Patent Application
20110038804
The invention relates to hyperpolarised 13C-α-ketoisocaproate, its use as imaging agent, an imaging medium comprising hyperpolarised 13C-α-ketoisocaproate and methods of 13C-MR detection wherein such an imaging medium is used. Further, the invention relates to methods of producing hyperpolarised 13C-α-ketoisocaproate.
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 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.
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 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 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.
Tumour tissue is often characterised by an increased perfusion and higher metabolic activity. The process of increasing the vascular bed, angiogenesis, is induced by cells that due to their higher metabolic needs and/or their larger distance from a capillary are not able to get enough substrates that can provide the energy needed to sustain energy homeostasis. It is in this area, where cells have problems in producing enough energy a marked change in metabolic pattern is expected. Tissue with problems sustaining energy homeostasis will alter its energy metabolism which in particular results in an increased lactate production. With the use of hyperpolarised 13C-pyruvate as an MR imaging agent, this higher metabolic activity can be seen by an increased production of 13C-lactate which can be detected by 13C-MR detection.
However, since the production of hyperpolarised 13C-pyruvate which is suitable as an in vivo imaging agent is not without challenges, there is a need of alternative hyperpolarised imaging agents which can be used to obtain information about metabolic activity, especially in the field of oncology.
We have now found that hyperpolarised 13C-α-ketoisocaproate may be used as such an imaging agent.
α-Ketoisocaproic acid is reversibly metabolized to leucine; the enzyme branched chain aminotransferase catalyses said reaction and glutamate/α-ketoglutarate is needed as co-substrates. Further, decarboxylation of α-ketoisocaproic acid by branched chain α-ketoacid dehydrogenase results in the formation of CO2 and subsequently bicarbonate. Both of these metabolic conversions of α-ketoisocaproic acid take place in the mitochondrion. Hence by using hyperpolarised 13C-α-ketoisocaproate as an imaging agent, the metabolic activity can be assessed.
Thus in a first aspect the invention provides an imaging medium comprising hyperpolarised 13C-α-ketoisocaproate
The term “imaging medium” denotes a liquid composition comprising hyperpolarised 13C-α-ketoisocaproate as the MR active agent, i.e. imaging agent.
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. of cell cultures, samples like for instance urine, saliva or blood, ex vivo tissue, for instance ex vivo tissue obtained from a biopsy or isolated organs, all of those derived from a living human or non-human animal body. In a preferred embodiment, the imaging medium used in the method of the invention may be used as an imaging medium for in vivo 13C-MR detection
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 “13C-α-ketoisocaproate” denotes a salt of 4-methyl-2-oxopentanoic acid, i.e. a salt comprising 4-methyl-2-oxopentanoate as an anion and said salt is isotopically enriched with 13C.
The isotopic enrichment of the hyperpolarised 13C-α-ketoisocaproate 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%. Generally, hyperpolarised 13C-α-ketoisocaproate according to the invention may be isotopically enriched at any carbon atom in the molecule. However, to achieve a long T1, it is preferred that 13C-α-ketoisocaproate is isotopically enriched with 13C at the C1-position (in the following denoted 13C1-α-ketoisocaproate) or at the C2-position (in the following denoted 13C2-α-ketoisocaproate) or in the C4-position (in the following denoted 13C4-α-ketoisocaproate). Multiple enrichment is also possible like isotopic enrichment at both the C1- and C2-position (in the following denoted 13C1-2-α-ketoisocaproate), at the C1- and the C4-position (in the following denoted 13C1-4-α-ketoisocaproate) or at the C1-, C2- and C4-position (in the following denoted 13C1,2,4-α-ketoisocaproate) Isotopic enrichment at the C1-position is preferred.
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-α-ketoisocaproate, e.g. solid hyperpolarised 13C-α-ketoisocaproate obtained by dynamic nuclear polarisation (DNP) of 13C-α-ketoisocaproate. 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-α-ketoisocaproate in the NMR spectrum is compared with signal intensity of 13C-α-ketoisocaproate 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 for dissolved hyperpolarised 13C-α-ketoisocaproate may be determined by liquid state NMR measurements. Again the signal intensity of the dissolved hyperpolarised 13C-α-ketoisocaproate is compared with the signal intensity of the dissolved 13C-α-ketoisocaproate before polarisation. The level of polarisation is then calculated from the ratio of the signal intensities of 13C-α-ketoisocaproate 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).
Hyperpolarised 13C-α-ketoisocaproate can be obtained by directly polarising 13C-α-ketoisocaproate or by polarisation of 13C-α-ketoisocaproic acid and subsequent conversion (neutralisation) of the acid to 13C-α-ketoisocaproate with a base. Since neutralisation with a base is an additional step, it is preferred to directly polarise 13C-α-ketoisocaproate. Suitable 13C-α-ketoisocaproates are commercially available or can be prepared from commercially available 13C-α-ketoisocaproic acid/13C-α-ketoisocaproates and will be discussed in detail in the following paragraphs.
One way for obtaining hyperpolarised 13C-α-ketoisocaproic acid/13C-α-ketoisocaproate 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-α-ketoisocaproic acid/13C-α-ketoisocaproate is preferred.
Another way for obtaining hyperpolarised 13C-α-ketoisocaproic acid/13C-α-ketoisocaproate 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-α-ketoisocaproic acid/13C-α-ketoisocaproate 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-α-ketoisocaproic acid/13C-α-ketoisocaproate. 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-α-ketoisocaproic acid/13C-α-ketoisocaproate. 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 of the compound to be polarised and a DNP agent 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 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.
In one embodiment, 13C-α-ketoisocaproic acid, preferably 13C1-α-ketoisocaproic acid is used as a starting material to obtain hyperpolarised 13C-α-ketoisocaproic acid by the DNP method which is then neutralised and converted to hyperpolarised 13C-α-ketoisocaproate with the help of a base. In another embodiment, 13C-α-ketoisocaproate, preferably 13C1-α-ketoisocaproate is used as a starting material to obtain hyperpolarised 13C-α-ketoisocaproate by the DNP method.
In a first embodiment, 13C-α-ketoisocaproic acid, preferably 13C1-α-ketoisocaproic acid is used as a starting material to obtain hyperpolarised 13C-α-ketoisocaproic acid by the DNP method which is then neutralised and converted to hyperpolarised 13C-α-ketoisocaproate with the help of a base. 13C-α-ketoisocaproic acid is a commercially available compound; alternatively 13C-α-ketoisocaproic acid may be prepared from commercially available sodium 13C-α-ketoisocaproate by conversion with an acid, a process which is well known in the art and illustrated in the Example part of this application.
In a second embodiment, 13C-α-ketoisocaproate, preferably 13C1-α-ketoisocaproate is used as a starting material to obtain hyperpolarised 13C-α-ketoisocaproate by the DNP method. Suitable 13C-α-ketoisocaproates are for instance sodium 13C-α-ketoisocaproate or 13C-α-ketoisocaproates 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, which is incorporated by reference herein. In another embodiment, 13C-α-ketoisocaproates of an organic amine or amino compound are used as a starting material, more preferably TRIS-13C-α-ketoisocaproate or meglumine-13C-α-ketoisocaproate acid. These salts are in detail described in WO-A-2007/069909, which is incorporated by reference herein.
The term “TRIS” denotes 2-amino-2-hydroxymethyl-1,3-propanediol and the term “TRIS-13C-α-ketoisocaproate” denotes a salt which contains a 13C-α-ketoisocaproate anion and a TRIS cation, i.e. TRIS ammonium (2-hydroxymethyl-1,3-propanedioyl ammonium).
For the hyperpolarisation of 13C-α-ketoisocaproic acid/13C-α-ketoisocaproate by the DNP method, a composition is prepared which comprises 13C-α-ketoisocaproic acid or 13C-α-ketoisocaproate 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-α-ketoisocaproic acid/13C-α-ketoisocaproate. 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-α-ketoisocaproic acid/13C-α-ketoisocaproate 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-α-ketoisocaproic acid or 13C-α-ketoisocaproate 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-α-ketoisocaproic acid/13C-α-ketoisocaproate, the trityl radical has to be stable and soluble in these compounds or solutions thereof to achieve said intimate contact between 13C-α-ketoisocaproic acid/13C-α-ketoisocaproate 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-α-ketoisocaproate is used as a starting material to obtain hyperpolarised 13C-α-ketoisocaproate 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-α-ketoisocaproic acid is used as a starting material to obtain hyperpolarised 13C-α-ketoisocaproate 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 radicals 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.
Generally, for the DNP process, a solution of the starting material, i.e. 13C-α-ketoisocaproic acid or 13C-α-ketoisocaproate (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 needs to be used to promote dissolution of the DNP agent and the sample. If the hyperpolarised 13C-α-ketoisocaproate 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. To be used as an in vivo imaging agent, the hyperpolarised 13C-α-ketoisocaproate 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-α-ketoisocaproic acid/13C-α-ketoisocaproate 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-α-ketoisocaproate used in the imaging medium of the invention is administered to a human or non-human animal being since they might not be physiologically tolerable.
If 13C-α-ketoisocaproic acid is used to obtain hyperpolarised 13C-α-ketoisocaproate via DNP, a solution of the DNP agent, preferably a trityl radical and more preferably a trityl radical of formula (1) in 13C-α-ketoisocaproic acid, which is a liquid at room temperature, is prepared. A glass former like for instance glycerol or glycol may optionally be added. Intimate mixing of the compounds can be promoted by several means known in the art, such as stirring, vortexing (whirl-mixing) or sonication and/or gentle heating.
If a 13C-α-ketoisocaproate like for instance TRIS-13C-α-ketoisocaproate is used as the starting material, it may be dissolved in a suitable solvent, preferably water, or solvent mixture and the DNP agent may be added to this solution. In another embodiment, the DNP agent is dissolved in a suitable solvent or solvent mixture and the 13C-α-ketoisocaproate is added to this solution. A glass former like for instance glycerol or glycol may optionally be added, for instance if sodium 13C-α-ketoisocaproate. Again intimate mixing of the compounds can be promoted by several means known in the art, such as stirring, vortexing or sonication and/or gentle heating.
If 13C-α-ketoisocaproates are used as a starting material which crystallize upon freezing, like for instance sodium 13C-α-ketoisocaproate, the addition of a glass former to the solvent or solvent mixture is preferred. A suitable solvent for 13C-α-ketoisocaproates is water, suitable glass formers are for instance glycol or glycerol. Thus in one embodiment 13C-α-ketoisocaproate is dissolved in a solvent or solvent mixture and the DNP agent and a glass former are added to this solution. In another embodiment, the DNP agent is dissolved in a solvent or solvent mixture and the 13C-α-ketoisocaproate and a glass former are added to this solution. In yet another embodiment the DNP agent is dissolved in a glass former and the 13C-α-ketoisocaproate and a solvent or solvent mixture is added to this solution.
The composition to be DNP-polarised comprising 13C-α-ketoisocaproic acid or 13C-α-ketoisocaproate 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 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 composition to be polarised.
As with the DNP agent described before, the 13C-α-ketoisocaproic acid/13C-α-ketoisocaproate to be polarised must be in intimate contact with the paramagnetic metal ion as well. The composition used for DNP comprising 13C-α-ketoisocaproic acid or 13C-α-ketoisocaproate, a DNP agent and a paramagnetic metal ion may be obtained in several ways.
If 13C-α-ketoisocaproic acid is used as a starting material, it is preferred to add the DNP agent to 13C-α-ketoisocaproic acid, either as a solid or in solution. If the trityl radical of formula (1) is used as DNP agent, it is preferably added 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. Alternatively, a solution of the DNP agent and paramagnetic metal ion may be prepared and 13C-α-ketoisocaproic acid is added to this solution or the solution is added to 13C-α-ketoisocaproic acid. Intimate mixing of the compounds can be promoted by several means known in the art, such as stirring, vortexing or sonication and/or gentle heating.
If a 13C-α-ketoisocaproate like TRIS-13C-α-ketoisocaproate is used as the starting material, it may be dissolved in a suitable solvent or solvent mixture and the DNP agent may be added to this solution. The DNP agent, preferably a trityl radical, might be added as a solid or in solution. In a subsequent step, the paramagnetic metal ion is added. Also the paramagnetic metal ion might be added as a solid or in solution. In another embodiment, the DNP agent and the paramagnetic metal ion are dissolved in suitable solvents or a suitable solvent and to this solution is added the 13C-α-ketoisocaproate. In yet another embodiment, the DNP agent (or the paramagnetic metal ion) is dissolved in a suitable solvent and added to the solid or dissolved 13C-α-ketoisocaproate. 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, the amount of solvent to dissolve all the compounds is kept to a minimum. As discussed before, if 13C-α-ketoisocaproates are used as a starting material which crystallize upon freezing, like for instance sodium 13C-α-ketoisocaproate, the addition of a glass former to the solvent or solvent mixture is preferred. Again intimate mixing of the compounds can be promoted by several means known in the art, such as stirring, vortexing or sonication and/or gentle heating.
If a trityl radical is used as DNP agent, a suitable concentration of such a trityl radical 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 0.3 to 4 mM is preferred.
After having prepared a composition comprising 13C-α-ketoisocaproic acid or 13C-α-ketoisocaproate, 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. In a preferred embodiment, the composition is 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-α-ketoisocaproic acid or 13C-α-ketoisocaproate are polarised, for instance when the compound is intended to be used 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 (e.g. 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. Ke1F (polychlorotrifluoroethylene) 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 94 GHz at 200 mW. The level of polarisation may be monitored 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 and/or sufficient 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-α-ketoisocaproic acid or 13C-α-ketoisocaproate is compared with the signal intensity of the 13C nuclei in 13C-α-ketoisocaproic acid or 13C-α-ketoisocaproate before DNP. The polarisation is then calculated from the ratio of the signal intensities before and after DNP.
After the DNP process, the solid composition comprising the hyperpolarised 13C-α-ketoisocaproic acid or 13C-α-ketoisocaproate is transferred from a solid state to a 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. 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-α-ketoisocaproate has been used as the starting material for the dynamic nuclear polarisation and if the solid composition comprising the hyperpolarised 13C-α-ketoisocaproate 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-α-ketoisocaproate is intended for use in an imaging medium for in vivo 13C-MR detection. The aqueous carrier may contain a base to adjust the pH of the final solution in such a way that it is suitable for in vivo administration. Suitable pH ranges from 6.8 to 7.8. 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. In another preferred embodiment, the aqueous carrier or the non aqueous solvents or solvent mixtures may further comprise one or more compounds which are able to bind or complex free paramagnetic ions, e.g. chelating agents like DTPA or EDTA.
If 13C-α-ketoisocaproic acid has been used as the starting material for the dynamic nuclear polarisation, the hyperpolarised 13C-α-ketoisocaproic acid obtained needs to be converted to 13C-α-ketoisocaproate. If the solid composition comprising the hyperpolarised 13C-α-ketoisocaproic acid is liquefied by dissolution, and the hyperpolarised 13C-α-ketoisocaproate is intended to be used in vivo, the dissolution medium is preferably an aqueous carrier, e.g. water or a buffer solution, preferably a physiologically tolerable buffer solution or it 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-α-ketoisocaproic acid into hyperpolarised 13C-α-ketoisocaproate, 13C-α-ketoisocaproic acid is generally reacted with a base. In one embodiment, 13C-α-ketoisocaproic acid is reacted with a base to convert it to 13C-α-ketoisocaproate. For in vivo intended use an aqueous carrier is subsequently added. In another preferred embodiment the aqueous carrier and the base are combined in one solution and this solution is added to 13C-α-ketoisocaproic acid, dissolving it and converting it into 13C-α-ketoisocaproate at the same time. In a preferred embodiment, the base is an aqueous solution of NaOH, Na2CO3 or NaHCO3, most preferred the base is NaOH.
In another preferred embodiment, the aqueous carrier buffer 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.
For in vitro applications of hyperpolarised 13C-α-ketoisocaproate 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 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-α-ketoisocaproic acid or 13C-α-ketoisocaproate. Removal of these compounds is preferred if the hyperpolarised 13C-α-ketoisocaproic acid or 13C-α-ketoisocaproate is intended for use in an imaging medium for in vivo use. If hyperpolarised 13C-α-ketoisocaproic acid was used as a starting material for DNP, it is preferred to first convert the hyperpolarised 13C-α-ketoisocaproic acid into 13C-α-ketoisocaproate and remove the DNP agent and the optional paramagnetic metal ion after said conversion has taken place.
Methods which are 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-α-ketoisocaproate of the imaging medium according to the invention is obtained by dynamic nuclear polarisation of a composition that comprises 13C-α-ketoisocaproic acid or TRIS-13C-α-ketoisocaproate or sodium 13C-α-ketoisocaproate, a trityl radical of formula (1) and optionally a paramagnetic chelate comprising Gd3+.
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, i.e. hyperpolarised 13C-α-ketoisocaproate, 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. hyperpolarised 13C-α-ketoisocaproate, 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 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. Exact dosage and concentration of the imaging medium will depend upon a range of factors such as toxicity and the administration route. Generally, the imaging medium is administered in a concentration of up to 1 mmol 13C-α-ketoisocaproate per kg bodyweight, preferably 0.01 to 0.5 mmol/kg, more preferably 0.1 to 0.3 mmol/kg. 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.
The imaging medium according to the invention is preferably used in a method of 13C-MR detection and such a method is another aspect of the invention.
Thus, in a second aspect the invention provides a method of 13C-MR detection using an imaging medium comprising hyperpolarised 13C-α-ketoisocaproate.
In a preferred first embodiment, the invention provides a method of 13C-MR detection using an imaging medium comprising hyperpolarised 13C-α-ketoisocaproate wherein signals of 13C-leucine and optionally of 13C-α-ketoisocaproate are detected.
In a preferred second embodiment, the invention provides a method of 13C-MR detection using an imaging medium comprising hyperpolarised 13C-α-ketoisocaproate wherein signals of 13CO2 and/or 13C-bicarbonate and optionally of 13C-α-ketoisocaproate are detected.
In a preferred third embodiment, the invention provides a method of 13C-MR detection using an imaging medium comprising hyperpolarised 13C-α-ketoisocaproate wherein signals of 13C-leucine and 13CO2 and/or 13C-bicarbonate and optionally of 13C-α-ketoisocaproate are detected.
The term “signals of 13C-leucine and optionally 13C-α-ketoisocaproate are detected” means that in the method of the invention, only the signal of 13C-leucine is detected or the signals of 13C-leucine and 13C-α-ketoisocaproate are detected.
The term “signals of 13CO2 and/or 13C-bicarbonate and optionally of 13C-α-ketoisocaproate are detected” means that in the method of the invention only the signal of 13CO2 or only the signal of 13C-bicarbonate is detected or that the signals of 13CO2 and 13C-bicarbonate are detected or that the signals of 13CO2 and 13C-α-ketoisocaproate or the signals of 13C-bicarbonate and 13C-α-ketoisocaproate or the signals of are detected or that the signals of 13CO2 and 13C-bicarbonate and 13C-α-ketoisocaproate are detected.
The term “signals of 13C-leucine and 13CO2 and/or 13C-bicarbonate and optionally of 13C-α-ketoisocaproate are detected” means that in the method of the invention the signals of 13C-leucine and 13CO2 or the signals of 13C-leucine and 13C-bicarbonate or the signals of 13C-leucine and 13CO2 and 13C-bicarbonate are detected. It further means that the signals of 13C-leucine and 13CO2 and 13C-α-ketoisocaproate or the signals of 13C-leucine and 13C-bicarbonate and 13C-α-ketoisocaproate or the signals of 13C-leucine and 13CO2 and 13C-bicarbonate and 13C-α-ketoisocaproate are detected
The term “13C-leucine” denotes 2-amino-4-methyl-pentanoic 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-leucine” denotes a compound which is 13C-enriched at the C1- and/or C2- and/or C4-position The position of the isotopic enrichment in 13C-leucine is of course dependent on the position of the isotopic enrichment in its parent compound 13C-α-ketoisocaproate. Thus, if for example hyperpolarised 13C1-α-ketoisocaproate was used in the imaging medium used in the method of the invention, the signal of 13C1-leucine is detected.
The term “13C-bicarbonate” denotes HCO3− that is isotopically enriched with 13C, i.e. in which the amount of 13C isotope is greater than its natural abundance. Likewise the term “13CO2” denotes carbon dioxide that is isotopically enriched with 13C, i.e. in which the amount of 13C isotope is greater than its natural abundance. Whether it is possible to detect 13C-bicarbonate and/or 13CO2 is of course dependent on the position of the isotopic enrichment in its parent compound 13C-α-ketoisocaproate. Only if 13C-α-ketoisocaproate which is 13C-enriched at the C1-position is used in the imaging medium used in the method of the invention, 13C-bicarbonate and 13CO2 is formed and thus may be detected by 13C-MR detection.
The metabolic conversion of α-ketoisocaproate acid to leucine and carbon dioxide is shown in scheme 1 for 13C1-α-ketoisocaproate; * denotes the 13C-label: 13C-α-ketoisocaproate is converted to 13C1-leucine by branched chain aminotransferase (BCAT, EC 2.6.1.42) and to 13CO2 (and subsequently 13C-bicarbonate) by branched chain alpha-keto acid dehydrogenase (BCKD, EC 1.2.4.4.).
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-leucine, 13CO2, 13C-bicarbonate and/or 13C-α-ketoisocaproate. 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-leucine, 13CO2, 13C-bicarbonate and/or 13C-α-ketoisocaproate 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 or volume of interest, i.e. a certain tissue, organ or part of said human or non-human animal body.
In another preferred embodiment of the method of the invention, the above-mentioned signals of 13C-leucine, 13CO2, 13C-bicarbonate and/or 13C-α-ketoisocaproate 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 derived from a human or non-human animal being. Said metabolic profile is then generated by in vitro 13C-MR detection.
Thus, in a preferred first embodiment, the invention provides a method of 13C-MR detection using an imaging medium comprising hyperpolarised 13C-α-ketoisocaproate wherein signals of 13C-leucine and optionally of 13C-α-ketoisocaproate are detected and wherein said signals are used to generate a metabolic profile.
In a preferred second embodiment, the invention provides a method of 13C-MR detection using an imaging medium comprising hyperpolarised 13C-α-ketoisocaproate wherein signals of 13CO2 and/or 13C-bicarbonate and optionally of 13C-α-ketoisocaproate are detected and wherein said signals are used to generate a metabolic profile.
In a preferred third embodiment, the invention provides a method of 13C-MR detection using an imaging medium comprising hyperpolarised 13C-α-ketoisocaproate wherein signals of 13C-leucine and 13CO2 and/or 13C-bicarbonate and optionally of 13C-α-ketoisocaproate are detected and wherein said signals are used to generate a metabolic profile.
In a more preferred first embodiment, the signals of 13C-leucine and 13C-α-ketoisocaproate are used to generate said metabolic profile.
In one embodiment, the spectral signal intensities of 13C-leucine and optionally 13C-α-ketoisocaproate are used to generate the metabolic profile. In another embodiment, the spectral signal integrals of 13C-leucine and optionally 13C-α-ketoisocaproate are used to generate the metabolic profile. In another embodiment, signal intensities from separate images of 13C-leucine and 13C-α-ketoisocaproate are used to generate the metabolic profile. In yet another embodiment, the signal intensities of 13C-leucine and optionally 13C-α-ketoisocaproate are obtained at two or more time points to calculate the rate of change of 13C-leucine and optionally the rate of change of 13C-α-ketoisocaproate.
In another embodiment the metabolic profile includes or is generated using processed signal data of 13C-leucine and optionally 13C-α-ketoisocaproate, 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.
Hence, in a preferred embodiment a corrected 13C-leucine signal, i.e. 13C-leucine to 13C-α-ketoisocaproate signal is included into or used to generate the metabolic profile. In a further preferred embodiment, a corrected 13C-leucine to total 13C-carbon signal is included into or used to generate the metabolic profile with total 13C-carbon signal being the sum of the signals of 13C-leucine and 13C-α-ketoisocaproate. In a more preferred embodiment, the ratio of 13C-leucine to 13C-α-ketoisocaproate is included into or used to generate the metabolic profile.
In a more preferred second embodiment, the signals of 13CO2 and/or 13C-bicarbonate and 13C-α-ketoisocaproate are used to generate said metabolic profile.
In one embodiment, the spectral signal intensities of 13CO2 and/or 13C-bicarbonate and optionally 13C-α-ketoisocaproate are used to generate the metabolic profile. In another embodiment, the spectral signal integrals of 13CO2 and/or 13C-bicarbonate and optionally 13C-α-ketoisocaproate are used to generate the metabolic profile. In another embodiment, signal intensities from separate images of 13CO2 and 13C-bicarbonate and optionally 13C-α-ketoisocaproate or separate images of 13CO2 and 13C-α-ketoisocaproate or separate images of 13C-bicarbonate and 13C-α-ketoisocaproate are used to generate the metabolic profile. In yet another embodiment, the signal intensities of 13CO2 and/or 13C-bicarbonate and optionally of 13C-α-ketoisocaproate are obtained at two or more time points to calculate the rate of change of 13CO2 and/or 13C-bicarbonate and optionally the rate of change of 13C-α-ketoisocaproate.
In another embodiment the metabolic profile includes or is generated using processed signal data of 13CO2 and/or 13C-bicarbonate and optionally 13C-α-ketoisocaproate, 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.
Hence, in a preferred embodiment a corrected 13CO2 and/or 13C-bicarbonate signal, i.e. 13CO2 to 13C-α-ketoisocaproate signal or 13C-bicarbonate to 13C-α-ketoisocaproate is included into or used to generate the metabolic profile. In a further preferred embodiment, a corrected 13CO2 and/or 13C-bicarbonate to total 13C-carbon signal is included into or used to generate the metabolic profile with total 13C-carbon signal being the sum of the signals of 13CO2 and/or 13C-bicarbonate signal and 13C-α-ketoisocaproate. In a more preferred embodiment, the ratio of 13CO2 and/or 13C-bicarbonate signal to 13C-α-ketoisocaproate signal is included into or used to generate the metabolic profile.
In a more preferred third embodiment, the signals of 13C-leucine and 13CO2 and/or 13C-bicarbonate and optionally of 13C-α-ketoisocaproate are used to generate a metabolic profile.
In one embodiment, the spectral signal intensities of 13C-leucine and 13CO2 and/or 13C-bicarbonate and optionally 13C-α-ketoisocaproate are used to generate the metabolic profile. In another embodiment, the spectral signal integrals of 13C-leucine and 13CO2 and/or 13C-bicarbonate and optionally 13C-α-ketoisocaproate are used to generate the metabolic profile. In another embodiment, signal intensities from separate images of 13C-leucine and 13CO2 and 13C-bicarbonate and optionally 13C-α-ketoisocaproate or separate images of 13C-leucine and 13CO2 and optionally 13C-α-ketoisocaproate or separate images of 13C-leucine and 13C-bicarbonate and optionally 13C-α-ketoisocaproate are used to generate the metabolic profile. In yet another embodiment, the signal intensities of 13C-leucine and 13CO2 and/or 13C-bicarbonate and optionally of 13C-α-ketoisocaproate are obtained at two or more time points to calculate the rate of change of 13C-leucine and 13CO2 and/or 13C-bicarbonate and optionally the rate of change of 13C-α-ketoisocaproate.
In another embodiment the metabolic profile includes or is generated using processed signal data of 13C-leucine and 13CO2 and/or 13C-bicarbonate and optionally 13C-α-ketoisocaproate, 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.
Hence, in a preferred embodiment corrected 13C-leucine and 13CO2 and/or 13C-bicarbonate signals, i.e. 13C-leucine to 13CO2 signal or 13C-leucine to 13C-bicarbonate signal and optionally 13C-leucine to 13C-α-ketoisocaproate signal is included into or used to generate the metabolic profile. In a further preferred embodiment, a corrected 13C-leucine and 13CO2 and/or 13C-bicarbonate to total 13C-carbon signal is included into or used to generate the metabolic profile with total 13C-carbon signal being the sum of the signals of 13C-leucine and 13CO2 and/or 13C-bicarbonate signal and optionally 13C-α-ketoisocaproate. In a more preferred embodiment, the ratio of 13C-leucine and 13CO2 and/or 13C-bicarbonate signal to 13C-α-ketoisocaproate signal is included into or used to generate the metabolic profile
The metabolic profile generated in the preferred embodiment of the method according to the invention provides information about the metabolic 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.
Such a disease is preferably cancer since tumour tissue is usually characterized by a higher metabolic activity than healthy tissue. Such a higher metabolic activity would be apparent from 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 the metabolic profile by high signals of 13C-leucine and/or 13CO2 and/or 13C-bicarbonate or by a high corrected 13C-leucine and/or 13CO2 and/or 13C-bicarbonate signal or a high ratio of 13C-leucine to 13C-α-ketoisocaproate signal and/or 13CO2 and/or 13C-bicarbonate to 13C-α-ketoisocaproate signal or a high ratio of 13C-leucine signal to total carbon signal and/or a high ratio of 13CO2 and/or 13C-bicarbonate signal to total carbon signal or a high metabolic rate of 13C-leucine and/or 13CO2 and/or 13C-bicarbonate build-up.
In cancer tissue the concentration of glutamate is often higher than in healthy tissue. This co-substrate will enable a high turn over of 13C-α-ketoisocaproate to 13C-leucine in the cancer tissue where the BCAT activity is high.
The term “high” is a relative term and it has to be understood that the “high signal, ratio, metabolic rate” etc. which is seen in a metabolic profile of a diseased tissue as described above is increased compared to the signal, ratio, metabolic rate etc. which is seen in a metabolic profile of a healthy tissue.
In hepatic steatosis, there is a lower activity of the enzyme BCKD in the liver and a 13C-breath test based on the decarboxylation of 13C-α-ketoisocaproate is used for diagnosing said disease state. In this test the exhaled 13CO2 is collected dynamically and quantified by methods in the art. Hence there is an indication that the information provided by the metabolic profile generated in the preferred embodiment of the method according to the invention may be used for identification of liver related diseases like fatty liver, liver fibrosis or liver cirrhosis. For such liver-related diseases, it can be assumed that they would manifest themselves in a metabolic profile of a diseased liver by a change in 13CO2 and/or 13C-bicarbonate signal and/or ratio of these metabolites to 13C-α-ketoisocaproate when compared to a metabolic profile of a healthy liver.
Yet another disease may be kidney related diseases since it is known that the activity of BCAT which catalyzes the conversion of 13C-α-ketoisocaproate to 13C-leucine is highly active in the kidneys. In kidney diseases which manifest themselves by a change in BCAT activity, it can be assumed that said change, which may be a change in 13C-leucine signal and/or a change in ratio of 13C-leucine to 13C-α-ketoisocaproate can be detected in a metabolic profile of a diseased kidney/diseased kidney tissue when compared to a metabolic profile of a healthy kidney/healthy surrounding kidney tissue.
Anatomical and/or—where suitable—perfusion information may be included in the method of the invention for identification of diseases. Anatomical information may for instance be obtained by acquiring a proton or 13C-MR image with or without employing a suitable contrast agent before or after the method of the invention.
In another preferred embodiment, the imaging medium comprising hyperpolarised 13C-α-ketoisocaproate is administered repeatedly, thus allowing dynamic studies. This is a further advantage of the method according to the invention compared to other MR detection methods using conventional MR contrast agents which—in higher doses—may show toxic effects. α-Ketoisocaproate is present in the human body and hyperpolarised 13C-α-ketoisocaproate was well tolerated in the animal models we have used and described in the Examples part of this application. Hence it is expected that hyperpolarised 13C-α-ketoisocaproate will be well tolerated in patients as well and thus administration of repeated doses of this compound should be possible.
As stated above, the metabolic profile provides information about the metabolic 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. However, a physician may also use this information in a further step to choose the appropriate treatment for the patient under examination.
Thus, said information may be used to monitor treatment response, e.g. treatment success of the above mentioned diseases, and its sensitivity makes the method especially suitable for monitoring early treatment response, i.e. response to treatment shortly after its commencement.
In yet another embodiment, the method of the invention may be used to assess drug efficacy. In said embodiment, potential drugs for curing a certain disease like for instance anti-cancer drugs, may be tested at a very early stage in drug screening, for instance in vitro in a cell culture which is a relevant model for said certain disease or in diseased ex vivo tissue or a diseased isolated organ. Alternatively, potential drugs for curing a certain disease may be tested at an early stage in drug screening in vivo, for instance in an animal model which is relevant for said certain disease. By comparing the metabolic profile of said cell culture, ex vivo tissue, isolated or test animal before and after treatment with a potential drug, the efficacy of said drug and thus treatment response and success can be determined which of course provides valuable information in the screening of potential drugs.
Yet another aspect of the invention is a composition comprising 13C-α-ketoisocaproic acid or 13C-α-ketoisocaproate, a DNP agent and optionally a paramagnetic metal ion. Said composition can be used for obtaining hyperpolarised 13C-α-ketoisocaproate by dynamic nuclear polarisation (DNP) which can be used as imaging agent (MR active agent) in the imaging medium according to the invention.
In one embodiment, the composition according to the invention comprises 13C-α-ketoisocaproic acid, a DNP agent and optionally a paramagnetic metal ion. In a preferred embodiment, said DNP agent is a trityl radical, more preferably a trityl radical of formula (1) and most preferably 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, preferably a salt or paramagnetic chelate comprising Gd3+ and more a paramagnetic chelate comprising Gd3+. Optionally, said composition further comprises a solvent or solvents and/or a glass former. In a preferred embodiment, the composition comprises a glass former like for instance glycerol. The aforementioned compositions can be used for obtaining hyperpolarised 13C-α-ketoisocaproic acid by dynamic nuclear polarisation (DNP) with a high polarisation level. Said hyperpolarised 13C-α-ketoisocaproic acid can be converted into hyperpolarised 13C-α-ketoisocaproate by dissolution with a base, e.g. NaOH, as described earlier in the application.
In another embodiment, said composition comprises 13C-α-ketoisocaproate, preferably TRIS-13C-α-ketoisocaproate or sodium 13C-α-ketoisocaproate, a DNP agent and optionally a paramagnetic metal ion. In a preferred embodiment, said DNP agent is a trityl radical, more preferably a trityl radical of formula (1) and most preferably 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, preferably a salt or paramagnetic chelate comprising Gd3+ and more a paramagnetic chelate comprising Gd3+. Suitably, said composition further comprises a solvent or solvents; preferably an aqueous carrier. Optionally, said composition comprises a glass former like for instance glycerol. The aforementioned compositions can be used for obtaining hyperpolarised 13C-α-ketoisocaproate by dynamic nuclear polarisation (DNP) with a high polarisation level.
Yet another aspect of the invention is a composition comprising hyperpolarised 13C-α-ketoisocaproic acid or hyperpolarised 13C-α-ketoisocaproate, a DNP agent and optionally a paramagnetic metal ion, wherein said composition is obtained by dynamic nuclear polarisation of a composition as described in the previous paragraphs.
Yet anther aspect of the invention is hyperpolarised 13C-α-ketoisocaproic acid or hyperpolarised 13C-α-ketoisocaproate, preferably TRIS-13C-α-ketoisocaproate or sodium 13C-α-ketoisocaproate. Preferred embodiments are hyperpolarised 13C1-α-ketoisocaproic acid or hyperpolarised 13C1-α-ketoisocaproate, preferably TRIS-13C1-α-ketoisocaproate or sodium 13C1-α-ketoisocaproate. The aforementioned compounds can be used as imaging agent (MR active agent) in the imaging medium according to the invention and said imaging medium can be used in the methods of 13C-MR detection according to the invention.
Yet another aspect of the invention is a method for producing hyperpolarised 13C-α-ketoisocaproic acid or hyperpolarised 13C-α-ketoisocaproate, the method comprising preparing a composition comprising 13C-α-ketoisocaproic acid or 13C-α-ketoisocaproate a DNP agent and optionally a paramagnetic metal ion and carrying out dynamic nuclear polarisation on said composition. The preparation of said composition and how to carry out dynamic nuclear on said composition is described in detail earlier in the application.
The invention is illustrated by the following non-limiting examples.
To a micro test tube were added sodium 13C1-α-ketoisocaproate (Cambridge Isotopes, 151.2 mg, 0.987 mmol), TRIS (156.7 mg, 0.994 mmol) and 2 ml methanol. The test tube was sonicated and a white powder (NaCl) precipitated. The supernatant was taken up in a syringe and filtered through a syringe filter (0.45 μm) into another test tube containing 35 ml diethyl ether. The precipitation was centrifuged and the ether was removed by vacuum.
The TRIS-13C1-α-ketoisocaproate obtained in Example 1a (73.2 mg, 0.29 mmol) was dissolved in 50.0 mg of a mixture 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; 44.0 mg, 30.8 μmol) which had been synthesised according to Example 7 of WO-A1-98/39277 (44.0 mg, 30.8 μmol) and the Gd-chelate of 1,3,5-tris-(N-(DO3A-acetamido)-N-methyl-4-amino-2-methyl-phenyl)-[1,3,5]tria-zinane-2,4,6-trione (paramagnetic metal ion; 2.30 mg, 1.1 μmol) which had been synthesised according to Example 4 of WO-A-2007/064226 in glycerol (1543 μl, 1948 mg). The resulting composition was sonicated and gently heated to dissolve all compounds. The composition (80 μl, 10 mM in trityl radical and 1 mM in Gd3+) was transferred with a pipette into a sample cup (probe-retaining container) which was quickly lowered into liquid nitrogen and then 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 (93.89 GHz). Polarisation was followed by solid state 13C-NMR and the solid state polarisation was determined to be 36%.
After 120 minutes of dynamic nuclear polarisation, the obtained frozen polarised composition was dissolved in 6 ml phosphate buffer (pH 7.3, 40 mM). The pH of the final solution containing the dissolved composition was 7.3. The TRIS-13C1-α-ketoisocaproate concentration in said final solution was 50 mM.
Liquid state polarisation was determined by liquid state 13C-NMR at 400 MHz to be 34%.
Sodium 13C1-α-ketoisocaproate (19.5 mg, 0.126 mmol) was dissolved in 50.0 mg of a mixture 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; 44.0 mg, 30.8 μmol) which had been synthesised according to Example 7 of WO-A1-98/39277 (44.0 mg, 30.8 μmol) and the Gd-chelate of 1,3,5-tris-(N-(DO3A-acetamido)-N-methyl-4-amino-2-methyl-phenyl)-[1,3,5]tria-zinane-2,4,6-trione (paramagnetic metal ion; 2.30 mg, 1.1 μmol) which had been synthesised according to Example 4 of WO-A-2007/064226 in glycerol (1543 μl, 1948 mg). To this solution 5 μl of water were added. The resulting composition was sonicated and gently heated to dissolve all compounds. The composition (110 μl, 12.5 mM in trityl radical and 1.3 mM in Gd3+) was transferred with a pipette into a sample cup which was quickly lowered into liquid nitrogen and then 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 (93.89 GHz). Polarisation was followed by solid state 13C-NMR and the solid state polarisation was determined to be approximately 30%.
After 120 minutes of dynamic nuclear polarisation, the obtained frozen polarised composition was dissolved in 6 ml of phosphate buffer (pH 7.3, 40 mM) The pH of the final solution containing the dissolved composition was 7.3. The sodium 13C1-α-ketoisocaproate concentration in said final solution was 50 mM.
Liquid state polarisation was determined by liquid state 13C-NMR at 400 MHz to be 29%.
Sodium 13C1-α-ketoisocaproate (210.0 mg, 1.37 mmol) was dissolved in a cooled solution of 500 μl concentrated H2SO4 in 2 ml water. The mixture was extracted 4 times with 6 ml diethyl ether. The organic phases were combined and dried over MgSO4. The dried solution was filtered (0.45 μm syringe filter) to remove grains of MgSO4 and the diethyl ether was removed under vacuum. 175 mg 13C1-α-ketoisocaproic acid (98%) were obtained.
The trityl radical tris(8-carboxy-2,2,6,6-(tetra(methoxyethyl)-benzo-[1,2-4,5′]-bis-(1,3)-dithiole-4-yl)-methyl sodium salt (1.18 mg, 0.74 mmol) which had been synthesised according to Example 1 of WO-A2-2006/011811 and 1.52 mg of an aqueous solution of the Gd-chelate of 1,3,5-tris-(N-(DO3A-acetamido)-N-methyl-4-amino-2-methyl-phenyl)-[1,3,5]tria-zinane-2,4,6-trione (paramagnetic metal ion, 14.5 μl/g solution) which had been synthesised according to Example 4 of WO-A-2007/064226 were dissolved in 49 μl 13C1-α-ketoisocaproic acid (50.5 mg, 0.19 mmol). The resulting composition was sonicated and gently heated to dissolve all compounds. The composition (42 μl, 14 mM in trityl radical and 1.5 mM in Gd3+) was transferred with a pipette into a sample cup which was quickly lowered into liquid nitrogen and then 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 (93.89 GHz). Polarisation was followed by solid state 13C-NMR and the solid state polarisation was determined to be 27%.
After 90 minutes of dynamic nuclear polarisation, the obtained frozen polarised composition was dissolved in 6 ml of a solution prepared from 5.97 ml phosphate buffer (pH 7.3, 40 mM) and 30 μl aqueous NaOH solution (12 M). The pH of the final solution containing the dissolved composition was 7.3. The sodium 13C1-α-ketoisocaproate concentration in said final solution was 50 mM.
Liquid state polarisation was determined by liquid state 13C-NMR at 400 MHz to be 25%.
175 μl of an imaging medium comprising hyperpolarised sodium 13C1-α-ketoisocaproate 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-α-ketoisocaproate concentration in said imaging medium was about 50 mM. A 12 mm surface coil (tuned for carbon) was positioned over the mouse to cover the liver region and signals of 13C-α-ketoisocaproate and 13C-leucine were detected by 13C-MR spectroscopy which was carried out in a 2.4 T Bruker spectrometer to generate a metabolic profile. A total of 10 13C-spectra were acquired with a repetition time of 5 s and 30 degree RF pulses. The result is shown in
2 ml of an imaging medium comprising hyperpolarised sodium 13C1-α-ketoisocaproate which was prepared as described in Example 2 was injected into a Wistar rat over a time period of 6 s. The sodium 13C1-α-ketoisocaproate concentration in said imaging medium was about 50 mM. A rat coil (tuned for carbon and proton) was positioned on the rat to cover the kidney region and the signals of 13C1-α-ketoisocaproate and 13C-leucine were detected by 13C-MR spectroscopy which was carried out using a 2.4 T Bruker spectrometer to generate a metabolic profile of the kidney region. A 13C-chemical shift image was acquired with the following parameters: FOV 12.6×12.6 mm2×12 mm, matrix size 18×18, 7 degree RF pulse, TR=66 ms. Total acquisition time was 15 seconds and the chemical shift imaging started 18 seconds after the start of the injection of the imaging medium. A high resolution proton image was acquired for referencing. The results are shown in
EL-4 cells were injected into a C57Bl/6 mouse to generate a subcutaneous mouse lymphoma. 175 μl of an imaging medium comprising hyperpolarised sodium 13C1-α-ketoisocaproate which was prepared as described in Example 2 was injected into the mouse over a time period of 6 s. The sodium 13Ci-α-ketoisocaproate concentration in said imaging medium was about 50 mM. A 20 mm surface coil (tuned for carbon) was positioned over the subcutaneous tumour and signals of 13C-α-ketoisocaproate and 13C-leucine were detected by 13C-MR spectroscopy which was carried out using a 2.4 T Bruker spectrometer to generate a metabolic profile of the tumour and the surrounding healthy tissue. A 13C-chemical shift image was acquired with the following parameters: FOV 35×35 mm2×10 mm, matrix size 16×16, 10 degree RF pulse, TR=35 ms. Total acquisition time was 11 seconds (die to triggering on breathing) and the chemical shift imaging started 15 seconds after the start of the injection of the imaging medium. Omniscan™ (GE Healthcare)—enhanced proton imaging was performed to confirm the tumour position and perfusion. The results are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 20076640 | Dec 2007 | NO | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2008/067986 | 12/19/2008 | WO | 00 | 9/28/2010 |
