18 F-click labeling and preclinical evaluation of a new 18 F-folate for PET imaging
© Schieferstein et al.; licensee Springer. 2013
Received: 15 July 2013
Accepted: 31 August 2013
Published: 16 September 2013
The folate receptor (FR) is a well-established target for tumor imaging and therapy. To date, only a few 18 F-folate conjugates via 18 F-prosthetic group labeling for positron emission tomography (PET) imaging have been developed. To some extent, they all lack the optimal balance between efficient radiochemistry and favorable in vivo characteristics.
A new clickable olate precursor was synthesized by regioselective coupling of folic acid to 11-azido-3,6,9-trioxaundecan-1-amine at the γ-position of the glutamic acid residue. The non-radioactive reference compound was synthesized via copper-catalyzed azide-alkyne cycloaddition of 3-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)prop-1-yne and γ-(11-azido-3,6,9-trioxaundecanyl)folic acid amide. The radiosynthesis was accomplished in two steps: at first a 18 F-fluorination of 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl-4-methylbenzenesulfonate, followed by a 18 F-click reaction with the γ-azido folate. The in vitro, ex vivo, and in vivo behaviors of the new 18 F-folate were investigated using FR-positive human KB cells in displacement assays and microPET studies using KB tumor-bearing mice.
The new 18 F-folate with oligoethylene spacers showed reduced lipophilicity in respect to the previously developed 18 F-click folate with alkyl spacers and excellent affinity (K i = 1.6 nM) to the FR. Combining the highly efficient 18 F-click chemistry and a polar oligoethylene-based 18 F-prosthetic group facilitated these results. The overall radiochemical yield of the isolated and formulated product averages 8.7%. In vivo PET imaging in KB tumor-bearing mice showed a tumor uptake of 3.4% ID/g tissue, which could be reduced by FR blockade with native folic acid. Although the new 18 F-oligoethyleneglycole (OEG)-folate showed reduced hepatobiliary excretion over time, a distinct unspecific abdominal background was still observed.
A new 18 F-folate was developed, being available in very high radiochemical yields via a fast and convenient two-step radiosynthesis. The new 18 F-OEG-folate showed good in vivo behavior and lines up with several recently evaluated 18 F-labeled folates.
Since the folate receptor (FR) is a well-established target in tumor imaging and tumor therapy, many radiofolates and chemotherapeutics based on the natural ligand folic acid have been developed and investigated . Folic acid is a vitamin essential for de novo DNA synthesis in eukaryotic cells where it is converted into the co-enzyme 5,6,7,8-tetrahydrofolate and acts as a carrier of C1 building blocks . The FR is a glycosyl phosphatidylinositol-anchored protein which has a high affinity for folic acid (K d ~ 1 nM) and is (over)expressed in many types of human tumors, e.g., ovarian cancer or endometrial cancer –. The expression of the FR in healthy tissues, directly accessible from the bloodstream, is limited to the proximal tubules of the kidneys, where it is involved in the recycling of folic acid from renal excretion [6, 7]. Hence, specific accumulation of an intravenously administered radiofolate is mostly associated with a pathophysiological cause. Therefore, many folate conjugates, featuring different radionuclides for various applications, have been developed and evaluated in the past two decades . The introduction of FR targeting to tumor diagnostics in the field of nuclear imaging goes back to 1981 using 125I-labeled pteroylglutamic acid (equals folic acid) , which was not particularly promising. In spite of that, a number of radiofolates have been reported, many of which feature radionuclides useful in single photon emission computed tomography. Examples are 111In-diethylenetriamine pentaacetic acid-folates, 99mTc-folates, and 67Ga-folates, which showed promising results in preclinical in vivo tumor targeting –. In 2006, Bettio and co-workers synthesized a 18 F-labeled folate for application in positron emission tomography (PET), formed by amide coupling of the prosthetic group 4-[18 F]fluorobenzylamine and native folic acid. The coupling afforded a mixture of α- and γ-regioisomers, which was not separated before in vivo animal PET studies . Good visualization of FR-positive tumors was achieved; however, one major drawback was the time-consuming multistep radiosynthesis, the regioisomeric mixture, and low radiochemical yields. To overcome the complicated radiosynthesis and provide a regioselective product, another radiofolate was developed using the copper-catalyzed azide-alkyne cycloaddition (CuAAC, click reaction). The structural isomerism was circumvented by a regioselective derivatization at the carboxylic acid in the γ-position of the folate precursor . The radio-CuAAC clearly simplified the radiosynthesis and gave the first 18 F-click-labeled folate in high radiochemical yields within ≤90 min. However, in vivo animal PET imaging revealed an unfavorable biological distribution profile with a poor signal-to-noise ratio and a very high abdominal background, which were assumed to be related to the loss of hydrophilicity of the tracer. To retain the polarity of the radiofolate, which is obviously necessary for favorable in vivo characteristics, a radiofolate was developed by coupling a folate carbohydrazide with 2-[18 F]fluoro-2-deoxy-d-glucose ([18 F]FDG) via oxime formation with the open-chain form of glucose . Another approach of Fischer and co-workers also used the efficiency of CuAAC for 18 F-radiolabeling in combination with the inevitable polarity of [18 F]FDG to enhance pharmacodynamics . In this case, a 18 F-labeled azido-FDG derivative was used as a prosthetic group and coupled via CuAAC to an alkyne-carrying γ-folate. This derivative gave promising results, with significantly enhanced tumor-to-background ratios due to a high tumor uptake and reduced background. An alternative to folate radioconjugates is the derivatives synthesized by direct 18 F-fluorination approaches, which led to the development of 2′-[18 F]fluorofolic acid, synthesized via a nucleophilic aromatic 18 F-flourination at the 2′-position of folic acid . Preclinical evaluation showed excellent in vivo behavior with a clear-cut visualization of FR-positive KB tumors and healthy tissues (kidneys). However, the direct 18 F-fluorination of folic acid requires protecting group chemistry with cleavage under harsh conditions, resulting in degradation and poor radiochemical yields. Very recently, an optimized version of 2′-[18 F]fluorofolic acid was reported . The intended aromatic ring was exchanged by a pyridine to further reduce the electron density in the 2′-position for the nucleophilic 18 F-fluorination. As a result, the radiochemical yield was significantly improved and, besides an increased liver uptake, the pharmacokinetic characteristics were excellent for in vivo PET imaging. In respect to the 18 F-folates via prosthetic group conjugates, a new 18 F-polyethylene glycol (PEG)-folate was developed very recently and published while this manuscript was in preparation. The new 18 F-PEG-folate was not primarily intended for tumor imaging, but for targeting the FR-β on activated macrophages in a rat model of arthritis . The major objective was to reduce background signal in the periarticular tissue by following a similar strategy as for the here presented work to improve pharmacokinetics by introducing oligoethylene glycol spacers .
The aim of this study was to investigate the influence of oligoethylene glycol spacers on the polarity of a conjugated 18 F-labeled radiofolate, when introduced at the γ-position of the glutamate residue, since many drugs showed improved pharmacokinetics due to PEGylation . In this paper, the synthesis, radiolabeling, and preclinical evaluation of a new 18 F-labeled γ-radiofolate are reported. The radiofolate features oligoethylene glycol spacers for enhanced polarity and is radiolabeled via the highly efficient radio-CuAAC reaction.
Reagents and solvents were purchased from Sigma-Aldrich Co. (St. Luois, MO, USA), Acros (Geel, Belgium), or Merck AG (Darmstadt, Germany) and used without further purification, unless otherwise stated. The building block N2-N,N-dimethylaminomethylene-10-formylpteoric acid was generously provided by Merck & Cie AG (Schaffhausen, Switzerland). 3H-folic acid was purchased from Moravek Biochemicals Inc. (Brea, CA, USA). Reactions were monitored by thin layer chromatography (performed on Merck silica gel 60 F254, not modified, pre-coated silica gel on aluminum-supported plates) or high-performance liquid chromatography (HPLC).
Radiosyntheses were performed either in a manipulator-equipped hot cell by conventional heating (starting activities >5 GBq [18 F]fluoride) or manually in a lead-shielded fume hood (starting activities ≤5 GBq [18 F]fluoride) using a focused laboratory microwave (CEM Discover, Matthews, NC, USA) in the following mode: 1-min pre-run, 10-min reaction time, and a maximum power of 300 W.
Information about compound characterizations and analytical or preparative HPLC as well as radio-HPLC conditions can be found in Additional file 1.
Small animal PET imaging was performed on a GE eXplore Vista PET/CT scanner (GE Healthcare, Little Chalfont, UK).
Synthesis of the 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (10)
The 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate was prepared using a modified method of that described by Li and co-workers . Briefly, 2-[2-(2-hydroxyethoxy)ethoxy]ethanol (7) (5 g, 33 mmol) was added to a suspension of sodium hydride (1.3 g, 33 mmol) in dimethylformamide (15 mL) cooled to 0°C, and propargyl bromide (3.5 mL, 33 mmol) was added. The reaction mixture was then allowed to warm to room temperature and stirred. After 24 h, the solvent was removed and the crude reaction mixture purified by silica gel column chromatography (ethyl acetate/n-hexane, 1:2) to give 8 as a pale yellow oil in 42% yield (2,6 g, 14 mmol).
Compound 8 (1 g, 5 mmol) and p-toluenesulfonyl chloride (1.9 g, 10 mmol) were dissolved in anhydrous dichloromethane (10 mL) and cooled to 0°C. 1,4-Diazabicyclo[2.2.2]octene (560 mg, 5 mmol), dissolved in 5 mL dichloromethane, was added dropwise to the solution. The reaction mixture was stirred for 2 h at room temperature and gave, after purification by column chromatography, 10 as a colorless oil (54%, 940 mg, 2.7 mmol).
Synthesis of 3-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)prop-1-yne (9)
Compound 8 (200 mg, 1 mmol) was dissolved in anhydrous dichloromethane (5 mL) and cooled to 0°C. To this solution, N,N-diaminosulfur trifluoride (132 μL, 1 mmol) was added, and the reaction mixture maintained at 0°C for 1 h, before allowing it to warm to room temperature and stirring for additional 5 h. After removal of the solvent in vacuo, the crude reaction mixture was purified via column chromatography to give 9 as a pale yellow oil (40%, 80 mg, 0.4 mmol).
Synthesis of γ-(11-azido-3,6,9-trioxaundecanyl)folic acid amide (5)
N-(tert-butoxycarbonyl)glutamic acid α-methyl ester (1) (200 mg, 0.7 mmol) was reacted with 11-azido-3,6,9-trioxaundecan-1-amine (152 mg, 0.7 mmol) using 298 mg (0.7 mmol) 1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate (COMU) as coupling agent and 2 eq. of 2,2,6,6-tetramethylpiperidine (TMP) as base. The reaction was performed in acetonitrile and stirred at room temperature for 16 h. The crude residue was re-dissolved in dichloromethane (20 mL) and washed successively with aqueous hydrochloric acid (0.1 M, 2 × 10 mL) and aqueous sodium hydrogen carbonate (0.1 M, 3 × 10 mL), dried over sodium sulfate, and filtered, and the solvent was removed under reduced pressure. Purification by silica gel column chromatography (ethyl acetate/hexane, 10:1) afforded a colorless oil (86%, 274 mg, 0.6 mmol). Compound 2 was deprotected using dichloromethane/trifluoroacetic acid (1:1, 10 mL) at room temperature for 12 h. The mixture was co-evaporated with toluene (3 × 5 mL) and used in the next step without further purification. After deprotection, 3 was coupled to the activated ester of protected pteroic acid (230 mg, 0.6 mmol) prepared by adding COMU (256 mg, 0.6 mmol) and TMP (2 eq.) in anhydrous dimethylformamide (5 mL) to give the activated ester complex. The deprotected 3 was added dropwise to the solution, and the mixture was stirred at 40°C for 12 h, after which the solvent was removed in vacuo. The crude reaction mixture was re-dissolved in dichloromethane and extracted analogously to the coupling reaction of 1 and 11-azido-3,6,9-trioxaundecan-1-amine. The mixture was purified first by aluminum oxide column chromatography (dichloromethane/methanol, 15:1) followed by a second flash chromatography on silica gel (ethyl acetate/methanol, 5:1), giving 103 mg (0.14 mmol) of 4 as a yellow powder.
Synthesis of the 16-(4-(((2-amino-4-oxo-3,4-dihyxdropteridin-6-yl)methyl)amino)benzamido)-1-(4-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)-1H-1,2,3-triazol-1-yl)-13-oxo-3,6,9-trioxa-12-azaheptadecan-17-oic acid (6).
Compound 5 (5 mg, 0.02 mmol), copper(I) iodide (0.5 eq.), and a mixture of diisopropylethyl amine (DIPEA)/2,6-lutidine (1 eq.) were dissolved in 2 mL acetonitrile. The reaction was allowed to react for 15 min before 15 mg (0.02 mmol) of 9, dissolved in 2 mL of 0.05 M phosphate buffer, was added in one portion to the reaction mixture. The mixture was reacted at 130°C for 9 min in a sealed vessel using a laboratory microwave at 55 W and purified by semi-preparative HPLC. The combined fractions were lyophilized and re-dissolved in water (1 mL) and the pH was adjusted to 2, leading to the precipitation of the product, which was separated by centrifugation (10,000 rpm, 8 min). The supernatant was removed and the precipitate was lyophilized, yielding 9 mg (0.01 mmol) of 6 as a yellow solid.
Synthesis of 16-(4-(((2-amino-4-oxo-3,4-dihydropteridin-6-yl)methyl)amino)benzamido)-1-(4-(2-(2-(2-[18 F]fluoroethoxy)ethoxy)ethoxy)-1H-1,2,3-triazol-1-yl)-13-oxo-3,6,9-trioxa-12-azaheptadecan-17-oic acid ([18 F]12).
No-carrier-added (n.c.a.) [18 F]fluoride was produced via the 18O(p,n)18 F nuclear reaction. Isotopically enriched [18O]water (97% enrichment) was irradiated by an 18-MeV proton beam and trapped on an anion exchange resin (Sep-Pak Light Waters Accell Plus QMA Cartridge, Waters Corporation, Milford, MA, USA), which was pre-conditioned with a 1-M potassium carbonate solution (10 mL) and rinsed with pure water (20 mL). The n.c.a. [18 F]fluoride was eluted with 1 mL of a methanolic tetrabutylammonium hydroxide solution (95.7 mg tetrabutylammonium hydroxide (TBA-OH) × 30 H2O in 2 mL methanol) into a 5-mL sealed reaction vial. After azeotropic drying using three portions of acetonitrile, 800 μL of acetonitrile was added to the dry [18 F]fluoride-base mixture. The 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate 10 (6 mg, 17 μmol) was dissolved in 200 μL acetonitrile and subsequently added to the [18 F]fluoride solution. The reaction time was 12 min at 110°C, followed by quenching with 5 mL of 50 mM phosphate buffer. After HPLC purification (t R([18 F]11), 15 min) the fraction of the 18 F-labeled prosthetic group was diluted by addition of 25 mL of water and trapped on a Phenomenex Strata X-C18 cartridge (Torrance, CA, USA). After washing with 5 mL of water, the final prosthetic group 11 was eluted into a 5-mL reaction vial with 800 μL acetonitrile, which was equipped with copper(II) acetate (1.5 mg in 500 μL), sodium ascorbate (9 mg in 500 μL of 0.05 M phosphate buffer), and 5 (2 mg in 500 μL of 0.05 M phosphate buffer). The click reaction was performed at 110°C. After 13 min, the reaction was quenched with 0.05 M phosphate buffer and filled to a final volume of 5 mL. Purification was performed using the semi-preparative radio-HPLC system described before. The product fraction was acidified by addition of 500 μL of a 1-M hydrochloric acid solution and passed through a Phenomenex Strata X-C cartridge. After washing with 5 mL of water, the final radiotracer was eluted with 2.5 mL phosphate-buffered saline containing 10% ethanol.
Relative lipophilicity (k’ value)
The relative lipophilicity of 6 was determined as capacity factor k’ (k’ = (t retention - t solvent) / t solvent) by reversed-phase HPLC using a methanol-phosphoric acid buffer eluent system at pH 2. Under these conditions, the retention time (t R) was 4.49 min, which equals a k’ value of 1.12. This method has already been described elsewhere .
In vitro binding affinity assays
Displacement studies using [3H]folic acid and the non-radioactive reference compound 6 were carried out according to the previously described procedure .
In vitro metabolite studies in fetal calf serum
A solution of [18 F]12 (50 μL, approximately 5 MBq) was incubated with 500 μL of fetal calf serum (FCS) at 37°C and the mixture shaken at 900 rpm. The time points were set between 0 and 90 min using 500 μL of FCS and 50 μL of the radiotracer for each time point. Plasma proteins were precipitated by addition of cold acetonitrile (300 μL), followed by centrifugation (10,000 rpm, 10 min). An aliquot of each time point (15, 30, 60, and 90 min, 100 μL) was injected into the analytical radio-HPLC system for analytics.
In vivo studies
All animal experiments were approved by the local veterinary department and complied with Swiss and local laws on animal protection. Female CD-1 nude mice (Charles River, Sulzfeld, Germany) were fed with a folate-deficient rodent diet (Harlan Laboratories, Indianapolis, IN, USA). After an acclimatization period of 5 to 7 days, human KB tumor cells (5 × 106 cells in 0.1 mL sterile phosphate-buffered saline (PBS)) were inoculated subcutaneously on both shoulders of each mouse. Twelve days later, the animals were intravenously injected with [18 F]12 (approximately 5 MBq, 100 μL). Blocking studies were performed with excess folic acid dissolved in PBS (100 μg in 100 μL) injected 5 min prior to [18 F]12. Animals were euthanized at the indicated time points, and selected organs and tissues were collected, weighed, and measured in a γ-counter. The incorporated radioactivity was expressed as percentage injected dose per gram (%ID/g) of tissue. PET/computed tomography (CT) experiments were performed with a dedicated small-animal PET/CT scanner (eXplore Vista PET/CT, Sedecal, Algete, Spain/GE Healthcare). Animals were intravenously injected with [18 F]12 (approximately 13 MBq, 100 μL). For scanning, mice were anesthetized with isoflurane in an air/oxygen mixture. The PET scans were acquired from 60 to 90 min post-injection (p.i.) followed by a CT. After acquisition, PET data were reconstructed in user-defined time frames, and the fused datasets of PET and CT were analyzed with PMOD software (version 3.4).
Results and discussion
Results of the CuAAC reaction for different catalysts and heating conditions
Cu(I)I + sodium
Cu(II)acetate + sodium
Cu(II)sulfate + sodium
60 ± 3
Relative lipophilicity (k’ value)
The capacity factor k’ (k’ = (t retention - t solvent) / t solvent) was determined using reversed-phase HPLC and enables different (radio)folates to be compared based on their polarity. The values determined can be used to give hint to the in vivo behavior in terms of the degree of hepatobiliary excretion by comparison with literature examples. The 18 F-click folate  and the 2-[18 F]fluorofolic acid  have k’ values of 2.28 and 0.53, respectively, of which the latter shows an excellent in vivo profile while the former is unfavorable in terms of its abdominal background. The determined k’ value of 1.12 for compound 12 was between these two values of the previously synthesized radiofolates, suggesting that it might be a promising candidate for in vivo imaging applications.
The established radio-CuAAC approach was used for labeling as it has been proven to produce high radiochemical yields and obviates the need for protecting groups. The prosthetic group [18 F]11 was synthesized by following a modified protocol of Li et al.  using tetrabutylammonium hydroxide as base during 18 F-fluorination instead of the Kryptofix 2.2.2/potassium carbonate system. In agreement with the findings of Li et al. , the radiofluorination showed a strong temperature dependency with a conversion of greater than 75% achieved with conventional heating at 110°C. The crude reaction mixture was purified by semi-preparative HPLC that resulted in 58% radiochemical yield (RCY). After dilution, the prosthetic group [18 F]11 was trapped on a Phenomenex Strata X-C18 cartridge and eluted directly into a vial containing the azido-folate 5, the copper catalyst, and sodium ascorbate. This final setup was a result of an extensive optimization process, as the conditions used during the non-radioactive reference synthesis could not be successfully translated into radiolabeling. The use of Cu(I)I and a mixture of DIPEA/2,6-lutidine led to significant degradation of the precursor 5 and thus resulted in poor RCYs of 5% to 10%. Therefore, the system described above with no additional base was found optimal. A much higher RCY (≥90%) was achieved through microwave-supported radio-CuAAC; however, such a protocol was not applicable in the manipulator-equipped hot cell used for productions of [18 F]12 for animal studies. For the radiolabeling of the prosthetic group by conventional heating, higher amounts of radioactivity (40 to 100 GBq) were used to compensate for reduced RCY (58%) and prolonged reaction times. Final purification of the radiofolate was achieved using a semi-preparative HPLC system, followed by acidification (pH 1) and subsequent fixation on a Phenomenex Strata X-C cartridge. The loaded cartridge was flushed with water and the product [18 F]12 eluted with PBS buffer containing 10% of ethanol. Sterile filtration of the eluate gave the final product [18 F]12 in a very high radiochemical purity of ≥97% and good overall RCY of 8.7% within a total radiosynthesis time of approximately 2.5 h (end of bombardment (EOB)) for the hot cell-based synthesis. The overall radiosynthesis time could be reduced to approximately 90 min (EOB) using microwave-supported hands-on synthesis in a lead-shielded hood.
In vitro metabolite studies in fetal calf serum
The stability of the new radiotracer, [18 F]12, was evaluated in the presence of FCS. For this purpose, [18 F]12 was added to FCS and incubated at 37°C. At certain time points (15, 30, 60, and 90 min), aliquots were extracted and proteins removed by precipitation. The integrity of the radiotracer was determined by analytical radio-HPLC. No degradation or defluorination of the radiotracer [18 F]12 was observed over 90 min, and therefore, [18 F]12 can be considered stable for the general duration of a microPET (μPET) scan.
In vitro binding affinity assays
Ex vivo biodistribution studies
Ex vivo biodistribution studies of 18 F-OEG-folate 12 in mice at various time points and 18 F-click folate
30 min p.i.
60 min p.i.
90 min p.i.
60 min p.i. blockade a
18 F-click folate 45 min p.i. b
(n = 3)
(n = 3)
(n = 3)
(n = 3)
(n = 4)
0.20 ± 0.02
0.18 ± 0.01
0.16 ± 0.02
0.04 ± 0.01
0.13 ± 0.01
1.81 ± 0.61
1.09 ± 0.07
0.79 ± 0.16
0.02 ± 0.01
0.90 ± 0.22
1.21 ± 0.15
1.02 ± 0.7
0.86 ± 0.17
0.04 ± 0.03
0.85 ± 0.04
0.53 ± 0.14
0.49 ± 0.04
0.43 ± 0.08
0.06 ± 0.03
0.33 ± 0.12
41.80 ± 2.58
40.77 ± 4.34
41.04 ± 7.04
0.26 ± 0.08
16.53 ± 2.22
2.18 ± 0.76
1.39 ± 0.13
0.96 ± 0.37
0.12 ± 0.11
2.50 ± 0.6
23.52 ± 7.37
4.56 ± 2.31
1.92 ± 0.52
14.72 ± 19.32
19.59 ± 5.26
164.2 ± 72.5
29.48 ± 19.04
11.36 ± 4.29
105.5 ± 128.7
56.00 ± 27.64
5.24 ± 1.57
4.05 ± 0.67
2.30 ± 0.54
0.21 ± 0.09
1.71 ± 0.14
309.3 ± 187.6
133.2 ± 67.38
55.13 ± n.d.
775.5 ± 206.5
667.4 ± 530.1
1.91 ± 0.25
1.64 ± 0.46
1.17 ± 0.03
0.08 ± 0.09
1.47 ± 0.05
1.13 ± 0.21
0.84 ± 0.16
0.06 ± 0.06
0.13 ± 0.01
8.01 ± 1.16
9.28 ± 1.68
7.03 ± 1.98
0.20 ± 0.18
3.39 ± 0.54
3.39 ± 0.44
3.54 ± 0.68
0.19 ± 0.07
3.13 ± 0.83
Ratio of tumor to organ or tissue
16.81 ± 3.13
18.95 ± 2.68
22.72 ± 6.73
24,08 ± 0.82
0.69 ± 0.22
0.86 ± 0.19
1.55 ± 0.15
1.18 ± 0.69
0.08 ± 0.02
0.08 ± 0.01
0.09 ± 0.01
0.19 ± 1.38
In vivo μPET studies
There is a demand for 18 F-labeled radiofolates with superior in vivo behavior, which can be produced using a facile and robust radiosynthesis transferrable into routine productions for clinical applications. To this end, a novel 18 F-radio folate, with high affinity to the folate receptor and increased polarity compared to the original lead compound (18 F-click folate), was developed. 18 F-labeling of the prosthetic group and the radio-CuAAC reaction were optimized to give excellent RCYs and purities within short reaction times. 18 F-click chemistry, by providing a facile and robust labeling procedure, again confirmed its outstanding potential and its particular suitability for 18 F-labeling of folate derivatives. Ex vivo biodistribution experiments showed a highly specific uptake in FR-positive human KB tumors and kidneys. Compared to the previously developed 18 F-click folate, the new radiofolate [18 F]12 showed significantly reduced hepatobiliary excretion while maintaining the tumor uptake. In in vivo μPET studies, human KB tumor xenografts were visualized, while a moderate tumor-to-background contrast was found for [18 F]12. Coronal slices of the PET imaging clearly showed a heterogeneous uptake of [18 F]12 in the outer rim of KB xenografts. In comparison to the previously developed [18 F]fluoro-deoxy-glucose folate, the new 18 F-OEG-folate showed similar background levels. On the other hand, the threefold higher tumor uptake of the [18 F]fluoro-deoxy-glucose folate with significantly increased contrast values led to a much better tumor visualization. However, the very recently reported 18 F-PEG-folate with structural similarities to the new 18 F-OEG-folate gave promising results in imaging FR-expressing activated macrophages in inflammatory diseases. This study confirms the suitability of such PEGylated 18 F-folates for in vivo PET imaging of FR-positive tissue and their broad potential. In summary, the newly developed 18 F-labeled radiofolate has excellent radiochemical availability and exhibits a high and specific affinity to the folate receptor.
The authors thank Dr. Cristina Müller and Stephanie Haller for performing biological in vitro assays and ex vivo biodistribution studies. We also thank Merck & Cie AG (Switzerland) for kindly providing the protected pteroic acid. This work is further supported by the research cluster SAMT of the Johannes Gutenberg University Mainz.
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