Simultaneous noninvasive quantification of redox and downstream glycolytic fluxes reveals compartmentalized brain metabolism

Abstract

Disclosed herein are compositions that include a first hyperpolarized probe, wherein the first hyperpolarized probe is a compound that includes dehydroascorbic acid, dehydroascorbate, or a mixture thereof. The composition may include a second hyperpolarized probe wherein the second hyperpolarized probe is a compound that includes pyruvic acid, pyruvate, or a mixture thereof. Also disclosed are methods that use the compositions disclosed herein, including methods of imaging and methods of measuring metabolic flux that include administering to a subject an effective amount of a composition disclosed herein and detecting by magnetic resonance the hyperpolarized probe(s) and / or one or more metabolites of the hyperpolarized probe(s).

Description

Atty. Dkt. No.: 115872-3458SIMULTANEOUS NONINVASIVE QUANTIFICATION OF REDOX AND DOWNSTREAM GLYCOLYTIC FLUXES REVEALS COMPARTMENTALIZED BRAIN METABOLISM CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No.63 / 789,231, filed April 15, 2025, the contents of which are incorporated herein by reference in its entirety for any and all purposes.GOVERNMENT SUPPORT

[0002] This invention was made with government support under CA252037 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD

[0003] The present technology is directed generally to molecular imaging and metabolic analysis, and more specifically to noninvasive methods and systems for probing cellular redox state and metabolic flux in biological tissues.SUMMARY

[0004] In one aspect, provided is a composition including a first hyperpolarized probe, wherein the first hyperpolarized probe is a compound that has been subjected to hyperpolarization and comprises dehydroascorbic acid, dehydroascorbate, or a mixture thereof. The first hyperpolarized probe may include [l-13C]dehydroascorbic acid, [1-13C]dehydroascorbate, [1-13C, 4-2H]dehydroascorbic acid, [1-13C, 4-2H]dehydroascorbate or a mixture of any two or more thereof.

[0005] The composition may also include a second hyperpolarized probe wherein the second hyperpolarized probe is a compound that has been subjected to hyperpolarization. The second hyperpolarized probe may include pyruvic acid, pyruvate, or a mixture thereof. The second hyperpolarized probe may include [l-13C]pyruvic acid, [2-13C]pyruvic acid, [1-13C]pyruvate, [2-13C]pyruvate, or a mixture of any two or more thereof.-1- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458

[0006] The first hyperpolarized probe may include [l-13C]dehydroascorbic acid, [1-13C]dehydroascorbate, [1-13C, 4-2H]dehydroascorbic acid, [1-13C, 4-2H]dehydroascorbate or a mixture of any two or more thereof, and the second hyperpolarized probe may include [1-13C]pyruvic acid, [2-13C]pyruvic acid, [l-13C]pyruvate, [2-13C]pyruvate, or a mixture of any two or more thereof.

[0007] The first hyperpolarized probe and / or the second hyperpolarized probe may include an NMR active nucleus, and the active nuclear may include1H,2H,13C,31P,15N, or19F.

[0008] The hyperpolarization of the first probe and / or hyperpolarization of the second hyperpolarized probe may performed via Para-Hydrogen Induced Polarization (PHIP), Dynamic Nuclear Polarization (DNP), Spin-Exchange Optical Pumping, or other Gas-Induced Polarization. The hyperpolarization of the first hyperpolarized probe and / or hyperpolarization of the second hyperpolarized probe may include introducing a radical source. The radical source may include an organic persistent free radical, photo-induced non-persistent radicals, nanodiamond, or metabolite which can form temporary radicals, silicon particles, or a mixture of any two or more thereof.

[0009] In any embodiments, the composition may further include a pharmaceutically acceptable carrier. The composition may be formulated for parenteral administration. The composition may be formulated for intravenous administration.

[0010] In another aspect, provided is a method of imaging using the probes disclosed herein and summarized above. The method of imaging includes administering to a subject an effective amount of a composition comprising a first hyperpolarized probe such that the first hyperpolarized probe contacts one or more mammalian cells; detecting by magnetic resonance the first hyperpolarized probe and / or one or more metabolites of the first hyperpolarized probe in the one or more mammalian cells. The method may also include administering to the subject an effective amount of a composition comprising a second hyperpolarized probe such that the second hyperpolarized probe contacts one or more mammalian cells; and detecting by magnetic resonance the second hyperpolarized probe and / or one or more metabolites of the second hyperpolarized probe in the one or more mammalian cells.-2- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458

[0011] The one or more metabolites of the first hyperpolarized probe may include vitamin C, including [l-13C]vitamin C and / or [1 -13C, 4-2H]vitamin C. The one or more metabolites of the second hyperpolarized probe may include lactate, including [l-13C]lactate.

[0012] The one or more mammalian cells may include one or more cancer cells. The cancer cells may include one or more of prostate, glioma, breast, cervical, renal, pancreatic, sarcoma, lung, oral, epithelial, or brain cancer cells. In some embodiments, the subject may be suspected of having a cancer, a traumatic brain injury, and / or a cardiovascular disease. The subject may be diagnosed as having a cancer, a traumatic brain injury, and / or a cardiovascular disease. The subject may be human.

[0013] In any embodiments, the first hyperpolarized probe and the second hyperpolarized probe may be copolarized.

[0014] The detecting by magnetic resonance may include simultaneously detecting one or more metabolites of the first hyperpolarized probe and one or more metabolites of the second hyperpolarized probe. The magnetic resonance may include2H magnetic resonance.

[0015] The first hyperpolarized probe and / or the second hyperpolarized probe may be deuterated where the deuterated probe exhibits a longer Ti relaxation time than its nondeuterated analog.

[0016] In another aspect, provided is a method of measuring metabolic flux in a subject using the probes disclosed herein and summarized above. The method includes administering to the subject an effective amount of a composition comprising a first hyperpolarized probe such that the first hyperpolarized probe contacts one or more mammalian cells; detecting by magnetic resonance at a plurality of timepoints the first hyperpolarized probe and / or one or more metabolites of the first hyperpolarized probe in the one or more mammalian cells; and determining a metabolic flux of the first hyperpolarized probe based on a rate of conversion of the first hyperpolarized probe to the one or more metabolites of the first hyperpolarized probe. The method may also include administering to the subject an effective amount of a composition comprising a second hyperpolarized probe such that the second hyperpolarized probe contacts one or more mammalian cells; and detecting by magnetic resonance at a plurality of timepoints the second hyperpolarized -3- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458probe and / or one or more metabolites of the second hyperpolarized probe in the one or more mammalian cells.

[0017] The one or more metabolites of the first hyperpolarized probe may include vitamin C, including [l-13C]vitamin C and / or [1 -13C, 4-2H]vitamin C. The one or more metabolites of the second hyperpolarized probe may include lactate, including [l-13C]lactate.

[0018] The one or more mammalian cells may include one or more cancer cells. The cancer cells may include one or more of prostate, glioma, breast, cervical, renal, pancreatic, sarcoma, lung, oral, epithelial, or brain cancer cells. In some embodiments, the subject may be suspected of having a cancer, a traumatic brain injury, and / or a cardiovascular disease. The subject may be diagnosed as having a cancer, a traumatic brain injury, and / or a cardiovascular disease. The subject may be human.

[0019] In any embodiments, the first hyperpolarized probe and the second hyperpolarized probe may be copolarized.

[0020] The detecting by magnetic resonance may include simultaneously detecting one or more metabolites of the first hyperpolarized probe and one or more metabolites of the second hyperpolarized probe. The magnetic resonance may include2H magnetic resonance.

[0021] The first hyperpolarized probe and / or the second hyperpolarized probe may be deuterated where the deuterated probe exhibits a longer Ti relaxation time than its nondeuterated analog.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIGs. 1A-1D show the chemistry of [l-13C]dehydroascorbate. FIG. 1A shows the stability of the dehydroascorbate (DHA) monomer at room temperature (RT; triangle), in aqueous solution (circle), and at -25 °C (square). FIG. IB shows13C NMR dynamic spectra of hyperpolarized [l-13C]pyruvate (171.1 ppm) and [1-13C]DHA formulation doped with 15 mM AHI 11501 trityl radical. Samples polarized at 5 T and 0.8 K, dissolved in D2O, and neutralized by 4 M NaOAc in D2O. FIG. 1C shows longitudinal13C spin-lattice relaxation time,13C Ti, of hyperpolarized (HP) [l-13C]pyruvate / [l-13C]DHA (6 / 4; v / v at 1 T) shown as a function of time and13C polarization. FIG. ID shows13C polarization and -4- 4921-1995-2792.7Atty. Dkt. No.: 115872-345813C Zi of [2-13C]dihydroxyacetone (DHAc; circle), [2-13C]glycerate (diamond), and [2-13C]fructose (square) in aqueous DHA at 1 T.

[0023] FIGs. 2A-2G demonstrate that dynamic HP MRS of HP [l-13C]pyruvate / [l-13C] DHA reveals rapid metabolic flux in the murine brain. FIG. 2A is a schematic diagram of the experimental timeline and setup. Fig. 2B provides an axial view of a mouse brain with accompanying slice selective excitation (15 mm in thickness covering the murine brain and the urea phantom) used for13C MRS in vivo. FIG. 2C shows the evolution of metabolic substrates and their metabolic products observed in a murine brain after injecting 300 pL of cohyperpolarized [l-13C]pyruvate and [1-13C]DHA in D2O in a nude mouse.13C NMR spectra, acquired on a murine brain with a temporal resolution of 1 s, show the dynamics of both [1-13C]DHA and [l-13C]pyruvate along with their metabolic products. Downstream metabolites derived from pyruvate (lactate, pyruvate hydrate, alanine, and bicarbonate) and DHA (VitC) are annotated on a representative13C NMR spectrum acquired 38 s after injection. FIG. 2D provides time course graphs of HP [l-13C]pyruvate (rhombus) and [1-13C]DHA (triangle). FIG. 2E provides time course graphs of the metabolic products of HP [l-13C]pyruvate and [1-13C]DHA. A.U., arbitrary units. The [l-13C]urea signal (filled circle) of the phantom placed adjacent to the mouse, and used as a reference, is shown in FIGs. 2D-2E. FIG. 2F provides a comparison between the calculated kinetic rate constants derived from the metabolic conversion of [l-13C]pyruvate to its products: [l-13C]lactate (APL), [l-13C]bicarbonate (APB), and [l-13C]alanine (APA). FIG. 2G provides the calculated kinetic rate constant deriving from the metabolic conversion of [1-13C]DHA to [l-13C]VitC ( DV). Values are reported as means ± SE (n = 3 biological replicates per group).

[0024] FIGs. 3A-3D demonstrate simultaneous spatially resolved metabolic conversion of both HP DHA and HP pyruvate in the murine brain. FIG. 3A is a schematic of the acquisition strategy, HP DHA / pyruvate injected over 10 s. After a delay of 15 s, a rapid multislice 2D EPSI acquisition was carried out, which covers the entire mouse brain. FIG.3B shows a representative coronal slice of the murine brain with the annotated corresponding13C MRSI grid from the stack of images acquired after infusion of HP DHA / pyruvate and an expanded grid of13C spectra demonstrating differential conversion of DHA and pyruvate in the slice. FIG. 3C shows representative13C spectrum with the peaks of interest annotated. FIG. 3D shows representative color overlay maps of the area under -5- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458the curve of each metabolite, demonstrating the distribution of HP substrates and products in the representative slice.

[0025] FIGs. 4A-4G show 2D multislice EPSI for the quantification of metabolism in different brain compartments. FIGs 4A-4C provide representative T2-weighted1H MRI of the murine brain in coronal (FIG. 4A), sagittal (FIG. 4B), and (FIG. 4C) axial planes annotating the segmentation of the WM (thin dashes) and GM (heavy dashes) for subsequent analysis. FIGs. 4D-4G show quantification of total carbon derived from HP pyruvate (Total13C Pyruvate) (FIG. 4D), total carbon derived from HP DHA (Total13C DHA) (FIG. 4E), ratio of lactate / total carbon derived from pyruvate (Lac / Total Carbon) (FIG. 4F), and ratio of VitC / total carbon derived from DHA (VitC / Total Carbon) (FIG. 4G), n = 4 biological replicates per group.

[0026] FIGs. 5A-5I show 2D multivoxel CSI with HP [l-13C]pyruvate, [2-13C]pyruvate, and [l-13C]dehydroascorbate (DHA) to infer regional metabolism in a mouse brain. FIG.5A is an overlay of a coronal multivoxel13C HP MRSI acquisition of a mouse brain on the corresponding morphological1H T2-weighted image. The shade of each voxel of the MRSI grid indicates the total13C signal in that voxel. The insets provide (TOP) a representative13C HP spectrum of a voxel in the CR of a mouse brain showing the main metabolite peaks ([l-13C]pyruvate at 171.1 ppm, [l-13C]bicarbonate at 161.1 ppm, [1-13C]pyruvate-hydrate at 179 ppm, [l-13C]lactate at 183 ppm, [2-13C]pyruvate at 206 ppm, [5-13C]glutamate at 181.4 ppm, [1-13C]DHA at 174 ppm, and [l-13C]VitC at 178 ppm), and (BOTTOM) a representative13C HP spectrum of the DGM region of a mouse brain. For both spectra, a quantitative estimate (dark gray line) is shown along with the residual line (light gray line). A magnification of the spectral regions where the metabolites of interest are observed (175.5 to 185 and 160 to 162.5 ppm) is shown for each spectrum. FIGs. 5B-5D provide graphs showing the quantification of total [l-13C]pyruvate delivered (FIG. 5B) and ratios of its products lactate (FIG. 5C) and bicarbonate (FIG. 5D) to total carbon. FIG.5E shows the quantification of total [1-13C]DHA. FIG. 5F shows the quantification of total [2-13C]pyruvate delivered, and FIG. 5G shows ratios of its products glutamate to total carbon. FIG. 5H shows the ratio of C5 glutamate to bicarbonate. FIG. 51 shows the ratio of [l-13C]VitC to total carbon derived from [1-13C]DHA. n = 4 biological replicates per group.-6- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458

[0027] FIGs. 6A-6D show representative13C dynamics of HP substrates prepared using DHA as a glassing agent, dissolved in D2O and dynamic measured at IT using a Magritek benchtop NMR spectrometer. FIG. 6A: HP [2-13C]Fructose; FIG. 6B: HP [2-13C]glycerate; FIG. 6C: HP [l-13C]pyruvate / [2-13C]pyruvate / [l-13C]DHA; and FIG. 6D: HP [2-13C]DHAC.

[0028] FIG. 7 A and FIG. 7B provide kinetic fits analyzed from the dynamic HP MRS of HP [l-13C]Pyruvate / [l-13C]DHA in the murine brain. FIG. 7 A provides kinetic fits of HP pyruvate conversion to its downstream metabolites lactate, bicarbonate, and alanine for three replicates. FIG. 7B provides kinetic fits of HP DHA conversion to vitamin C (VitC), n = 3 biological replicates.

[0029] FIGs. 8A-8F show 2D-Multi Voxel Chemical Shift Imaging (CSI) with HP [1-13C]pyruvate and [l-13C]dehydroascorbate (DHA) for the quantification of metabolism in U87 glioma xenograft. FIG. 8A provides an overlay of a coronal multi-voxel13C HP CSI acquisition of a murine brain with U87 xenograft on the corresponding morphological T2-weighted image. FIG. 8B shows a representative13C HP spectrum of a voxel in the tumor area showing the main metabolite peaks ([l-13C]pyruvate at 171.1 ppm, [1-13C]DHA at 174 ppm, [l-13C]vitamin C at 178 ppm, [l-13C]pyruvate hydrate at 179 ppm and [l-13C]lactate at 183 ppm). FIGs. 8C-8F show quantification of [l-13C]pyruvate (FIG. 8C), [1-13C]DHA (FIG. 8D), [l-13C]lactate (FIG. 8E), and [l-13C]vitamin C (FIG. 8F) in tumor and contralateral region (Brain).

[0030] FIGs. 9A-9C show [l-13C]Urea phantom MR scans acquired to normalize the13C signal of each hyperpolarized metabolite. FIG. 9A shows a representative coronal T2-weighted image of a mouse brain adjacent to the cylindrical phantom containing a 6M [1-13C]urea solution, used as a reference. The overlay of the corresponding13C MRSI grid from the stack images acquired using the multi-slice echo planar spectroscopic imaging (EPSI) scan is shown for the HP EPSI scan acquired on the mouse brain and the EPSI scan acquired on the phantom during the same MRI session. The inset on the right shows a magnification of the overlayed grid on the mouse brain with the13C EPSI spectra showing the spatial distribution of the substrates and downstream metabolites. The bottom inset provides a magnification of the overlayed grid on the phantom showing the spatial-7- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458distribution of [l-13C]urea signal over the phantom. FIG. 9B is a representative13C HP spectrum of the expanded grid (FIG. 9A, right inset, dark line) showing the main metabolite peaks ([l-13C]pyruvate at 171.1 ppm, [1-13C]DHA at 174 ppm, [l-13C]vitamin C at 178 ppm, [l-13C]pyruvate hydrate at 179 ppm and [l-13C]lactate at 183 ppm). FIG. 9C shows the central [l-13C]urea spectrum of the grid (FIG. 9A, bottom inset, dark line) showing the maximum [l-13C]urea signal on the phantom.

[0031] FIGs. 10A-10C show 2D multi -voxel Chemical Shift Imaging (CSI) with HP [1-13C]pyruvate and [ 1 -13C] dehydroascorbate (DHA) to infer regional metabolism in a mouse brain. FIG. 10A shows a representative T2-weighted coronal image of a mouse brain showing the cortical (CR) region-of-interest (ROI) (heavy dashes) and the deep grey matter (DGM) ROIs (thin dashes). The image was acquired on a mouse brain with in-plane resolution of 102 x 102 pm2. FIG. 10B is an overlay of a coronal multi -voxel13C HP MRSI acquisition of a mouse brain on the corresponding morphological1H T2-weighted image. The shade of each voxel of the MRSI grid indicates the total13C signal in that voxel. The top inset provides a representative13C HP spectrum of a voxel in the cortical region (CR) of a mouse brain showing the main metabolite peaks ([l-13C]pyruvate at 171.1 ppm, [1-13C]bicarbonate at 161.1 ppm, [1-13C] pyruvate hydrate at 179 ppm, [l-13C]lactate at 183 ppm, [1-13C]DHA at 174 ppm and [l-13C]vitamin C at 178 ppm). The bottom inset provides a representative13C HP spectrum of the deep grey matter region (DGM) of a mouse brain showing the main metabolites peaks. The [5-13C]glutamate signal at 181.4 ppm is not visible in both cortical and DGM regions (dark arrow in insets). For the spectra in the insets, a quantitative estimate obtained using AMARES (Advanced Method for Accurate, Robust, and Efficient Spectral fitting; jMRUI) is shown along with the residual line. A magnification of the spectral regions where the metabolites of interest are observed (175.5 - 185 ppm and 160 -162.5 ppm) is shown for each spectrum. FIG. 10C provides heatmaps showing visualization of glutamate, glutathione, and ascorbate from a sagittal section of a mouse brain obtained using MALDI-IMS. The heatmaps display the relative levels of each metabolite in the murine brain.

[0032] FIGs. HA and 11B provide hyperpolarization of [1-13C]DHA and [l-13C]ascorbic acid. Both the samples were polarized on SpinLab (5.0 T, 0.8 K) and dissolved in D2O. Dynamic spectra were measured on 1 T NMR spectrometer (Magritek) and polarization and -8- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458Ti was calculated using MATLAB. FIG. 11A provides polarization and13C Ti of [1-13C]DHA (4M) / [l-13C]pyruvate (8.4M) doped with 15 mM AHI 11501 trityl radical. FIG.11B provides 2.2M [1-13C] Ascorbic acid in glycerol / water (2 / 8; v / v) mixture doped with 15 mM 0X063 radical. For non-deuterated DHA and Vitamin C, polarization of -30% (5-6 times increase than the previously reported methods) and about 18% (about 2-3 times increase than previously reported values), respectively, was achieved and the liquid state13C Tiof labelled carbon (Cl) was calculated to be about 106 s for HP [1-13C]DHA and about 63 s for HP [l-13C]ascorbic acid solution.

[0033] FIGs. 12A and 12B provide hyperpolarization of C-4 deuterated [l-13C]Ascorbic acid (mixture of both deuterated isomers) polarized on SpinLab (5.0 T, 0.8 K) and dissolved in D2O. Dynamic spectra were measured on 1 T NMR spectrometer (Magritek) and polarization and Ti was calculated using MATLAB. FIG. 12A provides the dynamic HP NMR signal decay. FIG. 12B shows the 13C Ti for both C-4 deuterated L-ascorbic acid and C-4 deuterated D-Isoascorbic acid. Deuteration of C-4 increases the 13 Ti of vitamin C significantly from about 63 s to about 100 s.

[0034] FIGs. 13A and 13B provide hyperpolarization of C-4 deuterated [1-13C]DHA (mixture of both deuterated isomers) polarized on SpinLab (5.0 T, 0.8 K) and dissolved in D2O. Dynamic spectra were measured on 1 T NMR spectrometer (Magritek) and polarization and Ti was calculated using MATLAB. FIG. 13A provides the dynamic HP NMR signal decay. FIG. 13B shows the 13C Ti for both C-4 deuterated L-DHA and C-4 deuterated D-IsoDHA. Deuteration of C-4 increases the 13 Ti of DHA significantly from about 105 s to about 112 s and longer for IsoDHA.

[0035] FIG. 14A provides HP dynamic spectra of UV-irradiated [l-13C]pyruvic acid / [l -13C]DHA sample at 6.7 T, 1.2 K, dissolved in D2O and acquired at 600 MHz NMR spectrometer. 20 beads of 7 pL were irradiated for 25 s to afford 40 mM of radical and sample was polarized at 6.7 T, 1.2 K polarizer. The polarization was measured to be -25 %. FIG. 14B shows the average13C Ti of hyperpolarized [l-13C]pyruvic acid / [l-13C]DHA in D2O sample measured on 600 MHz NMR spectrometer.

[0036] FIG. 15 provides a graph showing body weight fluctuation of B6 female mice after intravenous administration of 187 mM sodium acetate, 85 mM13C-dehydroascorbic acid -9- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458(13C-DHAA), 57 mM13C-DHAA, 187 mM13C-pyruvate, 85 mM13C-DHAA + 187 mM13C-pyruvate, 57 mM13C-DHAA + 125.4 mM13C-pyruvate, or 40 mM13C-DHAA + 88 mM13C-pyruvate.

[0037] FIG. 16 provides a schematic overview of a human clinical experiment to determine proper dosing of13C-DHAA and13C-pyruvate.DETAILED DESCRIPTION

[0038] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s).

[0039] In various aspects, the present technology provides compounds and methods of imaging using a hyperpolarized probe. The compounds provided herein can be formulated into pharmaceutical compositions that are useful in the disclosed methods. Also provided is the use of the compounds as hyperpolarized probes.

[0040] The following terms are used throughout as defined below.

[0041] As used herein and in the appended claims, singular articles such as “a” and “an” and “the” and similar referents in the context of describing the elements (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments and does not pose a limitation on the scope of the claims unless otherwise stated. No language in the specification should be construed as indicating any non-claimed element as essential.-10- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458

[0042] As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art, given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term - for example, “about 10 wt.%” would be understood to mean “9 wt.% to 11 wt.% .” It is to be understood that when “about” precedes a term, the term is to be construed as disclosing “about” the term as well as the term without modification by “about” - for example, “about 10 wt.%” discloses “9 wt.% to 11 wt.%” as well as disclosing “10 wt.%.”

[0043] The phrase “and / or” as used in the present disclosure will be understood to mean any one of the recited members individually or a combination of any two or more thereof -for example, “A, B, and / or C” would mean “A, B, C, A and B, A and C, B and C, or the combination of A, B, and C.”

[0044] Generally, reference to a certain element such as hydrogen or H is meant to include all isotopes of that element. For example, if an R group is defined to include hydrogen or H, it also includes deuterium and tritium. Compounds comprising radioisotopes such as tritium,14C,32P, and35S are thus within the scope of the present technology. Procedures for inserting such labels into the compounds of the present technology will be readily apparent to those skilled in the art based on the disclosure herein.

[0045] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 atoms refers to groups having 1, 2, or 3-11- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458atoms. Similarly, a group having 1-5 atoms refers to groups having 1, 2, 3, 4, or 5 atoms, and so forth.

[0046] Pharmaceutically acceptable salts of compounds described herein are within the scope of the present technology and include acid or base addition salts which retain the desired pharmacological activity and is not biologically undesirable (e.g., the salt is not unduly toxic, allergenic, or irritating, and is bioavailable). When the compound of the present technology has a basic group, such as, for example, an amino group, pharmaceutically acceptable salts can be formed with inorganic acids (such as hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, and phosphoric acid), organic acids (e.g. alginate, formic acid, acetic acid, trifluoroacetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalene sulfonic acid, andp-toluenesulfonic acid) or acidic amino acids (such as aspartic acid and glutamic acid). When the compound of the present technology has an acidic group, such as for example, a carboxylic acid group, it can form salts with metals, such as alkali and earth alkali metals (e.g. Na+, Li+, K+, Ca2+, Mg2+, Zn2+), ammonia or organic amines (e.g. dicyclohexylamine, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine) or basic amino acids (e.g. arginine, lysine and ornithine). Such salts can be prepared in situ during isolation and purification of the compounds or by separately reacting the purified compound in its free base or free acid form with a suitable acid or base, respectively, and isolating the salt thus formed.

[0047] Those of skill in the art will appreciate that compounds of the present technology may exhibit the phenomena of tautomerism, conformational isomerism, geometric isomerism and / or stereoisomerism. As the formula drawings within the specification and claims can represent only one of the possible tautomeric, conformational isomeric, stereochemical or geometric isomeric forms, it should be understood that the present technology encompasses any tautomeric, conformational isomeric, stereochemical and / or geometric isomeric forms of the compounds having one or more of the utilities described herein, as well as mixtures of these various different forms.-12- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458

[0048] “ Tautomers” refers to isomeric forms of a compound that are in equilibrium with each other. The presence and concentrations of the isomeric forms will depend on the environment the compound is found in and may be different depending upon, for example, whether the compound is a solid or is in an organic or aqueous solution. For example, in aqueous solution, quinazolinones may exhibit the following isomeric forms, which are referred to as tautomers of each other:As another example, guanidines may exhibit the following isomeric forms in protic organic solution, also referred to as tautomers of each other:

[0049] Because of the limits of representing compounds by structural formulas, it is to be understood that all chemical formulas of the compounds described herein represent all tautomeric forms of compounds and are within the scope of the present technology.

[0050] Stereoisomers of compounds (also known as optical isomers) include all chiral, diastereomeric, and racemic forms of a structure, unless the specific stereochemistry is expressly indicated. Thus, compounds used in the present technology include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions. Both racemic and diastereomeric mixtures, as well as the individual optical isomers can be isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and these stereoisomers are all within the scope of the present technology.

[0051] The terms “cancer” and “tumor,” used interchangeably herein, refer to cells or tissues that exhibit an aberrant growth phenotype characterized by a significant loss of -13- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458control of cell proliferation. The methods and compositions of this disclosure apply to malignant, pre-metastatic, metastatic, and non-metastatic cells.

[0052] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. Also within this disclosure are Arabic numerals referring to referenced citations, the full bibliographic details of which are provided preceding the claims. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure.

[0053] The Present Technology

[0054] One of the most profound developmental evolutionary changes that separate humans from lower species is the substantial increase in size and complexity of the human brain. Decades of research have illuminated the intricate connections of cells in the brain, much of which is driven by metabolic pathways that operate both for neurotransmission and at a fundamental level for energy production (1, 2). Different regions of the brain are known to use differential metabolism to facilitate brain function, although changes in these metabolic processes are difficult to visualize (3). Moreover, concomitant metabolic processes including the generation of reactive oxygen species (ROS) and the change in brain redox biochemistry are exceedingly challenging to quantify in the brain. In disease, changes in redox have been implicated from brain tumors to neurodegeneration, including oncogenic transformation of low-grade gliomas with mutant isocitrate dehydrogenase, a redox pathway consuming reduced form of NADP+(nicotinamide adenine dinucleotide phosphate) (NADPH), and the putative pathogenesis of Alzheimer’s disease (4-8).

[0055] Although many metabolic methods have been pioneered in the past two decades, it remains challenging to access the metabolism of the brain using these predominantly destructive approaches (9). Traditional imaging approaches have been explored including positron emission tomography and magnetic resonance imaging (MRI) (10, 11), but they suffer from the inability to discern a metabolic product from a substrate and lack the sensitivity to measure a real-time metabolic flux. Moreover, translational methods are needed to truly understand beyond metabolic pool sizes and provide insights into metabolic fluxes in human brain biology.-14- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458

[0056] Isotope tracing methods with high sensitivity ex vivo analytical analysis have provided a means to estimate the contribution of substrates to their respective metabolite pools, revealing a wealth of information about the origins of a nutrient (12). These methods, although, fall short in the ability to measure a rate given the need to repeat the sample in the same region in time. The ability to investigate metabolism in real time and quantify dynamic changes soon after a rapid infusion of an isotopically enriched substrate has been enabled by boosting the intensity of the magnetic resonance signal, achieved via hyperpolarization (HP) (13, 14). HP MRI has revolutionized the field of biomedical imaging by providing a noninvasive way to visualize in vivo metabolic processes, which allow the identification of metabolic aberrations in pathogenesis. HP MRI provides crucial and clinically relevant information for early-stage detection of disease and monitoring the therapy response. However, the potential of HP MRI has been limited due to the lack of efficient HP methods for key substrates of interest. After nearly two decades of scientific research in the field of HP13C magnetic resonance, HP13C-pyruvate remains the only metabolic probe translated to humans for investigating brain biochemistry (15-19).Although novel molecules have been developed for use in preclinical models (20), their use has been limited by the physical and chemical properties of the substrate as well as the feasibility of the clinical translation of HP methods (14). To exploit the vast potential of HP MRI in biomedical imaging, an HP approach characterized by a substrate with an appropriately long13C spin-lattice relaxation time (7i ) and no associated toxicity, high13C polarization, and feasibility of clinical implementation is required.

[0057] Oxidative stress is typically the result of an imbalance between the cells generation of ROS and its ability to reduce them. Changes in oxidative stress have been associated with oncogenesis (21), but it also plays a crucial role in the pathogenesis of chronic diseases, such as cardiovascular diseases, diabetes, and neurodegenerative diseases (8, 22). Therefore, an efficient HP probe that allows for the noninvasive in vivo investigation of oxidative stress could provide valuable insights into its role in disease development and progression. Such a probe could aid in early disease detection and improve current cancer therapies targeting oxidative stress [i.e., vitamin C (VitC) cancer therapy and radiation therapy] (23, 24). Dehydroascorbic acid (DHA), which is the oxidized form of VitC, has been shown to rapidly transport into cells via the SLC2A family of solute carriers, predominantly SLC2A1 (GLUT1) (25). Hyperpolarized DHA can report on the redox state -15- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458in vivo and act as a specific probe to assess oxidative stress in vivo (26). Conversion of DHA to VitC is a sensitive indicator of oxidative stress, making DHA an appropriate biomarker capable of differentiating between the oxidative stress in pathogenic and healthy tissues (27). However,13C-DHA suffers from associated toxicity and a poor achievable13C polarization due to its solubility in non-biocompatible organic solvents (28). With several promising ongoing clinical trials targeting or leveraging oxidative stress for response, gaining key insights into DHA / VitC metabolism not only can improve the current understanding of the role of oxidative stress in pathogenesis but also has the potential to improve the development of therapies.(0058] To address this, a previously unidentified hyperpolarized formulation of DHA leveraging its monomeric form was developed. The polarization of13C-DHA was drastically improved by the newly created, clinically translatable formulation, which uses removable trityl radical, and has no associated toxicity. When using DHA as a solvent, the ability to codissolve it with pyruvic acid (PA) was demonstrated by establishing a simultaneous copolarization approach to monitor fluxes through both pathways simultaneously in the murine brain. This approach was extended to the copolarization of several endogenous substrates, making it a vehicle for the translation of other crucial HP substrates. To demonstrate the utility of such an approach, the metabolic flux of HP DHA and pyruvate was simultaneously probed, measuring metabolic flux rates in vivo. These fluxes were subsequently spatially resolved, and differential metabolic fluxes across regions of the murine brain were measured. To strengthen the value of the proposed HP approach, a xenograft mouse model of glioblastoma was investigated to evaluate the differential metabolism of the tumor lesion and the contralateral healthy tissue. Furthermore, the extension of this approach was demonstrated by spatially measuring multiple stages of pathway flux in the murine brain using multiple isotopic positions simultaneously, thus providing insights into the time-dependent compartmentalization of brain biochemistry.

[0059] Thus, the present technology may contain two components in a single intravenous injection: hyperpolarized [ 1 -13C] pyruvate (HP -pyruvate) and hyperpolarized [ 1 -13C] dehydroascorbic acid (HP-DHAA), each of which are shown in Scheme 1. Both pyruvate and DHAA are endogenous biomolecules.-16- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458Scheme 1. Structures of HP-pyruvate (left) and HP-DHAA (right).

[0060] Hyperpolarization is a process that polarizes nuclear spins, yielding compounds with improved signal in magnetic resonance imaging (MRI) compared to non-HP counterparts (46). Hyperpolarization is not expected to introduce new toxicities since it does not alter intrinsic chemistry or biological activity, and hyperpolarized compounds are non-radioactive.

[0061] HP-pyruvate serves as a probe for cancer glycolysis, enabling real-time in vivo assessment of metabolic processes that are central to cancer and other diseases (46). Upon administration, HP-pyruvate is taken up by cells and metabolizes to several products (lactate, alanine, bicarbonate) that can be quantified in near real-time with magnetic resonance spectroscopic imaging (MRSI) to measure glycolytic flux. The preferential conversion of pyruvate to lactate is characteristic of the Warburg effect, a hallmark of aggressive cancers wherein aerobic glycolysis and lactic acid fermentation dominate energy production even in an abundance of oxygen. An increase in bias toward lactate product, measurable with MRSI, is consistent with higher tumor grade (47). HP-pyruvate has been extensively studied non-clinically (including General Electric’s IND#71651) and is under multiple clinical investigations (IND#129547, NCT02421380, NCT05041166, and NCT06305728).

[0062] HP-DHAA is a novel probe designed to detect oxidative stress in tumor cells (48). DHAA (or DHA) is the oxidized form of vitamin C (reduced form: ascorbic acid, AA). Upon administration, DHAA is rapidly taken into cells by glucose transporters (GLUT 1, 3 and 4) and reduced to AA by intracellular reducing agents (ex. glutathione), as demonstrated in preclinical animal models of prostate cancer (48). Reduction of HP-DHAA to AA indicates oxidative stress which can be quantified with MRSI. Many tumors upregulate antioxidant systems (redox adaptation) and overexpress GLUT transporters;-17- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458therefore, HP-DHAA specifically accumulates in tumor cells, and changes in DHAA to AA conversion could indicate tumor aggressiveness and response to therapy (48).Administration of HP-DHAA with HP-pyruvate allows for simultaneous interrogation of oxidative stress with glycolytic flux, constituting a powerful noninvasive probe into tumor biology.

[0063] The present technology provides probes and compositions containing such probes, where the probes and compositions may be used for many purposes, including, but not limited to, imaging (in vitro and / or in vivo), quantifying metabolic flux, investigating metabolic processes (including probing brain metabolism), and diagnosing particular diseases or conditions.

[0064] In one aspect, the present technology provides a hyperpolarized probe that includes dehydroascorbic acid, dehydroascorbate, or a mixture thereof, where the dehydroascorbic acid, dehydroascorbate, or a mixture thereof has been subjected to hyperpolarization. The hyperpolarized probe may be deuterated. The hyperpolarized probe may be [1 -13C]dehydroascorbic acid, [l-13C]dehydroascorbate, [ 1-13C, 4-2H]dehydroascorbic acid, [1-13C, 4-2H]dehydroascorbate, or a mixture of any two or more thereof.

[0065] In another aspect, provided is a composition including a first hyperpolarized probe. The first hyperpolarized probe may be a compound that has been subjected to hyperpolarization and comprises dehydroascorbic acid, dehydroascorbate, or a mixture thereof. The first hyperpolarized probe may include [l-13C]dehydroascorbic acid, [1-13C]dehydroascorbate, [1-13C, 4-2H]dehydroascorbic acid, [1-13C, 4-2H]dehydroascorbate, or a mixture of any two or more thereof.

[0066] The composition may, in any embodiments, further include a second hyperpolarized probe wherein the second hyperpolarized probe is a compound that has been subjected to hyperpolarization. The second hyperpolarized probe may include pyruvic acid, pyruvate, or a mixture thereof. In some embodiments, the second hyperpolarized probe includes [1-13C]pyruvic acid, [2-13C]pyruvic acid, [l-13C]pyruvate, [2-13C]pyruvate, or a mixture of any two or more thereof.-18- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458

[0067] In any embodiments, the composition may include a first hyperpolarized probe and a second hyperpolarized probe, where the first hyperpolarized probe may include [1-13C]dehydroascorbic acid, [l-13C]dehydroascorbate, [ 1-13C, 4-2H]dehydroascorbic acid, [1-13C, 4-2H]dehydroascorbate, or a mixture of any two or more thereof and the second hyperpolarized probe may include [l-13C]pyruvic acid, [2-13C]pyruvic acid, [1-13C]pyruvate, [2-13C]pyruvate, or a mixture of any two or more thereof.

[0068] The hyperpolarized probes (including the first hyperpolarized probe and the second hyperpolarized probe) may include an active nucleus, including any NMR active nucleus. The active nucleus may be1H,2H,13C,31P,15N, or19F. In some embodiments, the active nucleus may be1H,2H, or13C.

[0069] Thus, the first hyperpolarized probe and / or the second hyperpolarized probe may be deuterated. The deuterated probe exhibits a longer Ti relaxation time than its nondeuterated analog.

[0070] Hyperpolarization of the first probe and / or hyperpolarization of the second hyperpolarized probe may be performed via Para-Hydrogen Induced Polarization (PHIP), Dynamic Nuclear Polarization (DNP), Spin-Exchange Optical Pumping, or other Gas-Induced Polarization.

[0071] In any embodiments, the composition may further include a pharmaceutically acceptable carrier. The composition may be formulated for various routes of administration, for example, by oral, parenteral, topical, rectal, nasal, vaginal administration, or via implanted reservoir. Parenteral or systemic administration includes, but is not limited to, subcutaneous, intravenous, intraperitoneal, and intramuscular, injections. In some embodiments, the composition is formulated for parenteral administration. In some embodiments, the composition is formulated for intravenous administration.

[0072] The hyperpolarized probes may be administered in an “effective amount.” “Effective amount” refers to the amount of a compound or composition required to produce a desired effect. One example of an effective amount includes amounts or dosages that-19- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458yield acceptable detection levels of the hyperpolarized probes and their metabolites using magnetic resonance.

[0073] In any embodiments, the hyperpolarized probes may be administered at a concentration of about 10 mM to about 500 mM, including from about 10 mM to about 300 mM, about 25 mM to about 250 mM, or about 40 mM to about 200 mM. The hyperpolarized probes may be administered at a concentration of about 10 mM, about 25 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200 mM, about 225 mM, about 250 mM, about 275 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, or about 500 mM.

[0074] In another aspect of the present technology, provided is a method of imaging that includes administering an effective amount of a composition that includes a first hyperpolarized probe such that the first hyperpolarized probe contacts one or more mammalian cells; detecting by magnetic resonance the first hyperpolarized probe and / or one or more metabolites of the first hyperpolarized probe in the one or more mammalian cells; wherein the hyperpolarized probe is a compound that has been subjected to hyperpolarization and includes dehydroascorbic acid, dehydroascorbate, or a mixture thereof. In any embodiments, the method may further include administering to the subject an effective amount of a composition comprising a second hyperpolarized probe such that the second hyperpolarized probe contacts one or more mammalian cells, and detecting by magnetic resonance the second hyperpolarized probe and / or one or more metabolites of the second hyperpolarized probe in the one or more mammalian cells, wherein the second hyperpolarized probe is a compound that has been subjected to hyperpolarization.

[0075] The first hyperpolarized probe and the second hyperpolarized probe may be present and administered in a single composition, or the first hyperpolarized probe and the second hyperpolarized probe may be present and administered in multiple compositions. In some embodiments, the composition (or multiple compositions) is formulated for parenteral administration. In some embodiments, the composition (or multiple compositions) is formulated for intravenous administration.-20- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458

[0076] The first hyperpolarized probe may include dehydroascorbic acid, dehydroascorbate, or a mixture thereof. The first hyperpolarized probe may include [l-13C]dehydroascorbic acid, [l-13C]dehydroascorbate, [1 -13C, 4-2H]dehydroascorbic acid, [1-13C, 4-2H]dehydroascorbate, or a mixture of any two or more thereof. The second hyperpolarized probe may include pyruvic acid, pyruvate, or a mixture thereof. In some embodiments, the second hyperpolarized probe includes [l-13C]pyruvic acid, [2-13C]pyruvic acid, [1-13C]pyruvate, [2-13C]pyruvate, or a mixture of any two or more thereof, the first hyperpolarized probe may include [l-13C]dehydroascorbic acid, [l-13C]dehydroascorbate, [ 1 -13C, 4-2H]dehydroascorbic acid, [ 1 -13C, 4-2H]dehydroascorbate, or a mixture of any two or more thereof and the second hyperpolarized probe may include [l-13C]pyruvic acid, [2-13C]pyruvic acid, [l-13C]pyruvate, [2-13C]pyruvate, or a mixture of any two or more thereof.

[0077] The person of ordinary skill in the art may readily determine which metabolite(s) to monitor. In some embodiments, a metabolite of the first hyperpolarized probe may include vitamin C, such as [l-13C]vitamin C and / or [ 1-13C, 4-2H]vitamin C. In some embodiments, a metabolite of the second hyperpolarized probe may include lactate, such as [l-13C]lactate.

[0078] The hyperpolarized probes (including the first hyperpolarized probe and the second hyperpolarized probe) may include an active nucleus, including any NMR active nucleus. The active nucleus may be1H,2H,13C,31P,15N, or19F. In some embodiments, the active nucleus may be1H,2H, or13C.

[0079] Thus, the first hyperpolarized probe and / or the second hyperpolarized probe may be deuterated. The deuterated probe exhibits a longer Ti relaxation time than its nondeuterated analog.

[0080] Hyperpolarization of the first probe and / or hyperpolarization of the second hyperpolarized probe may be performed via Para-Hydrogen Induced Polarization (PHIP), Dynamic Nuclear Polarization (DNP), Spin-Exchange Optical Pumping, or other Gas-Induced Polarization. Hyperpolarization may include introducing a radical source, and the radical source may include an organic persistent free radical, photo-induced non-persistent radicals, nanodiamond, or metabolite which can form temporary radicals, silicon particles, or a mixture of any two or more thereof. The first hyperpolarized probe and the second hyperpolarized probe may be copolarized.-21- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458

[0081] The one or more mammalian cells may include one or more cancer cells. The cancer cells may include one or more of prostate, glioma, breast, cervical, renal, pancreatic, sarcoma, lung, oral, epithelial, or brain cancer cells.

[0082] In any embodiments, the subject may be suspected of having a cancer, a traumatic brain injury, and / or a cardiovascular disease. The subject may be diagnosed as having a cancer, a traumatic brain injury, and / or a cardiovascular disease. In any embodiments, the subject may be human.

[0083] Imaging may be obtained from detection of the hyperpolarized probe (e.g., detecting the presence of13C) using magnetic resonance imaging (MRI) or magnetic resonance spectroscopic imaging (MRSI). In any embodiments, detecting by magnetic resonance may include simultaneously detecting two or more of the first hyperpolarized probe, one or more metabolites of the first hyperpolarized probe, the second hyperpolarized probe, and one or more metabolites of the second hyperpolarized probe. The magnetic resonance may includeJH magnetic resonance,2H magnetic resonance,13C magnetic resonance,31P magnetic resonance,15N magnetic resonance, and / or19F magnetic resonance.

[0084] In another aspect of the present technology, disclosed is a method of measuring metabolic flux in a subject wherein the method includes administering to the subject an effective amount of a composition comprising a first hyperpolarized probe such that the first hyperpolarized probe contacts one or more mammalian cells, detecting by magnetic resonance at a plurality of timepoints the first hyperpolarized probe and / or one or more metabolites of the first hyperpolarized probe in the one or more mammalian cells, an determining a metabolic flux of the first hyperpolarized probe based on a rate of conversion of the first hyperpolarized probe to the one or more metabolites of the first hyperpolarized probe, wherein the first hyperpolarized probe is a compound that has been subjected to hyperpolarization and comprises dehydroascorbic acid, dehydroascorbate, or a mixture thereof. In any embodiments, the method may also include administering to the subject an effective amount of a composition comprising a second hyperpolarized probe such that the second hyperpolarized probe contacts one or more mammalian cells, and detecting by magnetic resonance at a plurality of timepoints the second hyperpolarized probe and / or one or more metabolites of the second hyperpolarized probe in the one or more mammalian-22- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458cells, wherein the second hyperpolarized probe is a compound that has been subjected to hyperpolarization.

[0085] The first hyperpolarized probe and the second hyperpolarized probe may be present and administered in a single composition, or the first hyperpolarized probe and the second hyperpolarized probe may be present and administered in multiple compositions. In some embodiments, the composition (or multiple compositions) is formulated for parenteral administration. In some embodiments, the composition (or multiple compositions) is formulated for intravenous administration.

[0086] The first hyperpolarized probe may include dehydroascorbic acid, dehydroascorbate, or a mixture thereof. The first hyperpolarized probe may include [l-13C]dehydroascorbic acid, [l-13C]dehydroascorbate, [1 -13C, 4-2H]dehydroascorbic acid, [1-13C, 4-2H]dehydroascorbate, or a mixture of any two or more thereof. The second hyperpolarized probe may include pyruvic acid, pyruvate, or a mixture thereof. In some embodiments, the second hyperpolarized probe includes [l-13C]pyruvic acid, [2-13C]pyruvic acid, [1-13C]pyruvate, [2-13C]pyruvate, or a mixture of any two or more thereof. The first hyperpolarized probe may include [l-13C]dehydroascorbic acid, [l-13C]dehydroascorbate, [ 1 -13C, 4-2H]dehydroascorbic acid, [ 1 -13C, 4-2H]dehydroascorbate, or a mixture of any two or more thereof and the second hyperpolarized probe may include [l-13C]pyruvic acid, [2-13C]pyruvic acid, [l-13C]pyruvate, [2-13C]pyruvate, or a mixture of any two or more thereof.

[0087] The person of ordinary skill in the art may readily determine which metabolite(s) to monitor. In some embodiments, a metabolite of the first hyperpolarized probe may include vitamin C, such as [l-13C]vitamin C and / or [ 1-13C, 4-2H]vitamin C. In some embodiments, a metabolite of the second hyperpolarized probe may include lactate, such as [l-13C]lactate.

[0088] The hyperpolarized probes (including the first hyperpolarized probe and the second hyperpolarized probe) may include an active nucleus, including any NMR active nucleus. The active nucleus may be1H,2H,13C,31P,15N, or19F. In some embodiments, the active nucleus may be1H,2H, or13C.-23- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458

[0089] Thus, the first hyperpolarized probe and / or the second hyperpolarized probe may be deuterated. The deuterated probe exhibits a longer Ti relaxation time than its nondeuterated analog.

[0090] Hyperpolarization of the first probe and / or hyperpolarization of the second hyperpolarized probe may be performed via Para-Hydrogen Induced Polarization (PHIP), Dynamic Nuclear Polarization (DNP), Spin-Exchange Optical Pumping, or other Gas-Induced Polarization. Hyperpolarization may include introducing a radical source, and the radical source may include an organic persistent free radical, photo-induced non-persistent radicals, nanodiamond, or metabolite which can form temporary radicals, silicon particles, or a mixture of any two or more thereof. The first hyperpolarized probe and the second hyperpolarized probe may be copolarized.

[0091] The one or more mammalian cells may include one or more cancer cells. The cancer cells may include one or more of prostate, glioma, breast, cervical, renal, pancreatic, sarcoma, lung, oral, epithelial, or brain cancer cells.

[0092] In any embodiments, the subject may be suspected of having a cancer, a traumatic brain injury, and / or a cardiovascular disease. The subject may be diagnosed as having a cancer, a traumatic brain injury, and / or a cardiovascular disease. In any embodiments, the subject may be human.

[0093] In some embodiments, the composition is administered to a mammal. As used herein, the term mammal includes, but is not limited to, a cat, dog, rodent, or primate. In one embodiment, the mammal is a human. Typically, the composition is administered to a human suffering from or suspected of suffering from cancer and / or a traumatic brain injury. As used herein, a “subject” or a “patient” is a mammal as described herein. The term “subject” and “patient” can be used interchangeably. A “subject” or “patient” is a mammal, such as a cat, dog, rodent or primate. Typically the subject is a human, and, preferably, a human suffering from or suspected of suffering from a cancer or a traumatic brain injury. The term “subject” and “patient” can be used interchangeably.

[0094] The pharmaceutical compositions and medicaments may be prepared by mixing one or more compounds of the present technology, pharmaceutically acceptable salts thereof, -24- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458stereoisomers thereof, tautomers thereof, or solvates thereof, with pharmaceutically acceptable carriers, excipients, binders, diluents or the like. The compounds and compositions described herein may be used to prepare formulations for imaging and for monitoring metabolic flux. Such compositions can be in the form of, for example, granules, powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions. The instant compositions can be formulated for various routes of administration, for example, by oral, parenteral, topical, rectal, nasal, vaginal administration, or via implanted reservoir. Parenteral or systemic administration includes, but is not limited to, subcutaneous, intravenous, intraperitoneal, and intramuscular, injections. The following dosage forms are given by way of example and should not be construed as limiting the present technology.

[0095] For oral, buccal, and sublingual administration, powders, suspensions, granules, tablets, pills, capsules, gelcaps, and caplets are acceptable as solid dosage forms. These can be prepared, for example, by mixing one or more compounds of the present technology, or pharmaceutically acceptable salts or tautomers thereof, with at least one additive such as a starch or other additive. Suitable additives are sucrose, lactose, cellulose sugar, mannitol, maltitol, dextran, starch, agar, alginates, chitins, chitosans, pectins, tragacanth gum, gum arabic, gelatins, collagens, casein, albumin, synthetic or semi-synthetic polymers or glycerides. Optionally, oral dosage forms can contain other ingredients to aid in administration, such as an inactive diluent, or lubricants such as magnesium stearate, or preservatives such as paraben or sorbic acid, or anti-oxidants such as ascorbic acid, tocopherol or cysteine, a disintegrating agent, binders, thickeners, buffers, sweeteners, flavoring agents or perfuming agents. Tablets and pills may be further treated with suitable coating materials known in the art.

[0096] Liquid dosage forms for oral administration may be in the form of pharmaceutically acceptable emulsions, syrups, elixirs, suspensions, and solutions, which may contain an inactive diluent, such as water. Pharmaceutical formulations and medicaments may be prepared as liquid suspensions or solutions using a sterile liquid, such as, but not limited to, an oil, water, an alcohol, and combinations of these. Pharmaceutically suitable surfactants, suspending agents, emulsifying agents, may be added for oral or parenteral administration.-25- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458

[0097] Suspensions may include oils. Such oils include, but are not limited to, peanut oil, sesame oil, cottonseed oil, corn oil and olive oil. Suspension preparation may also contain esters of fatty acids such as ethyl oleate, isopropyl myristate, fatty acid glycerides and acetylated fatty acid glycerides. Suspension formulations may include alcohols, such as, but not limited to, ethanol, isopropyl alcohol, hexadecyl alcohol, glycerol and propylene glycol. Ethers, such as but not limited to, poly(ethyleneglycol), petroleum hydrocarbons such as mineral oil and petrolatum; and water may also be used in suspension formulations.

[0098] Injectable dosage forms generally include aqueous suspensions or oil suspensions which may be prepared using a suitable dispersant or wetting agent and a suspending agent. Injectable forms may be in solution phase or in the form of a suspension, which is prepared with a solvent or diluent. Acceptable solvents or vehicles include sterilized water, Ringer's solution, or an isotonic aqueous saline solution. An isotonic solution will be understood as isotonic with the subject. Alternatively, sterile oils may be employed as solvents or suspending agents. Typically, the oil or fatty acid is non-volatile, including natural or synthetic oils, fatty acids, mono-, di- or tri-glycerides.

[0099] For injection, the pharmaceutical formulation and / or medicament may be a powder suitable for reconstitution with an appropriate solution as described above. Examples of these include, but are not limited to, freeze dried, rotary dried or spray dried powders, amorphous powders, granules, precipitates, or particulates. For injection, the formulations may optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers and combinations of these.

[0100] Compounds of the present technology may be administered to the lungs by inhalation through the nose or mouth. Suitable pharmaceutical formulations for inhalation include solutions, sprays, dry powders, or aerosols containing any appropriate solvents and optionally other compounds such as, but not limited to, stabilizers, antimicrobial agents, antioxidants, pH modifiers, surfactants, bioavailability modifiers and combinations of these. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aqueous and nonaqueous (e.g., in a-26- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458fluorocarbon propellant) aerosols are typically used for delivery of compounds of the present technology by inhalation.[01011 Dosage forms for the topical (including buccal and sublingual) or transdermal administration of compounds of the present technology include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, and patches. The active component may be mixed under sterile conditions with a pharmaceutically-acceptable carrier or excipient, and with any preservatives, or buffers, which may be required. Powders and sprays can be prepared, for example, with excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. The ointments, pastes, creams and gels may also contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Absorption enhancers can also be used to increase the flux of the compounds of the present technology across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane (e.g., as part of a transdermal patch) or dispersing the compound in a polymer matrix or gel.

[0102] Besides those representative dosage forms described above, pharmaceutically acceptable excipients and carriers are generally known to those skilled in the art and are thus included in the instant present technology. Such excipients and carriers are described, for example, in “Remington’s Pharmaceutical Sciences” Mack Pub. Co., New Jersey (1991), which is incorporated herein by reference.

[0103] The formulations of the present technology may be designed to be short-acting, fastreleasing, long-acting, and sustained-releasing as described below. Thus, the pharmaceutical formulations may also be formulated for controlled release or for slow release.

[0104] The instant compositions may also comprise, for example, micelles or liposomes, or some other encapsulated form, or may be administered in an extended release form to provide a prolonged storage and / or delivery effect. Therefore, the pharmaceutical formulations and medicaments may be compressed into pellets or cylinders and implanted intramuscularly or subcutaneously as depot injections or as implants such as stents. Such implants may employ known inert materials such as silicones and biodegradable polymers.-27- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458

[0105] Specific dosages may be adjusted depending on conditions of disease, the age, body weight, general health conditions, sex, and diet of the subject, dose intervals, administration routes, excretion rate, and combinations of drugs. Any of the above dosage forms containing effective amounts are well within the bounds of routine experimentation and therefore, well within the scope of the instant present technology.

[0106] Those skilled in the art are readily able to determine an effective amount by simply administering a compound of the present technology to a patient in increasing amounts until, for example, metabolic activity may be monitored. The compounds of the present technology can be administered to a patient at dosage levels in the range of about 0.1 to about 1,000 mg per day. For a normal human adult having a body weight of about 70 kg, a dosage in the range of about 0.01 to about 100 mg per kg of body weight per day is sufficient. The specific dosage used, however, can vary or may be adjusted as considered appropriate by those of ordinary skill in the art. For example, the dosage can depend on a number of factors including the requirements of the patient and the pharmacological activity of the compound being used. The determination of optimum dosages for a particular patient is well known to those skilled in the art.

[0107] A compound of the present technology can also be modified, for example, by the covalent attachment of an organic moiety or conjugate to improve pharmacokinetic properties, toxicity or bioavailability (e.g., increased in vivo half-life). The conjugate can be a linear or branched hydrophilic polymeric group, fatty acid group or fatty acid ester group. A polymeric group can comprise a molecular weight that can be adjusted by one of ordinary skill in the art to improve, for example, pharmacokinetic properties, toxicity or bioavailability. Exemplary conjugates can include a polyalkane glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone and a fatty acid or fatty acid ester group, each of which can independently comprise from about eight to about seventy carbon atoms. Conjugates may include polyethylene amine (PEI), polyglycine, hybrids of PEI and polyglycine, polyethylene glycol (PEG) or methoxypolyethylene glycol (mPEG). Conjugates for use with a compound of the present technology can, in one aspect, improve in vivo half-life. Other exemplary conjugates for use with a compound of the present technology as well as-28- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458applications thereof and related techniques include those generally described by U.S. Patent No. 5,672,662, which is hereby incorporated by reference herein.

[0108] The present technology provides methods of identifying a target of interest including contacting the target of interest with a detectable or imaging effective quantity of a compound or composition of the present technology. A detectable or imaging effective quantity is a quantity of a compound of the present technology necessary to be detected by the detection method chosen. For example, a detectable quantity can be an administered amount sufficient to enable detection of binding of the labeled compound to a target of interest including, but not limited to, glioma, cervical carcinoma, vulvar carcinoma, endometrial carcinoma, primary ovarian carcinoma, metastatic ovarian carcinoma, nonsmall cell lung cancer, small cell lung cancer, bladder cancer, colon cancer, primary, gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, and / or prostate cancer. Upon binding of the labeled compound to the target of interest, the target may be isolated, purified and further characterized such as by determining the amino acid sequence of a protein to which the compound of the present technology is bound.

[0109] The examples herein are provided to illustrate advantages of the present technology and to further assist a person of ordinary skill in the art with preparing or using the compounds of the present technology or salts, pharmaceutical compositions, derivatives, solvates, metabolites, prodrugs, racemic mixtures or tautomeric forms thereof. The examples herein are also presented in order to more fully illustrate the preferred aspects of the present technology. The examples should in no way be construed as limiting the scope of the present technology, as defined by the appended claims. The examples can include or incorporate any of the variations, aspects or aspects of the present technology described above. The variations, aspects or aspects described above may also further each include or incorporate the variations of any or all other variations, aspects or aspects of the present technology.EXAMPLES

[0110] Example 1. Materials and Methods.-29- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458

[0111] Reagents. Isotopically enriched [l-13C]ascorbic acid, [1-13C]PA and [2-13C]PA along with deuterium oxide (D2O), sodium acetate, activated charcoal, and methanol (ACS reagent, >99.8%) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Radical AHI 11501 was purchased from GE Healthcare (Chicago, IL, United States). All other reagents and solvents were used directly as supplied by the chemical manufacturers.

[0112] Synthesis Characterization. The DHA synthesis was carried out in oven-dried glassware. 'H and13C nuclear magnetic resonance (NMR) spectroscopy was performed on DHA solutions in D2O using a Bruker AVANCE III 600 MHz (14.1 T) vertical bore NMR spectrometer (Bruker BioSpin, Billerica, MA, United States) at 25°C. NMR spectra of DHA solutions are described by the following nomenclature: 8 = shift and multiplicity s = singlet, m = multiplet, t = triplet, d = doublet, q = quartet, and dd = doublet of doublets) with coupling constants reported in hertz. The melting point was measured using the OptiMelt automated melting point system (Stanford Research System, Sunnyvale, CA, United States). The pH of aqueous solutions was measured using a pH meter (Thermo Fisher Scientific, Waltham, MA). NMR data were processed using the Mnova Software Suite (version 14.3.2, Santiago de Compostela, Spain).

[0113] HP Methods. One hundred microliters of [1-13C]DHA / [1-13C]PA (40 / 60; v / v) doped with 15 mM AHI 11501 trityl radical was prepared. The sample was polarized for 1.5 to 2 hours on a SpinLab hyperpolarizer (5 T, 0.8 K, GE Healthcare) by irradiating it at a 139.88-GHz microwave frequency. After polarization, the frozen sample was dissolved in 9.0 ml of D2O and pyruvate was neutralized by adding 216 pl of a 4 M sodium acetate solution (in D2O) to the dissolute to afford the HP solution of pH ~ 5.5. The HP solution contained 40 mM [ 1-BC] DHA and 90 mM [1-13C]PA. Approximately 700 pl of the HP solution was transferred to a 5-mm NMR tube and loaded to a 1-T Spinsolve Be NMR spectrometer (Magritek, Wellington, NZ) for Be NMR acquisition. NMR spectra were acquired with a flip angle excitation of 5° every 5 s over 4 min. The spin-lattice relaxation time (Ti) was estimated by a monoexponential fit and corrected for the flip angle. Thermal polarization was determined from the average spectrum of 256 scans acquired with a flip angle of 90° every 10 s. Final polarization values, corrected for the flip angles and the apparent Ti were 5 to 10% for all dissolutions. The concentration of13C-DHA and13C-PA in the HP solution-30- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458was measured by Be NMR at 600 MHz in the presence of 6 pl of 1 mM Gd-DOTA and 100 pl of 100 mM [1 -13C] lactate standard added to 500 pl of the HP solution.

[0114] Animals. All animal experiments were conducted under the approval of the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center in accordance with the standards for animal care and use, as set by the federal law under the Animal Welfare Act (13-12-019). Eight female athymic nude mice (the Jackson Laboratory, Bar Harbor, ME, United States) aged 16 to 20 weeks and weighting 28 to 36 g and four female C57BL / 6J mice (the Jackson Laboratory) aged 22 weeks and weighting 30 to 37 g were used in this study . For tumor imaging, three female athymic Foxnlnu mice (Charles River, Wilmington, MA, United States) aged 8 to 10 weeks and weighting 22 to 25 were investigated. Mice were housed in groups of up to five mice per cage with free access to food and water, a 12-hour:12-hour light:dark cycle, temperature of 18° to 23°C, and humidity of 40 to 60%. Before imaging, each mouse underwent tail vein cannulation using a 23 -gauge rodent tail-vein catheter (Braintree Scientific, Braintree, MA, United States) filled with heparin (1 U / ml; Fresenius Kabi, Lake Zurich, IL, United States) in 0.9% of sodium chloride. Anesthesia was performed with inhaled isoflurane mixed with oxygen delivered at a rate of 0.5 to 1 liter / min. Isoflurane concentration was 3% during induction (2 min) and 1.0 to 2% through acquisition. Anesthetized mice were laid prone on a dedicated holder that ensured minimal motion during scans. Respiration rate and body temperature were monitored through an MR-compatible system (Model 1025, SA Instruments Inc., Stony Brook, NY, United States) and maintained at 85 ± 20 breaths / min and 37° ± 0.5°C by regulating the isoflurane concentration and circulating warm water system, respectively.

[0115] Five minutes before the injection of the cohyperpolarized13C probes, the isoflurane flow was turned off to perform the hyperpolarized scan on the awake mouse. Immediately after the hyperpolarized scan, the anesthetic regime was restored until the end of the MRI session.

[0116] Glioma Model — Cell Line. Human U87 glioblastoma cell line (American Type Culture Collection, Manassas, VA, United States) was used for orthotopic xenografts in nude mice. Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Gibco BRL, Thermo Fisher Scientific), penicillin (50 U / ml) and-31- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458streptomycin (250 pg / ml; Gibco) in a humidified 5% CC>2 / 95% air at 37°C. Cells were split when reaching -70% confluence and used until passage 10.

[0117] Glioma Model — Tumor Implantation. Orthotopic human gliomas were implanted in three athymic Foxnlnu mice. Anesthetized mice were placed into a stereotaxic frame (Stoelting Co., Wood Dale, IL, United States), and 1 x 105cells dissociated into a singlecell suspension were injected in a volume of 2 pL of phosphate-buff ered saline via a stereotaxic injector at a flow rate of 2 pL / min (Quintessential Stereotaxic Injector) into the right striatum of the mice (coordinates from bregma: 0.0-mm posterior, 2.0-mm lateral, and 3.0-mm ventral).

[0118] In vivo MRI and Spectroscopy. Experiments were performed on a Bruker BioSpec 3.0-T / 18-cm horizontal bore system (Bruker BioSpin, Billerica, MA, United States) equipped with a high-performance gradient set (max gradient strength: 900 mT / m; slew rate: 4200 mT / m per s). A dual-tunedJH / 13C transmit / receive radio frequency volume coil (30 mm in inner diameter; Bruker BioSpin MRI GmbH, Ettlingen, Germany) was used. After collecting scout images to assess the position of the mouse brain at the magnet isocenter, multislice T2-weighted fast spin echo (TSE) images of mouse brain were acquired in the coronal orientation as an anatomical reference [repetition time (TR) / effective echo time (TEeff) = 2500 / 33 ms, echo train length = 8, field of view = 32 * 32 mm2, matrix size = 128 x 128, in-plane resolution of 250 x 250 pm2, 21 slices of 1 mm in thickness covering the whole mouse brain, two averages, and scan time of 100 s]. Static magnetic field homogeneity was optimized using a localized field-map shimming routine (ParaVision 360, v2.0 pl 1, Bruker BioSpin), resulting in water linewidths of 15 to 23 Hz over the selected volume of the mouse brain. The hyperpolarized13C MRSI scan was acquired on awake mice 25 s after starting the intravenous injection (10-s duration) of the cohyperpolarized13C probes (details on the MR sequence used in each specific experiment are reported below). For quantification purposes, a cylindrical phantom (radius = 0.5 cm; height = 3 cm) containing a 6 M (unless specified)13C-labeled urea solution was placed adjacent to the mouse brain, included in the field of view of T2-weighted and13C MRSI scan.

[0119] In vivo13CNMR Spectroscopy. To trace, in real time, the metabolic flux in a mouse brain through hyperpolarized13C MRS, three independent experiments were-32- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458performed on athymic nude mice (n = 3). Mice were intravenously injected over 10 s with 300 pL of cohyperpolarized 40 mM [1-13C]DHA and 100 mM[l-13C]pyruvate. Following the mouse setup as described above, a13C slice-localized single-pulse-and-acquire sequence (128-ps block pulse at a 15° flip angle, 10-kHz bandwidth, 5-kHz spectral width, 2048 points, one average, 240 repetitions, TR = 1000 ms, 15-mm slice thickness to cover the whole brain, and scan time of 4 min) was used. To monitor the evolution of the metabolic products and substrates in the mouse brain ([l-13C]pyruvate at 171.1 parts per million (ppm), [l-13C]bicarbonate at 161 ppm, [l-13C]alanine at 176 ppm, [1-13C]DHA at 174 ppm, and [l-13C]VitC at 178 ppm), the start of the dynamic13C MRS scan was set immediately after the dissolution DNP of the hyperpolarized substrates and 10 to 15 s before injection of the hyperpolarized13C probes. For each experiment, a cylindrical 4 M urea phantom (163 ppm) was placed adjacent to the mouse brain as a reference for the13C signal normalization.

[0120] In vivo13C EPSI. When studying the regional differences of metabolite conversion in a mouse brain, four independent experiments were performed on C57BL / 6J (n = 4) mice. Following the mouse positioning and shimming optimization, mice were intravenously injected with 250 pL of cohyperpolarized 40 mM [1-13C]DHA and 100 mM [l-13C]pyruvate for 10 s. The13C hyperpolarized scan was acquired in the coronal orientation of the mouse brain using a multislice EPSI sequence (TR / TEeff = 900 / 3.525 ms, field of view = 32 * 32 mm2, flip angle of 41°, image size of 12 x 12, in-plane resolution of 2.667 x 2.667 mm2, six slices of 3.5-mm thickness covering the whole brain, one average, and scan time of 11 s). The13C hyperpolarized EPSI scan was performed 25 s after the start of the injection time, which was found to be optimal for metabolite production in the mouse brain. To normalize the13C signal of the metabolic substrates and products in the mouse brain, the13C EPSI scan was repeated to image the 6 M13C-labeled urea phantom after optimizing the homogeneity of the magnet field over the phantom volume.

[0121] In vivo13C CSI To investigate whether the copolarization of multiple isotope positions, [l-13C]pyruvate and [2-13C]pyruvate along with [1-13C]DHA, could give insights into the compartmentalization of metabolites in the murine brain, four independent experiments were performed on athymic nude mice (n = 4). Following the mouse setup, each mouse underwent the13C hyperpolarized MRI scan 25 s after injecting 250 pL of cohyperpolarized 40 mM [1-13C]DHA, 50 mM [l-13C]pyruvate, and 88 mM [2- -33- 4921-1995-2792.7Atty. Dkt. No.: 115872-345813C]pyruvate. The13C hyperpolarized scan was acquired in the coronal orientation using a 2D multivoxel CSI sequence (TR / TEeff = 141.4 / 5 ms, field of view = 32 * 32 mm2, flip angle of 20°, in-plane resolution of 2.667 x 2.667 mm2, one slice of 15 mm in thickness covering the whole brain, one average, and scan time of 20.4 s). For each mouse, the13C CSI sequence was repeated to image the13C urea phantom for signal normalization purposes, as previously described.

[0122] To investigate brain tumor metabolism, U87 glioma-bearing mice with comparable tumor size (tumor volume ranging from 14 to 20 mm3) were injected with 300 pL of cohyperpolarized 40 mM [1-13C]DHA and 100 mM[l-13C]pyruvate over 10 s. Mice were imaged using the same MRI protocol previously described except for the 5-mm thickness of the CSI, which covered the entire tumor and was centered in it.

[0123] In vivo13C NMR Spectroscopic Data Analysis. First, a Lorentzian apodization of 10 Hz was applied to13C NMR. For each time point, each peak of every spectrum was integrated to generate time varying data, as previously described (42). Experimental signal integrals of all metabolites for each hyperpolarized13C experiment (n = 3) were quantitatively modeled using a custom MATLAB script (MATLAB R2021b, The MathWorks). Kinetic rate constants related to the conversion of [l-13C]pyruvate to [1-13C]lactate (APL), [l-13C]pyruvate to [l-13C]alanine (ATA), [l-13C]pyruvate to [1-13C]bicarbonate (A B), and [1-13C]DHA to [l-13C]VitC (ADV) were calculated using an “inputless” (42). The estimated longitudinal relaxation time (Ti) of each metabolite was set to 50 s with a fitting range extended from 10 to 80 s.

[0124] In vivo13C EPSI Data Analysis. ROIs were defined on the coronal TSE T2-weighted images (FIG. 4A) of a mouse brain. ROIs were drawn symmetrically in the right and left hemispheres of the mouse brain, ensuring their position within the corpus callosum, the largest WM structure (FIGs. 4A-4C) in the brain, and the thalamus, which has a GM structure (FIGs. 4A-4C). For each mouse, T2-weighted images and corresponding ROIs were overlaid on the related MRSI data and metabolic maps, which were calculated using the Spectroscopic Imaging, Visualization and Computing (Sivic 0.9) software package (43). Before computing the metabolite maps of the metabolites ([l-13C]pyruvate and [1-13C]DHA) and downstream metabolites ([l-13C]lactate and [ 1 -13C] VitC), a preprocessing-34- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458step applied to each hyperpolarized13C spectra per slice [Lorentzian apodization of 5 Hz, first-order and second-order phase, and baseline correction; zero-filling interpolation (ZFP) resulting in an image size of 24 x 24; and in-plane resolution of 1.333 x 1.333 mm2] was performed using a custom MATLAB script (MATLAB R2021b, The MathWorks). For each mouse, an ROI-based analysis was performed on each metabolite map overlaid on the corresponding T2-weighted image. The quantified value of each metabolite per brain region was normalized to the quantified value of the 6 M13C-labeled urea phantom, used as a reference.

[0125] In vivo13C CSI Data Analysis. ROIs were defined on the coronal TSE T2-weighted image (FIG. 8A) of a mouse brain. ROIs were drawn to ensure their position within the cortical region (CR) of the mouse brain (FIG. 8A, dark ROI) and the thalamus nuclei, which were named DGM (FIG. 8A, light ROIs) to differentiate from the GM of the CR. For each mouse, the T2-weighted image and corresponding ROIs were overlaid on the multivoxel CSI data, which were analyzed using the jMRUI (44) software package version 7.0 build0.04. A first preprocessing step was applied to each13C spectrum of the hyperpolarized13C MRSI scan (Lorentzian apodization of 3 Hz, first-order and second-order phase, and baseline correction; ZFP resulting in an image size of 36 x 36; and inplane resolution of 0.889 x 0.889 mm2). Quantification of13C NMR spectral peak areas was performed for each spectrum in the CR and DGM brain regions in time domain using the AMARES (Advanced Method for Accurate, Robust, and Efficient Spectral fitting) (45) algorithm of jMRUI, which provides the estimated fit for each individual component, the overall spectrum, and the residual line. For each experiment, the mean values of each quantified metabolite per ROI was determined and normalized to the 6 M13C urea phantom. The same analysis was performed for13C MRSI data acquired on a mouse injected only with [l-13C]pyruvate and [1-13C]DHA to evaluate differences with the metabolite production, especially [5-13C]glutamate in the CR and DGM brain regions.

[0126] Matrix-Assisted Laser Desorption Ionization Imaging Mass Spectrometry.Following in vivo hyperpolarized MRI, mice were euthanized for MALDI (Matrix- Assisted Laser Desorption Ionization Imaging Mass Spectrometry) imaging mass spectrometry (IMS) to assess the glycolytic metabolism. Dissected brains were snap-frozen in 2-Methylbutane (Cat # M0167; Sigma Aldrich, St Louis, MA, USA), placed in dry-ice, and -35- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458pre-cooled at -80 °C. Frozen brains were cryo-sectioned (12-m thick) in the sagittal orientation at -18 °C using a Leica CM 1950 Cryostat (Leica Microsystem GmbH, Wetzlar, Germany) and then thaw mounted onto indium tin oxide (ITO, Delta Technologies, Limited - Loveland, CO, USA) coated glass slides and stored at -80 °C. Prior matrix deposition with N-(l -naphthyl) ethylenediamine dinitrate (NEDN) in negative ion mode and matrix sprayer HTX TM-Sprayer™ (HTX Technologies LLC, NC, USA), ITO-slides were dried for 30 min under a vacuum chamber. Immediately after, they were loaded on a 7T scimaX-MRMS (Magnetic Resonance Mass Spectrometry) system (Bruker Daltonics Inc, Billerica, USA) equipped with a SmartBeam II laser and a MALDI source. Data were acquired with a spatial resolution of 80 pm x 80 pm and acquisition time of 2.5 minutes per mm2. Data visualization and analysis was performed using Scils Lab software (SCiLS GmBH, Bremen, Germany).

[0127] Normalization of13C-labelled Metabolite Signal. For each hyperpolarized session, the MRI protocol included the MRSI phantom scan, performed to normalize the13C signal of each hyperpolarized 13 C-labelled metabolite to the [l-13C]urea signal, used as a reference. During animal positioning, a cylindrical phantom (1 cm diameter, 3 cm heigh) containing a 6M [l-13C]urea solution was placed adjacent to the mouse's head (FIG. 9A) with the main axis parallel to the slice-selection gradient (z-axis of the scanner) in order to cover the full length of the mouse head. Following the13C hyperpolarized MRI scan on the mouse brain (FIGs. 9A-9C) and the shim optimization on the phantom, the MRSI scan was repeated to acquire the13C signal deriving from [l-13C]urea phantom (FIGs. 9A-9C). The13C signal of each hyperpolarized13C-labelled metabolite was normalized for the integrated peak of the urea (FIG. 9C) and corrected for the percentage of the dynamic nuclear polarization determined after dissolution.

[0128] Statistical Analysis. Statistical analysis was performed using GraphPad Prism version 10.0.3 (GraphPad Software, La Jolla, CA, United States). Values are reported as means ± SD for all dataset unless specified otherwise. For slab dynamic,13C MRS experiments (n = 3) were used. For the hyperpolarized13C multislice EPSI experiments (n = 4), statistically significant differences of the mean metabolite values in the WM and GM brain regions were determined by a two-tailed, paired, Student’s t test. The same statistical analysis was applied to the mean metabolite values determined in the CR and -36- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458DGM brain regions when multivoxel hyperpolarized13C CSI experiments (n = 4) were considered. In all statistical analysis, P < 0.05 was taken as significant.

[0129] Example 2. Synthesis of Dehydroascorbic Acid (DHA).[01301 Activated charcoal (7.0 g) was suspended in methanol (250 mL) in a 500 mL Erlenmeyer flask and the charcoal suspension was stirred at room temperature for 1 hour. After an hour, the charcoal suspension was filtered using a Buchner funnel, washed with 30 mL of methanol and dried.

[0131] A 5.0 g of [l-13C]ascorbic acid was taken in a 500 mL Erlenmeyer flask and dissolved in 250 mL of methanol followed by the addition of 6.5 g (1.3 x weight of ascorbic acid) of dried activated charcoal powder to the flask. The resulting mixture was then stirred at room temperature overnight (14 hours). After 14 hours, the complete conversion of ascorbic acid to dehydroascorbic acid was confirmed using13C NMR spectroscopy. After the complete oxidation of ascorbic acid, the reaction mixture was filtered using a Buchner funnel to remove the charcoal and washed with 50 mL of methanol. The resulting filtrate was concentrated on vacuo and dried overnight on high vacuum to remove the traces of methanol to afford dehydroascorbic acid (DHA) as a white crystalline semi-solid (4.5 g, 91.09 % yield). The product was characterized using13C and 'H NMR spectroscopy (600 MHz, Bruker NMR spectrometer) in D2O and stored at -25 °C.1H NMR (600 MHz, D2O): 5 4.65 (d, J = 1.4 Hz, 1H), 4.48 (dd, J = 5.4, 2.6 Hz, 1H), 4.17 (dd, J = 10.4, 5.4 Hz, 1H), 4.06 (dd, J= 10.4, 2.6 Hz, 1H).13C NMR (151 MHz, D2O): 5 173.33, 105.39 (d, J = 11.2 Hz), 91.04 (d, J = 67.6 Hz), 87.29, 75.94, 72.60 (d, J = 1.4 Hz). Melting point of the product was measured to be 29.5±0.6 °C.

[0132] Examples. Stability of DHA.

[0133] Post demonstrating the DHA monomer as an efficient HP13C probe for assessing brain redox, the ability of DHA as a glassing solvent for preparing new HP solution with other crucial metabolites to investigate various metabolic pathways (Table 1) was explored. HP probes were dissolved in aqueous DHA or only DHA or in the DHA / PA (40 / 60; v / v) cocktail to produce the HP formulation consisting of a high substrate concentration and better glassing properties. All the formulations, listed in Table 1, created using DHA, aq.-37- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458DHA or DHA / PA cocktail formed a glass when frozen in liquid N2 which is crucial for getting efficient spectral diffusion and polarization transfer. The stability of dehydroascorbic acid in solution and different temperatures evaluated by13C NMR spectroscopy, which showed formation of impurities via DHA degradation (data not shown).Table 1. Generality of DHA as a co-solvent and substrate in creating efficient HP formulations for multiple crucial metabolites of interest.MetabolicSubstrate Reference Formulation Formulation Activity2- KIC / Glycerol KIC / DHA (70 / 30) ketoisocaproic BCAT activity(5M) (5.907 M) acidkBA / (Glycerol / water; kBA / DHA / PA 2-ketobutyricLDH 50 / 50) (60 / 16 / 24) acid(3.4 M) (7.08 M) 3- Ketone body HBA / DHA (70 / 30) hydroxybutyric Not Reportedmetabolism (7.56 M) acidCellularCholine CHCl / Water CHCI / DHA / H2O phospholipidchloride (~4M) (4.4 M) metabolismFructose / water Fructose / DHATLO D-Fructose Fructolysis(4M) (5.5 M) Glycerate / (Glycerol / water;Glycerate / DHATLO Glycerate C2 cycle 3 / 2)(4M) (3M)Dihydroxy DHAc / Water DHAC / DHA / H2O Gluconeogenesisacetone (8.0 M) (9.26 M)-38- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458

[0134] Example 4. Generality of DHA as a Glassing Agent and Copolarization Substrate for HP Studies.

[0135] To demonstrate the glassing ability of DHA, HP formulations of [2-13C]dihydroxyacetone, [2-13C]glycerate, and [2-13C]fructose in aq. DHA were tested (FIGs.6A-6D) and resulted in significant increase in the13C polarization as compared to previous work. Formulation of 5.5M [2-13C]Fructose in DHA / H2O (FIG. 6A) provided 2.5x fold increase in13C polarization compared to previously obtained polarization of 12% for 4M fructose formulation. For [2-13C]dihydroxyacetone (FIG. 6D),13C polarization increased from 16% to about 19.0% along with the substrate concentration increase of 9.26 M from 8 M. 4M [2-13C]glycerate formulation in DHA / H2O provided the polarization of -15%. It is evident that DHA as a glassing solvent for preparing HP formulations is beneficial for not only achieving higher polarization but also eliminating exogeneous glassing solvents.Additionally, without being bound by theory, DHA can be used both as a glassing solvent as well as a HP13C substrate providing a possibility of investigating multiple metabolic pathways simultaneously.

[0136] Examples. Co-hyperpolarized [1-13C] pyruvate and [1-13C] dehydroascorbate (DHA) to Investigate Metabolism in a Glioma Model.

[0137] To simultaneously evaluate glycolytic metabolism and oxidative stress in a disease state using co-polarized [l-13C]pyruvate and [1-13C]DHA, orthotopic xenografts of U87 glioblastoma were used. Mice underwent a stereotactic orthotopic implantation of U87 glioblastoma cells in the striatum (caudate putamen). Tumor growth was monitored twice a week through MRI. Once tumors reached at least 3 mm in diameter (approximately a volume of 14.2 mm3) mice underwent the13C HP scan. Mice were injected via tail vein over 10s with 300pL of co-hyperpolarized 40 mM [1-13C]DHA and 100 mM [1-13C]pyruvate. Both substrates were co-hyperpolarized for two hours through the SpinLab polarizer (GE, Healthcare) and dissolved in D2O. MR experiments were performed using a 3T MRI system (B inker) equipped with a quadrature double-tuned 1 H / 13C volume coil. The MRI protocol included a T2-weighted sequence for anatomical reference and 13C Chemical Shift Imaging (CSI) sequence (25 x 25 mm2field-of-view, 5 mm thickness to cover the entire tumor) run 25s after substrate injection. Spectroscopic data were processed-39- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458using a custom MATLAB script and analyzed in SIVIC software. Normalization for polarization and concentration were performed using the 6M urea phantom. Comparison between metabolite concentrations in tumor lesion and contralateral brain were compared using a two-tailed, paired, Student’s Ltest.

[0138] Compared with the contralateral brain, increased mean concentration of both substrates, [l-13C]pyruvate (FIG. 8C) and [1-13C]DHA (FIG. 8D), and downstream metabolites, [l-13C]lactate (FIG. 8E) and [l-13C]vitamin C (FIG. 8F), was found. A significantly higher lactate production in tumor lesion than healthy contralateral brain shows the sensitivity and feasibility of the method to detect tumor.

[0139] Example 6. Monomeric Formulation of Dehydroascorbate Facilitates Enhanced HP and Copolarization with Multiple Substrates.

[0140] Conventionally, the bench-stable dimeric form of DHA has been used for studying the properties and activity of DHA using HP13C magnetic resonance spectroscopy (MRS). However, the dimeric form of DHA is not readily soluble at high concentrations in physiological or aqueous solvents. The poor solubility of the DHA dimer in its standard formulation leads to a poor achievable13C polarization (<7%), making the assessment of the in vivo metabolic transformation challenging (26). The DHA dimer is chemically synthesized from the oxidation of ascorbic acid, and the synthesis of the DHA dimer goes through the DHA monomer as an intermediate compound, which is then converted to the bench-stable DHA dimer because the monomeric form of DHA has been perceived as being unstable. The limitations of the dimeric form were addressed by pursuing the possibility of generating a monomeric formulation. In our approach, the reaction intermediate, DHA monomer, was isolated as a colorless semisolid as shown in Scheme 2.Scheme 2. Formation of DHA monomer.HO HO. *■ ? Ck AHO OH Ki OH QH . , .. < 6 14 11 “sX | as£«Bsc acta | l«s^'Cjiddwd rasssss' bfe seid0a gdttte)-40- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458Additionally, the stability of the reaction intermediate (DHA monomer) was investigated (FIG. 1A). At room temperature, degradation of the DHA monomer was observed after 3 hours when in aqueous solution, whereas it was less than 2% up to 4 hours for solid DHA (FIG. 1A). When DHA was kept stored at -25°C, no degradation was observed for up to 2 months (FIG. 1A). Although the DHA monomer is highly soluble in water (up to 8 M), it was dissolved with neat PA in 60 / 40 (PA / DHA; v / v), achieving a homogeneous glassforming formulation, which resulted in high liquid-state polarization for both DHA and pyruvate (-30% for both molecules) with no change in13C 7i (FIGs. 1B-1C). Typically, a neat PA sample polarizes to -35% on our current system; the slight reduction in achieved polarization could be attributed to the dilution of PA from neat (14 M) to 8.8 M in the reported formulation. This approach was further extended to the combination of [1-13C]pyruvate and [2-13C]pyruvate with [1-13C]DHA, finding no loss in performance (FIG.6C). Given the planar geometry of DHA, the inventors hypothesized that, in this neat formulation, it would be amenable to many chemical structures, providing a unique solvent to facilitate efficient polarization transfer. To this end, a range of chemical space was pursued to assess the ability of DHA to be used as a dynamic nuclear polarization (DNP) solvent (FIG. ID and Table 1) Monomeric DHA was found able to solubilize several molecules of biochemical importance to a higher concentration than previously described (Table 1) Moreover, it facilitates significantly higher polarizations than those achieved previously under comparable polarization conditions, thus enabling the simultaneous metabolic investigation of the substrate along with DHA (29-33).

[0141] Example 7. Dynamic HP13C MRS Measures Quantitative Metabolic Fluxes Derives from HP DHA and HP Pyruvate Simultaneously In Vivo.

[0142] Overcoming the limitations of previous attempts to trace the metabolism of HP DHA provides the means of studying the redox-mediated process in the murine brain. Wild-type mice were injected with an HP solution containing [1-13C]DHA and [1-13C]pyruvate, and dynamic13C MRS was performed on the mouse brain (FIGs. 2A-2B).Representative in vivo data demonstrate the delivery and conversion of both substrates to their known products (FIG. 2C). Kinetics of delivery demonstrates comparable time of arrival and sustained metabolic conversion (FIGs. 2D-2E and 7A-7B). With this enhanced sensitivity afforded by the increased polarization, the kinetic rates were fit for each of the -41- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458metabolic fluxes allowing for the ability to compare these pathways in vivo. Given that they are in the same bolus, their direct comparison can be considered. Both An. and PB are defined as standard kinetic rates measured in the human brain. The measured rates in the combined formulation are comparable to previously published measurements of HP pyruvate (FIG. 2F) (15, 18, 19), whereas DV is in line with previous measurements in rat models of HP DHA (FIG. 2G) (34). These findings suggest that the conversion rate is adequate for in vivo detection in humans at the considered concentration and provide support for clinical translation of DHA as an HP imaging agent.

[0143] To further demonstrate the applicability of this approach, simultaneous injection of HP copolarized [l-13C]pyruvate and [1-13C]DHA was applied to a murine U87 glioblastoma model (FIGs. 8A-8F). Both substrates, [l-13C]pyruvate and [1-13C]DHA, show slightly increased delivery to the tumor as expected from a previous work in rat models (FIGs. 8C-8D) (35). Although VitC levels were modestly increased in the tumor, a significant elevation in [l-13C]lactate was observed (FIGs. 8E-8F) analogous to previous reports. This further supports the ability to simultaneously measure these metabolic conversions in both the normal brain and tumor models, here in the mouse brain.

[0144] Example 8. Spatially Resolved HP MRI Reveals Differential Redox Compartmentalization.

[0145] Although exciting as a redox probe, it has been challenging to assess the compartmentalization of these pairs in the murine brain in vivo. Leveraging this previously unknown approach, measurable differences in the gray matter (GM) and white matter (WM) for these fluxes were investigated. To determine these changes, a second cohort of mice was imaged using a two-dimensional (2D) multislice echo-planar spectroscopic imaging (EPSI) approach, allowing for the assessment of all metabolites throughout the brain (FIG.3A). Representative13C spectra demonstrate the ability to spectrally resolve the metabolites and visualize conversions throughout the brain (FIGs. 3B-3C). Integrating these peaks allows for the generation of spatial maps for each metabolite in the simultaneous experiment (FIG. 3D)

[0146] Because data are multislice, they can be reconstructed into a 3D dataset and used to segment regions of GM and WM, providing a direct comparison of relative delivery and -42- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458fluxes in both compartments (FIGs. 4A-4C). By normalizing to the urea phantom (FIGs.9A-9C), the concentrations of HP substrates and products were estimated in vivo (FIGs. 4D-4G, and FIGs. 9A-9B) The calculated absolute concentrations shed light on the achieved substrate concentration in the brain for each molecule, given a bolus injection of them combined. These data would suggest that, although -300 pM pyruvate and DHA are achieved in the GM, a much higher concentration of DHA is achieved in the WM (-500 pM). Given transport dynamics across the blood-brain barrier (BBB) and ultimate uptake in the cell, availability of the substrate locally in time will play an important role in understanding future metabolic fluxes. A relatively increased conversion of pyruvate to lactate was found in the WM, whereas the reverse was seen for DHA with increased VitC localized in the GM (FIG. 4F and FIG. 4G)

[0147] Example 9. Copolarization of Multiple Isotope Positions Annotates Spatially Defined Metabolic Fluxes in the Murine Brain.

[0148] Regional differences in metabolic pools reflect the generation and accumulation of metabolites in the brain. One such metabolic flux is the generation of glutamate in the brain from glycolytic flux to facilitate neurotransmission. To determine whether pyruvate utilization through multiple pathways along with DHA could provide insights into this compartmentalization, the copolarization of [l-13C]pyruvate, [2-13C]pyruvate, and [1-13C]DHA was used. To capture the formation of both13C-bicarbonate from [l-13C]pyruvate and [5-13C]glutamate from [2-13C]pyruvate, imaging was performed within 15 s after injection (FIG. 5A). Moreover, to provide the necessary spectral resolution, a 2D chemical shift imaging (CSI) approach was used. All three products in the murine brain (bicarbonate, glutamate, and VitC) were visualized (see insets of FIG. 5A) . To further confirm that the resonance annotated and adjacent to lactate was C5 glutamate, complimentary in vivo experiments were conducted with only hyperpolarized [l-13C]pyruvate and [1-13C]DHA to demonstrate that the peak was undetected (Fig. 10A).

[0149] Similar to the previous multislice experiment, both Cl and C2 pyruvate were similarly distributed throughout the brain. In contrast, the products Cl lactate and Cl VitC were respectively higher in the cortex and deep GM (DGM) brain regions. Using those metabolites as spatial landmarks, the distribution of other quantified metabolites could be-43- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458compared. Unexpectedly, although bicarbonate was observed in both the cortex and DGM to a similar degree, C5 glutamate appears to only be significantly produced in the cortex region (FIGs. 5B-5I). To determine what mechanistically drives these metabolic fluxes, matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI) (28, 29) was conducted on murine brains (Fig. IOC) (36, 37). By measuring glutamate and lactate pool sizes, a colocalization of these pools with the HP pyruvate-derived conversions was observed, suggesting these pool sizes as the result of metabolic flux in those areas. In contrast, VitC was accumulated to a much higher degree in the cortex, whereas the conversion of HP DHA to VitC occurred more markedly in the DGM (Fig. 10B). Given the mechanism by which DHA is converted to VitC (26), cofactor availability that actually drives its conversion was postulated. The predominant cofactor for DHA reduction in the brain is glutathione (GSH). Moreover, MALDI MSI data revealed high GSH concentrations in the DGM (FIG. 10B), suggesting that DHA was first converted to VitC in the GM and then potentially shuttled to other brain regions outside of the HP timescale. Together, this approach provides a means of annotating a completely different metabolic parameter, spatially defined metabolic flux, as compared to pool sizes measured by traditional methods.

[0150] Example 10. Multi-voxel Chemical Shift Imaging In Vivo with HP [1-13C]DHA and [l-13C]pyruvate.

[0151] To further confirm our findings on the injection of copolarized HP [1-13C]DHA, [1-13C]pyruvate and [2-13C]pyruvate, a 2D multi-voxel Chemical Shift Imaging (CSI) experiment was performed in vivo on an athymic nude mouse by injecting only copolarized HP [1-13C]DHA and [l-13C]pyruvate. The experiment was carried out following the same animal procedures and MRI protocol used for the mice injected with the three probes.Results showing the metabolites peaks detected in the cortical (CR) and deep gray matter (DGM) regions are shown in FIGs. 9A-9B. In both brain regions the [5-13C]glutamate peak at 181.4 ppm is not visible (red arrow). On the contrary, [5-13C]glutamate was detected and quantified in the cortical brain region when mice were injected with the co-polarized three probes (FIG. 5B).-44- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458

[0152] Example 11. Formulation Enables Monitoring of Spatially Defined Metabolic Fluxes in the Murine Brain and is Compatible with Human Studies.

[0153] Given the putative direct link between bioenergetics and brain function as well as the dysregulation of these processes in disease, our understanding of this link in the brain is still poorly understood in humans (1,2). The regulation of brain redox along with the response to oxidative stress in different brain regions both implicated in several brain diseases (e.g., cancer and neurodegenerative diseases) requires new clinical translational methods to measure metabolic fluxes in vivo. Moreover, the degree of compartmentalized metabolism conducted by brain regions is of great interest. To this end, a previously unidentified approach capable of studying brain redox and glycolytic metabolism noninvasively was developed and used to characterize the spatial distribution of metabolic flux.

[0154] The rationale behind this study exploits the possibility of investigating different aspects of brain redox homeostasis through two reactions: the conversion of pyruvate to lactate and DHA to VitC. Both can be traced simultaneously in the brain when investigated with the two HP copolarized probes. Pyruvate and DHA cross the BBB via monocarboxylate transporters and GLUT1 transporters, respectively. Pyruvate is converted to lactate by LDH (lactate dehydrogenase) enzyme, which requires reduced form of NAD+ [nicotinamide adenine dinucleotide] (NADH) oxidation, whereas DHA is reduced to VitC through a reaction dependent on both GSH and NADPH. Therefore, quantification of the metabolites involved to both reactions provide insights into the redox status in the brain by simultaneously sampling both the NAD+ / NADH and NADPH / NADP / GSH pools. In the intact brain, comparable transport dynamics are seen for pyruvate and DHA, whereas in comparison to tumor-bearing mice with orthotopic xenografts, increased pyruvate delivery to the brain is observed with minimal change in DHA. This suggests that pyruvate transport is rate limited by the BBB and DHA transport is not. Differential transporter expression has been demonstrated in models of disease beyond cancer, for example, in loss of neuronal pyruvate dehydrogenase (38), making it extremely important for understanding brain biology.-45- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458

[0155] Using this approach, it was feasible to spatially resolve brain compartments capable of driving more pyruvate reduction to lactate in contrast to regions where more DHA was reduced to ascorbate. Given the redox cofactors and mechanisms that drive each metabolic flux, this approach suggests that the DGM of the murine brain more significantly accumulates ascorbate on this timescale, likely as the site of increased reducing potential. In these brain regions, correlative MALDI MSI also demonstrated larger pool sizes of GSH, the cofactor predominantly responsible for DHA reduction. In contrast, a slightly higher lactate production was found in the murine cortex and WM. These findings are supported by recent translational human studies performed with hyperpolarized Cl pyruvate, where high levels of HP lactate were found in the normal appearing WM (17, 39, 40). However, these differences appeared to be minor (19) when data were normalized to pyruvate delivery (19).

[0156] Using three HP probes simultaneously, the differential pyruvate metabolism along with the DHA compartmentalization was quantified. It was observed that, whereas bicarbonate production resulting from the oxidation of HP Cl pyruvate, such as lactate, appears throughout the murine brain, C5 glutamate production resulting from either the oxidation or multiple tricarboxylic acid (TCA) cycle steps of HP C2 pyruvate predominantly occurs in the cortex although virtually absent in the DGM. The latter corresponds to those brain regions with high glutamate pool size, as shown in MALDI MSI data, suggesting that the metabolic flux through the TCA cycle to this pool is reflective of the brain regions that are responsible for high net flux to glutamate. This is well aligned with both recent studies using13C isotope tracing in the murine brain as well as a recent HP C2 pyruvate study in humans that suggests localization to cortex (41). This highlights the nature of the proposed approach and the uniqueness of the data generated from real-time metabolic flux imaging. Like isotope tracing, this rapid HP MRI approach reveals where first-pass metabolic conversion happens in a spatial region, which may not be correlated to where a metabolite pool size is large. Moreover, given the rapid development of methods to spectroscopically image and encode HP metabolites throughout the brain, application of such approaches to humans could yield an unparalleled amount of human brain metabolic flux data in a single imaging session.-46- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458

[0157] A limitation of the current study is linked to the acquisition strategy used and its parameters (e.g., in-plane nominal resolution and slice thickness) for both multislice EPSI and multivoxel CSI data acquisition. With standard preprocessing, including zero filling, the nominal reconstructed resolution was 1.3 x 1.3 mm2(EPSI) and 0.9 x 0.9 mm2(CSI). To best estimate the metabolite contribution to specific brain regions, high-resolution T2-weighted images were used for segmentation and pixels from the13C data that were predominantly in the region of interest (ROI) were included in analysis to minimize partial volume effects.

[0158] This work suggests the feasibility of such an approach and provides a formulation and methodology to conduct such clinical trials. Previous formulations of DHA were incompatible with clinical translation due to the solvent system used and the inability to generate a polarization state and dose necessary for human studies. The formulation achieved here overcomes all of these limitations, creating a neat DHA formulation.

[0159] Example 12. Synthesis of Deuterated Compounds.

[0160] As described in more detail below, specific deuterated compounds were also explored as probes.

[0161] Synthesis of C-4 Deuterated Ascorbic Acid. Ascorbic acid was synthesized according to Scheme 3, where reaction conditions and yields are provided in Table 2.Scheme 3: Synthetic scheme to achieve the deuteration of ascorbic acid.Base D2O. T ®C in e seated tubeL- Ascorbic AcidTable 2: Reaction parameters and results to achieve deuteration of ascorbic acid.StartingMaterial T (°C) Base Time Deuteration-47- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458L-VitC 105 KOH (2.3 eq) 48 h 76%L-VitC 103 KOH (2.5 eq) 24 h + 8 h 95%L-VitC 101 KOH (2.1 eq) 8 h + 8 h + 8 h 84%L-VitC 110 KOH (2.3 eq) 16 h + 6 h+ 6 h 87%D-IsoVitC 103 KOH (2.3 eq) 5 h + 5 h+ 5 h 83%D-IsoVitC 103 NaOH (3 eq) 26 h 95%L-VitC 101 NaOH (3 eq) 16 h 96%

[0162] Separation of Isomers. Separation of both deuterated isomers using fractional separation by mixing the crude deuterated mixture in boiling acetonitrile and filtrate was kept in the fume hood to cool down and precipitate. The remaining solid was again mixed with boiling acetonitrile and similarly other fraction was collected. This process was repeated several times and about 10 fractions were collected. The collected filtrates were left in a fume hood and allowed to cool down. The precipitation of single isomers in different fractions was seen and verified by13C NMR (data not shown).

[0163] Synthesis of C-4 Deuterated Dehydroascorbic Acid. C-4 deuterated [1-13C] ascorbic acid was synthesized according to Scheme 4.Scheme 4. Synthesis of C-4 deuterated dehydroascorbic acid.Activated Charcoal Methanol 15 h, RT

[0164] The spin-lattice relaxation time of carbon atoms on different compounds was evaluated, and is provided in Table 3 and Table 4.-48- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458Table 3. Spin lattice relaxation time (Ti, seconds) of all carbons for L-ascorbic acid, D-isoascorbic acid, LDHA, D-isoDHA and their C-4 deuterated analogues in D2O. Tis were evaluated using inversion recovery NMR experiment at 600 MHz NMR spectrometer. The Ti elongation for C-4 carbon in all cases can be clearly seen due to the effect of deuteration.D- D- D- L-VitC L-DHACarb L- IsoVitC L- D- IsoDHA IsoVit (deuterat (deuteraton # VitC (deuterat DHA IsoDHA (deuterate C ed) ed)ed) d)Cl 8.181 7.82 7.9 8.07 10.06 8.04 9.66 8.76C2 9.677 9.166 9.61 10.8 35.8 28.53 53.1 44C3 5.933 5.5 6.33 6.99 60.45 54.58 62.83 60.21C4 1.26 1.181 10.43 9.576 1.506 1.355 11 12.8C5 1.193 1.13 1.21 1.302 0.961 0.784 0.78 0.903C6 0.726 0.7 0.711 0.8 1.609 1.44 1.496 1.521Table 4. Spin lattice relaxation time (Ti, seconds) of all carbons for L-ascorbic acid, D-isoascorbic acid, LDHA, D-isoDHA and their C-4 deuterated analogues in D2O. Tis were evaluated using inversion recovery NMR experiment at 600 MHz NMR spectrometer. The Ti elongation for C-4 carbon in all cases can be clearly seen due to the effect of deuteration.L-VitC D-IsoVitC D- Carbon # L-VitC D-IsoVitC L-DHA (deuterated) (deuterated) IsoDHACl 8.02 9.18 7.66 8.6 10.02 9.02-49- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458C2 7.53 8.32 6.95 6.785 19.05 17.34C3 5.11 5.65 5.34 5.345 16.83 16.19C4 1.29 1.42 8.94 9.5 1.59 1.567C5 1.21 1.33 1.188 1.3 1.03 0.95C6 0.755 0.87 0.736 0.798 1.71 1.64

[0165] Synthesis of C-4 Deuterated [I-13C] Ascorbic Acid. C-4 Deuterated [1-13C] ascorbic acid was synthesized as shown below in Scheme 5. Percentage deuteration was calculated using13C NMR spectroscopy (600 MHz) (spectra not shown). The reaction results in two deuterated isomers of vitamin C, L-ascorbic acid (54.1%) and D-isoascorbic acid (45.9%). The deuteration for L-isomer was calculated to be 96.5% and for D-iso isomer 95.6% .Scheme 5. Synthetic scheme to achieve C-4 deuteration of ascorbic acid.NaOH DaO, WI’C 14 h tn a sealed tab®54.% «.S%

[0166] Hyperpolarization Methods for [I-13C] Ascorbic Acid, [1-13C] DHA, C-4 Deuterated [I-13C] Ascorbic Acid, and C-4 Deuterated [1-13C] DHA . As shown by FIGs.11A-11B, 12A-12B, and 13A-13B, [1-13C] ascorbic acid, [1-13C] DHA, C-4 deuterated [1-13C] ascorbic acid, and C-4 deuterated [ 1 -13C] DHA were successfully hyperpolarized, and deuteration of C-4 increases the Ti.

[0167] Example 13. UV-DHA / PA.-50- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458

[0168] The source of electron polarization in conventional DNP sample is trityl, which is a stable radical and remains in the solution post dissolution. Post dissolution, the radical in the solution accelerates the13C polarization decay via electron nuclear dipolar coupling. The presence of radical in the sample force the immediate dissolution of the polarized sample. With radical present, the sample cannot be polarized in advance or transported to the remote location lacking the DNP infrastructure. To achieve a polarized DNP sample that could be stored or transported to other facilities, non-persistent radicals can be employed instead of stable trityl radicals for 13C spin polarization. Using UV-induced nonpersistent radicals as electron source over stable free radicals provides several advantages, such as (i) UV-induced non persistent radical recombines during the dissolution step destroying themselves (ii) Since the radicals are already quenched, it is not required to be removed from the final HP solutions prior to injection (iii) Prevents the loss of a significant part of the polarization during the transfer of hyperpolarized solutions into MRI scanners (iv) UV-radicals can be destroyed post polarization while the polarized sample is still inside the hyperpolarized (UV-radical quenches -195K-200K), therefore providing a polarized sample which could be stored or transported without any significant loss of polarization.

[0169] Preparation of UV-radical based DNP sample. Generating a UV-DNP sample is a two-step process. (1) Making of the formulation - The DNP sample is prepared in a similar fashion as for conventional DNP with persistent radicals by dissolving the substrate of interest in a glassing matrix without doping the sample with trityl. (2) In situ generation of the radical - Once the radical free formulation is achieved, the radicals are generated via UV-photolysis of the sample at 77 K.

[0170] Process of Generating UV Radicals. Once the radical free DNP sample is made, the radicals are generated by following the procedure given below.

[0171] (i) Using a pipette, a 6-10 pL of the solution is dropped into a dewar filled with liquid nitrogen. The dropped solution freezes and forms a spherical glassing bead.

[0172] (ii) The frozen glassing sample beads are then transferred into a quartz dewar filled with liquid nitrogen.-51- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458

[0173] (iii) The quartz dear containing the frozen sample beads (number of beads depends on the amount of sample needed for the experiments).

[0174] (iv) The sample beads are then irradiated using a broadband UV-source (280nm-450 nm) for 10 s and then ESR spectroscopy is performed to evaluate the generated radical concentration.

[0175] (v) Step IV is repeated until the radical concentration reaches a plateau. Fitting the radical concentration vs irradiation time afford the radical generation buildup time constant and maximum concentration generated via the UV-photolysis of the sample.

[0176] Hyperpolarization of a VV-DNP Sample. The UV-irradiated sample beads are transferred to the sample vial, precooled in liquid nitrogen, and the cold sample vial is then lowered into the polarizer for hyperpolarization process. UV radicals are irradiated using microwave frequency to promote efficient polarization transfer.

[0177] Hyperpolarization ofW-irradiated[l-13C]pyruvic acid / [l-13C]DHA. The UV sample is prepared as described above. The sample was dissolved in 5 mL D2O (100 mg / L EDTA) and HP dynamic spectra were acquired on 600 MHz NMR spectrometer. The delay between dissolution and NMR acquisition was between 20-30 s. The polarization of the sample was measured to be 25 %, at the time of first measurement, which is comparable to the polarization achieved from the same sample polarized using trityl radical (FIGs. 14A-14B)

[0178] Example 14. Safety and Tolerability of HP-DHAA / HP-pyruvate in Patients with Brain Tumors.

[0179] The safety and tolerability of HP-DHAA / HP-pyruvate will be evaluated in patients with brain tumors, following a 3+3 design. Dose escalation will include patients with any intracranial mass. Dose expansion would include (1) patients with any grade histologically proven primary glioma or radiographic suspicion for glioma with plan for tissue sampling, and (2) measurable disease as defined by: bidimensional lesions with clearly defined margins (note: lesions do not have to be contrast enhancing), with 2 perpendicular diameters of at least 10 mm, visible on >2 axial slices.-52- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458

[0180] Three intravenous dose levels will be evaluated: 1 mg / kg HP-DHAA with 1.9 mg / kg HP-pyruvate, 2.5 mg / kg HP-DHAA with 4.7 mg / kg HP -pyruvate, and 5 mg / kg HP-DHAA with 9.4 mg / kg HP-pyruvate. DHAA will be co-polarized with pyruvate and prepared as a solution of 7.53 mg / kg HP-DHAA and 14.02 mg / kg HP-pyruvate (HP -DHAA / HP -pyruvate ratio of 0.53).

[0181] Hyperpolarization will take place on the clinical SpinLab hyperpolarizer. The dose used in the expansion cohort will be the maximum safe dose from the dose escalation cohorts. HP-DHAA / HP-pyruvate will be administered as a single intravenous bolus dose.

[0182] Example 15. Clinical Experience with HP-pyruvate, DHAA, and AA.

[0183] Clinical Experience with HP-pyruvate. HP-pyruvate is under investigation in over 30 clinical trials including healthy volunteers, cancer patients (prostate, glioma, breast, cervical, renal, and pancreatic), patients with traumatic brain injury, and patients with cardiovascular disease. MSK holds an IND for the evaluation of HP-pyruvate in patients with histologically confirmed malignant solid tumors and in healthy subjects (IND #125947, NCT02421380 and NCT05041166). HP-pyruvate is well tolerated at doses used for imaging and is rapidly metabolized to naturally occurring compounds. HP-pyruvate has been administered at a dose of 9.5 mg / kg in MSK-sponsored INDs (NCT02421380 and NCT05041166).

[0184] Clinical experience with DHAA. Oral administration of DHAA has been reported twice in studies to evaluate vitamin C levels in the blood and urine following DHAA or AA dosing. In a study evaluating alternating 8-day cycles of daily oral doses of 75 mg or 110 mg per day of DHAA or AA, plasma levels of AA were comparable, while urinary excretion suggested slightly lower utilization of DHAA compared to AA (50). In a study evaluating a single 300 mg dose of DHAA or AA, excretion of AA increased to a similar degree following administration of DHAA or AA, while excretion of DHAA did not increase. Increase in the blood levels of vitamin C were similar, but more rapid in subjects administered DHAA compared to AA (51).

[0185] The proportion of DHAA in foods has not been reported, and quantification of vitamin C does not typically discern between DHAA and AA, though in any solution of-53- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458vitamin C at steady state, there exists a small percentage of DHAA in equilibrium. The recommended dietary allowance of vitamin C is 90 mg / day and 75 mg / day for men and women, respectively. Pregnancy and lactation increase the allowance in women to 85 mg / day and 120 mg / day, and smokers are recommended to add 35 mg / day additional vitamin C is approved for injection at a maximum dose of 200 mg (33 mg / min infusion rate) once daily up to 7 days for the treatment of scurvy in adult and pediatric patients.

[0186] Based on this knowledge, plasma samples from patients with resectable or metastatic solid tumor malignancies that received a high intravenous dose of vitamin C (NCT03146962, IND#132719) were examined. Patients in this study received 4 doses of 1.25 g / kg vitamin C (3 hour infusion time) per week for a minimum of one week and a maximum of six months. Plasma samples, taken immediately after 1.25 g / kg vitamin C infusion, were determined to have DHAA concentrations ranging from 0.7-1.2 mg / mL (Table 5), which corresponds to approximately 27-96 mg / kg. Because samples were taken immediately after vitamin C dosing, oxidation of vitamin C to DHAA is not expected to contribute to measured values of DHAA. The mean value of 61.0mg / kg is 61x our proposed starting dose of 1 mg / kg.Table 5. DHAA values measured in plasma of patients receiving vitamin C infusions.Sample DHAA Cone. Patient DHAA Cone, Name (mg / mL) Weight (kg) (mg / kg)PT 04 Post 1.23 88 69.8PT 05 Post 1.08 94.8 56.7PT 06 Post 0.68 122.5 27.8PT 07 Post 0.72 64 56.1PT 11 Post 0.53 95.3 27.6PT 13 Post 0.81 49.4 82.3PT 15 Post 0.68 62.6 54.5PT 16 Post 0.92 67.1 68.3PT 17 Post 0.76 54 70.6PT 19 Post 1.00 52.2 96.0-54- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458Average 0.84 61.0

[0187] Example 16. Nonclinical Safety and Toxicity Data.

[0188] DHAA has been administered to several animal species in doses with human equivalents far exceeding the proposed human starting dose of 1 mg / kg HP -DHAA.Serious toxicities were observed at very high or repeated doses exceeding 1000 mg / kg; DHAA is otherwise well tolerated with transient side effects such as salivation, lacrimation, and hyperactivity at high doses. These studies are summarized in Table 6.Table 6. Animal studies of injected DHAA.Injected DHAA dose Animal Human Equivalent Dose Reference 40, 250, or 500 mg / kg (IV) Mouse 3.3 mg / kg, 20.3 mg / kg, 5240.6 mg / kg100 mg / kg (IP) Rat 16.1 mg / kg 54 666.67 mg / kg / day x 3 days, 107.5 mg / kg / day x 3 days, 55 Ratl.lg / kg, 1.5g / kg (IV) 177.4 mg / kg, 241 mg / kg200+700 mg / kg (IV) Rat 32.3+112.9 mg / kg 53 800 to 1650 mg / kg (IV) Rat 129 to 266.1 mg / kg 56 700 to 800 mg / kg / day x 2 days Rat 112.9 to 129 mg / kg / day x 2 57 (IV) days500 mg / kg (IV, over 30 min) Baboon 278 mg / kg 58

[0189] In a study of DHAA as a potential cerebroprotectant for ischemic stroke, mice were injected via tail vein with 40, 250, or 500 mg / kg of AA, DHAA, or vehicle before or after cerebral ischemia. No side effects were reported (52). A study of ischemic stroke in rats utilized a single intraperitoneal dose of 100 mg / kg / mL DHAA via intraperitoneal injection. Side effects were not reported (54). In a study on the effects of DHAA on the central nervous system, Sprague-Dawley rats were injected intravenously with 200 mg / kg DHAA followed by 700 mg / kg DHAA after 20-30 minutes. The first dose caused a short period of hyperactivity, followed by salivation and lacrimation in two-thirds of rats. The second dose -55- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458caused salivation and lacrimation in 92% and 77% of rats, respectively (53). Several studies have reported diabetogenesis in rats following intravenous injection of very high doses of DHAA (53 and 55-57). Among these, the lowest diabetogenic single dose was 1,100 mg / kg, equivalent to a human dose of 177.4 mg / kg (55). Others utilized repeat doses such as -666.67 mg / kg / day for 3 days or 700 mg / kg / day for 2 days. These studies noted increased accumulation of DHAA in the liver and pancreatic islets of adult mice and rats.

[0190] A postischemic stroke study was conducted in adult male baboons. Cohorts of baboons (n=18) received either vehicle or 500 mg / kg DHAA 3 hours after ischemic stroke. The study was terminated at day 10 post-ischemia due to the results of a futility analysis. Side effects were not reported (58). The dose of 500 mg / kg is equivalent to a human dose of 278 mg / kg, a safety factor of 278x above our proposed starting dose of 1 mg / kg.

[0191] A non-GLP toxicology study in mice in the MSK Antitumor Assessment Core was conducted. Cohorts of 6 animals were euthanized 24 hours after a single injection of13C-DHAA,13C-pyruvate or13C-DHAA+13C-pyruvate to assess potential acute toxicity and cohorts of 3 animals were monitored for 2 additional weeks for reversible or delayed toxicity (Table 7). There were no significant differences in body weights (FIG. 15), hematology, and chemistry laboratory serum testing. At interim and final sacrifice, there were no pathologic findings that were attributed to the test articles. Mice from all treatment groups were noted to have dark spots on their spleens. Further pathology testing determined that the spots did not correlate to any histopathological findings and were determined to be consistent with normal background findings for the mouse strain. This study demonstrated safety at mouse dose up to lOOmg / kg DHAA, supporting a human equivalent dose of 8.1 mg / kg.Table 7. Non-GLP toxicology study in mice.ConcentrationGroup Solution Dose (mg / kg) Survival (mM)Vehicle Control1 187 mM N / A 6 / 6(Sodium Acetate)213C-DHAA High Dose 85 mM 150 mg / kg 6 / 6-56- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458313C-DHAA Mid Dose 57 mM 100 mg / kg 6 / 6 413C-pyruvate 187 mM 164.6 mg / kg 5 / 6150 mg / kg +513C-DHAA +13C-pyruvate 85 mM + 187 mM 5 / 6164.6 mg / kg57 mM + 125.4 100 mg / kg +613C-DHAA +13C-pyruvate 6 / 6 mM 110.3 mg / kg70 mg / kg +713C-DHAA +13C-pyruvate 40 mM + 88 mM 6 / 677.4 mg / kg

[0192] Example 17. Toxicology Studies of HP-DHAA / HP-pyruvate in Rats.[01931 A GLP-compliant toxicology study will be performed on rats as summarized in Table 8. The DHAA dose levels will be equivalent to human doses of 1 mg / kg, 5 mg / kg, and 10 mg / kg, and will be injected with a pyruvate dose consistent with the DHAA / pyruvate ratio planned in humans. Each treatment and control condition will include groups sacrificed at two weeks post-injection (Final Sacrifice) and 24 hours postinjection (Interim Sacrifice), and an additional group for toxicokinetic analysis (TK). Groups are summarized in Table 9. Parameters to be evaluated in the study are summarized in Table 10.Table 8. GLP toxicology study in rats.Compliance GLPSpecies / Strain Rat / Sprague-Dawley, male and female, age 6-9 weeks Dosing regimen and Single dose, IVroute of administrationTreatment groups 6.2 mg / kg DHAA + 11.7 mg / kg pyruvate (low), 31 mg / kg DHAA + 58.5 mg / kg pyruvate (med), 62 mg / kg DHAA + 117 mg / kg pyruvate (high)Control groups Vehicle, 62 mg / kg DHAA (high)Table 9. Treatment and control groups for GLP toxicology study.-57- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458Group Animals / group Sex Interim Sacrifice (24 hour)1. Vehicle Control 10 M 2. Vehicle Control 10 F 3. DHAA - high dose 10 M 4. DHAA - high dose 10 F 5. DHAA / pyruvate - low dose 10 M 6. DHAA / pyruvate - low dose 10 F 7. DHAA / pyruvate - med dose 10 M 8. DHAA / pyruvate - med dose 10 F 9. DHAA / pyruvate - high dose 10 M 10. DHAA / pyruvate - high dose 10 F Final Sacrifice (two week)11. Vehicle Control 5 M 12. Vehicle Control 5 F 13. DHAA - high dose 5 M 14. DHAA - high dose 5 F 15. DHAA / pyruvate - low dose 5 M 16. DHAA / pyruvate - low dose 5 F 17. DHAA / pyruvate - med dose 5 M 18. DHAA / pyruvate - med dose 5 F 19. DHAA / pyruvate - high dose 5 M 20. DHAA / pyruvate - high dose 5 F TK / Cytokine Assessment21. Vehicle Control 3 M 22. Vehicle Control 3 F 23. DHAA - high dose 3 M 24. DHAA - high dose 3 F 25. DHAA / pyruvate - low dose 3 M 26. DHAA / pyruvate - low dose 3 F 27. DHAA / pyruvate - med dose 3 M-58- 4921-1995-2792.7Atty. Dkt. No.: 115872-345828. DHAA / pyruvate - med dose 3 F29. DHAA / pyruvate - high dose 3 M30. DHAA / pyruvate - high dose 3 F

[0194] Table 10. Parameters for evaluation.All animals observed for mortality / moribundity once Mortality / moribunditydailyDetailed clinical observations will be recorded at least Clinical observations twice weekly before test article administration, weekly during the recovery period, and once prior to necropsy. Once immediately following dosing (within 15 minutes), Cage-side observationsthen once dailyBody weights will be recorded at least twice weekly Body weight before test article administration, weekly during the recovery period, and once prior to necropsy.Hematology, clinical chemistry, coagulation, urinalysis Clinical pathologyonce prior to necropsyBlood collected at 8 time points post-injection in TK treatment groups and 2 time points post-injection in TK Toxicokineticscontrol groups. TK groups will be discarded after euthanasia without further evaluation.Interim sacrifice one day post-injection and final Necropsysacrifice two weeks post-injectionLiver, spleen, testes / ovaries, adrenals, kidneys, heart, Organ weightsthymus, brainAll tissues from control groups (vehicle and DHAA- high) and DHAA / pyruvate (high dose only), including Microscopic evaluation interim and final sacrifice animals, will be processed to slides and evaluated by a pathologist. Test article related findings in the DHAA / pyruvate (high) group will be-59- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458subsequently evaluated in the DHAA / pyruvate (mid) group following by DHAA / pyruvate (low, if necessary).

[0195] Example 18. Dosing Studies.

[0196] Dosing studies will begin with a dose of 1 mg / kg HP-DHAA, which is a conservative dose, with a safety factor 6 lx lower than the measured level of DHAA in patients administered high doses of vitamin C and >5x lower than the highest dose that can be made with the instrument (7.53 mg / kg HP-DHAA). The highest proposed dose of 5 mg / kg HP-DHAA will maintain a ~12x safety factor below this measured level of DHAA.

[0197] Hyperpolarized (HP) [ 1 -13C] pyruvate is a non-radioactive investigational MRI imaging agent of cancer glycolysis (MSK trials NCT02421380, NCT05041166). This study will evaluate the safety of a single injection of HP [13C] pyruvate combined with HP [ 1 -13C] dehydroascorbate, a new magnetic resonance imaging (MRI) probe developed at MSK that can image oxidative stress. The broader goal of this study is to develop a sensitive and precise probe for cancer progression and response to treatment.

[0198] This will be a single-center, open label, first in human study of HP [ 1 -13C] pyruvate / dehydroascorbate for injection in patients with intracranial tumors. HP [ 1 -13C] dehydroascorbate will be co-polarized with [ 1 -13C] pyruvate under GMP compliant conditions, weight-adjusted, and administered by bolus intravenous injection. Changes in pyruvate, dehydroascorbate and their metabolites will be evaluated by non-invasive MRI. This research MRI procedure will be performed at the time of the standard of care MRI planned for these patients, adding approximately 15 min to the procedure, and will not require patients to return for another MRI.

[0199] The dose escalation phase of this study will include cohorts of up to 6 patients that will receive escalating, single dose amounts of HP -DHAA / HP -pyruvate up to a maximum of 18 patients. Dose levels will be 1 mg / kg HP-DHAA with 1.9 mg / kg HP-pyruvate, 2.5 mg / kg HP-DHAA with 4.7 mg / kg HP-pyruvate, and 5 mg / kg HP-DHAA with 9.4 mg / kg HP-pyruvate. Adverse events will be monitored. No blood samples will be drawn. Dose escalation will be followed by a dose expansion phase which will consist of 10 patients with -60- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458primary gliomas, 5 IDH-mutant and 5 IDH-wildtype. During this phase, spectroscopic imaging parameters will be collected to assess the rate of dehydroascorbate conversion to Vit C in spatially resolved areas of normal brain and tumor.

[0200] The study will proceed as shown in the scheme shown in FIG. 16. The study will include patients age 18 or older with any intracranial mass that are negative for pregnancy (blood or urine) two weeks prior to study enrollment, and that have not received radiotherapy 1 week prior or less to HP-pyruvate / HP-DHAA injection.

[0201] In the dose expansion phase, patients with any grade of histologically proven primary glioma or radiographic suspicion for glioma with plan for tissue sampling, and with measurable disease (bidimensional lesions with clearly defined margins, with 2 perpendicular diameters of at least 10 mm, visible on >2 axial slices) will be included.

[0202] Patients with acute major illness associated with unstable cardiac condition, renal creatinine >1.5 x ULN or creatinine clearance < 60 mL / min within 3-4 weeks prior to study enrollment or with a history of clinically significant arrhythmias such as sustained ventricular tachycardia, persistent atrial fibrillation not adequately controlled with medication, uncontrolled supraventricular tachycardia, or recent (within 3 months) episodes of polymorphic ventricular tachycardia, cardiac arrest, or structural heart disease that could contribute to arrhythmia development will be excluded. Prolonged QTc and PR intervals on screening EKG will be assessed individually and only considered if repeat testing is within normal limits.

[0203] This first human study of hyperpolarized [1-13C] dehydroascorbic acid atMSKCC will be an open-label study. Patients with intracranial tumors of any kind are eligible to enroll into this study, providing all other eligibility criteria are met. The human pharmacokinetics and metabolism will be studied through MRI based imaging studies in the region of interest.

[0204] If the standard clinical MRI scan includes contrast enhanced MRI, this will be conducted after the HP MR study. The research scan will be conducted analogously to the clinical scan. For all studies, the clamshell RF transmitter will be used to excite the MR signal. Reception will be conducted using the appropriate MR head coil. In the case of -61- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458patients who already have completed a clinical staging MRI examination, HP-DHAA / HP-pyruvate imaging will be done as a standalone research examination including anatomic MRI.

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Metabolic imaging of patients with prostate cancer using hyperpolarized [1-13C] pyruvate. Sci TranslMed 5(198). 2013. doi.org / 10.1126 / scitranslmed.3006070.50. Sabry JH, Fisher KH, Dodds ML. Human utilization of dehydroascorbic acid. JNutr 64(3). 1958. doi.org / 10.1093 / jn / 64.3.457.51. Teruuchi J, Mochizuki H. Metabolism of dehydroascorbic acid in human subjects. J Vitaminol 6. 1960. Doi.org / 10.5925 / jnsvl954.6.163.-66- 4921-1995-2792.7Atty. Dkt. No.: 115872-345852. Huang J, Agus DB, Winfee CJ, Kiss S, Mack WJ, McTaggart RA, Choudhri TF, Kim LJ, Mocco J, Pinsky DJ, Fox WD, Israel RJ, Boyd TA, Golde DW, Connolly ES. Dehydroascorbic acid, a blood-brain barrier transportable form of vitamin C, mediates potent cerebroprotection in experimental stroke. Proc Natl Acad Sci 98(20). 2001. Doi.org / 10.1073 / pnas.171325998.53. Patterson J, Mastin DW. Some effects of dehydroascorbic acid on the central nervous system. Am J Phsyiol 167(1). 1951.Doi.org / 10.1152 / ajplegacy.1951.167.1.119.54. Song J, Park J, Kim JH, Choi JY, Kim JY, Lee KM, Lee JE. Dehydroascorbic acid attenuates ischemic brain edema and neurotoxicity in cerebral ischemia: an in vivo study. Exp Neurobiol 24(1). 2015. Doi. org / 10.5607 / en.2015.24.1.41.55. Patterson JW. The diabetogenic effect of dehydroascorbic and dehydroisoascorbic acids. J Biol Chem 183(1). 1950. doi.org / 10.1016 / S0021-9258(18)56445-0.56. Bhattacharya SK, Robson JS, Stewart CP. The glutathione content of blood and tissues following alloxan and dehydroascorbic acid injections. Biochem J 62(1).1956. Doi. org / 10.1042 / bj 0620012.57. MacDonald MK, Bhattacharya SK. Histological changes in rats rendered hyperglycaemic by injection of dehydroascorbic acid. Q J Exp Physiol CognMed Sci 41(2). 1956. Doi.org / 10.1113 / expphysiol.1956. spOOl 172.58. Ducruet AF, Mack WJ, Mocco J, Hoh DJ, Coon AL, D’ Ambrosio AL, Winfree CJ, Pinsky DJ, Connolly ES. Preclinical evaluation of postischemic dehydroascorbic acid administration in a large-animal stroke model. Transl Stroke Res 2. 2011. Doi.org / 10.1007 / sl2975-011-0084-2.

[0206] While certain embodiments have been illustrated and described, a person with ordinary skill in the art, after reading the foregoing specification, can effect changes, substitutions of equivalents and other types of alterations to the compounds of the present technology or salts, pharmaceutical compositions, derivatives, prodrugs, metabolites, tautomers or racemic mixtures thereof as set forth herein. Each aspect and embodiment described above can also have included or incorporated therewith such variations or aspects as disclosed in regard to any or all of the other aspects and embodiments.

[0207] The present technology is also not to be limited in terms of the particular aspects described herein, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the -67- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458art. Functionally equivalent methods within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. It is to be understood that this present technology is not limited to particular methods, reagents, compounds, compositions, labeled compounds or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. Thus, it is intended that the specification be considered as exemplary only with the breadth, scope and spirit of the present technology indicated only by the appended claims, definitions therein and any equivalents thereof.

[0208] The embodiments, illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. Additionally, the phrase “consisting essentially of’ will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase “consisting of’ excludes any element not specified.

[0209] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0210] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any-68- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member.[02111 All publications, patent applications, issued patents, and other documents (for example, journals, articles and / or textbooks) referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

[0212] The present technology may include, but is not limited to, the features and combinations of features recited in the following lettered paragraphs, it being understood that the following paragraphs should not be interpreted as limiting the scope of the claims as appended hereto or mandating that all such features must necessarily be included in such claims:A. A composition comprising a first hyperpolarized probe, whereinthe first hyperpolarized probe is a compound that has been subjected to hyperpolarization and comprises dehydroascorbic acid, dehydroascorbate, or a mixture thereof.B. The composition of Paragraph A, further comprising a second hyperpolarized probe wherein the second hyperpolarized probe is a compound that has been subjected to hyperpolarization.C. The composition of Paragraph B, wherein the second hyperpolarized probe comprises pyruvic acid, pyruvate, or a mixture thereof.-69- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458D. The composition of any one of Paragraphs A-C, wherein the hyperpolarization of the first probe and / or hyperpolarization of the second hyperpolarized probe is performed via Para-Hydrogen Induced Polarization (PHIP), Dynamic Nuclear Polarization (DNP), Spin-Exchange Optical Pumping, or other Gas-Induced Polarization.E. The composition of any one of Paragraphs A-D, wherein the hyperpolarization of the first probe and / or hyperpolarization of the second hyperpolarized probe is performed via Dynamic Nuclear Polarization (DNP).F. The composition of any one of Paragraphs A-E, wherein the first hyperpolarized probe and / or the second hyperpolarized probe comprises an NMR active nucleus.G. The composition of Paragraph F, wherein the active nucleus comprises 'H,2H,13C,31P,15N, or19F.H. The composition of Paragraph F or Paragraph G, wherein the active nucleus comprises 'H,2H, or13C.I. The composition of any one of Paragraphs A-H, wherein the first hyperpolarized probe comprises [l-13C]dehydroascorbic acid, [l-13C]dehydroascorbate, [1-13C, 4-2H]dehydroascorbic acid, [1 -13C, 4-2H]dehydroascorbate or a mixture of any two or more thereof.J. The composition of any one of Paragraphs B-I, wherein the second hyperpolarized probe comprises [l-13C]pyruvic acid, [2-13C]pyruvic acid, [l-13C]pyruvate, [2-13C]pyruvate, or a mixture of any two or more thereof.K. The composition of any one of Paragraphs A-J comprising[l-13C]dehydroascorbic acid, [l-13C]dehydroascorbate, [1-13C, 4-2H]dehydroascorbic acid, [1 -13C, 4-2H]dehydroascorbate, or a mixture of any two or more thereof; and[l-13C]pyruvic acid, [2-13C]pyruvic acid, [l-13C]pyruvate, [2-13C]pyruvate, or a mixture of any two or more thereof.L. The composition of any one of Paragraphs A-K, further comprising a pharmaceutically acceptable carrier.-70- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458M. The composition of any one of Paragraphs A-L, formulated for parenteral administration.N. The composition of any one of Paragraphs A-M, formulated for intravenous administration.O. A method of imaging, the method comprising:administering to a subject an effective amount of a composition comprising a first hyperpolarized probe such that the first hyperpolarized probe contacts one or more mammalian cells;detecting by magnetic resonance the first hyperpolarized probe and / or one or more metabolites of the first hyperpolarized probe in the one or more mammalian cells;wherein the first hyperpolarized probe is a compound that has been subjected to hyperpolarization and comprises dehydroascorbic acid, dehydroascorbate, or a mixture thereof.P. The method of Paragraph O, further comprisingadministering to the subject an effective amount of a composition comprising a second hyperpolarized probe such that the second hyperpolarized probe contacts one or more mammalian cells; anddetecting by magnetic resonance the second hyperpolarized probe and / or one or more metabolites of the second hyperpolarized probe in the one or more mammalian cells;wherein the second hyperpolarized probe is a compound that has been subjected to hyperpolarization.Q. The method of Paragraph P, wherein the second hyperpolarized probe comprises pyruvic acid, pyruvate, or a mixture thereof.R. The method of any one of Paragraphs O-Q, wherein the hyperpolarization of the first hyperpolarized probe and / or hyperpolarization of the second hyperpolarized probe is performed via Para-Hydrogen Induced Polarization (PHIP), Dynamic Nuclear-71- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458Polarization (DNP), Spin-Exchange Optical Pumping, or other Gas-Induced Polarization.S. The method of any one of Paragraphs O-R, wherein the hyperpolarization of the first hyperpolarized probe and / or hyperpolarization of the second hyperpolarized probe is performed via Dynamic Nuclear Polarization (DNP).T. The method of any one of Paragraphs O-S, wherein the hyperpolarization of the first hyperpolarized probe and / or hyperpolarization of the second hyperpolarized probe comprises introducing a radical source.U. The method of Paragraph T, wherein the radical source comprises an organic persistent free radical, photo-induced non-persistent radicals, nanodiamond, or metabolite which can form temporary radicals, silicon particles, or a mixture of any two or more thereof.V. The method of any one of Paragraphs O-U, wherein the first hyperpolarized probe and / or the second hyperpolarized probe comprises an NMR active nucleus.W. The method of Paragraph V, wherein the active nucleus comprises1H,2H,13C,31P,15N, or19F.X. The method of Paragraph V or Paragraph W, wherein the active nucleus comprises1H,2H, or13C.Y. The method of any one of Paragraphs O-X, wherein the first hyperpolarized probe comprises [l-13C]dehydroascorbic acid, [l-13C]dehydroascorbate, [1-13C, 4-2H]dehydroascorbic acid, [1 -13C, 4-2H]dehydroascorbate, or a mixture of any two or more thereof.Z. The method of any one of Paragraphs P-Y, wherein the second hyperpolarized probe comprises [l-13C]pyruvic acid, [2-13C]pyruvic acid, [l-13C]pyruvate, [2-13C]pyruvate, or a mixture of any two or more thereof.AA. The method of any one of Paragraphs O-Z, wherein the administering comprises parenteral administration.-72- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458AB. The method of any one of Paragraphs O-AA, wherein the administering comprises intravenous administration.AC. The method of any one of Paragraphs O-AB, wherein the one or more metabolites of the first hyperpolarized probe comprises vitamin C, optionally wherein the one or more metabolites of the first hyperpolarized probe comprises [l-13C]vitamin C and / or [1-13C, 4-2H]vitamin C.AD. The method of any one of Paragraphs P-AC, wherein the one or more metabolites of the second hyperpolarized probe comprises lactate, optionally wherein the one or more metabolites of the second hyperpolarized probe comprises [l-13C]lactate.AE. The method of any one of Paragraphs O-AD, wherein the one or more mammalian cells comprises one or more cancer cells.AF. The method of Paragraph AE, wherein the one or more cancer cells comprises one or more of prostate, glioma, breast, cervical, renal, pancreatic, sarcoma, lung, oral, epithelial, or brain cancer cells.AG. The method of any one of Paragraphs O-AF, wherein the subject is suspected of having a cancer, a traumatic brain injury, and / or a cardiovascular disease.AH. The method of any one of Paragraphs O-AG, wherein the subject is diagnosed as having a cancer, a traumatic brain injury, and / or a cardiovascular disease.Al. The method of any one of Paragraphs P-AH, wherein the first hyperpolarized probe and the second hyperpolarized probe are copolarized.AJ. The method of any one of Paragraphs P-AI, wherein detecting by magnetic resonance comprises simultaneously detecting one or more metabolites of the first hyperpolarized probe and one or more metabolites of the second hyperpolarized probe.AK. The method of any one of Paragraphs O-AJ, wherein the magnetic resonance comprises2H magnetic resonance.-73- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458AL. The method of any one of Paragraphs O-AK, wherein the first hyperpolarized probe and / or the second hyperpolarized probe is deuterated, and wherein the deuterated probe exhibits a longer Ti relaxation time than its nondeuterated analog.AM. The method of any one of Paragraphs O-AL, wherein the subject is human.AN. A method of measuring metabolic flux in a subject, the method comprising:administering to the subject an effective amount of a composition comprising a first hyperpolarized probe such that the first hyperpolarized probe contacts one or more mammalian cells;detecting by magnetic resonance at a plurality of timepoints the first hyperpolarized probe and / or one or more metabolites of the first hyperpolarized probe in the one or more mammalian cells; anddetermining a metabolic flux of the first hyperpolarized probe based on a rate of conversion of the first hyperpolarized probe to the one or more metabolites of the first hyperpolarized probe;wherein the first hyperpolarized probe is a compound that has been subjected to hyperpolarization and comprises dehydroascorbic acid, dehydroascorbate, or a mixture thereof.AO. The method of Paragraph AN, further comprisingadministering to the subject an effective amount of a composition comprising a second hyperpolarized probe such that the second hyperpolarized probe contacts one or more mammalian cells; anddetecting by magnetic resonance at a plurality of timepoints the second hyperpolarized probe and / or one or more metabolites of the second hyperpolarized probe in the one or more mammalian cells;wherein the second hyperpolarized probe is a compound that has been subjected to hyperpolarization.AP. The method of Paragraph AO, wherein the second hyperpolarized probe comprises pyruvic acid, pyruvate, or a mixture thereof.-74- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458AQ. The method of any one of Paragraphs AN-AP, wherein the hyperpolarization of the first hyperpolarized probe and / or hyperpolarization of the second hyperpolarized probe is performed via Para-Hydrogen Induced Polarization (PHIP), Dynamic Nuclear Polarization (DNP), Spin-Exchange Optical Pumping, or other Gas-Induced Polarization.AR. The method of any one of Paragraphs AN-AQ, wherein the hyperpolarization of the first hyperpolarized probe and / or hyperpolarization of the second hyperpolarized probe is performed via Dynamic Nuclear Polarization (DNP).AS. The method of any one of Paragraphs AN-AR, wherein the hyperpolarization of the first hyperpolarized probe and / or hyperpolarization of the second hyperpolarized probe comprises introducing a radical source.AT. The method of Paragraph AS, wherein the radical source comprises an organic persistent free radical, photo-induced non-persistent radicals, nanodiamond, or metabolite which can form temporary radicals, silicon particles, or a mixture of any two or more thereof.AU. The method of any one of Paragraphs AN- AT, wherein the first hyperpolarized probe and / or the second hyperpolarized probe comprises an NMR active nucleus.AV. The method of Paragraph AU, wherein the active nucleus comprises 'H,2H,13C,31P,15N, or19F.AW. The method of Paragraph AU or Paragraph AV, wherein the active nucleus comprises 'H,2H, or13C.AX. The method of any one of claims Paragraphs AN-AW, wherein the first hyperpolarized probe comprises [l-13C]dehydroascorbic acid, [1-13C]dehydroascorbate, [1-13C, 4-2H]dehydroascorbic acid, [1-13C, 4-2H]dehydroascorbate, or a mixture of any two or more thereof.AY. The method of any one of Paragraphs AM- AX, wherein the second hyperpolarized probe comprises [l-13C]pyruvic acid, [2-13C]pyruvic acid, [l-13C]pyruvate, [2-13C]pyruvate, or a mixture of any two or more thereof.-75- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458L. The method of any one of Paragraphs AN-AY, wherein the administering comprises parenteral administration.BA. The method of any one of Paragraphs AN-AZ, wherein the administering comprises intravenous administration.BB. The method of any one of Paragraphs AN-BA, wherein the one or more metabolites of the first hyperpolarized probe comprises vitamin C, optionally wherein the one or more metabolites of the first hyperpolarized probe comprises [l-13C]vitamin C and / or [1-13C, 4-2H]vitamin C.BC. The method of any one of Paragraphs AN-BB, wherein the one or more metabolites of the second hyperpolarized probe comprises lactate, optionally wherein the one or more metabolites of the second hyperpolarized probe comprises [l-13C]lactate.BD. The method of any one of Paragraphs AN-BC, wherein the one or more mammalian cells comprises one or more cancer cells.BE. The method of Paragraph BD, wherein the one or more cancer cells comprises one or more of prostate, glioma, breast, cervical, renal, pancreatic, sarcoma, lung, oral, epithelial, or brain cancer cells.BF. The method of any one of Paragraphs AN-BE, wherein the subject is suspected of having a cancer, a traumatic brain injury, and / or a cardiovascular disease.BG. The method of any one of Paragraphs AN-BF, wherein the subject is diagnosed as having a cancer, a traumatic brain injury, and / or a cardiovascular disease.BH. The method of any one of Paragraphs AN-BG, wherein the first hyperpolarized probe and the second hyperpolarized probe are copolarized.BI. The method of any one of Paragraphs AN-BH, wherein detecting by magnetic resonance comprises simultaneously detecting one or more metabolites of the first hyperpolarized probe and one or more metabolites of the second hyperpolarized probe.-76- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458BJ. The method of any one of Paragraphs AN-BI, wherein the magnetic resonance comprises2H magnetic resonance.BK. The method of any one of Paragraphs AN-BJ, wherein the first hyperpolarized probe and / or the second hyperpolarized probe is deuterated, and wherein the deuterated probe exhibits a longer Ti relaxation time than its nondeuterated analog.BL. The method of any one of Paragraphs AN-BK, wherein the subject is human.-n- 4921-1995-2792.7

Claims

Atty. Dkt. No.: 115872-3458WHAT IS CLAIMED IS:

1. A composition comprising a first hyperpolarized probe, whereinthe first hyperpolarized probe is a compound that has been subjected to hyperpolarization and comprises dehydroascorbic acid, dehydroascorbate, or a mixture thereof.

2. The composition of Claim 1, further comprising a second hyperpolarized probe wherein the second hyperpolarized probe is a compound that has been subjected to hyperpolarization.

3. The composition of Claim 2, wherein the second hyperpolarized probe comprises pyruvic acid, pyruvate, or a mixture thereof.

4. The composition of Claim 2, wherein the hyperpolarization of the first probe and / or hyperpolarization of the second hyperpolarized probe is performed via ParaHydrogen Induced Polarization (PHIP), Dynamic Nuclear Polarization (DNP), Spin- Exchange Optical Pumping, or other Gas-Induced Polarization.

5. The composition of Claim 1, wherein the hyperpolarization of the first probe and / or hyperpolarization of the second hyperpolarized probe is performed via Dynamic Nuclear Polarization (DNP).

6. The composition of Claim 2, wherein the first hyperpolarized probe and / or the second hyperpolarized probe comprises an NMR active nucleus.

7. The composition of Claim 6, wherein the active nucleus comprises 'H,2H,13C,31P,15N, or19F.

8. The composition of Claim 6, wherein the active nucleus comprises 'H,2H, or13C.

9. The composition of Claim 1, wherein the first hyperpolarized probe comprises [1-13C]dehydroascorbic acid, [l-13C]dehydroascorbate, [1-13C, 4-2H]dehydroascorbic acid, [1 -13C, 4-2H]dehydroascorbate or a mixture of any two or more thereof.-78- 4921-1995-2792.7Atty. Dkt. No.: 115872-345810. The composition of Claim 2, wherein the second hyperpolarized probe comprises [1-13C]pyruvic acid, [2-13C]pyruvic acid, [l-13C]pyruvate, [2-13C]pyruvate, or a mixture of any two or more thereof.

11. The composition of any one of Claims 2-8 comprising[l-13C]dehydroascorbic acid, [l-13C]dehydroascorbate, [1-13C, 4-2H]dehydroascorbic acid, [1 -13C, 4-2H]dehydroascorbate, or a mixture of any two or more thereof; and[l-13C]pyruvic acid, [2-13C]pyruvic acid, [l-13C]pyruvate, [2-13C]pyruvate, or a mixture of any two or more thereof.

12. The composition of Claim 1, further comprising a pharmaceutically acceptable carrier.

13. The composition of Claim 1, formulated for parenteral administration.

14. The composition of Claim 1, formulated for intravenous administration.

15. A method of imaging, the method comprising:administering to a subject an effective amount of a composition comprising a first hyperpolarized probe such that the first hyperpolarized probe contacts one or more mammalian cells;detecting by magnetic resonance the first hyperpolarized probe and / or one or more metabolites of the first hyperpolarized probe in the one or more mammalian cells;wherein the first hyperpolarized probe is a compound that has been subjected to hyperpolarization and comprises dehydroascorbic acid, dehydroascorbate, or a mixture thereof.

16. The method of Claim 15, further comprisingadministering to the subject an effective amount of a composition comprising a second hyperpolarized probe such that the second hyperpolarized probe contacts one or more mammalian cells; anddetecting by magnetic resonance the second hyperpolarized probe and / or one or more metabolites of the second hyperpolarized probe in the one or more mammalian cells;-79- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458wherein the second hyperpolarized probe is a compound that has been subjected to hyperpolarization.

17. The method of Claim 16, wherein the second hyperpolarized probe comprises pyruvic acid, pyruvate, or a mixture thereof.

18. The method of Claim 16, wherein the hyperpolarization of the first hyperpolarized probe and / or hyperpolarization of the second hyperpolarized probe is performed via Para-Hydrogen Induced Polarization (PHIP), Dynamic Nuclear Polarization (DNP), Spin-Exchange Optical Pumping, or other Gas-Induced Polarization.

19. The method of Claim 15, wherein the hyperpolarization of the first hyperpolarized probe and / or hyperpolarization of the second hyperpolarized probe is performed via Dynamic Nuclear Polarization (DNP).

20. The method of Claim 15, wherein the hyperpolarization of the first hyperpolarized probe and / or hyperpolarization of the second hyperpolarized probe comprises introducing a radical source.

21. The method of Claim 20, wherein the radical source comprises an organic persistent free radical, photo-induced non-persistent radicals, nanodiamond, or metabolite which can form temporary radicals, silicon particles, or a mixture of any two or more thereof.

22. The method of Claim 15, wherein the first hyperpolarized probe and / or the second hyperpolarized probe comprises an NMR active nucleus.

23. The method of Claim 22, wherein the active nucleus comprises 'H,2H,13C,31P,15N, or19F.

24. The method of Claim 22, wherein the active nucleus comprises 'H,2H, or13C.

25. The method of Claim 15, wherein the first hyperpolarized probe comprises [1-13C]dehydroascorbic acid, [l-13C]dehydroascorbate, [1-13C, 4-2H]dehydroascorbic acid, [1 -13C, 4-2H]dehydroascorbate, or a mixture of any two or more thereof.-80- 4921-1995-2792.7Atty. Dkt. No.: 115872-345826. The method of Claim 16, wherein the second hyperpolarized probe comprises [1-13C]pyruvic acid, [2-13C]pyruvic acid, [l-13C]pyruvate, [2-13C]pyruvate, or a mixture of any two or more thereof.

27. The method of Claim 15, wherein the administering comprises parenteral administration.

28. The method of any one of Claims 25-27, wherein the administering comprises intravenous administration.

29. The method of Claim 15, wherein the one or more metabolites of the first hyperpolarized probe comprises vitamin C, optionally wherein the one or more metabolites of the first hyperpolarized probe comprises [l-13C]vitamin C and / or [1-13C, 4-2H]vitamin C.

30. The method of Claim 16, wherein the one or more metabolites of the second hyperpolarized probe comprises lactate, optionally wherein the one or more metabolites of the second hyperpolarized probe comprises [l-13C]lactate.

31. The method of Claim 15, wherein the one or more mammalian cells comprises one or more cancer cells.

32. The method of Claim 31, wherein the one or more cancer cells comprises one or more of prostate, glioma, breast, cervical, renal, pancreatic, sarcoma, lung, oral, epithelial, or brain cancer cells.

33. The method of Claim 15, wherein the subject is suspected of having a cancer, a traumatic brain injury, and / or a cardiovascular disease.

34. The method of Claim 15, wherein the subject is diagnosed as having a cancer, a traumatic brain injury, and / or a cardiovascular disease.

35. The method of Claim 16, wherein the first hyperpolarized probe and the second hyperpolarized probe are copolarized.-81- 4921-1995-2792.7Atty. Dkt. No.: 115872-345836. The method of Claim 16, wherein detecting by magnetic resonance comprises simultaneously detecting one or more metabolites of the first hyperpolarized probe and one or more metabolites of the second hyperpolarized probe.

37. The method of Claim 15, wherein the magnetic resonance comprises2H magnetic resonance.

38. The method of Claim 16, wherein the first hyperpolarized probe and / or the second hyperpolarized probe is deuterated, and wherein the deuterated probe exhibits a longer Ti relaxation time than its nondeuterated analog.

39. The method of any one of Claims 15-38, wherein the subject is human.

40. A method of measuring metabolic flux in a subject, the method comprising:administering to the subject an effective amount of a composition comprising a first hyperpolarized probe such that the first hyperpolarized probe contacts one or more mammalian cells;detecting by magnetic resonance at a plurality of timepoints the first hyperpolarized probe and / or one or more metabolites of the first hyperpolarized probe in the one or more mammalian cells; anddetermining a metabolic flux of the first hyperpolarized probe based on a rate of conversion of the first hyperpolarized probe to the one or more metabolites of the first hyperpolarized probe;wherein the first hyperpolarized probe is a compound that has been subjected to hyperpolarization and comprises dehydroascorbic acid, dehydroascorbate, or a mixture thereof.

41. The method of Claim 40, further comprisingadministering to the subject an effective amount of a composition comprising a second hyperpolarized probe such that the second hyperpolarized probe contacts one or more mammalian cells; anddetecting by magnetic resonance at a plurality of timepoints the second hyperpolarized probe and / or one or more metabolites of the second hyperpolarized probe in the one or more mammalian cells;-82- 4921-1995-2792.7Atty. Dkt. No.: 115872-3458wherein the second hyperpolarized probe is a compound that has been subjected to hyperpolarization.

42. The method of Claim 41, wherein the second hyperpolarized probe comprises pyruvic acid, pyruvate, or a mixture thereof.

43. The method of Claim 41, wherein the hyperpolarization of the first hyperpolarized probe and / or hyperpolarization of the second hyperpolarized probe is performed via Para-Hydrogen Induced Polarization (PHIP), Dynamic Nuclear Polarization (DNP), Spin-Exchange Optical Pumping, or other Gas-Induced Polarization.

44. The method of Claim 40, wherein the hyperpolarization of the first hyperpolarized probe and / or hyperpolarization of the second hyperpolarized probe is performed via Dynamic Nuclear Polarization (DNP).

45. The method of Claim 41, wherein the hyperpolarization of the first hyperpolarized probe and / or hyperpolarization of the second hyperpolarized probe comprises introducing a radical source.

46. The method of Claim 45, wherein the radical source comprises an organic persistent free radical, photo-induced non-persistent radicals, nanodiamond, or metabolite which can form temporary radicals, silicon particles, or a mixture of any two or more thereof.

47. The method of Claim 41, wherein the first hyperpolarized probe and / or the second hyperpolarized probe comprises an NMR active nucleus.

48. The method of Claim 47, wherein the active nucleus comprises 'H,2H,13C,31P,15N, or19F.

49. The method of Claim 47, wherein the active nucleus comprises 'H,2H, or13C.

50. The method of Claim 40, wherein the first hyperpolarized probe comprises [1-13C]dehydroascorbic acid, [l-13C]dehydroascorbate, [1-13C, 4-2H]dehydroascorbic acid, [1 -13C, 4-2H]dehydroascorbate, or a mixture of any two or more thereof.-83- 4921-1995-2792.7Atty. Dkt. No.: 115872-345851. The method of Claim 41, wherein the second hyperpolarized probe comprises [1-13C]pyruvic acid, [2-13C]pyruvic acid, [l-13C]pyruvate, [2-13C]pyruvate, or a mixture of any two or more thereof.

52. The method of Claim 40, wherein the administering comprises parenteral administration.

53. The method of any one of Claims 50-52, wherein the administering comprises intravenous administration.

54. The method of Claim 40, wherein the one or more metabolites of the first hyperpolarized probe comprises vitamin C, optionally wherein the one or more metabolites of the first hyperpolarized probe comprises [l-13C]vitamin C and / or [1-13C, 4-2H]vitamin C.

55. The method of Claim 41, wherein the one or more metabolites of the second hyperpolarized probe comprises lactate, optionally wherein the one or more metabolites of the second hyperpolarized probe comprises [l-13C]lactate.

56. The method of Claim 40, wherein the one or more mammalian cells comprises one or more cancer cells.

57. The method of Claim 56, wherein the one or more cancer cells comprises one or more of prostate, glioma, breast, cervical, renal, pancreatic, sarcoma, lung, oral, epithelial, or brain cancer cells.

58. The method of Claim 40, wherein the subject is suspected of having a cancer, a traumatic brain injury, and / or a cardiovascular disease.

59. The method of Claim 40, wherein the subject is diagnosed as having a cancer, a traumatic brain injury, and / or a cardiovascular disease.

60. The method of Claim 41, wherein the first hyperpolarized probe and the second hyperpolarized probe are copolarized.-84- 4921-1995-2792.7Atty. Dkt. No.: 115872-345861. The method of Claim 41, wherein detecting by magnetic resonance comprises simultaneously detecting one or more metabolites of the first hyperpolarized probe and one or more metabolites of the second hyperpolarized probe.

62. The method of Claim 40, wherein the magnetic resonance comprises2H magnetic resonance.

63. The method of Claim 41, wherein the first hyperpolarized probe and / or the second hyperpolarized probe is deuterated, and wherein the deuterated probe exhibits a longer Ti relaxation time than its nondeuterated analog.

64. The method of any one of Claims 40-63, wherein the subject is human.-85- 4921-1995-2792.7

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