Radiolabeled Phosphonium Salts as Mitochondrial Voltage Sensors for Positron Emission Tomography Myocardial Imaging Agents

Abstract

Despite substantial advances in the diagnosis of cardiovascular disease, ¹⁸F-labeled positron emission tomography (PET) radiopharmaceuticals remain necessary to diagnose heart disease because clinical use of current PET tracers is limited by their short half-life. Lipophilic cations such as phosphonium salts penetrate the mitochondrial membranes and accumulate in mitochondria of cardiomyocytes in response to negative inner-transmembrane potentials. Radiolabeled tetraphenylphosphonium cation derivatives have been developed as myocardial imaging agents for PET. In this review, a general overview of these radiotracers, including their radiosynthesis, in vivo characterization, and evaluation is provided and clinical perspectives are discussed.

Introduction

Since the 1980s, single photon emission computed tomography (SPECT) has played a key role in assessing myocardial blood flow (MBF) and in diagnosing coronary artery disease (CAD) [1–3]. Notably, SPECT agents such as ^99mTc-sestamibi, ^99mTc-tetrofosmin, or ²⁰¹Tl are the mainstay of myocardial perfusion imaging (MPI) [4, 5]. However, the technical limitations of SPECT imaging, such as the absence of a standardized method for correction of photon attenuation and significant uptake of SPECT tracers in organs adjacent to the heart, may compromise the delineation of small lesions and limit the diagnostic accuracy of SPECT [6]. Positron emission tomography (PET) has several technical advantages over SPECT. Not only does PET imaging have higher spatial resolution and a standardized method for the correction of photon attenuation, but PET also enables quantitative measurements of MBF because it allows for accurate determination of rapidly changing tissue counts over time. Therefore, PET allows for quantitative measures of myocardial tracer uptake and regional MBF [7, 8]. However, the PET tracers currently used for MPI have a short half-life (¹³N-NH₃: 9.97 min; ⁸²Rb: 1.27 min; ¹⁵O-H₂O: 2.04 min), which limits widespread clinical use of PET because of the need for an on-site cyclotron or generator [9, 10]. Thus, use of a radioisotope that has proper half-life for clinical use to label myocardial perfusion agents could overcome these limitations and simplify clinical protocols [7, 11].

The Rationale for ¹⁸F-Labeled Tetraphenylphosphonium Cation Derivatives

The mitochondrial membrane potential (MMP) is higher in cardiomyocytes than in normal epithelial cells, and loss of MMP is an early event in cell death caused by myocardial ischemia [12, 13]. Similar to SPECT agents such as ^99mTc-sestamibi and ^99mTc-tetrofosmin, tetraphenylphosphonium cation (TPP) derivatives penetrate the hydrophobic barriers of the plasma and mitochondrial membranes and accumulate in mitochondria of cardiomyocytes [14–18]. This accumulation occurs because the ^99mTc-sestamibi, ^99mTc-tetrofosmin, and TPP derivatives move into mitochondrium in response to its high density and the negative inner transmembrane potentials (−150 to −170 mV, negative inside) in cardiomyocytes [17]. Therefore, radiolabeled TPP derivatives are promising candidates for myocardial imaging [19, 20].

Table 1 shows the radiolabeled TPP derivatives that have been developed [15, 16, 21–36]. The chemical structures of radiolabeled TPP derivatives are shown in Fig. 1. Among them, the first reported was ¹¹C-triphenylmethylphosphonium cation (¹¹C-TPMP), which serves as a mitochondrial membrane voltage sensor detectible via PET [21]. However, ¹¹C-TPMP use is limited to use widely because of the short half-life of ¹¹C (20 min) despite good in vivo characteristics. To provide a tracer similar to ¹¹C-TPMP but with a longer half-life, the synthesis and evaluation of ¹⁸F-labeled TPP derivatives were investigated. Ravert et al. reported that 3-¹⁸F-fluoropropyltriphenylphosphonium, 4-¹⁸F-fluorobenzyltriphenylphosphonium (¹⁸F-FBnTP), and 4-¹⁸F-fluorobenzyltris 4-dimethylaminophenylphosphonium cations could be synthesized in multistep reactions with no carrier added (nca) ¹⁸F [37]. Among these cations, ¹⁸F-FBnTP showed promise as a myocardial perfusion agent. ¹⁸F-FBnTP is metabolically stable and performs well as a cardiac imaging agent in healthy and CAD models [15, 16, 27]. In a separate study, direct fluorination of TPP was investigated [23]. However, these ¹⁸F-labeled tracers needed improved in vivo characteristics, such as higher uptake and longer retention of tracer in the heart and more rapid wash out from liver to be used as myocardial imaging agents. Thus we developed novel ¹⁸F-labeled tetraphenylphosphonium cation derivatives, fluoroalkyltriphenylphosphonium cations (FATPs). Alkyl chain conjugated triphenylphosphonium cation, have improved characteristics through lipophilicity control [28–32].

Table 1.

The radiolabeled tetraphenylphosphonium cation derivatives

Radioisotope	Radiotracer	Target	Reference
11C	11C-triphenylmethylphosphonium cation (11C-TPMP)	heart, brain tumor	21, 22
18F	(4-18F -fluorophenyl)triphenylphosphonium (18F-TPP)	heart	23, 24, 25
	4-18F-fluorobenzyltriphenylphosphonium (18F-FBnTP)	heart, brown adipose tissue	15, 16, 26, 27
	(5-18F-fluoropentyl)triphenylphosphonium cation (18F-FPTP)	heart	31, 33, 34
	(6-18F-fluorohexyl)triphenylphosphonium cation (18F-FHxTP)	heart	29
	(7-18F-fluoroheptyl)triphenylphosphonium cation (18F-FHtTP)	heart	32
	(8-18F-fluorooctyl)triphenylphosphonium cation (18F-FOTP)	heart	32
	(2-(2-18F-fluoroethoxy)ethyl)triphenylphosphonium cation (18F-FETP)	heart	28
	(2-(2-18F-fluoroethoxy)ethyl)tris (4-methoxyphenyl)phosphonium cation (18F-FETMP)	heart	30
64Cu	64Cu-(4-((4,10-bis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1 yl)methyl)benzyl)triphenylphosphonium cation	glioma	35
	64Cu-(2-(diphenylphosphoryl)ethyl)diphenyl(4-((4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)methyl)benzyl)phosphonium cation	glioma	36

Fig. 1.

Schematic structure of radioisotope labeled tetraphenylphosphonium cation derivatives. a ¹¹C-triphenylmethylphosphonium cation (¹¹C-TPMP), b (4-¹⁸F-fluorophenyl)triphenylphosphonium cation (¹⁸F-TPP), c (3-¹⁸F-fluoropropyl)triphenylphosphonium cation, d 4-¹⁸F-fluorobenzyltriphenylphosphonium cation (¹⁸F-FBnTP), e 4-¹⁸F-fluorobenzyltris 4-dimethylaminophenylphosphonium cation, f (5-¹⁸F-fluoropentyl)triphenylphosphonium cation (¹⁸F-FPTP), g (6-¹⁸F-fluorohexyl)triphenylphosphonium cation (¹⁸F-FHxTP), h (7-¹⁸F-fluoroheptyl)triphenylphosphonium cation (¹⁸F-FHtTP), i (8-¹⁸F-fluorooctyl)triphenylphosphonium cation (¹⁸F-FOTP), j (2-(2-¹⁸F-fluoroethoxy)ethyl)triphenylphosphonium cation (¹⁸F-FETP), k (2-(2-¹⁸F-fluoroethoxy)ethyl)tris(4-methoxyphenyl)phosphonium cation (¹⁸F-FETMP), l ⁶⁴Cu-(4-((4,10-bis(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1 yl)methyl)benzyl)triphenylphosphonium cation, m ⁶⁴Cu-(2-(diphenylphosphoryl)ethyl)diphenyl(4-((4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)methyl)benzyl)phosphonium cation

Myocardial selectivity and hepatic clearance depend on the lipophilicity and functional groups of the radiotracer. Although little is known regarding the optimal lipophilicity needed for high myocardial selectivity, cationic radiotracers with log P values in the range of 0.5–1.3 are particularly useful for imaging organs with high mitochondrial density, as a result of their fast membrane-penetration kinetics [36]. Furthermore, the lipophilic interaction between the triphenylphosphonium cation and the lipid layer is attractive because the alkyl group increases lipophilicity [38, 39].

Radiosynthesis of ¹⁸F-Labeled Tetraphenylphosphonium Cation Derivatives

We described radiolabeling procedures of representative radiolabeled phosphonium salts including ¹¹C-TPMP, ¹⁸F-labeled TPP, ¹⁸F-labeled TPP derivatives and ¹⁸F-labeled fluoroalkylphosphonium cations (¹⁸F-FATPs) among reported radiolabeled phosphonium salts. Because ¹¹C-TPMP and ¹⁸F-labeled TPP showed the potential of phosphonium salts as MPI agents for PET, the first structure of ¹⁸F-FATPs was modified based on ¹⁸F-labeled TPP derivatives.

¹¹C-TPMP was prepared by methylation of triphenylphosphonium with ¹¹C-methyl iodide (¹¹C-CH₃I) [21]. ¹¹C-CH₃I is a very reactive structure and generally is used in ¹¹C chemistry. ¹¹C-TPMP was synthesized with a 9.4–12.5 % radiochemical yield (RCY, non-decay corrected) within 30 minutes and the specific activity was 14.8-33.3 GBq/μmol.

Direct ¹⁸F-labeling of TPP (¹⁸F-TPP) has been achieved by two methods (Fig. 2) [23–25]. The first method used 4-nitrophenyltriphenylphosphonium as the ¹⁸F-labeling precursor for a one step nucleophilic substitution reaction. This precursor was prepared first by following the procedure of Rieke et al. [40]. The diazonium chloride intermediate was formed by slowly adding hydrochloric acid to 4-nitroaniline. The formation of 4-nitrophenyltriphenylphosphonium was obtained by the addition of triphenylphosphine to the diazonium chloride intermediate, which was then extracted in aqueous solution and precipitated with sodium iodide (NaI) to generate the pure precursor. Cheng used potassium carbonate (K₂CO₃) and the phase transfer agent Kryptofix 222, and Shoup used ammonium hydroxide after counter ion exchange of 4-nitrophenyltriphenylphosphonium changed nitrate with ¹⁸F for labeling [23, 25]. The second method used 4-iodophenyltrimethylammonium iodide as a precursor [24]. After ¹⁸F-labeling of 4-iodophenyltrimethylammonium iodide, 1-¹⁸F-fluoro-4-iodobenzene was made to react with triphenylphosphine to yield the final labeled compound. The RCY of the two methods was 10–15 % and the specific activity was >18.5 TBq/mmol at the end of synthesis. The total synthesis time was less than 60 minutes, and the radiochemical purity above 95 %.

Fig. 2.

Different radiosynthetic methods of ¹⁸F-TPP. a Direct ¹⁸F labeling method using 4-nitrophenyltriphenylphosphonium b Two step ¹⁸F labeling method using 4-iodophenyltrimethylammonium iodide

¹⁸F-labeling of TPP derivatives was reported by Ravert et al. He created a functional group between the triphenylphosphonium and ¹⁸F [37]. The radiosynthesis of 3-¹⁸F-fluoropropyltriphenylphosphonium cation was performed in a single vessel. After drying the nca ¹⁸F in the presence of Kryptofix and K₂CO₃, propylene glycol ditosylate in acetonitrile was added and the solution heated. Triphenylphosphine in toluene was added subsequently. After the solution was heated to boiling, the toluene evaporated and purified by high pressure liquid chromatography (HPLC). They used 0.1 M ammonium formate as an HPLC eluent, but a buffer change was needed, such as injectable water, saline or phosphate buffer saline to use the cation for in vivo experiments due to the toxicity of ammonium formate. The synthesis, purification and formulation were completed in 56 minutes (n = 10). The RCY was 12 % (non-decay corrected) and the specific activity was 15.5 GBq/μmol.

The radiosynthesis of ¹⁸F-FBnTP was more complicated than that of the (3-¹⁸F-fluoropropyl)triphenylphosphonium cation. ¹⁸F-FBnTP was synthesized using a four-step procedure. As a precursor, 4-trimethylammoniumbenzaldehyde was used to create a third intermediate, 4-¹⁸F-fluorobenzyl bromide, which was reacted with triphenylphosphine to yield ¹⁸F-FBnTP. Finally, ¹⁸F-FBnTP was purified using a preparative HPLC system. A radiochemically pure product (>99 %) was obtained with a 6 % yield (non-decay corrected) with an average synthesis time of 82 minutes (n = 20) and a specific radioactivity of 16.7 GBq/μmol. The radiosynthesis of the 4-¹⁸F-fluorobenzyltris-4-dimethylaminophenylphosphonium cation was performed in the same manner as the 4⁻¹⁸F-FBnTP described above except that tris-dimethylaminophosphine was substituted for triphenylphosphine in the last synthetic step. The radiochemically pure radioproduct (>99 %) was obtained with a 15 % yield (non-decay corrected) with an average synthesis time of 80 minutes (n = 6) and an average specific radioactivity of 12.6 GBq/μmol.

Finally, ¹⁸F-FATPs, including (5-¹⁸F-fluoropentyl)triphenylphosphonium cation (¹⁸F-FPTP), (6-¹⁸F-fluorohexyl)triphenylphosphonium cation (¹⁸F-FHxTP), (7-¹⁸F-fluoroheptyl)triphenylphosphonium cation (¹⁸F-FHtTP), (8-¹⁸F-fluorooctyl)triphenylphosphonium cation (¹⁸F-FOTP), (2-(2-¹⁸F-fluoroethoxy)ethyl)triphenylphosphonium cation (¹⁸F-FETP), and (2-(2-¹⁸F-fluoroethoxy)ethyl)tris(4-methoxyphenyl)phosphonium cation (¹⁸F-FETMP), were investigated to assess the appropriate range of lipophilicity suitable for PET myocardial imaging agents. ¹⁸F-FATPs were prepared as described previously [28–32]. It took a total reaction time of less than 60 minutes for ¹⁸F-FATPs to be ready for tracer injection. A radiochemically pure radioproduct (>98 %) was obtained with a 10-20 % yield (non-decay corrected) and the average specific radioactivity was >5.9 Tbq/μmol.

Biodistribution studies of ¹⁸F-Labeled Tetraphenylphosphonium Cation Derivatives

Biodistribution results of radioisotope labeled TPP derivatives are summarized in Table 2. ¹⁴C-TPMP distribution was evaluated in mice and rats. ¹⁴C-TPMP had maximum cardiac uptake two minutes after injection (16.53 ± 1.90 % ID/g) in mice and its level remained nearly constant for 60 min. The heart-to-blood ratios were 31, 54, 86, and 153 at 2, 10, 30, and 60 min, respectively. The small intestine, liver, and muscle values also were relatively constant for 60 min, but lower than that of the heart. The kidney showed an extremely high concentration at 2 min, which then decreased rapidly over time. In rat studies, the heart concentration was also high at two minutes (3.05 ± 0.16 % ID/g) and remained almost constant for 60 min. The heart-to-blood ratios were 93 and 154 at 30 and 60 min, respectively. The distribution pattern was nearly identical to that of the murine study. The in vivo biodistribution of ¹⁴C-TPMP demonstrated that the cation first became concentrated in the myocardium and then remained constant with high heart to-blood ratios. The ¹⁴C-TPMP concentration in the kidney was initially high and then decreased rapidly [21]. High radioactivity was observed in the urinary bladder, and relatively high activity in the lumen of the intestine. These results suggest that ¹⁴C-TPMP is excreted mainly through the kidney and, to some extent, through the hepatobiliary system, because a high gallbladder concentration was observed in the preliminary study. Hepatobiliary secretion of the cation may interfere with interpretation of heart images adjacent to the liver. However, the heart-to-liver ratios were higher than two in murine studies with heart imaging.

Table 2.

Normal biodistribution at 60 minutes after i.v. injection of radioisotope labeled TPP derivatives into mice

	14C-TPMP*	18F-TPP*	18F-FBnTP	18F-FPTP	18F-FHxTP	18F-FETP	18F-FETMP
Blood	0.027 ± 0.002	0.02 ± 0.003	0.05 ± 0.01	0.04 ± 0.01	0.19 ± 0.03	0.23 ± 0.02	0.73 ± 0.63
Heart	4.144 ± 0.827	1.57 ± 0.18	12.16 ± 2.4	21.23 ± 3.60	23.20 ± 2.70	19.96 ± 2.08	29.88 ± 7.59
Lung	1.071 ± 0.108	0.38 ± 0.11	1.82 ± 0.3	2.96 ± 0.63	3.70 ± 0.12	4.01 ± 0.74	6.38 ± 1.21
Liver	1.049 ± 0.269	0.17 ± 0.03	8.09 ± 3.9	3.05 ± 0.92	1.30 ± 0.12	2.03 ± 0.17	11.22 ± 1.48
Kidney	1.288 ± 0.185	1.75 ± 0.38	24.70 ± 4.1	11.68 ± 3.73	12.68 ± 3.13	16.86 ± 2.73	41.10 ± 9.94
Muscle	0.648 ± 0.111	0.26 ± 0.11	1.27 ± 0.4	6.16 ± 1.78	4.53 ± 0.54	4.42 ± 1.15	5.41 ± 1.93
Heart-to-liver	3.95	8	1.50	7.25	17.83	9.87	2.74
Heart-to-lung	3.89	4	6.68	7.28	6.27	5.05	4.75
Heart-to-blood	153.48	75	243	523.34	126.76	86.78	61.07

^*: biodistribution in Sprague–Dawley rats, Data are expressed as the percentage of administered activity (injected dose) per gram of tissue

¹⁸F-TPP demonstrated an 11-fold higher accumulation in the heart at five minutes (1.64 % ID/g) relative to the blood and a five-fold higher accumulation relative to the liver [25]. At 30 min, the radioactivity in the heart was 1.51 % ID/g and the heart-to-blood ratio was 75. Blood activity changed significantly from 5 to 60 minutes, dropping from 0.15 to 0.02 % ID/g. At 5, 30, and 60 minutes, lung activity was 0.69, 0.36, and 0.38 % ID/g, respectively, whereas liver uptake was 0.34, 0.18, and 0.17 % ID/g, respectively. Heart-to-lung ratios at 5, 30, and 60 minutes were 2, 5, and 4, respectively. Bone accumulation, an indication of defluorination, was minimal: 0.33 % ID/g at 5 minutes and 0.39 % ID/g at 60 minutes.

Among 3-¹⁸F-fluoropropyltriphenylphosphonium, ¹⁸F-FBnTP, and 4-¹⁸F-fluorobenzyltris 4-dimethylaminophenylphosphonium cations, murine biodistribution was reported only for ¹⁸F-FBnTP [16]. ¹⁸F-FBnTP biodistribution in mice was organ specific, with the strongest uptake in the kidney, followed by the heart and liver, and very low uptake in the lung at 60 min. ¹⁸F-FBnTP demonstrated a 240-fold higher accumulation in the heart at 60 minutes (12.16 % ID/g) relative to the blood (0.05 % ID/g) and a 1.5-fold higher accumulation relative to the liver (8.09 % ID/g).

¹⁸F-FATPs showed superior heart-specific uptake and wash out from liver than those reported for radioisotope labeled TPP derivatives with modifications of lipophilicity and functional groups of ¹⁸F-FATPs. The lipophilicity and functional group changes between triphenylphosphonium and ¹⁸F of ¹⁸F-FATPs are summarized in Table 3. Although there is little information available with regard to the optimal lipophilicity needed for high myocardial selectivity, cationic radiotracers with log P values in the range of 0.5–1.3 are particularly useful for imaging organs with high mitochondrial density as a result of their fast membrane-penetration kinetics [36]. Furthermore, the lipophilic interaction between the triphenylphosphonium cation and the lipid layer is attractive because the alkyl group increases lipophilicity [38, 39]. In vivo biodistribution of ¹⁸F-FATPs was examined in BALB/c mice at 10, 30, 60, and 120 minutes after intravenous injection. A high level of radioactivity accumulation in the heart with rapid clearance from the liver and blood. The myocardial uptake of ¹⁸F-FATPs was more than 20 % ID/g at 10, 30, 60, and 120 minutes after radiotracer injection. The heart-to-liver ratio and heart-to-blood ratio was over 4 and 30, respectively, at 10 minutes. However, clearance of ¹⁸F-FETMP from the liver occurred later than for other organs due to the methoxy functional group of triphenylphosphonium (heart-to-liver ratio at 10 min: 2.33 ± 0.36).

Table 3.

Comparison of lipophilicity and functional groups between triphenylphosphonium and ¹⁸F of ¹⁸F-FATPs

PET imaging studies of ¹⁸F-Labeled Tetraphenylphosphonium Cation Derivatives

PET imaging studies of ¹¹C-TPMP were performed in normal dog models [21, 41]. ¹¹C-TPMP concentrated in the myocardium within a few minutes with very rapid clearance from the blood. The concentration varied from 0.038 to 0.082 % dose/ml and the heart-to-blood ratios were 28–53 and 49–105 at 20 and 60 minutes, respectively. In addition, these studies measured the ¹¹C-TPMP concentration in the heart after injection of potassium chloride, which resulted in a reduced heart rate, arrhythmia, and ventricular fibrillation. When ventricular fibrillation occurred, a decrease of about 40-50 % in the ¹¹C-TPMP concentration was observed. The rapid decrease of ¹¹C-TPMP might indicate an irreversible depolarization of the myocardium.

In vivo characterization of ¹⁸F-TPP was performed in normal rat and coronary occlusion rabbit models. ¹⁸F-TPP showed intense cardiac uptake with an initial spike in activity corresponding to blood flow followed by a plateau after 1–2 min. The cardiac uptake was 1.5 % ID/g in rats, which is in the range for that of ^99mTc-tetrofosmin or -sestamibi. The time-activity curve (TAC) generated from PET imaging of rats showed that ¹⁸F-TPP uptake by the heart wall was approximately two-fold higher than blood uptake for 60 minutes. Similarly, rabbit images indicated rapid blood clearance and clear delineation of the plateau of heart activity for the hour-long scanning period. The uptake and kinetics of ¹⁸F-FBnTP were evaluated in murine and normal canine models. ¹⁸F-FBnTP is metabolically stable and demonstrated excellent characteristics as a cardiac imaging agent in both models. The myocardium-to-liver uptake ratio in the TAC reached 1 at approximately 25 minutes after intravenous injection of ¹⁸F-FBnTP in mice. Furthermore, the myocardium-to-liver uptake ratio was 1 in dog models for 90 minutes after ¹⁸F-FBnTP injection.

¹⁸F-FATPs showed excellent heart-background contrast in microPET imaging of murine models at each time point and afforded better in vivo characterization than ¹³N-NH_3, which is the clinical gold standard myocardial imaging agent for PET [28–31, 34]. Static microPET images of normal rats after intravenous (i.v.) injection of ¹⁸F-FATPs or ¹³N-NH₃ are shown in Fig. 3. The ¹⁸F-FATP images demonstrated good visualization of the heart, with excellent heart-to-liver and heart-to-lung discrimination and high contrast between the myocardium and liver or lung. By contrast, ¹³N-NH₃ images showed higher liver uptake than heart uptake until 30 minutes after tracer injection. Intense liver uptake, caused by prominent hepatobiliary excretion, is frequently observed on ^99mTc-based myocardial imaging [42, 43]. High liver uptake is the cause of photon scatter that may mask the detection of flow abnormalities, particularly in the inferior and inferoapical left ventricular wall [43, 44]. The ¹⁸F-FATP TACs demonstrated rapid accumulation in the myocardium (1–2 minutes), with stable retention for at least 60 minutes. The myocardium-to-liver ratios of ¹⁸F-FPTP, ¹⁸F-FHTP, and ¹⁸F-FETP were 1.67 ± 0.21, 2.49 ± 0.08, and 2.6 ± 0.85, respectively, whereas myocardium-to-lung ratios were 3.65 ± 0.2, 5.67 ± 0.52, and 6.31 ± 0.8, respectively, 1 minutes after i.v. injection. By contrast, ¹³N-NH₃ TAC revealed that the myocardium-to-liver ratio and the myocardium-to-lung ratio were 0.8 ± 0.14 and 0.93 ± 0.07, respectively at 5 minutes after injection. Thereafter, liver and lung uptake of ¹³N-NH₃ was higher than that of the myocardium. The myocardium-to-liver ratios of ¹⁸F-FPTP, ¹⁸F-FHTP, and ¹⁸F-FETP were over seven-, 17-, and ten-fold higher, whereas myocardium-to-lung ratios were two-, three-, and three-fold higher, respectively, than those of ¹³N-NH₃ at 30 minutes after injection. On the basis of the TAC from microPET images, ¹⁸F-FATPs demonstrated high heart-to-liver and heart-to-lung ratios 10 minutes after injection. These results are consistent with the high-quality myocardial images obtained with ¹⁸F-FATPs.

Fig. 3.

Coronal microPET images of normal rats after intravenous injection of 37 MBq of a ¹³N-NH₃, b ¹⁸F-FPTP, c ¹⁸F-FHTP, d ¹⁸F-FETP and e ¹⁸F-FETMP. The heart is visible, with excellent heart-to-background contrast at each time point after radiotracer injection. H: heart; L: liver; SUV: standardized uptake value. (Reprinted with permission of Kim et al. [28–31, 34])

In addition, ¹⁸F-FATPs were compared with ¹³N-NH₃ in myocardial infarction (MI) models. Representative images of MI rats acquired after acute ligation of the left coronary artery (LCA) in the short-, vertical long-, and horizontal long-axes and a polar map collected 10 and 30 minutes after injection of ¹⁸F-FATPs or ¹³N-NH₃ are shown in Fig. 4. Sharply defined myocardial defects were present with ¹⁸F-FATPs or ¹³N-NH₃ (10 min). However, the ¹³N-NH₃ images significantly underestimated infarct size early after tracer injection (0–10 min, P = 0.027), which might be due to spillover of radioactivity into the left ventricular or right ventricular myocardium [34]. To confirm the spillover effect of ¹³N-NH₃ early after i.v. injection, we compared polar map images acquired from zero to three minutes and three to six minutes. The defect size and contrast ratio of ¹³N-NH₃ images at zero to three minutes were significantly lower than those at three to six minutes (P = 0.043), whereas ¹⁸F-FATPs showed similar values for defect size and contrast ratio at zero to three and three to six minutes. The defect size for MI generated from microPET polar map imaging of ¹⁸F-FPTP or ¹³N-NH₃ was compared with 2,3,5-triphenyltetrazolium chloride (TTC) staining to measure the coefficient of determination [33]. The correlation coefficient between TTC staining and the polar map of ¹⁸F-FPTP or ¹³N-NH₃ was 0.89 and 0.84, respectively (threshold: 60 %). The strong correlation demonstrated that noninvasive imaging results obtained by ¹⁸F-FPTP or ¹³N-NH₃ small-animal PET can serve as a surrogate for histology to quantify infarct size.

Fig. 4.

Short-, vertical long-, and horizontal long-axis and polar map images of a ¹³N-NH₃, b ¹⁸F-FPTP, c ¹⁸F-FHTP, or d ¹⁸F-FETP in each representative MI model. Data were collected between 0 to 10 and 20 to 30 minutes after radiotracer injection (37 MBq). (Reprinted with permission of Kim et al. [34])

Perfused Isolated Rat Heart Study

The first-pass extraction fraction (EF) values of ¹⁸F-FATPs or ¹³N-NH₃ were measured in isolated rat hearts perfused by the Langendorff method [34]. Despite the limitations of the Langendorff method, including the absence of normal humoral influences and neuronal regulation, a variety of cardiovascular researchers still use this technique because it has many advantages, such as simplicity, measurement accuracy, high reproducibility, and relatively low cost [45]. EF values for all flow rates are shown in Table 4. Lower EFs were found at higher flow rates, which was true for both ¹⁸F-FATPs and ¹³N-NH₃. The EF values of ¹⁸F-FPTP, ¹⁸F-FHTP, and ¹⁸F-FETP were significantly higher than those of ¹³N-NH₃ at flow rates of 4, 8, and 16 mL/minute (P < 0.05). At the lower flow rate of 0.5 mL/min, there were no significant differences between ¹⁸F-FPTP, ¹⁸F-FHTP, and ¹³N-NH₃. Only ¹⁸F-FETP demonstrated a significantly lower EF value than ¹³N-NH₃ at a flow velocity of 0.5 mL/min. The flow rate tested in this study (0.5–16 mL/min) would be relevant because the expected flow rate of the Langendorff method is 7–9 mL/minutes for rats and the MBF of normal rats under isoflurane anesthesia is 4.2 ± 0.9 mL/minutes [46]. Generally, flow rates in isolated rat hearts are considerably higher than those under physiologic conditions and lower EFs have been found at higher flow rates. Thus, perfusion tracers showed considerably lower values in the isolated heart model [7]. The SPECT agent ^99mTc-sestamibi demonstrated an average myocardial EF of 38 % (SD = 9 %) for a flow range of 0.52–3.19 mL/min/g in isolated rabbit hearts. In another study, ⁸²Rb exhibited a value of 42 % (SD = 6 %), which was observed after injection into the femoral vein of mongrel dogs at normal resting flow rates (0.75–1.5 mL/min/g) [47, 48]. The ¹⁸F-FATPs demonstrated similar EF values to ^99mTc-sestamibi and ⁸²Rb.

Table 4.

First-pass extraction fraction values (EF, %) of ¹⁸F-FATPs and ¹³N-NH₃ at different flow rates

	18F-FPTP	18F-FHTP	18F-FETP	13N-NH3
0.5 mL/min (n = 2)	41.26 ± 11.51	45.30 ± 8.78	33.89 ± 10.30*	49.30 ± 4.05
4.0 mL/min (n = 2)	30.18 ± 8.90†	31.59 ± 8.73†	24.35 ± 4.38*	15.18 ± 7.29
8.0 mL/min (n = 2)	23.21 ± 6.62†	22.57 ± 6.47*	19.70 ± 3.05*	12.89 ± 6.30
16 mL/min (n = 2)	18.50 ± 4.65†	18.43 ± 3.57†	13.94 ± 1.72†	7.38 ± 3.80

Data are expressed as mean ± SD. EF values were measured three times in each isolated heart. ^* P < 0.05 vs ¹³N-NH₃ by Mann–Whitney test. ^† P < 0.01 vs ¹³N-NH₃ by Mann–Whitney test. (Reprinted with permission of Kim et al. [34])

Limitations

Some limitations of radiolabeled TPP derivatives should be considered in view of the results so far achieved. First, although the results using radiolabeled TPP derivatives suggest that the agents used in the current preclinical studies may be suitable for clinical studies (because of the stable uptake and excellent pharmacokinetics), these results were obtained only in an experimental study using small animal models. Further preclinical application is being evaluated using a pig model that more closely resembles the human heart in its size, heart rate, myocardial blood flow, and mitochondrial density. Second, these results were limited to acute MI with permanent LCA ligation. This model is well-suited to determine myocardial defects but is not identical to the clinical situation in which hemodynamically relevant stenosis is unmasked by a stress-induced increase of myocardial blood flow. Further studies are needed to validate radiolabeled TPP derivatives for the detection of small areas of myocardial ischemia and scars from chronic infarctions.

Clinical Perspective and Conclusion

Despite numerous groundbreaking advances in the field, cardiovascular disease (CVD) remains the leading cause of mortality and morbidity worldwide [49, 50]. Cardiac PET imaging with novel radiotracer can display biological processes directly at the cellular and molecular level within the intact and living heart, and is becoming an indispensable tool in CVD diagnosis. The excellent performance of radiolabeled TPP derivatives as mitochondrial voltage sensors suggests their suitability for detecting CVD. This hypothesis is supported by previously reported studies that evaluated in vivo characterization of these agents in animal models. Among radiolabeled TPP derivatives, ¹⁸F-FATPs provided excellent cardiac PET image quality. In vivo characteristics of ¹⁸F-FATPs was significantly better than the current gold standard tracer, ¹³N-NH₃. In addition, ¹⁸F-FATPs overcame the limitation to widespread use of tracers in clinical PET because the half-life of ¹⁸F is 109.8 min. Further studies are needed to find the best ¹⁸F-FATP derivative. Nevertheless, ¹⁸F-FATPs are promising ¹⁸F-labeled radiopharmaceuticals for the evaluation of myocardial perfusion by PET. The use of ¹⁸F-FATP will support high-throughput clinical protocols as well as the widespread application of PET MPI in hospitals that do not have a cyclotron.

Compliance with Ethical Standards

Conflict of Interest

Dong-Yeon Kim and Jung-Joon Min declare that they have no conflict of interest.

Ethical Statement

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.