Alex X. Li

Four‐pool modeling of proton exchange processes in biological systems in the presence of MRI–paramagnetic chemical exchange saturation transfer (PARACEST) agents

Signal loss due to magnetization transfer (MT) from the macromolecular protons of biological tissues is an important consideration for the in vivo detection of paramagnetic chemical exchange saturation transfer (PARACEST) agents. In this study, a four‐pool model is presented that is based on the modified Bloch equations and incorporates terms for the proton exchange processes that occur in biological systems in the presence of MRI‐PARACEST contrast agents. The effect of the exchangeable proton chemical shift and PARACEST agent concentration are modeled in the presence of macromolecule‐derived MT. Experimental validation of the model was performed at 9.4 Tesla using Eu3+‐DOTAM‐glycine (Gly)‐phenylalanine (Phe) in both aqueous solution and samples containing 10% bovine serum albumin (BSA). The model was then used to measure the agent‐bound‐water chemical shift and the transverse relaxation time of macromolecular protons of a sample of Vero (nonhuman primate) cells labeled with Eu3+‐DOTAM‐Gly‐Phe and a phantom containing mouse brain tissue and 7 mM Eu3+‐DOTAM‐Gly‐Phe. In the brain tissue phantom, a chemical shift map with standard deviation (SD) < 0.7 ppm and a T2 map with SD < 0.6 μs were obtained. The results demonstrate the feasibility of in vivo temperature measurement based on the bound‐water chemical shift of Eu3+‐DOTAM‐Gly‐Phe in combination with this four‐pool model despite the inherent MT effect. Magn Reson Med 60:1197–1206, 2008.

A sensitive PARACEST contrast agent for temperature MRI: Eu3+-DOTAM-glycine (Gly)-phenylalanine (Phe)

Tissue temperature is a fundamental physiological parameter that can provide insight into pathological processes. The purpose of this study was to develop and characterize a novel paramagnetic chemical exchange saturation transfer (CEST) agent suitable for in vivo temperature mapping at 9.4T. The CEST properties of the europium (Eu3+) complex of the DOTAM‐Glycine (Gly)‐Phenylalanine (Phe) ligand were studied in vitro at 9.4T as a function of temperature, pH, and agent concentration. The transfer of magnetization (CEST effect) from the bound water to bulk water pools was ∼75% greater for Eu3+‐DOTAM‐Gly‐Phe compared to Eu3+‐DOTAM‐Gly at physiologic temperature (38°C) and pH (7.0 pH units) when using power level sufficiently low for in vivo imaging. Unlike Eu3+‐DOTAM‐Gly, whose CEST effect decreased with increasing temperature in the physiologic range, the CEST effect of Eu3+‐DOTAM‐Gly‐Phe was optimal at body temperature. A strong linear dependence of the chemical shift of the bound water pool on temperature was observed (0.3 ppm/°C), which was insensitive to pH and agent concentration. Temperature maps with SDs < 1°C were acquired at 9.4T in phantoms containing: 1) phantom A, an aqueous solution of 10 mM Eu3+‐DOTAM‐Gly‐Phe; 2) phantom B, 5% bovine serum albumin (BSA) with 15 mM Eu3+‐DOTAM‐Gly‐Phe; and 3) phantom C, mouse brain tissue with 4 mM Eu3+‐DOTAM‐Gly‐Phe. The temperature sensitivity combined with the high CEST effect observed at low concentration using low saturation power (B1) suggests this compound may be a good choice for in vivo temperature mapping at 9.4T. Magn Reson Med 59:374–381, 2008.

Quantitative Tissue Ph Measurement during Cerebral Ischemia Using Amine and Amide Concentration-Independent Detection (AACID) with MRI

Tissue pH is an indicator of altered cellular metabolism in diseases including stroke and cancer. Ischemic tissue often becomes acidic due to increased anaerobic respiration leading to irreversible cellular damage. Chemical exchange saturation transfer (CEST) effects can be used to generate pH-weighted magnetic resonance imaging (MRI) contrast, which has been used to delineate the ischemic penumbra after ischemic stroke. In the current study, a novel MRI ratiometric technique is presented to measure absolute pH using the ratio of CEST-mediated contrast from amine and amide protons: amine/amide concentration-independent detection (AACID). Effects of CEST were observed at 2.75 parts per million (p.p.m.) for amine protons and at 3.50 p.p.m. for amide protons downfield (i.e., higher frequency) from bulk water. Using numerical simulations and in vitro MRI experiments, we showed that pH measured using AACID was independent of tissue relaxation time constants, macromolecular magnetization transfer effects, protein concentration, and temperature within the physiologic range. After in vivo pH calibration using phosphorus (31P) magnetic resonance spectroscopy (31P-MRS), local acidosis is detected in mouse brain after focal permanent middle cerebral artery occlusion. In summary, our results suggest that AACID represents a noninvasive method to directly measure the spatial distribution of absolute pH in vivo using CEST MRI.

Elimination of the vesicular acetylcholine transporter in the striatum reveals regulation of behaviour by cholinergic-glutamatergic co-transmission.

A novel mouse model that eliminates cholinergic neurotransmission in the striatum while leaving glutamate release intact reveals differential effects on cocaine-induced behavior and dopaminergic responses.

Simultaneous in vivo pH and temperature mapping using a PARACEST‐MRI contrast agent

Altered tissue temperature and/or pH is a common feature in pathological conditions, where metabolic demand exceeds oxygen supply such as in tumors and following stroke. Therefore, in vivo tissue temperature and pH may become valuable biomarkers for disease detection and the monitoring of disease progression or treatment response in conditions with altered metabolic demand. In this study, pH is measured using the amide protons of a thulium (Tm3+) complex with a DOTAM‐Glycine‐Lysine (ligand: Tm3+‐DOTAM‐Gly‐Lys). The pH was uniquely determined from the linewidth of the asymmetry curve of the chemical exchange saturation transfer spectrum, independent of contrast agent concentration, or temperature for a given saturation pulse. pH maps with an inter‐pixel standard deviation of less than 0.1 pH units were obtained in 10 mM Tm3+‐DOTAM‐Gly‐Lys solutions with pH ranging from 6.0 to 8.0 pH units at 37°C. Temperature maps were simultaneously obtained using the chemical shift of the chemical exchange saturation transfer peak. Temperature and pH maps are demonstrated in the mouse leg (N = 3), where the mean and standard deviation for pH was 7.2 ± 0.2 pH unit and temperature was 37.4 ± 0.5°C. Magn Reson Med, 70:1016–1025, 2013.

A paramagnetic chemical exchange-based MRI probe metabolized by cathepsin D: design, synthesis and cellular uptake studies.

Overexpression of the aspartyl protease cathepsin D is associated with certain cancers and Alzheimers disease; thus, it is a potentially useful imaging biomarker for disease. A dual fluorescence/MRI probe for the potential detection of localized cathepsin D activity has been synthesized. The probe design includes both MRI and optical reporter groups connected to a cell penetrating peptide by a cathepsin D cleavable sequence. This design results in the selective intracellular deposition (determined fluorimetrically) of the MRI and optical reporter groups in the presence of overexpressed cathepsin D. The probe also provided clearly detectable in vitro MRI contrast by the mechanism of paramagnetic chemical exchange effects (OPARACHEE).

In vivo detection of MRI-PARACEST agents in mouse brain tumors at 9.4 T.

Paramagnetic chemical exchange saturation transfer (PARACEST) contrast agents are under development for biological target identification by magnetic resonance imaging. Image contrast associated with PARACEST agents can be generated by radiofrequency irradiation of the chemically shifted protons bound to a PARACEST contrast agent molecule or by direct irradiation of the on‐resonance bulk water protons. The observed signal change in a magnetic resonance image after the administration of a PARACEST contrast agent is due to both altered relaxation time constants and the CEST effect. Despite high sensitivity in vitro, PARACEST agents have had limited success in vivo where sensitivity is reduced by the magnetization transfer effect from endogenous macromolecules. The purpose of this study was to demonstrate the in vivo detection of a PARACEST contrast agent using the on‐resonance paramagnetic chemical exchange effect (OPARACHEE) in a mouse glioblastoma multiforme tumor model and to isolate the OPARACHEE effect from the changes in relaxation induced by the PARACEST agent. Three mice with tumors were imaged on a 9.4 T MRI scanner following tail vein injection of 150 μL 50 mM Tm3+‐DOTAM‐glycine‐lysine. A fast low angle shot pulse sequence with a low power radiofrequency pulse train (WALTZ‐16) as the preparation pulse was used to generate OPARACHEE contrast. To study the dynamics of agent uptake, reference images (without the preparation pulse) and OPARACHEE images were acquired continuously in an alternating fashion before, during and after agent injection. Signal intensity decreased by more than 10% in tumor in the control images after agent administration. Despite these changes, a clear OPARACHEE contrast of 1–5% was also observed in brain tumors after contrast agent injection and maintained in the hour following injection. This result is the first in vivo observation of OPARACHEE contrast in brain tumors with correction of T1 and T2 relaxation effects. Magn Reson Med, 2011.

Analogs of Eu3+ DOTAM-Gly-Phe-OH and Tm3+ DOTAM-Gly-Lys-OH: Synthesis and magnetic properties of potential PARACEST MRI contrast agents

Chelated lanthanide ions, especially gadolinium, have found wide use as contrast agents in magnetic resonance imaging. A new paradigm for generating contrast, termed PARACEST, was recently described that requires the slow exchange of water or other exchangeable protons present in the ligand framework. In previous work, we have described a synthetic method for the preparation of dipeptide conjugates of DOTAM for use as PARACEST agents. Two compounds possessed interesting magnetic properties: the Eu(3+) complex of DOTAM-Gly-Phe-OH and the Tm(3+) complex of DOTAM-Gly-Lys-OH. To understand the relationship between the structure of these complexes and their magnetic properties, we have expanded our synthetic methodology and prepared several new complexes. Ligands have been prepared in which the terminal phenylalanine moieties have been replaced with tryptophan or tyrosine, the distance to the amino acid residue possessing an alpha-substituent has been changed, or phenylalanine and lysine have been combined in the peptide sequence. The preparation of lanthanide(III) complexes of these ligands has been achieved and their PARACEST properties have been determined.

In vivo detection of PARACEST agents with relaxation correction

Several pulse sequences have been used to detect paramagnetic chemical exchange saturation transfer (PARACEST) contrast agents in animals to quantify the uptake over time following a bolus injection. The observed signal change is a combination of relaxation effects and PARACEST contrast. The purpose of the current study was to isolate the PARACEST effect from the changes in bulk water relaxation induced by the PARACEST agent in vivo for the fast low‐angle shot pulse sequence. A fast low‐angle shot–based pulse sequence was used to acquire continuous images on a 9.4‐T MRI of phantoms and the kidneys of mice following PARACEST agent (Tm3+‐DOTAM‐Gly‐Lys) injection. A WALTZ‐16 pulse was applied before every second image to generate on‐resonance paramagnetic chemical exchange effects. Signal intensity changes of up to 50% were observed in the mouse kidney in the control images (without a WALTZ‐16 preparation pulse) due to altered bulk water relaxation induced by the PARACEST agent. Despite these changes, a clear on‐resonance paramagnetic chemical exchange effect of 4‐7% was also observed. A four‐pool exchange model was used to describe image signal intensity. This study demonstrates that in vivo on‐resonance paramagnetic chemical exchange effect contrast can be isolated from tissue relaxation time constant changes induced by a PARACEST agent that dominate the signal change. Magn Reson Med 63:1184–1192, 2010.

Imaging chemical exchange saturation transfer (CEST) effects following tumor‐selective acidification using lonidamine

Increased lactate production through glycolysis in aerobic conditions is a hallmark of cancer. Some anticancer drugs have been designed to exploit elevated glycolysis in cancer cells. For example, lonidamine (LND) inhibits lactate transport, leading to intracellular acidification in cancer cells. Chemical exchange saturation transfer (CEST) is a novel MRI contrast mechanism that is dependent on intracellular pH. Amine and amide concentration‐independent detection (AACID) and apparent amide proton transfer (APT*) represent two recently developed CEST contrast parameters that are sensitive to pH. The goal of this study was to compare the sensitivity of AACID and APT* for the detection of tumor‐selective acidification after LND injection. Using a 9.4‐T MRI scanner, CEST data were acquired in mice approximately 14 days after the implantation of 105 U87 human glioblastoma multiforme (GBM) cells in the brain, before and after the administration of LND (dose, 50 or 100 mg/kg). Significant dose‐dependent LND‐induced changes in the measured CEST parameters were detected in brain regions spatially correlated with implanted tumors. Importantly, no changes were observed in T1‐ and T2‐weighted images acquired before and after LND treatment. The AACID and APT* contrast measured before and after LND injection exhibited similar pH sensitivity. Interestingly, LND‐induced contrast maps showed increased heterogeneity compared with pre‐injection CEST maps. These results demonstrate that CEST contrast changes after the administration of LND could help to localize brain cancer and monitor tumor response to chemotherapy within 1 h of treatment. The LND CEST experiment uses an anticancer drug to induce a metabolic change detectable by endogenous MRI contrast, and therefore represents a unique cancer detection paradigm which differs from other current molecular imaging techniques that require the injection of an imaging contrast agent or tracer. Copyright