Preparation of the hypoxia imaging PET tracer [ 18F]FAZA: reaction parameters and automation

Preparation of the hypoxia imaging PET tracer [ 18F]FAZA: reaction parameters and automation
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  Applied Radiation and Isotopes 62 (2005) 897–901 Preparation of the hypoxia imaging PET tracer [ 18 F]FAZA:reaction parameters and automation G. Reischl a,  , W. Ehrlichmann a , C. Bieg a , C. Solbach a , P. Kumar b ,L.I. Wiebe b , H.-J. Machulla a a PET-Zentrum, Radiopharmazie, Universita¨tsklinikum Tu¨bingen, Ro¨ntgenweg 15, Tu¨bingen D-72076, Germany b Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Canada Received 24 November 2004; accepted 15 December 2004 Abstract 18 F-labeling of the nitroimidazole nucleoside analogue 1-(5-fluoro-5-deoxy- a - D -arabinofuranosyl)-2-nitroimidazole(FAZA) was developed to use this tracer in PET for detection of hypoxia. Parameters for labeling and hydrolysis wereoptimized with regard to amount of precursor, temperature and time. Labeling yields reached a maximum of 62 7 4% at100 1 C within 5min using 5mg of precursor. Hydrolysis was best performed with 1mL of 0.1N NaOH at 20 1 C for2min. Transfer of these conditions to an automated synthesizer resulted in an overall radiochemical yield of 20.7 7 3.5%. Absolute yields at EOS were 9.8 7 2.3GBq of [ 18 F]FAZA ready for injection ( n ¼ 21; 50min after EOB;irradiation parameters: 35 m A, 60min). Thus, a convenient approach suitable for large-scale production of [ 18 F]FAZAwas developed by an automated process. r 2005 Elsevier Ltd. All rights reserved. Keywords:  Hypoxia; Nitroimidazole; [ 18 F]FAZA; Positron emission tomography 1. Introduction Hypoxic tumor cells are known to be more resistant toradiation therapy than those with normal oxygenationlevels (Adams and Dewey, 1963; Teicher, 1995). Identification of tumor tissue hypoxia may be relevantfor individual treatment planning and monitoring aswell as predicting prognosis (Dehdashti et al., 2003).Radiolabeled 2-nitroimidazoles were proposed for non-invasive hypoxia imaging by Chapman (Chapman, 1979;for review ref. cit. Machulla, 1999).[ 18 F]FMISO is a nitroimidazole derivate that has beenestablished for detecting tissue hypoxia with positronemission tomography (PET) (Rasey et al., 1987;Rajendran et al., 2003, 2004). The tracer’s primarycellular uptake results from diffusion and partition-based retention in lipophilic tissues such as brain, inaddition to hypoxia-based retention. The azomycinnucleosides were developed as highly diffusible but lesslipophilic radiotracers in order to reduce partition-baseduptake. The most prominent example of this class of substances is the radioiodinated 1-(5-iodo-5-deoxy- a - D -arabinofuranosyl)-2-nitroimidazole (IAZA) (Mannanet al., 1991). For application in PET, 1-(5-fluoro-5-deoxy- a - D -arabinofuranosyl)-2-nitroimidazole (FAZA)was developed (Kumar et al., 1999, 2002). First resultsin animal studies with [ 18 F]FAZA proved its value as a ARTICLE IN PRESS www.elsevier.com/locate/apradiso0969-8043/$-see front matter r 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.apradiso.2004.12.004  Corresponding author. Tel.: +4970712987443;fax: +497071295264. E-mail address:  gerald.reischl@uni-tuebingen.de(G. Reischl).  new PET tracer for detecting hypoxia (Piert et al., 2001,2002).Recently, two comparative studies of [ 18 F]FMISOand [ 18 F]FAZA both in vitro and in various murinetumor models demonstrated the oxygenation depen-dency of [ 18 F]FAZA uptake and its superior biokineticsover [ 18 F]FMISO (Sorger et al., 2003; Piert et al., 2005). These results make [ 18 F]FAZA a promising PET tracerfor the visualization of tumor hypoxia.To perform clinical studies with [ 18 F]FAZA, theoptimal synthesis conditions had to be developedthat would result in a labeling procedure with goodyields and high reproducibility. For this purpose, thenucleophilic substitution reaction of no-carrier-added[ 18 F]fluoride with 1-(2,3-di- O -acetyl-5- O -tosyl- a - D -ara-binofuranosyl)-2-nitroimidazole as precursor was cho-sen as a general protocol (Reischl et al., 2002) (Fig. 1). 2. Materials and methods  2.1. General  Acetonitrile for azeotropic distillation was obtainedfrom Merck (for DNA synthesis; Darmstadt, Germany).As the solvent for the radiolabeling reaction, DMSO(dried over molecular sieve) was used from Fluka,Germany. Kryptofix 2.2.2. was purchased from Merck(Darmstadt, Germany). The precursor was synthesizedas described previously (Kumar et al., 2001, 2002).All other chemicals and solvents (Merck or Fluka)were of the highest purity available and used asreceived.  2.2. Production of [ 18 F]fluoride [ 18 F]Fluoride was produced at the PETtrace cyclotron( E  p ¼ 16.5MeV, General Electric Healthcare, Uppsala,Sweden) by irradiation of enriched [ 18 O]water (1.5mL;Rotem, Germany, 94–96% enrichment) via the 18 O(p,n) 18 F nuclear reaction. For clinical applications,irradiations were performed for 60min with a current of 35 m A resulting in 70–80GBq of [ 18 F]fluoride.  2.3. Labeling In a 5mL vial closed with a septum, 100–1300 m L of water containing [ 18 F]fluoride was evaporated at 140 1 Cfor 10min together with 15mg (40 m mol) of Kryptofix2.2.2. and 50 m L of 0.5M of aqueous solution of potassium carbonate (25 m mol). With the aid of anargon sparge line, residual water was removed byrepeated azeotropic distillation at 140 1 C after adding1mL of acetonitrile. The labeling reaction was carriedout in 1mL of DMSO wherein different amounts of precursor were dissolved. Reactions were performed at40, 50, 70, 80, 100 and 120 1 C. To determine the timedependence in reactions at 100 1 C, the reaction mixturewas analyzed after 5, 10, 20, 40 or 60min.  2.4. Hydrolysis The reaction solution was diluted with 1mL of aqueous NaOH (0.05, 0.1 or 0.2N) and stirred at20 1 C (50, 100 1 C) for 2min. The impact of the durationof alkaline hydrolysis on the radiochemical yield of [ 18 F]FAZA was investigated using 0.1N NaOH at 1, 2,5, 10 and 20min at 20 1 C. Afterwards, the solution wasneutralized with 0.5mL of 0.4N aqueous NaH 2 PO 4 .  2.5. Automated production Fig. 2 shows a schematic diagram of the automatedsystem for the [ 18 F]FAZA synthesis (modified TRA-CERlab FX F  N , General Electric Healthcare, Mu ¨nster,Germany). In the first step, [ 18 F]fluoride was adsorbedon an ion exchange cartridge (SEP-PAK light, WatersAccell Plus QMA, USA) preconditioned with 10mL of 1N aqueous NaHCO 3 , 10mL of water and 5mL of acetonitrile. [ 18 F]fluoride was eluted and flushed into thereaction vial with a mixture of 900 m L of acetonitrile and100 m L of water wherein 3.5mg (25 m mol) of potassiumcarbonate and 15mg (40 m mol) of Kryptofix 2.2.2. weredissolved. The solution was dried under vacuum(approx. 12mbar) at 60 1 C for 5min and then at120 1 C for additional 5min. Labeling was carried outin 1mL of DMSO with 5mg of precursor under stirringat 100 1 C within 5min. Hydrolysis was achieved with1mL of 0.1N NaOH at 20 1 C in 2min. For neutraliza-tion of the reaction mixture, 0.5mL of 0.5N aqueousNaH 2 PO 4  was added. The unreacted [ 18 F]fluoride wasremoved by passing the reaction solution through anAl 2 O 3 -cartridge (SEP-PAK light, Alumina N, Waters,USA) preconditioned with 10mL of water. Afterwards,the cartridge was flushed with 1mL of HPLC eluent andthe combined fractions were injected onto the HPLCcolumn for separation. A Phenomenex Luna pre-columnC18/2, 50  10mm; 5 m m with a Phenomenex NucleosilC18, 250  10mm; 5 m m column and a mixture of  ARTICLE IN PRESS O CH 2 OAc AcO TsO NN NO 2 OCH 2 OHHO 18 FNNNO 2 1. [K / 2.2.2.] 18 F, DMSO2. NaOH Fig. 1. Synthesis of 1-(5-[ 18 F]fluoro-5-deoxy- a - D -arabinofura-nosyl)-2-nitroimidazole ([ 18 F]FAZA) by nucleophilic substitu-tion and subsequent hydrolysis. G. Reischl et al. / Applied Radiation and Isotopes 62 (2005) 897–901 898  ethanol/10mM NaH 2 PO 4  (5/95, v/v) as eluent wereused. At a flow rate of 4mL/min, the product had aretention time of 26–27min ( k  0 ¼ 5.6), detected with UV(220nm) and radio detectors. The volume of the productpeak was approximately 10mL. Finally, the product wassterile filtered through a 0.22 m m filter (Millex-GS,Millipore, USA). The overall synthesis time was50min. Activity was determined and a sample wastaken for quality control.  2.6. Analytical procedure For identification of the radiofluorinated FAZA andanalysis of chemical impurities, the product was co-eluted with standard FAZA on an HPLC systemthat used a C18-column (Supelcosil ABZ+, 250mm  4.6mm, 5 m m, Supelco, USA) and ethanol/10mMNaH 2 PO 4  (5/95, v/v) as eluent at a flow rate of 2mL/min. FAZA had a retention time of 3.4min ( k  0 ¼ 2.8).UV detection was performed at 254 and 220nm for thedetection of DMSO impurities since they were betterdetected at the lower wavelength. In parallel, theradioactivity assay was performed by NaI(Tl) scintilla-tion detection.For quantification of the radiochemical product yieldin the development of the labeling as well as for testingradiochemical purity in production runs, tlc separationon silica gel plates (POLYGRAM SIL G/UV 254 ,40  80mm, Macherey&Nagel, Du ¨ren, Germany) withethylacetate was performed. The radioactive spots werequantitatively assessed by means of an electronicautoradiograph (InstantImager, Canberra Packard,USA).  R f   values were 0.00, 0.62 and 0.77 for [ 18 F]fluor-ide, [ 18 F]FAZA and diacetyl-[ 18 F]FAZA, respectively.Tests on Kryptofix 2.2.2, endotoxines, pH-value,clarity, colorlessness and bacteria were performedaccording to the monograph ‘‘Fludeoxyglucose’’ in theEuropean Pharmacopoea (Ph Eur). 3. Results and discussion The synthesis of   18 F-labeled FAZA started by introdu-cing [ 18 F]fluorine to the precursor 1-(2,3-di- O -acetyl-5- O -tosyl- a - D -arabinofuranosyl)-2-nitroimidazole via stan-dard nucleophilic substitution conditions followed byhydrolysis of the protective acetyl groups (Fig. 1).Dependencies of labeling yields on reaction time,temperature and amount of precursor were determinedfirst.As shown in Fig. 3, the labeling process obviouslydepended on temperature. The product yield increasedfrom 8.8 7 4.2% at 40 1 C to 60.7 7 6.3% at 100 1 C.Although the yield was slightly higher at 120 1 C(62.1 7 2.1%) than at 100 1 C, the temperature of choicefor routine production was 100 1 C since at highertemperature the reaction solution turned dark brown,making the HPLC purification more difficult. ARTICLE IN PRESS aV10V31V11V20V9 2 3 4 5 61 ExhaustV8V7V17V19a bH 216 O H 218 OVacuumpumpExhaustV21abF Sep.Cartridge 18 PS-HCO –  3 K 2 CO 3  EluentMeCN/ H 2 ONaH 2 PO 4 NaoH Prec.DMSOV3 V4 V5 V6V14 V15 ba bV2V10.2 µ WasteFluidHPLC Pump HeV30Gamma Det.UV DetectorAutoZeroLampaa bbV13WasteComp.AirEluent 70% EtOH0.2 µ 0.2 µ V12V16He Power HeliumComp. AirPressureWaiting for StatusElapsed timenuclear interface [ 18 F ]FAZA- Synthesis HeV18Al 2 O 3 fromTarget Fig. 2. Scheme for the automated synthesis of [ 18 F]FAZA (modified TRACERlab FX F  N , GE Healthcare, Germany). G. Reischl et al. / Applied Radiation and Isotopes 62 (2005) 897–901  899  When using 1mg of precursor, the labeling yield was27.6 7 2.8% and it increased to 62.6 7 7.0% using 20mg(Fig. 4). For routine production, the amount of precursor was set to 5mg with an average labeling yieldof 57.9 7 5.6%. This amount combines the advantages of sufficient yield with a low consumption of precursor andavoids an overloading of the HPLC purification column.At a reaction temperature of 100 1 C with 10mg of precursor the labeling yield reached 60.7 7 6.3% within5min and remained at this level at longer reaction times(Fig. 5).Additionally, the hydrolysis parameters were opti-mized relative to time, temperature and concentration of NaOH. With 0.1N NaOH the hydrolysis step wasperformed best at 20 1 C, as shown in Table 1. Withrising temperature the [ 18 F]FAZA yield decreased due todecomposition of the product. Therefore, the vessel hadto be cooled down to 20 1 C before adding the base. Theinvestigated range of NaOH concentration from 0.05 to0.2N NaOH had no significant influence on thehydrolysis step (Table 2). The effect of hydrolysis timeis negligible, as well. In the range between 1 and 20min,the radiochemical yields were stable at ca. 60%.Surprisingly, for a quantitative deprotection (hydro-lysis), no conditions could be found (Table 3).To produce high amounts of [ 18 F]FAZA for clinicalstudies, the synthesis, using these optimized reactionparameters, was transferred to an automated synthesi-zer. Productions carried out after irradiations of 1h at35 m A beam resulted in a non-decay-corrected overallyield of the automated synthesis of 20.7 7 3.5% ( n ¼ 21).Absolute yields were in the range of 9.8 7 2.3GBq[ 18 F]FAZA 50min after EOB, ready for intravenousinjection. ARTICLE IN PRESS 0 20 40 60 80 100 120 140010203040506070 Temperature / ° C    R  a   d   i  o  c   h  e  m   i  c  a   l   Y   i  e   l   d   /   % Fig. 3. Dependence of the radiochemical yield of the labelingstep on temperature (10mg precursor, 5min reaction time; n ¼ 3). 00 2 4 6 8 10 12 14 16 18 20 2210203040506070 Precursor / mg (3)(3)(6)(6) (3)    R  a   d   i  o  c   h  e  m   i  c  a   l   Y   i  e   l   d   /   % Fig. 4. Labeling yield dependence on the amount of precursor(100 1 C, 5min reaction time). 0102030405060700 10 20 30 40 50 60 Time / min    R  a   d   i  o  c   h  e  m   i  c  a   l   Y   i  e   l   d   /   % Fig. 5. Time dependence of the radiochemical labeling yield(10mg precursor, 100 1 C;  n ¼ 3).Table 2Yield dependence of [ 18 F]FAZA on concentration of base in thehydrolysis step (labeled diacetyl intermediate ¼ 100%; 1mL,20 1 C, 2min)NaOH (mol/L) Radiochemical yield (%)  n 0.05 65.5 7 5.4 30.1 61.5 7 8.9 40.2 63.6 7 0.1 3Table 1Radiochemical yields of [ 18 F]FAZA from the protected labeledcompound after hydrolysis at different temperatures (1ml of 0.1N NaOH, 2min)Temperature ( 1 C) Radiochemical yield (%)  n 20 61.5 7 8.9 450 53.7 7 13.9 3100 5.0 7 1.2 3 G. Reischl et al. / Applied Radiation and Isotopes 62 (2005) 897–901 900  4. Conclusion The optimal labeling conditions were found to be5mg of precursor, 100 1 C reaction time for 5min with alabeling yield of higher than 60%. Deprotection wasbest achieved in 1mL of 0.1N NaOH at 20 1 C for 2min.The procedure was successfully transferred to anautomated, commercially available module for highlyreliable production of [ 18 F]FAZA in high amounts asnecessary for clinical studies. Acknowledgments This work was supported in part by Grant RI-14 fromthe Alberta Cancer Board. References Adams, G.E., Dewey, D.L., 1963. Hydrated electrons andradiobiological sensitization. Biochem. Biophys. Res. Com-mun. 12, 473–477.Chapman, J.D., 1979. Hypoxic sensitizers—implications forradiation therapy. N. Engl. J. Med. 301, 1429–1432.Dehdashti, F., Mintun, M.A., Lewis, J.S., 2003. In vivoassessment of tumor hypoxia in lung cancer with  60 Cu-ATSM. Eur. J. Nucl. Med. 30, 844–850.Kumar, P., Stypinski, D., Xia, H., McEwan, A.J., Machulla,H.-J., Wiebe, L.I., 1999. Fluoroazomycin arabinoside(FAZA): synthesis,  2 H and  3 H-labelling and preliminarybiological evaluation of a novel 2-nitroimidazole marker of tissue hypoxia. J. Labelled Compd. Radiopharm. 42, 3–16.Kumar, P., Wiebe, L.I., Atrazheva, E., Tandon, M., 2001. Animproved synthesis of   a -AZA,  a -AZP and  a -AZG, theprecursors to clinical markers of tissue hypoxia. TetrahedonLett. 42, 2077–2078.Kumar, P., Wiebe, L.I., Asikoglu, M., Tandon, M., McEwan,A.J., 2002. Microwave-assisted (radio)halogenation of nitroimidazole-based hypoxia markers. Appl. Radiat. Isot.57, 697–703.Machulla, H.-J. (Ed.), 1999, Imaging of Hypoxia—TracerDevelopments. Kluwer Academic Publishers, Dordrecht,Netherlands.Mannan, R.H., Somayaji, V.V., Lee, J., Mercer, J.R., Chap-man, J.D., Wiebe, L.I., 1991. Radioiodinated 1-(5-iodo-5-deoxy-beta- D -arabinofuranosyl)-2-nitroimidazole (iodoazo-mycin arabinoside IAZA): a novel marker of tissue hypoxia.J. Nucl. Med. 32, 1764–1770.Piert, M., Machulla, H.-J., Kumar, P., Link, T., Wiebe, L.I.,2001.  18 F labeled fluoroazomycin arabinoside (FAZA): anovel marker of tumor tissue hypoxia. J. Nucl. Med. 42, 279.Piert, M., Machulla, H.-J., Reischl, G., Ziegler, S., Kumar, P.,Wiebe, L.I., Schwaiger, M., 2002. Fluorine-18 labeledfluoroazomycin arabinoside (FAZA): imaging murine tumorhypoxia with improved biokinetics. J. Nucl. Med. 43, 278.Piert, M., Machulla, H.-J., Picchio, M., Reischl, G., Ziegler, S.,Kumar, P., Wester, H.-J., Beck, R., McEwan, A.J.B.,Wiebe, L.I., Schwaiger, M., 2005. Hypoxia-specific tumorimaging with  18 F fluoroazomycin arabinoside. J. Nucl. Med.46, 106–113.Rajendran, J.G., Wilson, D.C., Conrad, E.U., Peterson, L.M.,Bruckner, J.D., Rasey, J.S., Chin, L.K., Hofstrand, P.D.,Grierson, J.R., Eary, J.F., Krohn, K.A., 2003. [ 18 F]FMISOand [ 18 F]FDG PET imaging in soft tissue sarcomas:correlation of hypoxia, metabolism and VEGF expression.Eur. J. Nucl. Med. Mol. Imaging 30, 695–704.Rajendran, J.G., Mankoff, D.A., O’Sullivan, F., Peterson,L.M., Schwartz, D.L., Conrad, E.U., Spence, A.M., Muzi,M., Farwell, D.G., Krohn, K.A., 2004. Hypoxia andglucose metabolism in malignant tumors: evaluation by[ 18 F]fluoromisonidazole and [ 18 F]fluorodeoxyglucose posi-tron emission tomography imaging. Clin. Cancer Res. 10,2245–2252.Rasey, J.S., Grunbaum, Z., Magee, S., Nelson, N.J., Olive,P.L., Durand, R.E., Krohn, K.A., 1987. Characterization of radiolabeled fluoromisonidazole as a probe for hypoxiccells. Radiat. Res. 111, 292–304.Reischl, G., Ehrlichmann, W., Bieg, C., Kumar, P., Wiebe, L.I.,Machulla, H.-J., 2002. Synthesis of [ 18 F]FAZA, a new tracerfor hypoxia. J. Nucl. Med. 43, 364.Sorger, D., Patt, M., Kumar, P., Wiebe, L.I., Barthel, H., Seese,A., Dannenberg, C., Tannapfel, A., Kluge, R., Sabri, O.,2003. [ 18 F]Fluoroazomycinarabinofuranoside ( 18 FAZA)and [ 18 F]Fluoromisonidazole ( 18 FMISO): a comparativestudy of their selective uptake in hypoxic cells and PETimaging in experimental rat tumors. Nucl. Med. Biol. 30,317–326.Teicher, B.A., 1995. Angiogenesis and cancer metastases:therapeutic approaches. Crit. Rev. Oncol. Hematol. 20,9–39. ARTICLE IN PRESS Table 3Time dependence of hydrolysis on radiochemical yield of [ 18 F]FAZA calculated from the protected intermediate with0.1N NaOH at 20 1 C ( n ¼ 3)Time (min) Radiochemical yield (%)1 61.5 7 0.92 62.8 7 0.35 63.3 7 0.510 62.8 7 0.320 59.9 7 2.9 G. Reischl et al. / Applied Radiation and Isotopes 62 (2005) 897–901  901

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