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Hyperpolarization without a polarizer: in vivo 13C-MRI using SAMBADENA

Andreas B. Schmidt^1,2, Stephan Berner^1,3,4, Moritz Braig¹, Mirko Zimmermann¹, Jürgen Hennig¹, Dominik von Elverfeldt¹, and Jan-Bernd Hövener^2,4

¹Radiology - Medical Physics, University Medical Center Freiburg, Freiburg, Germany, ²Radiology and Neuroradiology, University Medical Center Schleswig-Holstein, Kiel, Germany, ³German Consortium for Cancer Research (DKTK), Heidelberg, Germany, ⁴German Cancer Research Center (DKFZ), Heidelberg, Germany

Synopsis

Hyperpolarization (HP) enhances the sensitivity of MR by several orders of magnitude and allows the detection of metabolism non-invasively and in vivo. However, well-established methods are costly, complex and require a dedicated “polarizer” next to the MRI system. SAMBADENA is, to date, the simplest and most cost-effective method to generate ¹³C-HP > 20 % for MRI. Within seconds HP is achieved within the MRI and little additional hardware is required. Here, the first in-vivo applications of SAMBADENA are reported, demonstrating its potential to be a fast, simple, low-cost alternative method for HP-MRI.

Introduction

The sensitivity of MRI to monitor metabolism non-invasively and in vivo can be dramatically enhanced by means of nuclear hyperpolarization (HP).^1–3 Dissolution dynamic nuclear polarization (dDNP¹) is well established and was used in human studies^2,3, but suffers from high cost, complexity and long HP cycles. Parahydrogen (pH₂)-based methods are less costly and faster but less advanced. Recently, a PASADENA (pH₂ and synthesis allow dramatically enhanced nuclear alignment⁴) experiment was realized on a commercial MRI system: by Synthesis Amid the Magnet Bore, A Dramatically Enhanced Nuclear Alignment (SAMBADENA)^5,6 with a ¹³C-polarization of P > 20% was achieved in vitro. Here, we present the first ¹³C-images acquired in vivo from SAMBADENA-hyperpolarized agents, putting the method to a real-life test with respect to the complex requirements of in-vivo imaging, namely anesthesia, animal monitoring, biocompatibility of the tracer solution (temperature, pressure) and well-controlled injection.

Methods

pH₂ was enriched to >90% as described elsewhere.⁶
Samples were prepared as before⁵ using aqueous ¹³C-, ²H₃-labeled hydroxyethyl-acrylate (HEA) at c_HEA=5.5mM, 22mM or 80mM and catalyst with concentration of c_cat=2mM or 4mM. pH₂, was catalytically added to HEA and ¹³C polarization was obtained by means of spin-order transfer (SOT) using the sequence l-PH-INEPT+.^8,9
Setup: For HP and MRI a 7T preclinical MRI system (Biospec 7/20,Bruker,Germany) was used in combination with a dual-resonant ¹H-¹³C-transmit-receive volume resonator (Rapid,Germany). A high-pressure and high-temperature reactor was custom-made to yield fast hydrogenation (inner volume ≈2ml, PSU 1000). The reactor was mounted to a custom-built mouse bed that allows animal heating, control and anesthesia (Fig. 1).
Hyperpolarization: The heated reactor (≈80°C) was filled with the hot precursor solution (≈80°C), closed and moved into the magnet. Hydrogenation was initialized by injecting pressurized pH₂ (p_pH2≈15-30bar) from the bottom of the reactor. After the variable hydrogenation time t_hthe SOT was executed. ¹³C-HP was quantified assuming 100% hydrogenation and pH₂-enrichment using a sample of thermally polarized n.a. acetone.
In-vivo SAMBADENA: The reactor was connected to the tail vein of a mouse (30g, 14 weeks old) that was placed next to it via a syringe using a catheter and a one-way check-valve (IDEX, USA). An HP-tracer solution was produced and ≈300µl were injected over a period of 10s. Another 5s later, the distribution of the tracer was imaged using a single-shot ¹³C-RARE sequence (90/180°, RARE-factor: 38, FOV: (8.4cm)², zerofilled to 128x96px, interpolated to 256x192px, one 6cm slice, TR=0.487s, TE=79ms, acquisition time:0.487s). ¹H-MRI was acquired for coregistration (¹H-Turbo-RARE,90/180°, RARE-factor:8, matrix:256², TR=2.5s, TE=33ms, acquisition time:161s). SNR was calculated as maximum intensity divided by standard deviation of the noise.

Results

HP at high tracer concentrations: HP was measured as function of c_HEA and an inverse correlation was observed (P=(22±2)%,(17±2)% and (7±1)% at c_HEA=5.5mM, 22mM and 80mM, respectively; pH₂ pressure of 15bar; N=2 each). The experiment was repeated with 80 mM solution at p_pH2=20bar, 25bar and 30bar and HP was quantified to (8±1)%, (8±1)% and 13%, respectively (Fig. 2). Note that the latter could not be repeated as some equipment was damaged by the elevated pressure.
In-vivo SAMBADENA: After the intravenous injection of HP HEP (300ml, c_cat=4mM, c_HEA=80mM, P≈7 %), strong ¹³C signal was imaged with an SNR of 168. The highest signal intensities originated from the vena cava, aorta and femoral arteries of the rodent (Fig. 3).

Discussion

For any biomedical application, highly concentrated, highly polarized samples are needed to achieve sufficient signal in vivo. While the received signal (~c_HEA*P) increased with the tracer concentration, the overall HP yield decreased suggesting incomplete hydrogenation. As expected and discussed before¹⁰, the HP yield was enhanced by supplying pH₂ at a higher pressure which increased the pH₂ available. While MRI definitely can be improved and it is expected that HP can be further enhanced along with dissolved pH₂ and by optimization of the reaction (temperature, c_cat) the HP levels were sufficient to demonstrate the in vivo applicability of SAMBADENA with a ¹³C-angiography of an adult mouse. The results show the potential of SAMBADENA to be a fast, simple and cost-efficient alternative method for the production and application of HP ¹³C-tracers. The HP of biomolecules like succinate^11,12, lactate^13–15, pyruvate or acetate¹⁶ is currently investigated.

Conclusion

In-vivo ¹³C-MRI of agents hyperpolarized by SAMBADENA without a dedicated polarizer was successfully demonstrated. With respect to recent advances in pH₂-based methods concerning metabolic tracers, SAMBADENA appears well placed to simplify the research on - and application of - HP metabolic tracers. The next crucial step will be to adapt SAMBADENA to these new methods and metabolically active molecules.

Acknowledgements

Support by the DFG (HO 4604/1-1 and HO 4604/2-1), the DKTK and the Heinrich-Böll-Stiftung (ABS, P131623) and the contribution of the COST Action TD1103 is greatly acknowledged. All animals were measured under the approval of the local ethics committee.

References

1. Ardenkjaer-Larsen, J. H. et al. Dynamic nuclear polarization polarizer for sterile use intent. NMR Biomed. 24, 927–932 (2011).

2. Nelson, S. J. et al. Metabolic Imaging of Patients with Prostate Cancer Using Hyperpolarized [1-13C]Pyruvate. Sci. Transl. Med. 5, 108 (2013).

3. Cunningham, C. H. et al. Hyperpolarized 13C Metabolic MRI of the Human Heart: Initial Experience. Circ. Res. CIRCRESAHA.116.309769 (2016). doi:10.1161/CIRCRESAHA.116.309769

4. Bowers, C. R. & Weitekamp, D. P. Parahydrogen and synthesis allow dramatically enhanced nuclear alignment. J. Am. Chem. Soc. 109, 5541–5542 (1987).

5. Schmidt, A. B. et al. Liquid-state carbon-13 hyperpolarization generated in an MRI system for fast imaging. Nat. Commun. (2017). doi:10.1038/ncomms14535

6. Berner, S. et al. Hyperpolarization of a biomolecule to 7% in an MRI using SAMBADENA. in EMIM 2017 (2017).

7. Hoevener, J.-B. et al. A continuous-flow, high-throughput, high-pressure parahydrogen converter for hyperpolarization in a clinical setting. NMR Biomed. 26, 124–131 (2013).

8. Haake, M., Natterer, J. & Bargon, J. Efficient NMR Pulse Sequences to Transfer the Parahydrogen-Induced Polarization to Hetero Nuclei. J Am Chem Soc 118, 88–91 (1996).

9. Bär, S. et al. On the spin order transfer from parahydrogen to another nucleus. J. Magn. Reson. 225, 25–35 (2012).

10. Gong, Q., Gordji-Nejad, A., Blümich, B. & Appelt, S. Trace Analysis by Low-Field NMR: Breaking the Sensitivity Limit. Anal. Chem. 82, 7078–7082 (2010).

11. Chekmenev, E. Y. et al. PASADENA Hyperpolarization of Succinic Acid for MRI and NMR Spectroscopy. J. Am. Chem. Soc. 130, 4212–4213 (2008).

12. Coffey, A. M. et al. Open-Source Automated Parahydrogen Hyperpolarizer for Molecular Imaging Using 13C Metabolic Contrast Agents. Anal. Chem. 88, 8279–8288 (2016).

13. Shchepin, R. V., Coffey, A. M., Waddell, K. W. & Chekmenev, E. Y. PASADENA Hyperpolarized 13C Phospholactate. J. Am. Chem. Soc. 134, 3957–3960 (2012).

14. Shchepin, R. V., Pham, W. & Chekmenev, E. Y. Dephosphorylation and biodistribution of 1-13C-phospholactate in vivo. J. Label. Compd. Radiopharm. 57, 517–524 (2014).

15. Cavallari, E., Carrera, C., Aime, S. & Reineri, F. 13C MR Hyperpolarization of Lactate by Using ParaHydrogen and Metabolic Transformation in Vitro. Chem. - Eur. J. 23, 1200–1204 (2017).

16. Reineri, F., Boi, T. & Aime, S. ParaHydrogen Induced Polarization of 13C carboxylate resonance in acetate and pyruvate. Nat. Commun. 6, 5858 (2015).

Figures

Figure 1: Hyperpolarization without a polarizer: The tracer is produced in situ within the MRI at the application site. The required components are a reactor, valves and tubing, pH₂, an animal bed, and a ¹³C-MRI system. The latter is used for the HP process and MRI.⁵

Figure 2: HP vs. pressure: HP was quantified with different concentrations of the precursor molecule HEA (squares: c_cat=2mM, c_HEA=5.5mM; diamonds: c_cat=4mM, c_HEA=22mM; asterisks: c_cat=4mM, c_HEA=80mM). When the concentration of the tracers is increased from 5.5mM to 80mM the HP drops from P=(22±2)% to P=(7±1)%, although the acquired signal (~c_HEP*P) is increasing. This effect suggests a lack of pH₂ and incomplete pH₂-hydrogenation. For the 80mM solution the HP could be increased again to P=13% by applying higher pH₂ pressures. Data points and error bars represent the mean value and st.dev. (N=2; at 30bar N=1 as equipment was damaged by high pressures).⁵

Figure 3: In vivo SAMBADENA: To demonstrate the in-vivo feasibility of SAMBADENA, an HP tracer solution was injected into the tail vein of an in-vivo mouse (300µl, concentration of tracer of c_HEP=80mM, P≈7%, in H₂O; 30g, 14 weeks old). About 5s later the distribution of the ¹³C-tracer was imaged using a single-slice, single-shot ¹³C-RARE sequence (a). Coregistration with a subsequently recorded ¹H-TurboRARE-3D MRI (b) revealed the tracer in the vascular system of the rodent, most prominently in the vena cava, aorta and femoral arteries ((c) and (d)). (d): www.biologycorner.com; CC BY-NC-SA 4.0.

Proc. Intl. Soc. Mag. Reson. Med. 26 (2018)

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