Bimodal MR–PET Agent for Quantitative pH Imaging (2010)

Two unknowns: The relaxivity of activatable magnetic resonance (MR) contrast agents depends on environmental factors, such as the pH value or enzymatic activity, but the MR signal depends on relaxivity and agent concentration. Simultaneous positron emission tomography (PET) and MR imaging (MRI) with the bimodal, pH-responsive MR–PET agent shown enabled the direct determination of concentration and T1, and hence the pH value. 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 agents1 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 study, Meade and co-workers demonstrated that the relaxivity of a specifically designed contrast agent could be changed in the presence of the enzyme β-galactosidase and thus created 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 that are responsive to other enzymes, to changes in the pH, to the concentration of specific metal ions, to partial oxygen pressure, or to temperature.5, 6 A smart agent to noninvasively map pH values with both high temporal and high spatial resolution would have broad utility. Decreased extracellular pH values are associated with cancer and ischemic diseases, such as stroke, ischemic heart disease, and kidney disease.7 The pH of tissue could be a very useful biomarker for the identification of disease and monitoring of response to therapy, but it remains a challenge to routinely assess pH values in vivo. The implantation of a pH electrode is invasive and offers little spatial information. 31P NMR spectroscopy can be used to measure pH values on the basis of the pH-dependent chemical shift of inorganic phosphate.8 The use of exogenous agents with pH-sensitive chemical shifts has also been described.7, 9 However, these NMR spectroscopic techniques are limited by low sensitivity, which results 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 agents are also required in millimolar concentrations for detection.10, 11 Recently, hyperpolarized 13C-carbonate MR was used to image pH values.12 There are several gadolinium-based smart agents whose relaxivity is pH-dependent owing to changes in complex hydration with changes in the pH.6, 7 To address the problem of complex concentration, Aime et al. proposed a R2/R1 ratiometric method,13 where R2 and R1 are the transverse and longitudinal relaxation rates of water protons; however, given the relatively large inherent R2 in living tissues relative to R1, the in vivo accuracy of such an approach has yet to be proven. A combination of fluorine MRI for quantification with a pH-sensitive gadolinium-based agent has also been suggested,14 although the sensitivity of 19F imaging is in the millimolar range. An early pH-sensitive agent was GdDOTA-4AMP.15 This agent was used to map pH values in vivo in renal-acidosis16 and brain-tumor17 models. To estimate the in vivo concentration of the agent, the investigators first injected GdDOTP, which shows pH-independent relaxivity, and imaged the regoin of interest. They assumed that the pharmacokinetics of GdDOTP was the same as for GdDOTA-4AMP, and that differences in the signal-versus-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 differences in pH. 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 possibility of absolute quantification. Quantitative PET imaging is routinely used in human and animal studies, for example, to measure neuroreceptor occupancy levels18 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 setup enables the simultaneous acquisition of PET and MR data, and makes it possible 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 the quantification of both concentration and relaxivity. This dual-label approach could lead to a range of quantitative smart probes for in vivo applications. Herein, we describe a bimodal MR–PET agent designed for quantitative pH imaging at concentrations commonly used for in vivo MRI (0.1–1 mM). The established pH-sensitive MR agent GdDOTA-4AMP was modified to incorporate a fluorine atom (either 18F or 19F). Because of the hydrophilic character of highly charged GdDOTA-4AMP, the 18F atom had to be introduced under aqueous conditions. We chose the versatile copper(I)-catalyzed Huisgen cycloaddition (“click reaction”) for this purpose.22 GdDOTA-4AMP-F was prepared in six steps in 19 % overall yield from an established bifunctional chelator, tBu-protected DOTAGA, 1 (Scheme 1).23 Compound 1 was activated and coupled with propargylamine, and subsequently deprotected in neat trifluoroacetic acid (TFA) to give 3. The phosphonate groups were introduced by coupling with aminomethylphosphonic acid diethyl ester, followed by mild deprotection of the phosphonate groups with trimethylsilyl bromide (TMSBr) in N,N-dimethylformamide (DMF). Finally, GdDOTA-4AMP-F was prepared from 5 in a one-pot process by treatment first with GdCl3 to form the GdIII complex 6 and then with 2-fluoroethylazide. [18F]Fluoroethylazide was prepared in two steps from 2-azidoethanol, whereas the isotopically unmodified compound was prepared in two steps from 2-fluoroethanol (see the Supporting Information).22 Synthesis of GdDOTA-4AMP-F and structure of GdDOTA-4AMP. Compounds 2 and 4 (not shown) can be found in the Supporting Information. HATU=O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, PyBOP=benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate. The introduction of the fluorine-containing moiety in GdDOTA-4AMP-F did not modify the pH dependence of the longitudinal relaxivity with respect to that of the parent compound GdDOTA-4AMP.24 GdDOTA-4AMP-F retained 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−1 s−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 result indicates little, if any, protein binding and suggests that the pH–relaxivity relationship will be valid in vivo. Relaxivity of GdDOTA-4AMP-F as a function of pH value (37 °C, 1.4 T) in the presence of NaCl (135 mM), KCl (5 mM), and CaCl2 (2.5 mM; •), and in rabbit plasma (○). The chemical concentration required for MR contrast is orders of magnitude higher than for PET imaging. For this reason, the 19F and 18F 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 values by using a clinical 3T magnetic resonance imager with an MR-compatible human PET scanner insert.21 Images were acquired by simultaneous MR–PET on two sets of phantoms: one in which T1 was varied (MR, Figure 2 A) and the probe concentration was constant (PET, Figure 2 B), and one in which T1 was constant (Figure 2 C) and the probe concentration was varied (Figure 2 D). Figure 2 eloquently displays the limitations of using an MR-responsive agent without independent knowledge of the agent concentration. In both sets of phantoms, the pH value varied. The only way to obtain pH values from the images is to combine both PET and MR datasets. A,C) T1-weighted MR images (the T1 values (ms) are given) and B,D) PET images (the PET intensities are given) 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 had the same concentration (0.45 mM); phantoms in images C and D had the following concentrations: 0.31 (1), 0.33 (2), 0.38 (3), 0.42 (4), 0.45 mM (5). Since the relationship between the PET signal and the radiochemical concentration is linear, 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 the pH can be measured (see Figure 1); this process was repeated at 3T, and a similar linear relationship between r1 and the pH was observed between pH 6 and 8.5. 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 the pH measured by a pH electrode and pH calculated from the MR and PET images. Graph showing pH values obtained by analysis of the MR–PET images versus pH values measured with a glass electrode. The solid line is a linear fit of the data; the dotted line represents a 1:1 correspondence. In conclusion, we have described a smart MR–PET agent that can quantitatively and noninvasively report pH values. Imaging data were obtained on a commercial clinical MR imager with a prototype human PET camera at agent concentrations routinely encountered in clinical MRI (0.1–1 mM), which augurs well for the application of GdDOTA-4AMP-F to image pH changes in vivo. The combination of PET for the quantification of concentration and MR for the quantification of T1 enables the simultaneous determination of relaxivity. With smart MR probes, whose relaxivity is proportional to an environmental stimulus, this bimodal imaging approach enables direct quantification of the stimulus, in this case the pH value. This bimodal MR–PET strategy is generally applicable to other smart MR probes. Detailed facts of importance to specialist readers are published as ”Supporting Information”. 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