U.S. patent application number 14/902447 was filed with the patent office on 2016-10-13 for hyperpolarized 1-13c-1,1-bis(acetoxy(methyl))-2,2'-cyclopropane as metabolic marker for mr.
This patent application is currently assigned to BRACCO IMAGING S.P.A. The applicant listed for this patent is BRACCO IMAGING SPA. Invention is credited to Claudia CABELLA, Sonia COLOMBO SERRA, Pernille Rose JENSEN, Magnus KARLSSON, Mathilde H. Lerche, Luigi MIRAGOLI, Roberta NAPOLITANO, Fabio TEDOLDI.
Application Number | 20160296645 14/902447 |
Document ID | / |
Family ID | 48698961 |
Filed Date | 2016-10-13 |
United States Patent
Application |
20160296645 |
Kind Code |
A1 |
Lerche; Mathilde H. ; et
al. |
October 13, 2016 |
HYPERPOLARIZED 1-13C-1,1-BIS(ACETOXY(METHYL))-2,2'-CYCLOPROPANE AS
METABOLIC MARKER FOR MR
Abstract
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane of formula
(I): The compound can be hyperpolarized and used as a contrast
agent in .sup.13C Magnetic Resonance diagnostic technique
(.sup.13C-MR) for the diagnosis of tumor.
Inventors: |
Lerche; Mathilde H.;
(Fredericksberg C, DK) ; JENSEN; Pernille Rose;
(Vaerlose, DK) ; KARLSSON; Magnus; (Malmo, SE)
; NAPOLITANO; Roberta; (Albiano D'Ivrea (Torino), IT)
; CABELLA; Claudia; (Pecco, IT) ; MIRAGOLI;
Luigi; (Dovera, IT) ; COLOMBO SERRA; Sonia;
(Vigliano Biellese, IT) ; TEDOLDI; Fabio;
(Marzano, IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BRACCO IMAGING SPA |
Milan |
|
IT |
|
|
Assignee: |
BRACCO IMAGING S.P.A
Milan
IT
|
Family ID: |
48698961 |
Appl. No.: |
14/902447 |
Filed: |
June 30, 2014 |
PCT Filed: |
June 30, 2014 |
PCT NO: |
PCT/EP2014/063842 |
371 Date: |
December 31, 2015 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07B 59/001 20130101;
A61K 51/0402 20130101; C07B 2200/05 20130101; A61K 49/10 20130101;
C07C 2601/02 20170501; A61B 5/055 20130101; A61B 5/4848 20130101;
A61K 2123/00 20130101; C07C 69/007 20130101 |
International
Class: |
A61K 51/04 20060101
A61K051/04; A61B 5/055 20060101 A61B005/055; A61B 5/00 20060101
A61B005/00; C07C 69/007 20060101 C07C069/007 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 1, 2013 |
EP |
13174504.4 |
Claims
1. The compound
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane, of formula
(I) ##STR00006## said compound being enriched above natural
abundance of .sup.13C in position 1 of the molecule.
2. The deuterated compound of claim 1 of formula (II):
##STR00007##
3. A compound according to claim 1 wherein said compound is
hyperpolarized.
4. An imaging medium comprising the compound according to claim
3.
5. (canceled)
6. (canceled)
7. (canceled)
8. (canceled)
9. A method of .sup.13C-MR detection of tumor comprising using the
imaging medium according to claim 4 and detecting signals of the at
least one hyperpolarized metabolic product of a carboxylesterase
conversion of said hyperpolarized compound.
10. The method according to claim 9, wherein said at least one
hyperpolarized metabolic product is
1-.sup.13C-1-(acetoxy(methyl))-1-(hydroxy(methyl))-2,2'-cyclopropane,
1-.sup.13C-1,1-(dihydroxy(methyl))-2,2'-cyclopropane or a mixture
thereof.
11. The method according to claim 9 wherein a first signal obtained
from a region of interest is compared with a second signal and the
comparison is used to determine a difference between tumor and
non-tumor tissue.
12. A method for operating an MRI system comprising the steps of:
a. submitting a subject who is affected or suspected to be affected
by a tumor, who has been positioned in an MRI system and treated
with the compound of claim 3, and wherein said compound has been
metabolically converted into the corresponding hyperpolarized
metabolic product(s) of a carboxylesterase conversion, to a
radiation having a frequency selected to excite nuclear spin
transitions in .sup.13C nuclei; b. recording an MR signal from said
excited nuclei; and c. comparing a first MR signal deriving from a
region of interest comprising said tumor or said suspected tumor
with a second MR signal deriving from said subject or from a sample
taken from said subject.
13. The method according to claim 12 further comprising the steps
of: d. determining a difference between said first signal and
second signal; e. comparing said difference of step d) with a
reference value, to produce a deviation value; and f. determining
if the deviation value is, in absolute value, higher than a
predetermined value.
14. The method according to claim 13, wherein said second signal is
determined on a non-tumor tissue, further comprising the step of:
g. providing an indication of possible tumor affection in case the
deviation value is in absolute value higher than said predetermined
value.
15. The method according to claim 13, wherein said second signal is
determined in the region of interest, at an earlier moment in time
with respect to the first signal, and optionally stored in the
system, said method further comprising the step of: g'. providing
an indication of tumor variation in case the deviation is in
absolute value higher than said predetermined value.
16. The method according to claim 13, wherein said subject has
undergone an anti-tumor treatment and wherein said second signal is
determined in the region of interest, at an earlier moment in time
with respect to said first signal, and optionally stored in the
system, said method further comprising the step of: g''. providing
an indication of efficacy of said treatment if this deviation is in
absolute value higher than a predetermined value.
17. The method according to claim 16 wherein said second signal is
determined before, after or at the beginning of the treatment.
18. A process for the preparation of the compound of claim 1
comprising the steps of: a) reacting
1-.sup.13C-1,1-Bis(hydroxy(methyl))-2,2'-cyclopropane with acetyl
chloride, and b) removing the excess of acetyl chloride and the
formed hydrochloric gas.
19. A compound according to claim 2 wherein said compound is
hyperpolarized.
20. An imaging medium comprising the compound according to claim
19.
21. A method of .sup.13C-MR detection of tumor comprising using the
imaging medium according to claim 20 and detecting signals of the
at least one hyperpolarized metabolic product of a carboxylesterase
conversion of said hyperpolarized compound.
22. The method according to claim 21, wherein said at least one
hyperpolarized metabolic product is
1-.sup.13C-1-(acetoxy(methyl))-1-(hydroxy(methyl))-2,2'-cyclopropane,
1-.sup.13C-1,1-(dihydroxy(methyl))-2,2'-cyclopropane or a mixture
thereof.
23. The method according to claim 21 wherein a first signal
obtained from a region of interest is compared with a second signal
and the comparison is used to determine a difference between tumor
and non-tumor tissue.
24. A method for operating an MRI system comprising the steps of:
a. submitting a subject who is affected or suspected to be affected
by a tumor, who has been positioned in an MRI system and treated
with the compound of claim 19, and wherein said compound has been
metabolically converted into the corresponding hyperpolarized
metabolic product(s) of a carboxylesterase conversion, to a
radiation having a frequency selected to excite nuclear spin
transitions in .sup.13C nuclei; b. recording an MR signal from said
excited nuclei; and c. comparing a first MR signal deriving from a
region of interest comprising said tumor or said suspected tumor
with a second MR signal deriving from said subject or from a sample
taken from said subject.
25. The method according to claim 24 further comprising the steps
of: d. determining a difference between said first signal and
second signal; e. comparing said difference of step d) with a
reference value, to produce a deviation value; and f. determining
if the deviation value is, in absolute value, higher than a
predetermined value.
26. The method according to claim 25, wherein said second signal is
determined on a non-tumor tissue, further comprising the step of:
g. providing an indication of possible tumor affection in case the
deviation value is in absolute value higher than said predetermined
value.
27. The method according to claim 25, wherein said second signal is
determined in the region of interest, at an earlier moment in time
with respect to the first signal, and optionally stored in the
system, said method further comprising the step of: g'. providing
an indication of tumor variation in case the deviation is in
absolute value higher than said predetermined value.
28. The method according to claim 25, wherein said subject has
undergone an anti-tumor treatment and wherein said second signal is
determined in the region of interest, at an earlier moment in time
with respect to said first signal, and optionally stored in the
system, said method further comprising the step of: g''. providing
an indication of efficacy of said treatment if this deviation is in
absolute value higher than a predetermined value.
29. The method according to claim 28 wherein said second signal is
determined before, after or at the beginning of the treatment.
30. A process for the preparation of the compound of claim 2
comprising the steps of: c) reacting the corresponding deuterated
analog of 1-.sup.13C-1,1-Bis(hydroxy(methyl))-2,2'-cyclopropane
with acetyl chloride, and d) removing the excess of acetyl chloride
and the formed hydrochloric gas.
Description
FIELD OF THE INVENTION
[0001] The invention relates to the field of Magnetic Resonance
(MR), in particular to novel diagnostic media comprising
hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane and to a
diagnostic method exploiting said molecule as MR tracer.
BACKGROUND OF THE INVENTION
[0002] Magnetic resonance imaging (MRI) is a technique that has
become particularly attractive to physicians as images of a
patient's body or parts thereof can be obtained in a non-invasive
way and without exposing the patient and the medical personnel to a
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 tumors or
metastases.
[0003] MRI using hyperpolarized molecules is an emerging technique.
WO 9935508 discloses a method of MR investigation of a patient
using a hyperpolarized solution of a high T.sub.1 agent as MRI
contrast agent. The term "hyperpolarization" means enhancing the
nuclear polarization of the NMR active nuclei present in the agent,
i.e. nuclei with non-zero nuclear spin, preferably .sup.13C- or
.sup.15N-nuclei, and thereby amplifying the MR signal intensity by
a factor of hundred and more. When using a hyperpolarized .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. The main difference
between conventional MRI contrast agents and these hyperpolarized
high T.sub.1 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. When hyperpolarization is obtained via a microwave
assisted transfer between unpaired electrons and the nuclei used as
MR probes, the techniques is referred as Dynamic Nuclear
Polarization (DNP).
[0004] A variety of possible high T.sub.1 agents for use as MR
imaging agents are disclosed in WO9935508, 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.
[0005] For example, WO 2009077575 discloses a method of .sup.13C-MR
detection using an imaging medium comprising hyperpolarized
.sup.13C-fumarate, in order to investigate both the citric acid and
the urea cycles by detecting .sup.13C-malate and optionally
.sup.13C-fumarate and/or .sup.13C-succinate signals. The metabolic
profile generated in a preferred embodiment of the method provides
information about the metabolic activity of the body and part of
the body under examination and said information may be used in a
subsequent step for, e.g. identifying diseases. Such a disease is
preferably cancer since tumor tissue is usually characterized by an
altered metabolic activity. As a technical aspect, if the compounds
to be polarized crystallize upon freezing or cooling their
solution, a glass-forming additive must be added to the
solution.
[0006] Dynamic nuclear polarization (DNP) has been applied recently
to magnetic resonance spectroscopy (MRS) in solution, where it can
be used to produce a large increase in sensitivity. Using this
technique, the metabolism of several .sup.13C-labeled compounds has
been observed and used to estimate rate constants for specific
enzyme-catalyzed reactions in vitro and in vivo (Day S E, Kettunen
M I, Gallagher F A, Hu D E, Lerche M, Wolber J, Golman K,
Ardenkjaer-Larsen J H, Brindle K M. Detecting tumor response to
treatment using hyperpolarized .sup.13C magnetic resonance imaging
and spectroscopy. Nat Med 2007; 13:1382-1387; Gallagher F A,
Kettunen M I, Hu D E, Jensen P R, Zandt R I, Karlsson M, Gisselsson
A, Nelson S K, Witney T H, Bohndiek S E, Hansson G, Peitersen T,
Lerche M H, Brindle K M. Production of hyperpolarized
[1,4-.sup.13C.sub.2]malate from [1,4-.sup.13C.sub.2]fumarate is a
marker of cell necrosis and treatment response in tumors. Proc Natl
Acad Sci USA 2009; 106:19801-19806). Furthermore, for some
hyperpolarized .sup.13C-labeled substrates there is sufficient
signal for the spatial distribution of both the substrate and its
metabolites to be imaged in vivo. As some of these substrates have
already been administered at relatively high concentrations in the
clinic, this technique has the potential to be translated into
clinical applications. To date, the most studied reactions have
been those involving hyperpolarized [1-.sup.13C]pyruvate: the
hyperpolarized label can be exchanged with either endogenous
lactate or alanine, or alternatively it can be irreversibly
converted to carbon dioxide, which is subsequently converted to
bicarbonate in the reaction catalyzed by carbonic anhydrase. These
metabolic reactions have been observed in tumors, in cardiac tissue
and in the liver (Merritt M E, Harrison C, Storey C, Jeffrey F M,
Sherry A D, Malloy C R. Hyperpolarized .sup.13C allows a direct
measure of flux through a single enzyme-catalyzed step by NMR. Proc
Natl Acad Sci USA 2007; 104:19773-19777; Schroeder M A, Swietach P,
Atherton H J, Gallagher F A, Lee P, Radda G K, Clarke K, Tyler D J.
Measuring intracellular pH in the heart using hyperpolarized carbon
dioxide and bicarbonate: a .sup.13C and .sup.31P MRS study.
Cardiovasc Res 2010; 86:82-91; Hu S, Chen A P, Zierhut M L, Bok R,
Yen Y F, Schroeder M A, Hurd R E, Nelson S J, Kurhanewicz J,
Vigneron D B. In vivo carbon-13 dynamic MRS and MRSI of normal and
fasted rat liver with hyperpolarized .sup.13C-pyruvate. Mol Imaging
Biol 2009; 11:399-407).
[0007] Recently, other endogenous molecules have been successfully
hyperpolarized: tumor pH has been measured in vivo from the
relative concentrations of .sup.13C-labeled bicarbonate and carbon
dioxide following the injection of hyperpolarized .sup.13C-labeled
bicarbonate (Gallagher F A, Kettunen M I, Day S E, Hu D E,
Ardenkjaer-Larsen J H, Zandt R, Jensen P R, Karlsson M, Golman K,
Lerche M H, Brindle K M. Magnetic resonance imaging of pH in vivo
using hyperpolarized .sup.13C-labelled bicarbonate. Nature 2008;
453:940-943); elevated levels of hyperpolarized malate have been
demonstrated in necrotic tumor tissue in vivo following the
injection of hyperpolarized .sup.13C-labeled fumarate (Gallagher F
A, Kettunen M I, Hu D E, Jensen P R, Zandt R I, Karlsson M,
Gisselsson A, Nelson S K, Witney T H, Bohndiek S E, Hansson G,
Peitersen T, Lerche M H, Brindle K M. Production of hyperpolarized
[1,4-.sup.13C.sub.2]malate from [1,4-.sup.13C.sub.2]fumarate is a
marker of cell necrosis and treatment response in tumors. Proc Natl
Acad Sci USA 2009; 106:19801-19806); the metabolism of glutamine to
glutamate, catalyzed by the mitochondrial enzyme glutaminase, has
been observed following administration of hyperpolarized
.sup.13C-labeled glutamine to cells in vitro (Gallagher F A,
Kettunen M I, Day S E, Lerche M, Brindle K M. .sup.13C MR
spectroscopy measurements of glutaminase activity in human
hepatocellular carcinoma cells using hyperpolarized
.sup.13C-labeled glutamine. Magn Reson Med 2008; 60:253-257); the
organ-specific metabolism of hyperpolarized .sup.13C-labeled
acetate to acetyl-CoA and acetyl carnitine has been observed in
vivo (Jensen P R, Peitersen T, Karlsson M, In't Zandt R, Gisselsson
A, Hansson G, Meier S, Lerche M H. Tissue-specific short chain
fatty acid metabolism and slow metabolic recovery after ischemia
from hyperpolarized NMR in vivo. J Biol Chem 2009;
284:36077-36082), and the metabolism of branched chain amino acids
has been observed in tumors following the addition of
hyperpolarized .sup.13C-labeled .alpha.-ketoisocaproate (Karlsson
M, Jensen P R, In't Zandt R, Gisselsson A, Hansson G, Duus J O,
Meier S, Lerche M H. Imaging of branched chain amino acid
metabolism in tumors with hyperpolarized .sup.13C ketoisocaproate.
Int J Cancer 2010; 127:729-736.10).
[0008] Although its etiology is lacking, cancer is
phenomenologically well characterized as a molecular disease.
Different kinds of cancers may have very different biochemical
forms, however they can share general molecular features.
[0009] Early diagnosis of cancer continues to be given large
attention since diagnosis at an early stage often increases the
chances of a successful treatment. In fact, early diagnosing cancer
and ensuring access to optimum treatment can lead to significant
improvements in survival.
[0010] Early diagnosing of cancer could be achieved by taking
advantage of a general molecular feature shared by different types
of cancer cells and whose alteration in cancer can be early
detected.
[0011] Carboxylesterases (CE, EC 3.1.1.1) are a family of enzymes
catalysing the chemical conversion of an ester in an acid and an
alcohol. A general reaction scheme is shown below:
##STR00001##
[0012] Carboxylesterases are ubiquitously expressed in mammalian
tissues. The many CE isoforms have been classified into 5 super
families (CE 1-5) based on amino acid homologies. The CE1 enzymes
are mainly localized to the liver, however they are also expressed
in most other tissue types. A rat specific CE1 isoform is secreted
from the liver to the blood in rats and mice and this iso-enzyme is
correlated to a high level of hydrolase activity detected in
rodents compared to humans (Yan, B. Dongfang Y., Bullock, P.,
Parkinson, A., Rat serum carboxylesterases, 1995, JBC, 32 (270):
19128-34; Rudakova, E V., Botneva N P., Makhaeva, G F. Comparative
analysis of esterase activities of human, mouse and rat blood,
2011, Bulletin of experimental biology and medicine, 152(1):
73-75). The other important isoform is the CE2 family, which is
also expressed in the human liver (approx. 4 times less than CE1)
as well as in most other tissues to a higher degree than CE1
(Talvar, S. The expression of human carboxylesterases in normal
tissues and cancer cell lines (2008), Master thesis).
[0013] The expression of carboxylesterases decreases in cancer in
both animal and human tissue. In particular, in hepatoma cells a 4
times decrease in the expression of carboxyl esterase has been
measured compared to normal hepatocytes. Dependent on the isoform
the expression is reported to be approx. 1.5-4 times higher in
normal tissue than in the corresponding malignant tissue (Talvar,
2008).
[0014] A number of studies have been reported on carboxylesterases
in cancer cells.
[0015] The expression of carboxylesterase was reported as
detectable in human cancer cells (HEPG2) and approx. 3-4 times
lower than the expression of carboxyl esterase in normal human
liver (hepatocytes) (Talvar, 2008). A patient study on non-tumor
and tumor tissues from liver cancer (HCC) patients showed an almost
3 times decrease in the expression of carboxyl esterase in the
tumor tissue (Na, K. et al., Human plasma carboxylesterase 1, a
novel serologic biomarker candidate for hepatocellular carcinoma
(2009), Proteomics, 9: 3989-99).
[0016] Another study showed that the carboxylesterase activity was
significantly lower in colon cancer xenografts compared to the
corresponding normal colon tissue in mice (Jansen et al., CPT-11 in
human colon cancer cell lines and xenografts: characterization of
cellular sensitivity determinants, 1997, Int. J. Cancer
70:335-40.)
[0017] A study has been reported on lung cancer patients where
carboxylesterase activity is correlated to esterase expression in
healthy and cancer lung tissue. In this study, they find that the
activity correlates well with the expression, which is shown to be
approx. 1.5 times higher in healthy tissue (Liewald F. et al,
Intracellular pH, esterase activity and DNA measurements of human
lung carcinomas by flow cytometry. 1990, Cytometry, 11: 341-48)
[0018] WO2012102773 discloses a method for the diagnosis and
treatment of cancer, in particular breast cancer, by measuring the
activity of the enzyme PMPMEase (human carboxylesterase 1). Said
activity is measured in a biological sample by assaying the enzyme
expression or enzymatic activity, in the last case through the
measurement of the consumption of a substrate or the production of
a product. It is only generally stated that the enzyme assay can be
performed in vivo.
[0019] U.S. Pat. No. 8,198,038 discloses a screening method to
distinguish healthy human beings from those with human liver cancer
(hepatocellular carcinoma; HCC) comprising the steps of collecting
human blood and detecting the presence of human liver
carboxylesterase 1 (hCE1) in the plasma, wherein the level of hCE1
protein is increased more in the plasma of patients with HCC than
in the plasma of healthy patients.
[0020] The conversion of an hyperpolarized ester, catalysed by
carboxylesterase, generates hyperpolarized metabolic products whose
MR signals are well distinguishable from each other and from the
injected substrate. Cancer cells and healthy cells convert a
hyperpolarized ester to a different degree, leading to differences
in said metabolic product signal amounts, therefore said difference
between the signals in tumor and non-tumor cells can be exploited
to identify cancer.
[0021] A common weakness of the endogenous molecules known in the
prior art, when used as hyperpolarized markers, is the relatively
fast vanishing of MR signals, of both the substrate itself and its
relevant metabolites. DNP in fact, as well as all other
hyperpolarization procedures, leads to a transient enhancement of
the nuclear magnetic order, that allows the in vivo visualization
of said moieties but, unfortunately, for a limited amount of time,
in the order of 3 to 5 times the longitudinal relaxation time of
the nuclei used as MR probe. Since MR imaging procedures require a
certain amount of time in order for the final image to be
reconstructed with acceptable sensitivity and resolution, markers
with slower nuclear relaxation, with respect to the product used so
far, are strongly desirable, also in view of inherent technical
features of the imaging procedure, such as dilution (upon injection
into patient) and metabolic conversion (time delay for achieving
detectable concentrations of metabolites).
[0022] Therefore, in order to be able to register in vivo metabolic
maps of tumour and healthy tissues, with superior sensitivity and
resolution with respect to the state of the art, novel metabolic
contrast agents with enhanced persistence of the hyperpolarized
signal (i.e. slower nuclear longitudinal relaxation) are strongly
needed
SUMMARY OF THE INVENTION
[0023] It has now been found that
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane is
characterized by a .sup.13C longitudinal relaxation time
significantly longer than any other known molecule used for
metabolic imaging.
[0024] Said 1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane
can be polarized by DNP in order to achieve a .sup.13C signal that
allows its detection in vivo over an extended acquisition window.
The conversion of said hyperpolarized ester, catalysed by
carboxylesterase, generates hyperpolarized metabolic products whose
MR signals are well distinguishable from each other and from the
injected substrate.
[0025] Cancer cells and healthy cells convert
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane to a
different degree leading to differences in said metabolic product
signal amounts, therefore said difference between the signals in
tumor and non-tumor cells can be exploited to identify cancer.
[0026] Within the context of cancer diagnosis, said difference in
the signals of hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane metabolized
by a carboxylesterase can be used for detecting the presence of a
tumor, for evaluating the efficacy of an anti-cancer therapy and/or
to determine a time evolution of a tumor.
[0027] It has also been found that the overall hyperpolarized
signal of the metabolites deriving from
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane is far more
intense than the hyperpolarized signal of the metabolites
generating from [1-.sup.13C]-pyruvate, the most used MR metabolic
agent, in particular in some cancer cell types.
[0028] Therefore, an object of the present invention is the
compound 1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane of
formula (I):
##STR00002##
[0029] said compound being enriched above natural abundance of
.sup.13C in position 1 of the molecule.
[0030] In a preferred embodiment, one or more hydrogen atoms of
said 1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane are
replaced by deuterium. More preferably, all the methylene groups of
the compound of formula (I) are deuterated, the compound being thus
identified as
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
and having the following structural formula (II):
##STR00003##
[0031] A process for the preparation of the new compound
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane comprising
the steps of: [0032] a) reacting
1-.sup.13C-1,1-Bis(hydroxy(methyl))-2,2'-cyclopropane with acetyl
chloride, and [0033] b) removing the excess of acetyl chloride and
the formed hydrochloric gas,
[0034] is also an object of the present invention.
[0035] The same process as above can be used for the preparation of
deuterated 1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane by
using in step a) deuterated
1-.sup.13C-1,1-Bis(hydroxy(methyl))-2,2'-cyclopropane.
[0036] The new compound can be hyperpolarized and used as a
contrast agent in .sup.13C Magnetic Resonance diagnostic technique
(.sup.13C-MR) for the diagnosis of tumor.
[0037] It is also an object of the present invention a method of
.sup.13C-MR detection of tumors using an imaging medium comprising
hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane, wherein
signals of the corresponding hyperpolarized metabolic products of
the carboxylesterase conversion are detected.
[0038] Said imaging medium comprising the hyperpolarized compound
of the invention is also an object of the invention.
[0039] In a preferred embodiment of the present invention, the
hyperpolarized metabolic products of carboxylesterase conversion of
the compound of the invention are
1-.sup.13C-1-(acetoxy(methyl))-1-(hydroxy(methyl))-2,2'-cyclopropane
and/or 1-.sup.13C-1,1-(dihydroxy(methyl))-2,2'-cyclopropane or a
mixture thereof.
[0040] In a preferred embodiment, wherein the
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane is
deuterated, its metabolic products are also deuterated and they are
indicated in the following description as
1-.sup.13C-1-(acetoxy(methyl-d.sub.2))-1-(hydroxy(methyl-d.sub.2))-2,2'-d-
.sub.4-cyclopropane and
1-.sup.13C-1,1-(dihydroxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane.
[0041] With respect to known hyperpolarized molecules used as
metabolic markers,
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane and its
metabolic products
1-.sup.13C-1-(acetoxy(methyl))-1-(hydroxy(methyl))-2,2'-cyclopropane
and 1-.sup.13C-1,1-(dihydroxy(methyl))-2,2'-cyclopropane have a
much slower .sup.13C MR signal decay, because of the unique
quaternary position of the observed .sup.13C nuclei which minimizes
any longitudinal relaxation effect due to dipolar interaction and
chemical shift anisotropy. This is a great advantage for metabolic
imaging based on hyperpolarized markers, that reflects into more
pronounced metabolite signals as well as on a broader time window
for signal detection.
[0042] In an embodiment of the present invention, in the method of
.sup.13C-MR detection a first signal obtained from a region of
interest is compared with a second signal (typically a signal
derived from a reference sample, e.g. a signal obtained from a
corresponding non-tumor/healthy tissue); said comparison is useful
to determine a difference between tumor and non-tumor tissue. Said
comparison can be used, for example, for the diagnosis of a tumor
or to provide a localization of a tumor.
[0043] Furthermore, when a first signal obtained from a region of
interest comprising a tumor tissue is compared with a second signal
obtained from the same region of interest at an earlier time, the
comparison between said first and said second signal can provide
information about the the tumor development over time, which can
also be an indication of aggressiveness, of the tumor and/or the
efficacy of a therapy when treating said tumor by
(immune)pharmacological and/or surgical and/or radio therapy.
[0044] In a preferred embodiment of the invention, said first
signal is the ratio between the signal of the metabolic product of
the carboxylesterase conversion and the signal of the administered
substrate (the hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane) detected in
the region of interest and said second signal is the same ratio but
detected in a reference sample, for example a corresponding
non-tumor/healthy tissue, or in the same region at an earlier
time.
[0045] Another object of the present invention is the above method
of .sup.13C-MR detection wherein said signals are used to generate
a metabolic profile, based on the metabolic carboxylesterase
conversion of hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane into the
corresponding hyperpolarized metabolic products, wherein said
metabolic profile is useful in detecting or providing indication of
a tumor.
[0046] In an embodiment of the present invention, said metabolic
profile is determined in a region of interest (where the presence
of a tumor tissue is known or suspected) and compared with a
metabolic profile of reference (e.g. relative to a corresponding
non-tumor tissue, typically a healthy tissue in the close proximity
of the tumor tissue).
[0047] Another object of the present invention is a method for
operating an MRI system comprising the steps of: [0048] a.
submitting a subject who is affected or suspected to be affected by
a tumor, who has been positioned in an MRI system and treated with
hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane, and wherein
said hyperpolarized .sup.13C ester has been metabolically converted
into the corresponding hyperpolarized metabolic product(s) of the
carboxylesterase conversion, to a radio frequency pulse having a
frequency selected to excite nuclear spin transitions in .sup.13C
nuclei; [0049] b. recording an MR signal from said excited nuclei;
and [0050] c. comparing a first MR signal deriving from a region of
interest comprising said tumor or said suspected tumor with a
second MR signal deriving from said subject or from a sample taken
from said object.
[0051] In an embodiment of the invention, said second signal is an
MR-signal deriving from a non-tumor tissue of said subject. In
another embodiment of the invention, said second signal is an
MR-signal which has been detected from the region of interest, at
an earlier time with respect to the first signal.
[0052] Another object of the invention is the above method further
comprising the steps of: [0053] d. determining a difference between
said first signal and second signal; [0054] e. comparing said
difference of step d) with a reference value, to produce a
deviation value; and [0055] f. determining if the deviation value
is, in absolute value, higher than a predetermined value.
[0056] Another object of the present invention is the above method,
wherein said second signal is determined on a non-tumor tissue,
further comprising the step of: [0057] g. providing an indication
of possible tumor affection in case the deviation value is in
absolute value higher than said predetermined value.
[0058] Another object of the present invention is the above method
for operating an MRI system comprising steps a) to f), wherein said
second signal is determined in the region of interest, at an
earlier moment in time with respect to the first signal, and
optionally stored in the system, said method further comprising the
step of: [0059] g'. providing an indication of tumor variation in
case the deviation is in absolute value higher than said
predetermined value.
[0060] Another object of the present invention is the above method
for operating an MRI system comprising steps a) to f), wherein said
subject has undergone an anti-tumor treatment and wherein said
second signal is determined in the region of interest, at an
earlier moment in time with respect to said first signal, and
optionally stored in the system, said method further comprising the
step of: [0061] g''. providing an indication of efficacy of said
treatment if this deviation is in absolute value higher than a
predetermined value.
[0062] In a preferred embodiment, said second signal is determined
before, after or at the beginning of the treatment, wherein the
effective time of detection of said second signal will be decided
by the person skilled in the art, according to patient's
conditions, kind of treatment, degree of severity of the disease
and any other clinical parameter within the general knowledge on
the matter. Examples of the time of determination of said second
signal are few days, e.g. 1 to 5, one or more weeks, one or more
months.
[0063] An MR system performing any of the methods above described
is also an object of the present invention.
[0064] The use of said MR system for providing an indication of the
presence of a tumor, of its grade of aggressiveness or for
monitoring the response to an antitumor therapy of a subject
affected by a tumor is also within the scope of the present
invention.
[0065] The present invention provides the advantages of making
available an imaging medium comprising hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane, which can be
used in MRI technique for the diagnosis of tumors with a selective
grade of distinction between tumor and non-tumor tissue.
[0066] A further advantage is represented by the possibility of
taking different registrations of the MR signals of the
hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane in a tumor
tissue, while an antitumor therapy is administered and to monitor
the progress of the therapy.
[0067] A further advantage is represented by the possibility of
detecting aggressive forms of tumors by monitoring the development
of the formation of the corresponding .sup.13C hyperpolarized
metabolic product(s) of the carboxylesterase conversion in a tumor
tissue.
[0068] These and other objects of the present invention and
advantages will be disclosed in detail in the following description
even by means of Figures and Examples.
FIGURES
[0069] FIG. 1. Dissolution spectrum of hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane.
[0070] FIG. 2. In cell DNP conversion of
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
and 1-.sup.13C pyruvate in rat liver cancer cells (rat hepatoma,
Morris). The DNP experiments were performed with 10 million cells.
A) Build-up of the metabolites
1-.sup.13C-1-(acetoxy(methyl-d.sub.2))-1-(hydroxy(methyl-d.sub.2))-2,2'-d-
.sub.4-cyclopropane and
1-.sup.13C-1,1-(dihydroxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
from injection of
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
and of 1-.sup.13C-lactate from injection of 1-.sup.13C-pyruvate,
respectively into whole Morris cells. B) Area under the curve of
the metabolites
1-.sup.13C-1-(acetoxy(methyl-d.sub.2))-1-(hydroxy(methyl-d.sub.2))-2,2'-d-
.sub.4-cyclopropane and 1-.sup.13C-lactate.
[0071] FIG. 3. In cell DNP conversion of
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
and 1-.sup.13C pyruvate in human prostate cancer cells (human
prostate carcinoma, PC-3). The DNP experiments were performed with
10 million cells. A) Build-up of the metabolites
1-.sup.13C-1-(acetoxy(methyl-d.sub.2))-1-(hydroxy(methyl-d.sub.2))-2,2'-d-
.sub.4-cyclopropane from injection of
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
and 1-.sup.13C-lactate from injection of 1-.sup.13C-pyruvate,
respectively into whole PC-3 cells. B) Area under the curve of the
metabolites
1-.sup.13C-1-(acetoxy(methyl-d.sub.2))-1-(hydroxy(methyl-d.sub.2))-2,2'-d-
.sub.4-cyclopropane and 1-.sup.13C-lactate.
[0072] FIG. 4. In cell DNP conversion of
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
in human prostate cancer cells (human prostate carcinoma, PC-3) and
in human prostate healthy cells (Immortalized human prostate cells,
PNT-1A)). The DNP experiments were performed with 10 million cells.
A) Build-up of the metabolite
1-.sup.13C-1-(acetoxy(methyl-d.sub.2))-1-(hydroxy(methyl-d.sub.2))-2,2'-d-
.sub.4-cyclopropane in PC-3 cells (filled squares) and in PNT-1A
cells (open circles). B) Area under the curve of the metabolite
1-.sup.13C-1-(acetoxy(methyl-d.sub.2))-1-(hydroxy(methyl-d.sub.2))-2,2'-d-
.sub.4-cyclopropane produced in PC-3 cells and in PNT-1A cells.
[0073] FIG. 5. .sup.13C NMR sum spectrum, obtained by integrating
over time the spectra of a time series acquired on the prostate of
a representative animal.
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
is labelled as A, whereas the metabolites signals are labelled as B
(1-.sup.13C-1-(acetoxy(methyl-d.sub.2))-1-(hydroxy(methyl-d.sub.2))-2,2'--
d.sub.4-cyclopropane) and C
(1-.sup.13C-1,1-Bis(hydroxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane).
[0074] FIG. 6. Time course of the .sup.13C NMR signals in rat
prostates (average over n=2 subjects) after injection of
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
(solid line). The evolution of the metabolites signals is
represented by a wide dashed line
(1-.sup.13C-1-(acetoxy(methyl-d.sub.2))-1-(hydroxy(methyl-d.sub.2))-2,2'--
d.sub.4-cyclopropane) and by a narrow dashed line
(1-.sup.13C-1,1-Bis(hydroxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane)
respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0075] Within the scopes of the present invention, the term MRI
means Imaging (typically for diagnostic purposes) by means of
Magnetic Resonance (MR) as commonly intended in the state of the
art and for example disclosed in WO200977575 and the references
cited therein.
[0076] Within the scopes of the present invention, the "imaging
medium" and "contrast agent" are used synonymously, as commonly
intended in the state of the art and for example disclosed in
WO200977575 and the references cited therein.
[0077] Within the scopes of the present invention, the terms
"hyperpolarization", "hyperpolarized" or similar mean enhancing the
nuclear polarization of NMR active nuclei present in the high
T.sub.1 agent as commonly intended in the state of the art and for
example disclosed in WO200977575 and the references cited
therein.
[0078] Within the scopes of the present invention, the term Dynamic
Nuclear Polarization (DNP) is a technique in Magnetic Resonance
Imaging as commonly intended in the state of the art and for
example disclosed in WO200977575 and the references cited
therein.
[0079] Within the meaning of the present invention, the term
"hyperpolarized" means the nuclear spin polarization of a compound
higher than thermal equilibrium.
[0080] Within the scope of the present invention "MRI system" means
apparatus, equipment and all features and accessories useful for
performing MR experiments, in particular for diagnostic
purposes.
[0081] Within the meaning of the present invention, "1-.sup.13C"
means that the labeled compound is enriched in .sup.13C in position
1 of the molecule. The term "enriched" means that the concentration
of the non-zero nuclear spin nuclei in the compound (in particular
of .sup.13C in position 1) is above the typical value of natural
abundance of said nuclei, preferably above at least 10% of natural
abundance, more preferably above at least 25%, and even more
preferably above at least 75% of its natural abundance and most
preferably above at least 90% of its natural abundance. Enrichment
can be achieved by chemical synthesis or biological labeling,
according to the prior art teachings. Enrichment of non-zero
nuclear spin nuclei over natural abundance may be determined, for
instance, on a reference amount of the material, e.g. at least 0.1
mmole, preferably at least 1 mmole of the material.
[0082] The new compound
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane is
synthetized starting from
1-.sup.13C-1,1-Bis(hydroxy(methyl))-2,2'-cyclopropane according to
methods known in the art, typically by esterifying the alcohol
groups of the starting compound and isolating the final compound by
any conventional means known in the art. For instance, acetyl
chloride is added, preferably in excess, to the starting compound
and the resulting mixture is stirred to obtain the desired product
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane. Preferably,
the excess of acetyl chloride and the formed hydrochloric gas are
removed from the mixture, and the final compound is isolated and
recovered from tho mixture.
[0083] The starting compound
1-.sup.13C-1,1-Bis(hydroxy(methyl))-2,2'-cyclopropane can be
obtained according to any preparation methods known in the art,
starting from commercially available .sup.13C-labelled diethyl
malonate.
[0084] For instance, .sup.13C-labelled diethyl malonate can be
subjected to a double alkylation by reacting it with
1,2-dibromethane and subsequent reduction of the ester groups with
LiAlH.sub.4, according to the following reaction scheme:
##STR00004##
as described by House, H. O. et al. . . . "The synthesis of
spiropentane-d8". J. Org. Chem. 1956, 21,1487-149. Alternatively,
diethyl malonate can be reacted with dihaloethane to provide a
malonic acid derivative with a cyclopropyl group in 2-position, as
described for instance by Singh R. K. and Danishefsky S., J. Org.
Chem. 1975, 40(20), 2969-2970. The malonic acid derivative is then
reduced as above described with LiAlH.sub.4to give the desired diol
compound.
[0085] In a preferred embodiment of the process, the starting
compound is deuterated
1-13C-1,1-Bis(hydroxy(methyl-d2))-2,2'-d4-cyclopropane which can be
obtained as described above by reacting from a corresponding
commercially available 13C-labelled diethyl malonate with
respective deuterated reactants, i.e. BrCD2CD2Br and LiAlD4, both
commercially available; according to the preparation methods
described above (where the hydrogen atoms of the respective
reactants are replaced by deuterium); the obtained product of the
invention, 1-13C-1,1-Bis(acetoxy(methyl-d2))-2,2'-d4-cyclopropane,
is thus also deuterated.
[0086] The 1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane is
hyperpolarized by Dynamic Nuclear Polarization (DNP), which is a
known method disclosed, for example, in WO9935508, and in
particular in WO2011124672. Hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane is obtained.
It can be used in an imaging medium in a method of .sup.13C-MR
detection.
[0087] The activity of the carboxylesterase isoforms CE1 and CE2 is
highly substrate dependent. In general substrates with a smaller
alcohol group than acid group are reported to have higher affinity
for the carboxylesterase isoform CE1 and the reverse class of
substrates with a larger alcohol group than acid group have higher
affinity for the CE2 enzyme (Imai, T. Human Carboxylesterase
Isozymes: Catalytic Properties and Rational Drug Design, (2006)
Drug Metab. Pharmacokinet 21(3): 173-85.).
[0088] The diacetate ester of the invention,
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane, is a
substrate fur the CE2 isoform.
[0089] It provides the advantage of being effectively hydrolyzed in
liver, prostate and breast cells, where the CE2 enzyme is highly
expressed. Especially in prostate cells, where the CE2 expression
is high.
[0090] 1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane also
provides good chemical and physical properties, as high solubility,
high polarization, very long T1 (as compared to other compounds
employed for metabolic imaging, illustrated in the following table
1), sufficient chemical shift separation between substrate and
product to detect the hydrolysis product in vivo.
TABLE-US-00001 TABLE 1 T1 of
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropan-
e compared with other metabolic imaging compounds Compound T1, 37
C, 14.1 T 1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))- 85 .+-. 4
2,2'-d.sub.4-cyclopropane 1-13C-pyruvate 45 .+-. 4 1-13C-acetate 50
.+-. 3 1.13C-lactate 40 .+-. 3 2-13C-1,1,2,2-d4-choline 43 .+-. 4
1,3-ethyl acetoacetate 27 .+-. 2 13C6-glucose 15 .+-. 2
1-13C-glucose 20 .+-. 2 1-13C-2-ketoisocaproate 35 .+-. 4
1-13C-alanine 31 .perp. 2 5-13C-glutamine 22 .+-. 2
1,4-13C2-Fumarate 35 .perp. 3 1,4-13C2-malate 30 .perp. 3
1-13C-bicarbonate 30 .+-. 2 1-13C-2-oxoglutarate 30 .+-. 3
[0091] A further advantage of the use of the compound of the
invention as a metabolic substrate is that its uptake into cells
takes place mainly by diffusion through the cell membrane.
Therefore, it is not uptake-limited and only the activity of the
metabolizing enzyme itself has influence on the amount of
hyperpolarized product that is produced. This means that the
detected signal in the present invention is highly representative
of the activity of the carboxylesterase, thus making said substrate
particularly useful as real time molecular contrast agents. On the
contrary, substrates like mono-carboxylic acids, e.g. pyruvic acid,
can stiffer the disadvantage of being uptake-limited; the signal of
their hyperpolarized product may therefore be not representative of
the activity of the specific enzyme to be detected.
[0092] The method of the present invention is a non-invasive
method, which allows a real time metabolic assessment of the
carboxylesterase activity in vivo. An image representative of said
activity is collected seconds to minutes following intravenous
injection of the substrate.
[0093] Essentially, the method of operating an MRI system according
to the present invention comprises the steps of a) recording an MR
signal from the excited nuclei; and b) comparing a first MR signal
deriving from the tumor or suspected tumor with a second MR signal
deriving from the same subject or from a sample thereof.
[0094] In a preferred embodiment of the invention, said first
signal deriving from said tumor is lower than said second MR
signal.
[0095] In an embodiment of the present invention, as shown in steps
d-f above, the MRI apparatus can process said first signal and said
second signal by comparing each other, calculating a difference
between the two signals and comparing said difference with a
reference value; as shown in step g above, if this comparison
provides a value which is, in absolute value, higher than a
predetermined value, then said MRI apparatus provides an indication
of possible tumor affection.
[0096] The use of said apparatus for monitoring the response of a
subject affected by a tumor to antitumor therapy (step g') or for
evaluating the aggressiveness of a tumor (step g'') are further
objects of the present invention.
[0097] Examples of said tumors are tumors selected from the group
consisting of liver, colon, prostate and breast. In a preferred
embodiment, the tumor is prostate tumor.
[0098] According to the present invention, the compound
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane can be
exploited as a marker of targeted therapies, where for targeted
therapy is intended the targeting of molecules important for the
carcinogenesis of the cancer cells.
[0099] In carrying out the methods of the present invention, the
first signal (S.sub.1), the second signal (S.sub.2) and the
reference value (R), depend on how the methods of the invention are
applied.
[0100] Typically, in order to have comparable data, the MR signals
obtained in the method of the invention are normalized with respect
to the corresponding signal of the
1-.sup.13C-1,1-Bis(acetoxy(methyl) 2,2' cyclopropane.
[0101] When the method of the present invention is performed to
provide an indication of possible tumor affection, said first
signal S.sub.1 is the ratio between the integral of the MR line of
the hyperpolarized metabolic product of the carboxylesterase
conversion and the integral of the MR line of the administered
substrate (the hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl)-2,2'-cyclopropane), detected in
the region of interest comprising the alleged tumor, while the
second signal S.sub.2 is the analogue ratio calculated in non-tumor
tissue; the reference value R is either equal to S.sub.2 or, in
case no signal of the hyperpolarized metabolic product is detected
in the healthy tissue under consideration, R is set to 3 times the
noise standard deviation divided by the substrate signal in the
same volume. Preferably, non-tumor tissue is surrounding the tumor,
so that the MR system can provide an accurate imaging of the tumor,
which is of great importance for the evaluation of surgical
intervention.
[0102] In case the purpose of the method is a follow-up of
antitumor therapy, said first signal corresponds to the metabolic
product signal detected in the tumor before, or at the start or at
a certain point after the beginning of therapy and said second
signal is the one produced by the same tumor after a certain period
subsequent to the detection of said first signal. The reference
value R is set equal to the first signal.
[0103] In case the purpose of the method is to determine
aggressiveness of a tumor, said first signal is the one produced by
the tumor at the start of the determination and said second signal
is the one produced by the same tumor after a certain period
subsequent to the detection of said first signal. Again the
reference value is set equal to the first signal.
[0104] The first and second MR signals can be obtained either as
single signals or calculated as a mean value of a plurality of
respective signals determined (from different voxels) in a selected
region of interest (S.sub.1) or in a non-tumor tissue
(S.sub.2).
[0105] In an embodiment of the invention, said first signal and
said second signal can be directly compared, either as single
signals or as mean values of a plurality of signals, to obtain the
desired information on the tumor tissue. In an alternative
embodiment of the invention, the signals can be used to generate a
parametric image and the comparison can be performed by comparing
the zones of said image corresponding to said first and said second
signal.
[0106] According to the present invention, a difference between
said first and said second signal is determined. This difference
(S.sub.1-S.sub.2) is important for the different scopes of the
present invention.
[0107] This difference is compared with the reference value to
produce a value representing the deviation (D) of said difference
from said reference value:
D=(S.sub.1-S.sub.2)/R.
[0108] If it is determined that this deviation provides a value
which is, in absolute value, higher than a predetermined value,
this deviation provides an indication of possible tumor affection,
of the efficacy of the antitumor therapy or of tumor
aggressiveness, depending on the purpose of the method of the
invention.
[0109] For instance, in an embodiment of the invention, said
predetermined value can be set at 2; accordingly, if the calculated
value "D" is equal or higher than 2, this can be indicative of a
possible presence of a tumor in the region of interest, of the
efficacy of the antitumor therapy or of tumor aggressiveness,
depending on the purpose of the method of the invention. Preferably
a deviation value D of from 2 to 10 can be indicative of said
presence, efficacy or aggressiveness, more preferably a deviation
from 2 to 20, even more preferably a deviation from 2 to 40,
particularly preferred is a deviation from 2 to 60, maximally
preferred is a deviation from 2 to 80, the most preferred is a
deviation from 2 to 100 or higher.
[0110] In an embodiment of the invention, the method is performed
on a subject who is suspected to suffer or suffers from a
tumor.
[0111] In another embodiment of the present invention, the above
method is performed on a subject who is undergoing or has been
subjected to an antitumor treatment and the reference value is the
signal of the hyperpolarized metabolic product(s) of the
carboxylesterase conversion in said region of interest determined
before, during or after said treatment. As above, if a deviation D
is calculated which is higher, in absolute value, than a
predetermined value (e.g. higher than 2, and preferably within the
above indicated ranges), this provides an indication of the
efficacy of the antitumor treatment.
[0112] In some embodiments, the present invention can be used in
the field of so-called "personalized medicine", or similarly
intended. As explained above, tumor therapy is affected by
variations in its efficacy even on the same type of tumor and with
the same anticancer therapeutic protocol. Such variations are due
to the different individual responses by the patients. Carrying out
the method of the present invention allows to monitor (follow-up)
the efficacy of a tumor therapy and, in case, allowing the doctor
to fit the therapy to the patient.
[0113] Typical metabolic imaging procedures with the compound of
the invention in human subjects should be performed at magnetic
fields .gtoreq.1 T. Field strengths of 1.5 T or higher are
preferred since the spectral separation between the injected
substrate (ester) and the observed metabolite (acid or alcohol)
scales linearly with the intensity of the applied field. The MR
scanner should be capable to detect .sup.13C signals in addition to
1H and although not always mandatory, surface or endoscopic
radiofrequency coils could allow achieving better results in
specific organs. For prostate investigation for instance, an
endorectal .sup.13C is expected to strongly increase the
sensitivity of the method with respect to a standard whole body
resonator. Being the hyperpolarized signals typically available for
a time range in the order of 3 to 5 times the longitudinal
relaxation rate of
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane, the total
acquisition time for a metabolic MR procedure will not exceed 5
min. Spectroscopic imaging sequences such as Single Voxel
Spectroscopy (SVS) or Chemical Shift Imaging (CSI) need to be used
in order to separate the signal coming from the substrate from that
coming from the hyperpolarized metabolic product. Fast
spectroscopic imaging sequences such as EPSI are preferred due to
the limited time available for the acquisition.
[0114] In order for the method to provide enough sensitivity,
1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2'-cyclopropane formulations
and dissolution/transport protocols which allow to maintain at
least 10% polarization at time of injection are preferred.
Preferably, at least of about 20% polarization is maintained, more
preferably at least of about 30% polarization is maintained, even
more preferably at least of about 60% polarization is maintained,
most preferably at least of about 80% polarization is maintained.
Examples of said dissolution/transport protocols are described, for
instance, in WO 02/36005.
[0115] The present invention will be further illustrated by the
following examples.
EXAMPLES
[0116] Where not otherwise specified, chemicals and reagents used
in the following examples are commercially available or can be
prepared according to methods well-known in the art.
Example 1
Synthesis of
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
[0117] 284 mg, 2.55 mmol of
1-.sup.13C-1,1-Bis(hydroxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane,
(prepared as described by House, H. O. et al. . . . "The synthesis
of spiropentane-d8". J. Org. Chem. 1956, 21,1487-149) were put in a
glass flask and cooled to 0.degree. C. on an ice bath. Acetyl
chloride (3 ml, 34 mmol) was added slowly while stirring. After
complete addition the mixture was allowed to warm to room
temperature and stirred for additionally 12 h. The excess acetyl
chloride and the formed hydrochloric gas were then removed in
vacuum. The compound of formula (II)
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
was recovered as a colorless oil; yield: 445 mg (2.27 mmol,
90%).
[0118] Spectral data are consistent with the expected structure, as
illustrated below:
##STR00005##
[0119] .sup.1H NMR (Acetone-d.sub.6, ppm): 2.01 (singlet)
[0120] .sup.13C NMR (D.sub.2O, ppm): 9.4 (multiplet, .beta.), 19.6
(singlet, .alpha., .sup.13C label), 21.4 (singlet, .epsilon.), 70.1
(multiplet, .gamma.), 175.8 (singlet, .delta.)
Example 2
Preparation of Hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
[0121] A) Finland radical, carboxylic acid form (0.7 mg, 0.67
.mu.mol) was dissolved in
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
(45 .mu.l, 50.7 mg, 0.27 mmol). To the solution was added a DMSO
solution of the gadolinium complex
([alfa1,alfa4,alfa7-tris[(phenylmethoxy)methyl]-1,4,7,10-tetraazacyclodod-
ecane-1,4,7,10-tetraacetato(4-)] gadolinate(1-)]hydrogen) (0.75 mg
of a 100 .mu.mol/g solution). The concentration of radical and
gadolinium were 15 mM and 1.6 mM respectively.
[0122] B) 30 .mu.mol of a
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
sample made following the description in example 2.A was
hyperpolarized. The composition was hyperpolarised under DNP
conditions at 1.2 K in a 3.35 T magnetic field under irradiation
with microwave (93.900 GHz). The polarization build-up constant was
750 s. The solid-state polarization was approx. 15%.
[0123] C) The sample was dissolved in 5 ml phosphate buffer (40 mM,
pH 7.3). The pH after dissolution was 7.3. The solution was
collected directly into a 10 mm NMR tube and transferred to a 14.1
T magnet (pH 7.2) where a time series of 5 degree 1D .sup.13C-NMR
spectra were recorded with a total delay between the pulses of 3 s.
The liquid state polarization was 13% (12 s after dissolution) and
the liquid state T.sub.1 was approx. 85 s at 14.1 T and 37 C. The
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
was not hydrolysed in the dissolution process, FIG. 1.
Example 3
Comparison Between Metabolism of Hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
and Hyperpolarized 1-.sup.13C-pyruvate in Rat Liver
(Morris7777)
[0124] Materials and Methods
[0125] The experiments were performed with a co-polarization of
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
and 1-.sup.13C-pyruvic acid in equal amounts of compounds (30
.mu.mol) resulting in a concentration of approx. 3.5 mM of each
substrate in the experiments. The DNP preparation of
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
was performed as described in example 2 and the DNP preparation of
1-.sup.13C-pyruvate was performed as described in WO 2006/011809.
The two substrates were co-polarized without mixing the
substrates.
[0126] Rat liver cancer cells (Morris7777) were grown in RPMI +10%
FBS and antibiotics. Following trypsin harvesting 10 million cells
were redissolved in 500 .mu.l phosphate buffer (PBS) and
transferred to a 10 mm NMR tube and placed with connecting tubing
in a 14.1 T magnet at 37 C.
[0127] Following dissolution in 5 ml phosphate buffer (40 mM pH
7.3) with addition of 2.5 .mu.l NaOH to neutralize the pyruvic acid
2 ml of the substrate mixture was injected into 10 million cells in
suspension. A series of 20 degree pulses every 2 s (56 scans in
total) was acquired. The acquisition was started just before
injection of the hyperpolarized substrate. Data are presented as
metabolite signals as a function of time or as area under the curve
of the metabolite signals.
[0128] Results
[0129] An account of produced hyperpolarized
1-.sup.13C-1-(acetoxy(methyl-d.sub.2))-1-(hydroxy(methyl-d.sub.2))-2,2'-d-
.sub.4-cyclopropane and 1-.sup.13C-lactate in rat liver cancer
cells are shown in FIG. 2 and Table 2.
TABLE-US-00002 TABLE 2 Area under the metabolic curves for the two
metabolites arising from
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
and from 1-.sup.13C-lactate in Morris7777 cells. AUC (arb.
Metabolite Units)
1-.sup.13C-1-(acetoxy(methyl-d.sub.2))-1-(hydroxy(methyl-d.sub.2))-
43480 2,2'-d.sub.4-cyclopropane
1-.sup.13C-1,1-(dihydroxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
1559 1-.sup.13C-lactate 282
[0130] It can be seen from this example that it is possible to
follow the build-up of both the mono ester,
1-.sup.13C-1-(acetoxy(methyl-d.sub.2))-1-(hydroxy(methyl-d.sub.2))-2,2'-d-
.sub.4-cyclopropane and
1-.sup.13C-1,1-(dihydroxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
resulting from the hydrolysis of hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
in whole Morris7777 cells. It can also be appreciated that the
metabolic conversion of hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
is approx. 15 times higher than that of hyperpolarized 1-.sup.13C
pyruvate when comparing the maximum metabolite signal. Due to the
very long T.sub.1 of
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
the signal area under the curve of the mono-ester metabolite is
significantly larger than the area under the curve of
1-.sup.13C-lactate. This enables a potentially high quality image
of the in vivo signal with the
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
metabolite exploring an AUC which is almost 30 times that of
1-.sup.13C-lactate.
Example 4
Carboxyl esterase CE-2 Activities Measured with Hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
in Human Prostate Cancer Cells (PC-3) in Comparison to
Hyperpolarized 1-.sup.13C-pyruvate
[0131] Materials and Methods
[0132] The experiments were performed with a co-polarization of
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
and 1-.sup.13C-pyruvic acid in equal amounts of compounds (30
.mu.mol) resulting in a concentration of approx. 3.5 mM of each
substrate in the experiments. The DNP preparation of
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
was performed as described in example 2 and the DNP preparation of
1-.sup.13C-pyruvate was performed as described in WO 2006/011809.
The two substrates were co-polarized without mixing the
substrates.
[0133] Human cancer cells (PC-3) were grown in RPMI +10% FBS and
antibiotics. Following trypsin harvesting 10 million cells were
redissolved in 500 .mu.l phosphate buffer (PBS) and transferred to
a 10 mm NMR tube and placed with connecting tubing in a 14.1 T
magnet at 37.degree. C.
[0134] Following dissolution in 5 ml phosphate buffer (40 mM pH
7.3) with addition of 2.5 .mu.l NaOH to neutralize the pyruvic acid
2 ml of the substrate mixture was injected into 10 million cells in
suspension. A series of 20 degree pulses every 2 s (56 scans in
total) was acquired. The acquisition was started just before
injection of the hyperpolarized substrate. Data are presented as
metabolite signals as a function of time or as area under the curve
of the metabolite signals.
[0135] Results
[0136] An account of produced hyperpolarized
1-.sup.13C-1-(acetoxy(methyl-d.sub.2))-1-(hydroxy(methyl-d.sub.2))-2,2'-d-
.sub.4-cyclopropane and 1-.sup.13C-lactate in human prostate cancer
cells are shown in FIG. 3 and Table 3.
TABLE-US-00003 TABLE 3 Area under the metabolic curves for
1-.sup.13C-1-(acetoxy(methyl-
d.sub.2))-1-(hydroxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane and
1-.sup.13C-lactate in PC-3 cells. AUC (arb. Metabolite Units)
1-.sup.13C-1-(acetoxy(methyl-d.sub.2))-1-(hydroxy(methyl-d.sub.2))-
20550 2,2'-d.sub.4-cyclopropane 1-.sup.13C-lactate 3766
[0137] It can be seen from this example that it is possible to
follow the build-up of the mono ester,
1-.sup.13C-1-(acetoxy(methyl-d.sub.2))-1-(hydroxy(methyl-d.sub.2))-2,2'-d-
.sub.4-cyclopropane resulting from the hydrolysis of hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
in whole PC-3 cells. It can also be appreciated that the metabolic
conversion of hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
is more than 3 times higher than that of hyperpolarized 1-.sup.13C
pyruvate when comparing the maximum metabolite signal. Due to the
very long T.sub.1 of
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
the signal area under the curve of the mono-ester metabolite is
significantly longer than the area under the curve of
1-.sup.13C-lactate. This enables a potentially high quality image
of the in vivo signal with the
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
metabolite exploring an AUC which is almost 6 times that of
1-.sup.13C-lactate.
Example 5
Carboxyl esterase CE-2 Activities Measured with Hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
in Human Prostate Cancer Cells (PC-3) in Comparison to Healthy
Human Prostate Cells (PNT-1A)
[0138] Materials and Methods
[0139] The experiments were performed with a polarization of
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
(30 .mu.mol) resulting in a concentration of approx. 3.5 mM of this
substrate in the experiments. The DNP preparation of
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
was performed as described in example 2.
[0140] Human prostate cancer cells (PC-3) or immortalized human
prostate healthy cells were grown in RPMI +10% FBS and antibiotics.
Following trypsin harvesting 10 million cells were redissolved in
500 .mu.l phosphate buffer (PBS) and transferred to a 10 mm NMR
tube and placed with connecting tubing in a 14.1 T magnet at
37.degree. C.
[0141] Following dissolution in 5 ml phosphate buffer (40 mM pH
7.3), 2 ml of the substrate mixture was injected into 10 million
cells in suspension. A series of 20 degree pulses every 2 s (56
scans in total) was acquired. The acquisition was started just
before injection of the hyperpolarized substrate. Data are
presented as metabolite signals as a function of time or as area
under the curve of the metabolite signals.
[0142] Results
[0143] An account of produced hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
in prostate cancer and prostate healthy cells is shown in FIG.
4
[0144] It can be seen from this example that it is possible to
follow the build-up of the mono ester,
1-.sup.13C-1-(acetoxy(methyl-d.sub.2))-1-(hydroxy(methyl-d.sub.2))-2,2'-d-
.sub.4-cyclopropane resulting from the hydrolysis of hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
in human prostate cancer PC-3 cells and in human healthy prostate
cells. It can also be appreciated that the metabolic conversion of
hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
is approx. 6 times higher in the diseased cells than in the healthy
cells. This difference suggests that a large contrast between
diseased and healthy tissue can be expected in a human cancerous
prostate.
Example 6
Conversion of Hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
into Hyperpolarized
1-.sup.13C-1-(acetoxy(methyl-d.sub.2))-1-(hydroxy(methyl-d.sub.2))-2,2'-d-
.sub.4-cyclopropane and
1-.sup.13C-1,1-Bis(hydroxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
in the Healthy Prostate of Living Rats.
[0145] Materials and Methods
[0146] DNP-MRI experiment has been performed on 2 healthy
Copenhagen rats, 9 weeks old, with average weight of 180 g.
[0147] 0.24 mmol of a
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
sample prepared following the description in Example 2.A was
hyperpolarized according to the conditions reported in example 2.B.
The solid sample was then dissolved in 5 ml TRIS buffer (100 mM, pH
7.7) to obtain a hyperpolarized solution with 48 mM substrate
concentration and a pH of 7.
[0148] 2.8 ml of the dissolved hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
solution was injected intravenously at an injection rate of about
0.25 m L/s through a catheter placed in the tail vein of the
animal, resulting in a total administered dose of about 0.4
mmol/kg. A time series of 64 NMR spectra separated by 3 s and
generated by 10.degree. radiofrequency pulses was acquired starting
from 15 s before injection of the hyperpolarized substrate, in
order to follow the whole metabolic fate of the molecules and the
decay of the hyperpolarized signal. The .sup.13C MR signal was
collected by a 20 mm surface coil placed around the prostate.
Spatial localization of the signal has been achieved by combining
the limited sensitivity volume of the receiving coil with a slice
selective spectroscopic sequence including a single gradient kept
on only during the excitation period.
[0149] Results
[0150] Hyperpolarized
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
is taken up by prostate cells and converted into its hyperpolarized
metabolites,
1-.sup.13C-1-(acetoxy(methyl-d.sub.2))-1-(hydroxy(methyl-d.sub.2))-2,2'-d-
.sub.4-cyclopropane and
1-.sup.13C-1,1-Bis(hydroxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane,
by a two steps metabolic process clearly observable, in vivo, on
the time scale of the DNP experiment. A .sup.13C NMR sum spectrum,
obtained by integrating over time all the non-vanishing spectra of
a time series acquired on a representative animal, is reported in
FIG. 5. The chemical shift differences between the injected
substrate and the relevant metabolic products are notable, allowing
an unassailable identification of the different .sup.13C labelled
species which are present in the tissue under investigation. The
time evolution of the hyperpolarized .sup.13C signals of individual
species is shown in FIG. 6. While the signal of
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclop-
ropane is decaying, the products signal firstly build up, due to
metabolic conversion of
1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2'-d.sub.4-cyclopropane
and then gradually decay according to their T.sub.1 relaxation
rate.
* * * * *