Available online at www.sciencedirect.com

Nuclear Medicine and Biology 38 (2011) 807 – 817
www.elsevier.com/locate/nucmedbio

New radiosynthesis of 2-deoxy-2-[18 F]fluoroacetamido-D-glucopyranoseand its evaluation as a bacterial infections imaging agent
Miguel E. Martíneza , Yasushi Kiyonoa,⁎, Sakon Norikib , Kunihiro Inaic , Katheryn S. Mandapa ,Masato Kobayashia,e , Tetsuya Moria , Yuji Tokunagad , Vijay N. Tiwaria , Hidehiko Okazawaa ,Yasuhisa Fujibayashia,f , Tatsuo Idoa,gaBiomedical Imaging Research Center, University of Fukui, Eiheiji, Yoshida, Fukui, 910-1193, Japanb
Department of Tumor Pathology, University of Fukui, Fukui, 910-1193, Japanc
Department of Molecular Pathology, University of Fukui, Fukui, 910-1193, Japand
Department of Materials Science and Engineering, University of Fukui, Fukui, 910-8507, Japane
School of Health Sciences, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, 920-0942, Japanf
Molecular Imaging Center, National Institute of Radiological Sciences, Chiba, 263-8555, Japang
Japan Radioisotope Association, Tokyo, 113-8941, Japan
Received 5 November 2010; received in revised form 25 January 2011; accepted 12 February 2011

Abstract
Introduction: The diagnosis of infection and the ability to distinguish bacterial infection from nonbacterial inflammation by positronemission tomography (PET) have gained interest in recent years, but still few specific radiopharmaceuticals are available for use. In thisstudy, we developed a new radiosynthesis method of 2-deoxy-2-[18F]fluoroacetamido-D-glucopyranose ([18F]FAG) by applying
microwave irradiation and demonstrated that [18F]FAG could be a potential radiopharmaceutical to distinguish bacterial infection from
nonbacterial inflammation.
Methods: 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-bromoacetamido-D-glucopyranose was used as precursor, and labeling was performed under
microwave irradiation conditions followed by alkaline hydrolysis and high-performance liquid chromatography (HPLC) purification. In vitro
uptake of [18F]FAG by Escherichia coli was performed. Tissue biodistribution of [18F]FAG was performed in mice. Moreover, PET imaging
acquisition of E. coli infection and nonbacterial inflammation models was performed in rats. Tissue radiotracer-accumulated sites wereanalyzed by hematoxylin and eosin staining and anti–E.coli immunostaining.
Results: The radiosynthesis of [18F]FAG was achieved with microwave irradiation, and the radiochemical yield was 9.7%±2.8% end ofbombardment (EOB); the radiochemical purity was more than 98%, and the total synthesis time was 62 min. Compared with control group, in
vitro uptake of [18F]FAG by E. coli was significantly decrease in inhibition group (Pb.05). Biodistribution studies in mice showed rapid
clearance of [18F]FAG from the animal body. [18F]FAG clearly visualized the infection areas but not nonbacterial inflammation areas in PET
studies. Quantitative analysis revealed that the uptake of [18F]FAG into infection areas was significantly higher than that of [18F]FAG into
inflammation areas (Pb.05). Histological analysis demonstrated the presence of bacterial cells at the sites of accumulation of [18F]FAG.
Conclusions: Using 1,3,4,6-tetra-O-acetyl-2-deoxy-2-bromoacetamido-D-glucopyranose as a precursor, the new radiosynthesis method of[18F]FAG was achieved in fewer steps and with a shorter synthesis time than previously reported. Furthermore, [18F]FAG was able todistinguish bacterial infection from nonbacterial inflammation.
© 2011 Elsevier Inc. All rights reserved.
Keywords: Infection; Inflammation; Bacterial imaging agent; PET; Glucosamine derivatives

1. Introduction⁎ Corresponding author. Tel.: +81 776 61 8420; fax: +81 776 61 8170.
E-mail address: ykiyono@u-fukui.ac.jp (Y. Kiyono).
0969-8051/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.nucmedbio.2011.02.006

Although there have been significant advances in our
understanding of the pathogenesis of infection, infection still
remains a major cause of patient morbidity and mortality

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M.E. Martínez et al. / Nuclear Medicine and Biology 38 (2011) 807–817

throughout the world [1]. It is essential for the efficient
treatment of infection and prevention of complications to
distinguish bacterial infection from nonbacterial inflammation[2]. Most infections are diagnosed by clinical history, physicalexamination, microbiological analyses of samples derived
from the infected tissue and imaging techniques. Althoughmodern imaging techniques such as computed tomography
and magnetic resonance imaging can provide excellent
structural resolution in chronic stages of infection, thesemodalities have limitations in distinguishing bacterial infection from nonbacterial inflammation [3]. Positron emission
tomography (PET) or single-photon emission computed
tomography can detect earlier the physiological and biochemical changes at the site of lesions. Therefore, nuclear medicine
with its powerful imaging techniques can help to distinguishbacterial infection from nonbacterial inflammation. The
development of new radiopharmaceuticals for PET may be
helpful in identifying infected structures, and the potential of
PET for quantification could have further implications forprediction and monitoring of therapeutic response [4].
Amino sugars are versatile components of the cell surface
structures of bacteria. They form the essential backbone of thepeptidoglycan in both gram-positive and gram-negative
bacteria and are also constituents of the outer membrane
lipopolysaccharide layer and the polysaccharide capsules ofgram-negative bacteria. The peptidoglycan is the main
component of the bacteria cell wall and is built from alternating
units of N-acetylglucosamine and N-acetylmuramic acid,cross-linked with oligopeptides at the lactic acid residue ofN-acetylmuramic acid. The pathway for the metabolism ofN-acetylglucosamine and the biosynthetic pathway producing
uridine diphosphate-N-acetylglucosamine for incorporation
into cell wall components have been described elsewhere [5,6].
The incorporation of a positron emitter into N-acetylglucosamine structure could make this compound a specific bacterial
imaging agent for PET. Although a variety of N-acetylglucosamine derivatives have been synthesized and studied for theirroles as plasma-membrane modifiers, tumor chemotherapeuticor imaging agents [7–9], there are no studies of bacterial
infections imaging by the use of positron-labeled N-acetylglucosamine derivatives [10].
In this study, we developed a new radiosynthesis methodof [18 F]FAG by applying microwave irradiation anddemonstrated that [18F]FAG could be a potential radiopharmaceutical to distinguish bacterial infection from nonbacterial inflammation.
2. Materials and methods
2.1. Reagents
All reagents, unless otherwise specified, were obtained
from Nacalai Tesque (Kyoto, Japan), and were used without
further purification. Anhydrous acetonitrile, anhydrous
pyridine, anhydrous dichloromethane, 1,10-diaza4,7,13,16,21,24-hexaoxabicyclo[8.8.8]hexacosane (K222),

potassium carbonate and D-glucosamine hydrochloride
were obtained from Sigma-Aldrich (St. Louis, MO, USA).
high-performance liquid chromatography-grade anhydrous
acetonitrile was obtained from Kanto Chemical (Tokyo,
Japan). Cyclophosphamide monohydrate (CPA) and anhydrous magnesium sulfate were purchased from WAKO Pure
Industries (Osaka, Japan).
2.2. Bacterial strain and culture
From the point of view of safety, availability and easy
handling, Escherichia coli (E. coli) was chosen as a model ofbacteria. E. coli (ATCC No. 8739) was purchased from Kanto
Chemical. A single colony of bacteria collected from an LuriaBertani (LB)' agar plate was first grown overnight in 5 ml of
LB' broth at 37°C and 200 rpm. Afterward, 50 μl of the aliquot
was resuspended in 2 ml of fresh heart infusion broth andincubated for additional 4 h until a logarithmic multiplication
was observed. Bacteria were centrifuged and washed withwarmed phosphate-buffered saline before being adjusted to a
final concentration of 1×107 colony-forming unit.
2.3. General methods
Melting points (uncorrected) were determined on a
ATM-01 flat type apparatus (As One, Tokyo, Japan).
Thin-layer chromatography (TLC) was performed on glass
back plates coated with silica gel 60 F254 (Merck,
Darmstadt, Germany). The following solvent systems wereused: (A) 9:1 CHCl3/CH3OH or (B) 5:4:2:1 n-butanol/
CH3OH/ammonia 28%/H2O. Samples were visualized byspraying the plates with 5% H2SO4 in ethanol and heating or
reading radioactivity in a AR-2000 Imaging scanner(Bioscan, Washington DC, USA). Flash chromatography
was performed on a Yamazen YFLC (Yamazen, Tokyo,
Japan) system. Infrared (IR) spectra were recorded on a
Horiba FT-720 spectrometer (Horiba, Tokyo, Japan) andnuclear magnetic resonance (NMR) measurements were
determined on a JEOL LA-500 spectrometer (JEOL, Tokyo,
Japan) (500 MHz for 1H and 125 MHz for 13C). Samples
were dissolved in CDCl3, and Me4Si was used as theinternal reference. Elemental analyses were performed on a
CHN CORDER MT-6 Elemental analyzer (Yanaco, Tokyo,
Japan). Reverse-phase HPLC was performed using a
Shimadzu (Shimadzu, Kyoto, Japan) system equiped witha refractive index detector and Ludlum 44-10 NaI (Tl)radioactive detector (Ludlum Measurements; Sweetwater,
TX, USA). A Capintec Radiosotope Calibrator (CRC-12
Capintec Instruments Inc., Ramsey, NJ, USA) was used for
all radioactivity measurements unless otherwise stated.
2.4. Synthesis of 1,3,4,6-tetra-O-acetyl-2-deoxy-2-bromoacetamido-D-glucopyranose
2.4.1. 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-amino-D-glucopyranose hydrochloride (4)1,3,4,6-Tetra-O-acetyl-2-deoxy-2-amino-D-glucopyranose hydrochloride was prepared following the method

M.E. Martínez et al. / Nuclear Medicine and Biology 38 (2011) 807–817

described by Myszka et al. [11]. mp 220°C (decomposed),
IR (KBr)ν: 3432 (NH2), 1751 (C=O, Ac) cm−1, 1H-NMR(D2O, 500 MHz) δ: 5.95 (d, 1 H, J 8.6 Hz), 5.47 (dd, 1H, J
10.7 and 9.2 Hz), 5.17–5.09 (m, 1H), 4.38–4.33 (m, 1H),
4.24–4.18 (m, 2H), 3.77 (dd, 1H, J 10.6 and 8.6 Hz), 2.21,
2.13, 2.089 and 2.086 (s, 3H×4).
2.4.2. 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-amino-D-glucopyranose (5)To a solution of 4 (7.2 g, 19 mmol) in water (85 ml) was
slowly added sodium acetate (1.86 g, 23 mmol) undervigorous stirring. After crystallization began, the mixture
was stirred for approximately 1.5 h at room temperature. The
precipitate was extracted with dichloromethane, the organic
phase was dried over MgSO 4 and the solvent wasevaporated. Addition of acetone to the residue followed by
Et2O caused precipitation of 5 as a white solid to give 5.7 g(81%), which was further recrystallized from ethanol. mp
149–150°C; IR (CHCl3)ν: 3363 (NH), 1741 (C=O, Ac)
cm−1, 1H-NMR (CDCl3, 500 MHz) δ: 5.51 (d, 1 H, J 8.6
Hz), 5.07–5.02 (m, 2H), 4.31 (dd, 1H, J 12.2 and 4.4 Hz),4.08 (dd, 1H, J 12.5 and 2.1 Hz), 3.85–3.80 (m, 1H), 3.10–
3.04 (m, 1H), 2.18, 2.09, 2.08 and 2.03 (s, 3H×4).
2.4.3. 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-(bromoacetamido)D-glucopyranose (6)To a cooled solution of 5 (0.7 g, 2.0 mmol) in a mixture ofdry dichloromethane (10 ml) and dry pyridine (1 ml) was
added dicyclohexylcarbodiimide (0.45 g, 2.2 mmol) followed by bromoacetic acid (0.3 g, 2.2 mmol). The mixture
was stirred in an ice/water bath for 2 h. After filtration, thefiltrate was discarded and the solution washed with water,
diluted hydrochloric acid and again with water. The organic
layer was dried over MgSO4 and filtered, the solvent wasevaporated to dryness and the residue was recrystallized
from ethanol. mp 169–170°C; IR (CHCl3)ν: 3357 (NH),1754 (C=O, Ac) cm−1, 1H-NMR (CDCl3, 500 MHz) δ: 6.56
(d, 1H, J 9.5 Hz), 5.79 (d, 1H, J 8.5 Hz), 5.30 (dd, 1H, J 10.7
and 9.2 Hz), 5.13 (t, 1H, J 9.6 Hz), 4.31–4.22 (m, 2H), 4.14
(dd, 1H, J 12.5 and 2.1 Hz), 3.90–3.85 (m, 1H), 3.78 (s, 2H),
2.12, 2.10, 2.061 and 2.057 (s, 3H×4). 13C-NMR (CDCl3,
125 MHz) δ: 170.96, 170.63, 169.33, 169.31, 166.33, 92.1,
72.89, 71.90, 67.85, 61.62, 53.52, 28.20, 20.87, 20.69,
20.64, 20.56. Anal. Calcd for C16H22BrNO10: C, 41.04; H,
4.74; N, 2.99. Found: C, 40.97; H, 4.68; N, 3.02.
With the purpose of use for analysis and quality controls
during radiosynthesis, 1,3,4,6-tetra-O-acetyl-2-deoxy-2fluoroacetamido-D-glucopyranose was synthesized by asimilar procedure as for compound 6 using fluoroacetic
acid generated in situ; mp 196–198°C; IR (KBr)ν: 1754
(C=O, Ac) cm−1, 1H−NMR (CDCl3, 500 MHz) δ: 6.57 (d,1H, J 9.5 Hz), 5.79 (d, 1H, J 8.5 Hz), 5.27 (dd, 1H, J 10.7
and 9.3 Hz), 5.15–5.08 (m, 1H), 4.78 (dd, 1H, J 17.7 and14.7 Hz), 4.68 (dd, 1H, J 17.7 and 14.7 Hz), 4.32–4.22 (m,2H), 4.11 (dd, 1H, J 12.5 and 2.1 Hz), 3.86–382 (m, 1H),
2.10; 2.07, 2.024 and 2.020 (s, 3H × 4). 13C-NMR (CDCl3,809

125 MHz) δ: 170.88, 170.64, 169.31, 168.01, 167.87, 92.1,
79.93 (d, JC−F 186 Hz), 72.82, 72.12, 67.80, 61.58, 52.59,
20.84, 20.70, 20.56 and 20.54.
2.5. Radiosynthesis of [18F]FAG
All reactions were performed in a semiautomated system[12] with a CEM Discover (Matthews, NC, USA) singlemode microwave reactor system. This system is capable todeliver continuously microwave power with selectable
power output (0–300 W) and a programmable temperaturefrom 25°C to 250°C. It is equipped with an infrared
temperature control system located below the microwave
cavity floor that monitors and controls the temperature
conditions of the reaction vessel. The temperature sensor isused in a feedback loop with the magnetron to regulate thepower output that maintains the temperature set point. No
carrier added [18F]fluoride was produced in a RDS eclipse
RD/HP cyclotron (Siemens/CTI, Knoxville, TN, USA) via
the 18O(p,n)18F reaction using 18O-enriched water. After
irradiation, the enriched water was trapped into a SepPak
Light QMA cartridge (Waters, Milford, MA, USA) pretreated with 1 ml of 0.2 M K2CO3 and 10 ml of water. The[18F]fluorine trapped on the column was eluted with asolution of K2CO3/K222 in acetonitrile/water (96:4) based ona previous report [13]. The resulting [K/K222]+18F− solution
was then evaporated at 110°C in a stream of argon gas (20ml/min) using an average microwave power of about 150 W.
To the dried [K/K222]+18F− complex, a solution of bromide
precursor (6) in anhydrous CH3CN was added, andexperiments were conducted (under closed system reaction)
to optimize different variables including labeling temperature, reaction time and precursor amount. During optimization of the radiofluorination, samples from the crude productwere measured for incorporation yield using radio TLC, andthe result obtained was corrected based on the percent
distribution of the radioactivity obtained in the reaction
mixture. The mixture was then evaporated at 70°C underargon flow until 0.1–0.2 ml remained. The hydrolysis
conditions at room temperature using 1 ml of sodiumhydroxide was optimized considering base concentration andreaction time. The final solution was neutralized by theaddition of 2 M hydrochloric acid before purification in the
HPLC system. The radioactive mixture was purified by
HPLC (Cosmosil sugar-D column, 10 mm internal diameter(ID)×250 mm, CH3CN/H2O 80:20, 3 ml/min, 210 nm), andthe radioactivity peak corresponding to [18F]FAG was thendirectly transferred to a solvent replacement unit (JFE
Holdings, Tokyo, Japan) wherein removal of the solvent
system was performed in vacuum. The residue was thenredissolved in 1 ml of normal saline solution. The final
radiochemical purity and stability of [18 F]FAG were
determined by radio TLC (solvent B) and HPLC (Cosmosil
sugar-D column 4.6 mm ID×150 mm, CH3CN/H2O 80:20, 1
ml/min, 210 nm). The Rf value of [18F]FAG was determined
at 0.30, and the retention time for the peak was detected at

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M.E. Martínez et al. / Nuclear Medicine and Biology 38 (2011) 807–817

3.2 min; both were confirmed with a FAG cold standard
synthesized in our laboratory.
2.6. In vitro E. coli uptake study
E. coli grown in medium heart infusion broth was divided
in control and inhibition groups, and aliquots of 1 ml by
triplicate were used for time points. In inhibition group,
glucosamine at a final concentration of 10 mg/ml was added.
After the addition of 0.37 MBq of [18F]FAG in saline,
solution samples were incubated at 37°C. After 30- and 120min incubation, samples were washed three times with icecold phosphate-buffered saline (−), and then 500 μl of lysis
buffer was added. Aliquots of each sample were counted in a1480 Automatic Gamma Counter (Perkin Elmer, Osaka,
Japan). The contents of protein was measured by bicinchoninic acid Protein Assay Kit following the instruction
manual (Pierce Biotechnology, Rockford, IL, USA).
2.7. Animal studies
All animal experiments were approved by the animal care
committee at the University of Fukui and conducted inaccordance with the international standards for animal
welfare and institutional guidelines. Adequated measures
were taken to minimize pain or discomfort. Animals werehoused under a 12-h light/12-h dark cycle with free access tofood and water.
2.8. Biodistribution study
For the biodistribution study, 0.37 MBq of [18F]FAG was
injected into male ddY mice (n=30, 28–30 g, 6 weeks; Japan
SLC, Shizuoka, Japan) via the tail vein. Following theinjection, animals were killed under light ether anesthesia,
and heparinized blood was obtained at 5, 10, 15, 30, 60 and
90 min (n=5 each point). The organs and tissues were
removed, weighed and counted in a 1480 Automatic GammaCounter (Perkin Elmer). Tissue radioactivity was expressedas percentage injected dose per gram of tissue:½counts of tissue counts=½injection counts%dose=g¼
×100½gram of tissue

2.9. Induction of E. coli infections in rats
Healthy male Sprague-Dawley rats (n=5, 80–90 g, 5
weeks; Japan SLC) were used for the induction ofexperimental infections. A week before PET acquisition
image, the animals were immunosuppressed with an
intraperitoneal injection of 180 mg/kg of CPA. Four days
later, approximately 1×107 colony-forming units of bacteria
were administered into the right front leg muscle followed byadditional immunosuppression using 90 mg/kg of CPA on
the next day. All animals developed a detectable abscess, and2 days later, animals were used for PET experiments.

2.10. Induction of inflammatory process in rats
Healthy male Sprague-Dawley rats (n=5, 140–160 g, 6
weeks; Japan SLC) were used for the induction ofexperimental inflammation. Aseptic inflammation was
induced 24 h before PET image acquisition with 100 μl of
100% pure turpentine oil inoculated into the right front leg
muscle under ether anesthesia.
Before PET image acquisition, animals were fasted withno food overnight but with water supplied ad libitum. All
animals developed an abscess detectable as a palpablefluctuant mass by either method bacterial infection or
aseptic inflammation. The abscess sizes in both groups
measured using a slide gauge were similar and within arange of 1.5–2.0 cm.
2.11. Small animal PET imaging
PET imaging of infection or inflammation in rat was
performed using a small animal PET scanner (SHR-41000;
Hamamatsu Photonics, Hamamatsu, Japan) [14]. The
scanner acquires 213 slices covering an axial length of 160
mm, with a three-dimension mode and achieving a resolutionof 2.0–2.5 mm full width at half maximum in the transaxial
direction and 2.8 mm full width at half maximum in the axial
direction. One hour before scanning, animals were injectedwith 16.7–22.2 MBq of radiotracer ([18F]FAG or [18F]FDG)
via the tail vein. The animals were placed in the prone
position in the PET scanner and then maintained under 2%–
3% isoflurane (Abbott, Tokyo, Japan) air inhalation during
image acquisition. Static PET images were acquired without
attenuation correction over a 30-min period. Then regions ofinterest in three consecutive frames were drawn manually
over the right front leg muscle and the contralateral nontarget
left front leg muscle for each animal. [18F]FAG and [18F]
FDG uptake over infected or inflamed areas was quantified
using Dr. View Software, version 2.0.0 (JAS, Tokyo, Japan).
Finally, utilizing the mean value, all regions of interests wereconverted to standardized uptake values (SUV).
SUV ¼½tissue acitivity concentrationð½injection dose = ½body weightÞAfter PET imaging, the animals were killed, and theresected tissues were fixed with 20% formalin for pathological examinations.
2.12. Histology
The slide sections thin-sliced from the paraffin-embedded
block were stained with hematoxylin and eosin dye or withprimary anti–E. coli polyclonal antibody (DAKO, Japan) as
described [15]. A Histofine SAB-PO kit (Nichirei, Tokyo,
Japan) was used for the secondary reaction of immunostaining. The obtained sections were pathologically evaluated by
two different pathologists specializing in the pathophysiological analysis of infectious diseases.

M.E. Martínez et al. / Nuclear Medicine and Biology 38 (2011) 807–817

811

Fig. 1. Synthesis scheme of TA-BrAG. Chemical names: D-glucosamine hydrochloride (1), 2-deoxy-2-[p-methoxybenzylidene(amino)]-D-glucopyranose (2),
1,3,4,6-tetra-O-acetyl-2-deoxy-2-[p-methoxybenzylidene(amino)]-D-glucopyranose (3), 1,3,4,6-tetra-O-acetyl-2-deoxy-2-amino-D-glucopyranose hydrochloride (4), 1,3,4,6-tetra-O-acetyl-2-deoxy-2-amino-D-glucopyranose (5), 1,3,4,6-tetra-O-acetyl-2-deoxy-2-(bromoacetamido)-D-glucopyranose (6). Reagents andconditions: aq NaOH, p-anisaldehyde (a); Py, Ac2O (b); acetone, 5 M HCl (c); water, AcONa (d); DCC, BrCH2COOH, CH2Cl2/Py (e).

2.13. Statistical analysis
Unpaired t test was applied for two group comparison in
E. coli uptake study. SUV differences between groups were
tested for statistical significance using one-way analysis ofvariance with a post hoc Newman–Keuls multiple-comparison test. A probability value of less than .05 was considered
to indicate a significant difference. Results are expressed as
mean±S.D.
3. Results
3.1. Radiosynthesis of [18F]FAG
The steps of the new synthesis scheme for 1,3,4,6-tetra-Oacetyl-2-deoxy-2-bromoacetamido-D-glucopyranose (TABrAG) are summarized in Fig. 1. The final TA-BrAG
obtained by this route was used as a precursor for thesynthesis of [18F]FAG by nucleophilic substitution with
[18F]fluoride anion (Fig. 2).
We investigated several microwave reaction conditions inorder to optimize the [18F]F− incorporation rate into TABrAG precursor, including the effects of reaction temperature, microwave irradiation time and amount of TA-BrAG
precursor (Table 1). First, using 29.4 mg (62.5 μmol) of TA-

BrAG dissolved in 0.5 ml of anhydrous acetonitrile, we
studied the incorporation yields at 90°C, 100°C and 110°C(series 1∼3). The optimum labeling temperature wasdetermined to be at 100°C with an average microwave
power of around 30 W. The lower yield observed at higher
temperatures may be attributed to degradation of TA-[18F]
FAG. Next, the optimal amount of precursor was evaluated(series 4∼8). Five different molar ratios of TA-BrAG
precursor/K222 were examined (0.5, 1, 2, 3 and 5). The
incorporation yield by varying the amount of TA-BrAG
precursor keeping the K222 constant showed no significant
increase in incorporation yield while varying the ratio of TABrAG precursor/K222 from 2 up to 5. In consequence, theminimal amount of TA-BrAG precursor, which corresponds
to an amount of 11.7 mg (TA-BrAG precursor/K222 ratio of2) was selected. Finally, we investigated the incorporation
yields using five different reaction times (1.5, 3, 5, 10 and 20min) (series 9∼13). A reaction time of 10 min was enough toobtain the highest incorporation yields. In summary, utilizing
11.7 mg of TA-BrAG in acetonitrile reacted at 100°C andusing an average microwave power of 30 W gave an
incorporation yield of 75.5%±4.2% (n=3) in 10-min time.
For optimization of the hydrolysis of the O-acetyl groupsof the TA-[18F]FAG, the reaction time and concentrations ofFig. 2. Radiosynthesis scheme of [18F]FAG.

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M.E. Martínez et al. / Nuclear Medicine and Biology 38 (2011) 807–817

Table 1
Summary of microwave fluorination optimization
Series

TA-BrAG (mg)

Reaction
temperature (°C)

Molar ratio
TA-BrAG/K222 sln

Reactiontime (min)%TA-[18F]FAG
yield (RTLC)a

1
2
3
4
5
6
7
8
9
10
11
12
13

29.4
29.4
29.4
2.9
5.8
11.7
17.5
29.4
11.7
11.7
11.7
11.7
11.7

90
100
110
100
100
100
100
100
100
100
100
100
100

5
5
5
0.5
1
2
3
5
2
2
2
2
2

10
10
10
10
10
10
10
10
1.5
3
5
10
20

63.7±0.775.4±1.8
57.9±11.4
48.3±7.862.8±5.4
75.5±4.2
76.6±7.577.3±2.558.6±2.961.3±1.664.4±5.1
75.5±4.2
70.4±2.2aValues represent the mean±S.D. (n=3).

sodium hydroxide were examined (Table 2). The hydrolysis
of TA-[18F]FAG was feasible in liquid phase at room
temperature. The maximum yield of [18F]FAG was obtainedwith 1 M sodium hydroxide for 1 min. The TA-[18F]FAG
was reproducibly converted into [18F]FAG under these
conditions with an average recovery of 30%–40%.
After the optimization described above, we developed thesequential radiolabeling and hydrolysis reactions in asemiautomated system, equipped with a microwave, designed
in our facilities for the synthesis of [18F]flumazenil [12]. Five
hot runs with 286.8–1953.6 MBq were performed. Following
basic hydrolysis and purification by HPLC, the radiochemicalyield of [18F]FAG obtained at the end of synthesis was 9.7%
±2.8% end of bombardment (EOB) with a radiochemical
purity of 98.7%±1.5% (Fig. 3). The total reaction time was
reduced to only 62 min. The specific activity for [18F]FAG
was 18.09±2.9 GBq/μmol end of synthesis (EOS), and for[18F]FDG, it was 1195±298 GBq/μmol (EOS).
3.2. In vitro E. coli uptake study
Fig. 4 shows the result of [18F]FAG uptake by E. coli. In
control group, the uptake of [18F]FAG was increased withtime. On the other hand, the uptake of [18F]FAG was notchanged in inhibition group. There was significant difference
in uptake of [18F]FAG between control and inhibition groups
at 120 min (Pb.05).

3.3. Biodistribution study
To compare the bioequivalence of [18F]FAG synthesizedby previous report [16], biodistribution study was performedin mice. Results show a biodistribution pattern of [18F]FAG
similar to previous report [16]. Fig. 5 shows the tissue
distribution over time postinjection of [18F]FAG. Radioactivity decreased rapidly with time in all tissues. For the first
60 min, radioactivity was concentrated in these organs
involved in excretion and metabolism.
3.4. Small animal PET
PET imaging studies were performed in rats to allow abetter visualization of abscess sites. In Fig. 6A, a red toyellow signal represents the accumulation of [18F]FAG
obtained in the right front leg muscle where E. coli was
inoculated. Similar results were seen using [18F]FDG. (Fig.
6B). When noninfectious inflammatory lesions were examined by the two radiocompounds, only [18F]FDG clearlyvisualized the lesions (Fig. 6C, D).
The SUV values of these tracers in both sites are
summarized in Table 3. The uptake of [18F]FAG into target
muscle (0.54±0.21, n=5) was significantly higher than thatof [18F]FAG into nontarget muscle (0.19±0.07 n=5) in
infection model rats (Pb.01). However, there was no
significant difference between the uptake of [18F]FAG into
target (0.32±0.03, n=5) and nontarget areas (0.23±0.03,

Table 2
Hydrolysis yields of [18F]FAG versus time at room temperature
NaOH concentration (M)0.5 min

1 min

1.5 min

2 min

5 min

10 min

0.25
0.50
0.75
1.00
1.50

26.0±3.435.9±3.6
38.3±1.941.2±3.1
34.5±6.1

27.3±2.833.8±4.638.1±1.939.9±4.731.8±4.6

25.4±3.3
34.3±4.438.1±1.938.5±4.7
33.0±7.0

27.6±5.1
34.0±4.3
36.5±0.938.1±5.0
30.6±5.3

28.6±2.3
31.3±4.0
33.5±1.235.4±5.6
28.4±3.4

27.3±7.2
32.7±6.3
34.5±1.433.2±6.4
26.2±3.2

Values represent the mean±S.D. (n=3).

M.E. Martínez et al. / Nuclear Medicine and Biology 38 (2011) 807–817

813

Fig. 3. HPLC chromatograms of [18F]FAG on Cosmosil sugar-D column 4.6 mm ID×150 mm (A) UV at 210 nm; radioactivity (B).

n=5) in inflammation model rats. Furthermore, the uptake of[18 F]FAG into infection areas (0.54±0.21, n=5) was
significantly higher than that of [18F]FAG into inflammation
areas (0.32±0.03, n=5, Pb.05). In contrast, the uptake of[18F]FDG into target muscle (infection: 1.58±0.12, n=5,
inflammation: 1.36±0.30, n=5) was higher than that of [18F]
FDG into nontarget muscle (infection: 0.22±0.06, n=5,
inflammation: 0.28±0.06, n=5) in both infection and inflammation model rats. Similarly, the uptake of [18F]FDG in
infection areas (1.58±0.12, n=5) was significantly higher
than that of [18F]FDG in inflammation areas (1.36±0.30,
n=5, Pb.05).

Fig. 4. Uptake of [18F]FAG by E. coli in control and inhibition groups. The
uptake was expressed as the percentage of the injected dose per miligram ofprotein (% ID/mg protein; mean±S.D, n=3).

3.5. Histology
As shown in Fig. 7A, small basophilic particles were
scattered in the tissue injected with E. coli. The particles wereconfirmed as E. coli by immunostaining using anti–E. coli
antibody (Fig. 8). Turpentine oil injection, a representative
noninfectious inflammatory lesion, induced muscular necrosis
with severe neutrophil infiltrations around the droplets (Fig. 7B).
4. Discussion
It has been an important issue in nuclear medicine todiscriminate bacterial infection from nonbacterial inflammation, and several studies have been conducted to detect
differences between molecular events in infection andinflammation in recent decades. Radiolabeled autologous
leucocytes using [111 In]indium–oxine or [99mTc]technetium–hexamethylpropylene amine oxime (HMPAO) are
the most common method for evaluating infection andinflammation in nuclear medicine [17,18]. However, it isdifficult to distinguish bacterial infection from nonbacterial
inflammation using these radiopharmaceuticals [1]. The
antimicrobial peptide 99mTc-labeled ubiquicidin 29–41 is apromising radiopharmaceutical for distinguishing bacterial
infection from nonbacterial inflammation [19]. The use ofradiolabeled antimicrobial peptides to images infection andthe suggestion that they can differentiate infection frominflammation have been reviewed by Nibbering et al. [20].
Among PET radiopharmaceuticals, [18F]FDG was applied
for bacterial infection because the processes of bacterial
infection show increased glycolysis. However, even when
[18F]FDG is exquisitely sensitive, its specificity is low, and it
accumulates in a variety of conditions including malignant
and benign neoplasm, fractures and nonbacterial inflammation as well as bacterial infection. Although F-18–labeled
ciprofloxacin has been reported as a infection-specific814

M.E. Martínez et al. / Nuclear Medicine and Biology 38 (2011) 807–817

Fig. 5. Tissue biodistribution of [18F]FAG in mice (n=5 for each time) by the dissection method. Radioactivity was expressed as the percentage of the injected
dose per gram of tissue or organ (% ID/g; mean±S.D, n=5).

radiopharmaceutical, contradicting results have also beenreported that [18F]ciprofloxacin had no specific binding tobacteria [21] and poor retention in bacteria-infected tissues inpatients [22]. Thus, the search for better radiopharmaceuticals to distinguish between infection and inflammation is

ongoing. In this study, we hypothesized that F-18–labeled
N-acetylglucosamine derivatives were suitable radiopharmaceuticals for distinguishing bacterial infection from nonbacterial inflammation, and [18F]FAG was designed as a
candidate radiopharmaceutical.

Fig. 6. Representative static maximun intensity projection (MIP) PET images on rats with an induced bacterial infection or nonbacterial inflammation in the rightfront leg muscle. Images were acquired 60 min after intravenous injection of 16.7–22.2 MBq of [18F]FAG or [18F]FDG. Arrow indicates infection or
inflammation sites (animals positioned prone on imaging table). (A) Infection model after injection of [18F]FAG. (B) Infection model after injection of [18F]FDG.
(C) Inflammation model after injection of [18F]FAG. (D) Inflammation model after injection of [18F]FDG. The same scale is used in the images of the same row.

M.E. Martínez et al. / Nuclear Medicine and Biology 38 (2011) 807–817
Table 3
SUV values of [18F]FAG and [18F]FDG in bacterial infection andnonbacterial inflammation areas
Model

Bacterial infection (SUV)
Target areas

Nontarget areas Target areas Nontarget areas[ F]FAG 0.54±0.21⁎,# 0.19±0.07[18F]FDG 1.58±0.12⁎⁎,# 0.22±0.06
18Nonbacterial
inflammation (SUV)

0.32±0.03
0.23±0.03
1.36±0.30⁎⁎ 0.28±0.06

Values represent the mean±S.D. (n=5).
⁎ Pb.01 vs. the nontarget areas in infection model.
⁎⁎ Pb.001 vs. the nontarget areas in infection or inflammation model.
#
Pb.05 vs. the target areas in inflammation model.

To realize [18F]FAG-PET in a clinical situation, wedeveloped a simple radiosynthesis method of [18F]FAG byusing a new precursor and microwave heating in this study.
The radiosynthesis of [18 F]FAG has been previously
reported for tumor imaging [23,24]. However, in theprevious method, the three-step reaction led to longer total
reaction times, and the complex purification procedure toeliminate pyridine made it difficult to apply for clinicalpurposes. We designed a two-step semiautomated radiosynthesis system consisting of incorporation of [18F]F− using

815

microwave irradiation and basic hydrolysis. After purification, radiochemical purity of [18F]FAG was more than 98%,
the radiochemical yield was about 9.7% (EOB) and the total
reaction time was 62 min. The obtained final radiochemicalyield of [18F]FAG was lower considering the incorporation
yields (75.5%±4.2%) and hydrolysis (30%-40%) yields.
Considerable radioactivity was remained inside the preparative column during purification of the crude product. Thismay be the main reason for this discrepancy. Althoughfurther investigation regarding purification would be needed,
these results allow us to obtain [18F]FAG.
Addition of glucosamine has been reported to inhibit
acetylglucosamine pathway [25]. After a 120-min incubation, the uptake of [18F]FAG by E. coli cells was reduced
significantly. This results support the hypothesis that [18F]
FAG can be incorporated into the bacterial cell walls.
In the biodistribution of [18F]FAG in mice, immediately
after injection of [18F]FAG, transient accumulations ofradioactivity in the kidney, liver and blood were observed,
but after 60 min, almost all of these radioactivities became
low. The highest uptake in the kidney at 5 min reflected therapid urinary excretion of [18F]FAG. The other organs
showed no significant uptake, and the rapid clearance offeredFig. 7. Hemotoxylin and eosin tissues staining from correspondingly resected muscular samples at radiotracer acummulation site. (A) Rat injected with E. coli inthe right front leg muscle. (B) Rat injected with turpentine oil in the right front leg muscle.

816

M.E. Martínez et al. / Nuclear Medicine and Biology 38 (2011) 807–817

Fig. 8. Detection of E. coli in the correspondingly resected muscular sample of a rat injected with the bacterial cells in the right front leg muscle. Anti–E. coli
immunostaining confirmed the existence of the microbes as brown-stained dots on the slide section.

lower background levels during image acquisition. The
biodistribution results indicated the bioequivalence of newly
synthesized [18F]FAG with N-acetyl-D-[1-14C]glucosamineand [18F]FAG as previously reported [16]. Based on thebiodistribution data, the accumulation pattern of [18F]FAG
would be suitable for distinguishing bacterial infection frominflammation due to its low background accumulation.
Finally, using small animal PET, we demonstrated that
[18F]FAG accumulation was significantly higher in bacterial
infection compared with nonbacterial inflammation. Actually, [18F]FAG clearly visualized the E. coli–induced infection
areas but not nonbacterial inflammation areas. These results
indicated that [18F]FAG had the ability to discriminate theinfection areas from the inflammation areas. The uptakes of[18F]FDG into both infection and inflammation areas weresignificantly higher than those of [18F]FDG into nontarget
areas. These results of [18F]FDG were consistent withprevious reports [26] and suggested that it was difficult todistinguish the infection areas from inflammation areas with
[18F]FDG.
In the histological analysis, the presence of E. coli was
confirmed in the infection areas but not in the inflammation
areas. This was consistent with the result of PET study of[18F]FAG and suggested that [18F]FAG could be incorporated into bacterial cells. Further histological analysis of theinfection areas showed gross infiltration of granulocytes andmacrophages, while turpentine oil-induced inflammation
areas showed abscess formation with neutrophils andmacrophages surrounding the abscess wall. This may be
the reason why [18F]FDG cannot easily distinguish infection
from inflammation areas [27].

5. Conclusion
Using TA-BrAG as a precursor, a new radiosynthesis
method of [18F]FAG was achieved with fewer steps and ashortened synthesis time than previously reported. Further-more, [18F]FAG was able to distinguish infection frominflammation. To best of our knowledge, present study is thefirst trial to evaluate the N-glucosamine derivative as abacterial infection PET imaging agent. The mechanisms of
uptake for [18F]FAG in infected tissue still require to bestudied in-depth, but these promising findings will encourage future works with N-glucosamine derivatives.
Acknowledgments
This study was supported by the 21st Century COE
program and research grants (09-92-181 and 20591199)
from the Ministry of Education, Culture, Sports, Science andTechnology of Japan. We thank Mr. Toshinao Nakakoji and
Mr. Akira Ito of CMI for carrying out the [18F]fluoride
production and Dr. Talakad Lohith for preliminary PET
study support.
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