INTRODUCTION: NMR hyperpolarization techniques transiently boost nuclear spin polarization (P) by several orders of magnitude. The enhanced P can be stored on relatively long-lived ¹³C carriers of hyperpolarization, allowing them to be used as metabolic contrast agents with 4-6 orders of magnitude gain in NMR signal. [1-¹³C]pyruvate is the leading hyperpolarized (HP) contrast agent. It is currently under investigation in 14 clinical trials for spectroscopic imaging of aberrant metabolism of cancer and other diseases. Such real-time metabolic studies became possible due to the pioneering work of Ardenkjaer-Larsen and co-workers who established dissolution Dynamic Nuclear Polarization (d-DNP) technology (ca. 2003). Despite major success in the research domain, the production of HP [1-¹³C]pyruvate via d-DNP is slow (tens of minutes or longer) and expensive ($2M+ for a clinical device), representing a substantial barrier for clinical translation of this otherwise revolutionary contrast agent and molecular imaging modality. A metabolic MR scan with HP [1-¹³C]pyruvate can take less than a minute and uses no ionizing radiation. This is in sharp contrast to clinical [¹⁸F]fluorodeoxyglucose Positron Emission Tomography scans, which require ~2 hours and expose subjects to 30-50 mSv radiation. More efficient and cost-effective approaches for production of HP [1-¹³C]pyruvate are needed to enable its widespread clinical use. Signal Amplification by Reversible Exchange (SABRE)¹ is a Parahydrogen-Induced Polarization (PHIP) technique variant that requires no hydrogenation—instead, SABRE relies on reversible exchange of parahydrogen (p-H₂) and the to-be-hyperpolarized substrate with a catalyst. Recently, after some success with indirect methods, Duckett and co-workers⁴ and Goodson and co-workers⁵ independently demonstrated that SABRE can be employed for direct hyperpolarization of ¹³C carboxylate moieties—including [1-¹³C]pyruvate (P_13C~1.85%).⁴ In this work, we describe recent advances in spin physics and instrumentation for clinical-scale production of highly polarized HP [1-¹³C]pyruvate and other metabolically relevant HP contrast agents.

METHODS: [1-¹³C]pyruvate forms a complex with the standard catalyst used for SABRE, [IrCl(COD)(IMes)] (IMes = 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene; COD = cyclooctadiene)], Figure 1). The efficient hyperpolarization transfer from p-H₂-derived hydrides to the ¹³C nuclear spins of [1-¹³C]pyruvate becomes possible by performing SABRE in sub-microtesla magnetic fields using SABRE in SHield Enables Alignment Transfer to Heteronuclei (SABRE-SHEATH).^2,3 The overall diagram of the hyperpolarizer is provided in Figure 2c. The device (61x47x47 cm size) operates with either a static magnetic field using an internal DC power supply or a pulsed variable field using a commercially available wave-form generator.

RESULTS AND DISCUSSION: Figure 2a shows a spectrum of a 6 mM Ir-[1-¹³C]pyruvate complex composed of both 3a and 3b with P_13C of 39% at the time of detection; a corresponding ¹³C reference spectrum from thermally polarized neat [1-¹³C]acetic acid is shown in Figure 2b. Because the HP sample depolarizes during the 3-5-second long sample transfer (due to sample exposure in the Earth’s field and the 1.4 T field of the NMR spectrometer), the P_13C value at the time of production (i.e., at the conclusion of the SABRE-SHEATH process) is estimated to be >50% on the catalyst-bound complexes 3a and 3b. Kinetically, the build-up to these high P_13C levels becomes possible because the ¹³C T₁ relaxation time of HP [1-¹³C]pyruvate in this complex at 0.30 microtesla is relatively long: 9.5 s (i.e., the spin relaxation rate is ~0.1 s^-1) even at the relatively large catalyst concentration of 6 mM. As a result, the effective ¹³C polarization build-up constant, T_b = 4.9 s, corresponds to a build-up rate of ~0.2 s^-1—substantially faster (Figure 3c) than the spin relaxation rate (Figure 3d), allowing one to achieve order-unity ¹³C polarization.
Optimization of the magnetic field and temperature of ¹³C SABRE-SHEATH for [1-¹³C]pyruvate (Figure 3) revealed the optimum polarization transfer field B_T of ~ 0.3 microtesla and the optimum polarization transfer temperature T_T of ~0 °C.Temperature has a profound effect on the exchange rates of [1-¹³C]pyruvate involving complex 3b, Figure 4a. Even though the ¹³C polarization was nearly fully locked in the bound 3b state at 0 °C (Figure 4b, blue trace), the ¹³C polarization bolus can be rapidly released if the HP solution’s container is rapidly warmed up after sample transfer from the shield, but before inserting the sample in the NMR detector. We also investigate the role of H₂O as a potential secondary co-ligand or substrate modifier in SABRE-SHEATH experiments. Indeed, systematic H₂O titrations revealed the dependence of P_13C not only on DMSO concentration, but also on water concentration. Figure 5 shows a clear P_13C modulation of [1-¹³C]pyruvate by varying the concentration of H₂O added to the sample. The presence of H₂O in an optimal concentration doubles P_13C (compared to no water added), reaching ~13% for the free substrate at the optimum temperature.

CONCLUSION: Although future systematic optimization studies are certainly warranted to further improve ¹³C polarization efficiency of HP [1-¹³C]pyruvate produced via SABRE-SHEATH, this report clearly demonstrates that order-unity ¹³C polarization is fundamentally attainable. Moreover, the P_13C value of ~13% for the free substrate is already useable for in vivo studies. Future in vivo feasibility studies will need to address remaining translational roadblocks by combining the present polarization techniques with ongoing efforts to achieve rapid SABRE catalyst removal and HP [1-¹³C]pyruvate reconstitution into biocompatible aqueous media of injectable solutions.