Quantitative MRI Pharmacokinetic Modeling and MALDI Mass Spectrometry Imaging Biodistribution of a pH Mapping MRI Contrast Agent.

Abstract

Chemical exchange saturation transfer (CEST) MRI enables noninvasive mapping of tissue pH, a key biomarker of renal physiology and disease. We report a streamlined, three-step synthesis of diGlu, an anionic imidazole-4,5-dicarboxamide bearing two glutamate substituents, obtained at ≥99.5% purity using SOCl₂/DMF activation, low-temperature amidation, and mild HCl deprotection-avoiding phenyl-ester intermediates and TFA. The molecule presents two exchangeable protons at 7.7 ppm (imidazole) and 4.5 ppm (amide) whose pH-dependent exchange rates enable a two-offset ratiometric, concentration-independent readout.

Methods: C57BL/6J mice received tail-vein injections of 250, 500, or 750 mg kg^-1 I45DC-diGlu for our imaging studies. We established a novel biodistribution and pharmacokinetic analysis methodology employing matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) for biodistribution, CEST imaging and region of interest (ROI)-wise pharmacokinetic (PK) modeling, fitting SE (%) time-courses with log-normal (LN) and Uptake-Plateau-Decay (UPD) models and selecting the model by AICc on the SE time course curves.

Results: MALDI-MSI confirmed selective renal localization and unmetabolized probe in urine, with negligible liver or pancreas accumulation. The LN model was best for fitting the CEST MRI data at low doses (unimodal kinetics), whereas UPD captured rise-hold-washout at 750 mg kg^-1, yielding interpretable biomarkers (SE_max, TTP, AUC_total, t_1/2). Robust renal CEST contrast was observed at ≥ 500 mg kg^-1. Voxel-wise pH maps reproduced the expected corticomedullary gradient (mean ± SD): cortex 6.86 ± 0.087, outer medulla 6.80 ± 0.073, inner medulla 6.61 ± 0.079, with pH estimates largely dose-independent.

Conclusions: Together, the iodine- and metal-free chemistry, dual-offset window, dose-robust pH mapping, and MALDI-validated biodistribution position I45DC-diGlu as a practical scaffold for quantitative renal pH imaging and as a foundation for translational studies for renal pathophysiology.