U.S. patent application number 11/572679 was filed with the patent office on 2008-04-24 for method of tumour imaging.
Invention is credited to Mikkel Thaning, Rene in't Zandt.
Application Number | 20080095713 11/572679 |
Document ID | / |
Family ID | 35058362 |
Filed Date | 2008-04-24 |
United States Patent
Application |
20080095713 |
Kind Code |
A1 |
Thaning; Mikkel ; et
al. |
April 24, 2008 |
Method of Tumour Imaging
Abstract
The invention relates to a method of producing a composition
comprising hyperpolarised .sup.13C-pyruvate, the composition and
its use as an imaging agent for MR imaging. ##STR00001##
Inventors: |
Thaning; Mikkel; (Oslo,
NO) ; Zandt; Rene in't; (Malmo, SE) |
Correspondence
Address: |
GE HEALTHCARE, INC.
IP DEPARTMENT, 101 CARNEGIE CENTER
PRINCETON
NJ
08540-6231
US
|
Family ID: |
35058362 |
Appl. No.: |
11/572679 |
Filed: |
July 28, 2005 |
PCT Filed: |
July 28, 2005 |
PCT NO: |
PCT/NO05/00281 |
371 Date: |
January 25, 2007 |
Current U.S.
Class: |
424/9.3 |
Current CPC
Class: |
C07D 519/00 20130101;
G01N 24/00 20130101; A61K 49/10 20130101; A61K 49/1815
20130101 |
Class at
Publication: |
424/9.3 |
International
Class: |
A61K 49/06 20060101
A61K049/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 30, 2004 |
NO |
200443226 |
Claims
1. A method for the discrimination between healthy and tumour
tissue, said method comprising (a) acquiring direct .sup.13C-MR
images of .sup.13C-pyruvate and its .sup.13C-containing metabolites
alanine, lactate and optionally bicarbonate from a subject
pre-administered with a composition comprising hyperpolarised
.sup.13C-pyruvate, (b) optionally correcting the lactate signal for
the amount of pyruvate and/or alanine to obtain a weighted lactate
over pyruvate and/or lactate over alanine image, wherein tumour
tissue in said .sup.13C-images is indicated by the highest lactate
signal and/or, if the correction in step (b) has been carried out,
by a high weighted lactate over pyruvate and/or lactate over
alanine signal.
2. A method according to claim 1 wherein the hyperpolarised
.sup.13C-pyruvate is obtained by hyperpolarising at least one of
.sup.13C-pyruvic acid and 13C-pyruvate by the DNP method.
3. A method according to claim 1 wherein the composition comprising
.sup.13C-pyruvate further comprises one or more buffers selected
from the group consisting of phosphate buffer
(KH.sub.2PO.sub.4/Na.sub.2HPO.sub.4), ACES, PIPES, imidazole/HCl,
BES, MOPS, HEPES, TES, TRIS, HEPPS and TRICIN.
4. A method according to claim 1 wherein imaging sequences that
make use of multiechoes to code for frequency information are used
for acquiring the direct .sup.13C-images in step a).
5. A method according to claim 1 wherein the direct .sup.13C-images
in step a) are acquired at less than 400 s after the administration
of the composition comprising .sup.13C-pyruvate.
6. A method according to claim 1 further comprising the step of
acquiring a proton image with or without a proton MRI contrast
agent.
7. A method according to claim 1 wherein step b) further comprises
correcting the lactate signal for the amount of bicarbonate to
obtain a weighted lactate over bicarbonate image and, wherein if
correction in step b) has been carried out, tumour tissue in said
13C-images is indicated by the highest lactate signal, by a high
weighted lactate over pyruvate and/or lactate over alanine and/or
lactate over bicarbonate signal.
8. A method according to claim 1 wherein step b) is mandatory.
9. A method according to claim 8 wherein said correction is carried
out by (i) normalizing the lactate and pyruvate and/or alanine
and/or bicarbonate images to the maximum value in each individual
image (ii) multiplying the normalized lactate image by the inverted
pyruvate and/or alanine and/or bicarbonate image; and (iii)
multiplying the results of step (ii) by the original lactate
image.
10. A method according to claim 1 wherein the tumour is a brain
tumour, breast tumour, colon tumour, lung tumour, kidney tumour,
head and neck tumour, muscle tumour, ovarian tumour, gastric
tumour, pancreatic tumour, esophageal tumour or prostate
tumour.
11. A method according to claim 1 for in vivo MR tumour therapy
monitoring and/or tumour staging.
Description
[0001] The invention relates to a method of producing a composition
comprising hyperpolarised .sup.13C-pyruvate, the composition and
its use as an imaging agent for MR imaging.
[0002] Magnetic resonance (MR) imaging (MRI) is a imaging technique
that has become particularly attractive to physicians as it allows
for obtaining images of a patients body or parts thereof in a
non-invasive way and without exposing the patient and the medical
personnel to potentially harmful radiation such as X-ray. Because
of its high quality images, MRI is the favourable imaging technique
of soft tissue and organs and it allows for the discrimination
between normal and diseased tissue, for instance tumours and
lesions.
[0003] MR tumour imaging may be carried out with or without MR
contrast agents. On an MR image taken without contrast agent,
tumours from about 1-2 centimetres in size and larger will show up
fairly clearly. However, contrast-enhanced MRI enables much smaller
tissue changes, i.e. much smaller tumours to be detected which
makes contrast-enhanced MR imaging a powerful tool for early stage
tumour detection and detection of metastases.
[0004] Several types of contrast agents have been used in MR tumour
imaging. Water-soluble paramagnetic metal chelates, for instance
gadolinium chelates like Omniscan.TM. (Amersham Health) 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) if administered into the vasculature. They are
also cleared relatively rapidly from the body. Gadolinium chelates
have been found to be especially useful in increasing the detection
rate of metastases, small tumours, and improving tumour
classification, the latter by allowing the differentiation of vital
tumour tissue (well perfused and/or impaired blood-brain-barrier)
from central necrosis and from surrounding oedema or
macroscopically uninvolved tissue (see for instance C. Claussen et
al., Neuroradiology 1985; 27: 164-171).
[0005] 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 for example as a result of angiogenesis.
[0006] Despite the undisputed excellent properties of the
aforementioned contrast agents their use is not without any risks.
Although paramagnetic metal chelate complexes 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.
[0007] WO-A-99/35508 discloses a method of MR investigation of a
patient using a hyperpolarised solution of a high T.sub.1 agent as
MR imaging agent. The term "hyperpolarisation" means enhancing the
nuclear polarisation of NMR active nuclei present in the high
T.sub.1 agent, i.e. nuclei with non-zero nuclear spin, preferably
.sup.13C- or .sup.15N-nuclei. Upon enhancing the nuclear
polarisation of NMR active nuclei, the population difference
between excited and ground nuclear spin states of these nuclei are
significantly increased and thereby the MR signal intensity is
amplified by a factor of hundred and more. When using a
hyperpolarised .sup.13C- and/or .sup.15N-enriched high T.sub.1
agent, there will be essentially no interference from background
signals as the natural abundance of .sup.13C and/or .sup.15N is
negligible and thus the image contrast will be advantageously high.
A variety of possible high T.sub.1 agents suitable for
hyperpolarisation and subsequent use as MR imaging agents are
disclosed including but not limited to non-endogenous and
endogenous compounds like acetate, pyruvate, oxalate or gluconate,
sugars like glucose or fructose, urea, amides, amino acids like
glutamate, glycine, cysteine or aspartate, nucleotides, vitamins
like ascorbic acid, penicillin derivates and sulfonamides. It is
further stated that intermediates in normal metabolic cycles such
as the citric acid cycle like fumaric acid and pyruvic acid are
preferred imaging agents for the imaging of metabolic activity.
[0008] It has to be stressed that the signal of a hyperpolarised
imaging agent decays due to relaxation and--upon administration to
the patient's body--dilution. Hence the T.sub.1 value of the
imaging agents in biological fluids (e.g. blood) must be
sufficiently long to enable the agent to be distributed to the
target site in the patient's body in a highly hyperpolarised state.
Apart from the imaging agent having a high T.sub.1 value, it is
extremely favourable to achieve a high polarisation level.
[0009] Several hyperpolarising techniques are disclosed in
WO-A-99/35508 one of them is the dynamic nuclear polarisation (DNP)
technique whereby polarisation of the sample is effected by a
paramagnetic compound, the so-called paramagnetic agent or DNP
agent. During the DNP process, energy, normally in the form of
microwave radiation, is provided, which will initially excite the
paramagnetic agent. Upon decay to the ground state, there is a
transfer of polarisation from the unpaired electron of paramagnetic
agent to the NMR active nuclei of the sample. 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 described in WO-A-98/58272 and in
WO-A-01/96895, both of which are included by reference herein.
[0010] The paramagnetic agent plays a decisive role in the DNP
process and its choice has a major impact on the level of
polarisation achieved. A variety of paramagnetic agents--in
WO-A-99/35508 denoted as "OMRI contrast agents"--is known, for
instance oxygen-based, sulfur-based or carbon-based organic free
radicals or magnetic particles referred to 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.
[0011] We have now surprisingly found an improved method for
producing a liquid composition comprising hyperpolarised .sup.13C-
pyruvate which allows for obtaining hyperpolarised .sup.13C-
pyruvate with a remarkably high polarisation level. It has further
been found that such a composition is especially suitable for in
vivo MR tumour imaging.
[0012] Thus, viewed from one aspect, the present invention provides
a method for producing a liquid composition comprising
hyperpolarised .sup.13C-pyruvate said method comprising [0013] a)
forming a liquid mixture comprising a radical of formula (1),
.sup.13C-pyruvic acid and/or .sup.13C-pyruvate and freezing the
mixture;
##STR00002##
[0013] where [0014] M represents hydrogen or one equivalent of a
cation; and [0015] R1 which is the same or different represents a
straight chain or branched hydroxylated and/or alkoxylated
C.sub.1-C.sub.4-hydrocarbon group [0016] b) enhancing the .sup.13C
nuclear polarisation of pyruvic acid and/or pyruvate in the mixture
via DNP; [0017] c) adding a buffer and a base to the frozen mixture
to dissolve it and to convert the .sup.13C-pyruvic acid into a
.sup.13C-pyruvate to obtain a liquid composition or, when only
.sup.13C-pyruvate is used in step a), adding a buffer to the frozen
mixture to dissolve it to obtain a liquid composition; and [0018]
d) optionally removing the radical and/or reaction products thereof
from the liquid composition.
[0019] The terms "hyperpolarised" and "polarised" are used
interchangeably hereinafter and denote a polarisation to a level
over that found at room temperature and 1 T.
[0020] A radical of formula (I) is used in the method of the
invention
##STR00003##
where [0021] M represents hydrogen or one equivalent of a cation;
and [0022] R1 which is the same or different represents a straight
chain or branched hydroxylated and/or alkoxylated
C.sub.1-C.sub.4-hydrocarbon group.
[0023] Hereinafter, the term "radical" is used for the radical of
formula (I).
[0024] 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.
[0025] In a further preferred embodiment, R1 is the same or
different and represents hydroxymethyl or hydroxyethyl. In another
preferred embodiment, R1 is the same or different and represents a
straight chain or branched alkoxylated C.sub.1-C.sub.4-hydrocarbon
group, preferably --CH.sub.2--O--(C.sub.1-C.sub.3-alkyl),
--(CH.sub.2).sub.2--O--CH.sub.3 or
--(C.sub.1--C.sub.3-alkyl)--O--CH.sub.3. In another preferred
embodiment, R1 is the same or different and represents a straight
chain or branched alkoxylated C.sub.1-C.sub.4-hydrocarbon group
carrying a terminal hydroxyl group, preferably
--CH.sub.2--O--C.sub.2H.sub.4OH or --C.sub.2H.sub.4--O--CH.sub.2OH.
In a more preferred embodiment, R1 is the same and represents a
straight chain alkoxylated C.sub.1-C.sub.4-hydrocarbon group,
preferably methoxy, --CH.sub.2--OCH.sub.3,
--CH.sub.2--OC.sub.2H.sub.5 or --CH.sub.2--CH.sub.2--OCH.sub.3,
most preferably --CH.sub.2--CH.sub.2--OCH.sub.3.
[0026] In a most preferred embodiment, M represents hydrogen or
sodium and R1 is the same and represents
--CH.sub.2--CH.sub.2--OCH.sub.3.
[0027] The synthesis of the radicals is known in the art and
disclosed in WO-A-91/12024, WO-A-96/39367, WO 97/09633 and
WO-A-98/39277. Briefly, the radicals may be synthesized by reacting
three molar equivalents of a metallated monomeric aryl compound
with one molar equivalent of a suitably protected carboxylic acid
derivative to form a trimeric intermediate. This intermediate is
metallated and subsequently reacted with e.g. carbon dioxide to
result in a tri-carboxylic trityl carbinol which, in a further
step, is treated with a strong acid to generate a triarylmethyl
cation. This cation is then reduced to form the stable trityl
radical.
[0028] The isotopic enrichment of the .sup.13C-pyruvic acid and/or
.sup.13C-pyruvate 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%..sup.13C-pyruvic
acid and/or .sup.13C-pyruvate may be isotopically enriched at the
C1-position (in the following denoted .sup.13C.sub.1-pyruvic acid
and .sup.13C.sub.1-pyruvate), at the C2-position (in the following
denoted .sup.13C.sub.2-pyruvic acid and .sup.13C.sub.2-pyruvate),
at the C3-position (in the following denoted .sup.13C.sub.3-pyruvic
acid and .sup.13C.sub.3-pyruvate), at the C1- and the C2-position
(in the following denoted .sup.13C.sub.1,2-pyruvic acid and
.sup.13C.sub.1,2-pyruvate), at the C1- and the C3-position (in the
following denoted .sup.13C.sub.1,3-pyruvic acid and
.sup.13C.sub.1,3-pyruvate), at the C2- and the C3-position (in the
following denoted .sup.13C.sub.2,3-pyruvic acid and
.sup.13C.sub.2,3-pyruvate) or at the C1-, C2- and C3-position (in
the following denoted .sup.13C.sub.1,2,3-pyruvic acid and
.sup.13C.sub.1,2,3-pyruvate); the C1-position being the preferred
one.
[0029] Several methods for the synthesis of .sup.13C.sub.1-pyruvic
acid are known in the art. Briefly, Seebach et al., Journal of
Organic Chemistry 40(2), 1975, 231-237 describe a synthetic route
that relies on the protection and activation of a
carbonyl-containing starting material as an S,S-acetal, e.g.
1,3-dithian or 2-methyl-1,3-dithian. The dithian is metallated and
reacted with a methyl-containing compound and/or .sup.13C0.sub.2.
By using the appropriate isotopically enriched .sup.13C-component
as outlined in this reference, it is possible to obtain
.sup.13C.sub.1-pyruvate, .sup.13C.sub.2-pyruvate or
.sup.13C.sub.1,2-pyruvate. The carbonyl function is subsequently
liberated by use of conventional methods described in the
literature. A different synthetic route starts from acetic acid,
which is first converted into acetyl bromide and then reacted with
Cu.sup.13CN. The nitril obtained is converted into pyruvic acid via
the amide (see for instance S. H. Anker et al., J. Biol. Chem. 176
(1948), 1333 or J . E. Thirkettle, Chem Commun. (1997), 1025).
Further, .sup.13C-pyruvic acid may be obtained by protonating
commercially available sodium .sup.13C-pyruvate, e.g. by the method
described in U.S. Pat. No. 6,232,497.
[0030] Whether .sup.13C-pyruvic acid and/or .sup.13C-pyruvate is
used in the method of the invention is mainly dependent on the
radical employed. If the radical is soluble in .sup.13C-pyruvic
acid, then .sup.13C-pyruvic acid is preferably used and a liquid
mixture, preferably a liquid solution is formed by the radical and
.sup.13C-pyruvic acid. If the radical is not soluble in
.sup.13C-pyruvic acid, then .sup.13C-pyruvate and/or
.sup.13C-pyruvic acid and at least one co-solvent are used to form
a liquid mixture, preferably a liquid solution. It has been found
that the success of the polarisation in step b) and thus the level
of polarisation is dependent on the compound to be polarised and
the radical being in intimate contact with each other. Hence the
co-solvent is preferably a co-solvent or co-solvent mixture that
dissolves both, the radical and .sup.13C-pyruvic acid and/or
.sup.13C-pyruvate. For .sup.13C-pyruvate water is preferably used
as a co-solvent.
[0031] Further, it has been found that higher polarisation levels
in step b) are achieved when the mixture upon cooling/freezing
forms a glass rather than a crystallized sample. Again, the
formation of a glass allows a more intimate contact of the radical
and the compound to be polarised. .sup.13C-pyruvic acid is a good
glass former and is therefore preferably used in the method of the
invention, whenever the radical is soluble in .sup.13C-pyruvic
acid. .sup.13C-pyruvate is a salt and a liquid mixture of an
aqueous solution of .sup.13C-pyruvate and a radical will result in
a crystallized sample upon freezing. To prevent this, it is
preferred to add further co-solvents which are good glass formers
like glycerol, propanediol or glycol.
[0032] Hence in one embodiment, .sup.13C-pyruvate is dissolved in
water to obtain an aqueous solution and a radical, glycerol and
optionally a further co-solvent are added to form a liquid mixture
according to step a) of the method of the invention. In a preferred
embodiment, .sup.13C-pyruvic acid, a radical and a co-solvent are
combined to form a liquid mixture according to step a) of the
method of the invention. In a most preferred embodiment,
.sup.13C-pyruvic acid and a radical are combined to form a liquid
mixture according to step a) of the method of the invention.
Intimate mixing of the compounds can be achieved by several means
known in the art, such as stirring, vortexing or sonification.
[0033] The liquid mixture of step a) according to the method of the
invention preferably contains 5 to 100 mM radical, more preferably
10 to 20 mM radical, especially preferably 12 to 18 mM radical and
most preferably 13 to 17 mM radical. It has been found that the
build-up time for polarisation in step b) of the method of the
invention is shorter using higher amounts of radical, however, the
achievable polarisation level is lower. Hence these two effects
have to be balanced against each other.
[0034] The liquid mixture in step a) of the method according to the
invention is frozen before the polarisation of step b) is carried
out. Cooling/freezing of the liquid mixture may be achieved by
methods known in the art, e.g. by freezing the liquid mixture in
liquid nitrogen or by simply placing it in the polarizer, where
liquid helium will freeze the sample.
[0035] In step b) of the method according to the invention, the
.sup.13C nuclear polarisation of .sup.13C-pyruvic acid and/or
.sup.13C-pyruvate is enhanced via DNP. As described previously,
dynamic nuclear polarisation (DNP) is a polarisation method where
polarisation of the compound to be polarised is effected by a DNP
agent, i.e. a paramagnetic compound. With respect to the method of
the invention, polarisation is effected by the radical employed.
During the DNP process, energy, preferably in the form of microwave
radiation, is provided, which will initially excite the radical.
Upon decay to the ground state, there is a transfer of polarisation
from the unpaired electron of the radical to the .sup.13C nuclei of
the .sup.13C-pyruvic acid and/or .sup.13C-pyruvate.
[0036] The DNP technique is for example 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 of the method of the invention,
the DNP process is carried out in liquid helium and a magnetic
field of about 1 T or above. Suitable polarisation units for
carrying out step b) of the method of the invention 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 .sup.13C nuclei to take place.
The sample bore is preferably sealable and can be evacuated to low
pressures, e.g. pressures in the order of 1 mbar or less. A sample
(i.e. the frozen mixture of step a) of the method of the invention)
introducing means such as a removable sample-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 sample introducing means is preferably
sealable at its upper end in any suitable way to retain the partial
vacuum in the bore. A sample-retaining container, such as a
sample-retaining cup, can be removably fitted inside the lower end
of the sample introducing means. The sample-retaining container is
preferably made of a light-weight material with a low specific heat
capacity and good cryogenic properties such, e.g. KelF
(polychlorotrifluoroethylene) or PEEK (polyetheretherketone). The
sample container may hold one or more samples to be polarised.
[0037] The sample is inserted into the sample-retaining container,
submerged in the liquid helium and irradiated with microwaves,
preferably at a frequency about 94 GHz at 200 mW. The level of
polarisation may be monitored by acquiring solid state .sup.13C-NMR
signals of the sample during microwave irradiation, thus the use of
a polarising unit containing means to acquire solid state
.sup.13C-NMR spectra in step b) is preferred. Generally, a
saturation curve is obtained in a graph showing .sup.13C-NMR signal
vs. time. Hence it is possible to determine when the optimal
polarisation level is reached.
[0038] In step c) of the method of the invention, the frozen
polarised mixture is dissolved in a buffer, preferably a
physiologically tolerable buffer, to obtain a liquid composition.
The term "buffer" in the context of this application denotes one or
more buffers, i.e. also mixtures of buffers.
[0039] 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
(KH.sub.2PO.sub.4/Na.sub.2HPO.sub.4), ACES, PIPES, imidazole/HCl,
BES, MOPS, HEPES, TES, TRIS, HEPPS or TRICIN. More preferred
buffers are phosphate buffer and TRIS, most preferred is TRIS. In
another embodiment, more than one of the aforementioned preferred
buffers, i.e. a mixture of buffers, is used.
[0040] When .sup.13C-pyruvic acid was used in the compound to be
polarised, step c) also encompasses the conversion of
.sup.13C-pyruvic acid to .sup.13C-pyruvate. To achieve this,
.sup.13C-pyruvic acid is reacted with a base. In one embodiment,
.sup.13C-pyruvic acid is reacted with a base to convert it to
.sup.13C-pyruvate and subsequently a buffer is added. In another
preferred embodiment the buffer and the base are combined in one
solution and this solution is added to .sup.13C-pyruvic acid,
dissolving it and converting it into .sup.13C-pyruvate at the same
time. In a preferred embodiment, the base is an aqueous solution of
NaOH, Na.sub.2CO.sub.3 or NaHCO.sub.3, most preferred the base is
NaOH. In a particularly preferred embodiment, a solution of TRIS
buffer containing NaOH is used to dissolve .sup.13C-pyruvic acid
and convert it into the sodium salt of .sup.13C-pyruvate.
[0041] In another preferred embodiment, the buffer or--where
applicable--the combined buffer/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. It has
been found that free paramagnetic ions may cause shortening of the
T.sub.1 of the hyperpolarised compound, which is preferably
avoided.
[0042] The dissolution may be carried out by preferably using the
methods and/or devices disclosed in WO-A-02/37132. Briefly, a
dissolution unit is used which is either physically separated from
the polariser or is a part of an apparatus that contain the
polariser and the dissolution unit. In a preferred embodiment, step
c) is carried out at an elevated magnetic field 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.
[0043] In the optional step d) of the method of the invention, the
radical and/or reaction products thereof are removed from the
liquid composition obtained in step c). The radical and/or reaction
products may be removed partially, substantially or ideally
completely, the complete removal is preferred when the liquid
composition is used in a human patient. Reaction products of the
radical might be esters which may be formed upon reaction of
pyruvic acid with radicals of formula (I) comprising hydroxy
groups. In a preferred embodiment of the method of the invention,
step d) is mandatory. Methods usable to remove the radical and/or
reaction products thereof are known in the art. Generally, the
methods applicable depend on the nature of the radical and/or its
reaction products. Upon dissolution of the frozen mixture in step
c), the radical might precipitate and it may easily be separated
from the liquid composition by filtration. If no precipitation
occurs, the radical may be removed by chromatographic separation
techniques, e.g. liquid phase chromatography like reversed phase or
ion exchange chromatography or by extraction.
[0044] As radicals of formula (I) have a characteristic UV/visible
absorption spectrum, it is possible to use UV/visible absorption
measurement as a method to check for its existence in the liquid
composition after its removal. In order to obtain quantitative
results, i.e. the concentration of the radical present in the
liquid composition, the optical spectrometer can be calibrated such
that absorption at a specific wavelength form a sample of the
liquid composition yields the corresponding radical concentration
in the sample. Removal of the radical and/or reaction products
thereof is especially preferred if the liquid composition is used
as an imaging agent for in vivo MR imaging of a human or non-human
animal body.
[0045] From a further aspect, the present invention provides a
composition comprising hyperpolarised .sup.13C-pyruvate, preferably
hyperpolarised sodium .sup.13C-pyruvate and a buffer selected from
the group consisting of phosphate buffer and TRIS.
[0046] In a preferred embodiment, the hyperpolarised
.sup.13C-pyruvate has a polarisation level of at least 10%, more
preferably at least 15%, particularly preferably at least 20% and
most preferably more than 20%.
[0047] It has been found that such compositions are excellent
imaging agents for in vivo MR imaging, especially for in vivo MR
studying of metabolic processes and for in vivo MR tumour imaging
and a composition comprising hyperpolarised .sup.13C-pyruvate and a
buffer selected from the group consisting of phosphate buffer and
TRIS for use as a MR imaging agent forms another aspect of the
invention.
[0048] The composition of the invention is preferably produced by
the method as claimed in claim 1, more preferably by using
.sup.13C-pyruvate in step a) of the method of claim 1 and a radical
of formula (I) where M is hydrogen or a physiologically tolerable
cation and R1 is the same and represents a straight chain or
branched alkoxylated C.sub.1-C.sub.4-hydrocarbon group, preferably
methoxy, --CH.sub.2--OCH.sub.3, --CH.sub.2--OC.sub.2H.sub.5 or
--CH.sub.2--CH.sub.2--OCH.sub.3 and step d) is mandatory. In a
particularly preferred embodiment, the composition of the invention
is produced by the method as claimed in claim 1 wherein in step a)
.sup.13C-pyruvate and a radical of formula (I) where M represents
hydrogen and R1 is the same and represents
--CH.sub.2--CH.sub.2--OCH.sub.3 are used and step d) is
mandatory.
[0049] Another aspect of the invention is the use of a composition
comprising hyperpolarised .sup.13C-pyruvate, preferably
hyperpolarised sodium .sup.13C-pyruvate and a buffer selected from
the group consisting of phosphate buffer and TRIS for the
manufacture of a MR imaging agent for in vivo studying of metabolic
processes in the human or non-human animal body.
[0050] Yet another aspect of the invention is the use of a
composition comprising hyperpolarised .sup.13C-pyruvate, preferably
hyperpolarised sodium .sup.13C-pyruvate and a buffer selected from
the group consisting of phosphate buffer and TRIS for the
manufacture of a MR imaging agent for in vivo tumour imaging in the
human or non-human animal body, preferably for in vivo tumour
diagnosis and/or tumour staging and/or tumour therapy monitoring,
more preferably for in vivo prostate tumour diagnosis and/or
prostate tumour staging and/or prostate tumour therapy
monitoring.
[0051] The composition according to the invention may be used as a
"conventional" MR imaging agent, i.e. providing contrast
enhancement for anatomical imaging. A further advantage of the
composition according to the invention is, that pyruvate is an
endogenous compound which is very well tolerated by the human body,
even in high concentrations. As a precursor in the citric acid
cycle, pyruvate plays an important metabolic role in the human
body. Pyruvate is converted into different compounds: its
transamination results in alanine, via oxidative decarboxylation,
pyruvate is converted into acetyl-CoA and bicarbonate, the
reduction of pyruvate results in lactate and its carboxylation in
oxaloacetate.
[0052] It has now been found that the conversion of hyperpolarised
.sup.13C-pyruvate to hyperpolarised .sup.13C-lactate,
hyperpolarised .sup.13C-bicarbonate (in the case of
.sup.13C.sub.1-pyruvate, .sup.13C.sub.1,2-pyruvate or
.sup.13C.sub.1,2,3-pyruvate only) and hyperpolarised
.sup.13C-alanine can be used for in vivo MR studying of metabolic
processes in the human body. This is surprising as one has to bear
in mind that the T.sub.1 of hyperpolarised compounds decays due to
relaxation and dilution. .sup.13C-pyruvate has a T.sub.1 relaxation
in human full blood at 37.degree. C. of about 42 s, however, the
conversion of hyperpolarised .sup.13C-pyruvate to hyperpolarised
.sup.13C-lactate, hyperpolarised .sup.13C-bicarbonate and
hyperpolarised .sup.13C-alanine has been found to be fast enough to
allow signal detection from the .sup.13C-pyruvate parent compound
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
.sup.13C-lactate, hyperpolarised .sup.13C-bicarbonate and
hyperpolarised .sup.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
.sup.13C-pyruvate to hyperpolarised .sup.13C-lactate,
hyperpolarised .sup.13C-bicarbonate and hyperpolarised
.sup.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.
[0053] It has been found that 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 and thus allows for
the discrimination between healthy tissue and tumour tissue. This
makes the composition according to the invention an excellent agent
for in vivo MR tumour imaging.
[0054] Generally, in order to carry out MR imaging with the
composition according to the invention, the subject under
examination, e.g. patient or an animal, is positioned in the MR
magnet. Dedicated .sup.13C-MR RF-coils are positioned to cover the
area of interest.
[0055] The composition according to the invention, i.e. the
composition comprising hyperpolarised .sup.13C-pyruvate and a
buffer selected from the group consisting of phosphate buffer and
TRIS is administered parenterally, preferably intravenously,
intraarterially or directly into the region or organ of interest.
Dosage and concentration of the composition according to the
invention will depend upon a range of factors such as toxicity, the
organ targeting ability and the administration route. Generally the
composition is administered in a concentration of up to 1 mmol
pyruvate per kg bodyweight, preferably 0.01 to 0.5 mmol/kg, more
preferably 0.1 to 0.3 mmol/kg. The administration rate is
preferably less than 10 ml/s, more preferably less than 6 ml/min
and most preferable of from 5 ml/s to 0.1 ml/s. 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 after the administration and most preferably
30 to 40 s after the administration, an MR imaging sequence is
applied that encodes the volume of interest in a combined frequency
and spatial selective way. This will result in metabolic images of
.sup.13C-lactate, .sup.13C-alanine and .sup.13C-pyruvate and more
preferably in metabolic images of .sup.13C-lactate,
.sup.13C-alanine, .sup.13C-bicarbonate and .sup.13C-pyruvate.
Within the same period of time, a proton image with or without a
proton MRI contrast agent may be acquired to obtain anatomical
and/or perfusion information.
[0056] The encoding of the volume of interest can be achieved by
using so-called spectroscopic imaging sequences as described in for
instance T. R. Brown et al., Proc. Natl. Acad. Sci. USA 79,
3523-3526 (1982); A. A. Maudsley, et al., J. Magn. Res 51,147-152
(1983). Spectroscopic image data contain a number of volume
elements in which each element contains a full .sup.13C-MR
spectrum. .sup.13C-pyruvate and its .sup.13C-metabolites all have
their unique position in a .sup.13C-MR spectrum and their resonance
frequency can be used to identify them. The integral of the peak at
its resonance frequency is directly linked to the amount of
.sup.13C-pyruvate and its .sup.13C-metabolites, respectively. When
the amount of .sup.13C-pyruvate and each .sup.13C-metabolite is
estimated using time domain fitting routines as described for
instance in L. Vanhamme et al., J Magn Reson 129, 35-43 (1997),
images can be generated for .sup.13C-pyruvate and each
.sup.13C-metabolite in which a colour coding or grey coding is
representative for the amount of .sup.13C-pyruvate and each
.sup.13C-metabolite measured.
[0057] Although spectroscopic imaging methods have proven their
value in producing metabolic images using all kind of MR nuclei
e.g. .sup.1H, .sup.31p, .sup.23Na, the amount of repetitions needed
to fully encode the spectroscopic image makes this approach less
suitable for hyperpolarized .sup.13C. Care has to be taken to
ensure hyperpolarized .sup.13C- signal is available during the
whole MR data acquisition. At the expense of a reduced signal to
noise, this can be achieved by reducing the RF-pulse angle that is
applied in every phase encoding step. Higher matrix sizes require
more phase encoding steps and longer scan times.
[0058] Imaging methods based on the pioneering work by P. C.
Lauterbur (Nature, 242, 190-191, (1973) and P. Mansfield (J. Phys.
C. 6, L422-L426 (1973)), implying applying a readout gradient
during the data acquisition, will allow for higher signal to noise
images or the equivalent, higher spatial resolution images.
However, these imaging methods in their basic form will not be able
to produce separate images for .sup.13C-pyruvate and its
.sup.13C-metabolites but an image containing the signals of
.sup.13C-pyruvate and all of its .sup.13C-metabolites, i.e. the
identification of specific metabolites is not possible.
[0059] In a preferred embodiment, imaging sequences are used that
will make use of multi- echoes to code for the frequency
information. Sequences that can produce separate water and fat
.sup.1H-images are for example described in G. Glover, J Magn Reson
Imaging 1991;1:521-530 and S. B. Reeder et al., MRM 51 35-45
(2004). Since the metabolites to be detected and as such their MR
frequencies are known, the approach discussed in the references
above can be applied to directly image pyruvate, alanine and
lactate and preferably pyruvate, alanine, lactate and bicarbonate
and makes more efficient use of the hyperpolarised .sup.13C-MR
signal, giving a better signal quality compared to the classical
spectroscopic imaging technique, a higher spatial resolution and
faster acquisition times.
[0060] 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. Surprisingly, it is possible to make this change in
metabolism visible using hyperpolarised .sup.13C-pyruvate within
the short MR imaging time window available, i.e. using the high
.sup.13C-lactate signal in the tumour area to discriminate the
tumour from healthy tissue. As the perfusion is heterogeneous in
tumour tissue, it is preferred to correct the 13C-lactate signal
for the amount of pyruvate (.sup.13C-pyruvate signal) available in
the same region. This will allow for emphasising regions in the
tissue with a relative high lactate signal with respect to the
pyruvate signal and thus improve the discrimination between tumour
tissue and healthy tissue.
[0061] To correct for the pyruvate signal, both lactate and
pyruvate images are normalized to the maximum value in each
individual image. Second, the normalized lactate image is
multiplied by the inverted pyruvate image, e.g. the maximum
pyruvate signal in the image minus the pyruvate level for every
pixel. As a last step, the intermediate result gained in the
operation above is multiplied by the original lactate image.
[0062] To emphasise regions with altered metabolism, the high
.sup.13C-lactate signal in connection with a reduced
.sup.13C-alanine signal can be used in a similar operation as
described in the paragraph above. Surprisingly, the identification
of the tumour area, i.e. the discrimination between tumour tissue
and healthy tissue is improved by this correction as well. To
correct for the alanine signal, both lactate and alanine images are
normalized to the maximum value in each individual image. Second,
the normalized lactate image is multiplied by the inverted alanine
image, e.g. the maximum alanine signal in the image minus the
alanine level for every pixel. As a last step, the intermediate
result gained in the operation above is multiplied by the original
lactate image. In a similar manner, the .sup.13C-bicarbonate signal
may be included in the analysis as well. Further a proton image
acquired with our without a proton MRI contrast agent may be
included in the analysis to obtain anatomical and/or perfusion
information.
[0063] In another preferred embodiment, the composition according
to the invention is administered repeatedly, thus allowing dynamic
studies. This is a further advantage of the composition in
comparison to other MR imaging agents which, due to their
relatively long circulation in the patient's body, do not allow
such dynamic studies.
[0064] The composition according to the invention is further useful
as an imaging agent for in vivo MR tumour staging. The same
metabolic images and/or metabolic ratio images as described in the
preceding paragraphs may be used for this purpose with appropriate
cut off categories defined dependent on tumour size and metabolic
activity.
[0065] Further, the composition according to the invention is
useful as an imaging agent for in vivo MR tumour therapy
monitoring, e.g. by monitoring direct changes in metabolism pattern
of tumours upon treatment with therapeutic antitumour agents and/or
radiation treatment or in connection with any type of
interventional techniques with or without any kind of ablation,
i.e. chemical ablation combined with radio frequencies, microwaves
or ultrasound.
[0066] Tumour MR imaging can be influenced and improved by
preparing the patient or the animal in a way that will perturb the
protein metabolism, lipid metabolism or energy metabolism in
general. Ways to achieve this are known in the art, e.g. by abrosia
(for instance over night), glucose infusion and the like.
[0067] In a preferred embodiment, the composition according to the
invention is useful as an imaging agent for in vivo MR tumour
imaging, tumour therapy monitoring and tumour staging of brain
tumours, breast tumours, colon/colo-rectal tumours, lung tumours,
kidney tumours, head and neck tumours, muscle tumours, gastric
tumours, esophageal tumours, ovarian tumours, pancreas tumours and
prostate tumours. It has further been found that the composition
according to the invention is especially useful as an imaging agent
for in vivo MR prostate tumour imaging, i.e. prostate tumour
diagnosis and/or prostate tumour staging and/or prostate tumour
therapy monitoring.
[0068] When a man presents to the doctor with symptoms of urinary
pain or discomfort, prostate cancer is suspected. If the man is
over 50 years, a Prostate Specific Antigen (PSA) test is performed.
Prostate cancer is suspected on the basis of an elevated PSA and/or
abnormal Digital Rectal Examination (DRE). If the PSA test is
positive, the patient is sent to a specialist (an urologist) for
diagnosis using ultrasound guided biopsy. Of the two million biopsy
procedures per year performed in the US and Europe, 5 out of 6 and
2 out of 3 are negative, respectively. When detected at an early
stage, the five-year survival rate for these patients is 100%. As
prostate cancer is the most common cancer and the second leading
cause of cancer death in men, there is a strong medical demand for
a method for the diagnosis of prostate tumours which is capable of
detecting prostate tumours at an early stage and which could help
to reduce the number of biopsy procedures.
[0069] The .sup.13C-imaging of the prostate requires a
transmit-receive volume .sup.13C-RF-coil, preferably, a transmit
volume .sup.13C-RF-coil in combination with a MR receive only
endorectal RF-coil is used and more preferably, a transmit-receive
phased array volume .sup.13C-RF-coil in combination with a MR
receive only endorectal .sup.13C-RF-coil is used. Especially
preferred are coils that make the acquisition of a .sup.1H-prostate
image possible after the .sup.13C-imaging.
[0070] Another aspect of the invention is a composition comprising
.sup.13C-pyruvic acid and/or .sup.13C-pyruvate and the radical of
formula (I).
[0071] In a preferred embodiment, said composition comprises a
radical of formula (I) where M represents hydrogen or one
equivalent of a physiologically tolerable cation. 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.
[0072] In a further preferred embodiment, said composition
comprises a radical of formula (I) where R1 is the same or
different and represents hydroxymethyl or hydroxyethyl. In another
preferred embodiment, R1 is the same or different and represents a
straight chain or branched alkoxylated C.sub.1-C.sub.4-hydrocarbon
group, preferably --CH.sub.2--O--(C.sub.1-C.sub.3-alkyl),
--(CH.sub.2).sub.2--O--CH.sub.3 or
--(C.sub.1-C.sub.3-alkyl)--O--CH.sub.3. In another preferred
embodiment, R1 is the same or different and represents a straight
chain or branched alkoxylated C.sub.1-C.sub.4-hydrocarbon group
carrying a terminal hydroxyl group, preferably
--CH.sub.2--O--C.sub.2H.sub.4OH or --C.sub.2H.sub.4--O--CH.sub.2OH.
In a more preferred embodiment, R1 is the same and represents a
straight chain alkoxylated C.sub.1-C.sub.4-hydrocarbon group,
preferably methoxy, --CH.sub.2--OCH.sub.3,
--CH.sub.2--OC.sub.2H.sub.5 or --CH.sub.2--CH.sub.2--OCH.sub.3,
most preferably --CH.sub.2--CH.sub.2--OCH.sub.3.
[0073] In a particularly preferred embodiment, said composition
comprises a radical of formula (I) where M represents hydrogen or
sodium and R1 is the same and represents
--CH.sub.2--CH.sub.2--OCH.sub.3.
[0074] In a further preferred embodiment, said composition
comprises .sup.3C-pyruvic acid and/or .sup.13C-pyruvate with an
isotopic enrichment of 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%.
.sup.13C-pyruvic acid and/or .sup.13C-pyruvate may be isotopically
enriched at the C1-position, at the C2-position, at the
C3-position, at the C1- and C2-position, at the C1- and
C3-position, at the C2- and C3-position or at the C1-, the C2- and
the C3-position, with the C1-position being the preferred one.
[0075] In a particularly preferred embodiment, said composition
comprises .sup.13C-pyruvic acid and the radical of formula (I)
where M represents hydrogen or sodium and R1 is the same and
represents --CH.sub.2--CH.sub.2--OCH.sub.3, most preferably said
composition contains .sup.13C-pyruvic acid and the radical of
formula (I) where M represents hydrogen or sodium and R1 is the
same and represents --CH.sub.2--CH.sub.2--OCH.sub.3.
[0076] The compositions according to the invention comprising
.sup.13C-pyruvic acid and/or .sup.13C-pyruvate and the radical of
formula (I) are particularly useful for the production of
hyperpolarised .sup.13C-pyruvate, for instance for the production
of hyperpolarised .sup.13C-pyruvate according to the method of the
invention. Hence another aspect of the invention is the use of a
composition comprising .sup.13C-pyruvic acid and/or
.sup.13C-pyruvate and the radical of formula (I) for the production
of hyperpolarised .sup.13C-pyruvate.
[0077] The radicals of formula (I) where M represents hydrogen or
sodium and R1 is the same and represents
--CH.sub.2--CH.sub.2--OCH.sub.3 were found to be particularly
favourable for use in the method according to the invention due to
the following properties: they are soluble in .sup.13C-pyruvic acid
and stable when dissolved therein. They further show high
polarisation efficiency in step b) of the method according to the
invention and are stable during the dissolution step c), also when
a base is used in this step. They can easily be removed in step d)
of the method of the invention by for instance filtration using a
hydrophobic filter material.
[0078] Those radicals are new, hence another aspect of the
invention are radicals of formula (I) where M represents hydrogen
or sodium and R1 is the same and represents
--CH.sub.2--CH.sub.2--OCH.sub.3.
[0079] The radicals of the invention may be synthesized as
described in Example 1. Briefly, the radicals may be synthesized by
reacting three molar equivalents of a metallated monomeric aryl
compound with one molar equivalent of a suitably protected
carboxylic acid derivative to form a trimeric intermediate. This
intermediate is metallated and subsequently reacted with e.g.
carbon dioxide to result in a tri-carboxylic trityl carbinol which,
in a further step, is treated with a strong acid to generate a
triarylmethyl cation. This cation is then reduced to form the
stable trityl radical.
[0080] Yet a further aspect of the invention is the use of the
radicals according to the invention as a paramagnetic agent for the
hyperpolarisation of compounds in a DNP process.
EXAMPLES
Example 1
Synthesis of
Tris(8-carboxy-2,2,6,6-(tetra(methoxyethyl)benzo-[1,2-4,5']bis-(1,3)dithi-
ole-4-yl)methyl Sodium Salt
[0081] 10 g (70 mmol)
Tris(8-carboxy-2,2,6,6-(tetra(hydroxyethyl)benzo-[1,2-4,5']-bis-(1,3)-dit-
hiole-4-yl)methyl sodium salt which had been synthesized according
to Example 7 of WO-A1-98/39277 were suspended in 280 ml
dimethylacetamide under an argon atmosphere. Sodium hydride (2.75
g) followed by methyl iodide (5.2 ml) was added and the reaction
which is slightly exothermic was allowed to proceed for 1 hour in a
34.degree. C. water bath for 60 min. The addition of sodium hydride
and methyl iodide was repeated twice with the same amounts of each
of the compounds and after the final addition, the mixture was
stirred at room temperature for 68 hours and then poured into 500
ml water. The pH was adjusted to pH>13 using 40 ml of 1 M NaOH
(aq) and the mixture was stirred at ambient temperature for 15
hours to hydrolyse the formed methyl esters. The mixture was then
acidified using 50 ml 2 M HCl (aq) to a pH of about 2 and 3 times
extracted the ethyl acetate (500 ml and 2.times.200 ml). The
combined organic phase was dried over Na.sub.2SO.sub.4 and then
evaporated to dryness. The crude product (24 g) was purified by
preparative HPLC using acetonitrile/water as eluents. The collected
fractions were evaporated to remove acetonitrile. The remaining
water phase was extracted with ethyl acetate and the organic phase
was dried over Na.sub.2SO.sub.4 and then evaporated to dryness.
Water (200 ml) was added to the residue and the pH was carefully
adjusted with 0.1 M NaOH (aq) to 7, the residue slowly dissolving
during this process. After neutralization, the aqueous solution was
freeze dried.
Example 2
Production of Hyperpolarised .sup.13C-Pyruvate using
.sup.13C-Pyruvic Acid and the Radical of Example 1
[0082] A 20 mM solution was prepared by dissolving 5.0 mg of the
radical of Example 1 in .sup.13C.sub.1-pyruvic acid (164 .mu.l).
The sample was mixed to homogeneity and an aliquot of the solution
(41 mg) was placed in a sample cup and inserted in the DNP
polariser.
[0083] The sample was polarised under DNP conditions at 1.2 K in a
3.35 T magnetic field under irradiation with microwave (93.950
GHz). After 2 hours the polarisation was stopped and the sample was
dissolved using a dissolution device according to WO-A-02/37132 in
an aqueous solution of sodium hydroxide and
tris(hydroxymethyl)-aminomethane (TRIS) to provide a neutral
solution of hyperpolarized sodium.sup.13C.sub.1-pyruvate. The
dissolved sample was rapidly analysed with .sup.13C-NMR to assess
the polarisation and a 19.0 % .sup.13C polarisation was
obtained.
Example 3
Production of Hyperpolarised .sup.13C-Pyruvate using
.sup.13C-Pyruvic Acid and the Radical of Example 1
[0084] A 15 mM solution was prepared by dissolving the radical of
Example 1 (209.1 mg) in a mixture of .sup.13C.sub.1-pyruvic acid
(553 mg) and unlabelled pyruvic acid (10.505 g). The sample was
mixed to homogeneity and an aliquot of the solution (2.015 g) was
placed in a sample cup and inserted in the DNP polariser.
[0085] The sample was polarised under DNP conditions at 1.2 K in a
3.35 T magnetic field under irradiation with microwave (93.950
GHz). After 4 hours the polarisation was stopped and the sample was
dissolved using a dissolution device according to WO-A-02/37132 in
an aqueous solution of sodium hydroxide and
tris(hydroxymethyl)aminomethane (TRIS) to provide a neutral
solution of hyperpolarized sodium .sup.13C.sub.1-pyruvate with a
total pyruvate concentration of 0.5 M in 100 mM TRIS buffer. In
series with the dissolution device a chromatographic column was
connected. The column consists of a cartridge (D=38 mm; h=10 mm)
containing hydrophobic packing material (Bondesil-C18, 40UM Part
#:12213012) supplied by Varian. The dissolved sample was forced
through the column which selectively adsorbed the radical. The
filtered solution was rapidly analysed with .sup.13C-NMR to assess
the polarisation, 16.5% .sup.13C polarisation was obtained. The
residual radical concentration was subsequently analysed with a UV
spectrophotometer at 469 nm and was determined to be below the
detection limit of 0.1 .mu.M.
Example 4
Production of Hyperpolarised .sup.13C-Pyruvate using
.sup.13C-Pyruvic Acid and
Tris(8-carboxy-2,2,6,6-tetra(hydroxyethoxy)methyl-benzo
[1,2-d:4,5-d']bis(1,3)dithiole-4-yl)methyl Sodium Salt
[0086]
Tris(8-carboxy-2,2,6,6-tetra(hydroxyethoxy)methyl-benzo[1,2-d:4,5-d-
']-bis-(1,3)-dithiole-4-yl)methyl sodium salt was synthesised as
described in Example 29 in WO-A-97/09633.
[0087] A 20 mM solution was prepared by dissolving
Tris(8-carboxy-2,2,6,6-tetra(hydroxyethoxy)methyl-benzo
[1,2-d:4,5-d']-bis-(1,3)-dithiole-4-yl)methyl sodium salt in
.sup.13C.sub.1-pyruvic acid (83.1 mg). The sample was mixed to
homogeneity, placed in a sample cup and inserted in the DNP
polariser. The sample was polarised under DNP conditions at 1.2 K
in a 3.35 T magnetic field under irradiation with microwave (93.950
GHz). The .sup.13C-NMR signal from the sample was acquired using a
Varian Inova-200 NMR spectrometer. The DNP enhancement was
calculated from a measurement of the thermal equilibrium
.sup.13C-NMR signal and the enhanced NMR signal. 16% .sup.13C
polarisation was obtained.
Example 5
Tumour Imaging Using Hyperpolarised .sup.13C-Pyruvate as Imaging
Agent
[0088] 5.1 Tumour animal model and tumour preparation
[0089] R3230AC is a rat mammary adenocarcinoma that can be
maintained in female Fischer 344 rats. To establish the animal
tumour model, a frozen vial of R32030 cells containing RPMI 1640,
10% FBS and 10% DMSO was rapidly thawed in 37.degree. C.
Thereafter, the cell solution was transferred to FBS and increasing
volumes of RPMI 1640 were added. Finally, the cell suspension was
transferred to a 25 cm.sup.2 growth flask and put into an incubator
at 37.degree. C., 5% CO.sub.2. Growth media were changed every
other day. At the day of rat infection, removal of cells was
carried out either by mechanical force or by means of trypsin.
Cells were washed using phosphate buffer lacking calcium and
magnesium. Trypsin (0.05% trypsin in 0.02% EDTA) was added for 2-5
min. Then, 5 ml FBS was added and the cells were transferred into a
beaker containing RPMI 1640 with FCS and antibiotics (100 IU/ml
penicillin, 100 IU/ml streptomycin and 2.5 .mu.g/ml amphotericin
B). The cell solution was centrifugated and the cell pellet was
resuspended in 20 ml RPMI with FBS and antibiotics, centrifugation
and resuspension was repeated. The cells were then aliquoted to
vials containing 4.times.10.sup.6 cells/ml RPMI 1640. To obtain
donor tumours, female Fischer 344 rats (Charles River, 180-200 g)
were anaesthetised and 0.3 ml of the cell suspension was
subcutaneously injected in the inguinal region on both sides. 15
and 22 days later, pieces of tumour were prepared as described in
F. A. Burgener et al., Invest Radiol 22/6 (1987), 472-478; S. Saini
et al., J. Magn. Reson. 129/1 (1997), 35-43). Two incisions were
made on the ventral abdomen of recipient female Fischer rats. A
tumour piece was inserted into each pocket and the incisions were
closed. Rats were brought to imaging 12-14 days after tumour
engrafting.
[0090] 5.2 Rat Preparation and Proton MR Imaging
[0091] Weighed rats were anaesthetised using isoflurane (2-3%) and
kept on a heated table to ensure a body temperature of about
37.degree. C. A catheter was introduced into the tail vein and into
the arteria carotis communis sinistra. The rats were transported to
the MR machine and placed on a home-built pad that was heated to
approx. 37.degree. C. by means of circulating FC-104 Fluorinert.
This liquid will not give rise to background signals in .sup.1H-
and .sup.13C-MR imaging. Anaesthesia was continued by means of 1-2%
isoflurane delivered via a long tube to an open-breathing system at
a rate of 0.4 L/min. The arterial catheter was connected via a
T-tube to a pressure recorder and a pump delivering saline (rate
0.15 L/min) to prevent catheter clotting. Rats were positioned in a
rat MR coil (Rapid Biomedical, Germany) and imaging using a
standard proton MR imaging sequence to get anatomical information
and to determine the location of the tumour.
[0092] 5.3 .sup.13C-MR Imaging
[0093] Based on the proton frequency found by the MR system the MR
frequency for .sup.13C.sub.1-alanine was calculated according to
the following equation:
[0094] Frequency .sup.13C.sub.1-alanine=0.25144.times.[(system
frequency proton.times.1.00021)-0.000397708]
[0095] The frequency calculated positioned the MR signal arising
from .sup.13C.sub.1-alanine on resonance with
.sup.13C.sub.1-lactate on the left and .sup.13C.sub.1-pyruvate
resonating on the right of .sup.13C.sub.1-alanine. An unlocalised
MR spectroscopy sequence was run to ensure that the 13C-MR coil and
the system MR frequency had been set up correctly. The
.sup.13C-image location was positioned to cover the tumour (slice
thickness 10 mm, in plane pixel size 5.times.5 mm.sup.2). In the
reconstruction phase, the image data was zero-filled to result in
2.5.times.2.5.times.10 mm.sup.3 resolution. .sup.13C.sub.1-pyruvate
in TRIS buffer (90 mM) was injected in a dose of 10 ml/kg during a
period of 12 s with a minimum volume of 2 ml into the tail vein and
30 s after the start of the injection (i.e. 18 s after finishing
the injection), the chemical shift .sup.13C-MR sequence was
started.
[0096] 5.4 Analysis of the MR Imaging Data
[0097] MR imaging resulted in a matrix containing 16.times.16
elements in which each element or voxel/pixel contains a
.sup.13C-MR spectrum. In the reconstruction phase, the matrix was
zero-filled to 32.times.32, a mathematical operation that helps to
improve the spatial resolution. The dataset to be analysed
contained 1024 spectra as was exported to Dicom.RTM. format (DICOM
is the registered trademark of the National Electrical
Manufacturers Association for its standards publications relating
to digital communications of medical information) for further
analysis. About half of these spectra did not contain MR signals as
the position of these voxels was outside the animal. A location
within the animal revealed voxels with high pyruvate signals and
negligible lactate and alanine signal (blood pool) while other
voxels showed pyruvate, alanine and lactate in about equal
intensity.
[0098] The amplitudes for pyruvate, alanine and lactate were
estimated using time domain fitting procedures which included the
following: the zero order phase is constant over the dataset, the
first order phase is 1.4 ms, the line width or damping in the time
domain is allowed to vary between 0.5 and 3 times the average line
width of the whole dataset for each metabolite independently and
the frequency is allowed to vary with 20 Hz in both directions with
respect to the average frequency found over the whole dataset for
the highest peak, which has to be identified by the user.
[0099] The amplitudes for lactate, alanine and pyruvate were
reordered in a matrix and resampled to match the resolution of the
proton anatomical MR image. The .sup.13C-MR images were projected
on the anatomical images using an automated procedure to obtain an
operator-independent result. The results were displayed in image
sets containing the anatomical proton image of the tumour in the
rat, the metabolic .sup.13C-image for pyruvate, lactate and alanine
projected onto the anatomical image, images showing for every pixel
[0100] a)
([lactate].sub.norm.times.([pyruvate].sub.max-[pyruvate]).sub.norm).times-
.[lactate] and [0101] b)
([lactate].sub.norm.times.([alanine].sub.max-[alanine]).sub.norm).times.[-
lactate] in which the term "[. . .]norm represents the normalised
amplitude, i.e. scaled to its highest value in the metabolic image
and [lactate] the amplitude calculated.
[0102] A successful result for the discrimination of tumour tissue
and healthy tissue in a metabolic .sup.13C-MR image was defined as
highest lactate signal in the tumour area or a high weighted ratio
lactate over pyruvate in the tumour area and a high weighted
lactate over alanine ratio in the same pixel location.
[0103] 5.5 Biological Analysis
[0104] Tumour sites were visually inspected to detect signs of
bleeding. Tumours were liberated from the rat bodies, weighed and
cut in half. Tumour interiors were inspected visually assessing
homogeneity, necrosis and bleeding. The tumour tissues were stored
in 4% formalin.
[0105] A tumour-bearing rat was considered to be appropriate for
evaluation if the following criteria were met: tumour weight>100
mg, no visible necrosis or cysts in the tumour interior, a body
temperature above 35.degree. C. and a mean arterial blood pressure
above 60 mm Hg at time of MR investigation.
[0106] 5.6 Results
[0107] In total 30 different tumours were imaged in 18 rats. 1 rat
failed and 3 tumours failed the biological criteria described in
the preceding paragraph 5.5. The remaining 26 tumours in 17 rats
were homogenous and had a massive non-necrotic interior. The
average polarisation of .sup.13C.sub.1-pyruvate at the time of
injection was 21.2.+-.2.9% (mean .+-.SD) and the pH was
8.08.+-.0.14 (mean.+-.SD).
[0108] FIG. 1 displays a typical set of images of one imaged rat
with (1) the proton reference image, wherein the arrows indicate
the tumour locations, (2) the .sup.13C-pyruvate image, (3) the
.sup.13C-lactate image (4) the .sup.13C-alanine image (5) the
.sup.13C-lactate image corrected for .sup.13C-pyruvate and (6) the
.sup.13C-lactate image corrected for .sup.13C-alanine. Images (2)
to (6) are fused with the proton reference image.
[0109] FIG. 2 displays the same set of images, however with images
(2) to (6) which are not fused with the anatomical proton
image.
[0110] As a result, tumour location is indicated by a high pyruvate
signal (2), due to high metabolic activity. However the lactate
signal (3) ultimately identifies the correct location of the
tumour. Alanine is visible in the skeletal muscle and is absent in
the tumour tissue (4). The pyruvate and alanine corrected lactate
images (5) and (6) result in an excellent contrast for the tumour
as well.
[0111] It was thus demonstrated that the tumour location in the
metabolic images is indicated by a high lactate signal, a high
lactate signal corrected for pyruvate and a high lactate signal
corrected for alanine.
[0112] The analysis of the metabolic .sup.13C-MR images revealed a
metabolic contrast in the tumour area in [0113] 24 out of 26
tumours for the lactate signal [0114] 26 out of 26 tumours for the
lactate signal, pyruvate corrected (5.5, a)) [0115] 26 out of 26
tumours for the lactate signal, alanine corrected (5.5, b))
[0116] The overall rate of success for this study was 26 out of 26,
or 100%.
[0117] With this study, it was demonstrated that the hyperpolarised
.sup.13C.sub.1-pyruvate reach the region of interest (tumour) in a
time period which makes it possible to image the compound, that the
compound and its metabolites can be imaged and that metabolic
contrast can be obtained.
* * * * *