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18F-glutathione conjugate as a PET tracer for imaging tumors that overexpress L-PGDS enzyme.

Huang HL, Huang YC, Lee WY, Yeh CN, Lin KJ, Yu CS - PLoS ONE (2014)

Bottom Line: The inhibition percentage of the production of PGD2 from PGH2 at the presence of 200 µM of FBuEA-GS and 4-Dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)butyl]piperidine (AT-56) were 74.1 ± 4.8% and 97.6 ± 16.0%, respectively. [18F]FBuEA-GS bound L-PGDS (16.3-21.7%) but not the isoform, microsomal prostaglandin E synthase 1.No binding to GST-alpha and GST-pi was observed.The contrasted images indicated that the radiotracer accumulation in tumor lesions is probably related to the overexpression of L-PGDS.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering and Environmental Sciences, National Tsinghua University, Hsinchu, Taiwan.

ABSTRACT
Lipocalin-type prostaglandin D synthase (L-PGDS) has been correlated with the progression of neurological disorders. The present study aimed at evaluating the imaging potency of a glutathione conjugate of fluorine-18-labeled fluorobutyl ethacrynic amide ([18F]FBuEA-GS) for brain tumors. Preparation of [18F]FBuEA-GS has been modified from the -4-tosylate derivative via radiofluorination in 5% radiochemical yield. The mixture of nonradioactive FBuEA-GS derived from a parallel preparation has be resolved to two isomers in a ratio of 9:1 using analytic chiral reversed phase high performance liquid chromatography (RP-HPLC). The two fluorine-18-labeled isomers purified through nonchiral semipreparative RP-HPLC as a mixture were studied by assessing the binding affinity toward L-PGDS through a gel filtration HPLC, by analyzing radiotracer accumulation in C6 glioma cells, and by evaluating the imaging of radiotracer in a C6 glioma rat with positron emission tomography. The inhibition percentage of the production of PGD2 from PGH2 at the presence of 200 µM of FBuEA-GS and 4-Dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)butyl]piperidine (AT-56) were 74.1 ± 4.8% and 97.6 ± 16.0%, respectively. [18F]FBuEA-GS bound L-PGDS (16.3-21.7%) but not the isoform, microsomal prostaglandin E synthase 1. No binding to GST-alpha and GST-pi was observed. The binding strength between [18F]FBuEA-GS and L-PGDS has been evaluated using analytic gel filtration HPLC at the presence of various concentrations of the cold competitor FBuEA-GS. The contrasted images indicated that the radiotracer accumulation in tumor lesions is probably related to the overexpression of L-PGDS.

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Purification using chiral analytic RP-HPLC and semipreparative RP-HPLC.(a) The racemic mixture of nonradioactive FBuEA-GS 3 was resolved to two components by chiral analytic RP-HPLC. The major peak A and the minor peak B represent the presence of two isomers. Injection volume: 0.01 mL from the sample with concentration of 1 mg/0.2 mL. (b) A typical chromatogram of [18F]FBuEA-GS 3 after purification with semipreparative RP-HPLC. Injection volume: 0.01 mL from the purified sample with concentration of 440 µCi/0.2 mL. (c) The HPLC chromatogram of the purified [18F]FBuEA-GS 3 co-mixed with the authentic sample using semipreparative RP-HPLC. Injection volume: 0.2 mL from authentic sample with concentration of 0.02 mg/0.2 mL.
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pone-0104118-g002: Purification using chiral analytic RP-HPLC and semipreparative RP-HPLC.(a) The racemic mixture of nonradioactive FBuEA-GS 3 was resolved to two components by chiral analytic RP-HPLC. The major peak A and the minor peak B represent the presence of two isomers. Injection volume: 0.01 mL from the sample with concentration of 1 mg/0.2 mL. (b) A typical chromatogram of [18F]FBuEA-GS 3 after purification with semipreparative RP-HPLC. Injection volume: 0.01 mL from the purified sample with concentration of 440 µCi/0.2 mL. (c) The HPLC chromatogram of the purified [18F]FBuEA-GS 3 co-mixed with the authentic sample using semipreparative RP-HPLC. Injection volume: 0.2 mL from authentic sample with concentration of 0.02 mg/0.2 mL.

Mentions: Preparation of [18F]FBuEA 2, the intermediate to [18F]FBuEA-GS 3, has been modified. In general, [18F]FBuEA 2 was obtained in radiochemical yields ranging from 20 to 30%, resulting from a batch of more than 15 experiments. In contrast to the usual fluorinating agent [18F]Bu4NF (TBAF), combination of K[18F]F and cryptand [2,2,2] was used. Furthermore, we found that although t-BuOH did not improve the radiochemical yield, no failure was encountered during the fluorination. [18F]FBuEA-GS 3 could be easily formed via the conjugation of [18F]FBuEA 2 with GSH by merely adjusting pH = 8.0. Nonradioactive FBuEA-GS 3 obtained from a parallel experiment could be resolved into two isomers in a ratio of 9∶1 using analytic chiral HPLC (Fig. 2a). For preparation purposes, a mixture of the two isomers of [18F]FBuEA-GS 3 obtained from semipreparative RP-HPLC purification was promptly used for all experiments, including radioligand enzymatic binding assays, cellular uptake study, ex vivo biodistribution experiments, and in vivo PET studies. No further isolation of the two isomers with chiral RP-HPLC was resumed. From a series of experiments, [18F]FBuEA-GS 3 was obtained from [18F]F− (end of bombardment, EOB), resulting in a radiochemical yield of 5%. Its specific activity and radiochemical purity were determined to be 33 GBq/µmol and 98% (Fig. 2b), respectively.


18F-glutathione conjugate as a PET tracer for imaging tumors that overexpress L-PGDS enzyme.

Huang HL, Huang YC, Lee WY, Yeh CN, Lin KJ, Yu CS - PLoS ONE (2014)

Purification using chiral analytic RP-HPLC and semipreparative RP-HPLC.(a) The racemic mixture of nonradioactive FBuEA-GS 3 was resolved to two components by chiral analytic RP-HPLC. The major peak A and the minor peak B represent the presence of two isomers. Injection volume: 0.01 mL from the sample with concentration of 1 mg/0.2 mL. (b) A typical chromatogram of [18F]FBuEA-GS 3 after purification with semipreparative RP-HPLC. Injection volume: 0.01 mL from the purified sample with concentration of 440 µCi/0.2 mL. (c) The HPLC chromatogram of the purified [18F]FBuEA-GS 3 co-mixed with the authentic sample using semipreparative RP-HPLC. Injection volume: 0.2 mL from authentic sample with concentration of 0.02 mg/0.2 mL.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4128654&req=5

pone-0104118-g002: Purification using chiral analytic RP-HPLC and semipreparative RP-HPLC.(a) The racemic mixture of nonradioactive FBuEA-GS 3 was resolved to two components by chiral analytic RP-HPLC. The major peak A and the minor peak B represent the presence of two isomers. Injection volume: 0.01 mL from the sample with concentration of 1 mg/0.2 mL. (b) A typical chromatogram of [18F]FBuEA-GS 3 after purification with semipreparative RP-HPLC. Injection volume: 0.01 mL from the purified sample with concentration of 440 µCi/0.2 mL. (c) The HPLC chromatogram of the purified [18F]FBuEA-GS 3 co-mixed with the authentic sample using semipreparative RP-HPLC. Injection volume: 0.2 mL from authentic sample with concentration of 0.02 mg/0.2 mL.
Mentions: Preparation of [18F]FBuEA 2, the intermediate to [18F]FBuEA-GS 3, has been modified. In general, [18F]FBuEA 2 was obtained in radiochemical yields ranging from 20 to 30%, resulting from a batch of more than 15 experiments. In contrast to the usual fluorinating agent [18F]Bu4NF (TBAF), combination of K[18F]F and cryptand [2,2,2] was used. Furthermore, we found that although t-BuOH did not improve the radiochemical yield, no failure was encountered during the fluorination. [18F]FBuEA-GS 3 could be easily formed via the conjugation of [18F]FBuEA 2 with GSH by merely adjusting pH = 8.0. Nonradioactive FBuEA-GS 3 obtained from a parallel experiment could be resolved into two isomers in a ratio of 9∶1 using analytic chiral HPLC (Fig. 2a). For preparation purposes, a mixture of the two isomers of [18F]FBuEA-GS 3 obtained from semipreparative RP-HPLC purification was promptly used for all experiments, including radioligand enzymatic binding assays, cellular uptake study, ex vivo biodistribution experiments, and in vivo PET studies. No further isolation of the two isomers with chiral RP-HPLC was resumed. From a series of experiments, [18F]FBuEA-GS 3 was obtained from [18F]F− (end of bombardment, EOB), resulting in a radiochemical yield of 5%. Its specific activity and radiochemical purity were determined to be 33 GBq/µmol and 98% (Fig. 2b), respectively.

Bottom Line: The inhibition percentage of the production of PGD2 from PGH2 at the presence of 200 µM of FBuEA-GS and 4-Dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)butyl]piperidine (AT-56) were 74.1 ± 4.8% and 97.6 ± 16.0%, respectively. [18F]FBuEA-GS bound L-PGDS (16.3-21.7%) but not the isoform, microsomal prostaglandin E synthase 1.No binding to GST-alpha and GST-pi was observed.The contrasted images indicated that the radiotracer accumulation in tumor lesions is probably related to the overexpression of L-PGDS.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering and Environmental Sciences, National Tsinghua University, Hsinchu, Taiwan.

ABSTRACT
Lipocalin-type prostaglandin D synthase (L-PGDS) has been correlated with the progression of neurological disorders. The present study aimed at evaluating the imaging potency of a glutathione conjugate of fluorine-18-labeled fluorobutyl ethacrynic amide ([18F]FBuEA-GS) for brain tumors. Preparation of [18F]FBuEA-GS has been modified from the -4-tosylate derivative via radiofluorination in 5% radiochemical yield. The mixture of nonradioactive FBuEA-GS derived from a parallel preparation has be resolved to two isomers in a ratio of 9:1 using analytic chiral reversed phase high performance liquid chromatography (RP-HPLC). The two fluorine-18-labeled isomers purified through nonchiral semipreparative RP-HPLC as a mixture were studied by assessing the binding affinity toward L-PGDS through a gel filtration HPLC, by analyzing radiotracer accumulation in C6 glioma cells, and by evaluating the imaging of radiotracer in a C6 glioma rat with positron emission tomography. The inhibition percentage of the production of PGD2 from PGH2 at the presence of 200 µM of FBuEA-GS and 4-Dibenzo[a,d]cyclohepten-5-ylidene-1-[4-(2H-tetrazol-5-yl)butyl]piperidine (AT-56) were 74.1 ± 4.8% and 97.6 ± 16.0%, respectively. [18F]FBuEA-GS bound L-PGDS (16.3-21.7%) but not the isoform, microsomal prostaglandin E synthase 1. No binding to GST-alpha and GST-pi was observed. The binding strength between [18F]FBuEA-GS and L-PGDS has been evaluated using analytic gel filtration HPLC at the presence of various concentrations of the cold competitor FBuEA-GS. The contrasted images indicated that the radiotracer accumulation in tumor lesions is probably related to the overexpression of L-PGDS.

Show MeSH
Related in: MedlinePlus