PROJECT SUMMARY:
Abnormal metabolism is associated with a number of metabolic diseases such as cancer, diabetes, and
cardiovascular diseases. Important markers for the altered metabolic states include changes in fuel
preferences and loss of acidity regulations in tissues. To date, routine assessments of energy
metabolism and acidity in viable tissues remain a challenge. Sensitive imaging modalities that can
provide accurate characterization of these important biomarkers are therefore highly desirable. In this
application, we aim to establish a series of hyperpolarized (HP) 13C-enriched molecules that can be
activated by an endogenous enzyme esterase to produce both pH and metabolic imaging probes in
tissues. Successful development of these HP 13C-enriched compounds will allow for tissue acidity and
metabolic information to be simultaneously characterized following an injection of a single imaging
probe. Molecular candidates for these applications must be chemically stable, can be highly polarized
by dynamic nuclear polarization (DNP), have long 13C T1’s, and be highly sensitive to esterase
hydrolysis. Once hydrolyzed, the molecules must quickly decompose to form a pair of pH imaging
probes, HP 13CO2 and HP H13CO3-, as well as a HP 13C-enriched metabolic substrate such as 13C-
pyruvate. Here, we propose a series of ethyl alkyl mixed anhydride carbonate compounds with 13C-
enrichments at both the carbonate and carboxyl carbons. These carbon centers have long T1 making
them suitable for HP 13C imaging applications. Moreover, these small organic molecules are expected
to be efficiently polarized by DNP with significantly improved 13C MR imaging sensitivity. Last but not
least, the ester functional group in these mixed anhydrides is expected to be highly susceptible to
hydrolysis by esterase. Once injected into the circulation, we expect that these HP 13C-mixed anhydride
carbonates will be rapidly hydrolyzed by esterase producing monoacyl carbonate molecules. These
intermediates are expected to decompose, producing HP 13CO2 and a HP 13C-enriched metabolic
substrate for pH measurement and metabolism analysis in tissues, respectively. In Aim 1 of this study,
we will synthesize 13C-enriched ethyl acyl mixed anhydride carbonates with 13C-acetate, 13C-pyruvate,
or 13C-butyrate as the acyl moiety. HP 13C parameters such as T1, signal enhancement and chemical
stability of these compounds will also be evaluated. In Aim 2, we will investigate the production of HP
13CO2/H13CO3- and HP 13C metabolic substrate by esterase hydrolysis in solutions of isolated esterase,
isolated rat plasma, blood, and homogenates of rat liver and heart tissues. Finally, we will evaluate the
acute toxicity as well as the in vivo imaging efficacy of these HP 13C probes in mice and rats (Aim 3).
Successful execution of this study could lead to a series of HP 13C imaging probes that allow for
simultaneous imaging of tissue pH and energy metabolism in tissues associated with metabolic
diseases.