Patent Application
20100196283
The invention relates to a method of 13C-MR detection using an imaging medium comprising hyperpolarised 13C-lactate and to an imaging medium containing hyperpolarised 13C1-lactate for use in said method.
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.
WO-A-99/35508 discloses a method of MR investigation of a patient using a hyperpolarised solution of a high T1 agent as MRI contrast agent. The term “hyperpolarisation” means enhancing the nuclear polarisation of NMR active nuclei present in the high T1 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 T1 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 T1 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 T1 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.
Pyruvate for instance 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. Hyperpolarised 13C-pyruvate may for instance be used as an MR imaging agent for in vivo tumour imaging as described in detail in WO-A-2006/011810 and for assessing the viability of myocardial tissue by MR imaging as described in detail in WO-A-2006/054903.
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 13C1-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 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.
However, the production of hyperpolarised 13C-pyruvate which is suitable as an in vivo imaging agent is not without challenges. Hyperpolarised 13C-pyruvate is preferably obtained by dynamic nuclear polarisation (DNP) of either 13C-pyruvic acid or a 13C-pyruvate salt as described in detail in WO-A1-2006/011809, which is incorporated herein by reference.
The use of 13C-pyruvic acid simplifies the polarisation process since it does not crystallize upon freezing/cooling (crystallization leads to low dynamic nuclear polarisation or no polarisation at all). As a consequence no solvents and/or glass formers are needed to prepare a composition for the DNP process and thus a highly concentrated 13C-pyruvic acid sample can be used. However, due to its low pH a DNP agent needs to be used which is stable in the strong pyruvic acid. Further, a strong base is necessary to dissolve and convert the solid hyperpolarised 13C-pyruvic acid after the polarisation to hyperpolarised 13C-pyruvate. Both the strong pyruvic acid and the strong base require careful selection of materials (e.g. dissolution medium reservoir, tubes, etc.) the compounds get in contact with.
Alternatively, a 13C-pyruvate salt may be used in the DNP process. Unfortunately, sodium 13C-pyruvate crystallizes upon freezing/cooling which makes it necessary to add glass formers. If the hyperpolarised 13C-pyruvate is intended to be used as in vivo imaging agent, the pyruvate concentration in the composition containing the pyruvate and glass formers is unfavourably low. Besides, the glass formers are to be removed for in vivo use as well.
Thus preferred salts which may be used for DNP are those 13C-pyruvates which comprise an inorganic cation from the group consisting of NH4+, K+, Rb+, Cs+, Ca2+, Sr2+ and Ba2+, preferably NH4+, K+, Rb+ or Cs+, more preferably K+, Rb+, Cs+ and most preferably Cs+, as in detail described in WO-A-2007/111515. Most of these salts are not commercially available and need to be synthesized separately. Further, if the hyperpolarised 13C-pyruvate is used in vivo MR imaging it is preferred to exchange the inorganic cation from the group consisting of NH4+, K+, Rb+, Cs+, Ca2+, Sr2+ and Ba2+ by a physiologically very well tolerable cation like Na+ or meglumine. Hence an additional step is required after liquefaction of the solid hyperpolarised 13C-pyruvate during which polarisation decays.
Other preferred salts are 13C-pyruvate of an organic amine or amino compound, preferably TRIS-13C1-pyruvate or meglumine-13C1-pyruvate, as in detail described in WO-A-2007/069909. Again these salts need to be synthesized separately.
We have now found that hyperpolarised 13C-lactate may be used as imaging agent in MR imaging and/or MR spectroscopy instead of hyperpolarised 13C-pyruvate.
Sodium 13C-lactate is a commercially available compound which may be directly used for DNP since it does not crystallize upon cooling/freezing. Since this eliminates the necessity of glass formers and/or high amounts of solvent(s) in the sample, a highly concentrated sample can be prepared and used in the DNP process. Further, sodium 13C-lactate samples are pH neutral and hence a variety of DNP agents can be used. Lactate is an endogenous compound and its concentration in human blood is fairly high (1-3 mM) with local concentrations of 10 mM and more. Hence, lactate is very well tolerated and using hyperpolarised 13C-lactate as an imaging agent is advantageous from a safety perspective.
Thus, in a first aspect the invention provides a method of 13C-MR detection using an imaging medium comprising hyperpolarised 13C-lactate.
The term “13C-MR detection” denotes 13C-MR imaging or 13C-MR spectroscopy or combined 13C-MR imaging and 13C-MR spectroscopy, i.e. 13C-MR spectroscopic imaging. The term further denotes 13C-MR spectroscopic imaging at various time points.
The term “imaging medium” denotes a liquid composition comprising hyperpolarised 13C-lactate as the MR active agent, i.e. imaging agent. The imaging medium according to the invention may be used as imaging medium in a method of 13C-MR detection.
The imaging medium used in the method of the invention may be used as an imaging medium for in vivo 13C-MR detection, i.e. in living human or non-human animal beings. Further, the imaging medium used in the method of the invention may be used as imaging medium for in vitro 13C-MR detection, e.g. in cell cultures, body samples like for instance urine, saliva or blood, ex vivo tissue, for instance ex vivo tissue obtained from a biopsy or isolated organs.
The terms “lactate” and “lactic acid”, unless specified otherwise, denote the L-isomer (L-lactate, L-lactic acid), the D-isomer (D-lactate, D-lactic acid) and mixtures of the L- and D-isomer (D/L-lactate and D/L-lactic acid), e.g. a racemic mixture of the D- and L-isomer. D-lactate and L-lactate are converted to pyruvate by different enzymes (i.e. D- and L-lactate dehydrogenase, respectively); however, the metabolites formed are pyruvate, lactate, alanine and bicarbonate for both of the isomers and hence both isomers can be used in the method of the invention.
The imaging medium according to the invention may thus comprise hyperpolarised 13C-L-lactate or hyperpolarised 13C-D-lactate or a mixture thereof, e.g. a racemic mixture of hyperpolarised 13C-D/L-lactate. In a preferred embodiment, the imaging medium according to the invention comprises hyperpolarised 13C-L-lactate or a mixture of hyperpolarised 13C-L-lactate and hyperpolarised 13C-D-lactate, more preferably a racemic mixture. In a most preferred embodiment, the imaging medium according to the invention comprises hyperpolarised 13C-L-lactate.
The term “13C-lactate” denotes a salt of 13C-lactic acid that is isotopically enriched with 13C, i.e. in which the amount of 13C isotope is greater than its natural abundance. Unless otherwise specified, the term “13C-lactate” and “13C-lactic acid” denote a compound which is 13C-enriched at any of the 3 carbon atoms present in the molecule, i.e. at the C1-position and/or the C2-position and/or the C3-position.
The isotopic enrichment of the hyperpolarised 13C-lactate used in the method of the invention 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%. 13C-lactate used in the method of the invention may be isotopically enriched at the C1-position (in the following denoted 13C1-lactate), at the C2-position (in the following denoted 13C2-lactate), at the C3-position (in the following denoted 13C3-lactate), at the C1- and the C2-position (in the following denoted 13C1,2-lactate), at the C1- and the C3-position (in the following denoted 13C1,3-lactate), at the C2- and the C3-position (in the following denoted 13C2,3-lactate) or at the C1-, C2- and C3-position (in the following denoted 13C1,2,3-lactate). Isotopic enrichment at the C1-position is the most preferred since 13C1-lactate has a higher, i.e. longer T1 relaxation in human full blood at 37° C. than 13C-lactate which is isotopically enriched at other C-positions.
In a preferred embodiment, the imaging medium according to the invention comprises hyperpolarised sodium 13C-lactate, more preferably sodium 13C1-lactate.
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% and most preferred in excess of 10%.
The level of polarisation may for instance be determined by solid state 13C-NMR measurements in solid hyperpolarised 13C-lactate, e.g. solid hyperpolarised 13C-lactate obtained by dynamic nuclear polarisation (DNP) of 13C-lactate. 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-lactate in the NMR spectrum is compared with signal intensity of 13C-lactate in a NMR spectrum acquired before the polarisation process. The level of polarisation is then calculated from the ratio of the signal intensities before and after polarisation.
In a similar way, the level of polarisation for dissolved hyperpolarised 13C-lactate may be determined by liquid state NMR measurements. Again the signal intensity of the dissolved hyperpolarised 13C-lactate is compared with the signal intensity of the dissolved 13C-lactate before polarisation. The level of polarisation is then calculated from the ratio of the signal intensities of 13C-lactate before and after polarisation.
Hyperpolarisation of NMR active 13C-nuclei may be achieved by different methods which are for instance described in described in WO-A-98/30918, WO-A-99/24080 and WO-A-99/35508, and which all are incorporated herein by reference and hyperpolarisation methods known in the art are polarisation transfer from a noble gas, “brute force”, spin refrigeration, the parahydrogen method and dynamic nuclear polarisation (DNP).
To obtain hyperpolarised 13C-lactate, it is preferred to polarise 13C-lactate directly. Also 13C-lactic acid may be polarised, however the polarised 13C-lactic acid needs to be converted to polarised 13C-lactate, e.g. by neutralisation with a base. 13C-lactate salts are commercially available, e.g. sodium 13C-lactate. 13C-lactic acid is commercially available as well; it can also be obtained by protonating commercially available 13C-lactate, e.g. commercially available sodium 13C-lactate.
One way for obtaining hyperpolarised 13C-lactate is the polarisation transfer from a hyperpolarised noble gas which is described in WO-A-98/30918. Noble gases having non-zero nuclear spin can be hyperpolarised by the use of circularly polarised light. A hyperpolarised noble gas, preferably He or Xe, or a mixture of such gases, may be used to effect hyperpolarisation of 13C-nuclei. The hyperpolarised gas may be in the gas phase, it may be dissolved in a liquid/solvent, or the hyperpolarised gas itself may serve as a solvent. Alternatively, the gas may be condensed onto a cooled solid surface and used in this form, or allowed to sublime. Intimate mixing of the hyperpolarised gas with 13C-lactate or 13C-lactic acid is preferred.
Another way for obtaining hyperpolarised 13C-lactate is that polarisation is imparted to 13C-nuclei by thermodynamic equilibration at a very low temperature and high field. Hyperpolarisation compared to the operating field and temperature of the NMR spectrometer is effected by use of a very high field and very low temperature (brute force). The magnetic field strength used should be as high as possible, suitably higher than 1 T, preferably higher than 5 T, more preferably 15 T or more and especially preferably 20 T or more. The temperature should be very low, e.g. 4.2 K or less, preferably 1.5 K or less, more preferably 1.0 K or less, especially preferably 100 mK or less.
Another way for obtaining hyperpolarised 13C-lactate is the spin refrigeration method. This method covers spin polarisation of a solid compound or system by spin refrigeration polarisation. The system is doped with or intimately mixed with suitable crystalline paramagnetic materials such as Ni2+, lanthanide or actinide ions with a symmetry axis of order three or more. The instrumentation is simpler than required for DNP with no need for a uniform magnetic field since no resonance excitation field is applied. The process is carried out by physically rotating the sample around an axis perpendicular to the direction of the magnetic field. The pre-requisite for this method is that the paramagnetic species has a highly anisotropic g-factor. As a result of the sample rotation, the electron paramagnetic resonance will be brought in contact with the nuclear spins, leading to a decrease in the nuclear spin temperature. Sample rotation is carried out until the nuclear spin polarisation has reached a new equilibrium.
In a preferred embodiment, DNP (dynamic nuclear polarisation) is used to obtain hyperpolarised 13C-lactate. 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-lactate. Generally, a moderate or high magnetic field and a very low temperature are used in the DNP process, e.g. by carrying out the DNP process in liquid helium and a magnetic field of about 1 T or above. 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.
To polarise a chemical entity, i.e. compound, by the DNP method, a composition comprising the compound to be polarised and a DNP agent is prepared which is then frozen and inserted into a DNP polariser for polarisation. After the polarisation, the frozen solid hyperpolarised composition is rapidly transferred into the liquid state either by melting it or by dissolving it in a suitable dissolution medium. Dissolution is preferred and the dissolution process of a frozen hyperpolarised composition 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.
In order to obtain a high polarisation level in the compound to be polarised said compound and the DNP agent 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 compounds need to be chosen for polarisation which do not crystallize upon being frozen but rather form a glass. Sodium 13C-lactate is especially preferred since compositions containing sodium 13C-lactate do not crystallize upon freezing/cooling.
In one embodiment, 13C-lactic acid, preferably 13C1-lactic acid is used as a starting material to obtain hyperpolarised 13C-lactate by the DNP method. Said 13C-lactic acid may be 13C-L-lactic acid, 13C-D-lactic acid or a mixture thereof, e.g. a racemic mixture of 13C-D/L-lactic acid. In a preferred embodiment, said 13C-lactic acid is 13C-L-lactic acid or a mixture of 13C-L-lactic acid and 13C-D-lactic acid, more preferably a racemic mixture. In a most preferred embodiment, said 13C-lactic acid is 13C-L-lactic acid.
In a preferred embodiment, 13C-lactate, preferably 13C1-lactate is used as a starting material to obtain hyperpolarised 13C-lactate by the DNP method. Said 13C-lactate may be 13C-L-lactate, 13C-D-lactate or a mixture thereof, e.g. a racemic mixture of 13C-D/L-lactate. In a preferred embodiment, said 13C-lactate is 13C-L-lactate or a mixture of 13C-L-lactate and 13C-D-lactate, more preferably a racemic mixture. In a most preferred embodiment, said 13C-lactate is 13C-L-lactate. Suitable 13C-lactates are sodium 13C-lactate and 13C-lactates which comprise an inorganic cation from the group consisting of NH4+, K+, Rb+, Cs+, Ca2+, Sr2+ and Ba2+. The latter salts are described in detail in WO-A-2007/111515 which is incorporated by reference herein. Alternatively, 13C-lactates of an organic amine or amino compound, preferably TRIS-13C-lactate or meglumine-13C-lactate, as in detail described in WO-A-2007/069909 and incorporated by reference herein. In a most preferred embodiment sodium 13C-lactate and more preferably sodium 13C1-lactate and most preferably sodium 13C1-L-lactate is used as a starting material to obtain hyperpolarised lactate by the DNP method.
For the hyperpolarisation of 13C-lactate by DNP, a composition is prepared which comprises C-lactate or 13C-lactic acid and a DNP agent.
The DNP agent plays a decisive role in the DNP process as its choice has a major impact on the level of polarisation that can be achieved in 13C-lactate. A variety of DNP agents—in WO-A-99/35508 denoted “OMRI contrast agents” —is known. The use of oxygen-based, sulphur-based or carbon-based stable trityl radicals as 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 has resulted in high levels of polarisation in a variety of different samples.
In a preferred embodiment, the hyperpolarised 13C-lactate used in the method of the invention is obtained by DNP and the DNP agent used is a trityl radical. As briefly mentioned above, the large electron spin polarisation of the DNP agent, i.e. trityl radical is converted to nuclear spin polarisation of 13C nuclei in 13C-lactate or 13C-lactic acid via microwave irradiation close to the electron Larmor frequency. The microwaves stimulate communication between electron and nuclear spin systems via e-e and e-n transitions. For effective DNP, i.e. to achieve a high level of polarisation in 13C-lactate or 13C-lactic acid the trityl radical has to be stable and soluble in these compounds to achieve intimate contact between 13C-lactate/13C-lactic acid and the trityl radical which is necessary for the aforementioned communication between electron and nuclear spin systems.
In a preferred embodiment, the trityl radical is a radical of the formula (1)
wherein
In a preferred embodiment, M represents hydrogen or one equivalent of a physiologically tolerable cation. The term “physiologically tolerable cation” denotes a cation that is tolerated by the human or non-human animal living body. Preferably, M represents hydrogen or an alkali cation, an ammonium ion or an organic amine ion, for instance meglumine. Most preferably, M represents hydrogen or sodium.
If 13C-lactate is used as a starting material to obtain hyperpolarised 13C-lactate by the DNP method, R1 is preferably the same, more preferably a straight chain or branched C1-C4-alkyl group, most preferably methyl, ethyl or isopropyl; or R1 is preferably the same, more preferably a straight chain or branched C1-C4-alkyl group which is substituted by one hydroxyl group, most preferably —CH2—CH2—OH; or R1 is preferably the same and represents —CH2—OC2H4OH.
If 13C-lactic acid is used as a starting material to obtain hyperpolarised 13C-lactate by the DNP method, R1 is the same or different, preferably the same and preferably represents —CH2—OCH3, —CH2—OC2H5, —CH2—CH2—OCH3, —CH2—SCH3, —CH2—SC2H5 or —CH2—CH2—SCH3, most preferably —CH2—CH2—OCH3.
The aforementioned trityl radical of formula (1) 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, WO-A-97/09633, WO-A-98/39277 and WO-A-2006/011811.
For the DNP process, a solution of the starting material 13C-lactic acid or 13C-lactate (in the following denoted “sample”) and the DNP agent, preferably a trityl radical, more preferably a trityl radical of formula (1) is prepared. A solvent or a solvent mixture may be used to promote dissolution of the DNP agent in the sample. However, if the hyperpolarised 13C-lactate is intended to be used as an imaging agent for in vivo 13C-MR detection, it is preferred to keep the amount of solvent to a minimum or, if possible, to avoid the use of solvents. To be used as an in vivo imaging agent, the polarised 13C-lactate is usually administered in relatively high concentrations, 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 composition containing the sample, i.e. DNP agent, sample and if necessary solvent, is 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 composition containing the hyperpolarised 13C-lactic acid or 13C-lactate after the DNP process into the liquid state, e.g. for using it as an imaging agent for 13C-MR detection. This is due to the fact that for a given volume of dissolution medium in the dissolution process, the mass of the composition to dissolution medium ratio decreases, when the mass of the composition increases. Further, using certain solvents may require their removal before the hyperpolarised 13C-lactate used as an MR imaging agent is administered to a human or non-human animal being since they might not be physiologically tolerable.
If 13C-lactic acid is used as a starting material to obtain hyperpolarised 13C-lactate via DNP, preferably a solution of the DNP agent, preferably a trityl radical and more preferably a trityl radical of formula (1) in 13C-lactic acid is prepared. Mixtures of 13C-L-lactic acid and 13C-D-lactic acid are either liquids at room temperature (the 13C-D/L-lactic acid racemic mixture has a melting point of about 17° C.) or have a melting point which is between the melting point of the pure isomer and the racemate, i.e. between 17° C.-53° C. If a mixture of 13C-L-lactic acid and 13C-D-lactic acid is used which is a liquid at room temperature, the DNP agent is preferably dissolved in said liquid without further addition of any solvents. However, if solvent(s) are added, it is preferred to use a solvent which is a good glass former, e.g. glycerol. If a mixture of 13C-L-lactic acid and 13C-D-lactic acid is used or if 13C-L-lactic acid or 13C-D-lactic acid are used (both have a melting point of about 53° C.), this mixture or the 13C-L-lactic acid or 13C-D-lactic acid are preferably melted under gentle warming and the DNP agent is dissolved in the melted mixture or 13C-L-lactic acid or 13C-D-lactic acid. Preferably, no solvents are added. However, if solvent(s) are added, it is preferred to either add little water and/or add a solvent which is a good glass former, e.g. glycerol. Intimate mixing of the compounds can be promoted by several means known in the art, such as stirring, vortexing (whirl-mixing) or sonication.
If a 13C-lactate which is a solid at room temperature is used as a starting material to obtain hyperpolarised 13C-lactate via DNP, a solvent has to be added to prepare a solution of the DNP agent and the 13C-lactate. Preferably an aqueous carrier and most preferably water is used as a solvent. In one embodiment, the DNP agent is dissolved and the 13C-lactate is subsequently dissolved in the dissolved DNP agent. In another embodiment, 13C-lactate is dissolved in the solvent and subsequently the DNP agent is dissolved in the dissolved 13C-lactate. If the 13C-lactates mentioned in the first paragraph on page 10, i.e. sodium 13C-lactate, 13C-lactates which comprise an inorganic cation from the group consisting of NH4+, K+, Rb+, Cs+, Ca2+, Sr2+ and Ba2+ and 13C-lactates of an organic amine or amino compound are used, no glass formers have to be added, since a composition containing these 13C-lactates does not crystallize upon cooling/freezing. Again intimate mixing of the compounds can be promoted by several means known in the art, such as stirring, vortexing or sonication.
If the hyperpolarised 13C-lactate used in the method of the invention is obtained by DNP, the composition to be polarised comprising 13C-lactic acid or 13C-lactate and a DNP agent 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 and paramagnetic chelates, i.e. chemical entities comprising a chelator and a paramagnetic metal ion, wherein said paramagnetic metal ion and said chelator form a complex.
In a preferred embodiment, the paramagnetic metal ion is a compound comprising Gd3+ as a paramagnetic metal ion, preferably a paramagnetic chelate comprising a chelator and Gd3+ as a paramagnetic metal ion. In a more preferred embodiment, said paramagnetic metal ion is soluble and stable in the composition to be polarised.
As with the DNP agent described before, the 13C-lactic acid or 13C-lactate to be polarised must be in intimate contact with the paramagnetic metal ion as well. The composition used for DNP comprising 13C-lactic acid or 13C-lactate, a DNP agent and a paramagnetic metal ion may be obtained in several ways. In a first embodiment the 13C-lactate is dissolved in a suitable solvent to obtain a solution; alternatively, liquid or melted 13C-lactic acid as discussed on the previous page is used. To this solution of 13C-lactate or to the liquid/melted 13C-lactic acid the DNP agent is added and dissolved. The DNP agent, preferably a trityl radical, might be added as a solid or in solution, preferably as a solid. In a subsequent step, the paramagnetic metal ion is added. The paramagnetic metal ion might be added as a solid or in solution, preferably as a solid. In another embodiment, the DNP agent and the paramagnetic metal ion are dissolved in a suitable solvent this solution is added to 13C-lactic acid or 13C-lactate. In yet another embodiment, the DNP agent (or the paramagnetic metal ion) is dissolved in a suitable solvent and added to 13C-lactic acid or 13C-lactate. In a subsequent step the paramagnetic metal ion (or the DNP agent) is added to this solution, either as a solid or in solution, preferably as a solid. Preferably, the amount of solvent to dissolve the paramagnetic metal ion (or the DNP agent) is kept to a minimum. Again intimate mixing of the compounds can be promoted by several means known in the art, such as stirring, vortexing or sonication.
If a trityl radical is used as DNP agent, a suitable concentration of such a trityl radical in the composition is 1 to 25 mM, preferably 2 to 20 mM, more preferably 10 to 15 mM in the composition used for DNP. If a paramagnetic metal ion is added to the composition, a suitable concentration of such a paramagnetic metal ion is 0.1 to 6 mM (metal ion) in the composition, and a concentration of 0.5 to 4 mM is preferred.
After having prepared a composition comprising 13C-lactic acid or 13C-lactate, the DNP agent and optionally a paramagnetic metal ion said composition is frozen by methods known in the art, e.g. by freezing it in a freezer, in liquid nitrogen or by simply placing it in the DNP polariser, where liquid helium will freeze it. The composition may optionally be frozen as “beads” before it is inserted into to 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 13C-lactic acid or 13C-lactate are polarised, for instance when it is intended to use the polarised 13C-lactate in an in vivo 13C-MR detection method.
If a paramagnetic metal ion is present in the composition said composition may be degassed before freezing, e.g. by bubbling helium gas through the composition (for instance for a time period of 2-15 min) but degassing can be effected by other known common methods.
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, a moderate or high magnetic field and a very low temperature are used in the DNP process, e.g. by carrying out the DNP process in liquid helium and a magnetic field of about 1 T or above. Alternatively, a moderate magnetic field and any temperature at which sufficient polarisation enhancement is achieved may be employed. In a preferred embodiment, the DNP process is carried out in liquid helium and a magnetic field of about 1 T or above. Suitable polarisation units are for instance described in WO-A-02/37132. In a preferred embodiment, the polarisation unit comprises a cryostat and polarising means, e.g. a microwave chamber connected by a wave guide to a microwave source in a central bore surrounded by magnetic field producing means such as a superconducting magnet. The bore extends vertically down to at least the level of a region P near the superconducting magnet where the magnetic field strength is sufficiently high, e.g. between 1 and 25 T, for polarisation of the sample nuclei to take place. The bore for the probe (i.e. the frozen composition to be polarised) is preferably sealable and can be evacuated to low pressures, e.g. pressures in the order of 1 mbar or less. 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 0.1 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, 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. KelF (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. The microwave frequency may be determined from the EPR line of the DNP agent, which depends on the magnetic field of the magnet as 28.0 GHz/T. The optimal microwave frequency may be determined by adjusting the frequency for maximal NMR signal. Preferably, the optimal microwave frequency is in the about 94 GHz for a magnet charged to 3.35 T, 110 GHz for a magnet charged to 4 T, 140 GHz for a magnet charged to 5 T and 200 GHz for a magnet charged to 7 T. The power may be chosen between 50 and 200 mW, dependent on the probe size. The level of polarisation may be monitored as earlier described by for instance acquiring solid state 13C-NMR signals of the probe during microwave irradiation. Generally, a saturation curve is obtained in a graph showing NMR signal vs. time. Hence it is possible to determine when the optimal polarisation level is reached. A solid state 13C-NMR measurement suitably consists of a simple pulse-acquire NMR sequence using a low flip angle. The signal intensity of the dynamic nuclear polarised nuclei, i.e. 13C nuclei in 13C-lactic acid or 13C-lactate is compared with the signal intensity of the 13C nuclei in 13C-lactic acid or 13C-lactate before DNP. The polarisation is then calculated from the ratio of the signal intensities before and after DNP.
After the DNP process, the frozen solid composition comprising the hyperpolarised 13C-lactic acid or 13C-lactate is transferred from the solid state to the liquid state, i.e. liquefied. This can be done by dissolution in an appropriate solvent or solvent mixture (dissolution medium) or by melting the solid composition, e.g. by applying energy in the form of heat. Dissolution is preferred and the dissolution process 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. 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 13C-lactate has been used as the starting material for the dynamic nuclear polarisation and if the solid composition comprising the hyperpolarised 13C-lactate is liquefied by dissolution, an aqueous carrier, preferably a physiologically tolerable and pharmaceutically accepted aqueous carrier like water, a buffer solution or saline is suitably used as a solvent especially preferably if the hyperpolarised 13C-lactate is intended for use in an imaging medium for in vivo 13C-MR detection. For in vitro applications also non aqueous solvents or solvent mixtures may be used for instance DMSO or methanol or mixtures comprising an aqueous carrier and a non aqueous solvent, for instance mixtures of DMSO and water or methanol and water.
If 13C-lactic acid has been used as the starting material for the dynamic nuclear polarisation, the hyperpolarised 13C-lactic acid obtained has to be converted to 13C-lactate. If the solid composition comprising the hyperpolarised 13C-lactic acid is liquefied by dissolution, the dissolution medium preferably is an aqueous carrier, e.g. water or a buffer solution, preferably a physiologically tolerable buffer solution or comprises an aqueous carrier, e.g. water or a buffer solution, preferably a physiologically tolerable buffer solution. The terms “buffer solution” and “buffer” are hereinafter used interchangeably. In the context of this application “buffer” denotes one or more buffers, i.e. also mixtures of buffers.
Preferred buffers are physiologically tolerable buffers, more preferably buffers which buffer in the range of about pH 7 to 8 like for instance phosphate buffer (KH2PO4/Na2HPO4), ACES, PIPES, imidazole/HCl, BES, MOPS, HEPES, TES, TRIS, HEPPS or TRICIN.
To convert hyperpolarised 13C-lactic acid into hyperpolarised 13C-lactate, 13C-lactic acid is suitably reacted with a base. In one embodiment, 13C-lactic acid is reacted with a base to convert it to 13C-lactate and subsequently an aqueous carrier is added. In another preferred embodiment the aqueous carrier and the base are combined in one solution and this solution is added to 13C-lactic acid, dissolving it and converting it into 13C-lactate at the same time. In a preferred embodiment, the base is an aqueous solution of NaOH, Na2CO3 or NaHCO3, most preferred the base is an aqueous solution of NaOH.
In another preferred embodiment, the aqueous carrier or—where applicable—the combined aqueous carrier/base solution further comprises one or more compounds which are able to bind or complex free paramagnetic ions, e.g. chelating agents like DTPA or EDTA.
If hyperpolarisation is carried out by the DNP method, the DNP agent, preferably a trityl radical and the optional paramagnetic metal ion may be removed from the liquid containing the hyperpolarised 13C-lactate. Removal of these compounds is preferred if the hyperpolarised 13C-lactate is intended for use in an imaging medium for in vivo use. If 13C-lactic acid was as a starting material for DNP, it is preferred to first convert the hyperpolarised 13C-lactic acid into 13C-lactate and remove the DNP agent and the optional paramagnetic metal ion after the conversion has taken place.
Methods useful to remove the trityl radical and the paramagnetic metal ion are known in the art and described in detail in WO-A2-2007/064226 and WO-A1-2006/011809.
In a preferred embodiment the hyperpolarised 13C-lactate used in the method of the invention is obtained by dynamic nuclear polarisation of a composition that comprises sodium 13C-lactate, preferably sodium 13C1-lactate and more preferably sodium 13C1-L-lactate, a trityl radical of formula (1) and optionally a paramagnetic chelate comprising Gd3+. In this preferred embodiment, a solution of the trityl radical and, if used, the paramagnetic chelate comprising Gd3+ is prepared. The dissolved trityl radical and the optional dissolved paramagnetic chelate are added to sodium 13C-lactate and the composition is preferably sonicated or whirl-mixed to promote intimate mixing of all the components.
The imaging medium according to the method of the invention may be used as imaging medium for in vitro 13C-MR detection, e.g. 13C-MR detection in cell cultures, body samples, ex vivo tissue or isolated organs derived from the human or non-human animal body. For this purpose, the imaging medium is provided as a composition that is suitable for being added to, for instance, cell cultures, samples like urine, blood or saliva, ex vivo tissues like biopsy tissues or isolated organs. Such an imaging medium preferably comprises in addition to the imaging agent, i.e. the MR active agent hyperpolarised 13C-lactate a solvent which is compatible with and used for in vitro cell or tissue assays, for instance DMSO or methanol or solvent mixtures comprising an aqueous carrier and a non aqueous solvent, for instance mixtures of DMSO and water or a buffer solution or methanol and water or a buffer solution. As it is apparent for the skilled person, pharmaceutically acceptable carriers, excipients and formulation aids may be present in such an imaging medium but are not required for such a purpose.
Further, the imaging medium according to the method of the invention may be used as imaging medium for in vivo 13C-MR detection, i.e. 13C-MR detection carried out on living human or non-human animal beings. For this purpose, the imaging medium needs to be suitable for administration to a living human or non-human animal body. Hence such an imaging medium preferably comprises in addition to the imaging agent, i.e. the MR active agent hyperpolarised 13C-lactate, an aqueous carrier, preferably a physiologically tolerable and pharmaceutically accepted aqueous carrier like water, a buffer solution or saline. Such an imaging medium may further comprise conventional pharmaceutical or veterinary carriers or excipients, e.g. formulation aids such as stabilizers, osmolality adjusting agents, solubilising agents and the like which are conventional for diagnostic compositions in human or veterinary medicine.
If the imaging medium used in the method of the invention is used for in vivo 13C-MR detection, i.e. in a living human or non-human animal body, said imaging medium is preferably administered to said body parenterally, preferably intravenously. Generally, the body under examination is positioned in an MR magnet. Dedicated 13C-MR RF-coils are positioned to cover the area of interest. Dosage and concentration of the imaging medium will depend upon a range of factors such as toxicity and the administration route. At less than 400 s after the administration, preferably less than 120 s, more preferably less than 60 s after the administration, especially preferably 20 to 50 s an MR imaging sequence is applied that encodes the volume of interest in a combined frequency and spatial selective way. The exact time of applying an MR sequence is highly dependent on the volume of interest and on the species.
In the 13C-MR detection method according to the invention, it is preferred to detect signals of 13C-lactate, 13C-pyruvate, 13C-alanine and 13C-bicarbonate. The MR detectable 13C-labelled compounds are identical when either hyperpolarised 13C-lactate or hyperpolarised 13C-pyruvate is used as imaging agent. This is shown for 13C1-lactate and 13C1-pyruvate in scheme 1, wherein * denotes the 13C-label: on the left of scheme 1, the MR detectable signals of hyperpolarised 13C1-pyruvate (bold, parent compound) and its metabolites 13C-lactate, 13C-alanine and 13C-bicarbonate are shown; on the right of scheme 1, the MR detectable signals of hyperpolarised 13C1-lactate (bold, parent compound) and its metabolite 13C-pyruvate are shown. The latter further metabolizes to 13C-alanine and 13C-bicarbonate.
Thus in a preferred embodiment it is provided a method of 13C-MR detection using an imaging medium comprising hyperpolarised 13C-lactate, wherein signals of 13C-lactate, 13C-pyruvate and 13C-alanine, preferably signals of 13C-lactate, 13C-pyruvate, 13C-alanine and 13C-bicarbonate are detected.
The term “signal” in the context of the invention refers to the MR signal amplitude or integral or peak area to noise of peaks in a 13C-MR spectrum which represent 13C-lactate, 13C-pyruvate, 13C-alanine or 13C-bicarbonate. In a preferred embodiment, the signal is the peak area.
In a preferred embodiment of the method of the invention, the above-mentioned signals of 13C-lactate, 13C-pyruvate, 13C-alanine and 13C-bicarbonate are used to generate a metabolic profile.
In embodiment, the above-mentioned signals of 13C-lactate, 13C-pyruvate, 13C-alanine and 13C-bicarbonate are used to generate a metabolic profile of a living human or non-human animal being. Said metabolic profile may be derived from the whole body, e.g. obtained by whole body in vivo 13C-MR detection. Alternatively, said metabolic profile is generated from a region of interest, i.e. a certain tissue, organ or part of said human or non-human animal body.
In another embodiment, the above-mentioned signals of 13C-lactate, 13C-pyruvate, 13C-alanine and 13C-bicarbonate are used to generate a metabolic profile of cells in a cell culture, of samples like urine, blood or saliva, of ex vivo tissue like biopsy tissue or of an isolated organ. Said metabolic profile is then generated by in vitro 13C-MR detection.
Thus in a preferred embodiment it is provided a method of 13C-MR detection using an imaging medium comprising hyperpolarised 13C-lactate, wherein signals of 13C-lactate, 13C-pyruvate and 13C-alanine, preferably signals of 13C-lactate, 13C-pyruvate, 13C-alanine and 13C-bicarbonate are detected and wherein said signals are used to generate a metabolic profile.
Suitably, the signals of 13C-lactate, 13C-pyruvate and 13C-alanine are used to generate said metabolic profile. In a preferred embodiment, the signals of 13C-lactate, 13C-pyruvate, 13C-alanine and 13C-bicarbonate are used to generate a metabolic profile. Hereinafter the term “13C-labelled compounds” is used to denote 13C-lactate and 13C-pyruvate and 13C-alanine and to denote the preferred embodiment 13C-lactate and 13C-pyruvate and 13C-alanine and 13C-bicarbonate.
In one embodiment, the spectral signal intensities of the 13C-labelled compounds are used to generate the metabolic profile. In another embodiment, the spectral signal integrals of the 13C-labelled compounds are used to generate the metabolic profile. In another embodiment, signal intensities from separate images of the 13C-labelled compounds are used to generate the metabolic profile. In yet another embodiment, the signal intensities of the 13C-labelled compounds are obtained at two or more time points to calculate the rate of change of the 13C-labelled compounds.
In another embodiment the metabolic profile includes or is generated using processed signal data of the 13C-labelled compounds, e.g. ratios of signals, corrected signals, or dynamic or metabolic rate constant information deduced from the signal pattern of multiple MR detections, i.e. spectra or images. Thus, in a preferred embodiment a corrected 13C-lactate signal, i.e. 13C-lactate to 13C-alanine signal and/or 13C-lactate to 13C-pyruvate signal and/or 13C-lactate to 13C-bicarbonate signal is included into or used to generate the metabolic profile. In a further preferred embodiment, a corrected 13C-lactate to total 13C-carbon signal is included into or used to generate the metabolic profile with the total 13C-carbon signal being the sum of the signals of 13C-lactate, 13C-pyruvate, 13C-alanine and optionally 13C-bicarbonate.
The metabolic profile generated in the preferred embodiment of the method according to the invention provides information about the metabolic status and activity of the body, part of the body, cells, tissue, body sample etc under examination and said information may be used in a subsequent step for, e.g. identifying diseases, monitoring the course of a disease and/or determining a disease state or for monitoring therapy success.
Such a disease may be a tumour since tumour tissue is usually characterized by a higher metabolic activity than healthy tissue. Such a higher metabolic activity can be determined by comparing the metabolic profile of a tumour or of an ex vivo sample of a tumour with the metabolic profile of healthy tissue (e.g. surrounding tissue or healthy ex vivo tissue) and may manifest itself in said metabolic profile by high signals of the 13C-labelled compounds or high corrected 13C-lactate signal or high metabolic rates.
Another disease may be ischemia in the heart since ischemic myocardial tissue is usually characterized by a lower metabolic activity than healthy myocardial tissue. Again such a lower metabolic activity can be determined by comparing the metabolic profile of ischemic myocardial tissue with the metabolic profile of healthy myocardial tissue.
Yet another disease may be liver related diseases, such as liver fibrosis or liver cirrhosis. 60% of all lactate metabolism occurs in the liver and it is expected that due to cell death in liver diseases the signal of the 13C-labelled lactate metabolites will decrease in diseased areas of the liver. Thus a metabolic profile of a diseased liver would show a significantly decrease of signals from 13C-alanine and optionally from 13C-pyruvate or high corrected 13C-alanine signal or high ratio of 13C-alanine to 13C-lactate or total carbon.
If D-lactate is used in the method of the invention, diseases like sepsis, ischemia and diabetes and conditions like trauma may be identified (see for instance S. M. Smith et al., J, Infect. Dis. 154, (1986), 658-664; M. J. Murray et al., Am. J. Surg. 167, (1994), 575-578; Z. Li et al., Chin. Med. Sci. J. 16, (2001), 209-213 and Y. Kondoh et al., Res. Exp. Med 192, (1992), 407-414.
Yet another aspect of the invention is a composition comprising sodium 13C1-lactate or 13C1-lactic acid, a trityl radical and optionally a paramagnetic metal ion.
In a first embodiment, said composition comprises sodium 13C1-lactate, a trityl radical and optionally a paramagnetic metal ion. In a preferred embodiment, said sodium 13C1-lactate is 13C1-L-lactate. In another preferred embodiment, said trityl radical is a trityl radical of formula (1) wherein M represents hydrogen or sodium and R1 is preferably the same, more preferably a straight chain or branched C1-C4-alkyl group, most preferably methyl, ethyl or isopropyl; or R1 is preferably the same, more preferably a straight chain or branched C1-C4-alkyl group which is substituted by one hydroxyl group, most preferably —CH2—CH2—OH; or R1 is preferably the same and represents —CH2—OC2H4OH.
In another preferred embodiment said composition comprises a paramagnetic metal ion, and said paramagnetic metal ion is preferably a compound comprising Gd3+ as a paramagnetic metal ion, preferably a paramagnetic chelate comprising a chelator and Gd3+ as a paramagnetic metal ion. In a most preferred embodiment, the composition according to the invention comprises sodium 13C1-L-lactate, a trityl radical of formula (1) and a paramagnetic metal ion. Suitably, said composition further comprises a solvent or solvents; preferably an aqueous carrier and most preferably water is used as a solvent. The aforementioned compositions can be used for obtaining hyperpolarised sodium 13C1-lactate by dynamic nuclear polarisation (DNP) with a high polarisation level. In a second embodiment said composition comprises 13C1-lactic acid, a trityl radical and optionally a paramagnetic metal ion. In a preferred embodiment, said 13C1-lactic acid 13C1-L-lactic acid. In another preferred embodiment, said trityl radical is a trityl radical of formula (1) wherein M represents hydrogen or sodium and R1 is the same or different, preferably the same and preferably represents —CH2—OCH3, —CH2—OC2H5, —CH2—CH2—OCH3, —CH2—SCH3, —CH2—SC2H5 or —CH2—CH2—SCH3, most preferably —CH2—CH2—OCH3. In another preferred embodiment said composition comprises a paramagnetic metal ion, and said paramagnetic metal ion is preferably a compound comprising Gd3+ as a paramagnetic metal ion, preferably a paramagnetic chelate comprising a chelator and Gd3+ as a paramagnetic metal ion. In a most preferred embodiment, the composition according to the invention comprises sodium 13C1-L-lactic acid, a trityl radical of formula (1) and a paramagnetic metal ion. Said composition may further comprise a solvent or solvents; preferably an aqueous carrier and most preferably water is used as a solvent. The aforementioned compositions can be used for obtaining hyperpolarised 13C1-lactic acid by dynamic nuclear polarisation (DNP) with a high polarisation level. Said hyperpolarised 13C1-lactic acid can be converted into hyperpolarised 13C1-lactate by dissolution with a base, e.g. NaOH.
Yet another aspect of the invention is a composition comprising hyperpolarised sodium 13C1-lactate or hyperpolarised 13C1-lactic acid, a trityl radical and optionally a paramagnetic metal ion, wherein said composition is obtained by dynamic nuclear polarisation. In a preferred embodiment, said hyperpolarised sodium 13C1-lactate is hyperpolarised sodium 13C1-L-lactate and said hyperpolarised 13C1-lactic acid is hyperpolarised 13C1-L-lactic acid.
Yet another aspect of the invention is hyperpolarised sodium 13C1-L-lactate or hyperpolarised sodium 13C1-D-lactate, preferably hyperpolarised sodium 13C1-L-lactate.
Yet another aspect of the invention is an imaging medium comprising hyperpolarised sodium 13C1-lactate and/or hyperpolarised sodium 13C1-D-lactate, preferably sodium 13C1-L-lactate.
The imaging medium according to the invention may be used as imaging medium in 13C-MR detection.
The imaging medium according to the invention may be used as imaging medium for in vitro 13C-MR detection, e.g. 13C-MR detection of cell cultures, samples, ex vivo tissue or isolated organs derived from the human or non-human animal body. For this purpose, the imaging medium is provided as a composition that is suitable for being added to, for instance, cell cultures, samples like urine, blood or saliva, ex vivo tissues like biopsy tissues or isolated organs. Such an imaging medium preferably comprises in addition to the imaging agent hyperpolarised 13C-lactate a solvent which is compatible with and used for in vitro cell or tissue assays, for instance DMSO or methanol or solvent mixtures comprising an aqueous carrier and a non aqueous solvent, for instance mixtures of DMSO and water or a buffer solution or methanol and water or a buffer solution. As it is apparent for the skilled person, pharmaceutically acceptable carriers, excipients and formulation aids may be present in such an imaging medium but are not required for such a purpose.
Further, the imaging medium according to the invention may be used as imaging medium for in vivo 13C-MR detection, i.e. 13C-MR detection carried out on living human or non-human animal beings. For this purpose, the imaging medium needs to be suitable for administration to a living human or non-human animal body. Hence such an imaging medium preferably comprises in addition to the imaging agent, i.e. the MR active agent 13C-lactate, an aqueous carrier, preferably a physiologically tolerable and pharmaceutically accepted aqueous carrier like water, a buffer solution or saline. Such an imaging medium may further comprise conventional pharmaceutical or veterinary carriers or excipients, e.g. formulation aids such as stabilizers, osmolality adjusting agents, solubilising agents and the like which are conventional for diagnostic compositions in human or veterinary medicine.
The invention is illustrated by the following non-limiting examples:
To a micro test tube was added sodium 13C1-L-lactate solution (78.5 mg, Aldrich, 50% w/w sodium 13C1-lactate). The cap of the tube was punctured with a needle and the solution was frozen in liquid nitrogen. The tube was put in a flask and connected to a freeze-dryer. After drying the tube contained 41 mg dried sodium 13C1-L-lactate (approx 0.36 mmol, sticky substance). A 145 mM aqueous solution of tris(8-carboxy-2,2,6,6-(tetra(hydroxyethyl)benzo-[1,2-4,5′]-bis-(1,3)-dithiole-4-yl)-methyl sodium salt (trityl radical) which had been synthesised according to Example 7 of WO-A1-98/39277 was prepared and 3.5 μl of this solution were added to the dried sodium 13C1-lactate in the tube. Further, a 5 mM aqueous solution of the Gd-chelate of 1,3,5-tris-(N-(DO3A-acetamido)-N-methyl-4-amino-2-methylphenyl)-[1,3,5]tria-zinane-2,4,6-trione (paramagnetic metal ion) which had been synthesised according to Example 4 of WO-A-2007/064226 was prepared and 2.0 μl of this solution was added to the test tube with the sodium 13C1-lactate and the trityl radical. The resulting composition was sonicated and whirl-mixed to dissolve all compounds. The composition was transferred from the tube to a sample cup and the sample cup was inserted into a DNP polariser. The composition was polarised under DNP conditions at 1.2 K in a 3.35 T magnetic field under irradiation with microwave (94 GHz). Polarisation was followed by solid state 13C-NMR and the solid state polarisation was determined to be 20%.
After 60 min dynamic nuclear polarisation, the frozen polarised composition obtained was dissolved in 6 ml phosphate buffer (20 mM, pH 7.4, 100 mg/l EDTA). The pH of the final solution containing the dissolved composition was 7.4±0.1. The sodium 13C1-L-lactate concentration in said final solution was 60±2 mM.
Liquid state polarisation was determined by liquid state 13C-NMR at 400 MHz to be 18-20%.
Example 2 was carried out as Example 1a, however, a water/glycerol mixture (75:25) was used to prepare the trityl and the Gd-chelate solutions. Solid state polarisation was determined to be 17-20%. The frozen polarised composition obtained was dissolved as described in Example 1b. Liquid state polarisation was determined to be 15-20%. The sodium 13C1-L-lactate concentration in the final solution was 30-50 mM.
Example 3 was carried out as Example 1a, however, a water/glycerol mixture (50:50) was used to prepare the trityl and the Gd-chelate solutions. Solid state polarisation was determined to be 25%. The frozen polarised composition obtained was dissolved as described in Example 1b. Liquid state polarisation was determined to be 25%. The sodium 13C1-L-lactate concentration in the final solution was 30 mM.
1.5 mmol sodium 13C1-L-lactate is dissolved in a cooled solution of 500 μl concentrated H2SO4 in 2 ml water. The resulting mixture is continuously extracted with diethyl ether, the organic phases are combined, dried over MgSO4 and filtered. The filtrate is concentrated in vacuo and 13C1-L-lactic acid is obtained.
13C1-L-lactic acid (0.4 mmol) is gently melted and tris(8-carboxy-2,2,6,6-(tetra(methoxyethyl)benzo-[1,2-4,5′]bis-(1,3)dithiole-4-yl)methyl sodium salt which was synthesized as described in Example 1 of WO-A-2006/011810 is added to result in a 10 mM concentration of the trityl radical in said 13C1-L-lactic acid. Further, a 5 mM aqueous solution of the Gd-chelate of 1,3,5-tris-(N-(DO3A-acetamido)-N-methyl-4-amino-2-methylphenyl)-[1,3,5]tria-zinane-2,4,6-trione (paramagnetic metal ion) which had been synthesised according to Example 4 of WO-A-2007/064226 is prepared and 2.0 μl of this solution is added to the test tube with the 13C1-L-lactic acid and the trityl radical. The resulting composition is sonicated and whirl-mixed to dissolve all compounds. The composition is transferred from the tube to a sample cup and the sample cup was inserted into a DNP polariser. The composition was polarised under DNP conditions at 1.2 K in a 3.35 T magnetic field under irradiation with microwave (94 GHz). Polarisation was followed by solid state 13C-NMR.
To a micro test tube was added 21.7 mg D-lactic acid (0.24 mmol) together with 4 μl water. A 139 mmol/g aqueous solution of tris(8-carboxy-2,2,6,6-(tetra(hydroxyethyl)benzo-[1,2-4,5′]-bis-(1,3)-dithiole-4-yl)-methyl sodium salt (trityl radical) which had been synthesised according to Example 7 of WO-A1-98/39277 was prepared and 2.9 mg of this solution were added to the micro test tube. Further a 14.6 μmol/g aqueous solution of the Gd-chelate of 1,3,5-tris-(N-(DO3A-acetamido)-N-methyl-4-amino-2-methylphenyl)-[1,3,5]tria-zinane-2,4,6-trione (paramagnetic metal ion) which had been synthesised according to Example 4 of WO-A-2007/064226 was prepared and 1.26 mg of this solution was added to the test tube with the D-lactic acid and the trityl radical. The resulting composition was sonicated and whirl-mixed to dissolve all compounds. The composition was transferred from the tube to a sample cup and the sample cup was inserted into a DNP polariser. The composition was polarised under DNP conditions at 1.2 K in a 3.35 T magnetic field under irradiation with microwave (94 GHz).
After an overnight dynamic nuclear polarisation, the frozen polarised composition obtained was dissolved in 6 ml phosphate buffer (40 mM, pH 7.3, osmolality match to 200 mM with NaCl, 100 mg/l EDTA, 1 eq. NaOH). The pH of the final solution containing the dissolved composition was 7.1. The D-lactate concentration in said final solution was 40 mM.
Liquid state polarisation was determined by liquid state 13C-NMR at 400 MHz to be 14%. The liquid state relaxation (T1 at 9.4 T) was determined to 44 s.
An imaging medium was prepared as described in Example 1 and 25 μl of the imaging medium (2.7 mM sodium 13C1-lactate) was mixed into 10 M Hep-G2 cells. A dynamic set of 13C-MR spectra was acquired every 5 s with a 15 degree RF pulse. 13C1-pyruvate was clearly building up over time. The average conversion was 0.3% with a peak conversion (0.4%) approximately 20 s into the experiment.
200 μl of an imaging medium which was prepared as described in Example 1 was injected into a C57Bl/6 mouse over a time period of 6 s. The sodium 13C1-lactate concentration in said imaging medium was 60-90 mM and 3 animals were used in the experiment. A rat size whole body coil (tuned for proton and carbon) was placed over the animal and 13C-MR spectroscopy was carried out in a 9.4 T magnet. A dynamic set of 13C-MR spectra (in total 30) was acquired every 3 s with a 15 degree RF pulse. A significant amount of metabolism was seen with 13C1-pyruvate (approximately 2% of the 13C1-lactate signal) being the earliest peak, followed by 13C1-alanine (approximately 1.5% of the 13C1-lactate signal) at a later point of time. 13C1-bicarbonate (approximately 0.5% of the 13C1-lactate signal) was observable at a similar peak time as 13C1-pyruvate (
200 μl of an imaging medium which was prepared as described in Example 1 was injected into a C57Bl/6 mouse over a time period of 6 s. The sodium 13C1-lactate concentration in said imaging medium was about 60 mM. A surface coil (tuned for proton and carbon) was positioned over the liver of the animal and 13C-MR spectroscopy was carried out in a 9.4 T magnet. A dynamic set of 13C-MR spectra (in total 20) was acquired every 5 s with a 30 degree RF pulse. Again a significant amount of metabolism was seen including 13C1-pyruvate (approximately 3% of the 13C1-lactate signal), followed by 13C1-alanine (approximately 3.5% of the 13C1-lactate signal) at a later point of time (
200 μl of an imaging medium which was prepared as described in Example 1 was injected into a C57Bl/6 mouse over a time period of 6 s. The sodium 13C1-lactate concentration in said imaging medium was about 60 mM and 2 animals were used in the experiment. A surface coil (tuned for proton and carbon) was positioned over the heart of the animal and 13C-MR spectroscopy was carried out in a 9.4 T magnet. A dynamic set of 13C-MR spectra (in total 20) was acquired every 5 s with a 30 degree RF pulse. Again a significant amount of metabolism was seen including 13C1-pyruvate (approximately 2% of the 13C1-lactate signal), followed by 13C1-alanine at a later point of time. 13C1-bicarbonate (approximately 0.5% of the 13C1-lactate signal) was observable at a similar peak time as 13C1-pyruvate (
| Number | Date | Country | Kind |
|---|---|---|---|
| 20073920 | Jul 2007 | NO | national |
| 20074887 | Sep 2007 | NO | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP08/59763 | 7/25/2008 | WO | 00 | 1/26/2010 |
