We report the imaging of β-cyclodextrin/benzoic acid binding at 14T using hyperpolarized 13C magnetic resonance (MR). Benzoic acid was polarized using a dynamic nuclear polarization (DNP) approach and combined with β-cyclodextrin in aqueous solution. As anticipated, decreases in the spin-lattice relaxation constant (T1) were observed with decreases in the ligand/ receptor ratio. The calculated log K was approximately 1.7, similar to previously reported binding constants. Hyperpolarized [1-13C] benzoic acid was used to interrogate solutions of variable β-cyclodextrin concentration, with the mixtures imaged at 14T using a 3D frequency-selective MR sequence. Differences in β-cyclodextrin concentration were easily visualized. These results suggest that hyperpolarized 13C MR could be used in vivo to determine the presence, and density of receptors for a given ligand-receptor pair

Keshari, Kayvan R.

Kurhanewicz, John

MacDonald, Jeffrey M.

Wilson, David M.

PubMed

Carolina Digital Repository

Generating Contrast in Hyperpolarized 13C MRI using Ligand-Receptor InteractionsKayvan R. Kesharia, John Kurhanewicza, Jeffrey M. Macdonaldb, and David M. WilsonaDavid M. Wilson: david.m.wilson@ucsf.eduaDepartment of Radiology, University of California San Francisco, San Francisco, United States.Tel: (415)353-1668bDepartment of Biomedical Imaging, University of North Carolina Chapel Hill (UNC-CH), ChapelHill, North Carolina, United StatesAbstractWe report the imaging of β-cyclodextrin/benzoic acid binding at 14T using hyperpolarized 13Cmagnetic resonance (MR). Benzoic acid was polarized using a dynamic nuclear polarization(DNP) approach and combined with β-cyclodextrin in aqueous solution. As anticipated, decreasesin the spin-lattice relaxation constant (T1) were observed with decreases in the ligand/ receptorratio. The calculated log K was approximately 1.7, similar to previously reported bindingconstants. Hyperpolarized [1-13C] benzoic acid was used to interrogate solutions of variable β-cyclodextrin concentration, with the mixtures imaged at 14T using a 3D frequency-selective MRsequence. Differences in β-cyclodextrin concentration were easily visualized. These resultssuggest that hyperpolarized 13C MR could be used in vivo to determine the presence, and densityof receptors for a given ligand-receptor pair.At the core of modern pharmacotherapy is molecular recognition between small moleculeligands and their protein targets (typically enzymes) in water. The hydrophobic effect iscritical in this process. The driving force for the hydrophobic phenomenon is primarilyentropic, where water must adopt an “organized” structure as it solvates non-polarmolecules, thereby reducing solvent entropy1. This effect has been studied extensively usingvarious complexes of β-cyclodextrin, a naturally occurring cyclic polysaccharide composedof β-D-glucose residues linked through α-1,4 glycosidic bonds2. These molecules have beenof interest as a result of their amphiphilic nature: while the hydroxyl groups confer highwater solubility, the cyclic arrangement of sugars creates a hydrophobic cavity. Thecyclodextrin ligand-receptor system has been used to mimic a variety of interactionsbetween proteins and small molecules3-5. Here, we report the imaging of this model systemusing hyperpolarized 13C MR, a technique that can be extended in vivo to a variety ofbiomolecules relevant to the treatment of human disease.Recent developments in the fields of low temperature physics, electron paramagneticresonance (EPR), and NMR have allowed the application of dynamic nuclear polarization(DNP) to traditional liquids NMR6,7. This process typically requires the sample of choice toexist as an amorphous solid at 1°K in the presence of a stable organic free radical. Thistechnique has been used to study in vivo metabolism by polarizing a metabolite, at close tophysiologic levels (in the mM range), and injecting it into a system to observe its© The Royal Society of Chemistry [year]†Electronic Supplementary Information (ESI) available: Spectroscopoic data for the described ligand-receptor binding experiments.This material is available free of charge via the internet at http://www.rsc.org. See DOI: 10.1039/b000000x/NIH Public AccessAuthor ManuscriptAnalyst. Author manuscript; available in PMC 2013 August 07.Published in final edited form as:Analyst. 2012 August 7; 137(15): 3427–3429. doi:10.1039/c2an35406c.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptconversion8-11 or distribution by MRI/MRSI7. Bench-top studies have also been conductedto investigate ways the hyperpolarized signal can be used indirectly. These have focused onobserving other nuclei, typically 1H via direct bond12, or secondarily labeling a compoundby way of chemical reaction13.It is still unclear how to interpret changes of hyperpolarized signal as a function of thenuclear environment. The dominant force associated with decay of the hyperpolarized signalis spin-lattice relaxation (or T1). The hyperpolarized signal of long T1 carbons lasts longerand this signal can be converted into an ordered spin state using an appropriate pulsesequence14. In most DNP studies, the T1 relaxation time is used as a parameter fordetermining what spins to label and observe. The goal of this study was to use T1 relaxationto understand the effect of hydrophobic binding on hyperpolarized signals, and use T1changes to image a model ligand-receptor system in aqueous solution.It is well known that in fast exchange systems, changes in parameters such as observedmagnetization, chemical shift, and spin relaxation rates (T1, T2, T1ρetc…) can be used toestimate equilibrium constants such as the binding constant in a ligand-receptor system15. Inthis case we used the well-characterized system of benzoic acid (ligand) and β-cyclodextrin(receptor)16,17. In the presence of β-cyclodextrin, the aromatic ring of benzoic acidpreferentially inserts itself into the inner-core of the cyclodextrin molecule, confirmed by x-ray crystallography (Figure 1a).18To explore this system, samples of natural abundance benzoic acid were dissolved indimethylacetamide (DMA) to a concentration of 3.6M. 2mg of the Finland radical19 per100mg of benzoic acid was used as the organic free radical. Prepared samples werepolarized for approximately 1 hour (at 94.094 Ghz) in a Hypersense® (Oxford Instruments,Oxford UK) and dissolved in 5mL of 100 mM phosphate buffer (pH=7.8). For bindingstudies, the hyperpolarized benzoic acid solution was mixed with 0.5mM β-cyclodextrin inthe same 100mM phosphate buffer. All NMR studies were conducted at 310K in a 10mmbroadband probe on a 500Mhz (125Mhz for 13C) Varian INOVA NMR spectrometer. Alldata were acquired using a 5° flip angle and 3s repetition time. Apparent T1 relaxation timeswere fit to a mono-exponential equation in Matlab (Mathworks) as previouslydescribed13,20,21.As seen in Figure 1b, all carbons of natural abundance benzoic acid were hyperpolarized,observed and resolved in one 5° degree pulse at a concentration of 36.4mM.Hyperpolarized benzoic acid was then mixed in increasing concentrations relative to 0.5 mMβ-cyclodextrin to assess the changes in T1. The hyperpolarized decay of the C1 and C2carbons of benzoic acid, as well as the effect of changing the ligand-receptor ratio are shownin Figure 2. Overall signal decreased more rapidly in time as a result of the interaction of thebenzoic acid carbons and β-cyclodextrin. For example, when benzoic acid β-cyclodextrinwere combined at concentrations of 36.4 and 0.5 mM respectively, the apparent T1decreased from 34.9 s and 19 s to 28.8 s and 16.4 s for the C1 and C2 carbons. Increasingconcentrations of benzoic acid relative to a constant β-cyclodextrin were used to generatethe plot depicted in Figure 2b, where ΔObs is defined as 1/T1obs-1/T1free. The y-intercept ofthe linear fit can be used to determine the log binding constant (log K) as a result of changesin the observed T1 for the C1 and C215. The log K derived from the fits of the C1 and C2changes were 1.74 and 1.68 respectively. These constants were within the range of reportedlog K measurements of benzoic acid (1.5-2.2) interacting with β-cyclodextrin16. Due tosignificantly lower T1, the hyperpolarized signal for the other benzoic acid carbons couldnot be used for reliable T1 calculations. This approach was further extended to naturalabundance 2-naphthaleneacetic acid, 6-methoxy-α-methyl-sodium salt (naproxen), a drugKeshari et al. Page 2Analyst. Author manuscript; available in PMC 2013 August 07.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptused to treat inflammation that is a known β-cyclodextrin guest. For these studies, 6.5mMhyperpolarized naproxen was mixed with 0.5 mM β-cyclodextrin. The T1 relaxation time ofthe free naproxen C1 was 14.8 ± 0.3 secs (n=3) and when in the presence of the host wasreduced ∼30% to 10.6 ± 0.4 secs (n=3) (Supplementary Figure 1). The other carbons werenot observed in the presence of the cyclodextrin host, in keeping with previous workshowing loss of hyperpolarized 13C signal with binding22.We hypothesized that benzoic acid- cyclodextrin binding could be used to create imagecontrast in a 13C MR experiment at 14T. This strategy would take advantage of the T1-dependent loss of hyperpolarized signal observed in the presence of the cyclodextrinreceptor, effectively creating negative contrast. A similar phenomenon would be expected invivo for a given ligand/ receptor pair. [1-13C] benzoic acid was polarized as describedpreviously for the natural abundance substrate. The enriched [1-13C] benzoic acid sampleswere polarized to 10%, corresponding to a signal enhancement of approximately 9000-foldat 14T. Imaging studies were performed on a 14T, 600WB micro-imaging spectrometerequipped with 100G/cm gradients (Varian Instruments). For hyperpolarized 13C imaging, a3D frequency-selective echo planar sequence was used to acquire a 3D image for [1-13C]benzoic acid with an acquisition time of approximately 180ms23. The final concentration of[1-13C] benzoic acid was 2.5 mM, combined with the β-cyclodextrin host at finalconcentrations of 0.5, 2.5, and 10 mM. These results are described in Figure 3, with loss ofhyperpolarized 13C signal observed with increasing concentrations of β-cyclodextrin. Figure3a shows both 1H and 13C imaging of the four cyclodextrin-containing solutions as well as a5M [1-13C] benzoic acid phantom (in 6mm glass tubes). 1H gradient echo imaging revealssimilar signal for the five samples, while the hyperpolarized MR image shows diminishedsignal corresponding to the concentration of the cyclodextrin host. The same 3D MRsequence was used to image the tubes at equilibrium, with only the 13C benzoic acidphantom visible. The 1H image was used to define regions of interest (ROI's) and themean 13C MR signal intensities for the cyclodextrin solutions were used to construct thegraph displayed in Figure 3b, providing a gross correlate to the T1 experiments conductedpreviously.In a typical modern 1H MRI exam, tissue contrast is obtained by differences between theintrinsic T1 and T2 of various tissues. Our results suggest a new mechanism of MR contrast,whereby changes in T1 of an administered hyperpolarized 13C probe can be correlated withthe concentration of a cellular target. As the arsenal of hyperpolarized 13C probes expands,numerous ligand-receptor pairs relevant to the treatment of human disease may beinvestigated simultaneously24, and non-invasively. In the context of a biologically relevantligand/ receptor pair, we would expect to observe T1- mediated loss of hyperpolarized signalin the presence of receptor, that in many cases would be clinically significant. Forexample, 13C steroid probes could be used to determine the estrogen/ progesterone receptorstatus in patients with breast cancer, or predict response to anti-androgen therapy in prostatecancer. This technique will benefit from the tighter binding seen in biologic systems,allowing receptors at smaller concentrations to be interrogated.Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.Notes and references1. Kyte J. Biophys Chem. 2003; 100:193–203. [PubMed: 12646366]2. Szejtli J. Chem Rev. 1998; 98:1743–1753. [PubMed: 11848947]3. Breslow R, Zhang XJ, Huang Y. J Am Chem Soc. 1997; 119:4535–4536.Keshari et al. Page 3Analyst. Author manuscript; available in PMC 2013 August 07.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript4. Breslow R, Hammond M, Lauer M. J Am Chem Soc. 1980; 102:421–422.5. Tabushi I, Kuroda Y, Yamada M, Higashimura H, Breslow R. J Am Chem Soc. 1985; 107:5545–5546.6. Ardenkjaer-Larsen JH, Fridlund B, Gram A, Hansson G, Hansson L, Lerche MH, Servin R, ThaningM, Golman K. Proc Natl Acad Sci USA. 2003; 100:10158–10163. [PubMed: 12930897]7. Golman K, Ardenkjaer-Larsen JH, Petersson JS, Mansson S, Leunbach I. Proc Natl Acad Sci USA.2003; 100:10435–10439. [PubMed: 12930896]8. Keshari KR, Kurhanewicz J, Bok R, Larson PE, Vigneron DB, Wilson DM. Proc Natl Acad Sci U SA. 2011; 108:18606–18611. [PubMed: 22042839]9. Albers MJ, Bok R, Chen AP, Cunningham CH, Zierhut ML, Zhang VY, Kohler SJ, Tropp J, HurdRE, Yen YF, Nelson SJ, Vigneron DB, Kurhanewicz J. Cancer Res. 2008; 68:8607–8615.[PubMed: 18922937]10. Chen AP, Kurhanewicz J, Bok R, Xu D, Joun D, Zhang V, Nelson SJ, Hurd RE, Vigneron DB.Magnetic resonance imaging. 2008; 26:721–726. [PubMed: 18479878]11. Gallagher FA, Kettunen MI, Day SE, Hu DE, Ardenkjaer-Larsen JH, in't Zandt R, Jensen PR,Karlsson M, Golman K, Lerche MH, Brindle KM. Nature. 2008; 453:940–U973. [PubMed:18509335]12. Merritt ME, Harrison C, Mander W, Malloy CR, Sherry AD. J Magn Reson. 2007; 189:280–285.[PubMed: 17945520]13. Wilson DM, Hurd RE, Keshari KR, Van Criekinge M, Chen AP, Nelson SJ, Vigneron DB,Kurhanewicz J. Proc Natl Acad Sci USA. 200914. Warren WS, Jenista E, Branca RT, Chen X. Science. 2009; 323:1711–1714. [PubMed: 19325112]15. Fielding L. Progress in Nuclear Magnetic Resonance Spectroscopy. 2007; 51:219–242.16. Rekharsky MV, Inoue Y. Chem Rev. 1998; 98:1875–1917. [PubMed: 11848952]17. Schneider HJ, Hacket F, Rudiger V, Ikeda H. Chem Rev. 1998; 98:1755–1785. [PubMed:11848948]18. Aree T, Chaichit N. Carbohydr Res. 2003; 338:439–446. [PubMed: 12559746]19. Cage B, McNeely JH, Russek SE, Halpern HJ. J Appl Phys. 2009; 10520. Deichmann R, Hahn D, Haase A. Magnetic Resonance in Medicine. 1999; 42:206–209. [PubMed:10398969]21. Keshari KR, Wilson DM, Chen AP, Bok R, Larson PE, Hu S, Van Criekinge M, Macdonald JM,Vigneron DB, Kurhanewicz J. J Am Chem Soc. 2009; 131:17591–17596. [PubMed: 19860409]22. Lerche MH, Meier S, Jensen PR, Baumann H, Petersen BO, Karlsson M, Duus JO, Ardenkjaer-Larsen JH. J Magn Reson. 2010; 203:52–56. [PubMed: 20022775]23. Lippert AR, Keshari KR, Kurhanewicz J, Chang CJ. J Am Chem Soc. 2011; 133:3776–3779.[PubMed: 21366297]24. Wilson DM, Keshari KR, Larson PE, Chen AP, Hu S, Van Criekinge M, Bok R, Nelson SJ,Macdonald JM, Vigneron DB, Kurhanewicz J. J Magn Reson. 2010; 205:141–147. [PubMed:20478721]Keshari et al. Page 4Analyst. Author manuscript; available in PMC 2013 August 07.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1.(a) Structure of the β-cyclodextrin- benzoic acid complex, as solved by X-raycrystallography in 2003 by Aree et al. (b) 13C NMR spectrum of natural abundancehyperpolarized benzoic acid, obtained in a single 5° pulse at 36.4 mM (310K, pH = 7.8). Allcarbons are observed: C1-176.6 ppm, C2-137.3 ppm, C3,C7- 129.8 ppm, C4,C6- 129.3 ppmand C5-132.2 ppm.Keshari et al. Page 5Analyst. Author manuscript; available in PMC 2013 August 07.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 2.(a) Hyperpolarized signal decay of the C1 carbon of benzoic acid without the β-cyclodextrinhost present (TR = 3 s, calculated T1 = 34.9 s at 11.7T). (b) Decay for the C2 carbon(calculated T1 = 19.0 s) (c) Plot of [β-cyclodextrin]/ΔObs as a function of increased benzoicacid [BA] concentration. The R2 of the C1 and C2 fits were 0.995 and 0.953 respectively.Keshari et al. Page 6Analyst. Author manuscript; available in PMC 2013 August 07.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 3.Hyperpolarized 13C MR imaging experiment conducted at 14T. (a) 1H imaging shows theorientation of tubes containing variable β-cyclodextrin concentration (0–10 mM finalconcentration). Also included is a [1-13C] benzoic acid phantom (5 M). Thehyperpolarized 13C MR imaging study was performed using a 3D frequency-selectivesequence after administration of hyperpolarized [1-13C] benzoic acid (final concentration2.5 mM), and demonstrates loss of signal corresponding with increasing [β-cyclodextrin].(b)Graph of hyperpolarized [1-13C] benzoic acid MR signals observed with variable [β-cyclodextrin], quantified for the imaging experiment.Keshari et al. Page 7Analyst. Author manuscript; available in PMC 2013 August 07.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

Generating contrast in hyperpolarized 13C MRI using ligand–receptor interactions

We report the imaging of β-cyclodextrin-benzoic acid binding at 14T using hyperpolarized (13)C magnetic resonance (MR). Benzoic acid was polarized using a dynamic nuclear polarization (DNP) approach and combined with β-cyclodextrin in aqueous solution. As anticipated, decreases in the spin-lattice relaxation constant (T(1)) were observed with decreases in the ligand-receptor ratio. The calculated log K was approximately 1.7, similar to previously reported binding constants. Hyperpolarized [1-(13)C] benzoic acid was used to interrogate solutions of variable β-cyclodextrin concentrations, with the mixtures imaged at 14T using a 3D frequency-selective MR sequence. Differences in β-cyclodextrin concentration were easily visualized. These results suggest that hyperpolarized (13)C MR could be used in vivo to determine the presence and density of receptors for a given ligand-receptor pair

Keshari, Kayvan R

Macdonald, Jeffrey M

Wilson, David M

eScholarship - University of California

UCSFUC San Francisco Previously Published WorksTitleGenerating contrast in hyperpolarized 13 C MRI using ligand –receptor interactionsPermalinkhttps://escholarship.org/uc/item/7g0729scJournalAnalyst, 137(15)ISSN0003-2654AuthorsKeshari, Kayvan RKurhanewicz, JohnMacdonald, Jeffrey Met al.Publication Date2012DOI10.1039/c2an35406c Peer reviewedeScholarship.org Powered by the California Digital LibraryUniversity of CaliforniaGenerating Contrast in Hyperpolarized 13C MRI using Ligand-Receptor InteractionsKayvan R. Kesharia, John Kurhanewicza, Jeffrey M. Macdonaldb, and David M. WilsonaDavid M. Wilson: david.m.wilson@ucsf.eduaDepartment of Radiology, University of California San Francisco, San Francisco, United States.Tel: (415)353-1668bDepartment of Biomedical Imaging, University of North Carolina Chapel Hill (UNC-CH), ChapelHill, North Carolina, United StatesAbstractWe report the imaging of β-cyclodextrin/benzoic acid binding at 14T using hyperpolarized 13Cmagnetic resonance (MR). Benzoic acid was polarized using a dynamic nuclear polarization(DNP) approach and combined with β-cyclodextrin in aqueous solution. As anticipated, decreasesin the spin-lattice relaxation constant (T1) were observed with decreases in the ligand/ receptorratio. The calculated log K was approximately 1.7, similar to previously reported bindingconstants. Hyperpolarized [1-13C] benzoic acid was used to interrogate solutions of variable β-cyclodextrin concentration, with the mixtures imaged at 14T using a 3D frequency-selective MRsequence. Differences in β-cyclodextrin concentration were easily visualized. These resultssuggest that hyperpolarized 13C MR could be used in vivo to determine the presence, and densityof receptors for a given ligand-receptor pair.At the core of modern pharmacotherapy is molecular recognition between small moleculeligands and their protein targets (typically enzymes) in water. The hydrophobic effect iscritical in this process. The driving force for the hydrophobic phenomenon is primarilyentropic, where water must adopt an “organized” structure as it solvates non-polarmolecules, thereby reducing solvent entropy1. This effect has been studied extensively usingvarious complexes of β-cyclodextrin, a naturally occurring cyclic polysaccharide composedof β-D-glucose residues linked through α-1,4 glycosidic bonds2. These molecules have beenof interest as a result of their amphiphilic nature: while the hydroxyl groups confer highwater solubility, the cyclic arrangement of sugars creates a hydrophobic cavity. Thecyclodextrin ligand-receptor system has been used to mimic a variety of interactionsbetween proteins and small molecules3-5. Here, we report the imaging of this model systemusing hyperpolarized 13C MR, a technique that can be extended in vivo to a variety ofbiomolecules relevant to the treatment of human disease.Recent developments in the fields of low temperature physics, electron paramagneticresonance (EPR), and NMR have allowed the application of dynamic nuclear polarization(DNP) to traditional liquids NMR6,7. This process typically requires the sample of choice toexist as an amorphous solid at 1°K in the presence of a stable organic free radical. Thistechnique has been used to study in vivo metabolism by polarizing a metabolite, at close tophysiologic levels (in the mM range), and injecting it into a system to observe its© The Royal Society of Chemistry [year]†Electronic Supplementary Information (ESI) available: Spectroscopoic data for the described ligand-receptor binding experiments.This material is available free of charge via the internet at http://www.rsc.org. See DOI: 10.1039/b000000x/NIH Public AccessAuthor ManuscriptAnalyst. Author manuscript; available in PMC 2013 August 07.Published in final edited form as:Analyst. 2012 August 7; 137(15): 3427–3429. doi:10.1039/c2an35406c.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptconversion8-11 or distribution by MRI/MRSI7. Bench-top studies have also been conductedto investigate ways the hyperpolarized signal can be used indirectly. These have focused onobserving other nuclei, typically 1H via direct bond12, or secondarily labeling a compoundby way of chemical reaction13.It is still unclear how to interpret changes of hyperpolarized signal as a function of thenuclear environment. The dominant force associated with decay of the hyperpolarized signalis spin-lattice relaxation (or T1). The hyperpolarized signal of long T1 carbons lasts longerand this signal can be converted into an ordered spin state using an appropriate pulsesequence14. In most DNP studies, the T1 relaxation time is used as a parameter fordetermining what spins to label and observe. The goal of this study was to use T1 relaxationto understand the effect of hydrophobic binding on hyperpolarized signals, and use T1changes to image a model ligand-receptor system in aqueous solution.It is well known that in fast exchange systems, changes in parameters such as observedmagnetization, chemical shift, and spin relaxation rates (T1, T2, T1ρetc…) can be used toestimate equilibrium constants such as the binding constant in a ligand-receptor system15. Inthis case we used the well-characterized system of benzoic acid (ligand) and β-cyclodextrin(receptor)16,17. In the presence of β-cyclodextrin, the aromatic ring of benzoic acidpreferentially inserts itself into the inner-core of the cyclodextrin molecule, confirmed by x-ray crystallography (Figure 1a).18To explore this system, samples of natural abundance benzoic acid were dissolved indimethylacetamide (DMA) to a concentration of 3.6M. 2mg of the Finland radical19 per100mg of benzoic acid was used as the organic free radical. Prepared samples werepolarized for approximately 1 hour (at 94.094 Ghz) in a Hypersense® (Oxford Instruments,Oxford UK) and dissolved in 5mL of 100 mM phosphate buffer (pH=7.8). For bindingstudies, the hyperpolarized benzoic acid solution was mixed with 0.5mM β-cyclodextrin inthe same 100mM phosphate buffer. All NMR studies were conducted at 310K in a 10mmbroadband probe on a 500Mhz (125Mhz for 13C) Varian INOVA NMR spectrometer. Alldata were acquired using a 5° flip angle and 3s repetition time. Apparent T1 relaxation timeswere fit to a mono-exponential equation in Matlab (Mathworks) as previouslydescribed13,20,21.As seen in Figure 1b, all carbons of natural abundance benzoic acid were hyperpolarized,observed and resolved in one 5° degree pulse at a concentration of 36.4mM.Hyperpolarized benzoic acid was then mixed in increasing concentrations relative to 0.5 mMβ-cyclodextrin to assess the changes in T1. The hyperpolarized decay of the C1 and C2carbons of benzoic acid, as well as the effect of changing the ligand-receptor ratio are shownin Figure 2. Overall signal decreased more rapidly in time as a result of the interaction of thebenzoic acid carbons and β-cyclodextrin. For example, when benzoic acid β-cyclodextrinwere combined at concentrations of 36.4 and 0.5 mM respectively, the apparent T1decreased from 34.9 s and 19 s to 28.8 s and 16.4 s for the C1 and C2 carbons. Increasingconcentrations of benzoic acid relative to a constant β-cyclodextrin were used to generatethe plot depicted in Figure 2b, where ΔObs is defined as 1/T1obs-1/T1free. The y-intercept ofthe linear fit can be used to determine the log binding constant (log K) as a result of changesin the observed T1 for the C1 and C215. The log K derived from the fits of the C1 and C2changes were 1.74 and 1.68 respectively. These constants were within the range of reportedlog K measurements of benzoic acid (1.5-2.2) interacting with β-cyclodextrin16. Due tosignificantly lower T1, the hyperpolarized signal for the other benzoic acid carbons couldnot be used for reliable T1 calculations. This approach was further extended to naturalabundance 2-naphthaleneacetic acid, 6-methoxy-α-methyl-sodium salt (naproxen), a drugKeshari et al. Page 2Analyst. Author manuscript; available in PMC 2013 August 07.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptused to treat inflammation that is a known β-cyclodextrin guest. For these studies, 6.5mMhyperpolarized naproxen was mixed with 0.5 mM β-cyclodextrin. The T1 relaxation time ofthe free naproxen C1 was 14.8 ± 0.3 secs (n=3) and when in the presence of the host wasreduced ∼30% to 10.6 ± 0.4 secs (n=3) (Supplementary Figure 1). The other carbons werenot observed in the presence of the cyclodextrin host, in keeping with previous workshowing loss of hyperpolarized 13C signal with binding22.We hypothesized that benzoic acid- cyclodextrin binding could be used to create imagecontrast in a 13C MR experiment at 14T. This strategy would take advantage of the T1-dependent loss of hyperpolarized signal observed in the presence of the cyclodextrinreceptor, effectively creating negative contrast. A similar phenomenon would be expected invivo for a given ligand/ receptor pair. [1-13C] benzoic acid was polarized as describedpreviously for the natural abundance substrate. The enriched [1-13C] benzoic acid sampleswere polarized to 10%, corresponding to a signal enhancement of approximately 9000-foldat 14T. Imaging studies were performed on a 14T, 600WB micro-imaging spectrometerequipped with 100G/cm gradients (Varian Instruments). For hyperpolarized 13C imaging, a3D frequency-selective echo planar sequence was used to acquire a 3D image for [1-13C]benzoic acid with an acquisition time of approximately 180ms23. The final concentration of[1-13C] benzoic acid was 2.5 mM, combined with the β-cyclodextrin host at finalconcentrations of 0.5, 2.5, and 10 mM. These results are described in Figure 3, with loss ofhyperpolarized 13C signal observed with increasing concentrations of β-cyclodextrin. Figure3a shows both 1H and 13C imaging of the four cyclodextrin-containing solutions as well as a5M [1-13C] benzoic acid phantom (in 6mm glass tubes). 1H gradient echo imaging revealssimilar signal for the five samples, while the hyperpolarized MR image shows diminishedsignal corresponding to the concentration of the cyclodextrin host. The same 3D MRsequence was used to image the tubes at equilibrium, with only the 13C benzoic acidphantom visible. The 1H image was used to define regions of interest (ROI's) and themean 13C MR signal intensities for the cyclodextrin solutions were used to construct thegraph displayed in Figure 3b, providing a gross correlate to the T1 experiments conductedpreviously.In a typical modern 1H MRI exam, tissue contrast is obtained by differences between theintrinsic T1 and T2 of various tissues. Our results suggest a new mechanism of MR contrast,whereby changes in T1 of an administered hyperpolarized 13C probe can be correlated withthe concentration of a cellular target. As the arsenal of hyperpolarized 13C probes expands,numerous ligand-receptor pairs relevant to the treatment of human disease may beinvestigated simultaneously24, and non-invasively. In the context of a biologically relevantligand/ receptor pair, we would expect to observe T1- mediated loss of hyperpolarized signalin the presence of receptor, that in many cases would be clinically significant. Forexample, 13C steroid probes could be used to determine the estrogen/ progesterone receptorstatus in patients with breast cancer, or predict response to anti-androgen therapy in prostatecancer. This technique will benefit from the tighter binding seen in biologic systems,allowing receptors at smaller concentrations to be interrogated.Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.Notes and references1. Kyte J. Biophys Chem. 2003; 100:193–203. [PubMed: 12646366]2. Szejtli J. Chem Rev. 1998; 98:1743–1753. [PubMed: 11848947]3. Breslow R, Zhang XJ, Huang Y. J Am Chem Soc. 1997; 119:4535–4536.Keshari et al. Page 3Analyst. Author manuscript; available in PMC 2013 August 07.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript4. Breslow R, Hammond M, Lauer M. J Am Chem Soc. 1980; 102:421–422.5. Tabushi I, Kuroda Y, Yamada M, Higashimura H, Breslow R. J Am Chem Soc. 1985; 107:5545–5546.6. Ardenkjaer-Larsen JH, Fridlund B, Gram A, Hansson G, Hansson L, Lerche MH, Servin R, ThaningM, Golman K. Proc Natl Acad Sci USA. 2003; 100:10158–10163. [PubMed: 12930897]7. Golman K, Ardenkjaer-Larsen JH, Petersson JS, Mansson S, Leunbach I. Proc Natl Acad Sci USA.2003; 100:10435–10439. [PubMed: 12930896]8. Keshari KR, Kurhanewicz J, Bok R, Larson PE, Vigneron DB, Wilson DM. Proc Natl Acad Sci U SA. 2011; 108:18606–18611. [PubMed: 22042839]9. Albers MJ, Bok R, Chen AP, Cunningham CH, Zierhut ML, Zhang VY, Kohler SJ, Tropp J, HurdRE, Yen YF, Nelson SJ, Vigneron DB, Kurhanewicz J. Cancer Res. 2008; 68:8607–8615.[PubMed: 18922937]10. Chen AP, Kurhanewicz J, Bok R, Xu D, Joun D, Zhang V, Nelson SJ, Hurd RE, Vigneron DB.Magnetic resonance imaging. 2008; 26:721–726. [PubMed: 18479878]11. Gallagher FA, Kettunen MI, Day SE, Hu DE, Ardenkjaer-Larsen JH, in't Zandt R, Jensen PR,Karlsson M, Golman K, Lerche MH, Brindle KM. Nature. 2008; 453:940–U973. [PubMed:18509335]12. Merritt ME, Harrison C, Mander W, Malloy CR, Sherry AD. J Magn Reson. 2007; 189:280–285.[PubMed: 17945520]13. Wilson DM, Hurd RE, Keshari KR, Van Criekinge M, Chen AP, Nelson SJ, Vigneron DB,Kurhanewicz J. Proc Natl Acad Sci USA. 200914. Warren WS, Jenista E, Branca RT, Chen X. Science. 2009; 323:1711–1714. [PubMed: 19325112]15. Fielding L. Progress in Nuclear Magnetic Resonance Spectroscopy. 2007; 51:219–242.16. Rekharsky MV, Inoue Y. Chem Rev. 1998; 98:1875–1917. [PubMed: 11848952]17. Schneider HJ, Hacket F, Rudiger V, Ikeda H. Chem Rev. 1998; 98:1755–1785. [PubMed:11848948]18. Aree T, Chaichit N. Carbohydr Res. 2003; 338:439–446. [PubMed: 12559746]19. Cage B, McNeely JH, Russek SE, Halpern HJ. J Appl Phys. 2009; 10520. Deichmann R, Hahn D, Haase A. Magnetic Resonance in Medicine. 1999; 42:206–209. [PubMed:10398969]21. Keshari KR, Wilson DM, Chen AP, Bok R, Larson PE, Hu S, Van Criekinge M, Macdonald JM,Vigneron DB, Kurhanewicz J. J Am Chem Soc. 2009; 131:17591–17596. [PubMed: 19860409]22. Lerche MH, Meier S, Jensen PR, Baumann H, Petersen BO, Karlsson M, Duus JO, Ardenkjaer-Larsen JH. J Magn Reson. 2010; 203:52–56. [PubMed: 20022775]23. Lippert AR, Keshari KR, Kurhanewicz J, Chang CJ. J Am Chem Soc. 2011; 133:3776–3779.[PubMed: 21366297]24. Wilson DM, Keshari KR, Larson PE, Chen AP, Hu S, Van Criekinge M, Bok R, Nelson SJ,Macdonald JM, Vigneron DB, Kurhanewicz J. J Magn Reson. 2010; 205:141–147. [PubMed:20478721]Keshari et al. Page 4Analyst. Author manuscript; available in PMC 2013 August 07.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 1.(a) Structure of the β-cyclodextrin- benzoic acid complex, as solved by X-raycrystallography in 2003 by Aree et al. (b) 13C NMR spectrum of natural abundancehyperpolarized benzoic acid, obtained in a single 5° pulse at 36.4 mM (310K, pH = 7.8). Allcarbons are observed: C1-176.6 ppm, C2-137.3 ppm, C3,C7- 129.8 ppm, C4,C6- 129.3 ppmand C5-132.2 ppm.Keshari et al. Page 5Analyst. Author manuscript; available in PMC 2013 August 07.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 2.(a) Hyperpolarized signal decay of the C1 carbon of benzoic acid without the β-cyclodextrinhost present (TR = 3 s, calculated T1 = 34.9 s at 11.7T). (b) Decay for the C2 carbon(calculated T1 = 19.0 s) (c) Plot of [β-cyclodextrin]/ΔObs as a function of increased benzoicacid [BA] concentration. The R2 of the C1 and C2 fits were 0.995 and 0.953 respectively.Keshari et al. Page 6Analyst. Author manuscript; available in PMC 2013 August 07.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptFigure 3.Hyperpolarized 13C MR imaging experiment conducted at 14T. (a) 1H imaging shows theorientation of tubes containing variable β-cyclodextrin concentration (0–10 mM finalconcentration). Also included is a [1-13C] benzoic acid phantom (5 M). Thehyperpolarized 13C MR imaging study was performed using a 3D frequency-selectivesequence after administration of hyperpolarized [1-13C] benzoic acid (final concentration2.5 mM), and demonstrates loss of signal corresponding with increasing [β-cyclodextrin].(b)Graph of hyperpolarized [1-13C] benzoic acid MR signals observed with variable [β-cyclodextrin], quantified for the imaging experiment.Keshari et al. Page 7Analyst. Author manuscript; available in PMC 2013 August 07.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript

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Generating contrast in hyperpolarized 13 C MRI using ligand –receptor interactions

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Generating contrast in hyperpolarized 13C MRI using ligand–receptor interactions

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