Optimizing an online SPE–HPLC method for analysis of (R)-[11C]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide [(R)-[11C]PK11195] and its metabolites in humans

doi:10.1016/j.nucmedbio.2004.12.005

Nuclear Medicine and Biology

Volume 32, Issue 3, April 2005, Pages 307-312

https://doi.org/10.1016/j.nucmedbio.2004.12.005 Get rights and content

Abstract

(R)-[¹¹C]PK11195 is used as a positron emission tomography tracer for activated microglia in several neurological disorders. Quantification of specific binding requires a metabolite-corrected plasma input function. In this study, a high-performance liquid chromatography (HPLC) procedure with online solid phase extraction was modified for analyzing (R)-[¹¹C]PK11195 plasma samples, yielding total sample recoveries of more than 98%. When applied to human studies, the use of two HPLC systems enabled analysis of up to seven plasma samples under regular conditions. Online radioactivity detection was compared with offline sample measurements of HPLC profiles. Offline measurements provided the most reliable results especially for late plasma samples. In 10 patients, an average decrease of parent compound from 94.6% at 2.5 min to 45.2% at 1 h after administration was observed.

Introduction

PK11195 [1-(2-chlorophenyl)-N-methyl-N-1-(1-methylpropyl]-3 isoquinolinecarboxamide) (CAS Registry No. 85532-75-8) is a highly selective ligand for the peripheral benzodiazepine receptor (PBR) binding site [1]. The PBR is located on the mitochondrial membrane and is present in high concentrations in peripheral organs such as the heart, lungs and adrenal glands [2]. In the brain, PBRs are mainly associated with activated microglia, which in turn are associated with the presence of neuronal injury [3]. Using ¹¹C-labeled PK11195, positron emission tomography (PET) studies have been performed in patients with stroke [4], [5], multiple sclerosis [6], Rasmussen's encephalitis [7] and Alzheimer's disease [8]. Use of [¹¹C]PK11195 for heart studies has also been described [9]. For quantification, a pure enantiomer should be used and the (R)-enantiomer is preferred because it has the highest affinity for binding to PBR sites [10].

To date, most PET studies have been analyzed using reference tissue models [8], [11]. Use of such a model, however, has not been validated for (R)-[¹¹C]PK11195, primarily because generation of a metabolite-corrected plasma curve has proven to be difficult. Validation of a reference tissue approach requires comparison with results obtained with a metabolite-corrected plasma input function [12].

(R)-[¹¹C]PK11195 is metabolized in vivo, resulting in labeled metabolites in plasma such as N-[¹¹C]methyl-sec-butylamine and [¹¹C]CH₂O [13]. Because of the short half-life of ¹¹C (t_1/2 = 20.3 min), a fast yet reproducible assay to determine the fraction of parent [¹¹C]PK11195 in plasma samples is needed. High performance liquid chromatography (HPLC) procedures for this purpose have been described, with both offline [13] and online [14] sample extraction procedures, using racemic mixtures.

Online solid phase extraction (SPE) with HPLC is preferred to minimize sample processing time with its associated loss in activity due to decay. Such a method has been described for several tracers [14], [15]. In the present study, this SPE procedure was modified and optimized for human (R)-[¹¹C]PK11195 studies. In addition, online radioactivity measurement was tested as an alternative to offline methods for obtaining HPLC activity plots.

Section snippets

Chemicals

Ammonium formate (BDH, Poole, England), di-ammonium hydrogen phosphate p.a. (Merck, Darmstadt, Germany) and methanol (BDH Hipersolv grade, BDH) were used to prepare the various HPLC buffers and eluent solutions. Reference PK11195 was obtained from ABX (Radeberg, Germany). (R)-[¹¹C]PK11195 was prepared as previously described [10].

Equipment

Two HPLC systems were used, each containing two pumps (Gyncotek P580 and Gyncotek 300, Separations, Hendrik Ido Ambacht, The Netherlands). The first pump supplied a

Results

The results of the experiments to optimize the SPE procedure are given in Table 1.

The settings specified in row (h) of Table 1 provided reproducible results for four successive analyses of (R)-[¹¹C]PK11195-containing plasma samples. Two separate experiments in which a batch of plasma was analyzed four times on the same HPLC system with the same SPE column yielded a parent percentage of 97.7±0.2% with a recovery of 96.9±4.9% and a parent percentage of 96.7±0.4% with a recovery of 100.1±4.4%.

Discussion

Implementation of SPE prior to the actual HPLC procedure of (R)-[¹¹C]PK11195-containing plasma samples was successful, yielding sample recoveries of >96%. A critical step within the SPE procedure was optimizing the flow rate of the plasma through the SPE column to enable quantitative trapping of the parent and nonpolar metabolites. Also, the time needed to elute activity from the SPE column was important. Increase in back pressure within SPE columns during the course of a study was observed. In

Conclusion

An HPLC method with an online SPE method for (R)-[¹¹C]PK11195 was optimized. Extraction efficiency was >96% and separation of the parent from metabolites was possible within a reasonable time frame, including the SPE procedure. This procedure is now in use for routine human (R)-[¹¹C]PK11195 studies, allowing for the analysis of up to seven metabolite samples per study.

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[¹¹C]Irinotecan was synthesized by two routes using [¹¹C]phosgene and [¹¹C]carbon dioxide fixation. Metabolites in the plasma of mice following injection of [¹¹C] irinotecan were investigated using a combination of column-switching high-performance liquid chromatography (HPLC) and on-line solid-phase extraction (SPE). Whole-body PET studies were conducted in wild-type mice and P-glycoprotein and breast cancer resistance protein (Pgp/Bcrp) knockout mice.
[¹¹C]Irinotecan was successfully synthesized by the two abovementioned routes. Decay-corrected radiochemical yields based on [¹¹C]carbon dioxide using [¹¹C]phosgene and [¹¹C]carbon dioxide fixation were 8.8 ± 2.0% (n = 8) and 16.9 ± 2.9 % (n = 5), respectively. Metabolite analysis of the plasma of mice following injection of [¹¹C]irinotecan was successfully performed using the column-switching HPLC and on-line SPE combination resulting in greater than 87 % recovery of radioactivity from HPLC. In the PET study in mice, the radioactivity levels in the brain, liver, and small intestine were slightly increased by inhibition of the Pgp/Bcrp function for more than 30 min after [¹¹C]irinotecan injection. This result demonstrated that in vivo behavior of [¹¹C] irinotecan and radioactive metabolites are influenced by the Pgp/Bcrp function.
PET studies using [¹¹C]irinotecan combined with metabolite analysis may be a useful tool for evaluating irinotecan pharmacokinetics and toxicity.
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The translocator protein 18 kDa (TSPO), although minimally expressed in healthy brain, is up-regulated in pathological conditions, coinciding with microglial activation. It is thereby a suitable in vivo biomarker of neuroinflammation for detection, evaluation and therapeutic monitoring of brain diseases. We aimed to estimate the radiation dosimetry of the positron emission tomography (PET) TSPO radioligand [¹⁸F]DPA-714, and we evaluated in healthy volunteers its whole-body uptake and cerebral kinetics.
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Nuclear Medicine and Biology

Abstract

Introduction

Section snippets

Chemicals

Equipment

Results

Discussion

Conclusion

References (18)

Peripheral benzodiazepine binding sites: effect of PK11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide: I. In vitro studies

Life Sci.

Peripheral-type benzodiazepine receptors

Life Sci.

Monitoring by PET of macrophage accumulation in brain after ischaemic stroke

Lancet

In-vivo measurement of activated microglia in dementia

Lancet

Synthesis of the enantiomers of [N-methyl-¹¹C]PK11195 and comparison of their behaviours as radioligands for PK binding sites in rats

Nucl. Med. Biol.

Simplified reference tissue model for PET receptor studies

Neuroimage

High-performance liquid chromatography determination of [¹¹C]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide in mouse plasma and tissue and in human plasma

J. Chromatogr. B.

Radioligands for PET studies of central benzodiazepine receptors and PK (peripheral benzodiazepine) binding sites — current status

Nucl. Med. Biol.

The peripheral benzodiazepine receptor ligand PK11195 binds with high affinity to the acute phase α1-acid glycoprotein: implications for the use of the ligand as a CNS inflammatory marker

Nucl. Med. Biol.

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Fluorine-18 Radiolabeled PET Tracers for Imaging Monoamine Transporters: Dopamine, Serotonin, and Norepinephrine