Evaluation of a Mitochondrial Voltage Sensor, (¹⁸F-Fluoropentyl)Triphenylphosphonium Cation, in a Rat Myocardial Infarction Model

Radiochemistry

¹⁸F-fluoride was produced by an ¹⁸O(p,n)¹⁸F reaction on a PETtrace cyclotron (GE Healthcare). Activity was extracted from H₂¹⁸O by an anion exchanger and then eluted by aqueous potassium carbonate (25.0 mmol) into the reaction vessel. The radioactive solution was dried together with 4.0 mg of Kryptofix 2.2.2 (Sigma-Aldrich) in 1.0 mL of acetonitrile under nitrogen at 100°C. The solution was evaporated at 100°C by bubbling nitrogen gas, and the residue was dried by azeotropic distillation with acetonitrile (1 mL, 3 times). Next, 4.0 mg of pentane-1,5-diyl bis(4-methylbenzenesulfonate) dissolved in 1.0 mL of anhydrous acetonitrile were added. Pentane-1,5-diyl bis(4-methylbenzenesulfonate) was prepared by modification of a previously reported method (21). The mixture was heated for 5 min at 90°C in the closed state. Radio–thin-layer chromatography showed a yield of more than 80% of the compound ¹⁸F-fluoropentyl 4-methylbenzenesulfonate. The solution was passed through a small silica Sep-Pak cartridge (Waters). Triphenylphosphine (6.0 mg) was dissolved in 1.0 mL of toluene, added to the reaction vessel, and heated to 220°C for 3 min with no separation step (Fig. 1) (22).

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FIGURE 1.

Radiosynthesis of (5-fluoropentyl)triphenylphosphonium salt. Reagents and conditions were as follows: (A) ¹⁸F-KF, acetonitrile, Kryptofix 2.2.2, 90°C, 5 min; (B) triphenylphosphine, toluene, 220°C, 3 min. RCY = radiochemical yield.

The solution was cooled and injected onto a semipreparative high-performance liquid chromatography (HPLC) column system for purification (acetonitrile:phosphate-buffered saline (PBS), 45:55; flow rate, 3 mL/min; ultraviolet, 254 nm; retention time, 21.8 min). For identification of the radioproduct, the collected HPLC fraction was coinjected with its nonradioactive compound. ¹⁸F-FPTP was dried, made isotonic with sodium chloride, and passed through a 0.20-μm membrane filter into a sterile multidose vial for in vitro and in vivo experiments.

Partition Coefficient and Stability Study

The log P was measured to compare the lipophilicities of ¹⁸F-FPTP, ³H-tetraphenylphosphonium (Moravek Biochemicals, Inc.), and ^99mTc-sestamibi. After the complete removal of volatiles of ¹⁸F-FPTP, ^99mTc-sestamibi and the residues were dissolved in a mixture of 3 mL of saline and 3 mL of n-octanol in a round-bottom flask. The mixture was vigorously stirred for 20 min at room temperature and was transferred to 15-mL Falcon conical tubes. The tubes were centrifuged at 3,000 rpm for 5 min. Samples in triplicate from n-octanol and aqueous layers were obtained and counted on a γ-counter (23). The partition coefficient of ³H-tetraphenylphosphonium was measured in the same manner and was counted on a liquid scintillation counter. The log P value was reported as the average of data obtained in 3 independent measurements (24).

For the labeling stability test, ¹⁸F-FPTP (0.37 MBq/100 μL) was incubated with human serum (1.0 mL) at 37°C in a water bath for 4 h and then analyzed by chromatography on instant thin-layer chromatography silica gel strips developed with a 9:2 ratio of methylene chloride:methanol. After they were developed, the chromatographic strips were scanned on an automatic thin-layer chromatography device. Thin-layer chromatography was performed at various time points. All experiments were done in triplicate (25). Also, for in vivo stability evaluation, 3 BALB/c mice (weight, 18–20 g; Orient) were used. After intravenous injection of ¹⁸F-FPTP (7.4 MBq), the mouse blood was collected at each time point (10, 30, 60, 120, and 240 min). The collected mouse serum was filtered and analyzed by HPLC.

Cell Lines, Culture Conditions, and Cell Uptake Studies

¹⁸F-FPTP accumulation was measured in cell culture with a rat embryonic cardiomyoblast cell line (H9c2). Human normal foreskin fibroblasts (HDFs) were used as a negative control. All cells were grown in deficient Dulbecco modified Eagle medium high glucose (containing 5.3 mM KCl and 110.34 mM NaCl; Life Technologies) plus 5% fetal bovine serum, 1% penicillin-streptomycin, and 1% l-glutamine. For the uptake test, cells were incubated at 37°C for 60 min with serum-free, radioactive culture medium. Radioactivity was measured after radioactive medium was removed. The wells were washed 3 times with cold PBS. The cells were harvested, and the cell-associated radioactivity was determined. The MMP-dependent cellular uptake of ¹⁸F-FPTP, ³H-tetraphenylphosphonium, and ^99mTc-sestamibi was assessed with H9c2 cells treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP), which is a protonophore that selectively abolishes the MMP (26). The compound was dissolved in dimethyl sulfoxide and diluted to the desired concentration with low K⁺ N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) buffer. The final concentrations of dimethyl sulfoxide were less than 0.1%. Different concentrations of CCCP solution (0.10, 0.50, 1.0, and 10.00 μM) were added to 5.0 × 10⁴ cells at 30 min before the start of the experiment. Radioactivity was determined by γ-counter or liquid scintillation counter after 1 h of incubation with ¹⁸F-FPTP, ³H-tetraphenylphosphonium, or ^99mTc-sestamibi (27). Triplicate samples were obtained for all uptake studies, and data are expressed as the net accumulation of probe, in (dpm cells/dpm medium/μg total protein) ± SE.

Animal Model and Murine Biodistribution Studies

Eight-week old male Sprague–Dawley rats (weight, 250–260 g; Orient) underwent left coronary artery (LCA) ligation, as previously described (28). Animal care, all experiments, and euthanasia were performed in accordance with protocols approved by the Chonnam National University Animal Research Committee and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (29). Animals underwent imaging studies at 24 h after LCA occlusion.

To confirm the activity of ¹⁸F-FPTP in vivo, biodistribution studies were performed with murine models. Biodistribution in different organs was assessed in BALB/c mice at 10, 30, 60, and 120 min after intravenous injection of 7.4 MBq of radiotracer (n = 6, each). Blood, heart, lung, liver, spleen, stomach, intestine, kidney, pancreas, muscle, and bone were sampled from the mice, and the radioactivity of each organ was measured with a γ-counter. Radioactivity determinations were normalized by the weight of the tissue and the amount of radioactivity injected to obtain the percentage injected dose per gram (%ID/g).

Small-Animal PET and Quantitative Analysis

A dedicated small-animal PET/CT scanner (Inveon; Siemens Medical Solutions) was used for in vivo imaging of ¹⁸F-FPTP kinetics. Normal or myocardial infarction (MI) rats were anesthetized with isoflurane, placed in a cradle, and equipped with masks for anesthesia gas supply and warm water pads at the tail veins for injection. Dynamic small-animal PET images were acquired for 60 min (40 s × 15 frames, 100 s × 30 frames) after injection of ¹⁸F-FPTP (37 MBq).

To determine pharmacokinetics, a region of interest was drawn around the heart. Time–activity curves of ¹⁸F-FPTP were generated to obtain the counts per pixel per second. Images obtained between 10 and 20 min after tracer injection were reconstructed using the 3-dimensional ordered-subset expectation maximization algorithm with 4 iterations. Reconstructed pixel sizes were 0.78 mm in both the transverse and the axial directions. The dimensions of the reconstructed images were 128 × 128 in each of the 159 transverse slices. Data were normalized and corrected for randoms, dead time, and decay.

Analysis of the small-animal PET images was performed with the PMOD software package (PMOD Technologies Ltd.) (4,30,31). To measure the average defect size, reconstructed PET data were reoriented into 20 segments of polar map images. Each polar map was normalized to its maximum. The defect area, which was defined as the fraction of polar map elements with reduced tracer uptake for more than 60% of the maximum, was expressed as the percentage of the left ventricular myocardium.

We compared the defect size on small-animal PET images with the gold standard 2,3,5-triphenyltetrazolium chloride (TTC)–stained images (32,33). In the presence of dehydrogenase enzyme that exists in viable myocardium, TTC is reduced and forms a formazan precipitate that makes viable tissue turn brick-red, whereas nonviable infarcted tissue without dehydrogenase activity remains pale. Staining was performed immediately after imaging to avoid further progression of MI. After the rats (n = 12) were euthanized, hearts were extracted and washed with 1× PBS. Each heart was frozen and divided perpendicularly into 4–5 cross sections of 1-mm thickness. These sections were incubated for 20 min with a solution of 1% TTC dissolved in 1×PBS at 37°C. Stained sections were washed twice with PBS and stored in 10% formaldehyde for 20 min. Specimens were imaged by a digital camera or placed between 2 glass slides and scanned. The size of the infarcted area (nonstained, pale white region) was measured on both sides, averaged for each slice, and summed from all slices using image processing software (ImageJ, http://rsb.info.nih.gov/ij/).

Statistical Analysis

Comparisons between 2 groups were made with a Student t test for independent samples with unequal variances. Comparisons among 3 groups were made with a 1-way ANOVA. P values of less than 0.05 were considered statistically significant. Correlation between infarct size measured using small-animal PET and TTC staining was calculated with a Pearson test (2-tailed, 95% confidence interval) using SPSS software (version 18.0; SPSS Inc.).