[.SUP.18.F]-labelled lactate derivative as pet radiotracer

Abstract

A positron emission tomography (PET) radiotracer for imaging lactate uptake, wherein the tracer is a [.sup.18F]-labelled lactate derivative which is [.sup.18F]-3-fluoro-2-hydroxypropionic acid: ##STR00001##
Also, a process for the radiosynthesis of the [.sup.18F]-labelled lactate derivative. Further, the use of the [.sup.18F]-labelled lactate derivative for imaging lactate uptake in living cells, especially in humans.

Claims

1. A compound which is [18F]-3-fluoro-2-hydroxypropionic acid: ##STR00011## or a pharmaceutically acceptable salt and/or solvate thereof.

2. The compound according to claim 1, wherein the salt is [.sup.18F]-3-fluoro-2-hydroxypropionate sodium salt.

3. A pharmaceutical composition comprising the compound according to claim 1, and at least one pharmaceutically acceptable excipient.

4. A medicament comprising the compound according to claim 1.

5. A method for predicting and/or monitoring if a tumor of an individual displays a therapeutic response to a treatment for modulating lactate uptake and/or lactate metabolism, comprising (1) when the method is a method for monitoring, administering to an individual a treatment for modulating lactate uptake and/or lactate metabolism; (2) administering to said individual an amount of the compound according to claim 1, sufficient to be detected by PET; (3) forming at least one PET image of the tumor; and (4) determining the lactate uptake of the tumor by observing the image.

6. The method according to claim 5, wherein the treatment for modulating lactate uptake is selected from drugs that inhibit monocarboxylate transporters.

7. The method according to claim 5, wherein the treatment for modulating lactate metabolism is selected from drugs that inhibit lactate dehydrogenase.

8. A method for in vitro detection of lactate uptake in a tissue, said method comprising (1) contacting said tissue with an amount of the compound according to claim 1, sufficient to be detected by PET; (2) forming at least one PET image; and (3) determining lactate uptake by observing the image.

9. A method for imaging cancer cells, said method comprising (1) administering to an individual with cancer an amount of the compound according to claim 1 sufficient to be detected by PET; and (2) forming at least one PET image showing the distribution of said compound, within the cancer cells of the individual.

10. A method for monitoring a cancer therapy in an individual, said method comprising (1) administering a cancer therapy to an individual; (2) administering to said individual an amount of the compound according to claim 1, sufficient to achieve imaging; and (3) performing imaging using PET by detecting a signal from said compound, within the individual, to follow the response of the individual to the therapy.

11. A process of manufacturing of [.sup.18F]-3-fluoro-2-hydroxypropionic acid or a pharmaceutically acceptable salt and/or solvate thereof, comprising the following steps: a) an epoxide-ring opening reaction on benzyl oxirane-2-carboxylate (II) ##STR00012## in presence of [.sup.18F]-fluoride, to afford [.sup.18F]-benzyl 3-fluoro-2-hydroxypropionate (III*) ##STR00013## and b) hydrolysis of [.sup.18F]-benzyl 3-fluoro-2-hydroxypropionate (III*) to afford [.sup.18F]-3-fluoro-2-hydroxypropionic acid or a pharmaceutically acceptable salt and/or solvate thereof.

12. The process according to claim 11, comprising a preliminary step of synthesis of benzyl oxirane-2-carboxylate (II) by epoxidation of benzyl acrylate.

13. The method according to claim 6, wherein the drug that inhibits monocarboxylate transporters is a drug that inhibits monocarboxylate transporter 1.

14. The method according to claim 7, wherein the drugs that inhibit lactate dehydrogenase are selected from drugs that inhibit lactate dehydrogenase B, mitochondrial pyruvate carrier or alanine transaminase.

15. A method to detect lactate uptake in a cell or a population of cells, said method comprising: (1) administering to a cell or a population of cells an amount of the compound according to claim 1 sufficient to be detected by PET; (2) forming at least one PET image showing the distribution of said compound within the cell or the population of cells; and (3) determining lactate uptake by observing the image.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. A, Co-elution spectra of ()-[.sup.18F]-benzyl 3-fluoro-2-hydroxypropionate (III*) and nonradioactive benzyl 3-fluoro-2-hydroxypropionate (III), and ()-[.sup.18F]-2-fluoro-3-hydroxybenzylacrylate (IV*) and nonradioactive 2-fluoro-3-hydroxybenzylacrylate (IV) on a Supelco Discovery C18 HPLC column equipped with UV (A1) and NaI -ray (A2) detectors. B, Elution spectrum of ()-[.sup.18F]-3-fluoro-2-hydroxypropionate (V*) on a Nucleosil C18 Pyramid HPLC column equipped with a NaI -ray detector.

(2) FIG. 2. Elution spectrum of ()-[.sup.18F]-benzyl 3-fluoro-2-hydroxypropionate (III*) and ()-[.sup.18F]-2-fluoro-3-hydroxybenzylacrylate (IV*) on a Supelco Discovery C18 HPLC column equipped with a NaI -ray detector, showing regioisomer ratio when tert-amyl alcohol was used as a fluorination solvent (Method II).

(3) FIG. 3. 3-fluoropyruvate can be reduced to 3-fluoralacate by lactate dehydrogenase (LDH). A, Scheme of the reaction used for 3-fluoropyruvate reduction. B, 3-fluorpyruvate and 3-fluoralacate detection using mass spectrometry after the reaction schematized in A (n=3; N=1).

(4) FIG. 4. Oxidative human cancer cells trap ()-[.sup.18F]-3-fluoro-2-hydroxypropionate (V*). A, Representative western blots showing MCT1 expression in SiHa, HeLa and SQD9 human cancer cells. B, The oxygen consumption rate (OCR) of SiHa, HeLa and SQD9 cells on a Seahorse bioanalyzer. Cells received either glucose (25 mM)+L-lactate (10 mM) or only L-lactate (10 mM) as oxidative fuels in DMEM containing 10% of dialyzed FBS (n=8, *P<0.05, ***P<0.005). C, Cancer cells (or empty wells; blanks) were incubated during 6 min in the presence of ()-[.sup.18F]-3-fluoro-2-hydroxypropionate (V*) (45 Ci/ml), washed, and intracellular .sup.18F activity was measured using a Wiper Gold -counter (n=12-14, N=2, ***P<0.001).

(5) FIG. 5. MCT1 inhibitor AR-C155858 blocks the in vivo uptake of ()-[.sup.18F]-3-fluoro-2-hydroxypropionate (V*) by SiHa tumors in mice. Mice were bearing 2 SiHa tumors expressing a control shRNA (shCTR) or a shRNA against MCT1 (shMCT1). A, Representative images of vehicle-pretreated mice showing the physiological distribution of ()-[.sup.18F]-3-fluoro-2-hydroxypropionate (V*) 10, 30 and 60 min after tail vein injection (215 Ci in 100 L). Color scale is normalized for the injected dose and animal weight. B, Western blot showing MCT1, MCT4, -actin and Hsp90 expression in SiHa cells infected with shCTR or shMCT1 (Representative of n=3). C, Same as in A, except that mice were pretreated with AR-C155858 (5 mg/Kg IV 10 min before tracer injection). The representative image shows the exact same mouse as in A (30 min tracer image), assessed the day after. The bladder is indicated. D, Quantification of A and C (n=6-7; N=2, ***P<0.001).

(6) FIG. 6. ()-[.sup.18F]-3-fluoro-2-hydroxypropionate allows to document an early response of SQD9 head and neck cancer to MCT1 inhibitors AR-C155858 and AZD3965. MCT1 inhibitors AR-C155858 or AZD3965 were injected intravenously at a dose of 5 mg/kg. ()-[.sup.18F]-3-fluoro-2-hydroxypropionate (250 Ci) was injected intravenously 10 minutes after injection. Images were acquired 30 minutes after tracer injection. Arrows indicate SQD9 tumor localization (n=6). ***P<0.005 compared to vehicle, using Student's t test.

EXAMPLES

(7) The present invention is further illustrated by the following examples.

I. Chemical Examples

I.1. Material and Methods

(8) Chemicals.

(9) [.sup.18O]H.sub.2O was from Rotem. Benzyl acrylate was from Alpha Aesar; DMSO and tetrabutylammonium bicarbonate (TBAHCO.sub.3) from ABX; H.sub.3PO.sub.4 from Riedel-de Hain; Kryptofix 2.2.2. from Merck; NaH.sub.2PO.sub.4 and HPLC acetonitrile from VWR; and CDCl.sub.3 and TMS from Euristop. All other reagents were from Sigma-Aldrich.

(10) High-Performance Liquid Chromatography (HPLC).

(11) HPLC was performed on Gilson equipment (305 and 302 pumps) equipped with UV/VIS-151 and -ray NaI detectors connected in series and monitored by a GABI Star interface module (Raytest). Columns were for semi-preparative HPLC: Dionex Supelco Discovery C18, 5 m, 25010 mm; for analytical HPLC: MN, 150/4.6 Nucleosil 100-5 C18, 150 mm, ID: 4.6 mm and IonPac AS15, Dionex.

(12) Production of [.sup.18F]-Fluoride.

(13) [.sup.18F]-fluoride was produced on a medical-isotope cyclotron (IBA Cyclone 18/9) using a [.sup.18O]H.sub.2O liquid target. After irradiation, the target water was passed through a Chromafix 30-PS-HCO.sub.3 (Macherey-Nagel) or Accel Plus QMA Sep Pak light cartridge (Waters) to trap the [.sup.18F]-fluoride.

(14) General Scheme of Synthesis:

(15) ##STR00010##

I.2. Synthesis of benzyl oxirane-2-carboxylate (II)

(16) 3-Chloroperoxybenzoic acid (14.04 g) was added to a solution of benzyl acrylate (I) (23.04 mmol in 90 mL of dry dichloromethane (DCM)). The reaction mixture was heated under reflux and stirring for 7 days. DCM (100 mL) was then added to the solution, and washed twice with a saturated aqueous solution of sodium carbonate. The remaining DCM fraction was concentrated to 30 mL (rotavapor vacuum), and ethyl acetate (150 mL) was added. This solution was again washed twice with a saturated aqueous solution of sodium carbonate, and the recombined organics layers were dried over sodium sulfate, filtered and concentrated to dryness under reduced pressure. The crude was finally purified by silica gel chromatography using cyclohexane/ethyl acetate (95/5, 100 mL; and 10/90, 800 mL), and remaining volatiles were removed under vacuum to yield the desired compound (II). Yield: 53%, .sup.1H NMR (CDCl.sub.3 with 0.03% v/v TMS, 400 MHz): 7.38 (5.29H, m, H.sub.d, H.sub.e and H.sub.f), 5.18-5.27 (2.7H, q, H.sub.c), 3.47-3.49 (1H, dd, H.sub.b), 2.94-3.01 (2.18H, qd, H.sub.a).

I.3. Synthesis of ()-[.SUP.18.F]-3-fluoro-2-hydroxypropionate (V*)

(17) Method I.

(18) A Chromafix 30-PS-HCO.sub.3 cartridge loaded with [.sup.18F]-fluoride was eluted in reverse order to a reaction vessel using a solution of 0.075 M of tetrabutylammonium bicarbonate (TBAHCO.sub.3, 80 L, 6 mol) in acetonitrile (0.9 mL). Anhydrous [.sup.18F]-fluoride was obtained by azeotropic distillation with acetonitrile at 95 C. under a stream of helium. [.sup.18F]-fluoride recovery was of more than 80%. Benzyl oxirane-2-carboxylate (II) (10 L) dissolved in anhydrous DMSO (1 mL) was added to the [.sup.18F]-fluoride and was reacted for 10 minutes at 120 C. After cooling, the reaction mixture was diluted by 3.5 mL water and passed through a neutral alumina cartridge (Waters) to discard unreacted [.sup.18F]-fluoride. It yield 2 regioisomers: ()-[.sup.18F]-benzyl 3-fluoro-2-hydroxypropionate (III*) and ()-[.sup.18F] benzyl 2-fluoro-3-hydroxypropionate (IV*). About 90% of the [.sup.18F] radioactivity incorporated in organic molecules was related to both regioisomers (III*) and (IV*), with an about 1/1 ratio (FIG. 1A2).

(19) Compounds (III*) and (IV*) (NaI detector) co-eluted with nonradioactive benzyl 3-fluoro-2-hydroxypropionate (III) and benzyl 2-fluoro-3-hydroxypropionate (IV) (UV detector), respectively (FIGS. 1A2 and 1A1).

(20) The ()-[.sup.18F]-benzyl 3-fluoro-2-hydroxypropionate (III*) was isolated by semi-preparative HPLC (20 mM NaH.sub.2PO.sub.4/CH.sub.3CN 70/30, 3 mL/min, retention time=21 min), diluted with water (1.5vol), and loaded on a conditioned C18 Sep-Pak cartridge (Waters). The cartridge was rinsed with 10 mL of water and then loaded with 0.5 N NaOH. After 5 minutes, ()-[.sup.18F]-3-fluoro-2-hydroxypropionate (III*) was eluted with 1 mL of water, and pH set to 7.0 by the addition of H.sub.3PO.sub.4. ()-[.sup.18F]-3-fluoro-2-hydroxypropionate (V*) was characterized by analytical HPLC (IonPac AS15, Dionex, 14 mM of NaOH as eluent), with a retention time of 5.25 min (FIG. 1B).

(21) Method II.

(22) A Chromafix 30-PS-HCO.sub.3 cartridge loaded with [.sup.18F]-fluoride was eluted in reverse order to a reaction vessel using a 30 L aqueous solution of 0.55 mg K.sub.2C.sub.2O.sub.4 (3.0 mol)/2.25 mg Kryptofix 2.2.2. (6.0 mol) diluted in 1 mL of Trace Select methanol. Anhydrous [.sup.18F]-fluoride was obtained by azeotropic distillation with acetonitrile at 95 C. under a stream of helium. Benzyl oxirane-2-carboxylate (II) (10 L) dissolved in anhydrous 2-methyl-2-butanol (1 mL) was added to the [.sup.18F]-fluoride. The vial was sealed and heated at 105 C. for 10 minutes. Solvent was then evaporated to dryness at 100 C. under a stream of helium. After cooling, the reaction mixture was diluted by 4.5 mL of an acetonitrile/water 1/2 solution, and passed through a neutral alumina cartridge to discard unreacted [.sup.18F]-fluoride. ()-[.sup.18F]-3-fluoro-2-hydroxypropionate (V*) was then prepared as in Method I.

(23) In method II, by conducting the radiofluorination reaction in 2-methyl-2-butanol, a protic solvent, the regioselectivity of the epoxide opening improved up to more than 80% for ()-[.sup.18F]-benzyl 3-fluoro-2-hydroxypropionate (III*) (FIG. 2), and the global fluorination yield slightly increased (15-20%).

I.4. Synthesis of Reference Compound benzyl 3-fluoro-2-hydroxypropionate (III)

(24) To a solution of benzyl oxirane-2-carboxylate (II) (1.12 g) in dry DCM (7.5 mL) cooled to 0 C. was added Olah's reagent (Hydrogen fluoride pyridine: pyridine30%, hydrogen fluoride70%, 3.4 mL) dropwise. After reaching room temperature, the mixture was stirred for 35 h. The biphasic solution was added on a silica suspension in DCM (100 ml), then filtered and washed with 50 mL of DCM. The desired benzyl 3-fluoro-2-hydroxypropionate (III) was further purified over silica gel chromatography. .sup.1H NMR (CDCl.sub.3 with 0.03% v/v TMS, 400 MHz): 7.35-7.39 (5.29H, m, H.sub.d, H.sub.e and H.sub.f), 5.28 (1.85H, s, H.sub.c), 4.59-4.76 (1.94H, dqd, H.sub.a), 4.35-4.44 (0.92H, dquint, H.sub.b). The undesired regioisomer benzyl 2-fluoro-3-hydroxypropionate (IV) was obtained under the form of traces (FIG. 1A1).

II. Biological Examples

(25) Statistics.

(26) Data were analyzed using GraphPad Prism version 6.04 for Windows. All results are expressed as meanSEM. N refers to the number of independent experiments and n to the total number of replicates per treatment condition. Error bars are sometimes smaller than symbols. Student's t test and one-way ANOVA was used where appropriate. P<0.05 was considered to be statistically significant.

II.1. Lactate Dehydrogenase Assay

(27) A potential reduction of 3-fluoropyruvate to 3-fluoro-2-hydroxypropionate by lactate dehydrogenase (LDH) was measured in vitro using a previously reported protocol (Goncalves et al., Tetrahedron: Asymmetry, 1996, 7, 1237-1240).

(28) Briefly, 14.6 mg of nonradioactive 3-fluorpyruvate were dissolved in 4 mL of double distilled water containing 10 IU of rabbit muscle LDH (Sigma) and 5.3 IU of formate dehydrogenase (Sigma). The reaction was started by adding NADH to a final concentration of 0.2 mM and sodium formate to a final concentration of 40 mM. The final volume was adjusted to 5 mL with double distilled water. The reaction was carried out at 37 C. for 24 h under constant, gentle shaking at 120 rpm. Then, solution was spun through a 10 kDa filter to remove enzymes, and 3-fluoro-2-hydroxypropionate was detected by HPLC-MS using an Accela U(HPLC) equipped with a Luna Phenomenex 250*4.60 HPLC column and an ThermoScientific LTQ-ORBITRAP-XL fitted an electrospray ionization source working in negative mode.

(29) Data of FIG. 3 support the possibility that ()-[.sup.18F]-3-fluorolactate could be metabolized to [.sup.18F]-3-fluoropyruvate by LDH, i.e., along the oxidative pathway of lactate in oxidative cancer cells.

II.2. Oxidative Cancer Cells Take Up and Trap ()-[.SUP.18.F]-3-fluoro-2-hydroxypropionate In Vitro

(30) Cells and Gene Silencing.

(31) HeLa and SiHa human cervix squamous cell carcinoma and SDQ9 human laryngeal squamous cell carcinoma were from ATCC. Cells were routinely cultured in DMEM (Thermo Fischer) containing glucose (4.5 g/L), Glutamax and 10% FBS. MCT1-deficient and control SiHa cells were produced as previously described (De Saedeleer et al., Oncogene, 2014, 33, 4060-4068), using the following vectors from Open Biosystems: TRCN0000038340 (shMCT1-1) and TRCN0000038339 (shMCT1-2). Control shRNA (shCTR) was Addgene plasmid 1864.

(32) Western Blotting.

(33) Western blotting was performed as previously described (Van He et al., Front Pharmacol, 2015, 6, 228). Primary antibodies were a rabbits polyclonals against MCT1 (Merck Millipore # AB3538P) and MCT4 (Corbet C et al., Cancer Res, 2014, 74, 5507-5519); and mouse monoclonals against Hsp90 (BD Bioscience #610419), CD147 (BD Bioscience #555961) and -Actin (Sigma # A5441).

(34) Oximetry.

(35) Basal oxygen consumption rates were determined on a Seahorse XF96 bioenergetic analyzer according to manufacturer's recommendations. Twenty thousand cells per well were plated 18-h before the experiment in DMEM without glucose and glutamine, containing 10% dialyzed FBS, and L-lactate (10 mM), D-glucose (25 mM). Data are normalized to cell number at the end of the experiment.

(36) In Vitro Tracer Uptake Assay.

(37) A modified version of the .sup.14C-Lactate uptake assay described by Draoui et al., (Draoui et al., Bioorg Med Chem, 2013, 21, 7107-7117) was used. Briefly, 250,000 cells were plated in flat-bottom 24 well plates (t=0). When cells were attached (t=6 h), medium was replaced by DMEM without glucose and glutamine, containing 10% dialyzed FBS and 10 mM of L-lactate, pH 7.0. Cells were then incubated overnight at 37 C., 5% CO.sub.2. On the day of experiment, (t=24 h), cells medium was removed and cells were briefly washed twice with a modified KREBS solution without glucose (HEPES 25 mM, NaCl 120 mM, KCl 4.8 mM, KH.sub.2PO.sub.4 1.2 mM, MgSO.sub.4 1.2 mM, CaCl.sub.2 2 mM). Where indicated, the cells were treated during 12 min with -cyano-4-hydroxycinnamte (CHC, 30 M), AR-C155858 (10 M) or vehicle in KREBS containing 10 mM of L-lactate. After incubation, the solution was replaced by the KREBS solution containing 10 mM of L-Lactate, pharmacological agents or vehicle and [.sup.18F]-3-fluoro-2-hydroxypropionate (45 Ci/ml). Cells were incubated for 10 min for ()-[.sup.18F]-3-fluoro-2-hydroxypropionate, after which the solution was removed and the cells were washed 3 times with an ice-cold KREBS solution containing L-Lactate (10 mM). Cells were lysed with NaOH 0.1 N, and .sup.18F activity was measured in the cell lysate using a Wiper Gold -counter (Laboratory Technologies). Activity is expressed as % of initial dose. For background determination, wells without cells were treated in the exact same way.

(38) Results.

(39) To evaluate ()-[.sup.18F]-3-fluoro-2-hydroxypropionate as a potential tracer of lactate uptake by oxidative cancer cells, SiHa, HeLa and SQD9 cells were selected for in vitro assays. Indeed, all 3 cell lines did express MCT1 (FIG. 4A), and oximetry on a Seahorse bioanalyzer confirmed that HeLa and SQD9 were at least as oxidative as SiHa cells in vitro (FIG. 4B). As previously reported for SiHa (Sonveaux et al., J Clin Invest, 2008, 118, 3930-3942), the cells could use lactate as an oxidative fuel in the absence of glucose (FIG. 4B). In vitro, they took up and trapped ()-[.sup.18F]-3-fluoro-2-hydroxypropionate 6 min after the delivery of 45 Ci/ml of the tracer (FIG. 4C). At this time, intracellular doses ranged from 0.1% for SiHa and HeLa to 0.3% of the initial dose for SQD9. It was thus considered that ()-[.sup.18F]-3-fluoro-2-hydroxypropionate can qualify as a tracer of lactate uptake by oxidative cancer cells.

II.3. Validation of ()-[.SUP.18.F]-3-fluoro-2-hydroxypropionate as a Tracer of Lactate Uptake by Tumors In Vivo

(40) In Vivo Tracer Uptake Assay.

(41) All in vivo experiments were performed with approval of UCL Comit d'Ethique pour l'Experimentation Animale (approval ID 2014/UCL/MD/014) according to national and European animal care regulations. To avoid inter-subject variability, 500,000 SiHa-shCTR and SiHa-shMCT1 cells in a HBSS:Matrigel 1:1 solution were respectively injected in the left and right flank of same 6.5 week-old male NMRI nude mice. In another model, 1,000,000 SQD9 cells in a HBSS:Matrigel 1:1 solution were injected on the back of same 6.5 week-old male NMRI nude mice. Experiments were performed on tumors of 10 mm in diameter, i.e., about 3 weeks tumor cell inoculation. For intravenous injection, MCT1 inhibitor AR-C155858 (Tocris) was dissolved in 0.9% NaCl with 10% (2-hydroxypropyl)--cyclodextrin at a concentration of 2.5 mg/ml (Vijay et al., Pharm Res, 2015, 32, 1894-1906). For intravenous injection, MCT1 inhibitor AZD3965 (Selleckchem) was first dissolved in pure ethanol at a concentration of 100 mg/ml, then diluted in 0.9% NaCl with 10% (2-hydroxypropyl)--cyclodextrin at a final concentration of 2.5 mg/ml. ()-[.sup.18F]-3-fluoro-2-hydroxypropionate (150-250 Ci) was injected in the tail vein of the animals 10 min after the delivery of AR-C155858 (5 mg/Kg), AZD3965 (5 mg/Kg), or vehicle (70 L). At indicated times, a whole body 10-min static PET imaging (small-animal Mosaic PET Scan system, Philips Medical Systems) directly followed by a 10 min transmission CT scan (NanoSPECT/CT Small Animal Imager, Bioscan; source: 370 MBq .sup.137Cs; X-Ray tube voltage: 55 kVp; number of projections: 180; exposure time 1000 ms) were performed on isoflurane-anesthetized mice kept at 35 C. PET images were corrected for attenuation and reconstructed using fully 3D iterative algorithm 3D-RAMLA in a 128128120 matrix, with a voxel size of 1 mm.sup.3. CT images were reconstructed with a voxel size of 0.2210.2210.221 mm.sup.3. 2D Regions of interest (ROIs) were manually delineated on PET images using the PMOD software version 3.5 (PMOD technologies Ltd). Tumor localization was determined on PET/CT fused images. Ribcage and skin were used as internal and external limits, respectively. Tracer uptake is expressed as standard uptake value (SUV) calculated on the mean value of voxels within the manually defined 3D volume of interest (VOI).

(42) Results.

(43) To validate ()-[.sup.18F]-3-fluoro-2-hydroxypropionate as a tracer of lactate uptake by tumors, mice bearing 2 SiHa tumors that expressed either shCTR or shMCT1 were used. This model is the original model in which the metabolic symbiosis based on lactate exchange was demonstrated (Sonveaux et al., J Clin Invest, 2008, 118, 3930-3942). ()-[.sup.18F]-3-fluoro-2-hydroxypropionate was administered IV at a dose of 150-250 Ci. PET/CT images revealed that tracer distribution was time-dependent, with best tumor contrast 30 min after tracer injection (FIG. 5A). Other organs known to express MCTs and to take up lactate, such as the gut and the liver, were also labeled. At this time point, there was no detectable bone labeling that could have indicated defluorination. Of note, 60 min after tracer injection, spinal and joint labeling was detected, indicating that some defluorination had occurred (FIG. 5A). At 30 min, there was no apparent discrimination of SiHa-shCTR and SiHa-shMCT1 by the tracer. This lack of difference was explained by a compensatory overexpression of MCT4 upon MCT1 silencing, which was detected by western blotting (FIG. 5B).

(44) It was therefore decided to evaluate the ability of ()-[.sup.18F]-3-fluoro-2-hydroxypropionate to detect a pharmacological inhibition of MCT1, thus recapitulating at best a clinical treatment. One day after the initial determination of tracer biodistribution, the same group of mice was treated with MCT1 inhibitor AR-C155858 (5 mg/Kg) administered IV 10 min before a second PET/CT scan. Images acquired 30 min after ()-[.sup.18F]-3-fluoro-2-hydroxypropionate delivery revealed that MCT1 inhibition by AR-C155858 induced a highly significant decrease in tracer uptake in the tumors, liver and gut (FIGS. 5C&D). The bladder, which was already apparent on pre-treatment images (FIG. 5A, 30 min), was much more positively marked after systemic MCT1 inhibition (FIG. 5C), indicating that urine is the preferred route for ()-[.sup.18F]-3-fluoro-2-hydroxypropionate clearance. Accordingly, kidneys, that preferentially express MCT2 for lactate clearance, were labelled after treatment. Note that for comparison, PET/CT scans of the same mouse is shown in FIGS. 5A&C at the 30 min acquisition time, with a color scale normalized for the injected dose.

(45) To confirm the ability of ()-[.sup.18F]-3-fluoro-2-hydroxypropionate to detect a pharmacological inhibition of MCT1, the experiment was repeated using SQD9 human laryngeal squamous cell carcinoma cells in mice. ()-[.sup.18F]-3-fluoro-2-hydroxypropionate was administered IV at a dose of 250 Ci to the tumor-bearing mice, and PET-CT images were acquired showing that the tumors captured and accumulated the tracer. On the next day using the same mice, MCT1 inhibitor AR-C155858 (5 mg/Kg) or AZD3965 (5 mg/Kg) were administered IV 10 min before a second PET/CT scan. Images acquired 30 min after ()-[.sup.18F]-3-fluoro-2-hydroxypropionate delivery revealed that MCT1 inhibition by AR-C155858 or, alternatively, AZD3965 induced a highly significant decrease in tracer uptake in the tumors and liver (FIG. 6).