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

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.

Figure 1. Preparation of [¹⁸F]FBuEA-GS 3 from tosylate 1 via a sequential two-step radiochemical synthesis followed by HPLC purification.

Figure 2. 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.

Figure 3. Inhibition of the formation of PGD₂ from PGH₂ in the presence of 200 µM of each test compound.

Results are the mean of duplicated measurements.

Figure 4. HPLC chromatograms for the mixture of [¹⁸F]FBuEA-GS 3 with different enzymes.

(a) mPGES-1, (b) L-PGDS (lipocalin-type; rat recombinant), (c) PGDS (lipocalin-type; human recombinant), (d) PGDS (lipocalin-type; mouse recombinant), (e) COX-1 (ovine), (f) COX-2 (ovine), (g) GSTA1-1 and (h) GSTP1. (b′)∼(f′) are chromatograms resolved from the radioactivity signals of (b)∼(f) using Origin software.

Figure 5. Inhibition curves of the binding of [¹⁸F]FBuEA-GS 3 to L-PGDS (human) at the presence of the inhibitor FBuEA-GS 3 in various concentrations. Data was obtained from single measurement.

(a) The plot of 5 sec. equilibrium. The maximal binding ratio of the control group was 16% and the non-specific binding (nsb) ratio was 6%. (b) The plot of 10 min. equilibrium. The maximal binding ratio was 13% and the non-specific binding (nsb) ratio was 5%.

Figure 6. Comparison between the cellular uptake of two radiotracers.

The radioactivity uptake of [¹⁸F]FBuEA-GS 3 (a) and [¹⁸F]FBuEA 2 (b) by C-6 tumor cells (redline) and fibroblasts (blue line).

Figure 7. Immunohistological staining of COX enzymes and L-PGDS in both tumor and normal tissues.

(a) COX1 staining- tumor, (b) COX2 staining- normal, (b′) COX2 staining- tumor, (c) L-PGDS staining- normal, (c′) L-PGDS staining- tumor.

Figure 8. Bar diagram for the radiometabolite analysis of the radiotracer using semipreparative RP-HPLC.

The blood samples were taken at various time points after [¹⁸F]FBuEA-GS 3 was injected intravenously.

Figure 9. Ex vivo analysis of the distribution of [¹⁸F]FBuEA-GS 3 in a rat.

Figure 10. Images of PET and MRI of brain of a C6-glioma rat.

(a) Dynamic PET images taken over 20–40 min at three cross sections. Lower right shows an MRI image. (b) Images of the coronal cross section at different time frames (10–30 min, 30–60 min, 60–90 min and 90–120 min) post injection of [¹⁸F]FBuEA-GS 3. PET scanner description: microPET R4; Concorde Microsystems Inc. Injection dose: 1.58 mCi/0.5 mL.