Purified signal-enhancing contrast agents for magnetic resonance imaging - Patents.com

Abstract

The present invention relates to an improved method for obtaining purified contrast agents suitable for magnetic resonance imaging. The contrast agents are prepared by methods such as Dynamic Nuclear Polarization (DNP), Parahydrogen Induced Polarization (PHIP) or Signal Amplification by Reversible Exchange (SABRE). High purity is achieved by performing an evaporation step to separate the signal enhancing precursor or contrast agent from the metal catalyst or radical source.

Description

[Technical field]

[0001] The present invention relates to an improved method for obtaining purified contrast agents suitable for magnetic resonance imaging. The contrast agents are prepared by methods such as dynamic nuclear polarization (DNP), parahydrogen induced polarization (PHIP) or Signal Amplification By Reversible Exchange (SABRE). High purity is achieved by performing an evaporation step to separate the signal enhancing precursor or contrast agent from the metal catalyst or radical source. [Background technology]

[0002] Magnetic resonance imaging (MRI) is a technique widely used in research laboratories and clinics to examine and diagnose disease patterns. To make the disease more visible, a contrast agent is injected into the organism. Worldwide, approximately 30 million contrast-based examinations are performed in clinics every year.

[0003] A special form of contrast agent that allows to directly observe chemical processes such as metabolism is based on the process of hyperpolarization. In the field of nuclear magnetic resonance (NMR), the phenomenon on which MRI is based, the hyperpolarization procedure increases the signal of the contrast agent by several orders of magnitude. Hyperpolarization is used, for example, to increase the signal of metabolites, which are then injected to visualize metabolism in tumors. This has already been shown in clinical studies in humans. In particular, it has been shown that such methods can be used to identify tumors and evaluate the effectiveness of treatment. In the future, they may replace positron emission tomography (PET) in cancer staging.

[0004] A common method of amplifying signals by hyperpolarization is based on the technique of dynamic nuclear polarization (DNP). To achieve the maximum possible signal amplification, contrast agents are usually hyperpolarized at cryogenic temperatures (i.e., temperatures below 2 K) in the presence of radicals using microwave radiation. This process takes tens of minutes to hours, making it very time-consuming and significantly limiting the use of this method. After signal amplification, contrast agents are heated and dissolved so that they can be injected, whereby radicals are typically filtered out.

[0005] Another method of hyperpolarization, which delivers an amplified signal within seconds, is based on parahydrogen or ortho-deuterium. The use of parahydrogen is the more common form of hyperpolarization. Parahydrogen is a spin isomer of hydrogen gas that is enriched by passing the corresponding gas over a catalyst (usually iron oxide or activated carbon) at low temperature. At 77 K, an enrichment degree of about 50% is achieved, and at 25 K, this is about 100%. If the catalyst is removed after enrichment, the gas can be bottled and stored at room temperature for days to weeks. In the parahydrogen-based hyperpolarization process, the spin order generated by the enrichment of parahydrogen is converted into an amplified signal from the molecule, which is then used as a contrast agent.

[0006] There are two ways in which parahydrogen can be combined with the substrate to achieve a hyperpolarized signal. On the one hand, parahydrogen can be added to the molecule by a catalyst, which changes the chemical structure of the substrate (hydrogenation parahydrogen induced polarization, PHIP). After the addition process, the spin order is converted into an amplified observable signal. On the other hand, parahydrogen can form a transiently stable complex with the catalyst and the compound of interest (e.g. a metabolite), where the spin order is converted into an amplified signal of the compound of interest. The unstable complex then dissociates again in its components, resulting in an unchanged signal-amplifying contrast agent. This non-hydrogenation PHIP method is called SABRE (signal amplification by reversible exchange, SABRE).

[0007] Overall, pyruvate, lactate, acetate, succinate, fumarate, acetoacetate and hydroxybutyrate and their potential alkyl esters are the most important contrast agents. These molecules can be signal enhanced via PHIP or SABRE by generating precursors with unsaturated moieties such as vinyl esters that are hydrogenated with parahydrogen and subsequently converted to metabolites, or by using them directly as esters (PHIP) or by potential direct signal enhancement via SABRE, but a catalyst is always required to generate the enhanced signal. When generating contrast agents with DNP, a radical generating source is required.

[0008] However, the catalysts are difficult to separate and usually consist of heavy metals such as rhodium, iridium, platinum, palladium, ruthenium and rhenium.Similarly, sufficient separation of the radical source is difficult to achieve.

[0009] So far, purification procedures have relied on filtration, precipitation and washing or liquid phase separation of organic solvents and water to deliver aqueous injectable solutions.

[0010] All these methods show difficulties in filtering out, for example, the last amounts of rhodium catalyst. The rhodium content is usually reduced by a factor of 100-1000 by these state-of-the-art techniques, leaving micromolar metal contents. Assuming an injection volume of 25 mL, typical in clinical studies using DNP techniques (CY Lee et al. Neuroimage 204, 116-202, 2020), this results in the injection of, for example, 10 μg of rhodium (5 μM metal concentration in 25 mL of injection solution, molecular weight of rhodium 103 g / mol). In comparison, these values ​​are about 1000 times higher than the metal base levels found in human blood, as suggested by recent studies (G. Rentschler et al. International Journal of Hygiene and Environmental Health 221, 223-230, 2018).

[0011] For better removal of catalyst or radical source, the purification described here is carried out by a rapid evaporation / distillation step. Hereby, evaporation can also mean using a stripping gas. Evaporation can be accelerated by applying a vacuum, with or without a stripping gas. The stripping gas can be any gas or vapor, preferably an inert gas, e.g. nitrogen, hydrogen. Stripping gas is more efficient when combined with a vacuum.

[0012] This evaporation step has been proven to further reduce the metal content by a factor of 100-1000 compared to state-of-the-art purification, as confirmed by ICP-MS analysis. Thus, for example, the rhodium content is in the nanomolar range.

[0013] Compared to other methods, the approach presented here is particularly suitable for large-scale production of clinic-applicable hyperpolarized contrast agents.

[0014] Based on the above-mentioned state of the art, the object of the present invention is to provide means and methods for purifying signal enhancing compounds more efficiently compared to the state of the art methods. This object is achieved by the subject matter of the independent claims herein as well as further advantageous embodiments described in the dependent claims herein, the examples, the figures and the general description. Summary of the Invention

[0015] The present invention relates to a method for preparing an imaging agent, characterized in that it comprises an evaporation step, in which a signal enhancing compound containing nuclear spins is separated from a liquid.

[0016] Terms and Definitions For purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the set forth definition shall control.

[0017] As used herein, the terms "comprising," "having," "containing," and "including," and other similar forms and their grammatical equivalents, are intended to be equivalent in meaning and open-ended in that the item or items following any one of these words are not meant to be an exhaustive enumeration of such item or items, or to be limited to only the item or items listed. For example, an article "comprising" components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can include not only components A, B, and C, but also one or more other components. Thus, "comprises" and similar forms and their grammatical equivalents are intended and understood to include the disclosure of embodiments that "consist essentially of" or "consist of."

[0018] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to one-tenth of the unit of the lower limit, between the upper and lower limits of that range, and any other stated or intervening value within that stated range, is encompassed within the disclosure, subject to any specifically excluded limits in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0019] Reference herein to "about" a value or parameter includes (and describes) the variation directed to the value or parameter itself. For example, a statement indicating "about X" includes the statement "X."

[0020] As used in this specification, including the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0021] The term "alkyl" in the context of this specification refers to saturated or unsaturated straight or branched chain hydrocarbons. For example, C1-C6 alkyl in the context of this specification refers to saturated or unsaturated straight or branched chain hydrocarbons having 1, 2, 3, 4, 5 or 6 carbon atoms. Non-limiting examples of C1-C6 alkyl include methyl, ethyl, propyl, 1-methylethyl (isopropyl), n-butyl, 2-methylpropyl, tert-butyl, n-pentyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, n-hexyl, 3-methyl-2-pentyl and 4-methyl-2-pentyl.

[0022] The term "signal enhanced compound" or "signal enhanced compound that comprises a nuclear spin" refers to a compound that comprises a hyperpolarized hydrogen atom or a hyperpolarized heteronucleus. The signal enhanced compound may be used as such as a purified imaging agent or may be modified prior to using the modified signal enhanced compound as a purified imaging agent. The modification may be a chemical transformation such as transfer of spin order or cleavage to a metabolite.

[0023] Contrast agents are purified versions of signal enhancing compounds that are potentially intended to be delivered to a biological sample (enzymes, cells) or to be injected into an organism.

[0024] The term "heteronuclear" refers to a nucleus other than hydrogen. The most suitable heteronuclei are 13 C or 15 N, especially 13 C

[0025] "Hyperpolarized H atom", "Hyperpolarized 13 C atom" or "hyperpolarized 15 The term "N atom" refers to an H atom with a hyperpolarized spin, 13 C atom or15 Refers to the N atom.

[0026] The term "liquid" refers to a liquid that contains a signal enhancing compound prior to the evaporation step. The liquid may further contain a neat catalyst, such as an ionic liquid, or a solvent and a catalyst, or a solvent and a radical source, whereby the solvent may be the signal enhancing compound. Additionally, the liquid may contain by-products and / or unreacted / unpolarized educt(s) and other impurities.

[0027] The signal enhancing compound is not gaseous under the conditions prior to the evaporation step.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0029] explanation Contrast agents suitable for use in clinical settings, for example for the diagnosis of diseases by MRI, or for preclinical studies, should ideally be free of reactants, by-products, and catalysts or radicals and generally free of sources of impurities. If the preparation is carried out in organic solvents, the contrast agent can be transferred to an aqueous solution that can be injected into the organism or applied to cells or enzymes.

[0030] The imaging agents can be prepared by methods utilizing radicals in combination with spontaneous reactions or transfer, pulsed NMR techniques, pulsed polarisation transfer, electric field cycling or microwave radiation. Well known methods include, for example, PHIP, SABRE or DNP.

[0031] PHIP is a hydrogenation procedure. The appropriate educt contains an unsaturated bond that is hydrogenated during the process in the presence of a metal catalyst. As a result, the educt changes its chemical structure during the hyperpolarization step. For example, a vinyl ester (educt) can be converted to a para-hydrogenated ethyl ester. The para-hydrogenated ethyl ester may be used as an imaging agent as such. Alternatively, the spin order of the para-hydrogenated ethyl ester can be modified using a 13 C or 15 It may be further transferred to a heteronucleus such as N and then used as an imaging agent. Optionally, the ethyl ester can be cleaved. For example, hyperpolarised ethyl pyruvate can be cleaved to give hyperpolarised pyruvate.

[0032] To obtain a highly purified contrast agent, at least one purification step is necessary, which can be carried out after the hydrogenation step and / or after the transfer of spin order to the heteronuclei.

[0033] SABRE is a non-hydrogenation procedure. In contrast to PHIP, para-hydrogen does not react in an addition reaction, but forms a temporarily stable complex with the catalyst and the educt (e.g., a metabolite such as pyruvate), in which the spin order of para-hydrogen is transferred to the hydrogen atom of the educt. The unstable complex then dissociates again in its components. Thus, the educt does not change its chemical structure in the hyperpolarization step. The resulting signal-enhancing compound may be used as a contrast agent, etc., or the spin order may be changed before the signal-enhancing compound is used as a contrast agent. 13 C or 15 It may optionally be further transferred to a heteronucleus such as N. In the particular case of SABRE, the spin order of the parahydrogen is directly transferred to the spin of the heteronucleus.

[0034] A purification step may be performed after the transfer of spin order of the parahydrogen to the educt and / or after further transfer of spin order to the heteronucleus.

[0035] Signal enhancing compounds may also be prepared by optimized PHIP or SABRE methods. Such a method is - providing a molecule comprising two protons and at least one heteronucleus (S3, S4), the protons having a nuclear spin coupled to the nuclear spin of the at least one heteronucleus; - exposing the protons and the at least one heteronuclease to a magnetic field (B0) in a z-direction, the z-direction forming a right-handed Cartesian coordinate system with the x- and y-directions; - applying a series of radio frequency pulses to the protons and to the at least one heteronucleus in order to transfer the 2-spin order to hyperpolarization of the at least one heteronucleus, the series of radio frequency pulses being comprised of a first, a second and a third group of 180° radio frequency pulses (N A , N B , N C ), a first group of 180° radio frequency pulses (N A ) for the first time interval (τ A ) between n A The second group of 180° radio frequency pulses (N B ) is the second time interval (τ B ) between n B A third group of 180° radio frequency pulses (N C ) is the third time interval (τ C ) between n C applied n times in succession A , n B , n C are integers, steps, Includes.

[0036] A purification step may be performed after the transition of the two spin orders or longitudinal orders.

[0037] In DNP, the higher polarization of electron spins is converted to hyperpolarized nuclear spins. Parahydrogen is not used at all. Instead, radicals are typically used as a source of spin order to signal enhance the molecule of interest. As a result, the educt does not change its chemical structure during the hyperpolarization step.

[0038] After hyperpolarization, the signal enhancing compound can be purified.

[0039] The purpose of the purification step is to separate the catalyst (PHIP, SABRE) or the radical source (DNP) from the signal enhancing compounds.

[0040] Typically, the proton longitudinal relaxation time of the signal enhancing compound is in the range of 1 to 15 seconds, e.g. 13 For a C spin this can be as long as 60 seconds. To maintain the signal, purification needs to be done quickly, i.e. in the range of seconds rather than minutes. Such rapid purification is achieved by a rapid evaporation step.

[0041] A first aspect of the invention relates to a method for preparing an imaging agent, characterized in that it comprises an evaporation step, in which a signal enhancing compound comprising nuclear spins is separated from a liquid.

[0042] In certain embodiments, the imaging agents are prepared by PHIP (parahydrogen induced polarization), SABRE (signal amplification by reversible exchange), or DNP (dynamic nuclear polarization).

[0043] The liquid refers to a liquid that contains a signal enhancing compound prior to the evaporation step. - Neat catalysts, such as ionic liquids, and signal enhancing compounds, or - Solvents, signal enhancing compounds and catalysts, or - Solvents, signal enhancing compounds and radical sources and the solvent may be a signal enhancing compound. Additionally, the liquid may contain by-products and / or unreacted / unpolarized educts and other impurities.

[0044] When performing PHIP or SABRE to prepare a signal enhancing compound, the liquid is a solution that includes a solvent, a signal enhancing compound, and a catalyst, whereby the solvent can be the signal enhancing compound.

[0045] When DNP is performed to prepare a signal enhancing compound, the liquid is a solution that includes a solvent, a signal enhancing compound, and a radical source, whereby the solvent can be the signal enhancing compound.

[0046] In certain embodiments, the liquid comprises a signal enhancing compound and a catalyst. The catalyst can be a solid catalyst dissolved in the signal enhancing compound. In certain embodiments, the liquid comprises a signal enhancing compound and a radical source.

[0047] Additionally, the liquid may contain by-products and / or unreacted / unpolarized educts and other impurities.

[0048] Depending on the boiling temperature, either the solvent of the liquid evaporates or the signal enhancing compound evaporates. If the liquid contains several components with different boiling points, e.g. solvent, signal enhancing compound and catalyst, two or more distillations may be required, e.g. evaporating the organic solvent first and then the signal enhancing compound. It may also be necessary to first evaporate the unreacted precursor compound before evaporating the solvent and / or the signal enhancing compound. Suitable solvents are, for example, aqueous solvents, methanol, chloroform or acetone. To allow a rapid evaporation step, the components may be selected such that the boiling point of the signal enhancing compound is the lowest, so that the signal enhancing compound can be distilled in one step from the liquid of the reaction vessel.

[0049] In certain embodiments, the liquid comprises a catalyst, a signal enhancing compound, and a water-immiscible organic solvent. Water is added prior to evaporation to form two phases. The signal enhancing compound is separated from the liquid by evaporating the organic solvent and transferring the signal enhancing compound to the aqueous phase to provide a purified signal enhancing compound in water.

[0050] The desired chemical transformation (e.g., cleavage) of the enhancing compound can occur during this process (e.g., if a cleavage agent such as a base has been added to the water), after which impurities can be filtered out of the aqueous solution.

[0051] In certain embodiments, the signal enhancing compound is separated from the liquid by evaporating the signal enhancing compound to provide a purified signal enhancing compound. In certain embodiments, the signal enhancing compound is separated from the liquid by first evaporating the organic solvent, and after separating the organic solvent, the signal enhancing compound is evaporated to provide a purified signal enhancing compound.

[0052] In certain embodiments, the signal enhancing compound is separated from the liquid by evaporating the signal enhancing compound to provide a purified signal enhancing compound, and the purified signal enhancing compound is modified by chemical transformation such as transfer of spin order and / or cleavage into metabolites, and post-modification purification of the signal enhancing compound is achieved by a further evaporation step of the signal enhancing compound.

[0053] Additional purification steps can be performed after any evaporation step, such as filtration, precipitation and washing or liquid phase separation to provide the contrast agent.

[0054] In certain embodiments, the signal enhancing compound is separated from the liquid by evaporating the signal enhancing compound to provide a purified signal enhancing compound, and the purified signal enhancing compound is modified by a chemical transformation such as a transfer of spin order or cleavage into a metabolite, and post-modification purification of the signal enhancing compound is achieved by further purification steps such as filtration, precipitation and washing or liquid phase separation.

[0055] To maintain the spin order of the signal enhancing compound, the evaporation step may be carried out in a static magnetic field.

[0056] In a particular embodiment, the evaporation step is carried out in a static magnetic field.

[0057] In a particular embodiment, the evaporation step is carried out in a static magnetic field having a field strength of at least 10 mT.

[0058] The longitudinal relaxation time depends on the temperature and pressure. The relaxation time in the gas phase is typically brought about by relaxation of spin rotation. For large molecules such as ethyl lactate, ethyl pyruvate or ethyl acetate, which are particularly suitable as precursors of signal-enhancing contrast agents or as contrast agents themselves (e.g., ethyl pyruvate), it is on the order of 1-15 seconds in the gas phase. Longer times are preferable and they can be obtained by increasing the pressure and / or temperature in the gas phase. Thus, fast distillation steps are feasible and can be used as an excellent mechanism during purification to maintain signal enhancement.

[0059] Evaporation can be further improved by using a co-solvent or a stripping gas.

[0060] In certain embodiments, a co-solvent or stripping gas is used during the evaporation step.

[0061] In certain embodiments, a stripping gas is used during the evaporation step.

[0062] In certain embodiments, the signal enhancing compound is evaporated at a pressure of at least 3 bar.

[0063] In a particular embodiment, the signal enhancing compound is evaporated under pressure, in particular at a pressure of at least 3 bar, and a stripping gas is used.

[0064] In certain embodiments, the signal enhancing compound is evaporated at a temperature of at least 390K.

[0065] The time required to evaporate a particular amount of the signal enhancing compound can be reduced by applying a vacuum during the evaporation step.

[0066] In certain embodiments, a vacuum is applied during the evaporation step. Evaporation can be facilitated by application of a vacuum, with or without a stripping gas.

[0067] In certain embodiments, the evaporation step with the stripping gas is facilitated by applying a vacuum.

[0068] In certain embodiments, the signal enhancing compound is evaporated using a stripping gas under pressure, the evaporation being enhanced by application of a vacuum, in particular by application of 10 mbar or less.

[0069] In a particular embodiment, the evaporation step is carried out at 10 mbar or less.

[0070] In a particular embodiment, the evaporation step is carried out below 10 mbar and no stripping gas is applied.

[0071] In particular, when preparing signal enhancing compounds via PHIP, unreacted unsaturated educts may still be present in the liquid. To prevent potential toxicity, the educts may be quenched. This can be done, for example, by adding an excess of a thiol-containing biomolecule, such as the amino acid cysteine, which reacts with the unsaturated precursor in a thiol-ene reaction. The reaction products need to be non-volatile, which is typically achieved, for example, when using salts of biomolecules. To speed up this process, additional catalysts may be added or radicals need to be introduced. However, it is important that they are not volatile and remain in the reaction vessel while the signal enhancing compounds evaporate and are transferred to another vessel.

[0072] In certain embodiments, prior to the evaporation step, the liquid unreacted educts are converted into non-volatile products, in particular non-volatile salts. This can be achieved, for example, by adding the amino acid cysteine ​​or another suitable molecule in the presence of a catalyst to trap the unreacted signal enhancing precursors by reacting with their double or triple C-C bonds.

[0073] In certain embodiments, the purified signal enhancing compound comprises - condensed after evaporation and then dissolved in an aqueous solvent, or - Directly captured in the aqueous medium after evaporation.

[0074] The solvent in which the purified signal enhancing compound is dissolved or directly entrapped can be a biocompatible solvent.

[0075] In certain embodiments, the signal enhancing compound is volatile.

[0076] In certain embodiments, a chelating molecule is added prior to the evaporation step that binds the catalyst and greatly reduces its volatility.

[0077] There must be a sufficient difference in boiling points between the liquid and the signal enhancing compound to allow efficient distillation to vaporize only the signal enhancing compound.

[0078] In certain embodiments, the signal enhancing compound has a boiling point that differs from that of the liquid by at least 20° C., particularly at least 40° C.

[0079] In certain embodiments, the signal enhancing compound is a hyperpolarized H atom or a hyperpolarized 13 C atoms or hyperpolarized 15 Contains N atoms.

[0080] Suitable signal enhancing compounds are hyperpolarized metabolites such as esters of amino acids with short side chains (e.g., alanine or serine), pyruvate or its alkyl esters, lactate, acetate, succinate or its alkyl esters, fumarate or its alkyl esters, acetoacetate or its alkyl esters, hydroxybutyrate or its alkyl esters, ketoisocaproate or its alkyl esters, malate or its alkyl esters, citrate or its alkyl esters, etc. These signal enhancing compounds can be obtained by PHIP, SABRE or DNP.

[0081] When performing SABRE to prepare signal enhancing compounds, heterocycles such as pyridine or nicotinamide can also be used as educts.

[0082] In certain embodiments, the signal enhancing compound is selected from a 5- or 6-membered heterocycle containing one or more N atoms, nicotinamide or a compound of formula I.

[0083] In certain embodiments, the signal enhancing compound is a compound of formula I, [ka] During the ceremony, R is H, -OH, -OX' or C 1-4 -alkyl, where alkyl is unsubstituted or substituted with -OH; Y is C 1-6 -Alkyl or C 1-6 -alkenyl, where the alkyl is unsubstituted or substituted with one or more substituents selected from NH, -OH, -COOH or -COOX'; X, X' and X' are each independently C 1-4 -alkyl, m, n and p are independently 0 or 1; The compound of formula I contains at least one hyperpolarized atom, in particular a hyperpolarized H atom or a hyperpolarized 13 C atoms or hyperpolarized 15 It includes N atoms, and in particular one, more or all H can be replaced with deuterium.

[0084] When PHIP is performed to obtain a signal enhancing compound, the educt contains a double bond. Suitable educts are compounds containing vinyl, allyl or propargyl moieties. These moieties are para-hydrogenated during PHIP. The para-hydrogenated moiety corresponds to moiety X.

[0085] In a particular embodiment, X, X' and X" are ethyl and each C atom is bonded to a hyperpolarized H atom.

[0086] In certain embodiments, the imaging agent is selected from hyperpolarized pyruvate or its alkyl esters, lactate, acetate, succinate or its alkyl esters, fumarate or its alkyl esters, acetoacetate or its alkyl esters, hydroxybutyrate or its alkyl esters, ketoisocaproate or its alkyl esters, malate or its alkyl esters, citrate or its alkyl esters.

[0087] In certain embodiments, the imaging agent is selected from hyperpolarized pyruvate or its alkyl esters, lactate, acetate, succinate or its alkyl esters, fumarate or its alkyl esters, acetoacetate or its alkyl esters, hydroxybutyrate or its alkyl esters.

[0088] In certain embodiments, the alkyl ester is 1-6 -Alkyl esters, especially C 1-4 Alkyl esters, more particularly ethyl esters.

[0089] When performing PHIP or SABRE to obtain a signal enhancing compound, a catalyst is required for the addition of parahydrogen or the transfer of spin order. Suitable catalysts are known to those skilled in the art.

[0090] In certain embodiments, the catalyst is selected from a rhodium catalyst, an iridium catalyst, a platinum catalyst, a palladium catalyst, a ruthenium catalyst, an osmium catalyst, or a rhenium catalyst.

[0091] Suitable rhodium catalysts are Wilkinson's catalyst or rhodium complexes containing phosphine ligands such as [1,4-bis-(diphenylphosphino)-butane]-(1,5-cyclooctadiene)-rhodium(I)-tetrafluoroborate.

[0092] Suitable ruthenium catalysts are cyclopentadiene complexes such as [Cp*Ru(MeCN)3]PF6: tris-(acetonitrile)-pentamethylcyclopentadienyl ruthenium(II)-hexafluorophosphato.

[0093] Iridium catalysts can be used in particular for SABRE. Suitable catalysts are Crabtree's catalyst or Ir complexes with N-heterocyclic carbene ligands such as [IrCl(cod)(IMes)], COD: cyclooctadiene, IMes: 1,3-bis(2,4,6 trimethylphenyl)imidazolinium chloride.

[0094] When DNP is used to prepare signal enhancing compound, radical source is used.Suitable radical is known to those skilled in the art.Non-limiting examples are nitroxide radical such as TEMPO, AMUPOL and TOTAPOL, or trityl radical such as Ox063.

[0095] The invention is further illustrated by the following examples and figures from which further embodiments and advantages can be derived, these examples are intended to illustrate the invention and are not intended to limit the scope of the invention. [Brief description of the drawings]

[0096] [Figure 1] An exemplary procedure for obtaining signal-enhanced pyruvate as a clean contrast agent is shown. [Diagram 2] 13 shows another exemplary procedure for obtaining signal-enhancing pyruvate as a clean contrast agent.

[0097] First, a volatile precursor of the contrast agent is required, which may be the final desired molecule (e.g. ethyl pyruvate, ethyl lactate, ethyl acetate, diethyl fumarate, ethyl acetoacetate or ethyl hydroxybutyrate). The latter is usually the case when SABRE is used. The metabolite of interest or its precursor is then reacted with the catalyst and parahydrogen inside the first compartment (reaction chamber), in a solvent or as a neat compound. See Figures 1 and 2 for details of an exemplary procedure. If a solvent is used, this is preferably water to avoid the use of organic solutions or solvents with a boiling point significantly different from that of the metabolite or its precursor. After the reaction, the compound is separated from the solvent by distillation and collected in the second compartment. Depending on the boiling points of the metabolite and the solvent, the compound is either distilled from the solvent or the solvent is first distilled off and the compound is distilled off in a second step.

[0098] The metabolites are condensed and then taken up by a biocompatible solvent (water, buffer, saline, etc.) or are directly captured in the aqueous medium. A stripping gas can be used additionally or as a co-solvent for simultaneous evaporation, but must be removed before in vivo application (potentially by further evaporation / distillation after converting the precursor to the desired contrast agent). To prevent partial distillation of the catalyst, heavy chelating molecules can be added to the solution to bind the metal before distillation begins. Further distillation may also be required if the original reaction in the first compartment is carried out in an organic solvent or if unreacted precursor molecules need to be removed. Elementary cleavage and neutralization of the precursor can occur in the second compartment.

[0099] example Example 1: A specific embodiment of the invention may be to place a first compartment in a low magnetic field, which may be the earth's field. A reaction between the pure contrast agent precursor and parahydrogen takes place in said low magnetic field, and the contrast agent is evaporated and transported to a second compartment placed inside a higher field magnet. The higher magnetic field may have a field strength of, for example, 100 mT to several Teslas. The magnetic field in the high field magnet should be high enough that the chemical shift in Hertz is larger than the J-coupling between the two parahydrogen-derived protons. At this higher magnetic field, a pulse sequence may be used to obtain in-phase proton magnetization. Heteronuclear ( 13 C or 15 If a contrast agent in which a nucleus other than a proton, such as N, is used, a pulse sequence must be applicable to transfer the proton polarization to said heteronuclei. This reduces the requirement for higher magnetic field homogeneity. The contrast agent is then evacuated or washed out of this chamber by physiological solution and is available for administration.

[0100] Example 1a: Example 1a is similar to Example 1, where the reaction is carried out in a first compartment located at a higher field ranging from 100 mT to several Tesla. Preparation of in-phase proton magnetization or polarization transfer to a different phase is carried out in the same compartment. The contrast agent is distilled into the second compartment.

[0101] Example 2: Example 2 is similar to Examples 1 and 1a, in which the contrast agent precursor is dissolved in a solvent with a higher boiling point than the contrast agent. After hydrogenation, the contrast agent is evaporated from the solvent into a second chamber. Preferably, the difference in boiling point between the contrast agent and the solvent is >20°C, more preferably >40°C.

[0102] Example 3: Example 3 is similar to Examples 1 and 1a, where the contrast agent precursor is dissolved in a solvent with a boiling point lower than that of the contrast agent. After hydrogenation, the separation is performed in two steps. During the first step, the temperature of both chambers is set below the boiling point of the solvent and the solvent is evaporated. In the second step, the temperature of the first chamber is set above or close to the boiling point of the contrast agent and then the contrast agent is distilled into the second chamber. Preferably, the difference between the contrast agent and the solvent is >20°C, more preferably >40°C.

[0103] Example 4: Example 4 is similar to Examples 1, 1a, 2, and 3, in that after hydrogenation, evaporation is promoted by flow of a carrier / stripping gas through the first chamber. A vacuum can be applied during distillation for the same purpose.

[0104] Example 5: Example 5 is similar to Examples 1, 1a, 2, 3 and 4 in that a chelating molecule is added to the reaction mixture after hydrogenation to bind the catalyst and greatly reduce its volatility.

[0105] Example 6: Example 6 is similar to Examples 1, 1a, 2, 3, 4, 5, in which the amino acid cysteine ​​or another suitable molecule is added in the presence of a catalyst to react with the double or triple C-C bonds, thereby trapping the unreacted imaging agent precursors and rendering them non-volatile.

[0106] Example 6a: Example 6a is similar to Examples 1, 1a, 2, 3, 4, and 5, in that the contrast agent precursor is a vinyl compound (e.g., vinyl pyruvate). The non-hydrogenated vinyl precursor is removed from the product contrast agent by distilling it off, since it has a lower boiling point than the product molecule. In Examples 1, 1a, and 2, an additional distillation step is required before distilling the product, whereas in Example 3, the vinyl compound is distilled along with the solvent.

[0107] Example 7: Example 7 is similar to the above examples, where the actual contrast agent is obtained by cleaving the distillation product after the hydrogenation reaction in the second compartment and after the pulse sequence has already been applied. The final contrast agent is obtained in the second compartment. The contrast agent can be purified from the cleaved waste by distilling it, as shown in examples 3 and 4 for the first compartment.

[0108] Example 8: Example 8 is similar to Example 7, in which the distilled molecules of the second compartment are dissolved in an amphiphilic solvent (eg, acetone, alcohol) to facilitate the cleavage reaction.

[0109] Example 9: Example 9 is similar to Examples 7 and 8 when the molecule requires a two-step cleavage: first, anhydrous base for cleavage in a suitable solvent (e.g. alcohol) is added, followed a few seconds later by the addition of water.

[0110] Example 10: The precursor molecules carrying the catalyst are parahydrogenated in a water-immiscible organic solvent. Water is then added and the mixture is vigorously mixed or ultrasonicated. The organic solvent is evaporated as in examples 3 and 4, and the enhanced molecules are transferred to the aqueous phase. The remaining catalyst is then filtered and the solution is ready for administration.

[0111] Example 11 Same as in Example 10, except that the precursor is vinyl pyruvate and a solution of carbonate or sodium hydroxide is added instead of water. Finally, an aqueous solution of cleaved pyruvate is obtained.

Claims

1. A method for preparing a contrast agent, characterized in that it comprises an evaporation step in which a signal enhancing compound containing nuclear spins is separated from a liquid.

2. The method according to claim 1, wherein the contrast agent is prepared by PHIP (parahydrogen induced polarization), SABRE (signal amplification by reversible exchange), or DNP (dynamic nuclear polarization).

3. The method according to claim 1 or 2, wherein the liquid contains a catalyst or a radical source.

4. The method according to claim 1 or 2, wherein the signal enhancing compound is separated from the liquid by evaporating the signal enhancing compound to provide a purified signal enhancing compound.

5. The method according to claim 1 or 2, wherein the evaporation step is carried out in a static magnetic field, in particular a static magnetic field having a magnetic field strength of at least 10 mT.

6. The method according to claim 1 or 2, wherein the signal enhancing compound evaporates at a pressure of at least 3 bar and / or a temperature of at least 390 K.

7. The method according to claim 1 or 2, wherein a co-solvent or a stripping gas is used during the evaporation step, in particular a stripping gas is used during the evaporation step.

8. The method according to claim 1 or 2, wherein a vacuum is applied during the evaporation step, in particular a vacuum of 10 mbar is applied.

9. The method according to claim 1 or 2, wherein prior to the evaporation step, unreacted free bodies in the liquid are converted into non-volatile products, in particular non-volatile salts.

10. The purified signal enhancing compound is condensed after evaporation and then dissolved in an aqueous solvent, or directly captured in an aqueous solvent after evaporation, according to the method of claim 4.

11. The method according to claim 1 or 2, wherein the signal enhancing compound is volatile.

12. The method according to claim 1 or 2, wherein the signal enhancing compound has a boiling point that is at least 20 °C, particularly at least 40 °C, different from that of the liquid.

13. The signal enhancing compound contains hyperpolarized H atoms or hyperpolarized １３ C atoms or hyperpolarized １５ N atoms, the method according to claim 1 or 2.

14. The signal enhancing compound is selected from a 5- or 6-membered heterocycle containing one or more N atoms, nicotinamide, or a compound of formula I, particularly a compound of formula I, 【Chemical 1】 wherein R is selected from H, -OH, -OX', or C １－４ -alkyl, wherein the alkyl is unsubstituted or substituted with -OH, Y is C １－６ -alkyl or C １－６ -alkenyl, wherein the alkyl is unsubstituted or substituted with one or more substituents selected from NH ２ ,, -OH, -COOH, or -COOX', X, X', and X" are independently of one another C １－４ -alkyl, particularly X, X', and X" are ethyl, and each C atom is bonded to a hyperpolarized H atom, m, n, and p are independently of one another 0 or 1, the compound of formula I contains at least one hyperpolarized atom, particularly a hyperpolarized H atom or hyperpolarized １３ C atom or hyperpolarized １５ N atom, and in particular one or more, or all, of the Hs can be exchanged with deuterium, the method according to claim 1 or 2.

15. The method according to claim 1 or 2, wherein the contrast agent is selected from hyperpolarized pyruvate or its alkyl ester, lactate, acetate, succinate or its alkyl ester, fumarate or its alkyl ester, acetoacetate or its alkyl ester, hydroxybutyrate or its alkyl ester, ketoisocaproate or its alkyl ester, malate or its alkyl ester, citrate or its alkyl ester, and / or the catalyst is selected from a rhodium catalyst, an iridium catalyst, a platinum catalyst, a palladium catalyst, a ruthenium catalyst or a rhenium catalyst.

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