WO2009150211A1

WO2009150211A1 - Dnp polariser and method of producing a hyperpolarised selected material

Info

Publication number: WO2009150211A1
Application number: PCT/EP2009/057254
Authority: WO
Inventors: Jan Henrik ARDENKJÆR-LARSEN; Jan Wolber; Haukur Johannesson; Werner Meyer; Gerhard Reicherz; Jörg HECKMANN
Original assignee: Ge Healthcare Limited
Priority date: 2008-06-13
Filing date: 2009-06-12
Publication date: 2009-12-17

Abstract

The invention relates to a DNP polariser for the polarisation of a sample comprising a trityl radical and a selected material comprising nuclei with a low gyromagnetic ratio. The invention further relates to a method of producing a hyperpolarised selected material by using the above-mentioned DNP polariser.

Description

DNP polariser and method of producing a hyperpolarised selected material

Magnetic resonance (MR) imaging (MRI) is a technique that has become particularly attractive to physicians as images of a patients body or parts thereof can be obtained in a non- invasive way and without exposing the patient and the medical personnel to potentially harmful radiation such as X-rays. Because of its high quality images and good spatial and temporal resolution, MRI is a favourable imaging technique for imaging soft tissue and organs.

MRI may be carried out with or without MR contrast agents. However, contrast- enhanced MRI usually enables the detection of much smaller tissue changes which makes it a powerful tool for the detection of early stage tissue changes like for instance small tumours or metastases.

Several types of contrast agents have been used in MRI. Water-soluble paramagnetic metal chelates, for instance gadolinium chelates like Omniscan™ (GE Healthcare) are widely used MR contrast agents. Because of their low molecular weight they rapidly distribute into the extracellular space (i.e. the blood and the interstitium) when administered into the vasculature. They are also cleared relatively rapidly from the body.

Blood pool MR contrast agents on the other hand, for instance superparamagnetic iron oxide particles, are retained within the vasculature for a prolonged time. They have proven to be extremely useful to enhance contrast in the liver but also to detect capillary permeability abnormalities, e.g. "leaky" capillary walls in tumours which are a result of tumour angiogenesis.

Despite the undisputed excellent properties of the aforementioned contrast agents their use is not without any risks. Although paramagnetic metal chelates have usually high stability constants, it is possible that toxic metal ions are released in the body after administration. Further, these type of contrast agents show poor specificity.

WO-A-99/35508 discloses a method of MR investigation of a patient using a hyperpolarised solution of a high Ti agent as MRI contrast agent. The term "hyperpolarisation" means enhancing the nuclear polarisation of NMR active nuclei present in the high Ti agent, i.e. nuclei with non-zero nuclear spin, preferably ¹³C- or ¹⁵N-nuclei. Upon enhancing the nuclear polarisation of NMR active nuclei, the population difference between excited and ground nuclear spin states of these nuclei is significantly increased and thereby the MR signal intensity is amplified by a factor of hundred and more. When using a hyperpolarised ¹³C- and/or ¹⁵N-enriched high Ti agent, there will be essentially no interference from background signals as the natural abundance of ¹³C and/or ¹⁵N is negligible and thus the image contrast will be advantageously high. The main difference between conventional MRI contrast agents and these hyperpolarised high Ti agents is that in the former changes in contrast are caused by affecting the relaxation times of water protons in the body whereas the latter class of agents can be regarded as non-radioactive tracers, as the signal obtained arises solely from the agent.

A variety of possible high Ti agents for use as MR imaging agents are disclosed in WO-A-99/35508, including non-endogenous and endogenous compounds. As examples of the latter intermediates in normal metabolic cycles are mentioned which are said to be preferred for imaging metabolic activity. By in vivo imaging of metabolic activity, information of the metabolic status of a tissue may be obtained and said information may for instance be used to discriminate between healthy and diseased tissue.

For instance pyruvate is a compound that plays a role in the citric acid cycle and the conversion of hyperpolarised ¹³C-pyruvate to its metabolites hyperpolarised ¹³C- lactate, hyperpolarised ¹³C-bicarbonate and hyperpolarised ¹³C-alanine can be used for in vivo MR studying of metabolic processes in the human body.

The metabolic conversion of hyperpolarised ¹³C-pyruvate to its metabolites hyperpolarised ¹³C-lactate, hyperpolarised ¹³C-bicarbonate and hyperpolarised ¹³C- alanine can be used for in vivo MR study of metabolic processes in the human body since said conversion has been found to be fast enough to allow signal detection from the parent compound, i.e. hyperpolarised ¹³Ci-pyruvate, and its metabolites. The amount of alanine, bicarbonate and lactate is dependent on the metabolic status of the tissue under investigation. The MR signal intensity of hyperpolarised ¹³C- lactate, hyperpolarised ¹³C-bicarbonate and hyperpolarised ¹³C-alanine is related to the amount of these compounds and the degree of polarisation left at the time of detection, hence by monitoring the conversion of hyperpolarised ¹³C-pyruvate to hyperpolarised ¹³C-lactate, hyperpolarised ¹³C-bicarbonate and hyperpolarised ¹³C- alanine it is possible to study metabolic processes in vivo in the human or non- human animal body by using non-invasive MR imaging and/or MR spectroscopy.

The MR signal amplitudes arising from the different pyruvate metabolites vary depending on the tissue type. The unique metabolic peak pattern formed by alanine, lactate, bicarbonate and pyruvate can be used as fingerprint for the metabolic state of the tissue under examination.

Hyperpolarised ¹³C-pyruvate may for instance be used as an MR imaging agent for assessing the viability of myocardial tissue by MR imaging as described in detail in WO-A-2006/054903 and for in vivo tumour imaging as described in detail in WO-A- 2006/011810.

A method to obtain hyperpolarised ¹³C-pyruvate or other hyperpolarised compounds is the so-called dynamic nuclear polarisation (DNP). In DNP, polarisation of MR active nuclei in a compound to be polarised is affected by a polarisation agent or so- called DNP agent, a compound comprising unpaired electrons. During the DNP process, energy, normally in the form of microwave radiation, is provided, which will initially excite the DNP agent. Upon decay to the ground state, there is a transfer of polarisation from the unpaired electron of the DNP agent to the NMR active nuclei of the compound to be polarised, e.g. to the ¹³C nuclei in ¹³C-pyruvate.

Generally, DNP is carried out in a DNP polariser, a device which provides the moderate or high magnetic field and the very low temperatures which are used in the DNP process. Alternatively, a moderate magnetic field and any temperature at which sufficient polarisation enhancement is achieved may be employed. The DNP technique is for example further described in WO- A-98/58272 and in WO-A- 01/96895, both of which are included by reference herein. The core components of a modern DNP polariser which is suitable for polarising compounds which are for instance to be used as in vitro or in vivo MR imaging agents are: a magnet for producing a suitable magnetic field, a microwave source which in connection with the magnetic field promotes nuclear polarisation (polarising means), a cryostat for providing a low temperature, a sample container which holds the sample, i.e. the composition of the compound to be polarised and the DNP agent and means to liquefy the frozen hyperpolarised sample, e.g. dissolution or melting means. Such modern DNP polarisers are for instance described in detail in WO-A-02/37132 and WO- A-02/36005, which are incorporated by reference or in J. Wolber et al, Nucl Instr Methods A 526 (2004), 173-181.

To polarise a chemical entity, i.e. compound, by the DNP method, a composition of the compound to be polarised and a DNP agent (i.e. a sample) is prepared which is then optionally frozen and inserted into a DNP polariser (where it will freeze if it has not been frozen before) for polarisation. After the polarisation, the frozen solid hyperpolarised sample is rapidly transferred into the liquid state either by melting it or by dissolving it in a suitable dissolution medium. The dissolution process of a frozen hyperpolarised sample and suitable devices therefore are described in detail in WO-A-02/37132. The melting process and suitable devices for the melting are for instance described in WO- A-02/36005.

Once the hyperpolarised sample has been transferred into the liquid state, the enhanced nuclear polarisation in the hyperpolarised compound decays due to relaxation and - for instance upon administration to the patient's body or to an in vitro NMR assay - dilution. The consequence is a continuous loss of signal. Hence it is extremely important and favourable to achieve high polarisation levels and various methods have been established to increase said level of polarisation. One approach is to add paramagnetic metal ions to the sample and said approach is described in detail in WO-A-2007/064226. Another method is to prevent the sample from crystallizing upon cooling/freezing, i.e. to use an amorphous frozen sample in the DNP polarisation process. This may be achieved by adding glass formers like for instance glycerol or glycol to the sample or to prepare a sample comprising the compound to be polarised as a specific chemical entity that does not form crystals upon cooling/freezing: in WO-A-2007/069909 compositions comprising carboxylates of organic amines or amino compounds are disclosed which allow the production of hyperpolarised carboxylates with high polarisation levels while in WO-A- 2007/111515 compositions comprising carboxylates or sulphonates of comprising inorganic cations from the group consisting of NH₄ , K , Rb , Cs , Ca , Sr and Ba²⁺ are used to prepare amorphous frozen samples which allow the production of hyperpolarised carboxylates or sulphonates with high polarisation levels.

Various parameters other than sample condition or presence of paramagnetic metal ions influence the level of polarisation that can be achieved by DNP, amongst those are temperature, magnetic field, microwave frequency and power, microwave irradiation time or the concentration of the DNP agent and all of these parameters interact in a complex way. For an industrial application of hyperpolarised compounds, e.g. as MR imaging agents or agents for in vitro NMR applications it is important to keep in mind that the polarisation time is a crucial factor: even though a long polarisation time is of benefit for the level of polarisation obtained, customer's demands usually mean that one is looking for obtaining the highest possible level of polarisation within a short polarisation time.

Methods and devices for producing hyperpolarised material by dynamic nuclear polarisation (DNP) are known in the art.

D. A. Hall et al., Science 276, 1997, 930-932 disclose dynamic nuclear polarisation of ¹³C- and/or ¹⁵N-labelled material at a magnetic field of 5 T. No trityl radical was used for the polarisation but the nitroxide free radical 4-amino TEMPO.

In examples 3 and 4 of WO-A-99/35508, DNP polarisation of ¹³C-labelled material at a magnetic field of 2.5 T is disclosed. No trityl radical was used in these examples.

In WO-A-02/37132 and WO-A-02/36005 DNP polarisers are disclosed which comprise dissolution means (WO-A-02/37132) or melting means (WO-A-02/36005) to liquefy the solid hyperpolarised material obtained by the DNP process in order to make it useful for MRI or NMR applications. It is disclosed that the magnet of such a DNP polariser is charged to a magnetic field of 0.1 to 25 T. In the example of WO- A-02/37132, it is disclosed that ¹³C-labelled material was DNP polarised in the presence of the trityl radical OX063 at a magnetic field of 3.35 T.

In WO-A-2006/011809, WO-A-2006/011810, WO- A-2006/011811, WO-A- 2006/054903, WO-A-2007/064226, WO-A-2007/069909, WO-A-2007/111515, WO-A-2008/020765 and WO-A-2008/026937, DNP polarisation of various material comprising nuclei with a low gyromagnetic ratio is disclosed. A DNP polariser was used for this purpose whose magnet was charged to a magnetic field of 3.35 T and various trityl radicals were used as DNP agents.

Although good polarisation has been obtained by the methods described in the references above, there is still a need to improve the level of polarisation in material which is for instance intended to be used as MRI agent in patients.

We have now found that polarisation of nuclei with a low gyromagnetic ratio, e.g. nuclei like ¹³C, ¹⁵N or D can be increased dramatically when the magnet of the DNP polariser is charged to a magnetic field ranging from about 4 T to about 7 T and a trityl radical is used as DNP agent.

When the magnetic field is raised or the temperature is lowered both the nuclear and electronic polarisation increases (Boltzmann distribution). However, the efficiency of the DNP process will depend on a number of factors that are unknown, and can have any dependence of the magnetic field. The magnetic field dependence of polarisation has previously been studied by J. Harmsen, "Chemisch dotiertes und elektronenbestrahltes 1-Butanol-dlO als polarisiertes Target fur teilchen- physikalische Experimente", Dissertation an der Fakultat fur Physik und Astronomie der Ruhr Universitat Bochum, 2002. These studies which were carried out on various samples using nitroxide radicals as DNP agents demonstrated that despite the higher Boltzman polarisation of the radical and nuclei the DNP enhanced nuclear polarisation was decreasing at 5 T over 3.5 T. Three field strengths were studied (2.5 T, 3.5 T and 5 T), and it was experimentally demonstrated for several samples that the maximum polarisation was obtained with a magnetic field in the range of 3 - 4 T. The conclusion from these studies was that for unknown reasons the DNP efficiency was strongly reduced at the higher field. It was therefore surprising for samples which are DNP polarised using a trityl radical as DNP agent that the polarisation would increase at higher magnetic field and even increase at a rate stronger than the Boltzman factor.

Thus in a first aspect the invention provides a method for producing a hyperpolarised sample comprising a hyperpolarised selected material comprising nuclei with a low gyromagnetic ratio and a trityl radical, wherein a DNP (dynamic nuclear polarisation) polariser is used which comprises a magnet which is charged to a magnetic field in the range from 4 T to 7 T.

The terms "hyperpolarised" and "polarised" are used interchangeably hereinafter and denote a nuclear polarisation level in excess of 0.1%, more preferred in excess of 1% even more preferred in excess of 10% and most preferred in excess of 20%.

The level of polarisation in the hyperpolarised selected material may for instance be determined by solid state NMR measurements. By way of example, if the hyperpolarised selected material is for instance solid hyperpolarised ¹³C-pyruvate obtained by the method according to the invention the level of polarisation may be determined by solid state ¹³C-NMR measurements. The solid state ¹³C-NMR measurement preferably consists of a simple pulse-acquire NMR sequence using a low flip angle. The signal intensity of the hyperpolarised ¹³C-pyruvate in the NMR spectrum is compared with signal intensity of ¹³C-pyruvate in a NMR spectrum acquired before the polarisation process. The level of polarisation is then calculated from the ratio of the signal intensities of before and after polarisation.

In a similar way, the level of polarisation in the hyperpolarised selected material may for instance be determined by liquid state NMR measurements. By way of example, the level of polarisation in hyperpolarised ¹³C-pyruvate obtained by the method of the invention and being in solution may be determined by liquid state NMR measurements. Again the signal intensity of the hyperpolarised ¹³C-pyruvate in solution is compared with the signal intensity of the ¹³C-pyruvate in solution when at thermal equilibrium in the field and temperature of the liquid state NMR spectrometer. The level of polarisation is then calculated from the ratio of the signal intensities of ¹³C-pyruvate after polarisation and at thermal equilibrium multiplied by the Boltzmann polarization at thermal equilibrium (given by magnetic field and temperature)..

The term "nuclei with a low gyromagnetic ratio" preferably denotes nuclei with a gyromagnetic ratio leading to a nuclear Larmor frequency less than the EPR line width of the trityl radical used in the method of the invention, i.e. a gyromagnetic ratio equal to or less than that of ¹³C, which is 2π x 10.705 x 10⁶ S ¹T^"1. Examples of preferred nuclei are ¹³C, ¹⁵N, D and ⁶Li and the selected material may comprise one or several of these nuclei, i.e. ¹³C and/or ¹⁵N and/or D and/or ⁶Li.

The term "hyperpolarised sample" denotes a composition comprising (a) a hyperpolarised selected material comprising nuclei with a low gyromagnetic ratio, (b) a trityl radical and (c) optionally other chemical entities like solvents and/or glass formers and/or paramagnetic metal ions. Further, in the context of the invention the term "sample" denotes a composition comprising (a) a selected material comprising nuclei with a low gyromagnetic ratio, (b) a trityl radical and (c) optionally other chemical entities like solvents and/or glass formers and/or paramagnetic metal ions.

A sample is used to obtain a hyperpolarised sample according to the method of the invention, i.e. a sample is prepared and inserted into a DNP polariser which comprises a magnet which is charged to a magnetic field in the range from 4 T to 7 T. The sample is polarised in said DNP polariser and a hyperpolarised sample is obtained.

The selected material comprising nuclei with a low gyromagnetic ratio (a) may in principle be any chemical entity that comprises nuclei with a low gyromagnetic ratio.

In a preferred embodiment, the hyperpolarised selected material is a material which will be used in MRI, magnetic resonance spectroscopy (MRS) or magnetic resonance spectroscopic imaging (MRSI). In such an embodiment, the hyperpolarised selected material may be used for in vivo MR detection, i.e. in living human or non-human animal beings. Further, the hyperpolarised selected material may be used for in vitro

MR detection, e.g. of cell cultures, samples derived from a human or non-human animal like for instance urine, saliva or blood, ex vivo tissue, for instance ex vivo tissue obtained from a biopsy, isolated organs, proteins and peptides like for instance antibodies or receptors and the like. The term "MR detection" denotes MR imaging or MR spectroscopy or MR spectroscopic imaging. The term further denotes MR spectroscopic imaging at various time points.

In a preferred embodiment, the selected material is a drug candidate, suitably a small organic molecule, e.g. less than 2000 Da, or a mixture of several drug candidates and the drug candidate(s) hyperpolarised by the method of the invention may be used in

MR assays for instance to determine binding affinity to a certain receptor or in enzyme assays. Such assays are described in WO-A-2003/089656 or WO-A-

2004/051300 and they are preferably based on the use of liquid state MR spectroscopy. The selected material may or may not be isotopically enriched.

In another preferred embodiment, the selected material is an imaging agent or a precursor thereof and the hyperpolarised selected material is used as the active ingredient (imaging agent) in an imaging medium used for MRI, MRS or MRSI. Preferred selected materials are those which contain polarised nuclei that exhibit slow longitudinal relaxation so that polarisation is maintained for a sufficient length of time for transfer into, e.g. a patient, animal or cell culture and subsequent MR detection. Preferred selected materials contain nuclei with longitudinal relaxation time constants (Ti) that are greater than 10 seconds, preferably greater than 30 seconds and even more preferably greater that 60 seconds. Such so called "high Ti agents" are for instance described in WO-A-99/35508. Alternatively, Ti values of possible selected materials may be found in the literature or may be determined by acquiring an NMR spectrum of the possible selected material.

In a more preferred embodiment, the selected material is an endogenous chemical entity. In the context of the invention, the term "endogenous" means originating from a human or non-human animal, preferably originating from a human. In a more preferred embodiment, the selected material is an endogenous chemical entity which is an intermediate in a metabolic pathway in humans or non- human animals, preferably in humans. Examples of such metabolic pathways are the Krebs cycle,

Cori cycle, alanine cycle, glyoxylate cycle, Calvin cycle and the like. Intermediates in such metabolic pathways may be used for in vivo MR imaging of metabolic activity to obtain information of the metabolic status of a tissue under examination. Said information may for instance be used to discriminate between healthy and diseased tissue. Examples of such intermediates are amino acids like alanine, glycine, glutamine, glutamic acid, cysteine, asparagine and aspartic acid, acetate, pyruvic acid, pyruvate, oxalate, malate, fumarate, lactate, lactic acid, citrate, bicarbonate, malonate, succinate, oxaloacetate, α-ketoglutarate, 3-hydroxybutyrate, isocitrate and urea.

In a very preferred embodiment, the aforementioned preferred endogenous selected materials are isotopically enriched and more preferably isotopically enriched with nuclei with a low gyromagnetic ratio. By way of example, pyruvate is a preferred endogenous selected material which comprises MR active ¹³C nuclei that have a low gyromagnetic ratio at natural abundance (about 1.1 %). If pyruvate is intended to be used as imaging agent to produce an imaging medium, it is preferred to use ¹³C- isotopically enriched pyruvate to obtain hyperpolarised ¹³C-pyruvate according to the method of the invention. All three carbon atoms in pyruvate may be ¹³C- isotopically enriched, either at only one position, e.g. at the Cl -carbon atom or at multiple positions, e.g. at the Cl-, C2- and C3-position. However, to achieve a long T₁, it is preferred that pyruvate is isotopically enriched with ¹³C at the Cl -position. Another example of preferred endogenous selected materials are amino acids, for instance alanine. Alanine comprises NMR active ¹³C and ¹⁵N nuclei that have a low gyromagnetic ratio at natural abundance (1.1 % and 0.37 %, respectively). If alanine is intended to be used as imaging agent to produce an imaging medium, it is preferred to use ¹³C-isotopically enriched and/or ¹⁵N-iso topically enriched alanine obtain hyperpolarised ¹³C-alanine or ¹⁵N-alanine or ¹³C/¹⁵N-alanine according to the method of the invention. Again all three carbon atoms in alanine may be ¹³C- isotopically enriched, either at only one position, e.g. at the Cl -carbon atom or at multiple positions, e.g. at the Cl-, C2- and C3-position. However, the longest Ti is achieved when alanine is isotopically enriched with ¹³C at the Cl -position. If isotopically enriched selected material is used in the method of the invention, the isotopic enrichment of the selected material is preferably at least 75%, more preferably at least 80% and especially preferably at least 90%, an isotopic enrichment of over 90% being most preferred. Ideally, the enrichment is 100%. The isotopic enrichment may include either selective enrichments of one or more positions within the molecular structure of the selected material, e.g. at one specific carbon atom, or uniform enrichment of all positions, e.g. enrichment of all carbon atoms. Enrichment can for instance be achieved by chemical synthesis or biological labelling, both methods are known in the art and appropriate methods may be chosen depending on the selected material to be isotopically enriched. The optimal position for isotopic enrichment is dependent on the relaxation time of the MR active nuclei. As discussed before, selected materials are preferably isotopically enriched in positions with long Ti relaxation time. By way of example, ¹³C-enriched selected materials that are enriched at a carboxyl-C-atom, a carbonyl-C-atom or a quaternary C-atom are isotopically enriched in such positions with long Ti relaxation time.

As mentioned earlier, the sample according to the invention comprises a trityl radical. The trityl radical serves as a polarising agent/DNP agent and the large electron spin polarisation of the trityl radical is converted to nuclear spin polarisation of nuclei within the selected material via microwave irradiation close to electron Larmor frequency. The microwaves stimulate communication between electron and nuclear spin systems via e-e and e-n transitions. For effective DNP the trityl radical has to be stable and soluble in the sample to be polarised to achieve intimate contact between the selected material and the trityl radical which is necessary for the aforementioned communication between electron and nuclear spin systems. Oxygen- based, sulphur-based or carbon-based stable trityl radicals are preferred and such trityl radicals are for instance described in WO-A-99/35508, WO-A-88/10419, WO- A-90/00904, WO-A-91/12024, WO-A-93/02711 or WO-A-96/39367.

The optimal choice of trityl radical depends on several aspects. As mentioned before, the trityl radical and the selected material must be in intimate contact during DNP in order to result in optimal polarisation levels in the selected material. Thus, in a preferred embodiment, the trityl radical is soluble in the selected material or in a solution of the selected material. To prepare such a solution, a solvent or a solvent mixture may be used to dissolve the selected material. However if the polarised selected material is used for in vivo applications like in vivo MR imaging it is preferred to keep the amount of solvent to a minimum or, if possible, to avoid the use of solvents. The latter might be possible if the selected material to be polarised is for instance a liquid or if the sample may be transferred into the liquid state, e.g. by gently heating. To be used as an in vivo imaging agent, the polarised selected material is usually administered in a relatively high concentration, i.e. a highly concentrated sample is preferably used in the DNP process and hence the amount of solvent is preferably kept to a minimum. In this context, it is also important to mention that the mass of the sample should be kept as small as possible. A high mass will have a negative impact on the efficiency of the dissolution process, if dissolution is used to convert the solid hyperpolarised sample after the DNP process into the liquid state, e.g. for using it as an MR imaging agent. It has been observed that the efficiency of the dissolution and thereby the preservation of the attained polarisation decreases as the mass of the sample increases. This is presumably due to the fact that the volume of the sample increases to the third power of linear dimensions whereas the surface of the composition increases to the second power of linear dimensions. Further, using certain solvents may require their removal before the hyperpolarised selected material can be used as an MR imaging agent, i.e. can be administered to a patient since they might not be physiologically tolerable.

If the selected material to be polarised is a lipophilic (hydrophilic) compound, the trityl radical should be lipophilic (hydrophilic) too. Lipophilicity or hydrophilicity of the trityl radical can be influenced by choosing suitable residues which render the trityl radical lipophilic or hydrophilic. Further, the trityl radical has to be stable in presence of the selected material. Hence if the selected material to be polarised is an acid (a base), the trityl radical should be stable under acidic (basic) conditions. If the selected material to be polarised contains reactive groups, a trityl radical should be used which is relatively inert towards these reactive groups. From the aforesaid it is apparent that the choice of trityl radical is highly dependent on the chemical nature of the selected material.

J. H. Ardenkjεer-Larsen et al, PNAS 100(18), 2003, 10158-10163 describe the successful DNP polarisation of ¹³C-labelled and unlabelled urea using the trityl radical (Tris{8-carboxyl-2,2,6,6-tetra[2-(l-hydroxyethyl)]-benzo (l,2-d:4,5-d') bis

(1,3) dithiole-4-yl}methyl sodium salt (further described in US patent no. 6,013,810) and glycerol as a solvent which resulted in high polarisation levels in urea.

In WO- A-2006/011811, trityl radicals are disclosed which are especially useful DNP agents for the DNP polarisation of acidic organic compounds like lactic acid or pyruvic acid.

The trityl radicals used in the method of the invention may be synthesized as described in detail in WO-A-88/10419, WO-A-90/00904, WO-A-91/12024, WO-A- 93/02711, WO-A-96/39367 and WO- A-2006/011811.

Suitable concentrations of trityl radical in the sample according to the invention are 1 to 25 mM, preferably 2 to 20 mM, more preferably 10 to 15 mM.

As mentioned earlier, in order to obtain a high polarisation level in the selected material to be polarised said selected material and the trityl radical need to be in intimate contact during the DNP process. This is not the case if the composition crystallizes upon being frozen or cooled. To avoid crystallization, either glass formers need to be present in the composition or selected materials need to be chosen for polarisation which do not crystallize upon being frozen but rather form a glass.

The term "glass former" in the context of this application means a chemical compound that, when added to the mixture of selected material, trityl radical and optionally solvent, promotes vitrification and prevents crystallization of said mixture when it is cooled or frozen. Examples of preferred glass formers in the context of the invention are glycols, i.e. alcohols containing at least two hydroxyl groups, such as ethylene glycol, propylene glycol and glycerol or DMSO.

The sample used in the method of the invention may further comprise a paramagnetic metal ion. It has been found that the presence of paramagnetic metal ions may result in increased polarisation levels in the compound to be polarised by DNP as described in detail in WO-A2-2007/064226 which is incorporated herein by reference. The term "paramagnetic metal ion" denotes paramagnetic metal ions in the form of their salts or in chelated form, i.e. paramagnetic chelates. The latter are chemical entities comprising a chelator and a paramagnetic metal ion, wherein said paramagnetic metal ion and said chelator form a complex, i.e. a paramagnetic chelate.

In a preferred embodiment, the paramagnetic metal ion is a salt or paramagnetic chelate comprising Gd³⁺, preferably a paramagnetic chelate comprising Gd³⁺. In a more preferred embodiment, said paramagnetic metal ion is soluble and stable in the sample.

If a paramagnetic metal ion is added to the sample, a suitable concentration of such a paramagnetic metal ion is 0.1 to 6 mM (metal ion) in the sample, and a concentration of 0.3 to 4 mM is preferred.

In another embodiment, the sample comprises one or more solvents and/or one or more glass formers and/or paramagnetic metal ions. For the method according to the invention, a sample is prepared which comprises the selected material to be polarised, the trityl radical and optionally other chemical entities like one or more solvents and/or one or more glass formers and/or paramagnetic metal ions. If the selected material is not a liquid, a solvent needs to be added or the selected material has to be transferred into the liquid state, e.g. by gentle heating. Detailed descriptions of how to prepare samples comprising various selected materials are disclosed in the art, for instance in WO-A-2006/011809 (for pyruvic acid and pyruvate as selected materials), WO-A-2007/069909 and WO-A-2007/111515 (for various selected materials containing carboxyl and/or sulphonyl groups) and WO-A-2007/064226.

After having prepared the sample, said sample is frozen by methods known in the art, e.g. by freezing it in a freezer, in liquid nitrogen or by simply adding it to a sample cup and placing the sample cup in the DNP polariser, where liquid helium will freeze the sample. In another embodiment, the composition is frozen as "beads" before it is added to the sample cup and inserted into the polariser. Such beads may be obtained by adding the composition drop wise to liquid nitrogen. A more efficient dissolution of such beads has been observed, which is especially relevant if larger amounts of sample are polarised, for instance when the hyperpolarised selected material is intended to be used in an in vivo MR imaging medium.

The DNP technique is for instance described in WO-A-98/58272 and in WO-A- 01/96895, both of which are included by reference herein. Generally, the dynamic nuclear polarisation is carried out at very low temperatures which are obtained by the use of a cryostat with liquid helium or by using a cryogen-free cooling device such as a cold finger or cold head (see for instance US-Al -2008/0104966). In the context of this invention, the DNP process is preferably carried out in liquid helium, i.e. at a temperature below 4.2 K and as low as obtainable by pumping on the liquid helium. The magnet of the DNP polariser is charged to a magnetic field of from 4 T to 7 T, preferably of from 4.5 T to 6 T and more preferably of from 4.6 T to 5.5 T. Suitable polarisation units are for instance described in WO-A-02/37132 and WO-A- 02/36005. In a preferred embodiment, the polariser used in the method of the invention comprises a cryostat containing liquid helium and polarising means, i.e. a microwave chamber connected by a wave guide to a microwave source in a central bore surrounded by a magnet such as a superconducting magnet which is charged to the above-mentioned magnetic field. The bore extends vertically down to at least the level of a region P near the magnet where the magnetic field is from 4 T to 7 T. The bore for the probe (i.e. the frozen sample comprising the selected material to be polarised) is preferably sealable and can be evacuated to low pressures, e.g. pressures in the order of 1 mbar or less (to lower the temperature of the helium bath). A probe introducing means such as a removable transporting tube can be contained inside the bore and this tube can be inserted from the top of the bore down to a position inside the microwave chamber in region P. Region P is cooled by liquid helium to a temperature low enough to for polarisation to take place, preferably temperatures of the order of 0.6 to 100 K, more preferably 0.5 to 10 K, most preferably 1 to 5 K. The probe introducing means is preferably sealable at its upper end in any suitable way to retain the partial vacuum in the bore. A probe-retaining container, such as a probe-retaining cup or sample cup, can be removably fitted inside the lower end of the probe introducing means. The probe-retaining container is preferably made of a light-weight material with a low specific heat capacity and good cryogenic properties such, e.g. KeIF (polychlorotrifluoro-ethylene) or PEEK (polyetheretherketone) and it may be designed in such a way that it can hold more than one probe.

The probe is inserted into the probe-retaining container, submerged in the liquid helium and irradiated with microwaves, preferably at a frequency of about 140 GHz (5 T) at about 60 mW. The power depends on technical details of the microwave delivery system in addition to the properties of the trityl and sample. The microwave frequency depends on the field strength and will thus be in the range 110 GHz (4 T) to 200 GHz (7 T). The level of polarisation may for instance be monitored by solid state NMR measurements as described earlier in this application. The microwave source frequency is determined from the EPR line of the trityl radical, which depends on the magnetic field as 28.0 GHz/T. The optimal microwave frequency is determined by adjusting the frequency for maximal NMR signal. This procedure can be used for all field strengths.

For a given application/use of the polarised selected material, a certain polarisation will be required. Hence if the polarised selected material is to be used as MR imaging agent in a patient, a high polarisation is favourable since it allows a longer MR imaging window, i.e. more time to collect MR data from the patient. For some applications/use the maximum possible polarisation is preferred, i.e. a polarisation as close to unity polarisation possible. This will mean that microwave irradiation at the optimal frequency and power continues for several time constants until the NMR signal approaches its asymptotic value. The time constant of the build up of polarisation depends on the static magnetic field strength, and it was observed that the increased DNP at higher magnetic fields was correlated with longer build up time constant. The consequence for the method according to the invention is that the range of magnetic field optimal for DNP polarisation of low gyromagnetic ratio nuclei and using a trityl radical as DNP agent is defined around an optimum in terms of polarisation required in a defined period of time. To the low end of the magnetic field range the polarisation is decreasing and the target polarisation cannot be achieved. To the high end of the magnetic field range the polarisation approaches unity, but the polarisation time continues to increase and the defined period of time for achieving the polarisation has to be exceeded. For use in an imaging medium, the frozen sample containing the hyperpolarised selected material needs to be transferred from the solid state to the liquid state, i.e. liquefied after the dynamic nuclear polarisation.

Liquefaction can be achieved by dissolution in an appropriate solvent or solvent mixture (dissolution medium) or by melting the solid frozen composition. Both methods are described in detail in WO-A-02/37132 and WO-A-02/36005. Briefly, a dissolution unit/melting unit is used which is either physically separated from the polariser or is a part of an apparatus that contains the polariser and the dissolution unit/melting unit. In a preferred embodiment, dissolution/melting is carried out at an elevated magnetic field, e.g. inside the polariser, to improve the relaxation and retain a maximum of the hyperpolarisation. Field nodes should be avoided and low field may lead to enhanced relaxation despite the above measures.

If dissolution is used to liquefy the frozen sample containing the hyperpolarised selected material, a hot dissolution medium is usually used which is brought into contact with the frozen sample. The dissolved sample is transferred into a receiver vessel. If the dissolved sample is to be used as MR imaging medium for in vivo MR detection, the temperature of the MR imaging medium needs to be as such that is in the range of the body temperature of the patient. This can be achieved by contacting the dissolved sample with a heat exchanger. Due to the magnetic properties of the dissolved sample, i.e. the presence of the hyperpolarised material, a metal heat exchanger cannot be used. Non-metals are slow heat conductors however the use of a glass heat exchanger with a large surface has worked well. In a preferred embodiment, such a glass heat exchanger consists of several chambers for the dissolved sample and the cooling liquid. Minimum gaps between the surfaces of the heat exchanger ensure maximum benefit of surface area vs. fluid volume and prevent gas bubbles in the dissolved sample as a result of capillary effects. In addition, visual observation is possible.

Subsequent to dissolution, the trityl radical may be removed from the liquid containing the hyperpolarised selected material and removal of the trityl radical is preferred if the hyperpolarised selected material is intended for use in an imaging medium for in vivo MR detection. Methods which are useful to remove the trityl radical are known in the art and described in detail inWO-A2-2007/064226 and WO-Al -2006/011809.

Another aspect of the invention is a DNP polariser for the polarisation of a sample comprising a trityl radical and a selected material comprising nuclei with a low gyromagnetic ratio wherein said polariser comprises a magnet which is charged to a magnetic field in the range from 4 T to 7 T.

In a preferred embodiment, said DNP polariser comprises a magnet which is charged to a magnetic field in the range from 4.5 T to 6 T and more preferably from 4.6 T to 5.5 T.

Preferred embodiments of the sample, the selected material and the trityl radical are discussed earlier in the application.

Brief description of the drawings:

FIG. 1 shows the ¹³C DNP polarisation as a function of microwave frequency for sample 1 in Example 1 , determined by two different experiments as described in the Example 1 (solid and dashed lines). Since the purpose was to find optimum microwave frequency, none of the points correspond to steady state conditions. Further, FIG. 1 shows the ¹³C DNP polarisation as a function of microwave frequency for sample 2 in example 1 (circles connected by dotted line).

The invention is illustrated by the following non-limiting examples.

Examples

The sodium salt of ¹³Ci -pyruvate (Cambridge Isotope Laboratories) was converted to ¹³Ci-pyruvic acid and purified by distillation. ¹³C enrichment was >99% according to the supplier, and the purity was >99% as determined by ¹H-NMR.

The trityl radical tris(8-carboxyl-2,2,6,6-tetra(2-(l-methoxy-2,2-d2-ethyl))- benzo[l,2-d:4,5-d']bis-(dithiole-4-yl)methyl sodium salt was synthesized as described in Example 1 in WO- A-2006/011811. The radical had a purity of 92.5 % (determined by HPLC). The term "trityl radical" is used in the following to denote this particular trityl radical.

The Gd-chelate of l,3,5-tris-(N-(DO3A-acetamido)-N-methyl-4-amino-2-methyl- phenyl)-[l,3,5]triazinane-2,4,6-trione was prepared as described in Example 4 of WO-A-2007/064226. The term "3-Gd" is used in the following to denote this particular Gd-chelate.

Example 1 Samples were prepared by weighing the required amounts of ¹³Ci-pyruvic acid and trityl radical, respectively. The density of liquid pyruvic acid, 1.26 g/cm , was used to calculate the volume of the sample neglecting any (small) volume effect of the trityl radical. The concentration of trityl molecules was calculated from the molecular weight with correction for the purity. Samples of two different concentrations of trityl radical (15 and 20 mM) were used in this study. They will in the following be referred to as samples 1 and 2, respectively.

The polarisation enhancement as a function of microwave frequency was determined for two of the samples by different methods. For sample 1, two sets of data were obtained using different methods. First the microwave frequency was applied at a value below the EPR resonance frequency of the trityl radical, and then stepped up through the resonance in increments of 2 MHz. For each frequency setting, a time of polarisation of 800 seconds was used, and the signal intensity was then measured at the end of each polarisation time interval. Subsequently, a measurement where the frequency was stepped down was performed, but now the ¹³C polarisation was saturated after each build up. Since the time used to polarise is short compared with the build-up time, the two methods will be prone to artifacts, especially in the wings where the build-up time is longer. The main purpose of this experiment was however to determine the optimum microwave frequency, which also should be well determined despite the mentioned limitations.

For sample 2 a polarisation time of 24000 seconds was used for every setting of the microwave frequency, and in the case where steady-state had not been reached, an extrapolation to the steady-state value was employed using an exponential fit. A maximum polarisation of 64 ± 5 % was obtained.

The polarisation of a sample similar to sample 1 was measured at 3.35 T at optimal microwave frequency and power and a polarisation of 27 ± 5 %

Example 2

A sample was prepared containing 20 mg of ¹³Ci-pyruvic acid and being 15 mM in trityl radical. The sample was polarised at 3.35 T (93.930 GHz, 60 mW) for one hour. The time constant of the polarisation was 900 s and the solid state polarisation was essentially complete. The sample was subsequently dissolved in 6 mL of 40 mM phosphate buffer with 21 μL of NaOH (0.1 M). A hyperpolarised ¹³C-NMR spectrum was measured on a Varian 9.4 T NMR spectrometer and a polarisation of 20 % was quantified.

A sample was prepared containing 20 mg of ¹³Ci-pyruvic acid and being 15 mM in trityl radical. The sample was polarised at 4.64 T (130.110 GHz, 60 mW) for one hour. The time constant of the polarisation was 2700 s and a solid state polarisation of 3/4 of the extrapolated maximum was obtained, i.e. full polarisation was 55% in solid state and 40% was reached just prior to dissolution. The sample was dissolved in 6 mL of 40 mM phosphate buffer and 20 μL NaOH (0.1 M). A hyperpolarised ¹³C-NMR spectrum was measured on a Varian 9.4 T NMR spectrometer and a polarisation of 35 % was quantified. In conclusion an improvement in the polarisation from 20% to 35% was achieved by increasing the polariser field strength from 3.35 T to 4.64 T for a similar sample and same polarisation time.

Example 3

A sample was prepared which consisted of 20 mg of ¹³Ci-ketoisocaproic acid being 15 mM in trityl radical and 0.5 mM 3-Gd. The sample was polarised at 3.35 T (93.930 GHz, 60 mW) for one hour. The time constant of the polarisation was 1400 s and the solid state polarisation was essentially complete. The sample was subsequently dissolved in 6 mL 40 mM phosphate buffer and 21 μl NaOH (0.1 M). A hyperpolarised ¹³C-NMR spectrum was measured on a Varian 9.4 T NMR spectrometer and a polarisation of 27 % was quantified.

A sample was prepared which consisted of 40 mg of ¹³Ci-ketoisocaproic acid being 15 mM in trityl radical and 0.5 mM 3-Gd. The sample was polarised at 4.64 T (130.110 GHz, 60 mW) for one hour. The time constant of the polarisation was 2300 s and a solid state polarisation of 0.9 of the extrapolated maximum was obtained, i.e. full polarisation was 76% in solid state and 68% was reached just prior to dissolution. The sample was dissolved in 6 mL of 40 mM phosphate buffer with 20 μL NaOH (0.1 M). A hyperpolarised ¹³C-NMR spectrum was measured on a Varian 9.4 T NMR spectrometer and a polarisation of 54 % was quantified.

In conclusion an improvement in the polarisation from 27% to 54% was achieved by increasing the polariser field strength from 3.35 T to 4.64 T for a similar sample and same polarisation time.

Example 4 A sample was prepared which consisted of 20 mg of [1,4-¹³C₂] fumaric acid being 5 mM in trityl radical and 0.5 mM in 3-Gd. The sample was polarised at 3.35 T (93.930 GHz, 60 mW) for one hour. The time constant of the polarisation was 1500 s and the solid state polarisation was essentially complete. The sample was subsequently dissolved in 6 mL 40 mM phosphate buffer and 40 μl NaOH (0.1 M). A hyperpolarised ¹³C-NMR spectrum was measured on a Varian 9.4 T NMR spectrometer and a polarisation of 25 % was quantified.

A sample was prepared which consisted of 40 mg of [1,4-¹³C₂] fumaric acid being 5 mM in trityl radical and 0.5 mM in 3-Gd. The sample was polarised at 4.64 T

(130.110 GHz, 60 mW) for one hour. The time constant of the polarisation was 4400 s and a solid state polarisation of app. 2/3 of the extrapolated maximum was obtained, i.e. full polarisation was 86% in solid state and 57% was reached just prior to dissolution. The sample was dissolved in 6 mL of 40 mM phosphate buffer with 40 μL NaOH (0.1 M). A hyperpolarised ¹³C-NMR spectrum was measured on a

Varian 9.4 T NMR spectrometer and a polarisation of 41 % was quantified.

In conclusion an improvement in the polarisation from 25% to 41% was achieved by increasing the polariser field strength from 3.35 T to 4.64 T for a similar sample and same polarisation time

Claims

1. DNP polariser for the polarisation of a sample comprising a trityl radical and a selected material comprising nuclei with a low gyromagnetic ratio wherein said polariser comprises a magnet which is charged to a magnetic field in the range from 4 T to 7 T.

2. DNP polariser according to claim 1 wherein said magnet is charged to a magnetic field in the range from 4.5 T to 6 T, preferably 4.6 T to 5.5 T.

3. DNP polariser according to claims 1 and 2 wherein the selected material is an imaging agent or a precursor thereof.

4. DNP polariser according to claims 1 to 3 wherein the selected material is an endogenous chemical entity which is an intermediate in a metabolic pathway in humans or non-human animals.

5. DNP polariser according to claims 1 to 4 wherein said nuclei with a low gyromagnetic ratio are ¹³C, ¹⁵N, D and/or ⁶Li.

6. DNP polariser according to claims 1 to 5 wherein the selected material is isotopically enriched, preferably isotopically enriched with said nuclei with a low gyromagnetic ratio.

7. DNP polariser according to claims 1 to 6 wherein the sample further comprises a paramagnetic metal ion.

8. DNP polariser according to claims 1 to 7 wherein the sample further comprises one or more glass formers and/or one or more solvents.

9. Method for producing a hyperpolarised sample comprising a hyperpolarised selected material comprising nuclei with a low gyromagnetic ratio and a trityl radical, wherein a DNP polariser is used which comprises a magnet which is charged to a magnetic field in the range from 4 T to 7 T.