters to longitudinal (r(1)) and transverse (r(2)) relaxivity as a function of applied field, and to obtain theoretical relaxivity maxima over a range of fields to appreciate what relaxivities can be achieved experimentally. The field-dependent relaxivities of a panel of gadolinium and manganese complexes with different molecular parameters, water exchange rates, rotational correlation times, hydration state, etc. were measured to confirm that measured relaxivities were consistent with theory. The design tenets previously stressed for optimizing r(1) at low fields (very slow rotational motion; chelate immobilized by protein binding; optimized water exchange rate) do not apply at higher fields. At 1.5 T and higher fields, an intermediate rotational correlation time is desired (0.5-4 ns), while water exchange rate is not as critical to achieving a high r(1). For targeted applications it is recommended to tether a multimer of metal chelates to a protein-targeting group via a long flexible linker to decouple the slow motion of the protein from the water(s) bound to the metal ions. Per ion relaxivities of 80, 45, and 18 mM(-1) s(-1) at 1.5, 3 and 9.4 T, respectively, are feasible for Gd(3+) and Mn(2+) complexes.

Biodistribution of gadolinium-based contrast agents, including gadolinium deposition

The biodistribution of approved gadolinium (Gd)‐based contrast agents (GBCAs) is reviewed. After intravenous injection GBCAs distribute in the blood and the extracellular space and transiently through the excretory organs. Preclinical animal studies and the available clinical literature indicate that all these compounds are excreted intact. Elimination tends to be rapid and, for the most part, complete. In renally insufficient patients the plasma elimination half‐life increases substantially from hours to days depending on renal function. In patients with impaired renal function and nephrogenic systemic fibrosis (NSF), the agents gadodiamide, gadoversetamide, and gadopentetate dimeglumine have been shown to result in Gd deposition in the skin and internal organs. In these cases, it is likely that the Gd is no longer present as the GBCA, but this has still not been definitively shown. In preclinical models very small amounts of Gd are retained in the bone and liver, and the amount retained correlates with the kinetic and thermodynamic stability of the GBCA with respect to Gd release in vitro. The pattern of residual Gd deposition in NSF subjects may be different than that observed in preclinical rodent models. GBCAs are designed to be used via intravenous administration. Altering the route of administration and/or the formulation of the GBCA can dramatically alter the biodistribution of the GBCA and can increase the likelihood of Gd deposition. J. Magn. Reson. Imaging 2009;30:1259–1267.

Primer on gadolinium chemistry

Gadolinium is widely known by all practitioners of magnetic resonance imaging (MRI) but few appreciate the basic solution chemistry of this trivalent lanthanide ion. Given the recent linkage between gadolinium contrast agents and nephrogenic systemic fibrosis, some basic chemistry of this ion must be more widely understood. This short primer on gadolinium chemistry is intended to provide the reader the background principles necessary to understand the basics of chelation chemistry, water hydration numbers, and the differences between thermodynamic stability and kinetic stability or inertness. We illustrate the fundamental importance of kinetic dissociation rates in determining gadolinium toxicity in vivo by presenting new data for a novel europium DOTA‐tetraamide complex that is relatively unstable thermodynamically yet extraordinarily inert kinetically and also quite nontoxic. This, plus other literature evidence, forms the basis of the fundamental axiom that it is the kinetic stability of a gadolinium complex, not its thermodynamic stability, that determines its in vivo toxicity. J. Magn. Reson. Imaging 2009;30:1240–1248.

EP-2104R: A Fibrin-Specific Gadolinium-Based MRI Contrast Agent for Detection of Thrombus

Thrombus (blood clot) is implicated in a number of life threatening diseases, e.g., heart attack, stroke, pulmonary embolism. EP-2104R is an MRI contrast agent designed to detect thrombus by binding to the protein fibrin, present in all thrombi. EP-2104R comprises an 11 amino acid peptide derivatized with 2 GdDOTA-like moieties at both the C- and N-terminus of the peptide (4 Gd in total). EP-2104R was synthesized by a mixture of solid phase and solution techniques. The La(III) analogue was characterized by and 1D and 2D NMR spectroscopy and was found to have the expected structure. EP-2104R was found to be significantly more inert to Gd(III) loss than commercial contrast agents. At the most extreme conditions tested (pH 3, 60 degrees C, 96 hrs), less than 10% of Gd was removed from EP-2104R by a challenge with a DTPA based ligand, while the commercial contrast agents equilibrated within minutes to hours. EP-2104R binds equally to two sites on human fibrin (Kd = 1.7 +/- 0.5 microM) and has a similar affinity to mouse, rat, rabbit, pig, and dog fibrin. EP-2104R has excellent specificity for fibrin over fibrinogen (over 100-fold) and for fibrin over serum albumin (over 1000-fold). The relaxivity of EP-2104R bound to fibrin at 37 degrees C and 1.4 T was 71.4 mM(-1) s(-1) per molecule of EP-2104R (17.4 per Gd), about 25 times higher than that of GdDOTA measured under the same conditions. Strong fibrin binding, fibrin selectivity, and high molecular relaxivity enable EP-2104R to detect blood clots in vivo.

Epidermal growth factor receptor inhibition attenuates liver fibrosis and development of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most rapidly increasing cause of cancer‐related mortality in the United States. Because of the lack of viable treatment options for HCC, prevention in high‐risk patients has been proposed as an alternative strategy. The main risk factor for HCC is cirrhosis and several lines of evidence implicate epidermal growth factor (EGF) in the progression of cirrhosis and development of HCC. We therefore examined the effects of the EGF receptor (EGFR) inhibitor erlotinib on liver fibrogenesis and hepatocellular transformation in three different animal models of progressive cirrhosis: a rat model induced by repeated, low‐dose injections of diethylnitrosamine (DEN), a mouse model induced by carbon tetrachloride (CCl4), and a rat model induced by bile duct ligation (BDL). Erlotinib reduced EGFR phosphorylation in hepatic stellate cells (HSC) and reduced the total number of activated HSC. Erlotinib also decreased hepatocyte proliferation and liver injury. Consistent with all these findings, pharmacological inhibition of EGFR signaling effectively prevented the progression of cirrhosis and regressed fibrosis in some animals. Moreover, by alleviating the underlying liver disease, erlotinib blocked the development of HCC and its therapeutic efficacy could be monitored with a previously reported gene expression signature predictive of HCC risk in human cirrhosis patients. Conclusion: These data suggest that EGFR inhibition using Food and Drug Administration‐approved inhibitors provides a promising therapeutic approach for reduction of fibrogenesis and prevention of HCC in high‐risk cirrhosis patients who can be identified and monitored by gene expression signatures. (Hepatology 2014;59:1577‐1590)

Thrombus imaging with fibrin-specific gadolinium-based MR contrast agent EP-2104R: results of a phase II clinical study of feasibility.

Purpose:To determine the feasibility of detecting thrombi using a fibrin-specific gadolinium-based magnetic resonance imaging contrast agent, EP-2104R. Methods:Subjects with confirmed thrombus in the venous system (n = 14), or in the heart, or arterial system (n = 38) were enrolled. Patients were imaged before and at various times following a 4 μmol/kg intravenous bolus injection of EP-2104R: <1 hour (N = 16), 2 to 6 hours (N = 36), and/or 20 to 36 hours (N = 33). Images were assessed by investigators at each site and by a single reader not affiliated with the sites to determine whether thrombi were visible, not visible, or further enhanced with EP-2104R. A subset of data was analyzed quantitatively by measuring a signal intensity relative to background tissue. Results:Overall, 29 thrombi were visible before contrast administration, 3 of 14 in the venous system, and 26 of 38 in the arteries and heart. Thrombi generally enhanced in signal after EP-2104R injection, and an additional 7 were visualized. After contrast, 4 of 14 thrombi were visible in the venous system, and 32 of 38 in the arteries and heart. Thrombi were more conspicuous when imaged at 2 to 6 hours post EP-2104R compared with within 1 hour, because of lower blood background. Quantitatively, the post: pre signal intensity ratio was 1.90 at 2 to 6 hours post injection (standard deviation = 1.08, N = 20, P < 0.001); and 2.04 (standard deviation = 1.29, N = 19, P < 0.0025) for the 20 to 36 hours time point. There were no serious adverse events considered related to study drug. Conclusion:EP-2104R enhanced magnetic resonance imaging detects thrombi not readily visible in precontrast screening and gives additional enhancement of thrombi that are visible in precontrast imaging.

Bimodal MR–PET Agent for Quantitative pH Imaging

The scope of Magnetic Resonance Imaging (MRI) is moving beyond anatomical and functional imaging to also convey information at the molecular level. Molecular MRI is enabled by the introduction of protein-targeted contrast agents[1] as well as “smart” or activatable contrast agents.[2] MR contrast agents induce relaxation of tissue water, and the extent of this relaxation enhancement, termed relaxivity (r1), depends on a number of molecular factors including the hydration state of the contrast agent and its rotational diffusion rate. In a seminal paper, Meade and colleagues demonstrated that the relaxivity of a specifically designed contrast agent could be changed in the presence of the enzyme beta-galactosidase, thereby creating an imaging agent whose signal was activated by the presence of the enzyme.[3, 4] Numerous publications have followed in the last decade that describe “smart” agents responsive to other enzymes, to pH, to specific metal ion concentrations, to partial oxygen pressure and to temperature.[5, 6] The impressive gains in smart agent development have been slow to make their way into in vivo imaging studies, however. This can be appreciated from equation 1 which relates the water relaxation rate (1/T1) to r1. MR signal is a function T1 (Eq. 1, 1/T10 = relaxation rate in absence of agent), which depends on both r1 and contrast agent concentration ([Gd]). In vitro, the gadolinium concentration is known and fixed; any signal change is due to relaxivity change. In vivo, the agent concentration is unknown, will change with time, and may vary in diseased versus normal tissue. 1T1=r1⋅[Gd]+1T10 (1) A smart agent to noninvasively map pH with both high temporal and spatial resolution would have broad utility. Decreased extracellular pH is associated with cancer and ischemic diseases such as stroke, ischemic heart disease, and kidney disease.[7] pH could be a very useful biomarker to identify disease and monitor response to therapy, but it remains a challenge to routinely assess pH in vivo. Implanting a pH electrode is invasive and offers little spatial information. 31P NMR can measure pH via the pH-dependent chemical shift of inorganic phosphate.[8] Other papers have described using exogenous agents with pH-sensitive chemical shifts.[7, 9] Yet, these NMR spectroscopic techniques are limited by low sensitivity resulting in trade offs in imaging time (longer, more averages required) and resolution (lower, bigger volume elements required). Chemical exchange saturation transfer (CEST) agents have also been described for pH imaging, but these also require millimolar concentrations for detection.[10, 11] Recently hyperpolarized 13C-carbonate MR was used to image pH.[12] There are several Gd-based smart agents whose relaxivity is pH dependent due to changes in complex hydration with pH.[6, 7] To address the problem of complex concentration, Aime et al. proposed a R2/R1 ratiometric method[13] but given the relatively large R2 present in living tissues compared to R1, the in vivo accuracy of such an approach has yet to be proven. Combining fluorine MRI for quantification with a pH sensitive Gd-based agent has also been suggested,[14] although the sensitivity of F-19 imaging is in the millimolar range. An early pH sensitive agent was GdDOTA-4AMP.[15] This agent was used to map pH in vivo in renal acidosis[16] and brain tumor[17] models. To estimate the in vivo concentration of agent, these investigators first injected GdDOTP, which has pH-independent relaxivity, and imaged. They assumed that the pharmacokinetics of GdDOTP was the same as for GdDOTA-4AMP and that differences in the signal vs time curves for GdDOTP and GdDOTA-4AMP were due to differences in relaxivity. These studies demonstrated the potential for in vivo pH mapping and showed that MRI with GdDOTA-4AMP was sensitive enough to detect pH differences. The limitations of this approach were the need for two sequential injections and the assumption that both contrast agents have identical pharmacokinetics. Positron emission tomography (PET) offers exquisite sensitivity and the ability to perform absolute quantification. Quantitative PET imaging is routinely used in human and animal studies, for example to measure neuroreceptor occupancy levels[18] or to measure tissue perfusion.[19] The recent application of MR-compatible avalanche photodiode detector technology has now made it possible to have a functioning PET detector inside the MR magnet.[20, 21] This allows for the simultaneous acquisition of PET and MR data, and ability to obtain both temporally and spatially registered imaging data sets. We hypothesized that simultaneous MR-PET imaging with a bimodal MR-PET smart agent would result in quantification of both concentration and relaxivity. This dual label approach could enable a range of quantitative smart probes for in vivo applications. Here, a bimodal MR-PET agent designed for quantitative pH imaging at concentrations commonly used for in vivo MRI (0.1–1 mM) is described. The established pH sensitive MR agent GdDOTA-4AMP was modified to incorporate a fluorine atom (either 18F or 19F). The highly charged, hydrophilic GdDOTA-4AMP necessitated a strategy to introduce the 18F atom under aqueous conditions, and we chose the versatile Cu(I)-catalyzed Huisgen cycloaddition (“click reaction”) for this purpose.[22] GdDOTA-4AMP-F was prepared in six steps (Scheme 1) with a 25% overall yield starting from an established bifunctional chelator, tBu protected DOTAGA, 1.[23] 1 was activated and coupled with propargylamine, and subsequently deprotected in neat TFA to give 3. The introduction of the phosphonate groups was accomplished by coupling with aminomethyl-phosphonic acid diethyl ester, followed by mild deprotection of the phosphonate groups with trimethylsilyl bromide in DMF. The formation of the Gd(III) complex from the chloride salt, followed by reaction of fluoroethylazide and the alkyne intermediate 6, was performed in one pot. [F-18]fluoroethylazide was prepared in two steps from 2-azidoethanol, while the [F-19] version was prepared in two steps from 2-fluoroethanol (see Supp. Info).[22] Scheme 1 Synthesis of Gd-DOTA-4AMP-F and structure of Gd-DOTA-4AMP. The introduction of the fluorine-containing moiety into GdDOTA-4AMP-F did not modify the pH dependence of the longitudinal relaxivity with respect to the parent compound GdDOTA4-AMP.[24] GdDOTA-4AMP-F retains a monotonic decrease in relaxivity between pH 6.0 and 8.5 (Figure 1). In this pH range the relaxivity varied between 7.4 and 3.9 mM−1s−1 (60 MHz, 37 °C) when measured in an isotonic salt mixture. When the relaxivity was measured in rabbit plasma, the profile was found to be very similar to the profile measured in the salt solution. This indicates little if any protein binding and suggests that the pH-relaxivity relationship will be valid in vivo. Figure 1 Relaxivity of GdDOTA-4AMP-F as a function of pH (37 °C, 1.4 T) in presence of 135 mM NaCl, 5 mM KCl, and 2.5 mM CaCl2 (filled diamonds), and in rabbit plasma (open circles). The chemical concentration required for MR contrast is orders of magnitude higher than for PET imaging. For this reason, F-19 and F-18 versions of the probe were prepared separately, and subsequently mixed to produce a low specific activity MR-PET agent. Simultaneous MR-PET imaging was performed on a series of samples with varying pH using a clinical 3T MRI with a MR-compatible human PET scanner insert.[21] Figure 2 shows simultaneous MR-PET images acquired on phantoms where the T1 varied (MR, 2A) but the probe concentration was constant (PET, 2B); or where T1 was constant (2C) but the probe concentration varied (2D). Figure 2 eloquently displays the limitations of using an MR responsive agent without independent knowledge of the agent concentration. Note that in both sets of phantoms, the pH is varied. The only way to obtain pH values from the images is to combine both the PET and MR datasets. Figure 2 T1-weighted MR images (A and C, T1 values (ms) listed) and PET images (B and D, PET intensities (a.u.) listed) of phantoms at pH 6.5 (tube 1), 6.8 (2), 7.1 (3), 7.4 (4), and 7.8 (5). Phantoms in images A and B have the same concentration (0.45 mM); phantoms ... Since the PET signal is linear with radiochemical concentration, the unknown agent concentration can be determined by comparing the PET images with a series of standards. For the MR data, the relationship between r1 and pH can be measured (see Fig 1) and this was repeated at 3T where a similar linear relationship between r1 and pH between pH 6 to 8.5 was obtained. From these two standard curves, the PET and MR imaging data can be analyzed to estimate the pH of the samples. Figure 3 shows the good correspondence between pH measured by electrode and pH calculated from the MR and PET images. Figure 3 pH obtained from PET-MR image analysis versus pH measured by a glass electrode. The solid line is a linear fit of the data, while the dotted line represents a 1:1 correspondence. In conclusion, this communication describes a smart MR-PET agent that can quantitatively and non-invasively report on pH. Imaging data were obtained on a commercial clinical MRI with a prototype human PET camera at agent concentrations routinely encountered in clinical MRI (0.1 – 1 mM). This augurs well for the application of GdDOTA-4AMP-F to image pH changes in vivo. The combination of PET for quantifying concentration and MR for quantifying T1 allows for the simultaneous determination of relaxivity. For smart MR probes where relaxivity is proportional to an environmental stimulus, this bimodal imaging approach enables direct quantification of the stimulus, pH in this case. We note that this bimodal MR-PET strategy is generally applicable to other smart MR probes.

Postinfarction Myocardial Scarring in Mice: Molecular MR Imaging with Use of a Collagen-targeting Contrast Agent

PURPOSE To prospectively evaluate a gadolinium-based collagen-targeting contrast agent, EP-3533, for in vivo magnetic resonance (MR) imaging of myocardial fibrosis in a mouse model of healed myocardial infarction (MI). MATERIALS AND METHODS All procedures were performed in accordance with protocols approved by the animal care and use committee. MI was induced in eight mice by means of occlusion of the left anterior descending coronary artery followed by reperfusion. Four MR examinations were performed in each animal: one examination before, one examination 1 day after, and two examinations 6 weeks after the MI. For the latter two examinations, electrocardiographically gated inversion-recovery gradient-echo MR images were acquired before and serially (every 5 minutes) after the intravenous injection of either gadopentetate dimeglumine or EP-3533. The image enhancement kinetic properties of the postinfarction scar, normal myocardium, and blood were compared. RESULTS Dynamic T1-weighted MR imaging revealed the washout time constants for EP-3533 to be significantly longer than those for gadopentetate dimeglumine in regions of postinfarction scarring (mean, 194.8 minutes +/-116.8 [standard deviation] vs 25.5 minutes +/- 4.2; P < .05) and in normal myocardium (mean, 45.4 minutes +/- 16.7 vs 25.1 minutes +/- 9.7; P < .05). Findings on postmortem histologic sections stained for collagen correlated well with EP-3533-enhanced areas seen on inversion-recovery MR images. Fifty minutes after EP-3533 injection, the postinfarction scar tissue samples, as compared with the normal myocardium, had a twofold higher concentration of gadolinium. CONCLUSION Use of the gadolinium-based collagen-targeting contrast agent, EP-3533, enabled in vivo molecular MR imaging of fibrosis in a mouse model of healed postinfarction myocardial scarring.