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): ##STR00001##
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.

Claims

1. A method for operating an MRI system comprising the steps of: a. administering a solution of hyperpolarized compound 1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2-cyclopropane to a subject who is affected or suspected to be affected by a tumor, submitting the subject to a radiation having a frequency selected to excite nuclear spin transitions in .sup.13C nuclei; b. detecting an MR signal from said excited nuclei of at least one hyperpolarized metabolic product selected from the group consisting of 1-.sup.13C-1-(acetoxy(methyl))-1-(hydroxy(methyl))-2,2-cyclopropane and 1-.sup.13C-1, 1-(dihydroxy(methyl))-2,2-cyclopropane; 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.

2. The method according to claim 1 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.

3. The method according to claim 2, 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.

4. The method according to claim 2, 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.

5. The method according to claim 2, 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.

6. The method according to claim 5 wherein said second signal is determined before, after or at the beginning of the treatment.

7. A method for operating an MRI system comprising the steps of: a. administering a solution of hyperpolarized deuterated compound 1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2-cyclopropane of formula (II): ##STR00007## to a subject who is affected or suspected to be affected by a tumor, submitting the subject to a radiation having a frequency selected to excite nuclear spin transitions in .sup.13C nuclei; b. detecting an MR signal from said excited nuclei of at least one hyperpolarized metabolic product selected from the group consisting of 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; 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.

8. The method according to claim 7 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.

9. The method according to claim 8, 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.

10. The method according to claim 8, 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.

11. The method according to claim 8, 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.

12. The method according to claim 11 wherein said second signal is determined before, after or at the beginning of the treatment.

Description

FIGURES

(1) FIG. 1. Dissolution spectrum of hyperpolarized 1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2-d.sub.4-cyclopropane.

(2) 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.

(3) 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.

(4) 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.

(5) 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))-2,2-d.sub.4-cyclopropane).

(6) 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(methy-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

(7) 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.

(8) 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.

(9) 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, agent as commonly intended in the state of the art and for example disclosed in WO200977575 and the references cited therein.

(10) 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.

(11) Within the meaning of the present invention, the term hyperpolarized means the nuclear spin polarization of a compound higher than thermal equilibrium.

(12) 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.

(13) 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.

(14) The new compound 1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2-cyclopropane is synthesized 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 the mixture.

(15) 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.

(16) 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:

(17) ##STR00005##
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.4 to give the desired diol compound.

(18) 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.

(19) 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.

(20) 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).

(21) The diacetate ester of the invention, 1-.sup.13C-1,1-Bis(acetoxy(methyl))-2,2-cyclopropane, is a substrate for the CE2 isoform.

(22) 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.

(23) 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.

(24) TABLE-US-00001 TABLE 1 T1 of 1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2-d.sub.4-cyclopropane 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 2 5-13C-glutamine 22 2 1,4-13C2-Fumarate 35 3 1,4-13C2-malate 30 3 1-13C-bicarbonate 30 2 1-13C-2-oxoglutarate 30 3

(25) 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 suffer 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.

(26) 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.

(27) 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.

(28) In a preferred embodiment of the invention, said first signal deriving from said tumor is lower than said second MR signal.

(29) 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.

(30) 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.

(31) 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.

(32) 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.

(33) 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.

(34) 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.

(35) 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.

(36) 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.

(37) 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.

(38) 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).

(39) 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.

(40) According to the present invention, a difference between said first and said second signal is determined. This difference (S.sub.1S.sub.2) is important for the different scopes of the present invention.

(41) 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.1S.sub.2)/R.

(42) 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.

(43) 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.

(44) In an embodiment of the invention, the method is performed on a subject who is suspected to suffer or suffers from a tumor.

(45) 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.

(46) 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.

(47) Typical metabolic imaging procedures with the compound of the invention in human subjects should be performed at magnetic fields 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.

(48) 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.

(49) The present invention will be further illustrated by the following examples.

EXAMPLES

(50) 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 1-13C-1,1-Bis(acetoxy(methyl-d2))-2,2-d4-cyclopropane

(51) 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 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%).

(52) Spectral data are consistent with the expected structure, as illustrated below:

(53) ##STR00006##

(54) .sup.1H NMR (Acetone-d.sub.6, ppm): 2.01 (singlet)

(55) .sup.13C NMR (D.sub.2O, ppm): 9.4 (multiplet, ), 19.6 (singlet, , .sup.13C label), 21.4 (singlet, ), 70.1 (multiplet, ), 175.8 (singlet, )

Example 2. Preparation of Hyperpolarized 1-13C-1,1-Bis(acetoxy(methyl-d2))-2,2-d4-cyclopropane

(56) A) Finland radical, carboxylic acid form (0.7 mg, 0.67 mol) was dissolved in 1-.sup.13C-1,1-Bis(acetoxy(methyl-d.sub.2))-2,2-d.sub.4-cyclopropane (45 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-tetraazacyclododecane-1,4,7,10-tetraacetato(4-)]gadolinate(1-)]hydrogen) (0.75 mg of a 100 mol/g solution). The concentration of radical and gadolinium were 15 mM and 1.6 mM respectively.

(57) B) 30 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%.

(58) 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 3Comparison Between Metabolism of Hyperpolarized 1-13C-1,1-Bis(acetoxy(methyl-d2))-2,2-d4-cyclopropane and Hyperpolarized 1-13C-pyruvate in Rat Liver (Morris7777)

(59) Materials and Methods

(60) 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 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.

(61) Rat liver cancer cells (Morris777) were grown in RPMI+10% FBS and antibiotics. Following trypsin harvesting 10 million cells were redissolved in 500 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.

(62) Following dissolution in 5 ml phosphate buffer (40 mM pH 7.3) with addition of 2.5 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.

(63) Results

(64) 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.

(65) TABLE-US-00002 TABLE 2 Area under the metabolic curves for for 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 Metabolite (arb. Units) 1-.sup.13C-1-(acetoxy(methyl-d.sub.2))-1- 43480 (hydroxy(methyl-d.sub.2))-2,2-d.sub.4-cyclopropane 1-.sup.13-1,1-(dihydroxy(methyl-d.sub.2))- 1559 2,2-d.sub.4-cyclopropane 1-.sup.13C-lactate 282

(66) 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(methy-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 1-13C-1,1-Bis(acetoxy(methyl-d2))-2,2-d4-cyclopropane in Human Prostate Cancer Cells (PC-3) in Comparison to Hyperpolarized 1-13C-pyruvate

(67) Materials and Methods

(68) 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 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.

(69) Human cancer cells (PC-3) were grown in RPMI+10% FBS and antibiotics. Following trypsin harvesting 10 million cells were redissolved in 500 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.

(70) Following dissolution in 5 ml phosphate buffer (40 mM pH 7.3) with addition of 2.5 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.

(71) Results

(72) 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.

(73) 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 Metabolite (arb. Units) 1-.sup.13C-1-(acetoxy(methyl-d.sub.2))-1- 20550 (hydroxy(methyl-d.sub.2))-2,2-d.sub.4-cyclopropane 1-.sup.13C-lactate 3766

(74) 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(methy-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 vive 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 1-13C-1,1-Bis(acetoxy(methyl-d2))-2,2-d4-cyclopropane in Human Prostate Cancer Cells (PC-3) in Comparison to Healthy Human Prostate Cells (PNT-1A)

(75) Materials and Methods

(76) 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 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.

(77) 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 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.

(78) 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.

(79) Results

(80) 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.

(81) 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 1-13C-1,1-Bis(acetoxy(methyl-d2))-2,2-d4-cyclopropane into Hyperpolarized 1-13C-1-(acetoxy(methyl-d2))-1-(hydroxy(methyl-d2))-2,2-d4-cyclopropane and 1-13C-1,1-Bis(hydroxy(methyl-d2))-2,2-d4-cyclopropane in the Healthy Prostate of Living Rats

(82) Materials and Methods

(83) DNP-MRI experiment has been performed on 2 healthy Copenhagen rats, 9 weeks old, with average weight of 180 g.

(84) 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 2A 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.

(85) 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 mL/s through a catheter placed in the tail vein of the animal, resulting in a total administered close of about 0.4 mmol/kg. A time series of 64 NMR spectra separated by 3 s and generated by 10 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.

(86) Results

(87) 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(methy-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 labeled 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-cyclopropane 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.