Radiopharmaceuticals: Recent Developments and Trends 247 15 : Recent Developments and Trends

Guy Bormans, Kristin Verbeke, and Alfons Verbruggen

CONTENTS 15.1 Production of Radiopharmaceuticals, 15.1 Production of Radiopharmaceuticals, Quality Control and Availability Quality Control and Availability 247 15.2 Perfusion Agents 248 15.2.1 Myocardial Perfusion Imaging Agents 248 used in nuclear medicine are mostly 15.2.2 Perfusion Imaging Agents 250 artificial ones. They are primarily produced in a 15.2.3 Lung Ventilation Imaging Agents 251 reactor or cyclotron and supplied by commercial 15.3 Metabolism Agents 252 companies to individual nuclear medicine depart- 15.3.1 Glucose Metabolism 252 15.3.2 Amino Acid Metabolism 253 ments and institutions. On the other hand, some 15.3.3 Nucleosides Metabolism 254 radionuclides, in particular short-lived ones, are 15.3.4 Hypoxia Imaging 254 available at any time due to the availability of 15.4 Peptides and Proteins 255 appropriate generators. By far the 15.4.1 Somatostatin Receptor Tracers 255 111 most important generator in nuclear medicine is the 15.4.1.1 In-Octreotide 255 99 99m 15.4.1.2 P587 and P829 256 Mo/ Tc generator, which has led to the almost 99m 15.4.2 Peptides for Thrombus Imaging 257 unlimited availability of Tc. Together with the 15.4.3 Annexin V 258 excellent radiation characteristics of 99mTc and the 15.4.4 Monoclonal Antibodies 259 commercial availability of efficient and licensed 15.5 Neurotransmitter Receptor and Transporter labeling kits, this generator lies at the base of con- Tracers 260 15.5.1 Benzodiazepine Receptor Agents 260 temporary nuclear medicine practice. 15.5.2 Serotonergic (5-HT) Receptor Tracers 261 The very short-lived positron-emitting radio- 11 15.5.3 Dopaminergic Receptor Tracers 262 nuclides used in clinical PET [ C (t½ = 20.38 min), 15.5.4 Transporter (DAT) 263 13 15 N (t½ = 9.96 min) and O (t½ = 122.2 s)] are only 15.5.5 Norepinephrine Transporter (NET) 264 available at or near institutions which have cyclo- 15.5.6 Transporter (SERT) 264 15.6 Amyloid Imaging 265 tron facilities and can not be supplied to remote 15.7 Newer Labeling Methods: 99mTc(I) Tricarbonyl institutions or hospitals due to their rapid decay. Complexes 266 Since facilities with a cyclotron and radiochemistry 15.8 Cell Labeling 267 laboratory are expensive, they are not evenly spread 15.8.1 Red Blood Cell Labeling 267 worldwide, although their number has increased 15.8.2 White Blood Cell Labeling 268 References 268 significantly in recent years. A steadily growing number of PET centers per- forms clinical imaging but does not have a cyclotron. These sites are mostly limited to the use of radio- tracers labeled with fluorine-18 (t½ = 109.8 min), provided by nearby facilities. Currently, mainly 18F- (18FDG) is produced in these facilities, increasingly under ‘good manufacturing practice’ (GMP) conditions, and dispensed to PET imaging facilities in the area. It can be foreseen that soon other fluorine-18 labeled PET radiopharma- G. Bormans, PhD Verbeke ceuticals will be distributed through the logistic K. , PhD 18 A. Verbruggen, PhD channels that have been set up for FDG distribu- UZ Gasthuisberg, Radiopharmacy Department, Herestraat 49, tion. There is a growing interest in using PET radio- 3000 Leuven, Belgium pharmaceuticals labeled with gallium-68, a radio- 248 G. Bormans et al. nuclide available from a germanium-68/gallium-68 cardial perfusion imaging. Currently, 99mTc-labeled generator (Hoffend et al. 2004; Meyer et al. 2004; sestamibi and 99mTc-tetrofosmin are in routine clini- Nakayama et al. 2003). cal use. Each of these agents is a lipophilic mono- Incorporation of the cyclotron-produced PET ionic cation (Fig. 15.1) and can be prepared in a radionuclides into radiopharmaceuticals is per- nuclear medicine department using commercially formed using dedicated radiochemistry synthesis available freeze-dried kits (Tables 15.1 and 15.2). modules. These units are designed as closed sys- 99mTc-sestamibi is a complex of Tc(I) with six tems to minimize radiation exposure, are computer molecules of 2-methoxy-2-isobutylisonitrile (MIBI). controlled and operate fully automatically. Quality The labeling kit contains the isonitrile metal bind- control of the end-products can be performed using ing ligand as its Cu(I) complex to reduce toxicity, radio GC or radio HPLC and should be carried out volatility and the foul odor of the isonitrile and allow under the supervision of a qualified person. lyophilization (Ramalingam 1989; Iqbal et al. 1989; Sachdev et al. 1990). The heating step during label- ing is required to lower the oxidation state of Tc to +1, and to effect ligand exchange from [Cu(I)(MIBI)4]+ 15.2 to [Tc(I)(MIBI)6]+. It has been shown (Crombez Perfusion Agents et al. 1991; Hung et al. 1991) that 99mTc-MIBI can also be prepared simply and efficiently by just heat- 15.2.1 ing the Cardiolite (DuPont Pharmaceutical, Medi- Myocardial Perfusion Imaging Agents cal Imaging Division, North Billerica, MA) vial for 10–13 s in a microwave oven after addition of the Mainly because of the nearly optimal physical prop- 99mTc generator eluate. However, extreme caution erties of the radionuclide, 99mTc-labeled perfusion in applying this technique is required as incidents agents offer several advantages over 201TlCl for myo- of breakage of 99mTc-MIBI vials during the micro-

+ + EtO R O OEt R R N P P OEt N N EtO Tc C C C P P EtO OEt O Tc EtO OEt C C C b N N N R R R

CH3

R = CH3OC Fig. 15.1a,b. Structures of 99mTc-labeled myocardial perfusion imaging agents: 99mTc-MIBI CH a 3 (a) and 99mTc-tetrofosmin (b)

Table 15.1. Properties of the commercially available labeling kits for preparation of 99mTc-labeled myocardial perfusion imaging agents Brand name of labeling kit Cardiolite Myoview Manufacturer Bristol-Myers Squibb Medical Imaging GE Healthcare (Amersham Health) Chemical name of the ligand 2-methoxy-2-isobutyl isonitrile (MIBI) 1,2-bis [bis(2-ethoxyethyl)-phosphino]ethane or tetrofosmin Other names of 99mTc-complex 99mTc-sestamibi 99mTc-tetrofosmin 99mTc-hexamibi Composition of labeling kit Cu(MIBI)4BF4 01.0 mg Tetrofosmin 0.23 mg SnCl2.2H2O 00.075 mg SnCl2.2H2O 0.030 mg L-cysteine HCl H2O 01.0 mg Disodium sulfosalicylate 0.32 mg Sodium citrate 2H2O 02.6 mg Sodium D-gluconate 1.0 mg Mannitol 20.0 mg Sodium hydrogen carbonate 1.8 mg Radiopharmaceuticals: Recent Developments and Trends 249

Table 15.2. Guidelines for labeling and characteristics of 99mTc-labeled myocardial perfusion imaging agents 99mTc-MIBI 99mTc-Tetrofosmin Oxidation state of 99mTc +1 +5 Charge of the complex +1 +1 Incubation 10 min 100ºC 15 min room temperature Maximum activity 5.56 GBq 8.8 GBq Volume 1–3 ml 4–8 ml Eluate restrictions None Generator last eluted within 72 h Eluate not older than 6 h pH after reconstitution 5.5 7.5–9.0 Stability after reconstitution 6 h 8 h wave heating process have been reported (Hung and in the myocardial kinetics and body distribution. Gibbons 1992). The function of individual compo- Unlike 201TlCl, the 99mTc-labeled perfusion agents nents in the Cardiolite kit formulation has not been accumulate in the myocardium by passive diffu- described, but it can be assumed that citrate is pres- sion and are not transported by the Na+-K+-ATPase ent as a buffer, cysteine forms a temporary chelate enzymatic pump and none of the 99mTc-labeled with reduced technetium, and mannitol serves as a agents redistributes. Myocardial uptake for each of filler and ‘accelerator’ (Tweedle 1983). the tracer agents is proportional to blood flow and 99mTc-tetrofosmin is a 99mTc(V)dioxo-phosphine amounts at 60 min post injection to about 1.0 % of cation which can be prepared by simple addition of injected dose for 99mTc-MIBI (Wackers et al. 1989) 99mTc generator eluate to the labeling vial. After incu- and 1.2% for 99mTc-tetrofosmin (Higley et al. 1993) bation at room temperature for 15 min, the radio- at rest and 1.4 % and 1.1 %, respectively, during exer- chemical purity (RP) of the labeled product exceeds cise. Heart to lung and heart to liver ratios of the two 95% (Higley et al. 1993). Gluconate, present in the 99mTc-labeled agents are presented in Table 15.4. kit, is necessary to keep technetium in a +5 oxidation Whereas these 99mTc-labeled agents were origi- state after reduction by forming a relatively weak nally developed as myocardial perfusion agents, 99mTc-gluconate intermediary complex. Sulfosali- they have been reported to be substrates for P-glyco- cylic acid presumably accelerates ligand exchange protein (Pgp), the product of the human multidrug from 99mTc(V)gluconate to 99mTc(V)dioxo-tetrofos- resistance gene (MDR1), which confers resistance to min. The can be administered drugs by transporting cytotoxic agents out of cells up to 8 h post-reconstitution (radiochemical purity (Piwnica-Worms et al. 1993; Ballinger et al. 1996, >90%). 1997). As a consequence, they can be used for func- To assess the radiochemical purity of the 99mTc- tional imaging of multidrug resistance in tumors. labeled agents, simple quality control procedures Both 99mTc-MIBI (for this indication marketed (thin layer chromatography or separation on a under the brand name Miraluma; Bristol-Myers SepPak-cartridge) are proposed by the manufactur- Squibb Medical Imaging, North Billerica, MA) and ers. Using the appropriate stationary and mobile 99mTc-tetrofosmin (Myoview; GE Healthcare) have phase (Table 15.3), the amounts of 99mTc-complex, been found useful as a second line diagnostic radio- 99m - 99m TcO4 and TcO2 can easily and rapidly be pharmaceutical after mammography to assist in the determined. evaluation of breast lesions in patients with an abnor- Although the images obtained with these tracer mal mammogram or a palpable breast mass (Cayre agents are generally similar, there are differences et al. 2004; Spanu et al. 2002). Uptake of these trac-

Table 15.3. Systems proposed by the manufacturer for rapid quality control of 99mTc-labeled myocardial perfusion imaging agents 99mTc-MIBI 99mTc-Tetrofosmin TLC TLC Stationary phase Baker-Flex aluminum oxide Gelman ITLC/SG Mobile phase Ethanol Acetone/dichloromethane (35/65) 99mTc-complex Rf = 1 Rf = 0.2–0.8 99mTcO4- Rf = 0 Rf = 1 99mTcO2 Rf = 0 Rf = 0 250 G. Bormans et al.

Table 15.4. Heart to lung and heart to liver activity ratios at to improve some technical aspects regarding their rest and during exercise at different time points after injec- preparation and stability and to ascertain and com- 99m tion of Tc-labeled myocardial perfusion imaging agents in pare the individual value of the established radio- humans pharmaceuticals, namely 99mTc-labeled d,l-HM-PAO 99m 99m Tc-Tetrofosmin Tc-MIBI and 99mTc-l,l-ECD (Fig. 15.2, Table 15.5). Rest Exercise Rest Exercise 99mTc-labeled d,l-HM-PAO (generic name = 99mTc- 15 min H/lung 1.9 ± 0.2 1.9 ± 0.3 - - exa meta zi me) ca n be prepa red by reconst itut ion of t he H/liver 0.7 ± 0.1 1.2 ± 0.3 - - commercially available Ceretec kit (GE Healthcare) 30 min H/lung 2.0 ± 0.4 2.2 ± 0.5 2.2 ± 0.1 2.3 ± 0.2 H/liver 1.0 ± 0.2 1.4 ± 0.3 0.5 ± 0.1 1.4 ± 0.2 with generator eluate. However, to ensure adequate 60 min H/lung 2.1 ± 0.3 2.1 ± 0.4 2.4 ± 0.1 2.4 ± 0.2 radiochemical purity of the resulting 99mTc-prepara- H/liver 1.3 ± 0.4 1.6 ± 0.4 0.6 ± 0.1 1.8 ± 0.3 tion, severe restrictions are imposed by the manufac- turer on the volume, radioactivity concentration and ers (low versus high) discriminates breast tumors age of the generator eluate and the total radioactiv- with different histopathological characteristics and ity added to the kit. Moreover, due to the chemical prognosis. The mechanism of localization of these instability of 99mTc-d,l-HM-PAO (conversion of the cationic compounds in various types of breast tissue lipophilic primary complex to a more hydrophilic 99m - (e.g., benign, inflammatory, malignant, fibrous) has secondary complex or directly to TcO4 ), the prep- not been established. aration should be administered within 30 min after reconstitution. Eluate age and radioactivity concen- tration influence the level of oxidants formed through 15.2.2 radiolysis. Due to the low amount of stannous ion in Brain Perfusion Imaging Agents the kit (7.6 µg SnCl2.2H2O at the time of formulation of the kit), only very small amounts of oxidants can Although in recent years no new radiopharmaceu- be tolerated. In addition, the primary complex is also ticals for brain perfusion imaging have emerged, susceptible to radiolysis (Tubergen et al. 1991) which considerable efforts have been made in this field explains the radioactivity concentration restrictions.

H3CCH3 H3CCH3 O C2H5OOC NNHCOOC2H5 Tc H C O CH H C O CH 3 N N 3 3 N N 3 SS Tc Tc b N N N N H3C CH3 H3C CH3 OO OO a H H

Fig. 15.2a,b. Structures of 99mTc-labeled brain perfusion imaging agents: 99mTc-d,l-HMPAO (mixture of d,d (=d) and l,l (=l) isomers) (a) and 99mTc-l,l-ECD (b)

Table 15.5. Properties of the commercially available labeling kits for preparation of 99mTc-labeled brain perfusion imaging agents d,l-HM-PAO l,l-ECD Brand name of kit Ceretec Neurolite Manufacturer GE Healthcare (Amersham Health) Bristol-Myers Squibb Medical Imaging Chemical name of ligand [RR,SS]-4,8-diaza-3,6,6,9-tetramethylundecane- N,N’-2-ethylenediylbis-l-cysteine diethyl ester 2,10-dione bisoxime Other names of ligand Exametazime Bicisate Hexamethyl propylene amine oxime l,l-ethylcysteinate dimer d,l-HM-PAO l,l-ECD Composition of labeling kit exametazime 0.5 mg A: ECD.2HCl 00.9 mg SnCl2.2H2O 7.6 µg SnCl2.2H2O 00.072 mg NaCl 4.5 mg Na2EDTA.2H2O 00.36 mg mannitol 24 mg B: Phosphate buffer pH 7.2–8.0 Radiopharmaceuticals: Recent Developments and Trends 251

To overcome these severe restrictions, several • Add 3.7 GBq generator eluate in 2 ml NaCl 0.9% to procedures for stabilization of the primary 99mTc- vial B (phosphate buffer) d, l-HM-PAO complex have been reported (Hung • Add 3 ml NaCl 0.9% to vial A (containing et al. 1989; Ballinger and Gulenchyn 1991; Bill- ECD.2HCl + SnCl2.2H2O + additives) and mix inghurst et al. 1991; Sampson and Solanki 1991; • Within 30 s, add 1 ml of the solution in vial A to Anon 1989; Lang et al. 1989). Two efficient stabi- vial B and mix lization methods merit special attention. Weisner • Incubate for 30 min et al. (1993) described a method for stabilizing 1.11 GBq-99mTc-d,l-HM-PAO preparations by addi- The need for a rather long incubation period tion of 200 µg cobalt chloride hexahydrate in 2 ml (30 min) is apparently caused by the formation of a water shortly (within 2 min) after reconstitution of weak intermediate complex between reduced 99mTc the kit with pertechnetate. In this way, a prepara- and mannitol which slowly converts to 99mTc-l,l- tion is satisfactory up to 5 h after reconstitution. ECD (Mang’era et al. 1996). In order to shorten Mang’era et al. (1994) studied this method in more the preparation time, alternative procedures for detail and found that also high activity preparations preparation of 99mTc-l,l-ECD have been evalu- (up to 5.55 GBq) can be stabilized using this method. ated. Hung et al. (1997) found that a 97.4% ± 0.5% Radiochemical purity (% of primary lipophilic com- radiochemical purity (RCP) could be obtained after plex) exceeds 85% and clinical usefulness for both an 8–s microwave heating time at 300 W (solution brain perfusion and white cell labeling is retained temperature at 69°C) and an average RCP value of up to 6 h after reconstitution. As a consequence, it 96.4% was maintained throughout the 24–h evalua- is possible to prepare a multi-dose preparation of tion period. To prevent the possibility of radioactiv- 99mTc-d, l-HM-PAO. ity spillage caused by either ejection of the rubber In the US, the package insert of Ceretec kits stopper or shattering of the glass vial (Hung and prescribes stabilizing the reconstituted 99mTc-d,l- Gibbons 1992), an acrylic plastic container should HM-PAO preparation with methylene blue and be used or, alternatively, incubation in a water bath adjustment of the pH with phosphate buffer. For at 69°C can replace the microwave heating. However, this purpose, a complete labeling set comprises in the latter case, the time gain is more limited. the labeling vial with the exametazime ligand plus Because of its higher stability, 99mTc-bicisate stannous chloride, a vial of methylene blue injection is the agent of choice for subtraction ictal SPECT USP 1% and a vial of 0.003 M monobasic sodium coregistered with MRI (SISCOM) to localize the ictal phosphate USP and dibasic sodium phosphate onset zone after self-injection by patients with medi- USP in 0.9 % sodium chloride injection USP. Up to cally refractory epilepsy (Oliveira et al. 1999; van 2.00 GBq (54 mCi) 99mTc-pertechnetate may be used Paesschen et al. 2000; van Paesschen 2004). for reconstitution of the kit. For brain imaging when using the stabilizing protocol, generator eluate less than 30 min old should be used; for white blood cell 15.2.3 labeling, generator eluate less than 2 h old should be Lung Ventilation Imaging Agents used. The final radiopharmaceutical preparation with methylene blue stabilizer may be used up to 4 h Radioactive gases with suitable physical characteris- after the time of reconstitution. tics for use as ventilation imaging agents are limited Unlike 99mTc-d,l-HM-PAO, 99mTc-labeled l,l- to xenon-133 and krypton-81m. Aqueous aerosols of ethylene cysteine dimer (99mTc-l,l-bicisate) is a technetium-99m have been widely developed and chemically stable 99mTc-complex which can be used refined as replacement for these gases, but suffer up to 8 h after reconstitution of the commercially from practical limitations such as suboptimal par- available Neurolite kit (Bristol-Myers Squibb). In ticle size and specific activity. Burch et al. (1984, the kit, ethylene cysteine dimer (ECD) is supplied 1986a,b) developed a technique to produce an ultra- in the form of the dihydrochloride for reasons of fine dispersion of 99mTc-labeled carbon particles stability. Because a neutral pH is required to enable which can be considered as a pseudogas and is called efficient labeling, a phosphate buffer pH 7.2–8.0 is Technegas. After inhalation, Technegas is distrib- supplied in a separate vial. The standard procedure uted in the lungs in proportion to regional ventila- for preparation of 99mTc-l,l-ECD, as described by tion (Lemb et al. 1993). It does not show central the manufacturer, involves the following manipu- deposition nor mucociliary clearance. Technegas is lations: produced using a commercially available generator 252 G. Bormans et al. by heating pertechnetate solution with high specific physiological concepts such as turnover of oxygen, activity (2.6–4.0 GBq/ml) in a crucible of ultrapure glucose, amino acids, fatty acids or DNA precur- (≥ 99.99%) graphite at 2500°C. During this heating sors. step, argon is present (and is consequently inhaled Whereas labeling of candidate tracer agents with by the patient) to provide a non-reactive, inert shield 11C can result in an unchanged substrate, labeling around the crucible. No evidence has been reported with 18F and especially with 123I necessitates chemi- of any compound having been formed with argon cal changes (F for H, F for OH, alkyl fluoride for H, I under any circumstances. for H, I for OH, I for CH3) which can alter the physi- When Technegas is prepared in a mixture of 97% ological properties of the tracer molecule due to argon and 3% oxygen, a pseudogas ‘Pertechnegas‘ is steric and electronic effects. For labeling with 99mTc, formed. Whereas Technegas particles remain in the the necessity to incorporate a bifunctional chelat- lungs for a long period of time, Pertechnegas rap- ing moiety renders it extremely difficult to predict idly disappears with a clearance half-life similar to preserved specificity for the metabolic process. As that of pertechnetate aerosol (Tominaga et al. 1995; a consequence, examples of metabolic imaging with Burch and Browitt 1996; Scalzetti and Gagne 99mTc-labeled tracer agents are rather scarce. 1995). Pertechnegas has been reported to be useful for examining the integrity of the alveolar capillary membrane (Monaghan et al. 1991) and for identi- 15.3.1 fying individuals with opportunistic infection or Glucose Metabolism other diffuse lung pathology. The exact size and structure of the ultrafine 2-Fluoro-2-deoxy-D-glucose (2-18F-FDG; Fig. 15.3), carbon particles is difficult to determine. Some the work horse in clinical PET, is used for the evalu- authors assume that Technegas might consist of ation of glucose metabolism. The energy metabo- small bucky balls (Burch et al 1986a; Mackey et lism of the brain is exclusively based on oxidation al. 1994) compatible with the buckminsterfullerene of glucose whereas the heart uses nonesterified fatty model C60 in which 99mTc atoms are trapped. Most acids as primary substrate for energy production results suggest now that Technegas and Pertechne- and only glucose in the case of low plasma levels of gas contain 99mTc-labeled agglomerated graphite fatty acid. However, after feeding or glucose loading, particles in the size range of 60–160 nm. glucose becomes the primary substrate. In addition, In a comparison of Technegas, krypton-81m gas also tumor cells show a higher glucose consumption and a 99mTc labeled aerosol as lung ventilation tracer compared with normal tissues. agents, Rizzo-Padoin et al. (2001) conclude that In most departments with dedicated radiochem- ventilation scintigraphy using krypon-81m clearly istry facilities, 18F-FDG is prepared by a nucleophilic yields images with the highest quality, followed by displacement reaction of 18F-fluoride on 1,3,4,6- Technegas scintigraphy, but the use of krypton- tetra-O-acetyl-2-O-trifluoromethane-sulfonyl-β- 81m gas is only affordable in terms of cost if at least D-mannopyranose followed by acidic or (now more four patients are examined daily. In a similar com- frequently) alkaline hydrolysis of the acetyl esters parison of 81mKr and Technegas, Hartmann et al. (Hamacher et al. 1986). (2001) conclude that Technegas does not result in After administration, FDG is taken up in the cells more false-positive V/Q lung scan results. The use where it is phosphorylated by hexokinase to FDG- of Technegas, however, increases the number of 6-phosphate. FDG-6-phosphate is not a substrate nondiagnostic V/Q lung scan results, which would for glycolysis and is not further metabolized but increase the demand for further additional testing remains trapped in the cells for several hours. The to confirm or refute pulmonary embolism. blood clearance is triexponential with components

15.3 H CH OH Metabolism Agents HO 2 O H H HO H 18 Metabolic imaging requires natural or exogenous H F OH radiolabeled substrates which participate in a meta- bolic process. The design of such tracers is based on Fig. 15.3. Structure of 18F-FDG Radiopharmaceuticals: Recent Developments and Trends 253

having half-lives of 0.2–0.3 min, 11.6±1.1 min, and of lesser importance (Wienhard et al. 1991). There- 88±4 min (Phelps et al. 1978). The brain uptake in fore, also amino acid analogs which are a substrate dogs is 2%–3.5% at 120 min (Gallagher et al. 1977) for amino acid transporters but are not incorporated whereas the uptake in the myocardium is about into proteins can be used for tumor visualization. In 1%–4%. view of the longer half-life of fluorine-18, enabling Despite an intensive search (e.g. Henry et al. distribution to satellite PET centers, several fluo- 1995; Dumas et al. 2001, 2003), at this moment no rine-18 labeled amino acid analogs have thus been sugar derivative labeled with either 123I or 99mTc is evaluated as brain tumor tracers. available as a SPET substitute for 18F-FDG. In a comparative study, the uptake of O-(2- [18F]fluoroethyl)-L-tyrosine (FET) in brain tumors was found very similar to that of l-[11C-methyl]- 15.3.2 methionine (Weber et al. 2000). FET can be prepared Amino Acid Metabolism in a high yield (40%) by nucleophilic substitution with [18F]fluoride on ethylene glycol-1,2-tosylate, Radiolabeled amino acids could be important tools yielding [18F]fluorethyltosylate, which is reacted for the quantitative assessment of protein synthesis with L-tyrosine (Wester et al. 1999). Alternatively, rate. This would allow diagnosis of various neuro- FET can be prepared by nucleophilic fluorination of logical diseases as well as tumor evaluation (diag- O-(2-tosyloxyethyl)-N-trityl-L-tyrosine t-butyles- nosis, tumor grading and prognosis and therapy ter, followed by removal of the trityl and ester pro- monitoring). Radiolabeled amino acids pass the tecting groups in acidic conditions (Hamacher and blood–brain barrier and are accumulated in tissues Coenen 2002). via a specific amino acid transport system. l-11C- For SPET studies, l-3-123I-iodo-α-methyltyrosine methyl-methionine (Fig. 15.4) is the most widely (123I-IMT) can be considered (Kloss and Leven used labeled amino acid because of the ease, reli- 1979). There is evidence suggesting that IMT is also ability and high yield of its preparation (Berger accumulated in the brain via a specific facilitating et al. 1979; Langström et al. 1987). However, in l-amino acid transport system. The carrier system the cells, the labeled methyl group is transferred to for large neutral amino acids which also transports a large number of non-protein acceptor molecules non-iodinated α-methyltyrosine is likely to be such as lipids and nucleic acids (Ishiwata et al. involved (Pardridge 1977). In analogy with FET, 1988). As a consequence, the non-protein metabo- IMT is not incorporated into proteins (Langen et al. lism is too complex to allow accurate calculation of 1990) and its uptake only reflects amino acid trans- protein synthesis rate. port (Kawai et al. 1991; Langen et al. 1991). IMT It was found that the accumulation of amino acids can be prepared by electrophilic substitution via in in cells is dominated by their cellular transport by situ oxidation of 123I-iodide by chloramine-T, hydro- specific amino acid transporters and that irrevers- gen peroxide, iodogen or iodate with radiochemical ible trapping due to incorporation into proteins is yields of 70%–80%.

H H S S 11 11 COOH COOH H3 C H3C NH NH a 2 2 b H H 11 COOH 11 H3C COOH

NH2 CH3 NH2 c HO d H CH3 123 COOH I COOH

NH2 NH2 HO 18 O e F f

Fig. 15.4a-f. Structures of radiolabeled amino acids: L-11C-methyl-methionine (a), L-[1-11C]methionine (b), L-[1-11C]tyrosine (c), L-[1-11C]leucine (d), L-[3-123I]iodo-α-methyltyrosine (e) and O-(2-[18F]fl uoroethyl)-L-tyrosine (f) 254 G. Bormans et al.

15.3.3 Patient studies have shown that FLT shows Nucleosides Metabolism uptake in bone marrow and liver and in a variety of tumors, although generally to a lower extent In analogy with FDG, nucleosides or nucleoside as compared to FDG (Mier et al. 2002). FLT can analogs can be transported across the cell mem- be synthesized by nucleophilic fluorination of brane by selective transporters (either equilibrative different precursors including 5’-O-(4,4’-dime- or concentrative) and can then be phosphorylated thoxytrityl)-2,3’-anhydrothymidine and 3-N-Boc- intracellularly by specific kinases to the correspond- 1-[5-O-(4,4’-dimethoxytrityl)-3-O-nosyl-2-deoxy- ing mono-, di- or triphosphate derivatives and ulti- β-D-lyxofuranosyl]thymine followed by the removal mately they can be incorporated into DNA. of the protecting groups in acidic conditions As thymidine kinase 1 (TK1) shows an S-phase (Martin et al. 2002). dependent expression, the intracellular accumula- Nucleoside derivatives that are selective sub- tion of labeled nucleosides that are substrates for strates for herpes simplex virus thymidine kinase TK1 reflects DNA synthesis and thus tumor prolif- (HSVtk) have been developed for the in vivo visu- eration. alization of transgene expression using the HSVtk Since incorporation of [3H]thymidine ([3H]TdR) is gene as a reporter gene. Two classes of substrates the gold standard for in vitro tumor cell proliferation, were developed: radioiodine labeled uracil deriva- both methyl[11C]thymidine and 2-[11C]thymidine tives (e.g. 2’-fluoro-2’-deoxy-1-β-D-arabinofurano- (Vander Borght et al. 1992) have been explored as syl-5-[124I]iodouracil, FIAU) and fluorine-18 labeled PET tracers but they suffer from rapid in vivo deg- acyclo guanosine derivatives (e.g. 9-(4-[18F]fluoro- radation by thymidine phosphorylase in plasma, 3-hydroxymethylbutyl)guanine, FHBG, Fig. 15.5.d). resulting in a number of labeled metabolites and a FHBG is prepared by nucleophilic fluorination low tumor uptake. As the presence of a 2’ or 3’ fluo- of N2-monomethoxytrityl-9-[(4-(tosyl)-3-monome- rine atom prevents the degradation of nucleosides thoxytrityl-methylbutyl]guanine and subsequent by thymidine phosphorylase, several fluorine sub- deprotection of the methoxytrityl groups in acidic stituted nucleosides have been evaluated (Fig. 15.5). conditions (Shiue et al. 2001). Grierson et al. (2004) compared the uptake and FIAU can be labeled with fluorine-18 (Mangner retention characteristics of thymidine with those of et al. 2003) or with radioiodine, for which both 131I 18F-labelled FLT (3’-deoxy-3’-[18F]fluorothymidine, (Tjuvajev et al. 2002) and 124I (β+ emitter, half life FMAU (2’-arabino-fluoro-5-fluoromethyl-2’-deoxy- 4.15 days, Bengel et al. 2003) have been used. uridine) and FIAU (2’-arabino-fluoro-5-iodo-2’- deoxyuridine). FLT lacks a hydroxyl group in the 2’ position and is not incorporated into DNA in 15.3.4 contrast to FMAU and FIAU which, however, have Hypoxia Imaging a lower affinity for TK1 resulting in a lower cellular retention compared to FLT. The uptake of FLT still Whereas up to now perfusion agents were often used remains a factor of 7 lower than that of thymidine. to identify tissues with reduced flow and hence, FLT is transported in the cell by the equilibrative reduced delivery of oxygen, a marker of decreased transporter hENT and in detailed analysis showed intracellular oxygen tension would be an effective that FLT is initially retained in the cell as its mono- indicator of tissue that is viable but dysfunctional phosphate and at later time points as its mono- and (hypoxic tissue). Visualization of hypoxia in tumors triphosphate (Grierson et al. 2004). can be predictive for response to therapy. Most

O O O O H C H C I 3 3 N NH NH NH HN HO HO HO N O N O O N O O N O N H2N F F

F OH OH acbdOH F

Fig. 15.5a-d. Structures of radiolabeled nucleosides and nucleoside analogs FLT (a), FMAU (b), FIAU (c) and FHBG (d) Radiopharmaceuticals: Recent Developments and Trends 255

hypoxia markers contain a nitroimidazole moiety copper-60 (t½ = 24.5 min) labeled ATSM for visual- as the reactive chemical species. Nitroimidazoles are ization of tumor hypoxia (Dehdashti et al. 2003). reduced intracellularly in all cells, but in the absence Although several 99mTc-based compounds includ- of an adequate supply of oxygen, they undergo fur- ing BMS181321, BMS194796 and 99mTc-HL91 have ther reduction to more reactive products which bind been designed and evaluated as tracer agents for to cell components. In this way, they are trapped in imaging hypoxia, none of them is or has been com- hypoxic tissue (for a review, see Ballinger 2001). mercially available. Fluoromisonidazole (FMISO, Fig. 15.6) is a fluo- Iodine-123 labeled iodoazomycin arabinoside rinated analogue of the chemical radiosensitizer (IAZA) has been validated in animal models and misonidazole and is radiolabeled with 18F by a tested preclinically (Stypinksi et al. 2001), but no nucleophilic fluorination on a tetrahydropyranyl clinical studies with this agent have been reported protected tosyl precursor followed by acidic depro- so far. tection (Grierson et al. 1989; Lin and Berridge 1993). As [18F]FMISO shows a slow blood clear- ance, more hydrophilic radiolabeled nitroimidazole derivatives have been developed such as [18F]fluor 15.4 oerythromisonidazole ([18F]FETNIM, Yang et al. Peptides and Proteins 1995; Fig. 15.6) and [18F]fluoroazomycinarabinofu ranoside ([18F]FAZA, Sorger et al. 2003; Fig. 15.6). 15.4.1 Direct comparison of [18F]FMISO and [18F]FAZA in Somatostatin Receptor Tracers a rat tumor model showed a higher tumor to muscle ratio for [18F]FMISO at both 1 h and 3 h p.i. despite Radiolabeled natural somatostatin can not be used a faster clearance of [18F]FAZA from muscle tissue as a tracer agent for visualization of somatostatin (Sorger et al. 2003). Comparison of [18F]FETNIM receptors due to the very short plasma half-life of and [18F]FMISO in an experimental mammary the peptide (2–4 min). Therefore, analogs have been carcinoma model in rats showed similar tumor to developed which are more stable against the action muscle ratios which correlated with the oxygenation of peptidases. status in the tumors (Gronroos et al. 2004). Several clinical studies aiming to visualize hypoxia in tumors in order to predict response 15.4.1.1 to radiotherapy have been performed using both 111In-Octreotide [18F]FMISO (Bruehlmeyer et al. 2004) and FETNIM (Lehtiö et al. 2004). One of the somatostatin analogs is octreotide, a Radiocopper-labeled diacetyl-bis(N4-methyl- cyclic octapeptide containing the physiologically thiosemicarbazone) (Cu-ATSM, Fig. 15.6) does not active four-amino acid sequence (Phe-D-Trp-Lys- contain a nitroimidazole group but also shows spe- Thr). In order to enable labeling of octreotide with cific retention in hypoxic tissue (Lewis et al. 1999). 111In, a diethylenetriaminepentaacetic acid (DTPA) α Several clinical studies have been performed using molecule has been conjugated to the -NH2 group of

OH OH

NN 18F NN 18F NO a 2 b NO2 OH

125I 18F O O HO HO N N N N N N Cu OH OH O N O N ce2 d 2 NHSS NH

Fig. 15.6a-e. Structure of hypoxia imaging agents [18F]MISO (a), [18F]FETNIM (b), [125I]IAZA (c), [18F]FAZA (d) and Cu-ATSM (e) 256 G. Bormans et al.

the N-terminal D-Phe residue (Bakker et al. 1991a). not to SSTR1 and SSTR4, research has continued to The generic name of this conjugate is pentetreotide develop other somatostatin analogues, which also (Fig. 12.4). Labeling is performed using the com- bind with high affinity to the other receptor sub- mercially available Octreoscan 111 kit (Mallinck- types. Lanreotide is an octapeptide which binds rodt-Tyco Healthcare, St. Louis, Mo.) which consists to SSTR2 through SSTR5 with high affinity and to of two vials. 111In-indium chloride (vial A) is added SSTR1 with low affinity. This peptide is modified to vial B containing the lyophilized pentetreotide, with DOTA (Fig. 15.7) and labeled with 111In in a after which the mixture is incubated for 30 min at similar way as octreotide. The high-affinity binding room temperature. Labeling yield is higher than of lanreotide to SSTR3 and SSTR4 makes it possible 97% and the preparation should be used within 2 h to visualize certain tumors such as intestinal adeno- after reconstitution. The pH of the reconstituted carcinomas which are not visualized by 111In-pente- product varies between pH 3.5–5.0. treotide scintigraphy (Virgolini et al. 1998). After intravenous administration, 111In-labeled pentetreotide is rapidly cleared from the blood (Krenning et al. 1992). Blood radioactivity 15.4.1.2 decreases within 10 min to 33% of injected dose. P587 and P829 Unlike the initially developed 123I-octreotide, which is eliminated to a high degree via the hepatobiliary Despite the encouraging results obtained with system (Bakker et al. 1991b), 111In-pentetreotide is 111In-labeled somatostatin analogs, a 99mTc-labeled mainly excreted into the urine (50% of injected dose SSTR-binding tracer agent is highly desirable for after 6 h, 85% after 24 h and 90% after 48 h). Up to routine nuclear medicine procedures because 99mTc 4 h after injection, radioactivity in plasma and urine is considerably less expensive than 111In. Also, 99mTc is predominantly intact 111In-pentetreotide. The rel- provides a higher photon flux per unit of absorbed atively long residence time of 111In-pentetreotide in radiation dose and better quality images. the kidneys suggests that following glomerular fil- Diatide Inc., now a division of Berlex laboratories tration, part of the label is actively reabsorbed into (Londonderry, NH), developed a number of synthetic the tubules (Bakker et al. 1991c). high-affinity SSTR-binding peptides, designed for Because pentetreotide only binds with high affin- labeling with technetium-99m. From these peptides, ity to the somatostatin receptor subtype SSTR2, 99mTc-P587 and 99mTc-P829 (Fig. 15.8) were selected with moderate affinity to SSTR3 and SSTR5 and to enter clinical trials. These are peptides designed in a cyclic configuration which is not susceptible to reductive cleavage, in order to avoid the incompat- COOH ibility of having a disulfide in a molecule that is to be HOOC COOH radiolabeled with 99mTc in reducing conditions. In N N N the peptides, a sequence is incorporated to provide D HOOC CO Phe Cys Phe a donor atom set which allows stable complexation D S Trp of technetium-99m (Vallabhajosula et al. 1996). S Lys Labeling is performed by ligand exchange from a Thr-ol Cys Thr 99mTc-glucoheptonate. Since the labeling sequence is in fact a diamide monoamine monothiolate che- lator (P829), or a triamide thiolate chelator (P587), HOOC COOH heat needs to be applied (15 min at 100 °C) to pro- N N mote subtraction of the amide protons and afford stable 99mTc-complexes. In this way, labeling yields N N >90% can be obtained. A labeling kit for convenient D HOOC CO β Nal Phe Cys Tyr preparation of 99mTc-P829 (which has as generic D S Trp name technetium-99m depreotide) has been devel- S Lys oped under the name NeoTect and clinical studies NH2 Thr Cys Val have been performed to establish the usefulness of Blum (βNal = β-Naphtyl-alanine) the tracer agent ( et al. 2000). Imaging using b 99mTc-depreotide was found to be useful for iden- Fig. 15.7a,b. Structures of ligands for somatostatin receptor tifying somatostatin receptor bearing pulmonary tracer agents: pentetreotide (a) and DOTA-lanreotide (b) masses in patients presenting with pulmonary Radiopharmaceuticals: Recent Developments and Trends 257

O ONH2 -1 O O H N O NN 2 NH Tc

N S

O S O NH2 NH O HN NH

O

O

NH N O NH

O HN

OH a

0 NH2

O ONH2 O

ONN O NH H2N Tc

NH2 S NH NH2 O S O NH O HN NH

O

O

NH N O NH

O HN

99m OH Fig. 15.8a,b. Structures of Tc-labeled somatostatin receptor tracer agents: b P587 (a) and P829 (b)

lesions on CT and/or chest X-ray who have known detection of deep venous thrombosis. P280 (now malignancy or are highly suspect for malignancy. called bibapcitide) is a small oligopeptide, con- sisting of two identical, linked, cyclic 13-amino acid monomers (Fig. 15.9). Each monomer (called 15.4.2 apcitide) contains a (S-aminopropyl)cysteine-Gly- Peptides for Thrombus Imaging Asp tripeptide sequence which mimics the RGD- sequence and a Cys(Acm)-Gly-Cys(Acm) tripep- Platelet activation and deposition, the initial events tide sequence, which forms after deprotection an 99m in active thrombus formation, involve expression of N2S2 diamidedithiol type chelator for Tc-label- the GPIIb/IIIa receptor, which recognizes proteins ing. Because of the presence of protecting groups and peptides bearing the Arg-Gly-Asp (RGD) tri- (acetamidomethyl, Acm) on the two thiol groups of peptide sequence. the diamidedithiol and the presence of two amide Lister-James and co-workers (1996; Pearson et protons, 99mTc-labeling of P280 requires heating at al. 1996) at Diatide Inc. developed a 99mTc-labeled 100°C for 15 min during this heating step, bibapcit- GPIIb/IIIa receptor antagonist for scintigraphic ide is split and a 99mTc-apcitide complex is formed. 258 G. Bormans et al.

H2CS (D-Tyr)-Apc-Gly-Asp-NH-CH-C-Gly-Gly-Cys(Acm)-Gly-Cys(Acm)-Gly-Gly-NH-CH-CO-NH2 O O S

O N O

O

O N O

S H2CS (D-Tyr)-Apc-Gly-Asp-NH-CH-C-Gly-Gly-Cys(Acm)-Gly-Cys(Acm)-Gly-Gly-NH-CH-CO-NH2 O Fig. 15.9. Structure of the thrombus O imaging peptide bibapcitide

99mTc-apcitide has been approved by the FDA and mercaptobutyryl side chain (Kemerink et al. 2001) a kit for the preparation of 99mTc-apcitide has been but the radiochemical purity (82%±12%) and sta- developed (AcuTect®). Besides 100 µg bibapcitide, bility of the 99mTc-i-AnxV were found to be subop- each vial contains 89 µg SnCl2.2H2O for reduction timal. of 99mTc-pertechnetate and 75 mg sodium gluco- In 99mTc-BTAP-Anx V (Apomate, Theseus Imag- heptonate which forms an intermediary 99mTc-glu- ing Corp, Cambridge, MA) the protein is modified coheptonate complex which exchanges for apcitide with an N2S2 bis(mercaptoacetyl)diaminopentan upon heating. After administration, 99mTc-apcitide oyl group (Kemerink et al. 2001b), resulting in a exhibits a fast plasma clearance and predominantly stable and well-defined 99mTc-complex. However, renal excretion which is favorable for rapid delin- 99mTc-BTAP-Anx V has to be prepared through a eation of thrombi. It is indicated for scintigraphic preformed chelate approach which is time-consum- imaging of acute venous thrombosis in the lower ing and hardly applicable to routine use. In addition, extremities in patients with signs and symptoms of after intravenous administration of the tracer agent, acute blood clots. activity had already appeared in the bowel within a few hours, precluding imaging of apoptosis in the abdomen. 15.4.3 In 99mTc-Hynic-Anx V, the protein is modified Annexin V with a hydrazino nicotinamide side chain (Blan- kenberg et al. 1998; Ohtsuki et al. 1999) after which Radiolabelled derivatives of Annexin V (Anx V) have labeling is performed by simple addition of Sn2+ ions been developed in order to enable in vivo imaging and pertechnetate in the presence of tricine as co- of apoptosis. Annexin V is a 36-kDa human pro- ligand (Verbeke et al. 2003). Biodistribution studies Ohtsuki Kemer- tein which binds with high affinity (Kd = 7 nmol) to in rats ( et al. 1999) and humans ( phosphatidylserine, a membrane-bound phospho- ink et al. 2003) indicated that 99mTc-Hynic-Anx V lipid which is redistributed from the inner to the is mainly excreted through the kidneys, giving it a outer leaflet of the plasma membrane, early in the clear advantage over 99mTc-BTAP-Anx V. apoptotic process (Reutelingsperger et al. 1988; More recently, a 18F-labeled Annexin V deriva- Martin et al. 1995). tive has been prepared using N-succinimidyl 4- Up to now, three 99mTc-labelled Annexin V com- [18F]fluorobenzoate as the 18F labeling reagent pounds, differing in the bifunctional chelating agent (Murakami et al. 2004). In rats, the accumulation used for complexation of 99mTc, have been studied in of 18F-Anx V and 99mTc-Anx V in the infarcted area humans. In 99mTc-i-Anx V (Mallinckrodt), Annexin was comparable whereas the uptake of 18F-Anx V V was derivatized with a monodentate n-1-imino-4- in liver, spleen and kidney was significantly lower Radiopharmaceuticals: Recent Developments and Trends 259 than that of 99mTc-Anx V. Data in humans are not yet ton, USA). It is provided in liquid form in 1-ml single available. An extensive review describing recently dose vials at a concentration of 0.5 mg/ml. Before developed radiolabeled Annexin V derivatives adding 111In-chloride to the antibody solution, the which might have potential for in vivo use (includ- isotope solution must be buffered with 0.5 M sodium ing annexin V labeled with carbon-11, radioiodine, acetate. indium-111, copper-64 and radiogallium) has been Indium-111 capromab pendetide is indicated for published by Lahorte et al. (2004). use in immunoscintigraphy in patients with biopsy- As Theseus Imaging Corp. has stopped its activi- proven prostate carcinoma who are at high risk for ties of developing radiolabeled Annexin derivatives, pelvic lymph node metastases. It is also indicated it is not clear at this moment whether or not further in patients who have undergone a prostatectomy, progress in this area may be expected. and have rising prostate-specific antigen values and equivocal or no evidence of metastatic disease on standard metastatic evaluation, but in whom there 15.4.4 is a high clinical suspicion of occult metastatic dis- Monoclonal Antibodies ease. Immunoscintigraphy using 111In-capromab is not indicated as a screening test for carcinoma of the Since the advent of hybridoma technology in 1975 prostate or for readministration in order to assess (Kohler and Milstein 1975), an enormous effort response to treatment. has been put into the development of radiolabeled Arcitumomab is a Fab’ fragment generated from monoclonal antibodies (MoAbs), mainly to be used IMMU-4, a murine IgG1 monoclonal antibody, by as in vivo tumor localizing agents. Only a few of enzymatic digestion with pepsin to produce F(ab’)2 these MoAbs have reached the point of proven clini- fragments which are further reduced to Fab’ frag- cal utility and have been approved for use in clinical ments. It is directed against a 200-kDa carcinoem- diagnosis. bryonic antigen (CEA) that is expressed on the cell Capromab (Mab 7E11-C5.3) is a murine mono- surface of numerous tumors, particularly of the gas- Patt clonal antibody of the IgG1K subclass which binds trointestinal tract ( et al. 1994). The Fab’ frag- specifically to a prostate-specific membrane gly- ment is used rather than the whole antibody because coprotein (PSMA), a cell surface antigen which is of its more favorable pharmacokinetics (faster blood expressed only by prostatic epithelial cells (benign clearance and minimal liver uptake) and because and malignant). The antibody is labeled with 111In it minimized the frequency of human anti-mouse through site-specific modification of the oligosac- antibody response. The rapid blood clearance of the charide moiety of the molecule with a linker-chela- Fab’ fragment makes it compatible for labeling with tor, glycyl-tyrosyl-(N,ε-diethylenetriaminepenta- a short-lived isotope such as 99mTc and allows imag- acetic acid)- or GYK-DTPA (Rodwell et al. ing within 2–5 h after injection of the labeled anti- 1986). Therefore, the carbohydrate moieties of the body. The Fab’ fragment does not require any fur- MoAb are oxidized with sodium periodate and after ther modification or derivatization before labeling purification, the aldehyde groups are reacted with and is able to bind up to 1.85 GBq of reduced 99mTc the α-amino group of GYK-DTPA. The Schiff base within 5 min (Hansen et al. 1990). After intrave- formed in this way is further stabilized by reduc- nous injection, blood levels were 63%, 23% and tion with sodium cyanoborohydride. The resul- 7% at respectively 1 h, 5 h and 24 h after infusion. tant MoAb 7E11-C5.3-GYK-DTPA is designated as Because of its low molecular weight (54 kDa), elimi- CYT-356 or capromab pendetide. Because of the nation proceeds mainly through the kidneys with 28 restricted localization of the glycosylation sites on % of the radiolabel excreted in the urine over the immunoglobulins, this approach offers the advan- first 24 h after administration. tage of modification of the antibody at a site distal Arcitumomab is provided as a lyophilized formu- to the antigen combining site resulting in modified lation containing 1.25 mg of Arcitumomab and stan- antibodies with the same homogeneous antigen- nous chloride (CEA-scan, Immunomedics, Morris binding property and affinity as the unmodified Plains, USA). Labeling with 99mTc is performed by antibody. In addition, the antibodies can be labeled simple reconstitution of the contents of the vial with with a greater number of chelators without loss of 1 ml of a pertechnetate solution (1.1 GBq 99mTc) and antigen-binding capability. incubation for 5 min at room temperature. The antibody conjugate is commercially available Imaging with 99mTc-arcitumomab, in conjunc- under the name ProstaScint (Cytogen Corp, Prince- tion with standard diagnostic modalities, e.g., com- 260 G. Bormans et al. puted tomography, is indicated to detect, locate, able , functional receptors and bind- and determine the extent of recurrent and/or meta- ing sites at low (nM) concentrations. In this way, static colorectal carcinoma involving the liver and the relationship between changes in receptor or the extrahepatic abdominal and pelvic regions in binding site occupancy or concentration and the patients with a histologically confirmed diagnosis progress of certain diseases can be elucidated. In of colorectal carcinoma. It also provides additional current research, interest has been shown mainly information in patients suspected of tumor recur- in peptide receptor tracer agents (which have been rence or metastasis who have elevated or rising discussed separately) and neuroreceptor and trans- serum carcinoembryonic antigen (CEA) but no evi- porter radioligands. dence of disease by standard diagnostic methods. Antistage specific embryonic antigen-1 (anti- SSEA-1) is an IgM monoclonal antibody with a high 15.5.1 specificity for a glycoprotein lacto-N-fucopentae- Benzodiazepine Receptor Agents ose-III which is expressed on human neutrophils, eosinophils and lymphocytes. The antigen is also Central benzodiazepine receptors have been stud- known as CD15 (Barclay et al. 1990). Despite its ied in relation to diseases such as epilepsy (Savic high molecular weight of 900 kDa, this antibody et al. 1988), hepatic encephalopathy (Samson et al. shows a fast blood pool clearance which justifies 1987), Alzheimer’s disease (Yamasaki et al. 1986), labeling with the short lived 99mTc. In order to enable Huntington’s disease (Hantraye et al. 1984) and direct labeling with 99mTc, the antibody is reduced to chronic (Litton et al. 1991). generate sulfhydryl groups. The reduced antibody Whereas diazepam and flunitrazepam were the is lyophilized using maltose as a stabilizer and is first classical benzodiazepines to be labeled with commercially available as NeutroSpec (Mallinck- carbon-11 in the N-methyl group (Mazière et al. rodt-Tyco Healthcare, St. Louis, Mo.). Labeling is 1980), [N-methyl-11C]flumazenil and its iodinated performed by addition of 740–1480 MBq 99mTc in analog 123I- (Fig. 15.10a,b) are currently 0.20–0.35 ml generator eluate and incubation for the radioligands of choice for studies of central ben- 30 min at 37°C after which sufficient ascorbic acid zodiazepine receptors with PET, respectively SPET. solution (500 mg/ml, Cenolate) is added to make They are high affinity benzodiazepine antagonists the final preparation volume up to 1 ml. Thakur whereas they lack major intrinsic pharmacologic et al. (1996) found that after intravenous injection effects. of 99mTc-fanolesumab, 15%–50% of the adminis- Radiosynthesis of [N-methyl-11C]flumazenil tered dose was associated with polymorphonuclear involves N-methylation of N-desmethyl-flumaze- neutrophils (PMNs) whereas the activity associ- nil with NCA [11C]iodomethane or [11C]methyl tri- ated with lymphocytes, platelets and erythrocytes flate. A recent report showed that no carrier added was generally low. This is considerably higher as [18F]flumazenil (Fig. 15.10c) can be synthesized compared to another 99mTc-labeled monoclonal by nucleophilic fluorination using a nitro-precur- antibody BW 250/183 where only 3%–10% of the sor (Krasikova et al. 2004). As an alternative 2’- administered dose was associated with PMNs. The [18F]fluoroflumazenil ([18F]FFMZ, Fig. 15.10d, Mit- - terhauser high affinity of anti-SSEA-1 for human PMNs (Kd 10 et al. 2004) has also been synthesized 11 M) together with the fast blood clearance may be and showed a comparable affinity for the GABA the primary reason for the excellent image quality. receptor as flumazenil. 99mTc labeled fanolesumab is indicated for imaging 123I-iomazenil is prepared with high specific equivocal appendicitis in patients 5 years or older. activity [>185 TBq (5000 Ci)/mmol] by oxidative radioiodination of the tributylstannyl precursor (Beer et al. 1990). After administration, the initial uptake of [N- 15.5 methyl-11C]flumazenil and 123I-iomazenil in the Neurotransmitter Receptor and Transporter brain is similar to the rCBF seen with 99mTc-HMPAO Tracers (Yamamoto et al. 1983; Bartenstein et al. 1991). Within 30 min post injection, unbound tracer agent Scintigraphic imaging and especially positron emis- is almost completely cleared from the brain and the sion tomography (PET) are powerful non-invasive distribution of both radioligands closely resembles techniques in clinical research to detect, using suit- the known biodistribution of benzodiazepine recep- Radiopharmaceuticals: Recent Developments and Trends 261

N O N O

N O N O

N F N 11 CH 123 CH3 O 3 I O a b

N O N O

18F N O N O

18 N N F F CH CH3 c O 3 O d

Fig. 15.10a-c. Structures of the benzodiazepine receptor agents [N-methyl-11C]fl umazenil (a), 123I-iomazenil (b) and [18F]fl uorofl umazenil (c)

tors. As compared to [11C]flumazenil, 123I-iomazenil For SPET studies, 123I-5-I-R91150 ([123I]-4-amino- has a tenfold higher affinity for the neuronal type N-[1-[3-(4-fluorophenoxy)-propyl]-4-methyl-4- benzodiazepine receptor and a slower brain wash- piperidinyl]-5-iodo-2-methoxybenzamide) has been out (Johnson et al. 1990). synthesized by Mertens et al. (1994) by electro- philic substitution on the 5-position of the methoxy benzamide group of R91150 (Fig. 15.12). It is a 5-HT2 15.5.2 antagonist with high affinity and selectivity for the Serotonergic (5-HT) Receptor Tracers 5-HT2A receptor (the main subtype of 5-HT2 recep- tors in the brain). The in vitro binding constant (Ki) The in vivo investigation of brain serotonergic 5- for 5-HT2 receptors is 0.2 nM whereas the selectivity α α HT receptors has been pursued for several years. to other neurotransmitter receptors (5-HTx, 1, 2, Terriere There is evidence suggesting a role for the 5-HT D1 and D2) is at least a factor of 50 ( et al. system, in particular 5-HT2 receptors in the regula- 1995). The results obtained with this tracer agent up tion of a wide range of central mechanisms (Cowen to now (e.g. Busatto et al. 1997; Peremans et al. Versijpt 1991). Abnormalities in 5-HT2 receptors have been 2003; et al. 2003) indicate that this radioli- proposed in several neuropsychiatric conditions gand might be very useful for in vivo 5-HT2A recep- including major depression, Alzheimer-type demen- tor mapping, especially since SPET is cheaper and tia, drug abuse and schizophrenia. more accessible than PET. A great number of ligands have been prepared and evaluated for probing 5-HT receptors. At this N CH moment, [18F] (Mazière et al. 1988) and S 3 18 Lemaire [ F] ( et al. 1991) are the only N 18F N radioligands used for PET-studies of 5-HT2 recep- tors (Fig. 15.11). Both can be obtained by a direct O one-pot nucleophilic substitution of the nitro-pre- cursor by n.c.a. [18F]fluoride. The binding affinity O a values for different neurotransmitter receptor sites N S have been reported by Leysen (1989) (Table 15.6). In rats, the frontal cortex-to- activity N 18F ratio, which is a good index for ligand specificity N O (5-HT2/D2), reached 2.56 at 1 h post injection for [18F]altanserin whereas this ratio did not exceed 1.18 for [18F]setoperone. Hence, [18F]altanserin appeared O b more selective for binding to 5-HT2 receptors than Fig. 15.11a,b. Structures of the 5-HT2 receptor tracers [18F]setoperone. [18F]setoperone (a) and [18F]altanserin (b) 262 G. Bormans et al.

Table 15.6. Binding affinity values (Ki, nM) of setoperone and Within 60 min after administration, the ratio of altanserin for different neurotransmitter receptor sites radioactivity uptake in receptor-rich medial tempo- α 5-HT2 D2 1 ral cortex to that in reaches a value of 25, [3H] [3H] [3H]WB-4101 indicating the very low non-specific binding of this Setoperone 0.37 25 13 . Plasma analysis showed rapid metabo- Altanserin 0.13 62 04.55 lism but only to very polar radioactive compounds (most probably [11C]cyclohexanecarboxylic acid and derivatives) which are not expected to enter the CH3O O brain nor to be pharmacologically active.

NH2 NH 15.5.3 123 I Dopaminergic Receptor Tracers N The brain dopaminergic system lies in a well-defined region in the striatum and localization is relatively

O easy. Hence, a large number of ligands for the dopa- minergic system, and in particular the D2 recep- tor, have been studied so far. The most prominent F reversible and high selective D2 receptor ligands are the benzamides of the salicylamide type such as 123 Fig. 15.12. Structure of the 5-HT2 receptor tracer I-5-I- raclopride and FLB 457, eticlopride and fallypride R91150 (Fig. 15.14). The latter three compounds have a 30- fold higher affinity for the D2-receptor than raclo- Halldin Mazière Receptors of the 5-HT1 family (5-HT1A and 5- pride ( et al. 1991; et al. 1992) and HT1D) are distributed among several cortical and can be used to visualize the extrastriatal dopamine subcortical structures where they mediate neuro- receptors (Olsson et al. 2004). For the four ligands, nal hyperpolarization and inhibit the release of the (S)-enantiomer is the most potent form (Hög- neurotransmitters (Hoyer et al. 1994). A selective berg et al. 1991). They can be radiolabeled with ligand with high potency, high selectivity and pure 11C by O-methylation of the desmethyl precursor 11 antagonistic action of 5-HT1A receptors is WAY- with C-methyl iodide. In the case of eticlopride, 100635 (Fig. 15.13). The ligand can be radiolabeled two different O-methylated products are obtained with carbon-11 in the O-methyl as well as in the carbonyl position. In primates, it was found that the descyclohexane carbonyl analogue [O-methyl- 11C]WAY-100634 is a major radiolabeled metabolite N of [O-methyl-11C]WAY-100635 (Osman et al. 1996).

WAY-100634 is known to have a high affinity for N α OCH N 11C 5-HT1A receptors and 1-adrenoreceptors, suggest- 3 ing that this radioactive metabolite contributes to N O brain radioactivity in human PET-studies (Osman et al. 1996). Therefore, labeling of WAY-100635 with a carbon-11 has been changed to the carbonyl position (Pike et al. 1996) to avoid formation of radioactive WAY-100634 or any other pharmacologically active N metabolite. 11 NH [Carbonyl- C]WAY-1003635 is prepared by OCH3 N 11 reaction of cyclohexane[carbonyl- C]chloride N with WAY-100634 and is obtained in a 30%-50% decay-corrected radiochemical yield from trapped [11C]carbon dioxide in 20 min from EOB. After semi- b preparative reversed phase HPLC, radiochemical Fig. 15.13a,b. Structure of the 5-HT1 receptor tracer [carbonyl- purity exceeds 99% (Pike et al. 1995). 11C]WAY-100635 (a) and its metabolite WAY-100634 (b) Radiopharmaceuticals: Recent Developments and Trends 263

OH O O 18F Cl NH NH N N H H 11 O CH3 OCH3 CH3 Cl OCH a 3 b 11 OH O H3 C O O

H C NH 3 NH N H3C H N H O11CH 3 OH CH3 Cl CH3 c Cl d

Fig. 15.14a-d. Structures of the dopaminergic tracer agents (S)-[O-methyl-11C]raclopride (a), (S)-[6-O-methyl-11C]eticlopride (b), (S)-[6-O-methyl-11C]eticlopride (c) and [18F]fallypride (d)

(Fig. 15.14) which have to be separated by HPLC. 15.5.4 Alternatively, eticlopride can be labeled in the N- Dopamine Transporter (DAT) ethyl position as well. The fluorine-18 labeled ana- logs of eticlopride have also been prepared but show Dopamine reuptake sites can be visualized using generally inferior binding properties. radioligands binding to the dopamine transporter, The fluorine-18 labeled derivative of fallypride, a protein which plays an important role in the however, has been used with success to visualize inactivation and recycling of dopamine released Slifs- both striatal and extrastriatal D2 receptors ( into the synaptic cleft. Measurement of decrease in tein et al. 2004). the dopamine transporters may be a useful indi- The favorable results obtained with these cator of dopamine neuronal loss in Parkinson’s PET tracers stimulated the design of a 123I- and other neurodegenerative diseases (Kaufman labeled analog for SPET, namely (S)-N-[(1-ethyl- and Madras 1991). Beta-carbomethoxy-3β-(4- 2-pyrrolidinyl)]methyl-2-hydroxy-3-iodo-6- iodophenyl)tropane (β-CIT, Fig. 15.16) is a more methoxybenzamide (123I-IBZM, Fig. 15.15) (Kung potent analog of the cocaine congener in the inhi- et al. 1988). It can be prepared in high yield and bition of dopamine uptake (Boja et al. 1990) and with high specific activity by oxidative iodination has been radiolabeled with 123I for use in SPET of the enantiomerically pure BZM precursor with imaging (Carroll et al. 1991). Alternatively, 11C- n.c.a. 123I-iodide via in situ formation of peracetic β-CIT can be used for PET-imaging (Laihinen et acid (Bobeldijk et al. 1990). Biodistribution studies al. 1995). In addition, the N-fluoroethyl (β-CIT-FE) demonstrated that the agent is concentrated in the and N-fluoropropyl analogs (β-CIT-FP) have been basal ganglia although some non-specific binding synthesized and radiolabeled with the relatively was noted in cerebral cortex and cerebellum (Kung long-lived radionuclides 123I and 76Br. Due to the et al. 1990). If receptor density or blockade is being high proportion of homology between the dopa- quantified, it is necessary to correct for this non- mine transporter (DAT), the specific binding. (SERT) and the norepinephrine transporter (NET), these ligands are not selective for DAT (Amara and Kuhar β 1993). Table 15.7 shows the Ki values of - OH O CIT, β-CIT-FE, β-CIT-FP and cocaine to inhibit the 3 3 3 123I uptake of [ H]dopamine, [ H]serotonin and [ H]1- NH norepinephrine (Okada et al. 1998). N H Meegalla et al. (1997) have synthesized a tropane O derivative, TRODAT-1, designed for labeling with CH 99m 3 Tc, by conjugation of an N2S2 diaminedithiol-type CH3 chelator with tropane at the 2β-position (Fig. 15.17). Fig. 15.15. Structure of the dopaminergic tracer agent 123I- Labeling of the conjugate by ligand exchange from IBZM 99mTc-glucoheptonate yields a neutral, lipophilic 264 G. Bormans et al.

CH CH CH F CH3 CH2CH2F 2 2 2 COOCH COOCH COOCH N 3 N 3 N 3 H H H I I I abH H cH

Fig. 15.16a-c. Structures of the dopamine reuptake tracer agents β-CIT (a) and its analogues β-CIT-FE (b) and β-CIT-FP (c)

Table 15.7. Ki values (nM) for inhibition of [3H]dopamine, [3H]serotonin or [3H]1-norepinephrine uptake Dopamine transporter Serotonin transporter 1-Norepinephrine transporter 45 nM [3H]dopamine 20 nM [3H]serotonin 50 nM [3H]1-norepinephtine β-CIT 06.34 ± 1.68 029.17 ± 06.40 032.77 ± 13.41 β-CIT-FE 90.88 ± 4.92 132.73 ± 31.90 130.45 ± 49.18 β-CIT-FP 27.97 ± 7.36 113.39 ± 63.77 070.42 ± 15.37

SSO (Schou et al. 2004) have recently been reported to Tc bind specifically to NET in the brain, The deriva- tives are obtained by respectively [11C]methylation NN with [11C]methyl triflate, [18F]fluoromethylation N with [18F]fluoromethyl bromide or dideutero- 18 Cl [ F]fluoromethyl bromide. The fluorine-18 labeled derivatives (S,S)-[18F]FMeNER and (S,S)- [18F]FMeNER-D-2 (Fig. 15.18) offer images with Fig. 15.17. Structure of the 99mTc-labeled dopamine reuptake a better signal/noise ratio than carbon-11 labeled tracer agent TRODAT-1 (S,S)-[11C]MeNER. The dideutero derivative (S,S)- [18F]FMeNER-D-2 showed reduced in vivo defluo- rination as compared to (S,S)-[18F]FMeNER and 99mTc-TRODAT-1 complex in high yield (RCP > 93%). therefore seems to be the tracer of choice for visu- In vitro binding of the dopamine transporter was alization of NET with PET. assessed using the surrogate Re-complex, Re- TRODAT-1, and a Ki value of 14 nM was obtained (Kung et al. 1997). As compared to iodinated recep- 15.5.6 tor ligands, the ratio of specific to non-specific bind- Serotonin Transporter (SERT) ing of 99mTc-TRODAT-1 appears to be lower than that of 123I-β-CIT (1.66 vs 7). Nevertheless, animal stud- The most frequently used medication for treat- ies and studies in normal humans (Kung et al. 1996; ment of depression are selective serotonin reuptake Chou et al. 2004; Huang et al. 2004) indicate that inhibitors (SSRI). Several radiotracers for the visu- 99mTc-TRODAT-1 is a useful SPET imaging agent for alization of SERT with SPECT or PET have been localization of dopamine transporters. described. Huang et al. (2002) compared the PET tracers [11C]McN 5652, [11C]ADAM, [11C]DASB, [11C]DAPA and [11C]AFM in baboons. The authors 15.5.5 concluded that [11C]DASB and [11C]AFM (Fig. 15.19) Norepinephrine Transporter (NET) have superior characteristics over the other tracers for in vivo visualization of SERT as they provide The norepinephrine transporter has been associated images of SERT availability, in a shorter scanning with the pathophysiology of depression (Klimek time or with a better signal to noise ratio. et al. 1997). Several labeled derivatives of S,S- PET with [11C]DASB has successfully been used reboxetin ((S,S)-[11C]MeNER (Schou et al. 2003), for visualization of serotonin transporter occupancy (S,S)-[18F]FMeNER and (S,S)-[18F]FMeNER-D-2 after administration of different doses of several Radiopharmaceuticals: Recent Developments and Trends 265

18 11 O CD2 F O CH3

O O H H

NH NH H H ab

Fig. 15.18a,b. Structure of Norepinephrine transporter [18F]FMeNER-D-2 (a) and [11C]MeNER (b)

NH NH2 2 S S

C F N N N 11 11CH CH3 ab3

Fig. 15.19a,b. Structure of serotonin transporter [11C]DASB (a) and [11C]AFM (b)

serotonin reuptake inhibitors in healthy volunteers ited, as can be expected on the basis of their high (Meyer et al. 2004). In this study, an occupancy of molecular weight (> 700 Da) and the presence of 80% was observed at minimum therapeutic doses. very polar functional groups. More promising radioactive compounds with smaller molecular size and increased lipophilicity, derived from the amyloid binding dye thioflavin 15.6 T, have been reported. As thioflavin T contains a Amyloid Imaging positively charged quaternary amine which likely will limit brain uptake, benzothiazoles which are Alzheimer’s disease (AD) is characterized by the uncharged at physiological pH were designed and presence of abundant senile plaques composed of tested for their amyloid-β binding properties and in amyloid-β (Aβ) peptides and neurofibrillary tangles vivo brain uptake. formed by filaments of highly phosphorylated tau Klunk and coworkers (2001) found attractive proteins (Selkoe 1999). In vivo assessment of the characteristics in a carbon-11 labeled benzothiazole, beta-sheet proteins deposited in amyloid plaques namely [N-methyl-11C]6-Me-BTA-1 (Fig. 15.20). This using a radiolabeled imaging agent with high affin- compound enters the brain at levels comparable ity for Aβ would allow localization and quantifica- to commonly used neuroreceptor imaging agents tion of senile plaques in a non-invasive way and (7.61% ID/g at 2 min post injection) and shows good possibly in an early stage of the disease. clearance (t½ = 20 min) of free and non-specifically Several derivatives of Congo red (this compound is bound radioactivity in normal rodent brain tissue. being used for fluorescent staining of senile plaques In addition, it shows a relatively high affinity for in post-mortem brain sections of AD patients) and amyloid (Ki = 20.2 nM, being 45-fold that of thiofla- chrysamine G labeled with radioiodine or techne- vin T) and has a specificity for staining plaques and tium-99m have been developed and evaluated as neurofibrillary tangles in post-mortem AD brain. potential Aβ aggregate specific imaging agents (e.g., The octanol-buffer partition coefficient is 2290 Han Dezutter Link et al. 1996; et al. 1999a,b; et (log Poct = 3.36). These characteristics suggest that al. 2001). Many of these compounds showed high in [N-methyl-11C]6-Me-BTA-1 holds potential as an in vitro binding affinity for amyloid fibrils, but their vivo beta-sheet imaging agent for positron emission penetration through the blood–brain barrier and as tomography, although its lipophilicity is rather high a consequence their uptake in brain was very lim- and its clearance from normal brain relatively slow. 266 G. Bormans et al.

S 11 I CH3 X N N H a N N b CN CN

HO S 11CH 3 18F N N N H c d

11CH 3 Fig. 15.20a-e. Structure of amyloid binding agents as potential tracers for N imaging Alzheimer’s disease, N-[11C]methyl-6-Me-BTA-1 (a), TZDM (X=S) H HO and IBOX (X=O) (b), [18F]FDDNP (c), [11C]methyl- 6-OH-BTA-1 (PIB-2) (d) e and N-[11C]methylamino-4’-hydroxystilbene (e)

For this reason, Mathis et al. (2003) developed more nation of load of senile plaques in the living human polar derivatives of which the 6-hydroxy analog brain with PET (Barrio et al. 1999; Agdeppa et al. ([11C]6-OH-BTA-1, also called Pittsburg compound- 2001c). The tracer agent binds to two kinetically dis- B or PIB, Fig. 15.20) was found to have the most tinguishable binding sites on Aß(1-40) fibrils with optimal pharmacokinetics. The first clinical studies apparent Kd values of 0.12 nM for the high-affinity using this tracer agent allowed to conclude that PET binding site and 1.86 for the low-affinity binding site, imaging with PIB can provide quantitative informa- as determined using fluorescence titrations. Specifi- tion on amyloid deposits in living subjects (Klunk cally, the presence of the dicyano group in FDDNP et al. 2004). appears critical for high-affinity binding. Detailed Kung and coworkers (Zhuang et al. 2001a,b) analysis of the distribution pattern in AD patients developed a series of radioiodinated benzothiazoles and normals (Shoghi-Jadid et al. 2002; Small et al. of which TZDM and especially IBOX (Fig. 15.20) 2002) has led to the hypothesis that [18F]FDDNP may show the most promising properties. Octanol-buffer be an in-vivo marker for neurofibrillary tangles as partition coefficients were 70 and 124 for TZDM and well as for diffuse and dense core Aß plaques. This IBOX, respectively, and they were found to bind could increase the potential ability of this tracer to Aß(1-40) aggregates with equal high potency to detect AD presymptomatically, but it also sug- 18 (KI = 1.9 nM and 0.8 nm, respectively). Both agents gests that [ F]FDDNP is not solely an Aß-specific also showed excellent autoradiographic labeling of radiotracer, complicating its use in monitoring the plaques in post-mortem brain sections of a con- effectiveness of Aß-reducing medication. A further firmed AD patient. In normal mice, brain peak intriguing finding is the fact that FDDNP competes uptake was superior for 125I-IBOX (2.08% of injected with some, but not all, non-steroidal anti-inflamma- dose at 30 min post-injection versus 1.57% at 60 min tory drugs for binding to Aß fibrils in vitro and to for 125I-TZDM) and its wash-out from brain was Aß plaques ex vivo (Agdeppa et al. 2003). much faster. Recent developments by the group of Kung led to the finding that a relatively simple carbon-11 labeled stilbene, (N-[11C]methylamino-4-hydroxystilbene 15.7 or [11C]SB-13, Fig. 15.20) is an effective and favor- Newer Labeling Methods: able PET tracer for fibrillar Aß imaging in vivo, with 99mTc(I) Tricarbonyl Complexes similar performance to [11C]PIB (Ono et al. 2003; Verhoeff et al. 2004). In view of the high stability and inertness of Tc(I) In a different approach, Agdeppa and coworkers compounds, considerable efforts have been made (2001a,b) developed a small but highly lipophilic to optimize methods for convenient synthesis of 99m (log Poct = 3.92) fluorine-18 labeled compound, such Tc-complexes. Extensive research by the [18F]FDDNP (Fig. 15.20) which crosses the blood– groups of Schubiger αnd Alberto (Alberto et al. brain barrier and allows localization and determi- 1998a) has resulted in the successful development Radiopharmaceuticals: Recent Developments and Trends 267 of a facile synthesis (30 min at 75°C) of the tricar- 15.8 + bonyl complex of fac-(Tc(I)[H2O]3[CO]3) via the Cell Labeling 99m reduction of Na TcO4 with sodium borohydride at pH 11 under an atmosphere of carbon monox- 15.8.1 ide. This cationic species readily undergoes ligand Red Blood Cell Labeling exchange of coordinated water with a wide variety of donor ligands, of which the tridentate ligands Several studies comparing the biological behavior such as histidine yield the most stable complexes of 99mTc-red blood cells (99mTc-RBCs) labeled via (Abram et al. 1992; Schibli et al. 1998, 2000). The the in vitro, in vivo or modified in vivo method net charge of the final complex can be –1, 0 or +1 all revealed higher heart-to-background ratios and depending on the nature of the coordinating ligand. better subjective images with in vitro labeled RBCs Moreover, these complexes can be formed with very (Hamilton and Alderson 1977; Hegge et al. 1978). small amounts of the ligand, resulting in prepara- A labeling kit for in vitro labeling of RBCs with tions with extremely high specific activity. Recently, 99mTc in whole blood has originally been developed the preparation of the intermediary tri-aqua tri- at Brookhaven National Laboratory (Srivastava et carbonyl Tc(I) core has been further optimized al. 1983) and is commercially available under the by using potassium boranocarbonate (K2BH3CO2) brand name Ultra-Tag. as both a solid source of CO and the reductant for Each kit consists of three separate nonradioactive labeling with 99mTc (Dyszlewski et al. 2001). This components with the following constituents: has allowed the production of a kit formulation for 1. Labeling vial: SnCl2.2H2O, 105 µg (50 µg stannous the preparation of the tricarbonyl Tc(I) intermedi- ion minimum); sodium citrate.2H2O, 3.67 mg; ate, which can then be further used for convenient dextrose, 5.50 mg; pH prior to lyophilization production of a wide diversity of Tc(I) tricarbonyl 7.1–7.2 compounds by ligand for water exchange. Exam- 2. Syringe I (to be protected from light): sodium ples of interesting biologically active agents labeled hypochlorite, 0.6 mg; water for injection, 0.6 ml; as a 99mTc(I) tricarbonyl complex include octreo- pH 11–13 Marmion tate ( et al. 2000), a 5-HT 1A receptor 3. Syringe II: citric acid.H2O, 8.7 mg; sodium Alberto Wust ligand ( et al. 1998b), steroids ( et al. citrate.2H2O, 32.5 mg; dextrose, 12.0 mg; water 1998), a cocaine derivative as ligand for the dopa- for injection, 1.0 ml; pH 4.5–5.5 mine transporter (Hoepping et al. 1998), glucose (Dumas et al. 2001) and peptides and recombinant Red blood cell labeling is started by the addition proteins containing C-terminal His5-tag or His6-tag of 1–3 ml of autologous whole blood, anticoagulated (Waibel et al. 1999). This exciting chemistry thus with heparin or anticoagulant citrate dextrose solu- can be assumed to allow technetium-99m labeling tion (ACD) to the labeling vial. In this vial, the citrate of any appropriately designed recombinant protein, complex stannous ion and the dextrose is present synthetic peptide and a wide variety of organic to sustain red cell metabolism, which improves synthetic compounds and holds a great potential RBC viability. After an incubation period of 5 min, for the development of a new generation of 99mTc- during which a portion of the stannous ion in the radiopharmaceuticals. First clinical studies using reaction vial diffuses across the RBC membrane and the Tc-tricarbonyl technique to label biomolecules is bound intracellularly, the hypochlorite solution of (Buchegger et al. 2003) also revealed the potential syringe I is added to the vial to oxidize the extra- clinical usefulness of such 99mTc-complexes. There cellular stannous ion. To render the extracellular remains, however, controversy about whether or not tin more readily available for oxidation by hypo- new radiopharmaceuticals on the basis of the Tc-tri- chlorite, the solution in syringe II is added. Finally, carbonyl chemistry will be introduced into routine a pertechnetate solution (0.5–3 ml, 370–3700 MBq clinical use (Alberto and Welch 2003). Reasons 99mTc) is added and the vial is incubated with occa- for some skepticism are the fact that the key patent sional gentle mixing for 20 min. holder (Mallinckrodt) apparently is not actively pur- A RBC labeling efficiency ≥95% is typically 99m suing new Tc(CO)3-radiopharmaceuticals, the obtained using this in vitro labeling procedure. As shift of interest and research focus towards clinical the yield can decrease in the presence of excessive PET and the regulatory hurdle and high costs associ- amounts of the long-lived daughter nuclide 99Tc, the ated with the approval of a new diagnostic drug as use of fresh (<24 h ingrowth time) generator eluate compared to the financial revenues. is recommended. 268 G. Bormans et al.

15.8.2 tests cannot discriminate between cell damage and White Blood Cell Labeling activation since the techniques themselves often activate the granulocytes. Radiolabeled autologous white cells offer the most The most sensitive and practical means of qual- widely accepted radioisotopic method for imaging ity control of radiolabeled granulocytes are based infection and inflammation. Methods for labeling on their in vivo distribution following injection. can be classified depending on the choice of the When radiolabeled granulocytes are damaged or radionuclide (indium-111 or technetium-99m) and activated, they are sequestrated in the lung micro- the choice of the cell preparation (pure granulocytes vasculature by a mechanism which is poorly under- or mixed white cell populations). stood. As a consequence, dynamic imaging over the Both mixed leukocytes and pure granulocytes lungs is a useful test. Alternatively, the radiolabeled have been labeled with 111In. Although pure granu- granulocyte recovery, defined as the fraction of locytes offer the advantage of theoretically better injected labeled granulocytes that are circulating in sensitivity and specificity and avoid the risk of blood, can be determined. Normal recovery factors malignant transformations of lymphocytes, mixed at 45 min p.i. are about 35% while these values fall leukocytes are more practical for routine use because to less than 5% when the cells are severely activated they can be prepared easier and faster while having a or damaged (Peters 1994). These tests need not be comparative sensitivity in acute infections (Schau- performed with every preparation but are useful wecker et al. 1988). Hydroxyquinoline (oxine) was when setting up a white cell labeling procedure. the first established and mostly used lipophilic che- The normal biodistribution of 99mTc-leukocytes is lating agent to transport 111In into the cell (McAfee not fully identical to that of 111In-labeled leukocytes. and Thakur 1976). Whereas 111In-leukocyte distribution at 18–24 h is Because of the more favorable physical charac- primarily confined to the reticuloendothelial system teristics of 99mTc, many attempts have been made to of liver, spleen, bone marrow and major blood ves- develop methods for labeling leukocytes with it. The sels (Seabold et al. 1997), non-specific bowel activ- first approaches to label leukocytes with techne- ity appears from about 3–4 h after administration tium-99m implied phagocytic engulfment of tech- of 99mTc-labeled white cells. This is due to elution of netium-labeled colloid (Schroth et al. 1981; Pull- secondary hydrophilic 99mTc-HMPAO species which man et al. 1986). However, this technique has not are excreted in the gut via biliary excretion and in the become established worldwide because, by deliber- urine (Peters et al. 1988). As a consequence, imag- ately inducing the cells to phagocytose, they become ing of inflammatory bowel disease (IBD) and intra- activated prior to injection (McAfee et al. 1984). abdominal abscesses using 99mTc-labeled leukocytes Successful labeling of leukocytes with 99mTc was needs to be performed within 2 h of injection. achieved in 1986 whit the use of 99mTc-HMPAO, like 111In-oxine a neutral lipophilic chelate capable of penetrating blood cells (Peters et al. 1986; Roddie et al. 1988). Since 99mTc-HMPAO is relatively selec- References tively taken up by granulocytes in mixed cell popu- lations and elutes more rapidly from lymphocytes Abram U, Abram S, Schibli R et al (1992) Synthesis and struc- and monocytes than from granulocytes, labeling tures of technetium (I) and rhenium (I) tricarbonyl com- plexes with bis(diphenylthiophosphoryl)amide, {M(CO) [ can be performed in a mixed white cell population 3 Ph2PS)2N](CH3CN)}(M=Tc, Re). Polyhedron 17:1303–1309 which avoids the need for complex and lengthy cell Agdeppa ED, Kepe V, Flores-Torees S et al (2001a) In vitro purification (Frier 1994). binding characteristics of FDDNP and a new analog for Leukocytes can be labeled with either 111In or synthetic [beta]-amyloid fibrils. J Nucl Med 42:64P 99mTc by a variety of accepted procedures (Datz Agdeppa D, Kepe V, Liu J et al (2001b) Binding characteris- Thakur Danpure tics of radiofluorinated 6-dialkylamino-2-naphtylethyli- 1993, 1994; et al. 1977; et al. 1988; dene derivatives as positron emission tomography imag- Dewanjee 1990; Mortelmans et al. 1989). ing probes for ß-amyloid plaques in Alzheimer’s disease. J A major concern when preparing radiolabeled Neurosci 21:RC189 (1–5) cells is the effect of the labeling procedure on granu- Agdeppa ED, Kepe V, Shogi-Jadid K et al (2001c) In vivo and locyte viability and function. In vitro tests examin- in vitro labeling of plaques and tangles in the brain of an Alzheimer’s disease patient: a case study. J Nucl Med ing chemotaxis, superoxide generation in response 42:65P to activation and morphological changes can be Agdeppa ED, Kepe V, Petri A et al (2003) In-vitro detection used to assess granulocyte function. However, these of (S)-naproxen and ibuprofen binding to plaques in the Radiopharmaceuticals: Recent Developments and Trends 269

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