Available online at www.sciencedirect.com

Nuclear Medicine and Biology 39 (2012) 447–460www.elsevier.com/locate/nucmedbio

Synthesis of oncological [ 11C]radiopharmaceuticals for clinical PETFilippo Lodia,⁎, Claudio Maliziaa, Paolo Castelluccic, Gianfranco Cicoriab,

Stefano Fantic, Stefano BoschiaaPET Radiopharmacy, Nuclear Medicine Unit, Azienda Ospedaliero Universitaria di Bologna, Policlinico S. Orsola-Malpighi, Bologna, Italy

bMedical Physics, Azienda Ospedaliero Universitaria di Bologna, Policlinico S. Orsola-Malpighi, Bologna, ItalycPET Center, Nuclear Medicine Unit, Azienda Ospedaliero Universitaria di Bologna, Policlinico S. Orsola-Malpighi, Bologna, Italy

Received 22 June 2011; received in revised form 14 October 2011; accepted 22 October 2011

Abstract

Positron emission tomography (PET) is a nuclear medicine modality which provides quantitative images of biological processes in vivo atthe molecular level. Several PET radiopharmaceuticals labeled with short-lived isotopes such as 18F and 11C were developed in order to tracespecific cellular and molecular pathways with the aim of enhancing clinical applications. Among these [11C]radiopharmaceuticals areN-[11C]methyl-choline ([11C]choline), L-(S-methyl-[11C])methionine ([11C]methionine) and 1-[11C]acetate ([11C]acetate), which havegained an important role in oncology where the application of 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG) is suboptimal. Nevertheless, theproduction of these radiopharmaceuticals did not reach the same level of standardization as for [18F]FDG synthesis. This review describes themost recent developments in the synthesis of the above-mentioned [11C]radiopharmaceuticals aiming to increase the availability and hencethe use of [11C]choline, [11C]methionine and [11C]acetate in clinical practice.© 2012 Elsevier Inc. All rights reserved.

Keywords: Clinical PET; Oncology; [11C]radiopharmaceuticals; Radiosynthesis; [11C]choline; [11C]methionine; [11C]acetate

1. Introduction

Positron emission tomography (PET) is a powerful nuclearmedicine modality which provides imaging of biologicalprocesses in vivo [1]. This technique is based on administra-tion and detection of the biodistribution of radiopharmaceu-ticals labeled with positron-emitting radionuclides, allowingbetter quality imaging than conventional single-photon-emitting tomography, with higher sensitivity and good spatialresolution; also PET allows an accurate quantification ofregional radiopharmaceutical concentration [2].

Several PET radiopharmaceuticals labeled with short-lived isotopes such as 18F (t1/2=109.8 min) and 11C(t1/2=20.4 min) were developed in order to visualize specificcellular and molecular pathways and then applied inoncology, neurology and cardiology [3–7]. In particular,11C is an attractive PET radionuclide because carbon is aubiquitous element in biomolecules thus, [11C]-labeling

⁎ Corresponding author.E-mail address: filippo.lodi@aosp.bo.it (F. Lodi).

0969-8051/$ – see front matter © 2012 Elsevier Inc. All rights reserved.doi:10.1016/j.nucmedbio.2011.10.016

does not change the chemical structure and the biochemicalproperties in vivo. Moreover, the possibility to choose fromdifferent labeling positions in the same molecule providesthe possibility to refine the radiopharmaceutical in terms ofmetabolic stability and nonspecific background ratio [8]. Theshort life of 11C also enables comparative PET studies withthe same [11C]radiopharmaceutical (multitracer studies) in ashort time frame with more favorable patient dosimetry [9].On the other hand, the production of these radiopharmaceu-ticals must be performed in PET facilities with on-sitecyclotrons and should be as fast as possible to reduce the lossof activity due to decay.

In the last few years, [11C]radiopharmaceuticals havegained increased importance in clinical PET, with relevantapplications mainly in clinical oncology, in case of limitationof the gold standard PET radiopharmaceutical 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG), a glucose analogue used forstaging, restaging and assessing the therapy response of avariety of tumors [10]. Among [11C]radiopharmaceuticals,N-[11C]methyl-choline ([11C]choline), L-(S-methyl-[11C])methionine ([11C]methionine) and 1-[11C]acetate ([11C]acetate) (Fig. 1) are widely used in clinical PET with

Fig. 1. Oncological [11C]radiopharmaceuticals in clinical PET.

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important applications in oncology [11]. Fast and efficientlabeling reactions and purification methods are needed tohave high radiochemical yield, resulting in high radiophar-maceutical activities available for clinical use. Severallabeling methods have been described in the literature tomeet the increasing demand of these radiopharmaceuticalsfor clinical routine. Each of them presents some advantagesand disadvantages as well as differences in radiochemicalyield, overall synthesis time, purification procedures,radiochemical and chemical purity of the final product andsuitability for process automation.

The aim of this paper is to review some of the most recentpublications about the synthesis of the widely usedoncological [11C]radiopharmaceuticals: [11C]choline, [11C]methionine and [11C]acetate.

ig. 2. Production of [11C]CH3I: (1) LiAl4/HI method (“wet chemistry”), (2)dination of [11C]CH4 (“gas phase chemistry”).

2. Production of [11C]radiopharmaceuticals

The first step of [11C]radiopharmaceuticals synthesis isthe radionuclide production. 11C is produced by cyclotron as[11C]CO2 or [11C]CH4 by 14N(p,α)11C nuclear reaction.[11C]CO2 is produced using a mixture of nitrogen with traceamount to 2% of oxygen, while [11C]CH4 is produced usinga mixture of nitrogen with 5%–10% of hydrogen as gastarget. Another way to produce [11C]CH4 is the reduction of[11C]CO2 with hydrogen on a nickel catalyst at hightemperature [12]. [11C]CO2 can be recovered from cyclotronand purified by means of cryogenic trapping with liquidnitrogen or by trapping on molecular sieves [13]. [11C]CH4

can be recovered and purified with a Porapak N trap [12].The use of in-target produced [11C]CH4 improves the

specific activity (SA) [14,15] but requires a long time toreach maximum yield, and in general, total obtained activityis lower compared to [11C]CO2 target [15]. [11C]CO2 and[11C]CH4 are usually employed for the preparation of morereactive [11C]-labeling agents which are directly involved in[11C]radiopharmaceuticals synthesis.

The most commonly used are [11C]methylating agentssuch as [11C]methyl iodide ([11C]CH3I) and [11C]methyltriflate ([11C]CH3OTf), which are employed in alkylationreactions ([11C]methylations) [16].

[11C]CH3I can be prepared by using two differentmethodologies: the “wet chemistry,” which is based on[11C]CO2 reduction by LiAlH4 and followed by iodinationwith hydroiodic acid (HI) [13,17,18], and the “gas phasechemistry,” which synthesizes [11C]CH3I from radicaliodination of [11C]CH4 by molecular iodine (I2) [12,19](Fig. 2).

These methods present some pros and cons in terms of[11C]CH3I radiochemical yield, SA, synthesis reagents andcleaning procedures after the production.

Compared to “gas phase chemistry,” the “wet chemistry”method generally provides [11C]CH3I in higher yields(almost twofold higher) and in shorter synthesis time: thesefeatures are advisable in [11C]-labeling and in clinicalpractice because of the short half-life of this radionuclide andfor the high activities needed to study several patients inshorter time. However, the use of reagents like HI andLiAlH4 makes difficult the managing of the synthesispreparation and cleaning procedures. Moreover, LiAlH4

represents a carrier of cold CO2 which may decrease the SAof [11C]CH3I. Average SA values reached with this methodare 1–5 Ci/μmol decay corrected (DC) at the end of synthesis(EOS). Low LiALH4 amount with freshly distilled solvent,low target volume and high-purity gas with traps for coldCO2 in the line from gas target to cyclotron are recom-mended to increase [11C]CH3I SA [13].

On the contrary, an advantage of the “gas phasechemistry” resides in the ease of use in cleaning proceduresbecause of HI elimination and in the possibility to run moresyntheses of [11C]CH3I without adding or changing re-agents. Furthermore, elimination of LiAlH4 contributes tothe higher SA of [11C]CH3I (even more than 15 Ci/μmol DCat EOS [12]), a clear advantage of this method when higherSA radiopharmaceuticals are needed (i. e. high-affinityreceptorial molecules). Although high SA is always a goodprerequisite, oncological [11C]radiopharmaceuticals such as[11C]choline and [11C]methionine are generally transportedinto the cell and are not receptorial affinity molecules,making SA a less critical factor.

Because of its higher chemical reactivity, [11C]CH3OTfenables fast and low-temperature labeling reactions withsmaller amount of desmethyl precursors. [11C]CH3OTf issynthesized by passing [11C]CH3I, through a silver triflate(AgOTf) column at high temperature (200°C) [20–22](Fig. 3).

[11C]methylation reaction can be performed by “bub-bling” technique or on solid-phase support. In the first

Fio

Fig. 3. Production of [11C]CH3OTf from [11C]CH3I.

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approach, the methylating agent ([11C]CH3I or [11C]CH3OTf) is bubbled in a solvent where desmethyl precursoris dissolved. At the end of reaction, solvent is removed byevaporation. In the latter approach, the methylating agentflows through a solid-phase extraction (SPE) column or aloop made of stainless steel or different plastic materials(Tefzel, Teflon, polyethylene, etc.) on which desmethylprecursor solution is loaded [23,24]. This synthesis methodprovides fast reaction time and easy workup and makesautomation straightforward. Furthermore, SPE columnscould be used for a partial purification of the product.

Final steps of [11C]radiopharmaceuticals synthesis arepurification of the product by means of semipreparativehigh-performance liquid chromatography (HPLC) or SPEtechniques, followed by formulation and sterilization by0.22-μm membrane filter.

[11C]CH3I and [11C]CH3OTf synthesis, [11C]-labeling

reactions and purification steps of the final product areperformed in automated closed systems called synthesismodules [25–29]. These devices provide the reduction ofoperator exposure to high level of radiations, enable toobtain high reproducibility of the synthesis processminimizing operator errors and could facilitate thecompliance with regulatory requirements for GoodManufacturing Practices (GMP).

3. Oncological [11C]radiopharmaceuticals

3.1. [11C]choline

Choline is a quaternary ammonium salt which isimportant for cellular metabolism. This molecule is involvedin cell signal transduction, in lipoprotein metabolism and inthe biosynthesis of phosphatidylcholine, the main compo-nent of eukaryotic cell membrane [30]. In the biosynthesis ofthis phospholipid, choline is phosphorylated by cholinekinase to phosphorylcholine which is converted to phospha-tidylcholine. Several malignant tumors have been shown tooverexpress choline kinase [31], resulting in increased levelsof phosphorylcholine by accumulation of free choline forcell membrane synthesis [32–34]. For this reason, [11C]choline was introduced as a radiopharmaceutical foroncological PET studies of a variety of malignant diseases[35–38]. Among these, the most important clinical applica-tion is in prostate cancer [39,40], where [18F]FDG has beendemonstrated to be not accurate due to low uptake [41,42],related to lower expression of glucose transport proteins andto the huge [18F]FDG urinary excretion which interfereswith the imaging in the pelvis area. [11C]choline is rapidlyaccumulated by prostate cancer, and the uptake remains

constant thereafter, allowing better visualization of this kindof tumor [43].

This radiopharmaceutical was employed for the detectionof local or distant tumor recurrence after radical prostatec-tomy (Fig. 4) or radiation therapy [44–47]. [11C]choline hasalso been used to stage prostate cancer [48–52], andpreliminary results are available regarding the use of [11C]choline in the study of bladder cancer [53].

Another application of [11C]choline is the imaging ofbrain tumors, where it provides better visualization ofneoplastic masses than [18F]FDG with clear tumor delinea-tion because of low physiological uptake in the gray matter[54,55].

3.2. [11C]methionine

[11C]methionine is a labeled analogue of the essentialamino acid methionine. This molecule enters in severalmetabolic pathways such as protein synthesis and conversionto S-adenosylmethionine, which is the main biologicalmethyl group donor and precursor of cysteine and de-rivatives. Tumor cells present an enhanced expression ofamino acid transporter systems as well as protein synthesiswith an increased demand of methionine [56]. For thesereasons, [11C]methionine was used as oncological radio-pharmaceutical in the study of brain tumors [57] since aminoacid transporters are overexpressed to increase amino acidsuptake [58,59]. Furthermore, since [11C]methionine showedlow uptake in normal brain tissue in comparison to [18F]FDG, the resulting low physiological uptake [60,61] allows abetter identification and delineation of low to intermediate-grade tumors. Time/activity curves in glioma tumors showedthat the uptake of [11C]methionine is maximum at 8–10 minand remained constant at least to 20 min [62].

[11C]methionine has found clinical applications in case ofsuspected recurrence of gliomas (Fig. 5) [63], for delineatingradiotherapy target volume [64], as guidance for stereotacticbrain biopsy [65], and for monitoring and predicting medicaltreatment response [66]. The use of [11C]methionine wasalso investigated in several applications such as hyperpara-thyroidism [67,68], head and neck tumors [69], and lungcancer [70].

3.3. [11C]acetate

Acetate is an important metabolite in the synthesis ofcholesterol and lipids. Acetate underwent an intracellularconversion to acetyl-CoA which is a substrate for Krebs'scycle (TCA) for cellular energy production and a metabolicintermediate for fatty acids and cholesterol synthesis aswell. [11C]acetate was initially employed for the study ofmyocardial metabolism [71,72] because lipid metabolismseems to be the mayor route for energy production insteadof glycolysis in normal myocardium [73]. More recently,the use of [11C]acetate was investigated in oncologybecause tumor cells show several alterations of lipidmetabolism like overexpression of fatty acid synthase

Fig. 4. A 74-year-old male with prostate cancer. PET/computed tomography (CT) images acquired 5 min after the administration of 400 MBq of [11C]choline.Patient suffering from biochemical relapse Prostate Specific Antigen (PSA) (3.0 ng/ml) after radical prostatectomy. CT, PET and fused image showing increaseduptake of [11C]choline in a metastatic right iliac lymph node.

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(FAS). This enzyme catalyzes the fatty acid synthesis fromacetyl-CoA which becomes incorporated into phospho-lipids and cellular membranes [74,75]. This was supportedby the fact that in vitro and in vivo pharmacological FASinhibition can reduce [11C]acetate uptake in tumor [76].Low urinary excretion and metabolic characteristics make[11C]acetate an important tracer for the imaging of prostatecancer [42,77,78]. It should be noted that because of [11C]acetate kinetic behavior of prostate cancer [79], delayedimage acquisition to 30–40 min after injection results inbetter tumor to background ratio [80] because TCA activitydrops to b20% of the peak at 20 min [79].

[ 11C]acetate was also employed in the study ofhepatocarcinoma (HCC) (Fig. 6) [81,82], lung cancer [83]and brain tumors [84,85].

4. Synthesis methods and quality control of [11C]choline,[11C]methionine and [11C]acetate

4.1. [11C]choline synthesis

Synthesis of this [11C]radiopharmaceutical is an N-[11C]methylation of amine precursor N,N′-dimethylaminoethanol(DMAE) [86,87] using [11C]CH3I or [11C]CH3OTf as

Fig. 5. A 32-year-old female with glioblastoma. PET images acquired 10 min after the administration of 360 MBq of [11C]methionine. PET shows an increaseduptake of the radiopharmaceutical in the postsurgical rim in the right temporoparietal lobe for the presence of a relapse of the disease.

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methylating agents (Fig. 7). During [11C]methylation,DMAE behaves as a [11C]choline precursor and reactionsolvent as well. However, in order to keep residual DMAE inthe product as low as possible because of competition with[11C]choline in the cell uptake in vivo and to improve thechemical purity of the product [88], DMAE can be alsodiluted with different solvents. [11C]methylation is thenfollowed by purification and formulation of the product inorder to obtain a sterile injectable solution for clinical usewith high chemical and radiochemical purity.

In Table 1 are reported some published recent synthesismethods for this radiopharmaceutical with methylating

agent. Many of these methods produced [11C]CH3I or[11C]CH3OTf using “wet chemistry” approach because SAis not a critical factor for oncological use of thisradiopharmaceutical. [11C]choline was synthesized usingbubbling and SPE [11C]methylation methods. Some effortsto improve the radiosynthesis were done to reduce the overalltime and the amount of precursor DMAE. Hara and Yuasa[90] described the synthesis of this radiopharmaceuticalusing [11C]CH3I as a methylating agent, produced with “wetchemistry” method. [11C]CH3I was bubbled in 0.5 ml ofprecursor DMAE followed by heating at 130°C for 5 min.The excess of precursor was removed by evaporation, and

Fig. 6. A 65-year-old male with HCC. PET/CT images acquired 10 min after the administration of 360 MBq of [11C]acetate. CT, PET and fused image showingincreased uptake of [11C]acetate in a metastatic lesion of a vertebral body. Maximum Intensity Projection (MIP) shows the presence of multiple bone lesions.

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[11C]choline was purified with a cation exchange SPEcolumn (Sep-Pak Accell Plus CM, Waters). Synthesis yield,based on [11C]CO2 produced, was high (86% DC at the endof bombardment EOB), as well as radiochemical purity.

Fig. 7. Radiochemical synthesis of [11C]choline.

Residual amount of DMAE was less than 0.2 μg/ml. Theoverall synthesis time was 20 min. The same [11C]cholinesynthesis method described by Hara and Yuasa was appliedby Mishani et al. [94] in a commercially available automatedmodule for the routine production of this radiopharmaceu-tical. This method employed 0.2 ml of DMAE for the [11C]CH3I reaction with a synthesis yield similar to that describedby Hara and Yuasa, and the radiochemical purity was 100%.

Zhang et al. [95] reported a synthesis based on [11C]CH3Ias methylating agent and bubbling method, performing thelabeling reaction in a small volume of acetone to reduce theamount of employed precursor. This paper also described the

Table 1Some recent synthesis methods for [11C]choline

Authors and year [11C]Methylationagent

Method ofsynthesis

Ref.

Pascali et al., 2000 [11C]CH3I SPE (on column) [89]Hara and Yuasa, 1999 [11C]CH3I Bubbling [90]Reischl et al., 2004 [11C]CH3I SPE (loop) [91]Quincoces et al., 2006 [11C]CH3I SPE (on column) [92]Kuznetsova et al., 2003 [11C]CH3I SPE (on column) [93]Mishani et al., 2001 [11C]CH3I Bubbling [94]Zhang et al., 2006 [11C]CH3I Bubbling [95]Roivainen et al., 2000 [11C]CH3OTf Bubbling [96]Zheng et al., 2004 [11C]CH3OTf Bubbling [97]Lodi et al., 2008 [11C]CH3I SPE (on column) [98]Smith et al., 2006 [11C]CH3I SPE (loop) [99]Shao et al., 2011 [11C]CH3I SPE (on column) [100]

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results of modifying several labeling parameters, such astemperature, reaction time and volume, quantity of precursorDMAE and pH, in order to optimize the [11C]cholinesynthesis reaction. The better reaction conditions found were2 mg of precursor in 50 μl of acetone at room temperature for2 min with a radiochemical yield, based on [11C]CH3Iproduced, of 96.5%±2.3% (n=3, DC, EOB). The totalsynthesis time was less than 20 min.

[11C]CH3OTf was employed in the synthesis of [11C]choline by Roivainen et al. [96] and Zheng et al. [97]. Themethods described by these groups take advantage of higherreactivity of [11C]CH3OTf in reducing the amount of DMAEduring the [11C]methylation reaction without heating. Roivai-nen et al. trapped [11C]CH3OTf in a reaction vessel containing25 μl of DMAE at room temperature. The vessel was thenwarmed at 100°C to remove both the precursor and themethylating agent by evaporation under nitrogen flow, and theresidue was collected with saline. [11C]choline was synthesizedin 25 min with radiochemical yield of 65% (based on produced[11C]CO2 DC, EOB), radiochemical purity higher than 99.9%and residual DMAE concentration b0.2 μg/ml. Zheng et al.used 10 μl of DMAE dissolved in 250 μl of acetonitrile in the[11C]CH3OTf reaction followed by purification with SPEtechnique. [11C]choline was obtained with high radiochemicalyield (60%–85% DC EOB, based on produced [11C]CO2) andhigh radiochemical purity (95%–99%) in 15–20 min.

Pascali et al. [89] introduced the solid-phase synthesisapproach in performing the [11C]methylation reaction ontoa C18 SPE column where DMAE was loaded. The realbreakthroughs of this method over conventional bubblingmethod were the easiness of the process and automation aswell as the reduction of overall synthesis time. [11C]CH3Iwas delivered onto a C18 Sep-Pak (Waters) column where60 μl of DMAE was loaded; the reaction occurred at roomtemperature, and the excess of precursor was removed bypassing ethanol and water through the column. [11C]cholinewas retained by another SPE cation exchange cartridge(Sep-Pak Accell Plus CM, Waters) and eluted with NaCl0.9% solution. Obtained yield, based on [11C]CO2

produced, was 87% (DC, EOB), and the radiochemical

purity was higher than 99.5%. The total synthesis time was12 min. The residual DMAE concentration in the productwas 28 μl/ml. This method was applied in the routineproduction of [11C]choline by Lodi et al. [98]. This groupanalyzed the reliability and the reproducibility of thismethod, reporting the results of long-term survey and themost frequent troubleshooting. Quincoces et al. [92] usedthis SPE technique to perform a simultaneous synthesis of[11C]choline and [11C]methionine in order to obtaindifferent [11C]radiopharmaceuticals for clinical studies ina short time frame.

Kuznetsova et al. [93] reported a modification of themethod described by Pascali et al. in reducing the precursoramount in the reaction. DMAE was diluted with ethanol indifferent proportions and loaded onto tC18 SPE columnwithdifferent amount of resin. Better [11C]methylation condi-tions found were 25/50 μl DMAE/ethanol loaded onto 0.1 gof SPE column resin. Residual amount of DMAE in the finalsolution after purification was low (1.6 μg/ml), and theradiochemical yield was 85% (DC) with average synthesistime of 13 min referring to the trapping of [11C]CH3I ontoSPE column. Radiochemical purity of [11C]choline wasgreater than 99.5%. Shao et al. [100] produced [11C]cholinewith “gas phase chemistry” using only one Sep-Pak CMcartridge for both reaction and purification in order to reducethe back-pressure of two adjacent Sep-Pak cartridges whichslowed down the washing process. The amount of DMAEloaded on Sep-Pak was 40 μl diluted with 20 μl of ethanol,and the mean radiochemical yield was 63.1% (nN10, DCEOB). Radiochemical purity of [11C]choline was 99.9%.The use of only one cartridge reduced the total synthesistime, and the elimination of C18 Sep-Pak minimized theresidual precursor in the final solution.

Another solid-phase synthesis approach for [11C]cholinewas the loop method where DMAE is loaded in plasticloop and [11C]CH3I is delivered through to react with theprecursor coated on the loop surface at room temperature.Reischl et al. [91] described in-loop synthesis of [11C]choline using a 1.0-m Tefzel tube loaded with differentamounts of DMAE using [11C]CH3I produced with “gasphase chemistry.” The product was then purified by meansof cation exchange cartridge. Sixty microliters was theamount of precursor chosen for the production because ofhigh yield, low residual DMAE concentration in the finalsolution (b10 ppm) and high radiochemical purity (N99%)in 20 min of synthesis. Smith et al. [99] reported anotheron-loop [11C]methylation with [11C]CH3I by using HPLCloop (2 ml) loaded with 10 μl of DMAE in 200 μl ofDMF. [11C]choline was synthesized in 25 min with 45%of yield (DC, EOB).

4.2. [11C]methionine synthesis

Synthesis of [11C]methionine is a [11C]methylation ofL-homocysteine sulfide anion obtained in presence of basefrom precursor L-homocysteine thiolactone [101] (Fig. 8).

Table 2Some recent synthesis methods for [11C]methionine

Authors and year [11C]Methylationagent

Method ofsynthesis

Ref.

Pascali et al., 1999 [11C]CH3I SPE (on column) [105]Gomez et al., 2008 [11C]CH3I SPE (loop) [106]Mitterhauser et al., 2005 [11C]CH3I SPE (on column) [29]Quincoces et al., 2006 [11C]CH3I SPE (on column) [92]Nagren and Halldin, 1998 [11C]CH3OTf Bubbling [107]Lodi et al., 2008 [11C]CH3I SPE (on column) [98]Gomzina and Kuznetsova,2011

[11C]CH3I SPE (on column) [108]

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[11C]CH3I is bubbled in the precursor solution followed byreaction at high temperature, purification with semiprepara-tive HPLC [101–103] and formulation in order to obtain asterile injectable solution of [11C]methionine for clinical use,with high chemical and radiochemical purity. Other [11C]methionine precursors were L-S-benzyl-homocysteine [102]and DL-homocysteine [101,103].

As for [11C]choline synthesis, some efforts were done toreduce the overall time to improve the radiochemical yieldand the enantiomeric purity of the L-form which is thepreferable stereoisomer because it is directly involved inprotein synthesis. Nevertheless, the presence of the D-formdoes not affect tumor uptake [104].

In Table 2 are reported some recent synthesis methods for[11C]methionine. Nagren et al. [107] reported [11C]methio-nine synthesis by bubbling [11C]CH3OTf instead of [11C]CH3I in the precursor solution. Taking advantage of higherreactivity of [11C]CH3OTf, the reaction was carried out withsmall amount of L-homocysteine thiolactone (1 mg) in 1 MNaOH (9 μl) and water (100 μl) at low temperature (60°C, 1min). The product was then purified by semipreparativeHPLC. Radiochemical yield (60%–70%, DC from produced[11C]CH3OTf) and radiochemical purity were high, and thefinal product showed a lower amount of the D-form (1%–2%).

More recently, SPE [11C]methylation approaches wereintroduced, improving the synthesis of this radiopharmaceu-tical. Advantages of these methods over conventionalbubbling method are the simplicity of the process andautomation (the reaction is carried out at room temperature),the shorter reaction time and the elimination of time-consuming HPLC purification. As for [11C]choline, Pascaliet al. [105] described the solid-phase synthesis method also for[11C]methionine. [11C]methylation with [11C]CH3I wasperformed at room temperature onto a Sep-PakC18 disposablecolumn loaded with the precursor L-homocysteine thiolactonehydrochloride with a good radiochemical yield and shortsynthesis time (11 min). Furthermore, the C18 columnfurnished a purification of the product which was obtainedwith a radiochemical purity N99%. Optimal concentration for

Fig. 8. Radiochemical synthe

precursor, NaOH and ethanol was investigated in order toreach high radiochemical yield and high enantiomeric purityof the L-form as well [103]. Following conditions for theproduction were used: 15.4 mg of precursor dissolved in asolution of 0.5 N NaOH in ethanol-H2O 50:50 (1 ml) and 210μl of this solution loaded into the column. The product wasthen eluted with 0.05 M NaH2PO4 buffer and diluted withNaCl 0.9% solution. Radiochemical yield of [11C]methionine,based on [11C]CO2 produced, was 78% (DC, EOB), and theenantiomeric purity of the L-form was 90%.

Mitterhauser et al. [29] presented a solid supportedregioselective synthesis of [11C]methionine which was animprovement of themethod described by Schmitz et al. [109]:[11C]CH3I was reacted with a precursor solution L-homo-cysteine (2 mg in 1 ml of ethanol) adsorbed in a suspensioncontaining 20 mg Al2O3/KF. This method avoids the time-consuming HPLC purification of the product using SPEcartridges. Enantiomeric purity (L-form) was higher than99%, and no radiochemical impurities were detected in theproduct solution. Radiochemical yield was 21.22%±7.9%(n=258, mean±S.D.) not corrected for decay.

On-loop synthesis of [11C]methionine was reported byGomez et al. [106] where [11C]CH3I was trapped in theL-homocysteine precursor solution loaded in HPLC loop(2 ml). Precursor amount was 1 mg dissolved in 80 μl of 0.5M NaOH solution in ethanol/water 50/50, and the reaction

sis of [11C]methionine.

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was carried out for 1 min at room temperature. The productwas then diluted with NaCl 0.9% solution, pulled to a C18SPE column for purification and neutralized with NaH2PO4

buffer. Radiochemical yield of [11C]methionine was 57.8%±6.2% (n=20, [11C]CO2 produced, DC, EOB) in 12 min ofsynthesis time, with a radiochemical purity of 99.9%±0.05%.Only the L-form was present in the final product.

As for [11C]choline, Lodi et al. [98] reported thereliability and the reproducibility of the on-column [11C]methylation method for [11C]methionine synthesis describedby Pascali et al. as a suitable and useful tool for routineclinical use. The paper reported the results of consecutiveruns, quality controls and clinical applications during yearsof [11C]methionine routine production.

Quincoces et al. [92] introduced the simultaneoussynthesis of [11C]choline and [11C]methionine with thison-column method to produce several [11C]radiopharma-ceuticals for clinical applications. Recently, Gomzina andKuznetsova [108] reported a synthesis modification in orderto improve the enantiomeric purity of [11C]methionineproduced with the on-column method [105]: the lactoneprecursor (2.5 mg) was dissolved in a solution of 0.5 MNaOH in ethanol-H2O 35:65 (instead of 50:50) loaded ontoSep-Pak tC18 column. This condition provided highenantiomeric purity (93.7%±0.5%) as well as high repro-ducible radiochemical yield (75%±3%, n=100, based onproduced [11C]CH3I) for clinical applications.

4.3. [11C]acetate synthesis

[11C]acetate is synthesized by means of [11C]carboxyl-ation reaction of Grignard reagent methylmagnesiumchloride or bromide (CH3MgCl, CH3MgBr) by cyclotron-produced [11C]CO2 (Fig. 9). Unlike for [11C]methylationreactions, the target product [11C]CO2 is directly employedin the labeling step without any further chemical conversion.[11C]carboxylation is then followed by hydrolysis andpurification of the product. A different procedure involvingthe hydrolysis of [11C]acetyl chloride to [11C]acetate wasalso reported by Oberdorfer et al. [110]. [11C]acetate wasthen formulated in order to have a sterile injectable solutionfor clinical use.

As regards the synthesis method, [11C]CO2 can bebubbled directly in the Grignard reagent or can be flushedand reacted into a loop of different tubing materialscontaining the immobilized reagent in the inner surface(solid-phase synthesis). Several synthetic approaches for

Fig. 9. Radiochemical synt

[11C]acetate have been reported in the literature based on[11C]carboxylation of Grignard reagent. Among these, themain differences concern the reaction method (bubbling or insolid phase), the concentration of Grignard reagent and themodality of [11C]acetate isolation and purification. Regard-ing the latter, first syntheses of this radiopharmaceutical werebased on liquid–liquid extraction [111,112] or via distilla-tion of [11C]acetate in buffer solution [113]. Nevertheless,most of the [11C]acetate synthesis methods employed ionexchange resin columns for product purification because offaster approach and more adaptability to synthesis automa-tion. In Table 3 are reported some recent methods for [11C]acetate production with purification procedures.

A fully automated synthesis of [11C]acetate by means ofrobotic device with an SPE purification of final product wasdescribed by Moerlein et al. [115]: in this work, [11C]CO2

was trapped and reacted with 3 ml of 0.05 M CH3MgBr indiethyl ether. After evaporation of the solvent, the reactionmixture was hydrolyzed with 2 ml of 4 N hydrochloric acid.The product was then diluted with water and purified by SPEwith AG 11A8 ion retardation resin and C18 resin. Overallradiochemical yield was 60%–69% (based on trapped [11C]CO2) in 23 min, with a radiochemical purity of 95%–98%.

Roeda et al. [114] applied the [11C]acetate synthesisreported by Kruijer et al. [119] by bubbling [11C]CO2 inCH3MgCl solution followed by hydrolysis and ion exchangecolumns purification, introducing some modifications inorder to improve the method. The authors replacedhomemade columns with commercial available cartridgesand reduced the amount of Grignard reagent for [11C]carboxylation to decrease the formation of [11C]-labeled by-products. Since target formed nitric oxides are detrimentalfor the synthesis, a column trap [120] for the purification of[11C]CO2 from nitric oxide and nitrogen dioxide producedduring target irradiation was also introduced. [11C]CO2

produced was trapped in 1 ml CH3MgCl 0.1 M in THF, andthe reaction mixture was hydrolyzed by 6 ml of 1 mMCH3COOH solution and pulled to three SPE cartridges: PS-H+, PS-Ag+ and PS-OH (Chromfix, Machereney Nagel).Columns were rinsed with 4 ml of 1 mM CH3COOHsolution and [11C]acetate, retained on PS-OH, was washedwith 20 ml of sterile water and eluted with isotonic citratebuffer (pH 4.7). Final product solution was bubbled toremove unreacted [11C]CO2. Radiochemical yield of [11C]acetate was N80% ([11C]CO2 produced, DC, EOB) in 12min, and no radioactive impurities were present in the finalproduct solution.

hesis of [11C]acetate.

Table 3Some recent synthesis methods for [11C]acetate

Authors and year Method ofsynthesis

Method of [11C]acetateisolation/purification

Ref.

Roeda et al., 2002 Bubbling SPE [114]Moerlein et al., 2002 Bubbling SPE [115]Mitterhauser et al., 2004 Bubbling Distillation [116]Soloviev and Tamburella, 2006 Loop SPE [117]Le Bars et al., 2006 Loop SPE [118]

456 F. Lodi et al. / Nuclear Medicine and Biology 39 (2012) 447–460

Mitterhauser et al. [116] described a fast and reliablemethod for routine preparation of [11C]acetate via distilla-tion using a sterile, inert and disposable apparatus. Thissystem allows to perform [11C]carboxylation and purifica-tion under inert conditions, avoiding long cleaning anddrying procedures needed for the high sensitivity of theGrignard reagent to moisture. [11C]CO2 was trapped anddelivered in a vial containing 1 ml of 150 mM CH3MgBr indiethyl ether/THF. The reaction was quenched with 50 μl ofwater and the vial was heated at 95°C to remove the solventsunder helium flow. Aqueous phosphoric acid was then addedand the vial was heated to 135°C for 1 min. [11C]acetate wasdistilled at 175°C into a vial containing 6 ml of 125 mMphysiological phosphate buffer passing through a C18 Sep-Pak column to reduce the residual THF and the radiochem-ical impurity [11C]acetone. Radiochemical yield was26.0%±3.9% (n=61, mean±S.D., not DC, based oncalculated [11C]CO2 EOB) in 13.6±1.7 min synthesistime, with a radiochemical purity higher than 98%.

Soloviev and Tamburella [117] reported a solid-phaseon-loop captive solvent synthesis of [11C]acetate [121]combined with SPE purification method described byRoeda et al., under GMP conditions. Different anion-exchange SPE cartridges for [11C]acetate retention in orderto separate the product from an unknown anionic radiola-beled impurity, formed during the synthesis in differentamounts, were also tested. Maxiclean SAX-Cl (Alltech) wasthe cartridge which allowed better purification and goodtrapping of [11C]acetate. This method also avoided acidicsolution for the hydrolysis step using sterile water and strongcation-exchange cartridge PS-H+. The reaction loop (poly-ethylene, 100-cm length, 1.5-mm internal diameter) wasfilled with a solution of CH3MgCl in THF and flushed withnitrogen flow in order to have about 10–50 μl (20–100 μmolof CH3MgCl) in the tube surface. [11C]CO2 was trapped inthe reagent loop, and 30–50 ml of sterile water was passedthrough to push the crude product to SPE columns PS-H+,PS-Ag+ and SAX-Cl. [11C]acetate was retained on SAX-Clcartridge and eluted with 10 ml of NaCl 0.9% solution. Meanradiochemical yield was 65% (DC, EOB) in 5 min ofsynthesis time with mean radiochemical purity of 98.6%±1.1% (n=30). Another on-loop synthesis approach of[11C]acetate with SPE purification was described by LeBars et al. [118]. [11C]CO2was reacted on loop (polyethylene1-m, 1.6-mm internal diameter) filled with 100 μl of 1.5 M

CH3MgBr in diethyl ether/THF solution. Two milliliters of 1mM CH3COOH was passed through the loop and on SPEcartridges IC-H/IC-Ag (mixed) and IC-OH (Maxi-clean,Alltech). The loop and the cartridges where washed with 5 mlof water, and IC-OHwith further 10 ml of water. [11C]acetatewas eluted with 5 ml of NaCl 0.9% solution in a vial with0.5 ml of 2 N HCl and bubbled with nitrogen flow to remove[11C]carbonate. The solution was finally neutralized with6 ml NaHCO3 1.4% solution and diluted with NaCl 0.9%solution. Obtained radiochemical yield was 60%–70% (DC,EOB) in less than 12 min with high radiochemical purity.

4.4. Quality control

Quality controls of these [11C]radiopharmaceuticalsshould be performed according to Pharmacopoeia mono-graphs if present or according to the general “Radiophar-maceuticals preparation” monograph. Currently, only [11C]methionine and [11C]acetate monographs are available onEuropean Pharmacopoeia (Eu. Ph.) [122]. No monograph for[11C]choline is available so far.

Quality control protocols should include visual inspec-tion and measurements of fundamental parameters such aspH, sterility, bacterial endotoxins, radionuclidic purity,identification, radiochemical and chemical purity. Sterilityand bacterial endotoxins should be carried out as describedin the general Eu. Ph. chapters. Radionuclidic purity isdetermined by gamma-ray spectrometry and by measuringthe radionuclide half-life.

Identification, radiochemical and chemical purity aredetermined by HPLC equipped with radiodetector (Radio-HPLC) with different methods for each radiopharmaceutical:for [11C]choline, these parameters could be determined byRadio-HPLC connected with a conductimeter, by using astrong cation exchange resin as stationary phase with acidicmobile phase [100,123,124], or with a refractometer, byusing a reverse-phase resin with mobile phase containing acounter ion like naphthalene-2-sulfonic acid [89,90,93], inorder to detect the mass signal of choline as well as theresidual precursor DMAE. DMAE concentration could bealso detected by using UV detector [93,97] and gaschromatography (GC) [89,91,92]. Residual organic solventsare carried out by GC.

Concerning [11C]methionine, these parameters are deter-mined by reverse-phase Radio-HPLC connected with UVdetector. Chemical purity concerns the detection of theamount of methionine and cold impurities as L-homocysteinethiolactone and DL-homocysteine. Residual organic solventsare measured by GC. Enantiomeric purity must to bedetermined as well by using a thin layer chromatography forchiral separation [29,109] or HPLC [105,106].

Furthermore, an important issue to consider in routineproduction of [11C]methionine is the control of the stability(not prescribed by Eu. Ph.) because this radiopharmaceuticalis very sensitive to radiolysis caused by its own radiation,with consequent decrease of radiochemical purity [125,126].

457F. Lodi et al. / Nuclear Medicine and Biology 39 (2012) 447–460

For [11C]acetate, Radio-HPLC is connected with UVdetector with strongly basic anion exchanger resin and 0.1 MNaOH as mobile phase. Chemical purity concerns thedetection of the mass of acetate and the residual organicsolvents by GC. Another method to measure the radiochem-ical and chemical purity of [11C]acetate reported in theliterature concerns the Aminex and reverse-phase resins withUV and refractometer detectors [114,116–118].

5. Conclusion

11C is an attractive PET radionuclide because it enablesthe labeling of biomolecules without changing the chemicalstructure and the biochemical properties in vivo. The shorthalf-life is also an advantage for reduced dosimetry to thepatient and it enables multitracer studies in a short timeframe; however, the production process of these radiophar-maceuticals should be as fast as possible to reduce the loss ofactivity due to decay.

The most commonly used method for [11C]-labeling is[11C]methylation reaction with [11C]CH3I and [

11C]CH3OTf.[11C]CH3I can be prepared by using the “wet chemistry” or“gas phase chemistry” approaches: the first one provideshigher radiochemical yields but lower specific activity valueswith respect to “gas phase chemistry.” High SA is mandatoryin the synthesis of high-affinity receptorial molecules.

[11C]radiopharmaceuticals have gained increased impor-tance in clinical PET, finding relevant applications inoncology. Beside [18F]FDG, which covers the main part ofoncological PET studies, [11C]choline, [11C]methionine and[11C]acetate have found increasing interest and severalapplications in clinical PET. These radiopharmaceuticalshave good characteristics in terms of metabolic propertiesand low patient dosimetry because of the short half-life of11C and, particularly, have gained a pivotal role in the studyof most frequent neoplastic diseases (i.e., [11C]choline forprostate cancer) where they may overtake some problemsrelated to [18F]FDGmetabolic behavior such as low tumor tobackground ratio and low tumor uptake. Nevertheless,preparation of these radiopharmaceuticals has not reachedthe same level of standardization as for [18F]FDG synthesis.

This review represents an overview of the most recentmethodologies for the synthesis of these [11C]radiopharma-ceuticals and could be useful for a better understanding andstandardization of these methods in order to contribute to thewidespread application in clinical practice.

[11C]choline and [11C]methionine were synthesized by[11C]methylation of the respective precursors using [11C]CH3I or [

11C]CH3OTf with bubbling, on-column and on-loop techniques. Concerning [11C]choline, bubbling methodwas the classic approach for [11C]methylation and allows tomodify labeling parameters such as temperature in order toreach high yields with small amount of precursor employed[90,95]. With this approach, the residual precursor can alsobe removed by evaporation [90]. Furthermore, the use of

[11C]CH3OTf instead [11C]CH3I allows to reduce theamount of precursor without heating or at low temperature[96]. On the other hand, [11C]methylation on SPE column orloop [89,91,93,99,100] improves the radiosynthesis bysimplifying the process and automation (the reaction iscarried out on a solid support at room temperature) in ashorter time, and maintaining the high radiochemical yieldand purity of the bubbling method. Excess of precursor isremoved by washing the SPE column.

Critical review of the literature evidenced that, at least inthe last few years, there has been a trend in performing [11C]methionine synthesis on a solid support (on-column and “on-loop”) [29,105,106] because of faster approach which avoidstime-consuming HPLC purification with high yield and highradiochemical purity as well.

[11C]acetate is produced by [11C]carboxylation of aGrignard reagent with cyclotron-produced [11C]CO2 usingbubbling or on-loop techniques. The use of a low amount ofreagent together with SPE columns purification provideshigh radiochemical purity, faster synthesis process and highradiochemical yield [114,117]. Furthermore, purification of[11C]acetate with SPE disposable columns provides theproduction of this [11C]radiopharmaceutical more adapt-able to synthesis automation, particularly coupled with theon-loop approach [117,118].

The choice of the methodology depends on many factors;most of these are intrinsic in each organization andlaboratory. Regulations, which are different in severalcountries, could influence the choice of the method, leadingto a strict “pharmaceutical” approach by using sterilecassettes or other pharmaceutical device. Nevertheless, abetter understanding of the 11C “universe” could help infinding the right and customized solution.

Acknowledgments

The authors would like to acknowledge Prof. EyalMishani for critical discussion and his valuable scientificcontribution.

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