Background and Motivation

Hyperpolarized (HP) ¹³C pyruvate MR is under clinical investigation as a diagnostic tool for characterizing cancer aggressiveness and monitoring therapeutic response¹. HP ¹³C MR has the unique capability of simultaneous multi-probe imaging, a key advantage compared to other molecular imaging techniques such as positron emission tomography (PET)². Previous preclinical studies have shown that combining HP ¹³C pyruvate with a perfusion imaging agent – HP ¹³C urea – could provide a more sensitive evaluation of cardiac physiology³, tumor biology^4,5, and response to therapy^6,7 (Figure 1). Moreover, the addition of an extracellular perfusion agent could potentially improve kinetic modeling of pyruvate metabolism by providing an independent measurement of vascular input and tissue distribution.

We aim to bring this dual-agent HP MRI to clinical research studies. Here, we present the technical developments towards the clinical translation of combined HP ¹³C pyruvate and urea MR for simultaneous metabolic and perfusion imaging.

Methods and Results

1. Formulation: In preclinical studies, pyruvic acid (PA) and urea are sequentially frozen with minimal or no contact²; this approach is incompatible with the clinical polarizer and fluid path designs. Additionally, urea requires a solvent and glassing agent such as glycerol, which is undesirable for human studies. With these considerations, urea was dissolved in water first, then mixed with neat PA, resulting in a homogeneous mixture contains approximately 10M [1-¹³C]PA, 2.5M [¹³C,¹⁵N₂]urea, and 12.5mM GE trityl radical (Figure 2A). [¹³C,¹⁵N₂]urea was used because of its prolonged T1 at low-field compared to [¹³C]urea, preserving polarization during sample transfer.

2. Characterization: Reproducible glass formation is critical for dynamic nuclear polarization. Using differential scanning calorimetry (DSC), we found that PA/urea/water mixtures have similar glass transition temperatures (Tg) to neat PA (ranging from -70 to -80 °C), a desirable glass material, and did not observe crystallizations for a wide range of water content in the formulation (Figure 2B).

To test polarization performance, approximately 1.1ml combined ¹³C PA and urea probe was polarized on a 5T SPINlab polarizer using 139.97 GHz microwave for 3-4 hours, then rapidly dissolved in 41ml super-heated (130 °C), pressurized sterile water and subsequently neutralized using equivalent NaOH and Tris buffer. This procedure produced approximately 150mM sodium pyruvate, 35 mM urea, and < 3 µM EPA, with liquid-state polarization of 39.9 ± 5.9 % for pyruvate, 39.1 ± 7.3 % for urea (n = 20). A representative ¹³C NMR spectrum of HP dissolution acquired on a 1.4T bench-top spectrometer is shown in Figure 3A.

3. Safety-related studies for clinical translation: We investigated potential impurities in the PA-urea mixture by leveraging high-resolution NMR and HPLC-MS. Three impurity compounds were identified in hyperpolarized and mock dissolution studies (Figure 3B). The total molar % concentration of impurities is 2.13% in the final dissolution based on MS and 2.5% based on NMR.

We then investigated any potential toxicity of co-polarized pyruvate and urea and the impurities identified. Sprague Dawley rats were injected with normal saline (n = 3), co-polarized dissolution (n = 10), or isolated impurity products (n =10). We did not observe any adverse responses in vital signs (pulse, respiratory rate, oxygen saturation, temperature, body weight), laboratory tests (complete blood counts, liver-kidney panel), or gross pathology of major organs.

Finally, we developed a Standard Operating Procedure (SOP) to produce a sterile co-polarized ¹³C pyruvate and urea solutions for human injection, then validated the SOP by three consecutive successful Performance Qualification (PQ) trials. The SOP can produce sterile products with probe concentrations, pH, and polarizations comparable to the aforementioned characterizations (Figure 4).

4. Metabolite-specific imaging for co-polarized pyruvate and urea: The split peaks of [¹³C,¹⁵N₂]urea (J_CN = 20Hz, Figure 2A) causes off-resonance artifacts when long readout trajectories were used such as Echo Planar Imaging (EPI) or spiral gradient echo (GRE) imaging. In this study, we developed a 3D balanced steady-state free precession (bSSFP) urea-specific imaging sequence: urea frequency-selective RF excitation pulse (Figure 5A), which consists of urea frequency-selective RF excitation pulses interleaved with stack-of-spiral readouts⁸. This sequence was designed to improve the efficient use of polarization, minimize perturbation to other metabolites, and mitigate off-resonance artifacts (each readout duration = 4 ms), and implemented on commercially available software. Compared to single-shot spiral GRE images of urea, image blurring is considerably reduced in bSSFP images (Figure 5B).

To achieve simultaneous metabolic and perfusion imaging, pyruvate, lactate, and alanine were imaged using multi-slice spiral GRE sequences as previously reported⁸, interleaved with urea 3D bSSFP sequence. Adult rats (n = 3) were injected with 2.5ml co-polarized pyruvate and urea produced using forementioned SOPs, then imaged with volumetric converge and 4.2s temporal resolution. A panel of representative images and HP signal dynamics are shown in Figure 5C, D.

Summary

We developed a co-polarization system and a standard operating procedure (SOP) that can reproducibly generate sterile HP ¹³C pyruvate and urea solutions for human injection, as well as a novel imaging approach for simultaneous metabolic and perfusion imaging. Upon FDA approval, this work would lead to the first-in-human dual-agent HP MR study and the first non-pyruvate HP clinical study.

Acknowledgements

This work was supported by research grants from the National Institute of Health (NIH): R01CA214554 (JK), P41EB013598(DV, JK). HQ acknowledges graduate fellowship support from UCSF Graduate Division.