WO2026039330A2 - Dispositifs, systèmes et procédés de synthèse et de purification de métabolites hyperpolarisés à partir de parahydrogène - Google Patents

Dispositifs, systèmes et procédés de synthèse et de purification de métabolites hyperpolarisés à partir de parahydrogène

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Publication number
WO2026039330A2
WO2026039330A2 PCT/US2025/041467 US2025041467W WO2026039330A2 WO 2026039330 A2 WO2026039330 A2 WO 2026039330A2 US 2025041467 W US2025041467 W US 2025041467W WO 2026039330 A2 WO2026039330 A2 WO 2026039330A2
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membrane
hyperpolarized
metabolite
solvent
solution
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WO2026039330A3 (fr
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Clifford R BOWERS
Yiheng YAN
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University of Florida
University of Florida Research Foundation Inc
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University of Florida
University of Florida Research Foundation Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J8/00—Chemical or physical processes in general, conducted in the presence of fluids and solid particles; Apparatus for such processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
    • C07B59/001—Acyclic or carbocyclic compounds

Definitions

  • Magnetic Resonance Imaging based on proton magnetic resonance is a non-ionizing technique with high anatomic contrast.
  • proton background signals water/fat
  • 1H chemical shift range render it unsuitable for chemically selective molecular imaging.
  • the present disclosure provides for devices, systems, and methods of making hyperpolarized metabolites.
  • the devices, systems, and methods of making hyperpolarized metabolite can be made in a continuous manner, which can be advantageous when collecting an MRI image of a subject.
  • the present disclosure provides for making the hyperpolarized metabolite and separating the hyperpolarized metabolite from the solvent used in the preparation of the hyperpolarized metabolite. The separation can be performed quickly and faster than other methods.
  • the present disclosure also provides for contrast agents and methods of imaging using contrast agents.
  • the present disclosure provides for continuous flow hydrogenation reactor devices, comprising: a hydrogenation reactor, wherein hydrogenation reactor is configured to continuously form a first mixture from a precursor solution and parahydrogen; a spin order transfer device, wherein the spin order transfer device is in fluidic communication with the hydrogenation reactor and is configured to receive the first mixture, wherein a hyperpolarized metabolite is produced in the spin order device from the first mixture, wherein a hyperpolarized metabolite solution includes the hyperpolarized metabolite; a reagent introduction device in fluidic communication with the spin order transfer device, wherein one or more reagents are added to the hyperpolarized metabolite solution to form a modified hyperpolarized metabolite liquid; one or more membrane separator devices in fluid communication with the spin order transfer device and the reagent introduction device, wherein the membrane separator device is configured to continuously receive the modified hyperpolarized metabolite solution and separate the solvent from the hyperpolarized metabolite solution to form a hyperpolarized metabolite aqueous
  • the present disclosure provides for methods for making a hyperpolarized metabolite aqueous solution, comprising: providing a modified hyperpolarized metabolite solution, wherein the modified hyperpolarized metabolite solution comprising a hyperpolarized metabolite, a solvent, and one or more reagents; separating the solvent from the hyperpolarized metabolite solution to form a hyperpolarized metabolite aqueous solution and a solvent phase, wherein the separation is performed using a membrane separator device, wherein the membrane separator device is configured to continuously receive the modified hyperpolarized metabolite solution and separate the solvent from the hyperpolarized metabolite solution to form a hyperpolarized metabolite aqueous solution and a solvent phase; a collecting the hyperpolarized metabolite aqueous solution.
  • contrast agents comprising the hyperpolarized metabolite as described herein and produced using the method as described herein.
  • the present disclosure provides for methods for detecting a disease state associated with abnormal concentration or abnormal activity of a precursor compound metabolite in a subject, the method comprising: (a) administering the contrast agent as described herein to the subject; and (b) detecting the contrast agent in the subject.
  • the present disclosure provides for methods that employ a hydrophilic membrane for liquid/vapor separation for continuous flow stripping of residual volatile organics dissolved in the aqueous buffer containing the hyperpolarized metabolite.
  • Fig. 1A illustrates a flow-chemistry process for synthesis of hyperpolarized metabolites incorporating hydrogenation of the ester precursor with pH2 in acetone spin order transfer (SOT), hydrolysis with NaOD, phosphate buffering, mixing with methyl t-butyl ether (MTBE), continuous-flow liquid-liquid separation and continuous-flow stripping of residual volatile organic compounds using a liquid-vapor membrane separator.
  • Fig. 1 B illustrates a diagram showing the operating flow path of the concentric annular liquid-liquid separator.
  • FIG. 1C illustrates a cross-sectional diagram of Zaiput Flow Technologies’ patented membrane separator, which can provide continuous separation of an immiscible phase (liquid-liquid or gas-liquid) by leveraging differences in wetting properties of the liquids onto a porous membrane.
  • Fig. 2 illustrates specific and generalized chemical structures of unsaturated side-arm ester precursors to which the devices, systems, and methods for the synthesis and purification of hyperpolarized metabolites from parahydrogen.
  • FIG. 3A illustrates timing diagrams for coherence transfer after adiabatic transport of ethyl or allyl ester adducts of pH2 from strong to weak coupling followed by selective INEPT or MINERVA NMR pulse sequences.
  • Fig. 3B illustrates hyperpolarized NMR spectra for APd, selectively deuterated allyl pyruvate ester (preliminary data), where a 13C signal enhancement of 14,500 was observed, corresponding to a spin polarization of 12% (24% for APd2).
  • Fig. 4 illustrates numerical density matrix simulations of 13C polarizations resulting from application of MINERVA or INEPT coherence transfer pulse sequences to various hydrogenation adduct molecules after PASADENA or ALTADENA preparation.
  • Fig. 5 illustrates comparison of the droplet size distribution resulting from passage of a liquid through either an ultrasonically vibrating nozzle or a conventional spray injection nozzle.
  • Fig. 6A illustrates a representative flow separation scheme combining three Zaiput membrane separation devices, each fitted with a hydrophobic membrane, where the water/acetone/metabolite solution is mixed with an extraction solvent (e.g., MTBE) before the first stage, and the liquid emerging from non-wetting port is mixed with fresh extraction ATTORNEY DOCKET NO. 222112-2510 solvent after each consecutive separation device. Residual volatile organic compounds are removed using a solvent stripping method such as the one described in part C.
  • an extraction solvent e.g., MTBE
  • Fig. 6B illustrates an alternative counter-current configuration using three stages of membrane separation which provides optimal extraction efficiency using a fixed volume of extraction solvent (MTBE).
  • MTBE extraction solvent
  • Fig. 6C illustrates a method for stripping of residual dissolved extraction solvent (e.g., MTBE) and acetone or other volatile compounds in the aqueous phase by mixing with nitrogen gas to form a biphasic gas/liquid mixture. After diffusion of the volatile organic compounds into the gas phase, the vapor is removed by a membrane separation device. In the scheme shown, where a hydrophilic membrane is employed, the vapor is the non-wetting phase, and the aqueous phase containing the metabolite is the wetting phase.
  • residual dissolved extraction solvent e.g., MTBE
  • FIG. 7 illustrates preliminary results demonstrating the similar performance in the extraction of acetone from a 1 :1 v/v acetone/water mixture containing 200 mM Na pyruvate into methyl t-butyl ether (MTBE) using (a) conventional gravimetric extraction (dashed curves) and (b) flow separation using a Zaiput SEP-10 device fitted with a hydrophobic membrane (solid curves). Syringe pump flow rates of 3 ml/min for MTBE and 1.5 ml/min for the 1 :1 v/v acetone/water mixture were used. The liquids were mixed using an IDEX mixing tee fitted with a 10 pm frit followed by a 1 m section of 1/16 in (O.D.) green PEEK tubing.
  • IDEX IDEX mixing tee fitted with a 10 pm frit followed by a 1 m section of 1/16 in (O.D.) green PEEK tubing.
  • Fig. 8A illustrates a generalized experimental flow chemistry system incorporating spin order transfer in NMR #1 and spectroscopic analysis in NMR #2.
  • VOCs volatile organic solvents
  • Fig. 8B illustrates separation of the hyperpolarized metabolite dissolved in the aqueous phase from the original using a hydrophobic membrane. Removal of the dispersing medium by diffusion across the membrane fosters coalescence of the dispersed phase, shown in blue.
  • Fig. 9A and 9B illustrate the 300 MHz 1H NMR spectra acquired after hydrolysis of ethyl pyruvate and extraction into the aqueous phase by either batch or continuous-flow separation processes.
  • Fig. 9A illustrates the pre- and post-hydrolysis spectra obtained after addition of aqueous sodium carbonate solution to the ethyl pyruvate solution (in acetone-d6).
  • Fig. 9B illustrates spectra of the aqueous fraction after addition of aqueous base and separation by either gravimetric (orange spectrum) or continuous-flow LL separation using a Zaiput SEP- 10 device fitted with a hydrophobic membrane.
  • Fig. 10 illustrates schemes 1-3. ATTORNEY DOCKET NO. 222112-2510
  • Figs. 11 A and 11 B illustrate gravimetric extraction of acetone from water-acetone mixture under various conditions.
  • the initial volume ratio of solvent to water to acetone was roughly 4:1 :1 .
  • Fig. 12A illustrates the residual organic solvent amounts (mass fractions) after extraction with different solvents at room temperature. Anisole performs best.
  • Fig. 11 A illustrates extraction with anisole at different temperatures. The performance improves with increasing temperature. An extrapolation (dashed black line) after two simulated extractions is overlaid on the plot.
  • Figure 12 illustrates hydrolysis of allyl acetate under various conditions. All samples were vigorously mixed twice to ensure reaching chemical equilibrium.
  • the present disclosure provides for devices, systems, and methods of making hyperpolarized metabolites.
  • the devices, systems, and methods of making hyperpolarized metabolites can be made in a continuous manner.
  • the present disclosure provides for making the hyperpolarized metabolite and separating the hyperpolarized metabolite from the solvent used in the preparation of the hyperpolarized metabolite.
  • the present disclosure provides for devices, systems, and methods of making hyperpolarized metabolites.
  • the present disclosure also provides for contrast agents and methods of imaging using contrast agents.
  • a heteronucleus As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a heteronucleus,” “a metabolite,” or “a parahydrogen atom,” include, but are not limited to, mixtures or combinations of two or more such heteronuclei, metabolites, or parahydrogen atoms, and the like.
  • heteronucleus refers to any atomic nucleus other than the proton; e.g., 13C, 15N, 31 P, 19F, 2H, or 29Si.
  • Polarization refers to the difference in fractional populations of two spin states (for example, the spin-up and spin-down quantum states of the proton, denoted Polar ization of a spin-1/2 particle is defined as: where A' ⁇ and
  • A' ⁇ are the numbers of particles in the spin-up and spin-down states, respectively (See Fig. 1D).
  • hypopolarization refers to a non-Boltzmann thermal equilibrium nuclear spin order resulting in nuclear magnetic resonance signal enhancement.
  • hyperpolarized fluid sample refers to a liquid or gas containing target molecules hosting hyperpolarized nuclear spins.
  • precursor solution refers to a liquid solvent or solvent mixture containing a catalyst compound or catalyst nanoparticles together with the hydrogenation substrate molecules (precursor compound) that incorporate at least one unsaturated chemical moiety (e.g. a double bond, a triple bond, a carbonyl group, or a hydroxy group) to which one or more protons of H2 may be transferred, or alternatively, that becomes hyperpolarized through a spin order transfer mechanism, e.g.
  • parahydrogen refers to the metastable spin isomer of dihydrogen with proton spins in a singlet state that is antisymmetric with respect to permutation of the two protons.
  • parahydrogen will in some cases, depending on the context, also refer to dihydrogen gas that is only partially enriched in the parahydrogen spin isomer content relative to normal hydrogen, which is about 25% parahydrogen and about 75% orthohydrogen (the triplet state, which is symmetric with respect to permutation of the two protons).
  • heteronuclei will refer to the spin-1/2 isotopes other than the proton, including carbon-13, nitrogen-15, fluorine-19, and phosphorus-31.
  • INEPT or “insensitive nuclei enhancement by polarization transfer” refers to a signal enhancement method used in nuclear magnetic resonance spectroscopy and magnetic resonance imaging.
  • An INEPT pulse sequence can be used to transfer nuclear spin polarization from protons, including, but not limited to, atoms originating in parahydrogen, to a heteronucleus.
  • MRI refers to magnetic resonance imaging, the processing of collecting the spatial distribution of nuclear spins in an object, specimen, or patient.
  • MINERVA or “(Maximizing Insensitive Nuclear Enhancement Reached Via para-hydrogen Amplification)” refers to a coherence transfer pulse sequence for converting bilinear spin order generated by the PASADENA, ALTADENA, or LACADENA effects into hyperpolarized magnetization of a heteronucleus.
  • “Side Arm” refers to an unsaturated molecule containing a double or triple bond that is attached by an ester linkage to another molecule, which may be a carboxylic acid (or carboxylate) metabolite.
  • the side arm provides the chemical unsaturation necessary for incorporation of parahydrogen by chemical hydrogenation reaction. Examples of side arms are provided in Scheme 1 (Fig. 10).
  • SAH Segment Arm Hydrogenation
  • the present disclosure provides for devices, systems, and methods of making hyperpolarized metabolites.
  • the devices, systems, and methods of making hyperpolarized metabolite can be made in a continuous manner, which is advantageous ATTORNEY DOCKET NO. 222112-2510 when collecting an MRI image of a subject (e.g., animal, mammal (e.g., dog, cat), human,).
  • the present disclosure provides for making the hyperpolarized metabolite and separating the hyperpolarized metabolite from the solvent used in the preparation of the hyperpolarized metabolite. The separation can be performed quickly.
  • the separation can be performed in about 5-15 seconds per extraction, which is between 1x and 10x, 2x to 10x, or 4x to 10x faster than other methods.
  • the present disclosure also provides for contrast agents and methods of imaging using contrast agents. Additional details are provided in the Examples and Attachment.
  • Nuclear spin hyperpolarization (HP) techniques have ushered in a new era of chemically selective magnetic resonance imaging (MRI) and localized nuclear magnetic resonance (NMR) spectroscopy.
  • MRI chemically selective magnetic resonance imaging
  • NMR nuclear magnetic resonance
  • metabolites and other biomolecules become visible despite their low in vivo concentrations, thereby enabling disease detection and treatment response monitoring without exposure to ionizing radiation.
  • Parahydrogen (pH2) is a convenient source of singlet nuclear spin order that can be rapidly transformed into MRI-observable proton hyperpolarization through symmetry-breaking hydrogenation chemistry.
  • the requisite unsaturation for pH2 addition can be incorporated by synthesis of the vinyl or propargyl ester, as well as those shown in Fig. 2.
  • the non-equilibrium spin order is transferred to the carbonyl 13C to render, after hydrolytic cleavage, a hyperpolarized molecule that was not directly producible as the product of pairwise hydrogenation.
  • the hyperpolarized metabolite can be separated from the solvent quickly and efficiently.
  • the metabolite NMR signals obtained from nuclear spins in this hyperpolarized state can be many orders of magnitude stronger than the signals obtained when the nuclear spins are initially at thermal equilibrium.
  • the present disclosure provides for a continuous flow hydrogenation reactor.
  • the continuous flow hydrogenation reactor will first be described generally and then in more detail.
  • the continuous flow hydrogenation reactor can include a hydrogenation reactor, a spin order transfer device, a reagent introduction system, one or more membrane separator devices, and a collection device.
  • Fig. 6A in the Example section illustrates an embodiment of the reactor.
  • the hydrogenation reactor is configured to continuously form a first mixture from precursor solution (e.g., a catalyst, precursor compound, a solvent) and parahydrogen.
  • precursor solution e.g., a catalyst, precursor compound, a solvent
  • parahydrogen can be introduced in one or more ways, for example using a spray injection nozzle as described herein.
  • the hydrogenation reactor can be configured as a homogeneous hydrogenation device or system or a heterogeneous ATTORNEY DOCKET NO. 222112-2510 hydrogenation device or system. Examples of each of these are described in the Example section.
  • the hydrogenation reactor is in fluidic communication with a spin order transfer device.
  • the spin order transfer device is configured to receive the first mixture.
  • the spin order transfer device is configured to form a hyperpolarized metabolite from the first mixture, where a hyperpolarized metabolite solution includes the hyperpolarized metabolite.
  • the hyperpolarization is described herein and in the Examples.
  • a reagent introduction device is in fluidic communication with the spin order transfer device.
  • One or more reagents are added to the hyperpolarized metabolite solution to form a modified hyperpolarized metabolite solution.
  • the one or more reagents can include acetone, an aqueous base or acid, a phosphate buffer, and a methyl t- butyl ether (MTBE) solvent. Additional details regarding the reagents are described below and in the Examples.
  • a membrane separator device (or a plurality of membrane separator devices in serial connection) is in fluid communication with the spin order transfer device and the reagent introduction device.
  • the membrane separator device includes one or more membrane separators.
  • the membrane separator device is configured to continuously receive the modified hyperpolarized metabolite solution and separate the solvent from the hyperpolarized metabolite solution to form a hyperpolarized metabolite aqueous solution and a solvent phase. Additional details regarding the membrane separator device are provided herein and in the Examples.
  • a collection system is in fluidic communication with the membrane separator device(s).
  • the collection system receives the hyperpolarized metabolite aqueous solution.
  • the collection system can independently collect the organic solvent phase, which is kept separate from the hyperpolarized metabolite aqueous solution.
  • Each of the components of the continuous flow hydrogenation reactor can be in fluidic communication as provided using tubing, flow meters, flow controllers, in-line pumps, syringe pumps, back-pressure regulators, check-valves, pneumatic switching valves, manual valves, and the like.
  • the membrane separator(s) can be a liquid-liquid membrane separator that separates the organic solvent(s) from the modified hyperpolarized metabolite aqueous solution by phase separation using differential surface wetting.
  • the membrane separator includes a membrane having hydrophobic surface with pores extending from one side of the membrane to the other side of the membrane so that fluid can pass through the membrane; optionally, wherein the fluid is the organic non-polar solvent phase.
  • the membrane separator includes a membrane having hydrophilic surface with pores extending from one side of the membrane ATTORNEY DOCKET NO.
  • the membrane separator device can include a pressure system or backpressure regulator that maintains a pressure differential across the membrane. The pressure differential is adjusted to maximize the fluid transport through the pores to produce the separation, while preventing unwanted breakthrough of the aqueous phase through the membrane. Additional details regarding the membrane separator device are provided in the Example.
  • the membrane separator device(s) is configured to separate the organic solvent from the hyperpolarized metabolite solution to form a purified aqueous solution of the hyperpolarized metabolite (also referred to as the “hyperpolarized metabolite aqueous solution”) and a separate solution containing organic solvents (e.g., MTBE, acetone), the spent side arm alcohol, and catalyst residues.
  • the liquid-liquid membrane separation can be performed about 1 to 10, about 2 to 10, about 4 to 10, about 6 to 10, about 8 to 10, or about 10 times faster than a continuous flow hydrogenation reactor having a gravimetric separation device (all else being equal with only the substitution of the gravimetric separation device for the membrane separator device).
  • the membrane separator device is configured to separate the solvent from the hyperpolarized metabolite solution to form a hyperpolarized metabolite aqueous solution and the solvent phase in about 2 to 15 seconds per extraction.
  • the spin order transfer device can include comprising an adiabatic transport tube, wherein the adiabatic transport tube is configured to receive the first mixture from the hydrogenation chamber.
  • the spin order transfer device can include one of the following: an NMR spectrometer configured for field cycling of the static magnetic field or radiofrequency field; an NMR spectrometer configured for coherence transfer radiofrequency pulse sequences; or an NMR spectrometer configured for adiabatic field cycling in conjunction with coherence transfer radiofrequency pulse sequences; or an NMR spectrometer configured for amplitude ramps and/or frequency sweeps of the radio-frequency fields [0058] Now having described the continuous flow hydrogenation reactor in general, methods of the present disclosure are described in general, where additional details are provided herein.
  • the present disclosure provides for a method for making a purified aqueous solution of hyperpolarized metabolites.
  • the method includes providing a modified hyperpolarized metabolite solution.
  • the modified hyperpolarized metabolite solution comprising a hyperpolarized metabolite (the solute), a solvent, and one or more reagents.
  • the method provides for the hyperpolarized metabolite solution and the first ATTORNEY DOCKET NO. 222112-2510 mixture as described herein, where the first mixture and hyperpolarized metabolite solution are processed to form the modified hyperpolarized metabolite solution.
  • the method includes separating the organic solvent phase from the hyperpolarized metabolite solution to form a hyperpolarized metabolite aqueous solution and a separated organic solvent phase.
  • the separation is performed using a membrane separator device as described above and herein.
  • the membrane separator device is configured to continuously receive the liquid mixture including the hyperpolarized metabolite molecule, the hydrogenated side-arm adduct, water, salts and buffers, and organic solvents.
  • the membrane separates this mixture into a modified hyperpolarized metabolite in aqueous solution and the non-polar organic solvent phase.
  • the method provides for separately collecting the hyperpolarized metabolite in aqueous solution as well as the solvent phase.
  • the present disclosure also provides for a contrast agent including the hyperpolarized metabolite described herein as well as the hyperpolarized metabolite produced using the methods described herein.
  • the present disclosure provides for methods that employ a hydrophilic membrane for liquid/vapor separation for continuous flow stripping of residual volatile organics (e.g., acetone, MTBE, or a combination thereof) dissolved in the aqueous buffer containing the hyperpolarized metabolite.
  • the membrane strictly separates liquid and gas phases and neither chemical reactions nor accumulation of solvents or solutes occurs inside the membrane pores.
  • a carrier gas e.g., nitrogen, N 2
  • N 2 is used that does not appreciably dissolve in water (and water does not appreciably diffuse into the gas).
  • the volatile organic solvents e.g., MTBE and acetone
  • the volatile organic solvents diffuse from water into the gas phase during their contact in the biphasic gas/liquid mixture, and then the gas is removed from the membrane separator device through the nonwetting outlet port, while the aqueous buffer containing the hyperpolarized metabolite is collected from the wetting port of the separator device with the hydrophilic membrane installed.
  • the hydrogenating can conducted in a magnetic field of from about 0 to 1 pT, or 1 pT to 100 pT, or 100 pT to 2 T, 0.25 to 2 T, or of about 0, 0.25, 0.5, 0.75, 1 , 1.25, 1.5, 1 .75, or about 2 T, or a combination of any of the foregoing values, or a range encompassing any of the foregoing values.
  • the hydrogenation reaction can include contacting the precursor compound and the supply of parahydrogen with a hydrogenation catalyst.
  • the hydrogenation catalyst can be selected from or include Pt, Pd, Cu, Au, Ag, Rh, Ru, Ir, Ni, Sn, Co, Zn, Ce, Ti, Al, Fe, Si, or any combination thereof.
  • the ATTORNEY DOCKET NO. 222112-2510 hydrogenation catalyst can be in nanoparticle form or in liquid form, as a suspension, dispersion, a colloid, or emulsion.
  • the precursor compound can be an unsaturated ester of a Cn (n>0) carboxylic acid, where n can be 2 to 20, 2 to 10, 2 to 8, 2 to 6, 4 to 10, or 4 to 20.
  • the unsaturated ester can be a vinyl ester or a propargyl ester.
  • the precursor compound can be vinyl acetate, propargyl pyruvate, or any combination thereof.
  • one or more protons of the precursor compound are replaced with deuteron(s) or R-groups to reduce or eliminate homonuclear spin couplings with the parahydrogen sourced protons.
  • At least one heteronucleus of the precursor compound can be a carbon-13 atom, nitrogen-15, phosphorous-31 , or fluorine-19. In a further aspect, at least one heteronucleus of the precursor compound can be a carbon atom double bonded to an oxygen atom. In some aspects, the precursor compound can further include at least one deuterium.
  • the ester in the disclosed method, can be hydrolyzed after hydrogenating the precursor compound.
  • spin is transferred from the first parahydrogen atom or the second parahydrogen atom or simultaneously from both hydrogen atoms to the third hydrogen atom using adiabatic passage through one or more anti-crossings of the nuclear spin energy levels.
  • adiabatic passage can be accomplished via exposing the precursor compound, after hydrogenation to form the precursor adduct molecule, to a continuously increasing magnetic field until the detection magnetic field is reached, with a strength greater than about 0.25 T.
  • spin can be transferred from the third hydrogen atom to at least one heteronucleus using a suitable coherence transfer pulse sequence, e.g., selective or non-selective insensitive nuclei enhancement by polarization transfer (INEPT) or the radio frequency pulse sequence known as MINERVA.
  • INEPT selective or non-selective insensitive nuclei enhancement by polarization transfer
  • MINERVA radio frequency pulse sequence
  • the process is at least 10% efficient, about 10 % to 80%, about 80% to 100%, 80% to 99%, 80% to 90%, about 90 to 100%.
  • precursor compounds including at least one hyperpolarized heteronucleus produced by the disclosed method or other spin order transfer method as well as contrast agents including the precursor compounds.
  • the hydrogenation reactor can include an ultrasonic nozzle.
  • a precursor solution can be introduced to the surface of the ultrasonic nozzle, where the ultrasonic nozzle is configured to produce droplets of the precursor solution.
  • the droplets of the precursor solution have an average diameter of from about 1 to about 50 pm, or of about 1 , 5, 10, 15, 20, 25, 30, 35, 40, 45, or about 50 pm, or a combination of any ATTORNEY DOCKET NO. 222112-2510 of the foregoing values, or a range (e.g., about 5 to 50 m, about 15 to 45 pm, about 5 to 20 pm, etc.) encompassing any of the foregoing values.
  • the droplets can have a droplet distribution range of about 25 pm or less.
  • the precursor solution can be introduced using a syringe pump configured to deliver an amount of the precursor solution to the surface of the ultrasonic nozzle.
  • parahydrogen gas can be introduced into a flowing stream of liquid precursor solution containing the dissolved precursor compound and dissolved or suspended catalyst using an TelfonTM AF 2400 (amorphous fluoroplastic resin, or similar) gas permeable membrane.
  • TelfonTM AF 2400 amorphous fluoroplastic resin, or similar
  • parahydrogen gas can be introduced by merging it with a stream of the liquid precursor solution containing the dissolved precursor compound using a mixing chamber or mixing tee connected to a check valve and a back-pressure regulator.
  • the mixing chamber or mixing tee may be followed by a section of tubing that is sufficiently long to allow thorough mixing and dissolution of the gas into the liquid precursor solution.
  • the hydrogenation reaction is catalyzed by a solid, insoluble heterogeneous catalyst material by passing the precursor solution containing the dissolved substrate molecule and dissolved parahydrogen through a packed bed reactor containing the solid catalyst.
  • the parahydrogen gas is introduced by bubbling the parahydrogen through a reservoir of the precursor solution or by passing the precursor solution through a TelfonTM AF 2400 (amorphous fluoroplastic resin, or similar) gas permeable membrane.
  • the fluid sample is a homogeneous fluid including the precursor compound and a catalyst.
  • the fluid sample is a heterogeneous fluid including the precursor compound and a particle that includes a catalyst.
  • the catalyst can be a Group VIII, IB, or I IB transition metal-based catalyst including at least two different metals, or may not include a metal.
  • the catalyst can include at least one of Pt, Pd, Cu, Au, Ag, Rh, Ru, Ir, Ni, Sn, Co, Zn, Ce, Ti, Al, Fe, Si, O, or any combination thereof in the form of an intermetallic compound or an alloy.
  • the particle may be of homogeneous composition or it may have a core-shell structure, where the core of the particle is of one composition, not necessarily catalytically active, and the outer layer with a different composition that exposes catalytically active sites to the solution.
  • the catalyst can be a nanoparticle having a diameter of a single metal atom to about 0.5 nm, or 0.5 nm to 1 nm, or 1 nm to 10 nm, or 10 nm to about 500 nm, or of about 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, or about 500 nm, or a combination of any of the foregoing values, or a range encompassing any of the foregoing values.
  • the catalyst can be in liquid form.
  • the catalyst can ATTORNEY DOCKET NO. 222112-2510 be a Rh compound, cluster, or nanoparticle, or a Rh alloy, that is supported, tethered, or ligand stabilized in solution, or any combination thereof.
  • the precursor compound can be a metabolite or a derivative thereof.
  • the metabolite or derivative thereof can be administered in vitro or to a subject in vivo.
  • the metabolite or derivative thereof can be an unsaturated ester of a C1 to C4 carboxylic acid or C1 to C8 carboxylic acid or C1 to C12 carboxylic acid.
  • the unsaturated ester can be a vinyl ester or a propargyl ester.
  • the metabolite or derivative thereof can be vinyl acetate, propargyl pyruvate, or any combination thereof.
  • at least one heteronucleus of the precursor compound is a carbon-13, nitrogen-15, phosphorous-31 , or fluorine-19 atom, or in general, any spin-1/2 isotope
  • the precursor solution in the disclosed device or system, can be introduced over a range of speeds, where a specific introduction speed can be selected prior to introducing the precursor solution, and where the introduction speed of the precursor solution is correlated to an amount of hyperpolarization in the fluid sample. In this way, the device or system can operate in a continuous manner.
  • a disease state associated with abnormal concentration or abnormal activity of a precursor compound metabolite in a subject including at least the steps of:
  • the method further includes the step of administering one or more additional contrast agents to the subject, wherein the one or more additional contrast agents can be administered sequentially or simultaneously with the contrast agent or target molecule.
  • detecting is accomplished using magnetic resonance imaging.
  • a hyperpolarized metabolite solution can be made by exposing a precursor solution (e.g., including a solvent, precursor compound, and a catalyst) to parahydrogen. The interaction and/or chemical reaction between the molecules in the precursor solution and the parahydrogen in the presence of an appropriate homogeneous or heterogeneous catalyst can produce a hyperpolarized metabolite solution that includes hyperpolarized protons or heteronuclei on the precursor compounds (e.g., metabolites).
  • the method of making the hyperpolarized metabolite solution can include making droplets of a particular size having a particular (e.g., narrow) size distribution using ultrasonic energy.
  • the droplets which have a very high surface area compared to those produced by bubbling or non-ultrasonic spray-injection methods, are then exposed to gaseous parahydrogen.
  • the precursor compound, catalyst, and parahydrogen interact ATTORNEY DOCKET NO. 222112-2510 and/or react to form the hyperpolarized fluid sample, where a portion (about 50 to 100%) of the precursor compounds are hyperpolarized.
  • the precursor compound acquires one or more magnetized protons or hydrogen nuclei from parahydrogen by chemical exchange.
  • the spin order of parahydrogen is transferred to the spins in a precursor compound without any chemical hydrogenation of the target molecule, as occurs in the hyperpolarization phenomena commonly known as SABRE (Signal Amplification by Reversible Exchange), SWAMP (Surface Waters Are Magnetized from Parahydrogen), NEPTUN (Nuclear Exchange Polarization by Transposing Unattached Nuclei), or proton chemical exchange, as in the literature methods referred to as PHIP-X or PHIP-RELAY.
  • SABRE Signal Amplification by Reversible Exchange
  • SWAMP Surface Waters Are Magnetized from Parahydrogen
  • NEPTUN Nuclear Exchange Polarization by Transposing Unattached Nuclei
  • proton chemical exchange as in the literature methods referred to as PHIP-X or PHIP-RELAY.
  • the reaction chamber can be operated at a temperature of about 25 °C to 300 °C and a pressure between about 1 bar to 100 bar.
  • the reaction chamber includes an ultrasonic nozzle that has a surface.
  • the precursor solution can be delivered to the surface of the ultrasonic nozzle.
  • An ultrasonic nozzle may include a titanium horn body, a crystal/ceramic element with piezoelectric properties protected within a stainless-steel rear and front housing.
  • Piezoelectric elements can include crystalline materials such as quartz, gallium orthophosphate, langasite, lithium tantalate, lithium niobate.
  • piezoelectric ceramics that have been reported are barium titanate, potassium niobate, sodium tungstate, and the most commonly used lead zirconate titanate. Active and ground electrodes on the faces of the piezoelectric elements allow for an electrical connection to an ultrasonic generator.
  • a liquid feed tube is situated through the titanium horn body to the tip of the atomizing surface.
  • the atomizing surface shape can be conical, focused, or flat depending on the desired spray pattern.
  • the production of the droplets results from the inverse piezoelectric effect, where high frequency (acoustic) sound waves are converted to mechanical energy to produce a vibrating surface.
  • the ultrasonic nozzle can operate at a frequency ranging from about 20 kHz to 3 MHz to produce droplets of the precursor solution having diameters between 1 and 50 microns, depending on the operating frequency of a given nozzle.
  • the high surface-to-volume ratio of droplets formed by the ultrasonic nozzle favors rapid diffusion of gaseous parahydrogen across the liquid/gas interface and into the interior of the liquid droplet within a relatively short timescale.
  • the droplet diameter and size distribution can be tailored to maximize the hydrogenation reaction rate, conversion, and resultant hyperpolarization level in the collected fluid sample.
  • the hydrogenation device can have a volume sufficient to produce the desired volume of hyperpolarized fluid sample for in- vivo use, for example. In this regard, the hydrogenation device has a volume of about 1 to ATTORNEY DOCKET NO. 222112-2510
  • the hydrogenation device can be made of materials such as stainless steel, aluminum, polysulfone, vespel, polytetrafluoroethylene (PTFE), perfluoroalkoxy alkane (PFA), or polyether ether ketone (PEEK).
  • the ultrasonic nozzle is located at the top of the hydrogenation device and a collection vessel is located at the bottom of the hydrogenation device to collect the droplets as they move through the reaction chamber.
  • the hydrogenation device can have a length of about 2 to 6 inches and a width or diameter of about 1 to 3 inches.
  • the parahydrogen is introduced to the reaction chamber using a gas introduction system that is in gaseous communication therewith.
  • the gas introduction system can be configured to controllably introduce parahydrogen into the reaction chamber.
  • the gas introduction device can include appropriate equipment to acquire (if part of a different system) and/or flow the parahydrogen to the reaction chamber.
  • the gas introduction system can include tubing, flow valves, pressure gauges, pressure regulators, syringe pumps, in-line pumps, HPLC pumps, peristaltic pumps, thermocouples, flow meters, and the like to control introduction into the holding vessel through the inlet port.
  • the sample can be a homogeneous fluid including the solvent, the precursor compound, and a catalyst, where the catalyst is dissolved in the fluid.
  • the sample is a heterogeneous fluid including the precursor compound and a particle comprising a catalyst, where the catalyst is insoluble in the fluid or is in the form of a solution, suspension, colloid, or emulsion of nanoparticles.
  • the fluid can be an aprotic solvent.
  • the aprotic solvent can include: dioxane, nitromethane, acetonitrile, acetone, dichloromethane, dimethylformamide (DMF), dimethylsulfoxide (DMSO) or a combination thereof.
  • the aprotic solvent can be a perdeuterated and partially deuterated form of each of the solvents listed above or herein.
  • the fluid is perdeuterated or partially deuterated water and is diluted in the aprotic solvent.
  • the fluid is biphasic, including immiscible polar and non-polar liquids.
  • the amount of catalyst in the precursor solution and in each droplet should be sufficient to provide enough active sites to accommodate hyperpolarization of the desired number of target molecules.
  • the total number of active sites of the catalyst in the volume of precursor solution should be sufficient to allow complete conversion of all precursor compounds to hyperpolarized molecules.
  • Concentrations of precursor compounds, parahydrogen, and catalyst are appropriate for hyperpolarization either by exchange of one or more magnetized protons from adsorbed parahydrogen or by non-hydrogenative mechanisms.
  • the amount of catalyst required can depend on the particle size, as the surface to volume ratio scales as 1/r, where r is the ATTORNEY DOCKET NO. 222112-2510 particle radius (assuming a spherical particle shape), the surface composition, and the rate of exchange, as well as the type of catalyst.
  • a further aspect includes from one particular value and/or to other particular value.
  • ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’.
  • the range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’.
  • the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’.
  • the phrase “about x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.
  • a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.
  • Magnetic Resonance Imaging is a non-ionizing technique with superior anatomic contrast compared to traditional clinical imaging techniques, such as positron emission tomography (PET).
  • PET positron emission tomography
  • the presence of strong proton background signals (water/fat) and the limited chemical shift range of 1 H MRI render it unsuitable for chemically selective molecular imaging of metabolic activity.
  • 18F PET is in widespread clinical use to detect anomalous metabolic uptake of fluorodeoxyglucose.
  • MRI based on carbon-13 (13C) would offer the requisite chemical specificity for imaging metabolic dysfunction, but magnetic field polarized 13C transitions are too weak to be detected due to the unfavorable Boltzmann distribution and low physiological concentrations.
  • the sensitivity limitation can be overcome by hyperpolarized MRI which can intensify 13C NMR signals by more than five orders of magnitude, 1-3 and parahydrogenbased hyperpolarization is faster, cheaper, and more reliable than competing hyperpolarization techniques while providing comparable molar polarization levels.
  • the present disclosure provides for a new methodology, assisted by specifically designed catalyst materials, for continuous synthesis of parahydrogen-hyperpolarized metabolites in aqueous buffer.
  • the present disclosure concerns the recent advances enabling pH2-based hyperpolarization of discrete batches of [1-13C] pyruvate and other metabolites. While the batch synthesis of hyperpolarized metabolites is largely a solved problem, continuous-flow hyperpolarization requires new methods for flow chemistry, spin order transfer, and liquidliquid extraction.
  • Fig. 1 A illustrates a flow-chemistry process for synthesis of hyperpolarized metabolites incorporating hydrogenation of the ester precursor with pH2 in acetone spin order transfer (SOT), hydrolysis with NaOD, phosphate buffering, mixing with methyl t-butyl ether (MTBE), continuous-flow liquid-liquid separation and continuous-flow stripping of residual volatile organic compounds using a liquid-vapor membrane separator.
  • SOT acetone spin order transfer
  • MTBE methyl t-butyl ether
  • Fig. 1 B illustrates a diagram showing the operating flow path of the concentric annular liquid-liquid separator.
  • the organic solvent is the wetting phase
  • the aqueous solution of the hyperpolarized metabolite is the non-wetting phase.
  • Pper permeate channel back pressure
  • Pret retentate channel back pressure.
  • Fig. 1C illustrates a cross-sectional diagram of Zaiput Flow Technologies’ patented membrane separator, which can provide continuous separation of an immiscible phase (liquid-liquid or gas-liquid) by leveraging differences in wetting properties of the liquids onto a porous membrane.
  • the aqueous phase consists of the hyperpolarized metabolite in the form of a carboxylate ion, while the wetting phase consisting of MTBE and acetone, is removed using a hydrophobic membrane
  • Hydrogen has two spin isomers: parahydrogen (pH2) and orthohydrogen (oH2).
  • pH2 and oH2 are associated with symmetric and antisymmetric rotational levels, respectively, 4 and because the energy splitting between the lowest two rotational levels is approximately 170 K, significant para-enrichment occurs even at moderately low temperatures.
  • x p « 0.5 while at 20 K, still above the normal boiling point of H2, x p « 0.99.
  • an ortho-para interconversion catalyst e.g., iron oxide
  • the para-enriched gas can be warmed to ambient temperature where the enrichment can persist for weeks.
  • SAH Side-arm hydrogenation
  • vital metabolites e.g., pyruvate
  • the pH2 spin order is transferred via the J coupling across the ester linkage to the carbonyl 13C.
  • Both propargylic and vinylic side arms (Scheme 1. Fig. 10) have been demonstrated.5-13 Vinyl affords the advantage of stronger J couplings (three or four bonds) to the 1-13C carbonyl, but these compounds are relatively unstable and challenging to synthesize.
  • Propargyl esters in contrast, are stable at ambient temperature, and their triple bonds afford higher rates of hydrogenation.
  • the pH2 sourced protons in allyl ester adducts are also further from the 13C target, and so the J-couplings are quite small.
  • FIG. 2 illustrates specific and generalized chemical structures of unsaturated side-arm ester precursors to which the devices, systems, and methods for the synthesis and purification of hyperpolarized metabolites from parahydrogen, as described herein, are applicable.
  • Adiabatic passage through a rotating frame LAC Adiabatic passage through a rotating frame LAC.
  • the SOT method employed in Nagel uses adiabatic RF field sweeps at /iT static field.19 Dilution of pH2 spin order due to state mixing with the methylene protons is overcome by deuteration of the propargylic side-arm (Scheme 2, Fig. 10). Cross- relaxation to 2H is suppressed by performing the RF sweeps at a high enough static magnetic field for deuterons and protons to be weakly coupled.
  • propargylic esters are employed because of their chemical stability, ease of synthesis, and higher rates of hydrogenation compared to vinyl esters20,21.
  • this process method relies on a long- range 1 H-13C J-coupling of only 0.4 Hz, which sets a correspondingly long timescale for SOT. Nevertheless, an impressive 20% 13C polarization level was achieved for purified [1- 13C] pyruvate.
  • aspects for the present disclosure provide for devices, systems, and methods for continuous flow synthesis of 13C hyperpolarized pyruvate and other metabolites in aqueous buffer that combine, in series: (i) efficient continuous-flow hydrogenation reactor technology; (ii) efficient spin order transfer for vinyl or propargyl esters, and (iii) novel liquid-liquid membrane wetting separator technology for continuous extraction of hyperpolarized metabolites into a biocompatible aqueous buffer.
  • the steps in the flow chemistry were presented in Fig. 1A.
  • Fig. 10 illustrates Scheme 3.
  • Scheme 3 illustrates the density matrix simulations of the 1H spin polarizations for hydrogenation with pH2 at low field (strong coupling) followed by adiabatic transport to high field for deuterated vinyl and propargyl esters.
  • the high H3 polarization in APd2 derives from adiabatic passage through a LAC.24
  • ALTADENA affords flexibility in the choice of coherence transfer pathways.
  • the MINERVA pulse sequence operates on the bilinear terms (f zl / z2 in EPd3, or f z2 7 z3 in APd2) while sINEPT operates on the Zeeman operators (i.e., 7 Z1 - l z2 in EPd3 or 7 z2 - i z3 in APd2).
  • a preliminary 13C spin polarization of P(13C) 12 % was observed for APd (without any optimization), corresponding to an anticipated polarization of 24 % for APd2, a promising initial result.
  • Fig. 4 presents Spinach25 density matrix calculations of P(13C) for allyl and ethyl adducts.
  • EPd3 MINERVA achieves P(13C) > 95 % for either ALTADENA or PASADENA preparation. Similar theoretical performance is obtained for sINEPT after ALTADENA preparation.
  • Selective 1 H excitation is also beneficial for carboxylic acids bearing protons with appreciable coupling to the carbonyl 13C (e.g., acetate).
  • the ALTADENA/LACADENA preparation of the spin system is inherently compatible with flow conditions, as adiabatic passage from low field (c.a. 0.05 mT) to high field (ca. > 10 mT) occurs as the fluid flows from the reactor at low field to the RF detection coil in the NMR probe at high field.
  • the flow rate and tubing diameter are selected to fulfill the adiabaticity criterion to the greatest possible extent while minimizing spin relaxation.
  • the coherence transfer pulse sequence will be applied to the liquid entering the RF coil of the NMR probe.
  • the pulse sequence will be applied repeatedly using a recycle time selected to match the residence time in the RF coil of the NMR flow cell. Flow rate is the key parameter.
  • FIG. 3 A illustrates timing diagrams for coherence transfer after adiabatic transport of ethyl or allyl ester adducts of pH2 from strong to weak coupling followed by selective INEPT or MINERVA NMR pulse sequences.
  • Fig. 3B Hyperpolarized NMR spectra for APd (preliminary data), where a 13C signal enhancement of 14,476 was observed, corresponding to a spin polarization of 12% (24% for APd2).
  • Fig. 4 illustrates the density matrix simulations of 13C polarizations for MINERVA or INEPT coherence transfer pulse sequences applied to various adducts after PASADENA or ALTADENA preparation. Losses due to spin relaxation are neglected.
  • AP allyl pyruvate
  • APd allyl pyruvate-d
  • APd2 allyl pyruvate-d2
  • EPd3 ethyl pyruvate-d3 (structures shown in Scheme 3, Fig. 10). Deuteration improves the efficiency across the AP series.
  • Hydrogenative PHIP experiments are typically initiated by bubbling of pH2 gas through a capillary tube immersed in the reactant solution. This is simple and effective, but not well-suited for chemical kinetics or quantification of PHIP signal enhancement due to changing reactant and product concentrations, ongoing accumulation of product molecules, and deactivation of dissolved catalysts. Estimation of the signal enhancement in a batch reactor is prone to large errors as the PHIP and thermally polarized signals are difficult to isolate for equal numbers of product molecules. Thus, batch mode experiments are not well- suited for quantitative comparison of SOT methods.
  • Continuous-flow PHIP is ideal for systematic study and quantification of conversion, signal enhancement, and SOT efficiency because the initial condition of each experiment is identical, there is no accumulation of products, and fresh catalyst solution is employed in each trial.
  • continuous flow PHIP requires a continuous-flow reactor.
  • An ultrasonic spray injection reactor suitable for dissolved or suspended catalysts;28-31 This reactor design is scalable and integrable into continuous or interrupted flow SAH-PHIP processes.
  • Fig. 5 illustrates the reactor for homogeneous hydrogenation. Comparison of the droplet size distributions produced by ultrasonic spray injection at a nozzle frequency of 180 kHz (green) and conventional spray injection
  • phase separation While continuous liquid-liquid extraction (phase separation) can be achieved using gravimetric settling vessels, 38 a more rapid phase separation can be achieved by leveraging differential surface wetting.
  • the organic phase preferentially wets a porous hydrophobic surface such as PTFE
  • the aqueous phase preferentially wets a hydrophilic surface such as glass or stainless steel or remains in the bulk phase.
  • aspects of the present disclosure utilize a microporous PTFE membrane for the removal of organic solvent from an emulsion with an aqueous phase in which the hyperpolarized molecules are preferentially dissolved.
  • the separation step is performed immediately after injection of an aqueous base or other aqueous hydrolysis catalysis into the hyperpolarized fluid and restoration of neutral pH by mixing with a buffer.
  • Continuous-flow liquid-liquid and liquid-gas separation devices incorporating a porous PTFE membrane are commercially available from Zaiput Flow Technologies, Inc. [see Fig. 1 C], These separators provide continuous separation of immiscible phases by leveraging differences in wetting properties of the liquids onto a porous membrane. Both hydrophilic and hydrophobic membranes are available. The membrane pores are filled with the wetting phase, and a pressure differential is maintained between the two sides of the membrane. This pressure differential is finely adjusted by an internal pressure controller to apply just enough pressure to “push” the wetting phase through without forcing the nonwetting phase through the pores.
  • An aspect of this technology is that it exploits differences in wettability and surface forces to accomplish separation; hence, the device can even separate liquids with the same density.
  • Applications include radioisotope purification, 33 continuous separation and purification of chemical species, 34 and on-demand flow production of pharmaceuticals.35-37
  • These devices are suitable for use in a multi-stage ATTORNEY DOCKET NO. 222112-2510 extraction configuration, in a counter- current configuration or in series, as shown in Fig. 6A- C for three stages
  • the schemes can be readily modified to include additional separation stages or fewer stages, as needed to achieve the desired final purity.
  • Fig. 6 illustrates a three stage in-series configuration of Zaiput membrane separation devices, each fitted with a hydrophobic membrane, where the water/acetone/metabolite solution is mixed with MTBE before the first stage, and the liquid emerging from non-wetting port is mixed with fresh MTBE after each consecutive separation device. Residual volatile organic compounds are removed using a solvent stripping method such as the one described in part C of this figure.
  • Fig. 6B Alternative counter-current configuration using three stages if membrane separation which provides optimal extraction efficiency using a fixed volume of extraction solvent (MTBE).
  • Fig. 6C Alternative counter-current configuration using three stages if membrane separation which provides optimal extraction efficiency using a fixed volume of extraction solvent (MTBE).
  • Method for stripping of residual dissolved MTBE and acetone or other volatile compounds in the aqueous phase by mixing with nitrogen gas to form a biphasic gas/liquid mixture.
  • the vapor is removed by a membrane separation device.
  • a hydrophilic membrane is installed in the separation device, the vapor is the non-wetting phase, and the aqueous phase containing the metabolite is the wetting phase.
  • Multistage Liquid Liquid Extraction is a process where multiple extraction steps are repeated in order to increase the recovery of a product. This process is required when, due to a small partition coefficient, the recovery in a single extraction step is insufficient.
  • LLE can be performed with a “countercurrent extraction” scheme that provides the smallest consumption of extractant. In this scheme, the aqueous raffinate from one stage is fed to a former stage as a feed while the organic phase is moved in the opposite direction. Hence, even if the recovery of product in each stage is small, the overall system can achieve a high level of recovery.
  • selectivity of the extraction and process yield are decoupled as the yield depends on the number of extraction stages used. As the number of stages increases, extraction efficiency increases, while still using the same amount of organic solvent that would be used if only one batch extraction stage was performed.
  • the parameters for selecting an appropriate membrane are the interfacial tension between the two phases and the viscosity of the permeating phase. In general, the lower the interfacial tension, the smaller the required pore size.
  • the interfacial tension of the non-polar component MTBE
  • Water soluble salts for example, NaCI, Na2CO3, phosphate buffers
  • can increase the interfacial tension resulting in improved liquid-liquid phase separation and improved membrane separation.
  • our experimental results conclusively demonstrate that separation by the Zaiput membrane device is improved by the addition of sodium carbonate salt to the acetone/water/MTBE mixture, resulting in lower mass fraction of acetone and MTBE in the aqueous phase. This effect is closely related to the “salting-out” effect that occurs when the solubility of a nonelectrolyte in water decreases after adding salt.
  • a metabolite molecule which is present in the form of a sodium carboxylate salt can, at sufficiently high concentration, improve the removal of acetone from the aqueous phase in the separation of a mixture of MTBE, acetone, and water, thus increasing the purity of the aqueous phase and lowering the final concentration of acetone in the extracted aqueous phase.
  • salts e.g. NaCI, Na 2 CO 3
  • the salt concentration in an aqueous solution of the hyperpolarized metabolite must not exceed established limits for safe in-vivo administration.
  • higher concentrations of metabolite, which is present in the form of a sodium carboxylate salt can be hyperpolarized.
  • the high concentration of the sodium carboxylate species will increase the partitioning of acetone into the organic phase, without the need for addition of other salts (e.g., NaCI, Na 2 CO 3 ) that cannot be readily removed from the aqueous solution, and the final concentration of acetone in the aqueous phase after gravimetric or membrane separation will be reduced.
  • the concentration of sodium pyruvate can be reduced by dilution with additional aqueous buffer to obtain the desired concentration that is safe for in- vivo administration.
  • Fig. 7 illustrates preliminary results that demonstrate the similar performance in the extraction of acetone from a 1 :1 v/v acetone/water mixture containing 200 mM Na pyruvate into methyl t-butyl ether (MTBE) using (a) conventional gravimetric extraction (dashed curves) and (b) flow separation using a Zaiput SEP-10 device fitted with a ATTORNEY DOCKET NO. 222112-2510 hydrophobic membrane (solid curves). Syringe pump flow rates of 3 ml/min for MTBE and 1.5 ml/min for the 1 :1 v/v acetone/water mixture. The liquids were mixed using an IDEX mixing tee fitted with a 10 pm frit followed by a 1 m section of 1/16 in green PEEK tubing to promote plug flow mixing
  • the first implementation for removing residual organic solvents uses a concentric annular liquid-liquid phase separator such as the one illustrated in Fig. 1B.
  • the interface between liquid and gas phases at the membrane pore entrance is flat, and the device operates isothermally.
  • the tube-in-tube device is comprised of concentric annular channels.
  • the device may be fabricated using SwagelokTM fittings without adhesives or O-ring seals.
  • a similar tube-in-tube device for dissolution and degassing of hydrogen has been demonstrated using Dupont Teflon AF2400 tubing, a porous PTFE membrane material.
  • a second method for stripping residual volatile organic solvents (acetone, MTBE) from the aqueous solution of hyperpolarized metabolites (as carboxylate species) uses a Zaiput membrane device (such as the Zaiput model SEP- 10) that is suitable for gasliquid separations.
  • a Zaiput membrane device such as the Zaiput model SEP- 10.
  • Mixing of the acetone/MTBE/water mixture with N2 gas to form a biphasic mixture is followed by diffusion of the volatile organics into the gas phase followed by removal of the aqueous phase from the mixture by its selective wetting and transport through a hydrophilic porous membrane.
  • the purified aqueous phase containing the hyperpolarized metabolite exits the device through the wetting side port.
  • FIG. 8A A block diagram of an embodiment of an experimental setup is shown in Fig. 8A.
  • Arbitrary mixtures of dissolved (or dispersed) catalyst, precursor, and solvent can be prepared by control of the infusion pump rates, enabling kinetics studies of the flow chemistry and achieving desired molar polarization.
  • Reactants are continuously infused into the reactor (ultrasonic spray injection or planar gas diffusion device) at a target flow rate of 5 ml/min.
  • Dissolved catalyst can be removed using a commercially available silica gel supported chelating agent (SiliCycle, Inc.).
  • the pH2 adducts flow adiabatically into the NMR #1 where the coherence transfer pulse sequence is applied.
  • the residence time in the active volume of the NMR flow cell is approximately 6 s.
  • pumps A, B, and C inject an aqueous base (e.g., NaOD, to induce hydrolysis), phosphate buffer, and MTBE solvent, respectively.
  • aqueous base e.g., NaOD
  • phosphate buffer e.g., phosphate buffer
  • MTBE solvent e.g., MTBE solvent
  • acetone and the hydrophobic side-arm partition into the organic phase, while the hyperpolarized carboxylate remains in the aqueous phase.
  • the emulsion is then separated into organic and aqueous fractions using a Zaiput model SEP-10 liquid-liquid separator which can operate at flow rates up to 10 ml/min.
  • NMR #2 refers to our Varian VNMRS 400 MHz NMR spectrometer fitted with a Varian flow probe which will be used to analyze the final composition and polarization of the emergent metabolite solution.
  • This setup will facilitate the systematic studies necessary to develop a comprehensive numerical model of the overall process.
  • a powerful feature of this experimental setup is its modularity, allowing the various system parameters to be isolated and separately characterized.
  • the flow chemistry can be studied without hyperpolarization to establish the reaction conditions and chemical kinetics leading to complete conversion and hydrolysis.
  • the hyperpolarization and SOT steps can be independently characterized.
  • the performance of LLS will be studied as a function of flow rate, relative fractions of aqueous and organic phases, solute concentrations, and membrane back-pressure. Isolating the various parameter subspaces will assist the development of a complete numerical model and a rational strategy to maximize the molar polarization in aqueous buffer.
  • Fig. 8A illustrates experimental flow chemistry system with SOT (NMR #1) and spectroscopic analysis (NMR #2).
  • VOCs volatile organic solvents
  • Fig. 8B Separation of the hyperpolarized metabolite in aqueous buffer using a hydrophobic membrane. Removal of the dispersing medium fosters coalescence of the dispersed phase, shown in blue.
  • Fig. 9A shows the 300 MHz 1 H NMR spectrum of the initial solution of ethyl pyruvate in acetone-d6, in blue, and the spectrum obtained after the addition of aqueous base (Na2CO3 in D2O), in orange.
  • aqueous base Na2CO3 in D2O
  • H A and H A refer to the ethyl pyruvate ester precursor and free pyruvate methyl group signals, respectively. Also visible are the ethyl ester side-arm and ethanol signals resulting from hydrolysis.
  • Fig. 9B shows the 300 MHz 1 H NMR spectra after mixing and extraction of the partially hydrolyzed solution in acetone-d6/D20 after a single-pass through the SEP- 10 continuous-flow membrane separator device (blue spectrum) or after traditional gravimetric extraction with a single mixing of MTBE.
  • the presence of strong pyruvate methyl signals (labelled H A ) in the blue and orange spectra with similar intensities shows that the efficacy of the two separation methods is similar.
  • Fig. 9A and 9B illustrate 300 MHz 1H NMR spectra comparing the hydrolysis of ethyl pyruvate and extraction into the aqueous phase by batch and continuous-flow processes.
  • Fig. 9A illustrates the pre- and post-hydrolysis spectra obtained after addition of aqueous sodium carbonate solution to the ethyl pyruvate solution (in acetone-d6).
  • Fig. 9B illustrates the spectra of the aqueous fraction after addition of aqueous base and single stage separation by gravimetric (orange spectrum) or continuous-flow Zaiput SEP- 10 device with the hydrophobic membrane installed.
  • the reaction temperature, catalyst concentration, and infusion rate are the operating parameters that can be varied to optimize the rate of production of the desired hydrogenation products (e.g. allyl acetate).
  • the ultrasonic frequency of the nozzle can also be adjusted to vary the droplet size and hence the liquid/gas interfacial surface area.
  • Rh(dppb) catalyst The activity and pairwise selectivity of the Rh(dppb) catalyst will be examined in mixtures with cyclohexane, DMSO, or other added ligands, and hydrogenation rate with co-solvent will be compared to the rate in pure acetone. [0197] If the addition of a cosolvent is found to improve catalytic activity and pairwise selectivity to levels comparable to acetone, hydrogenation in the co-solvent system will be tested in the ultrasonic spray injection reactor and the operating parameter systematically varied to optimize conversion to the desired product.
  • Acetone is an outstanding solvent for hydrogenation of unsaturated ester precursors using the Rh(dppb) catalyst. After hydrogenation, aqueous base and buffer solutions are added to induced hydrolysis and restore the pH, respectively. Since acetone is miscible with water at all compositions, another organic solvent (immiscible with water) is required for extraction of acetone from the aqueous solution. The process described in Nagel employed MTBE to extract acetone. [14] Based on our preliminary data, we claim that anisole (and perhaps other solvents with similar polarity) is a better solvent for extracting acetone from acetone-water mixtures, as seen in Figure 12a. Moreover, the efficacy of extraction improves with increasing temperature.
  • Rh(dppb) catalyst in a co-solvent system (such as cyclohexane- acetone) can match hydrogenation in acetone, it would greatly simplify purification, as the solubility of cyclohexane (55 ppm) is much lower than ICH’s limit of 3370 ppm [31], rendering multi-stage extraction unnecessary.
  • the SEP-10 membrane device was shown to be able to remove emulsion of organic solvents, ensuring low organic solvent content, making residual solvent stripping unnecessary. These simplifications could reduce the extraction time by up to 40 s, thereby reducing the relaxation losses by about 20%.

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Abstract

La présente divulgation concerne des dispositifs, des systèmes et des procédés de fabrication de métabolites hyperpolarisés. Les dispositifs, les systèmes et les procédés de fabrication de métabolites hyperpolarisés peuvent être mis en œuvre de manière continue, ce qui peut être avantageux lors de la collecte d'une image IRM d'un sujet. La présente divulgation concerne la fabrication du métabolite hyperpolarisé et la séparation du métabolite hyperpolarisé du solvant utilisé au cours de la préparation du métabolite hyperpolarisé. La séparation peut être effectuée promptement et plus rapidement que d'autres procédés. La présente divulgation concerne également des agents de contraste et des procédés d'imagerie utilisant des agents de contraste.
PCT/US2025/041467 2024-08-13 2025-08-11 Dispositifs, systèmes et procédés de synthèse et de purification de métabolites hyperpolarisés à partir de parahydrogène Pending WO2026039330A2 (fr)

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