A simplified method for the measurement of nonmetabolized 2-[18F]F-A-85380 in blood plasma using solid-phase extraction

doi:10.1016/j.nucmedbio.2006.12.001

Nuclear Medicine and Biology

Volume 34, Issue 2, February 2007, Pages 221-228

https://doi.org/10.1016/j.nucmedbio.2006.12.001 Get rights and content

Abstract

Quantification of α₄β₂* nicotinic acetylcholine receptors using 2-[¹⁸F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-[¹⁸F]FA) and positron emission tomography (PET) imaging requires measurement of nonmetabolized radioligand in blood plasma, which was previously accomplished using high-performance liquid chromatography (HPLC). Here, we introduce a one-step solid-phase extraction (SPE) method for measuring the concentration of nonmetabolized 2-[¹⁸F]FA. This method allows many samples to be processed in a short period of time. SPE effectively separated 2-[¹⁸F]FA from radioactive metabolites typically observed in blood plasma after administration of radioligand in humans. Measurements of the 2-[¹⁸F]FA parent fraction in healthy human volunteers obtained using the SPE method were nearly identical to those obtained using HPLC (1.3±5% average underestimation of SPE), and reproducibility was good within and between runs (2% and 6% coefficient of variation, respectively). SPE recovery of 2-[¹⁸F]FA from blood plasma was not appreciably diminished (3±0.6%) by a larger volume of blood plasma loaded onto the cartridge, suggesting the possibility of increasing the plasma sample volume at later times in a PET study to improve measurement sensitivity. 2-[¹⁸F]FA was stable in blood stored on ice over 8 h and in saline at low concentrations (<2 MBq/ml) but not at high concentrations (ca. 130 MBq/ml). Using SPE, the elimination half-life and full body distribution volume of 2-[¹⁸F]FA in healthy human volunteers were estimated as 4.2±0.8 h and 220±70 L, respectively. These results suggest that SPE is the method of choice for the determination of the plasma 2-[¹⁸F]FA concentration when measurement of individual metabolites is not required.

Introduction

The recent development of 2-[¹⁸F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-[¹⁸F]FA) for use as a positron emission tomography (PET) imaging radioligand allowed in vivo visualization of α₄β₂* nicotinic acetylcholine receptors (nAChRs) in the human brain for the first time [1], [2] and subsequent quantification [3], [4]. The designation “α₄β₂*” suggests that in addition to at least one α₄ subunit and one β₂ subunit, the pentameric receptor complex may contain other subunits as well [5]. The α₄β₂* nAChR is one of two predominant subtypes expressed in the mammalian brain [6]. Because α₄β₂* receptors have a higher affinity for nicotine than the other predominant subtype, α₇ receptors [6], the α₄β₂* nAChRs are the most likely target for nicotine from tobacco smoke, and these receptors probably play a critical role in nicotine addiction [7], [8]. Additionally, α₄β₂* nAChRs have been implicated in Parkinson's disease, Alzheimer's disease, autism and epilepsy (reviewed in Gotti et al. [9]). Noninvasive imaging of α₄β₂* nAChRs using 2-[¹⁸F]FA has become widely used for studies of these conditions [10], [11], [12], [13], [14], [15], [16] and could be extremely useful to delineate the specific role of α₄β₂* nAChRs in nicotine addiction and neurodegenerative disorders.

Quantifying α₄β₂* nAChRs in vivo with 2-[¹⁸F]FA involves measurement of total radioactivity in the brain as well as the concentration of nonmetabolized radioligand in arterial blood plasma throughout the scanning period. To avoid the complexity of arterial blood sampling and analysis, one could use a reference region approach that does not require an arterial input function. The cerebellum has been used as a reference region in rhesus monkeys [17], but the use of the cerebellum as a reference region in humans, baboons and rats is limited [4], [18], [19]. Thus, measurement of nonmetabolized 2-[¹⁸F]FA in blood plasma is critical for quantification of PET data from humans and animals.

Previously [17], [18], separation of 2-[¹⁸F]FA from its radiometabolites in blood plasma has been accomplished using high-performance liquid chromatography (HPLC). However, measurement of plasma 2-[¹⁸F]FA with HPLC is time intensive. For example, a 7- to 8-h PET study with 2-[¹⁸F]FA involves analysis of 15 to 22 plasma samples, requiring 10 to 11 h processing time with HPLC. Taking into account the half-life of [¹⁸F]fluorine (1.8 h) and the elimination rate of 2-[¹⁸F]FA from blood in vivo (6 h in humans [3], ∼2 h in rhesus monkey [17]), one may encounter great difficulty in accurately measuring the amount of nonmetabolized radioligand remaining at later time points of a 7- to 8-h PET study using HPLC. In PET studies with small animals, analysis using HPLC is further limited by low radioactivity, which is a result of small sample volumes.

The purpose of the present study was to develop a new method for measuring the concentration of nonmetabolized 2-[¹⁸F]FA in blood plasma during PET studies. Because 2-[¹⁸F]FA is an organic cation, we chose a mixed-mode reversed-phase/cation exchange cartridge for solid-phase extraction (SPE). To test the possibility of SPE as an alternative to the HPLC method, we first evaluated the ability of the SPE method to separate 2-[¹⁸F]FA from its radiometabolites in blood plasma. Measurements of the parent fraction (the percentage of radioactivity in total plasma attributable to nonmetabolized radioligand) obtained using both methods were then compared, and reproducibility of SPE measurements was examined. In addition, using the SPE method, we assessed the in vitro stability of 2-[¹⁸F]FA in saline and blood and assayed dynamic changes of 2-[¹⁸F]FA concentration after bolus administration of radioligand to healthy human volunteers.

Section snippets

Subjects and blood sampling

2-[¹⁸F]FA was synthesized using a semiautomated method that was a modification of a previously published procedure [20], [21]. Data from human plasma were obtained in conjunction with human PET studies. Administration of 2-[¹⁸F]FA to human volunteers was performed under an Investigational New Drug application to the Food and Drug Administration. The study design was approved by the Institutional Review Board of the National Institute on Drug Abuse Intramural Research Program. All participants

Separation of 2-[¹⁸F]FA in blood plasma using SPE

In preliminary studies, we demonstrated that 90% to 95% of 2-[¹⁸F]FA standard loaded onto SPE cartridges in saline was retained on the matrix. Large washing volumes (8 ml) of deionized water, sodium bicarbonate with a concentration up to 0.5 M, acetic acid with a concentration of up to 1% and absolute ethanol each eluted a negligible amount of 2-[¹⁸F]FA standard (<2%). Taking into consideration the compatibility of the washing reagents, the sequence chosen was as follows: loading the sample

Discussion

Results from the present study suggest that SPE is an acceptable alternative to HPLC for the measurement of the 2-[¹⁸F]FA parent fraction in blood plasma during PET studies. SPE is a one-step procedure for measuring the concentration of nonmetabolized 2-[¹⁸F]FA in full blood plasma. SPE measurements were reproducible and nearly equivalent to those obtained with the HPLC method. Consistent with our results, blood samples containing 2-[¹⁸F]FA could be stored on ice over 8 h without any

Acknowledgments

This study was supported by the Intramural Research Program of the National Institute on Drug Abuse, NIH, DHHS and ONDCP. We thank Dr. Alane Kimes, who was the principal investigator on the human subject protocol, and Ms. Susan S. Vaupel, Institute for Laboratory Animal Research, The National Academies, for editorial assistance.

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Technical noteA simplified method for the measurement of nonmetabolized 2-[18F]F-A-85380 in blood plasma using solid-phase extraction

Abstract

Introduction

Section snippets

Subjects and blood sampling

Separation of 2-[18F]FA in blood plasma using SPE

Discussion

Acknowledgments

Nucl Med Biol

Neuroimage

Neuroimage

Neuroimage

Neuroimage

Neuroimage

Neuroimage

Nucl Med Biol

Nucl Med Biol

Biodistribution and radiation dosimetry of 18F-fluoro-A-85380 in healthy volunteers

J Nucl Med

2-[18F]F-A-85380: PET imaging of brain nicotinic acetylcholine receptors and whole body distribution in humans

FASEB J

Technical note
A simplified method for the measurement of nonmetabolized 2-[¹⁸F]F-A-85380 in blood plasma using solid-phase extraction

Separation of 2-[¹⁸F]FA in blood plasma using SPE

Biodistribution and radiation dosimetry of ¹⁸F-fluoro-A-85380 in healthy volunteers

2-[¹⁸F]F-A-85380: PET imaging of brain nicotinic acetylcholine receptors and whole body distribution in humans