Biodistribution and stability studies of [18F]Fluoroethylrhodamine B, a potential PET myocardial perfusion agent

doi:10.1016/j.nucmedbio.2009.12.005

Nuclear Medicine and Biology

Volume 37, Issue 3, April 2010, Pages 365-370

https://doi.org/10.1016/j.nucmedbio.2009.12.005 Get rights and content

Abstract

Introduction

Fluorine-18-labeled rhodamine B was developed as a potential positron emission tomography (PET) tracer for the evaluation of myocardial perfusion, but preliminary studies in mice showed no accumulation in the heart suggesting that it was rapidly hydrolyzed in vivo in mice. A study was therefore undertaken to further evaluate this hypothesis.

Methods

[¹⁸F]Fluoroethylrhodamine B was equilibrated for 2 h at 37°C in human, rat and mouse serum and in phosphate-buffered saline. Samples were removed periodically and assayed by high-performance liquid chromatography. Based on the results of the stability study, microPET imaging and a biodistribution study were carried out in rats.

Results

In vitro stability studies demonstrated that [¹⁸F]fluoroethylrhodamine B much more stable in rat and human sera than in mouse serum. After 2 h, the compound was >80% intact in rat serum but <30% intact in mouse serum. The microPET imaging and biodistribution studies in rats confirmed this result showing high and persistent tracer accumulation in the myocardium compared with the absence of uptake by the myocardium in mice thereby validating our original hypothesis that ¹⁸F-labeled rhodamines should accumulate in the heart.

Conclusions

[¹⁸F]Fluoroethylrhodamine B is more stable in rat and human sera than it is in mouse serum. This improved stability is demonstrated by the high uptake of the tracer in the rat heart in comparison to the absence of visible uptake in the mouse heart. These observations suggest that ¹⁸F-labeled rhodamines are promising candidates for more extensive evaluation as PET tracers for the evaluation of myocardial perfusion.

Introduction

There are currently several positron emission tomography (PET) tracers available for myocardial perfusion imaging (MPI) including [¹⁵O]H₂O, [¹³N]NH₃ and ⁸²Rb. However, the short half-lives of ¹⁵O (2 min) and ¹³N (10 min) limit their availability to those institutions with onsite cyclotrons. Rubidium-82 (t_1/2=76 s) is more widely available because it is produced by the ⁸²Sr/⁸²Rb generator, but high patient throughput is required to offset the high cost of the generator. These limitations have spurred interest in the development of an ¹⁸F-labeled MPI radiopharmaceutical [1], [2]. Fluorine-18 offers the advantages of high positron yield (97%), its half-life (110 min) allows repeated studies within the same day, and the distribution networks that have been established for [¹⁸F]FDG have demonstrated that production of ¹⁸F-labeled radiopharmaceuticals at central sites is a reasonable alternative to onsite production.

A number of ¹⁸F compounds have been proposed as possible myocardial perfusion agents including quaternary ammonium salts [3], tetraphenylphosphonium compounds [4], [5], [6], [7], [8] rotenone [9] and BMS-747158-02, a pyridazinone analog [10]. Of these, the most promising is perhaps BMS-747158-02, which was optimized to target mitochondrial complex I [11], [12] and shows better correlation between extraction and flow than the single-photon tracers ^99mTc-MIBI (^99mTc-MIBI = [^99mTc(hexakis(2-methoxyisobutylisonitrile))]¹⁺ and ²⁰¹Tl or the PET tracer [¹³N]NH₃ [12]. One possible limitation to this compound may be in vivo defluorination, although this is apparently lower in non-human primates than in rodents [13].

We recently reported the synthesis of ¹⁸F-labeled rhodamine B (Fig. 1) as potential myocardial perfusion agent [14], [15], [16], [17]. The compound is a lipophilic cation as are the single-photon myocardial perfusion agents ^99mTc-MIBI and ^99mTc-tetrofosmin and several of the ¹⁸F-labeled compounds listed above. Non-radiolabeled rhodamines are known to accumulate in the mitochondria in proportion to mitochondrial membrane potential [18], [19], [20], [21] and are substrates for Pgp, a protein-implicated in multidrug resistance [22], [23]. Vora and Dhalla [24] previously reported that unlabeled rhodamine 123 accumulated in the mouse heart. However, in our previous studies of 2′-[¹⁸F]fluoroethylrhodamine B, we observed no accumulation of the tracer in the mouse heart and suggested that this may be due to in vivo hydrolysis of the 2-fluoroethylester [16]. In the present work, we further examine this hypothesis by measuring the stability of 2′-[¹⁸F]fluoroethylrhodamine B in various sera. Based on the results of these in vitro studies, we evaluated the biodistribution of 2′-[¹⁸F]fluoroethylrhodamine B in a rat and found significant and persistent uptake in the myocardium.

Section snippets

General

Rhodamine B lactone (>97%) was purchased from MP Biomedicals (Solon, OH, USA). Ethylene glycol ditosylate (>97%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Extra dry reagent grade acetonitrile and Kryptofix (K2.2.2) (98%) were purchased from Fluka (St. Louis, MO, USA). Potasssium carbonate (99.997%) was purchased from Alfa Aesar (Ward Hill, MA, USA). Human, rat and mouse sera were purchased from Sigma-Aldrich. Other solvents and reagents were of the highest grade commercially

Synthesis of ¹⁸F-labeled rhodamine B

The radiosynthesis of [¹⁸F]FERhB was accomplished using the previously described one-pot synthesis [16]. This produced the desired product in a decay-corrected yield of 35% and 97% radiochemical purity. The specific activity was 2.5 GBq/μmol, and the total synthesis time was approximately 90 min. The apparent specific activity of the product is higher than that previously reported because the purification of the [¹⁸F]fluoroethyl tosylate to remove unreacted 1,2-ditosylethane reduced

Conclusion

Having previously described the synthesis of ¹⁸F-ethyl-rhodamine B and observed that the compound was apparently rapidly hydrolyzed in vivo in the mouse resulting in high gall bladder uptake and no visible accumulation in the heart, we carried out an in vitro study that confirmed that the compound is rapidly hydrolyzed in mouse serum and also observed that it is significantly more stable in rat (and human) serum. Based on these results, a microPET study was carried out that confirmed the

Acknowledgments

These studies were supported by the Children's Hospital Boston Radiology Foundation and NIH grant #5 R01 CA94338 (ABP).

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Myocardial perfusion imaging (MPI) is one of the most commonly performed investigations in nuclear medicine studies. Due to the physical properties of the positron emitter gallium-68 (⁶⁸Ga) and its availability from ⁶⁸Ge/⁶⁸Ga generator, together with the well-known coordination chemistry, we have synthesized ⁶⁸Ga-NOTA- and ⁶⁸Ga-NODAGA-rhodamine conjugates using a straightforward and a one-step simple reaction. Radiochemical yields were greater than 95% (decay corrected), with total synthesis time of less than 30 min. Radiochemical purities were always greater than 98% as assessed by TLC and HPLC. These synthetic approaches hold considerable promise as simple method for ⁶⁸Ga-rhodamine conjugates preparation, with high radiochemical yield and purity. Biodistribution studies in normal Fischer rats at 60 min post-injection, demonstrated significant (~3% ID/g) uptake and favorable biodistribution profile for ⁶⁸Ga-NOTA-rhodamine over ⁶⁸Ga-NODAGA-rhodamine conjugate. These results demonstrate that ⁶⁸Ga-NOTA-rhodamine conjugate may be useful as probe for the PET evaluation of myocardial perfusion.
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We recently reported the development of the [¹⁸F]fluorodiethylene glycol ester of rhodamine B as a potential positron emission tomography (PET) tracer for myocardial perfusion imaging (MPI). This compound was developed by optimizing the ester moiety on the rhodamine B core, and its pharmacokinetic properties were found to be superior to those of the prototype ethyl ester. The goal of the present study was to optimize the rhodamine core while retaining the fluorodiethyleneglycol ester prosthetic group.
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As was the case with the different prosthetic groups, we found that the rhodamine core has a significant effect on the in vitro and in vivo properties of this series of compounds. Of the rhodamines evaluated to date, the pharmacologic properties of the ¹⁸F-labeled diethylene glycol ester of rhodamine 6G are superior to those of the ¹⁸F-labeled diethylene glycol esters of rhodamine B, rhodamine 101, and tetramethylrhodamine. As with ¹⁸F-labeled rhodamine B, [¹⁸F]rhodamine 6G was observed to localize in the mitochondria of isolated rat cardiomyocytes.
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¹: Current address: Molecular Imaging Research, Bayer Schering Pharma, Muellerstrasse 178, 13353 Berlin, Germany.

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Biodistribution and stability studies of [18F]Fluoroethylrhodamine B, a potential PET myocardial perfusion agent

Abstract

Introduction

Methods

Results

Conclusions

Introduction

Section snippets

General

Synthesis of 18F-labeled rhodamine B

Conclusion

Acknowledgments

Nuc Med Biol

J Nucl Cardiol

J Nucl Cardiol

Appl Radiat Isot

J Biochem Biophys Methods

Biochem Pharmacol

Nuc Med Biol

Semin Nucl Med

Chem Phys Lett

Cardiovascular nuclear imaging: from perfusion to molecular function

Heart

A welcomed new myocardial perfusion imaging agent for positron emission tomography

Circulation

Radiosynthesis of 3-[18F]fluoropropyl and 4-[18F]fluorobenzyl triarylphosphonium ions

J Labelled Comp Radiopharm

Characterization of uptake of the new PET imaging compound 18F-fluorobenzyl triphenyl phosphonium in dog myocardium

J Nucl Med

Assessment of severity of coronary artery stenosis in a canine model using the PET agent 18F-fluorobenzyl triphenyl phosphonium: comparison with 99mTc-tetrofosmin

J Nucl Med

18F-Fluorobenzyltriphenyl phosphonium PET detects area-specific apoptosis in the aging myocardium

Evaluation of (4-[18F]fluorophenyl)triphenylphosphonium ion as a potential myocardial blood flow agent for PET

Biodistribution and stability studies of [¹⁸F]Fluoroethylrhodamine B, a potential PET myocardial perfusion agent

Synthesis of ¹⁸F-labeled rhodamine B

Radiosynthesis of 3-[¹⁸F]fluoropropyl and 4-[¹⁸F]fluorobenzyl triarylphosphonium ions

Characterization of uptake of the new PET imaging compound ¹⁸F-fluorobenzyl triphenyl phosphonium in dog myocardium

Assessment of severity of coronary artery stenosis in a canine model using the PET agent ¹⁸F-fluorobenzyl triphenyl phosphonium: comparison with ^99mTc-tetrofosmin

¹⁸F-Fluorobenzyltriphenyl phosphonium PET detects area-specific apoptosis in the aging myocardium

Evaluation of (4-[¹⁸F]fluorophenyl)triphenylphosphonium ion as a potential myocardial blood flow agent for PET