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Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2011.

Bookshelf ID: NBK83154PMID: 22238803

Ytterbium chelated to 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid,10-orthoaminoanilide

Yb-DO3A-oAA

Mark Pagel, PhD and Arvind Chopra, PhD.

Author Information
Mark Pagel, PhD
University of Arizona
mpagel/at/u.arizona.edu
Arvind Chopra, PhD
National Center for Biotechnology Information, NLM, Bethesda, MD 20894
micad/at/ncbi.nlm.nih.gov

Created: November 26, 2011; Last Update: January 5, 2012.

Chemical name:Ytterbium chelated to 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid,10-orthoaminoanilideImage YbDO3AoAA.jpg
Abbreviated name:Yb-DO3A-oAA
Synonym:Yb-DO3A-orthoaminoanilide
Agent Category:Compound
Target:Non-targeted
Target Category:Non-targeted
Method of detection:PARACEST magnetic resonance imaging (MRI)
Source of signal / contrast:Yb3+
Activation:No
Studies:

  • Checkbox Rodents

Structure of Yb-DO3A-oAA

Background

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[PubMed]

Many pathologies and biological processes are associated with changes in extracellular pH (pHe). For example, the pHe of the tumor microenvironment can be acidic (pH 6.5–6.9) relative to pHe in normal tissues (pH 7.2–7.4) due to aerobic glycolysis that produces excess lactic acid (1). Accurately measuring tumor pHe may be used to diagnose tumors. Furthermore, acidic tumors are chemoresistant against weak-base chemotherapies such as doxorubicin, and they may be more sensitive to weak-acid chemotherapies (2). Accurately measuring tumor pHe may provide patients and physicians with the ability to select the best chemotherapy for the tumor in order to provide personalized medicine to the patient. Clinical drug trials with experimental weak-base and weak-acid chemotherapies may benefit by stratifying patients on the basis of their tumor pHe. Treatments that modulate pHe, such as bicarbonate, have been shown to reduce metastases and increase survival in mouse models of human mammary carcinoma (3). Chronic administration of excessive bicarbonate, however, may lead to alkalosis of normal tissues, so a method to monitor tissue pHe is needed to support studies of pHe-modulating treatments. Many imaging methods have been developed to measure tumor pHe, including optical imaging, electron paramagnetic resonance (EPR) imaging, positron emission tomography imaging, magnetic resonance (MR) spectroscopy, and hyperpolarized MR imaging (MRI), but these methods suffer from poor depth of penetration, lack of accuracy or precision, produce images with poor spatial resolution, and/or require specialized hardware that is not readily available in the imaging clinic (4).

Chemical exchange saturation transfer (CEST) is a novel MRI contrast technique (5) that is an attractive alternative to the T1 and T2 contrast techniques, particularly at high magnetic fields (6). CEST agents possess a hydrogen proton with a moderate to slow exchange rate with water. The concentration required for in vivo imaging is in the ~10 mM range. The specific minimum concentration for each biomedical study depends on characteristics of the tissue, such as the endogenous T1 relaxation time of the tissue and the concentration of water in the tissue that is accessible to the agent. Selective saturation of the MR frequency of this proton, followed by exchange with solvent water, reduces the MR signal of the water. Agents that include a paramagnetic lanthanide ion in the structure shift the MR frequencies of the exchangeable proton to unique values to facilitate selective detection and are known as paramagnetic CEST (PARACEST) agents (7, 8). Endogenous MR contrast may be continually monitored in the presence of PARACEST agents by neglecting to saturate the MR frequency of the exchangeable proton (assuming that the T1 relaxation of the PARACEST agent is negligible).

The chemical exchange of a hydrogen atom from the CEST agent to a water molecule is base-catalyzed; therefore, CEST from these chemical functional groups is dependent on the pH of the environment. The possibility of measuring pH with a CEST MRI contrast agent was recognized along with the initial reports of diamagnetic CEST (DIACEST) agents (9). This methodology was subsequently extended to PARACEST agents that contained amide groups (8). The ratio of a pH-dependent CEST effect to a pH-independent CEST effect is used to measure the pH and does not depend on the concentration of the agent (10). Ytterbium chelated to 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid,10-orthoaminoanilide (Yb-DO3A-oAA) is an extension of the pioneering work with CEST MRI. This compound can be used to measure the pH-dependent CEST effects from an amide and an amine of the agent, and the ratio of the CEST effects can then be used to measure the pH in a concentration-independent manner (11).

The selective saturation of CEST MRI studies is typically applied for 1 to 5 seconds to generate a steady state of saturation (12), which greatly lengthens the time required for in vivo detection of PARACEST MRI contrast agents. Fast imaging methods, such as Rapid Acquisition with Relaxation Enhancement (RARE) and Fast Low Angle SHot (FLASH), reduce the total acquisition time and can be used to offset the long time needed for CEST MRI. The CEST-RARE MRI protocol can acquire an image as quickly as 5.5 seconds, and the CEST-FLASH MRI protocol can acquire an image within 13 seconds, depending on the endogenous T1 relaxation time of the tissue (13). In addition, a second "control" MR image is typically acquired to account for the direct saturation of water by applying selective saturation at a MR frequency with an opposite sign relative to the saturation frequency of the first MR image, whch doubles the total acquisition time to 11 seconds and 26 seconds for CEST-RARE and CEST-FLASH, respectively. This method does not account for B0 inhomogeneity or T2 relaxation effects. Alternatively, MR CEST spectroscopic imaging can be performed by acquiring a series of MR images while iterating the MR frequency of the selective saturation in order to create a CEST spectrum (also known as a Z-spectrum) for each pixel in the image. This method accounts for B0 inhomogeneity and T2 relaxation effects and can more accurately evaluate magnetization transfer effects (14). Typical in vivo CEST spectra require 27 to 61 saturation frequencies for acceptable spectral resolution. The need for multiple CEST MR images to create a CEST spectrum further lengthens the the time required for in vivo detection of CEST agents. However, extremely fast MR acquisition methods, such as Fast Imaging with Steady State Precession (FISP), can be used to acquire a single MR image within 1.3-5.3 seconds (comprising of 1 to 5 seconds for saturation and 0.3 seconds to acquire the image). This fast rate can acquire a series of 27 to 61 CEST MR images within 0.6–5.4 minutes (15). The CEST-FISP MRI protocol was used to detect the accumulation of the PARACEST agent Yb-DO3A-oAA within the tumor tissue of mice bearing human mammary carcinoma MDA-MB-231 cell tumors (16).

Synthesis

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[PubMed]

Yb-DO3A-oAA was synthesized using previously published methods (17). Briefly, DO3A-oAA was synthesized by the acylation of o-nitroaniline, and the product was coupled to 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) followed by reduction (hydrogenation) of the nitrobenzene group in the structure to obtain DO3A-oAA (yield, 90%). Yb-DO3A-oAA was obtained by incubating an aqueous mixture (pH 6.0) of DO3A-oAA and ytterbium chloride (ratio of 1:1.01) for 2 h at 60°C. The pH was then increased to 8.0 by the addition of 1 M sodium hydroxide, and the incubation was continued for another 0.5 h. Excess Yb(III) was precipitated from the solution by adjusting the pH to 12.0, and the precipitate was removed with centrifugation and filtration. The final solution was freeze dried, and the concentration of Yb-DO3A-oAA was determined with inductively coupled plasma mass spectrometry analysis. The final product was determined to contain Na+ and Cl– ions, but their presence did not influence the results obtained from subsequent studies.

In Vitro Studies: Testing in Cells and Tissues

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[PubMed]

No publication is currently available.

Animal Studies

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Rodents

[PubMed]

Prior to in vivo imaging, a ratio of the two CEST effects from Yb-DO3A-oAA was correlated with pH using phantoms with pH values that ranged between 6.35 and 7.57. This correlation showed an excellent measurement precision of 0.21 pH units using CEST MRI with Yb-DO3A-oAA (11). The pH measurements with CEST MRI using Yb-DO3A-oAA were compared with pH measurements with MR spectroscopy using 2-imidazole-1-yl-3-ethoxycarbonylpropionic acid at a concentration of 40 mM which showed an outstanding accuracy of 0.09 pH units and a R2 correlation coefficient of 0.99. This CEST-pH calibration was used for subsequent in vivo studies.

A SCID mouse model with a subcutaneous flank tumor of MDA-MB-231 cells was successfully used in a preliminary CEST MRI study (16). The in vivo CEST-FISP MRI study applied selective saturation with 300 Hz bandwidth and 20 mT power for 4.714 s, with an interpulse delay of 1 ms and a gradient spoiling applied at the end of the saturation period. FISP acquisition parameters included TR = 2.33 ms, TE = 1.16 ms, excitation flip angle = 60°, number of averages = 1, matrix = 64 × 64, field of view = 4 × 4 cm, in-plane spatial resolution = 625 × 625 mm, and slice thickness = 3 mm. These parameters were optimized with chemical solutions of the agent prior to the in vivo study. A single axial slice was acquired to visualize the tumor or leg. The temporal resolution of acquiring one image with one selective saturation frequency was 5.10 s. A series of 61 images was acquired, with selective saturation applied from +30 to −30 ppm in increments of 1 ppm, which required 5.2 min. After acquiring one image series, a 50-μL solution of 100 mM Yb-DO3A-oAA in saline was directly injected into the subcutaneous tumor in one mouse and into the leg muscle in another mouse within 30 s at a tissue depth of 2–3 mm using a 28 g syringe, and then six series of 61 images (for a total of 366 images) were acquired to create six CEST spectra, which required a total acquisition time of 31.2 min.

To obtain a CEST spectrum from a series of CEST-FISP MR images, a region of interest (ROI) was manually selected for the in vivo tumor or leg muscle tissue. Pixel-wise pH maps within the tissue ROI were also calculated. The CEST spectra were fit using a model function with a Lorentzian line shape and a super-Lorentzian line shape using Matlab R2009B (Mathworks, Natick, MA) (11). Some CEST spectra contained "salt and pepper" noise artifacts (16). These CEST spectra were median-filtered by substituting the value of each data point with the median value of a 61-point range around the data point (also known as a three-point median filter). The filtering affected the center point of the direct saturation of water in the CEST spectrum, but this original point was restored in the spectrum after filtering.

The bright image contrast generated after an intravenous (IV) injection and without selective saturation of the agent suggested pooling of the injection volume within the tumor tissue. The CEST spectrum of the ROI from this bright region showed strong CEST effects from the amide and amine that exceeded the 99% probability threshold, indicating that the change in image contrast was real (13). Both CEST effects were consistent throughout the 30-min scan session after injection of the agent, and CEST effects were not observed in the bladder or tissues surrounding the tumor, which supports tumor retention of the agent. The average CEST effects from the six CEST-FISP image series were 11.1 ± 2.0% and 18.9 ± 1.4% for the amide and amine, respectively. The CEST-pH correlation was used to translate the CEST measurements into a pHe measurement for the tumor ROI. The average pH of these six measurements was 6.82 ± 0.21, suggesting that the subcutaneous tumor had a lower pHe relative to normal tissues. The standard deviation of the pH measurements indicated that the precision of in vivo pHe measurements was comparable to the precision of pH measurements obtained with the solution-state phantoms. A similar CEST-to-pH translation was performed to generate a pixel-wise pH map of the tumor ROI at 23 min after injection, which showed an average pHe of 6.8 ± 0.4.

Yb-DO3A-oAA was also injected directly into a mouse thigh muscle. Similar to the study of the tumor tissue, the CEST-FISP MR image with no saturation of the agent showed a bright region after injection. The CEST effects within this bright region exceeded the 99% probability threshold and were attributed to the Yb-DO3A-oAA contrast agent. The average CEST effects from the four image series of muscle translated to an average pHe of 7.26 ± 0.14 within the muscle. Both CEST effects showed a small decrease during the first 18.2 min, suggesting some washout of the agent from the tissue, although this washout had little or no influence on the pH measurement as exemplified by the small standard deviation of the four measurements. The pixel-wise pHe map of the muscle ROI at 23 min after injection showed an average pHe of 7.2 ± 0.4. These results validated the fact that the pH of the tumor tissue was lower than the pH of the normal tissues.

In conclusion, the CEST effects of Yb-DO3A-oAA can measure pH throughout the physiological pH range of 6.35–7.57 at 20 mT saturation power and 300 MHz magnetic field strength. Salt and pepper artifacts can be removed from in vivo CEST spectra with median filtering. Although a DO3A-oAA metal chelate shows excellent retention in tumor tissue, which facilitates pH measurements from CEST spectra, typical IV injection concentrations used in this study were insufficient to generate CEST effects from Yb-DO3A-oAA in tumor tissue. However, direct injection of Yb-DO3A-oAA into tumor and muscle tissues circumvented the problem of poor CEST detection sensitivity. The pHe measurements showed excellent precision during the in vivo MRI scan session, and extracellular pH was lower in the tumor relative to muscle.

Other Non-Primate Mammals

[PubMed]

No publication is currently available.

Non-Human Primates

[PubMed]

No publication is currently available.

Human Studies

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[PubMed]

No publication is currently available.

Supplemental Information

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[Disclaimers]

No information is currently available.

NIH Support

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Studies presented in chapter were supported by National Institutes of Health grants R24CA110943 and R21CA133455.

References

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