Di-Pyridine-Containing Macrocyclic Triamide Fe(II) and Ni(II) Complexes as ParaCEST Agents.

Fe(II) and Ni(II) paraCEST contrast agents containing the di-pyridine macrocyclic ligand 2,2',2″-(3,7,10-triaza-1,5(2,6)-dipyridinacycloundecaphane-3,7,10-triyl)triacetamide (DETA) are reported here. Both [Fe(DETA)]2+ and [Ni(DETA)]2+ complexes were structurally characterized. Crystallographic data revealed the seven-coordinated distorted pentagonal bipyramidal geometry of the [Fe(DETA)]·(BF4)2·MeCN complex with five coordinated nitrogen atoms from the macrocyclic ring and two coordinated oxygen atoms from two amide pendant arms. The [Ni(DETA)]·Cl2·2H2O complex was six-coordinated in nature with a distorted octahedral geometry. Four coordinated nitrogen atoms were from the macrocyclic ring, and two coordinated oxygen atoms were from two amide pendant arms. [Fe(DETA)]2+ exhibited well-resolved sharp proton resonances, whereas very broad proton resonances were observed in the case of [Ni(DETA)]2+ due to the long electronic relaxation times. The CEST peaks for the [Fe(DETA)]2+ complex showed one highly downfield-shifted and intense peak at 84 ppm with another shifted but less intense peak at 28 ppm with good CEST contrast efficiency at body temperature, whereas [Ni(DETA)]2+ showed only one highly shifted intense peak at 78 ppm from the bulk water protons. Potentiometric titrations were performed to determine the protonation constants of the ligand and the thermodynamic stability constant of the [M(DETA)]2+ (M = Fe, Co, Ni, Cu, Zn) species at 25.0 °C and I = 0.15 mol·L-1 NaClO4. Metal exchange studies confirmed the stability of the complexes in acidic medium in the presence of physiologically relevant anions and an equimolar concentration of Zn(II) ions.

Tumour acidosis evaluated in vivo by MRI-CEST pH imaging reveals breast cancer metastatic potential.

BACKGROUND: Tumour acidosis is considered to play a central role in promoting cancer invasion and migration, but few studies have investigated in vivo how tumour pH correlates with cancer invasion. This study aims to determine in vivo whether tumour acidity is associated with cancer metastatic potential. METHODS: Breast cancer cell lines with different metastatic potentials have been characterised for several markers of aggressiveness and invasiveness. Murine tumour models have been developed and assessed for lung metastases and tumour acidosis has been assessed in vivo by a magnetic resonance imaging-based chemical exchange saturation transfer (CEST) pH imaging approach. RESULTS: The higher metastatic potential of 4T1 and TS/A primary tumours, in comparison to the less aggressive TUBO and BALB-neuT ones, was confirmed by the highest expression of cancer cell stem markers (CD44+CD24-), highlighting their propensity to migrate and invade, coinciding with the measurement obtained by in vitro assays. MRI-CEST pH imaging successfully discriminated the more aggressive 4T1 and TS/A tumours that displayed a more acidic pH. Moreover, the observed higher tumour acidity was significantly correlated with an increased number of lung metastases. CONCLUSIONS: The findings of this study indicate that the extracellular acidification is associated with the metastatic potential.

Evaluation of a similarity anisotropic diffusion denoising approach for improving in vivo CEST-MRI tumor pH imaging.

PURPOSE: Chemical exchange saturation transfer MRI provides new approaches for investigating tumor microenvironment, including tumor acidosis that plays a key role in tumor progression and resistance to therapy. Following iopamidol injection, the detection of the contrast agent inside the tumor tissue allows measurements of tumor extracellular pH. However, accurate tumor pH quantifications are hampered by the low contrast efficiency of the CEST technique and by the low SNR of the acquired CEST images, hence in a reduced detectability of the injected agent. This work aims to investigate a novel denoising method for improving both tumor pH quantification and accuracy of CEST-MRI pH imaging. METHODS: An hybrid denoising approach was investigated for CEST-MRI pH imaging based on the combination of the nonlocal mean filter and the anisotropic diffusion tensor method. The denoising approach was tested in simulated and in vitro data and compared with previously reported methods for CEST imaging and with established denoising approaches. Finally, it was validated with in vivo data to improve the accuracy of tumor pH maps. RESULTS: The proposed method outperforms current denoising methods in CEST contrast quantification and detection of the administered contrast agent at several increasing noise levels with simulated data. In addition, it achieved a better pH quantification in in vitro data and demonstrated a marked improvement in contrast detection and a substantial improvement in tumor pH accuracy in in vivo data. CONCLUSION: The proposed approach effectively reduces the noise in CEST images and increases the sensitivity detection in CEST-MRI pH imaging.

Investigating plasma volume expanders as novel macromolecular MRI-CEST contrast agents for tumor contrast-enhanced imaging.

PURPOSE: The aim of this study was to investigate two clinically approved plasma volume expanders (dextran 70 and voluven) as macromolecular MRI-chemical exchange saturation transfer (CEST) contrast agents to assess tumor vascular properties. METHODS: CEST contrast efficiency of both molecules (6% w/v) was measured in vitro at various irradiation saturation powers (1-6 µT for 5 s) and pH values (range, 5.5-7.9) and the exchange rate of hydroxyl protons was calculated. In vivo studies in a murine adenocarcinoma model (n = 4 mice for each contrast agent) upon i.v. injection provided CEST-derived perfusion tumor properties that were compared with those obtained with a gadolinium-based blood-pool agent (Gd-AAZTA-Madec). RESULTS: In vitro measurements showed a marked CEST contrast dependency to pH, with higher CEST contrast at lower pH values for both molecules. The measured prototropic exchange rates confirmed a base-catalyzed exchange rate that was faster for dextran 70 in comparison to voluven. Both molecules showed a similar CEST contrast increase (ΔST% > 3%) in the tumor tissue up to 30 min postinjection, with heterogeneous accumulation. In tumors receiving both CEST and T1 -weighted agents, a voxel-by-voxel analysis indicated moderate spatial correlation of perfusion properties between voluven/dextran 70 and Gd-AAZTA-Madec, suggesting different distribution patterns according to their molecular size. CONCLUSIONS: The obtained results showed that both voluven and dextran 70 can be exploited as MRI-CEST contrast agents for evaluating tumor enhancement properties. Their increased accumulation in tumors and prolonged contrast enhancement promote their use as blood-pool MRI-CEST agents to examine tumor vascularization.

A fast multislice sequence for 3D MRI-CEST pH imaging.

PURPOSE: Chemical exchange saturation transfer MRI can provide accurate pH images, but the slow scan time (due to long saturation periods and multiple offsets sampling) reduce both the volume coverage and spatial resolution capability, hence the possibility to interrogate the heterogeneity in tumors and organs. To overcome these limitations, we propose a fast multislice CEST-MRI sequence with high pH accuracy and spatial resolution. METHODS: The sequence first uses a long saturation pulse to induce the steady-state CEST contrast and a second short saturation pulse repeated after each image acquisition to compensate for signal losses based on an uneven irradiation scheme combined with a single-shot rapid acquisition with refocusing echoes readout. Sequence sensitivity and accuracy in measuring pH was optimized by simulation and assessed by in vitro studies in pH-varying phantoms. In vivo validation was performed in two applications by acquiring multislice pH images covering the whole tumors and kidneys after iopamidol injection. RESULTS: Simulated and in vivo data showed comparable contrast efficiency and pH responsiveness by reducing saturation time. The experimental data from a homogeneous, pH-varying, iopamidol-containing phantom show that the sequence produced a uniform CEST contrast across slices and accurate values across slices in less than 10 minutes. In vivo measurements allowed us to quantify the 3D pH gradients of tumors and kidneys, with pH ranges comparable with the literature. CONCLUSION: The proposed fast multislice CEST-MRI sequence allows volumetric acquisitions with good pH sensitivity, accuracy, and spatial resolution for several in vivo pH imaging applications.

Dual assessment of kidney perfusion and pH by exploiting a dynamic CEST-MRI approach in an acute kidney ischemia-reperfusion injury murine model.

Several factors can lead to acute kidney injury, but damage following ischemia and reperfusion injuries is the main risk factor and usually develops into chronic disease. MRI has often been proposed as a method with which to assess renal function. It does so by measuring the renal perfusion of an injected Gd-based contrast agent. The use of pH-responsive agents as part of the CEST (chemical exchange saturation transfer)-MRI technique has recently shown that pH homeostasis is also an important indicator of kidney functionality. However, there is still a need for methods that can provide more than one type of information following the injection of a single contrast agent for the characterization of renal function. Herein we propose, for the first time, dynamic CEST acquisition following iopamidol injection to quantify renal function by assessing both perfusion and pH homeostasis. The aim of this study is to assess renal functionality in a murine unilateral ischemia-reperfusion injury model at two time points (3 and 7 days) after acute kidney injury. The renal-perfusion estimates measured with iopamidol were compared with those obtained with a gadolinium-based agent, via a dynamic contrast enhanced (DCE)-MRI approach, to validate the proposed method. Compared with the contralateral kidneys, the clamped ones showed a significant decrease in renal perfusion, as measured using the DCE-MRI approach, which is consistent with reduced filtration capability. Dynamic CEST-MRI findings provided similar results, indicating that the clamped kidneys displayed significantly reduced renal filtration that persisted up to 7 days after the damage. In addition, CEST-MRI pH imaging showed that the clamped kidneys displayed significantly increased pH values, reflecting the disturbance to pH homeostasis. Our results demonstrate that a single CEST-MRI contrast agent can provide multiple types of information related to renal function and can discern healthy kidneys from pathological ones by combining perfusion measurements with renal pH mapping.

Noninvasive evaluation of renal pH homeostasis after ischemia reperfusion injury by CEST-MRI.

Acute kidney injury (AKI) in mice caused by sustained ischemia followed by reperfusion is associated with acute tubular necrosis and renal dysfunctional blood flow. Although the principal role of the kidney is the maintenance of acid-base balance, current imaging approaches are unable to assess this important parameter, and clinical biomarkers are not robust enough in evaluating the severity of kidney damage. Therefore, novel noninvasive imaging approaches are needed to assess the acid-base homeostasis in vivo. This study investigates the usefulness of MRI-chemical exchange saturation transfer (CEST) pH imaging (through iopamidol injection) in characterizing moderate and severe AKI in mice following unilateral ischemia reperfusion injury. Moderate (20 min) and severe (40 min) ischemia were induced in Balb/C mice, which were imaged at several time points thereafter (Days 0, 1, 2, 7). A significant increase of renal pH values was observed as early as one day after the ischemia reperfusion damage for both moderate and severe ischemia. MRI-CEST pH imaging distinguished the evolution of moderate from severe AKI. A recovery of normal renal pH values was observed for moderate AKI, whereas a persisting renal pH increase was observed for severe AKI on Day 7. Renal filtration fraction was significantly lower for clamped kidneys (0.54-0.57) in comparison to contralateral kidneys (0.84-0.86) following impairment of glomerular filtration. The severe AKI group showed a reduced filtration fraction even after 7 days (0.38 for the clamped kidneys). Notably, renal pH values were significantly correlated with the histopathological score. In conclusion, MRI-CEST pH mapping is a valid tool for the noninvasive evaluation of both acid-base balance and renal filtration in patients with ischemia reperfusion injury.

L-DOS47 Elevates Pancreatic Cancer Tumor pH and Enhances Response to Immunotherapy.

Acidosis is an important immunosuppressive mechanism that leads to tumor growth. Therefore, we investigated the neutralization of tumor acidity to improve immunotherapy response. L-DOS47, a new targeted urease immunoconjugate designed to neutralize tumor acidity, has been well tolerated in phase I/IIa trials. L-DOS47 binds to CEACAM6, a cell-surface protein that is highly expressed in gastrointestinal cancers, allowing urease to cleave endogenous urea into two NH4+ and one CO2, thereby raising local pH. To test the synergetic effect of neutralizing tumor acidity with immunotherapy, we developed a pancreatic orthotopic murine tumor model (KPC961) expressing human CEACAM6. Using chemical exchange saturation transfer-magnetic resonance imaging (CEST-MRI) to measure the tumor extracellular pH (pHe), we confirmed that L-DOS47 raises the tumor pHe from 4 h to 96 h post injection in acidic tumors (average increase of 0.13 units). Additional studies showed that combining L-DOS47 with anti-PD1 significantly increases the efficacy of the anti-PD1 monotherapy, reducing tumor growth for up to 4 weeks.

Intraperitoneal Delivery of Iopamidol to Assess Extracellular pH of Orthotopic Pancreatic Tumor Model by CEST-MRI.

The extracellular pH (pHe) of solid tumors is often acidic, as a consequence of the Warburg effect, and an altered metabolic state is often associated with malignancy. It has been shown that acidosis can promote tumor progression; thus, many therapeutic strategies have been adopted against tumor metabolism; one of these involves alkalinization therapies to raise tumor pH to inhibit tumor progression, improve immune surveillance, and overcome resistance to chemotherapies. Chemical exchange saturation transfer-magnetic resonance imaging (CEST-MRI) is a noninvasive technique that can measure pH in vivo using pH-sensitive contrast agents. Iopamidol, an iodinated contrast agent, clinically used for computed tomography (CT), contains amide group protons with pH-dependent exchange rates that can reveal the pHe of the tumor microenvironment. In this study, we optimized intraperitoneal (IP) delivery of iopamidol to facilitate longitudinal assessments of orthotopic pancreatic tumor pHe by CEST-MRI. Following IV-infusion and IP-bolus injections, we compared the two protocols for assessing tumor pH. Time-resolved CT imaging was used to evaluate the uptake of iopamidol in the tumor, revealing that IP-bolus delivered a high amount of contrast agent 40 min postinjection, which was similar to the amounts reached with the IV-infusion protocol. As expected, both IP and IV injection protocols produced comparable measurements of tumor pHe, showing no statistically significant difference between groups (p=0.16). In addition, we showed the ability to conduct longitudinal monitoring of tumor pHe using CEST-MRI with the IP injection protocol, revealing a statistically significant increase in tumor pHe following bicarbonate administration (p < 0.001). In conclusion, this study shows the capability to measure pHe using an IP delivery of iopamidol into orthotopic pancreatic tumors, which is important to conduct longitudinal studies.

L-DOS47 enhances response to immunotherapy in pancreatic cancer tumor.

Acidosis is an important immunosuppressive mechanism that leads to tumor growth. Therefore, we investigated the neutralization of tumor acidity to improve immunotherapy response. L-DOS47, a new targeted urease immunoconjugate designed to neutralize tumor acidity, has been well tolerated in phase I/IIa trials. L-DOS47 binds CEACAM6, a cell surface protein highly expressed in gastrointestinal cancers, allowing urease to cleave endogenous urea into two NH4+ and one CO2, thereby raising local pH. To test the synergetic effect of neutralizing tumor acidity with immunotherapy, we developed a pancreatic orthotopic murine tumor model (KPC961) expressing human CEACAM6. Our results demonstrate that combining L DOS47 with anti-PD1 significantly increases the efficacy of anti-PD1 monotherapy, reducing tumor growth for up to 4 weeks.

Effect of Esomeprazole Treatment on Extracellular Tumor pH in a Preclinical Model of Prostate Cancer by MRI-CEST Tumor pH Imaging.

Novel anticancer treatments target the pH regulating system that plays a major role in tumor progression by creating an acidic microenvironment, although few studies have addressed their effect on tumor acidosis. In this study, we investigated in vivo several proton pump inhibitors (PPIs) targeting NHE-1 (Amiloride and Cariporide) and V-ATPase (Esomeprazole and Lansoprazole) proton transporters in the DU145 androgen-insensitive human prostate cancer model. In cellulo results showed that DU145 are sensitive, with decreasing efficacy, to Amiloride, Esomeprazole and Lansoprazole, with marked cell toxicity both in normoxia and in hypoxia, with almost any change in pH. In vivo studies were performed upon administration of Esomeprazole to assess both the acute and chronic effects, and Iopamidol-based tumor pH imaging was performed to evaluate tumor acidosis. Although statistically significant tumor pH changes were observed a few hours after Esomeprazole administration in both the acute study and up to one week of treatment in the chronic study, longer treatment resulted in a lack of changes in tumor acidosis, which was associated to similar tumor growth curves between treated and control groups in both the subcutaneous and orthotopic models. Overall, this study highlights MRI-CEST tumor pH imaging as a valid approach to monitoring treatment response to PPIs.

In Vivo MRI-CEST Tumor pH Imaging Detects Resistance to Proton Pump Inhibitors in Human Prostate Cancer Murine Models.

The tumor microenvironment acidification confers treatment resistance; therefore, the interference with pH regulating systems is considered a new therapeutic strategy. In this study, two human prostate cancer cell lines, PC3 and LNCaP, have been treated in vitro with proton pump inhibitors (PPIs), namely Lansoprazole, Esomeprazole (V-ATPases-inhibitors), Cariporide, and Amiloride (NHE1-inhibitors). The cell viability and pH were assessed at several drug concentrations either at normoxic or hypoxic conditions. Since Esomeprazole showed the highest toxicity towards the PC3 cancer cells compared to LNCaP ones, athymic nude mice bearing subcutaneous or orthotopic PC3 tumors were treated with Esomeprazole (dose: 2.5 mg/kg body weight) for a period of three weeks-and tumor growth was monitored. MRI-CEST tumor pH imaging with Iopamidol was performed upon treatment at 3 h, 1 week (in combination with FDG-PET), and after 2 weeks for evaluating acute, early, and late responses. Although acute tumor pH changes were observed in vivo, long-term studies on both PC3 prostate cancer models did not provide any significant change in tumor acidosis or tumor growth. In conclusion, this work shows that MRI-CEST tumor pH imaging is a valuable tool for assessing the in vivo treatment response to PPIs.

Renal pH Imaging Using Chemical Exchange Saturation Transfer (CEST) MRI: Basic Concept.

Magnetic Resonance Imaging (MRI) has been actively explored in the last several decades for assessing renal function by providing several physiological information, including glomerular filtration rate, renal plasma flow, tissue oxygenation and water diffusion. Within MRI, the developing field of chemical exchange saturation transfer (CEST) has potential to provide further functional information for diagnosing kidney diseases. Both endogenous produced molecules as well as exogenously administered CEST agents have been exploited for providing functional information related to kidney diseases in preclinical studies. In particular, CEST MRI has been exploited for assessing the acid-base homeostasis in the kidney and for monitoring pH changes in several disease models. This review summarizes several CEST MRI procedures for assessing kidney functionality and pH, for monitoring renal pH changes in different kidney injury models and for evaluating renal allograft rejection.This chapter is based upon work from the COST Action PARENCHIMA, a community-driven network funded by the European Cooperation in Science and Technology (COST) program of the European Union, which aims to improve the reproducibility and standardization of renal MRI biomarkers. This introduction chapter is complemented by two separate chapters describing the experimental procedure and data analysis.

Dynamic Contrast Enhanced (DCE) MRI-Derived Renal Perfusion and Filtration: Experimental Protocol.

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) can provide a noninvasive way for assessing renal functional information following the administration of a small molecular weight gadolinium-based contrast agent. This method may be useful for investigating renal perfusion and glomerular filtration rates of rodents in vivo under various experimental (patho)physiological conditions. Here we describe a step-by-step protocol for DCE-MRI studies in small animals providing practical notes on acquisition parameters, sequences, T1 mapping approaches and procedures.This chapters is based upon work from the COST Action PARENCHIMA, a community-driven network funded by the European Cooperation in Science and Technology (COST) program of the European Union, which aims to improve the reproducibility and standardization of renal MRI biomarkers. This experimental protocol chapter is complemented by two separate chapters describing the basic concept and data analysis.

Renal pH Mapping Using Chemical Exchange Saturation Transfer (CEST) MRI: Experimental Protocol.

Chemical exchange saturation transfer (CEST) is recognized as one of the premier methods for measuring pH with this environmental variable expected to be an excellent biomarker for kidney diseases. Here we describe step-by-step CEST MRI experimental protocols for producing pH and perfusion maps for monitoring kidney pH homeostasis in rodents after administering iopamidol as contrast agent. Several CEST techniques, acquisition protocols and ratiometric approaches are described. The impact of length of acquisition time on the quality of the maps is detailed. These methods may be useful for investigating progression in kidney disease in vivo for rodent models.This chapter is based upon work from the COST Action PARENCHIMA, a community-driven network funded by the European Cooperation in Science and Technology (COST) program of the European Union, which aims to improve the reproducibility and standardization of renal MRI biomarkers. This experimental protocol is complemented by two separate chapters describing the basic concepts and data analysis.

Analysis Protocol for Dynamic Contrast Enhanced (DCE) MRI of Renal Perfusion and Filtration.

Here we present an analysis protocol for dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) data of the kidneys. It covers comprehensive steps to facilitate signal to contrast agent concentration mapping via T1 mapping and the calculation of renal perfusion and filtration parametric maps using model-free approaches, model free analysis using deconvolution, the Toft's model and a Bayesian approach.This chapter is based upon work from the COST Action PARENCHIMA, a community-driven network funded by the European Cooperation in Science and Technology (COST) program of the European Union, which aims to improve the reproducibility and standardization of renal MRI biomarkers. This analysis protocol chapter is complemented by two separate chapters describing the basic concept and experimental procedure.

Dynamic Contrast Enhancement (DCE) MRI-Derived Renal Perfusion and Filtration: Basic Concepts.

Dynamic contrast-enhanced (DCE) MRI monitors the transit of contrast agents, typically gadolinium chelates, through the intrarenal regions, the renal cortex, the medulla, and the collecting system. In this way, DCE-MRI reveals the renal uptake and excretion of the contrast agent. An optimal DCE-MRI acquisition protocol involves finding a good compromise between whole-kidney coverage (i.e., 3D imaging), spatial and temporal resolution, and contrast resolution. By analyzing the enhancement of the renal tissues as a function of time, one can determine indirect measures of clinically important single-kidney parameters as the renal blood flow, glomerular filtration rate, and intrarenal blood volumes. Gadolinium-containing contrast agents may be nephrotoxic in patients suffering from severe renal dysfunction, but otherwise DCE-MRI is clearly useful for diagnosis of renal functions and for assessing treatment response and posttransplant rejection.Here we introduce the concept of renal DCE-MRI, describe the existing methods, and provide an overview of preclinical DCE-MRI applications to illustrate the utility of this technique to measure renal perfusion and glomerular filtration rate in animal models.This publication is based upon work from the COST Action PARENCHIMA, a community-driven network funded by the European Cooperation in Science and Technology (COST) program of the European Union, which aims to improve the reproducibility and standardization of renal MRI biomarkers. This introduction is complemented by two separate publications describing the experimental procedure and data analysis.

Non-invasive Investigation of Tumor Metabolism and Acidosis by MRI-CEST Imaging.

Altered metabolism is considered a core hallmark of cancer. By monitoring in vivo metabolites changes or characterizing the tumor microenvironment, non-invasive imaging approaches play a fundamental role in elucidating several aspects of tumor biology. Within the magnetic resonance imaging (MRI) modality, the chemical exchange saturation transfer (CEST) approach has emerged as a new technique that provides high spatial resolution and sensitivity for in vivo imaging of tumor metabolism and acidosis. This mini-review describes CEST-based methods to non-invasively investigate tumor metabolism and important metabolites involved, such as glucose and lactate, as well as measurement of tumor acidosis. Approaches that have been exploited to assess response to anticancer therapies will also be reported for each specific technique.

Orthotopic induction of CH157MN convexity and skull base meningiomas into nude mice using stereotactic surgery and MRI characterization.

Meningioma in vivo research is hampered by the difficulty of establishing an easy and reproducible orthotopic model able to mimic the characteristics of a human meningioma. Moreover, leptomeningeal dissemination and high mortality are often associated with such orthotopical models, making them useless for clinical translation studies. An optimized method for inducing meningiomas in nude mice at two different sites is described in this paper and the high reproducibility and low mortality of the models are demonstrated. Skull base meningiomas were induced in the auditory meatus and convexity meningiomas were induced on the brain surface of 23 and 24 nude mice, respectively. Both models led to the development of a mass easily observable by imaging methods. Dynamic contrast enhanced MRI was used as a tool to monitor and characterize the pathology onset and progression. At the end of the study, histology was performed to confirm the neoplastic origin of the diseased mass.

Flip-angle based ratiometric approach for pulsed CEST-MRI pH imaging.

Several molecules have been exploited for developing MRI pH sensors based on the chemical exchange saturation transfer (CEST) technique. A ratiometric approach, based on the saturation of two exchanging pools at the same saturation power, or by varying the saturation power levels on the same pool, is usually needed to rule out the concentration term from the pH measurement. However, all these methods have been demonstrated by using a continuous wave saturation scheme that limits its translation to clinical scanners. This study shows a new ratiometric CEST-MRI pH-mapping approach based on a pulsed CEST saturation scheme for a radiographic contrast agent (iodixanol) possessing a single chemical exchange site. This approach is based on the ratio of the CEST contrast effects at two different flip angles combinations (180°/360° and 180°/720°), keeping constant the mean irradiation RF power (Bavg power). The proposed ratiometric approach index is concentration independent and it showed good pH sensitivity and accuracy in the physiological range between 6.0 and 7.4.