24 Current Radiopharmaceuticals, 2009, 2, 24-31

GABAA Receptor Specific as Potential Imaging Agents: In Vivo Characteristics of a New 18F-labelled Indiplon Derivative

W. Deuther-Conrad*,1, S. Fischer1, M. Scheunemann1, A. Hiller1, M. Diekers2, A. Friemel1, F. Wegner3, J. Steinbach1, A. Hoepping2 and P. Brust1

1Institute of Interdisciplinary Isotope Research, Leipzig, Germany, 2ABX advanced biochemical compounds GmbH, Radeberg, Germany and 3Department of Neurology, University of Leipzig, Leipzig, Germany

Abstract: The design of radioligands selective for particular GABAA receptor isoforms is challenging because of their great potential for the diagnostic imaging of neuropsychiatric diseases such as epilepsy and sleep disorders. A subtype specificity higher than that of the classical makes the novel indiplon an attractive candidate structure. In the present work, N-(3-[18F]fluoro-propyl)-N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]pyrimi- dine-7-yl]-phenyl}-acetamide ([18F]fluoro-propyl-indiplon; [18F]FPI) was characterized and validated as a candidate tracer for positron emission tomography imaging of 1-GABAA receptors. In vitro, receptor affinity and autoradiography of [18F]FPI were assessed in cerebellar homogenates and sagittal brain slices of rats, respectively. The regional brain and or- gan uptake was investigated in NMRI mice at various times after i.v. application of [18F]FPI by ex vivo autoradiography and dissection, respectively. Radiometabolites of [18F]FPI were determined in blood, brain, and urine samples by high- 18 performance liquid chromatography. [ F]FPI binds to cerebellar receptors with high affinity (KD 2.93 nM), and the in vi- 3 tro labelling pattern appears to be more selective for 1-containing GABAA receptors than [ H]. Intravenous application of [18F]FPI revealed (i) a strong biotransformation, (ii) low brain-to-plasma ratios of 18F (<0.4 at all times after tracer injection), (iii) homogenous and non-specific 18F distribution in the rat brain, and (iv) high radioactivity uptake in liver and femur. Thus, despite the promising results obtained by in vitro evaluation, [18F]FPI is excluded from further de- velopment as PET tracer.

Key Words: GABAA receptor, 1 subunit, organ distribution, metabolism, ex vivo autoradiography, positron emission tomo- graphy (PET).

1. INTRODUCTION the  subunit variant [12]. In particular, alterations in the mRNA and protein expression of 1 subunit have been de- The fast postsynaptic inhibition of signal transduction in tected in experimental Parkinson’s disease models [13], the mature nervous system, mainly mediated by ubiquitously amyotrophic lateral sclerosis [14], or chronic ethanol con- distributed GABAA receptors, is one of the physiological functions of the neurotransmitter GABA [1,2]. Among the sumption [15]. Furthermore, this specific subunit mediates sedation, anterograde amnesia and is partially involved in various GABA receptor isoforms,    is the most preva- A 1 2 2 seizure protection [3]. Despite the great variety of com- lent subtype followed by 232, 3n2, and diverse 4-6- containing isoforms [3]. Imbalances in neuronal inhibition pounds that bind to the BZ binding site, only few ligands affect not only learning, motivation, and movement [4] but show high subtype selectivity [16]. Hence, neuroimaging of may result in excitation, convulsion, anxiety, high blood 1-containing GABAA receptor isoforms is hampered by the pressure, restlessness, or [5]. Impaired integrity of lack of subtype specificity of the clinically applied BZ de- rived [11C]flumazenil [17]. Therefore, novel pyrazolo[1,5- the pentameric GABAA receptor is assumed to be one of the reasons for ineffective GABAergic neurotransmission. This a]pyrimidines such as the 1 selective ligands indiplon is supported by postmortem evidence of distinct expression [18,19] or an azaisostere of [20] could promote the patterns of various , , and  subunit proteins in epileptic development of 1 subunit-selective tracers applicable in patients [6,7] and in respective animal models [8-10]. The positron emission tomography (PET). The validity of this unique properties of synaptic GABAA receptors make them approach was recently confirmed by our group, as various important pharmacological targets, and agents acting on al- new F-substituted indiplon derivatives with high GABAA losteric binding sites such as , neuroactive ster- receptor affinity and 1 specificity were synthesised [21,22]. oids [11], or (BZ) type ligands are used fre- However, since the first 18F-labelled indiplon derivative, quently as anaesthetics, anticonvulsants, anxiolytics, or seda- 2-[18F]fluoro-N-methyl-N-{3-[3-(thiophene-2-carbonyl)- tive- [5]. The functional and pharmacological di- pyrazolo[1,5-a]pyrimidin-7-yl]-phenyl}-acetamide [23] re- versity of the GABAA receptor subtypes is associated with vealed unfavourable in vivo metabolism, further structural modifications were required. The aim of the current study was to enhance the meta- *Address correspondence to this author at the Winnie Deuther-Conrad, Institute of Interdisciplinary Isotope Research, Permoserstr. 15, 04318 bolic stability of potential indiplon derived PET tracers by Leipzig, Germany; Tel: +49-341-2352636; Fax: +49-341-2352731; further modification of the N-methyl-acetamide part of the E-mail: [email protected]

1874-4710/09 $55.00+.00 © 2009 Bentham Science Publishers Ltd. GABAA Receptor Specific Pyrazolopyrimidines as Potential Imaging Agents Current Radiopharmaceuticals, 2009, Vol. 2, No. 1 25

18 18 TosO O [ F]F /K2CO3/K222 F O N S DMF (8' / 135°C) N S N N N N N O N O

Scheme 1. Synthesis of [18F]FPI starting from the tosylate precursor. molecule. The poor stability of the [18F]fluoroacetamide [3H]flunitrazepam, similar brain structures were labelled on group [23] prompted us to introduce the fluorine at the end pig brain slices (Fig. 2E). The highest density of specific of the N-alkyl chain. We investigated the binding behaviour, binding sites was detected in the cortical and hippocampal the regional brain and organ uptake, and the metabolic pro- structures, in particular the dentate gyrus, as well as in the file of the newly synthesised N-{3-[18F]fluoro-propyl)-N-{3- superior and inferior colliculi. Moderate to low labelling was [3-(thiophene-2-carbonyl)-pyrazolo[1,5-a]pyrimidin-7-yl]-phe- observed in the cerebellum, the thalamus, and the striatum. nyl}-acetamide, [18F]FPI, by in vitro binding assays and autoradiography as well as ex vivo biodistribution and radi- ometabolite analyses. In addition to a high in vitro GABAA receptor affinity and 1 subunit specificity of FPI [22], the increased length of the N-alkyl group (from CH3 in indiplon to C3H6F in FPI) was assumed to hinder the cytochrome P450 3A4/5 catalysed N-demethylation which was reported for indiplon [24,25]. Moreover, the suitability of an N- [18F]fluoro-propyl-labelled PET ligand has been proven by studies of [18F]beta-CIT-FP in monkeys [26]. Although the novel compound shows favourable in vitro data and demon- strates improved pharmacokinetic properties, the high amount of blood-brain barrier permeable radiometabolites makes it an inappropriate candidate for further radiotracer development. 2. RESULTS 2.1. Radioligand Fig. (1). Competition of [18F]FPI by FPI in cerebellar membranes [18F]FPI was synthesised according to the route in of adult rats. Increasing concentrations of FPI (0.01 nM to 0.3 M) Scheme 1 in a total synthesis time of 90-115 min with an compete for more than 99% of total specific binding sites of overall decay-corrected radiochemical yield of 30-40%, a [18F]FPI (0.02 nM working concentration) at 60 min incubation radiochemical purity of  99%, and a specific radioactivity time in 50 mM TRIS-HCl, pH 7.4. Nonspecific binding of [18F]FPI of  250 GBq/mol (EOS). was determined with 10 M zolpidem. The equilibrium dissociation constant K of FPI was calculated by the equation: K = IC - [ra- 2.2. In Vitro Equilibrium Dissociation Constant of D D 50 18 dioligand]. The points represent the mean ± SD of one of two inde- [ F]FPI pendent experiments, each in triplicate. 18 The in vitro affinity of [ F]FPI in cerebellar membranes 2.4. Ex Vivo Organ Distribution and Brain Autoradio- of adult rats was estimated in two independent homologous graphy in Mice competition experiments, each in triplicate (Fig. 1). The 18 mean value of the equilibrium dissociation constant KD of The data on the F uptake in various organs of NMRI [18F]FPI was 2.93 nM (single values: 3.84 nM and 2.01 nM). mice at 5, 15, 30, 60, and 120 min p.i. of 200-300 kBq [18F]FPI are given in Table 1. 2.3. Comparative In Vitro Autoradiography on Rat and Pig Brain The uptake was highest at 5 min p.i. in the brain and cerebellum, as well as in blood, plasma, heart, lung, stomach, In vitro distribution of [18F]FPI binding sites was studied 3 liver, kidney, spleen, thymus, pancreas, adrenal glands, in comparison to [ H]flunitrazepam on sagittal brain slices of uterus/ovary, and muscle, and declined in all organs except adult rats and juvenile pigs (Fig. 2). the stomach by 90-97% at the end of the study. The washout In both species, the spatial pattern of [18F]FPI labelling in the stomach was slower and the uptake was reduced by was comparable (Fig. 2A, 2C). Specific binding was espe- 62% at 120 min p.i. Contrary to the described decline, the cially high in the olfactory bulb and the ventral pallidus of intestine and the femur steadily accumulated 18F, reaching a the rat, and in the inferior colliculi of both rat and pig. Mod- value of 21.2 and 4.91 %ID/g at 120 min p.i. erate to low densities were detected in rat and pig cortex, To identify the anatomical regions in the brain labelled various regions of the thalamus, the cerebellar layers, the by [18F]FPI in vivo, sagittal brain slices of NMRI mice in- superior colliculi, and the hippocampal formation. The den- jected with 16 MBq [18F]FPI were obtained at 20 (Fig. 3A), tate gyrus was detectable in particular in the pig brain. By 60 (Fig. 3B), and 120 (Fig. 3C) min p.i. The ex vivo autora- 26 Current Radiopharmaceuticals, 2009, Vol. 2, No. 1 Deuther-Conrad et al.

Fig. (2). In vitro digital autoradiographs of sagittal rat and pig brain slices labelled with [18F]FPI (A and B, and C and D, respectively) and [3H]flunitrazepam (E, F), respectively. Brain sections (12 m rat, 20 m pig) were analysed after 60 min incubation with either 0.8 nM [18F]FPI (specific activity ~ 120 GBq/mol EOS) or 1 nM [3H]flunitrazepam in 50 mM TRIS-HCl, pH 7.4, and exposed to 18F- and 3H- sensitive film plates for 60 min and 14 d, respectively. Nonspecific binding of [18F]FPI (B, D) and [3H]flunitrazepam (F) was determined by co-incubation with 10 M zolpidem. Standards on the lower left hand side indicate high and low radioactivity concentration. diographs in Fig. 3 show a homogenous distribution of 18F subunit mRNA in these structures in the rat brain [29,30]. radioactivity at each time point. This correlation is furthermore indicated by the high density of [18F]FPI binding sites in the ventral pallidum, which is 2.5. Ex Vivo Brain, Plasma, and Urine Radiometabolites characterised by intense 1 subunit mRNA and protein ex- The ex vivo components of 18F radioactivity were ana- pression [31-33]. The hippocampal structures, in particular lysed by radio-HPLC in brain and plasma samples at 15 and the dentate gyrus, are stronger labelled by the benzodi- 30 min (Fig. 4A and 4B, and 4C and 4D, respectively), and azepine [3H]flunitrazepam, most probably due to the high urine samples at 30 min p.i. of 50-200 MBq [18F]FPI (Fig. expression of 2 subunit mRNA in this region [3,29,32]. By 4E). The percentages of the separated radioactive com- contrast, the more intense cerebellar labelling by [18F]FPI pounds were calculated from the values of the -counting of could reflect the almost exclusive expression of 1 subunit the fractions collected from radio-HPLC. mRNA in that structure of the adult rat brain [29]. Thus, the 18 autoradiographic data of our study support the recently pro- [F]FPI is metabolised fast. Brain and plasma contained posed 1 specificity of FPI, proven indirectly in competition 66% and 56% of parent compound at 15 min p.i., and 68% assays with native 1- and 2/3-containing receptors [22]. and 27% at 30 min p.i. In the urine, the major fraction of total 18F was labelled metabolites and only 1% of parent Encouraged by the favourable in vitro data, the potential 18 compound was detectable at 30 min p.i. of [ F]FPI for the mapping of GABAA receptors was evalu- ated in vivo. The radioactivity level in the brain of NMRI 3. DISCUSSION mice after intravenous injection of [18F]FPI was the highest In the current study on the development of a new 18F- at 5 min p.i. and decreased continuously from 1.04 to 0.09 labelled indiplon derivative for in vivo mapping of GABAA %ID/g between 5 and 120 min p.i. A comparable peak in receptors, the equilibrium binding constant of the potential brain uptake at 5 min p.i. was described in a recently pub- PET ligand [18F]FPI in cerebellar membranes of adult rats lished biodistribution study of 11C-labelled zolpidem in rat proved the recently determined high in vitro affinity of FPI [34]. The low brain-to-plasma ratio of 18F, i.e. <0.4 at all 18 (Ki = 2.36 nM; [22]). times after injection of the indiplon analogue [ F]FPI, con- trasts the high brain uptake of indiplon with a brain-to- The [3H]flunitrazepam binding in pig brain slices in the plasma ratio peak of 1.7 at 30 min post oral application of 4 current study corresponds to the labelling shown recently in mg/kg in mouse [19]. Furthermore, in the current study no rat (Fig. 3A in [27]). Therefore, pig is assumed to be a valu- specific binding in different brain structures could be dem- 18 18 able model for the evaluation of cerebral GABAA receptor onstrated after [ F]FPI injection because F radioactivity is properties initially in vitro as well as in preclinical PET stud- homogenously distributed in ex vivo brain-autoradiographs. ies. In vitro, the labelling pattern of [18F]FPI is comparable The radioactivity uptake is high in the liver and increases in rat and pig brain slices and resembles the distribution of constantly in the femur. Hence, a strong biotransformation of and Ro15-4513 in rat [3,28]. Thus, [18F]FPI bind- [18F]FPI is indicated with [18F]F- as one important bone- 18 - ing is related to the GABAA receptor benzodiazepine binding trapped radiometabolite. Since [ F]F is not blood-brain bar- site, and the strong labelling of the olfactory bulb and the rier permeable it accounts for brain-to-plasma ratios of less inferior colliculi corresponds to the high expression of 1 than unity. In the brain tissue obtained at 15 and 30 min after GABAA Receptor Specific Pyrazolopyrimidines as Potential Imaging Agents Current Radiopharmaceuticals, 2009, Vol. 2, No. 1 27

Table 1. 18F Organ Distribution in NMRI Mice at 5, 15, 30, 60, and 120 min Post Intravenous Injection of [18F]FPI (200-300 kBq; Specific Activity ~ 250 GBq/mol)

Time Post Injection

Organ 5 min 15 min 30 min 60 min 120 min

Blood 2.81 ± 0.63 2.07 ± 0.70 1.26 ± 0.53 0.67 ± 0.17 0.25 ± 0.09

Plasma 3.65 ± 0.44 3.08 ± 0.75 1.63 ± 0.65 0.77 ± 0.19 0.26 ± 0.10

Brain 1.04 ± 0.14 0.51 ± 0.14 0.27 ± 0.08 0.14 ± 0.07 0.09 ± 0.03

Cerebellum 1.01 ± 0.06 0.56 ± 0.11 0.29 ± 0.09 0.12 ± 0.01 0.09 ± 0.02

Heart 3.60 ± 0.55 2.08 ± 0.47 0.99 ± 0.26 0.45 ± 0.14 0.11 ± 0.06

Lung 6.98 ± 3.44 3.45 ± 1.93 1.38 ± 0.32 0.77 ± 0.41 0.23 ± 0.05

Stomach 1.76 ± 0.93 1.71 ± 0.82 1.17 ± 0.55 0.96 ± 0.61 0.66 ± 0.60

Intestine 5.23 ± 1.15 11.5 ± 2.79 13.7 ± 3.40 18.6 ± 3.04 21.2 ± 4.88

Liver 23.3 ± 5.34 22.3 ± 3.30 11.8 ± 1.82 5.49 ± 1.77 2.84 ± 1.23

Kidney 9.21 ± 0.99 6.32 ± 0.88 3.39 ± 0.87 1.46 ± 0.36 0.38 ± 0.12

Spleen 2.83 ± 0.57 1.42 ± 0.20 0.65 ± 0.17 0.42 ± 0.31 0.14 ± 0.15

Thymus 2.42 ± 0.68 1.55 ± 0.44 0.68 ± 0.20 0.33 ± 0.17 0.10 ± 0.05

Pancreas 4.09 ± 0.55 2.25 ± 0.38 0.89 ± 0.18 0.70 ± 0.48 0.14 ± 0.04

Adrenal glands 6.77 ± 1.80 2.87 ± 1.08 1.13 ± 0.25 0.59 ± 0.16 0.19 ± 0.05

Uterus/Ovary 3.52 ± 1.71 1.68 ± 0.68 0.66 ± 0.25 0.38 ± 0.25 0.10 ± 0.08

Muscle 1.53 ± 0.67 0.94 ± 0.16 0.49 ± 0.14 0.21 ± 0.07 0.07 ± 0.04

Femur 1.14 ± 0.62 1.85 ± 0.66 2.83 ± 1.58 3.95 ± 1.32 4.91 ± 1.42

Results are presented as means ± SD values of the percentage of the injected dose per gram of tissue (n=5-7). injection, a high amount of radiometabolites of [18F]FPI, reactions are the two major biodegradation pathways affect- constituting for >30% of the total 18F radioactivity, has been ing the stability of indiplon by cytochrome P450 3A4/5 and detected. Accordingly, the 18F uptake in the brain after intra- microsomal esterases, respectively [24,25]. The latter reac- venous application of [18F]FPI does reflect not solely the tion was supposed to account for the in vivo instability of the specific uptake of [18F]FPI but of blood-brain barrier (BBB) recently synthesised [18F]fluoro-indiplon [23]. In the current permeable radiometabolites as well. The constant uptake of study, the blocking of the N-dealkylation was attempted by radioactivity in the intestine indicates the preference of hepa- the substitution of the N-methyl with the spatially larger N- tobiliary over renal excretion of [18F]FPI or radiometabolites propyl group. The rationale was to interfere with the cyto- or both, comparable to the significant first-pass hepatic me- chrome P450-catalyzed hydroxylation of sp3-C-atoms bear- tabolism reported for , another hypnosedative of the ing an N-heteroatom, which results in N-dealkylation and - pyrazolopyrimidine class [35,36]. The 18F detected in the deamination [37], by hindering the docking of [18F]FPI to the urine is related mainly to radiometabolites of [18F]FPI while catalytic site of cytochrome P450 isoenzyme(s). However, the bile excreted 18F labelled compounds were not investi- the actually observed biodegradation of [18F]FPI in mice gated by radio-HPLC. indicates substantial N-dealkylation. Although the structure 18 18 of [ F]FPI metabolites could not be elucidated in the ra- Although the metabolic stability of [ F]FPI is higher diotracer experiments, data on in vitro metabolism of in- than of [18F]fluoro-indiplon (~2% parent compound in the diplon [24,25] and 18F-FECNT [38] make the formation of brain at 5 min p.i.; [23]), which indicates the general suitabil- BBB permeable [18F]-3-fluoropropionaldehyde (with alde- ity of the hitherto structural modifications, the enzymatic hyde, , and carboxylate metabolically interchangeable susceptibility of the N-alkyl-acetamide moiety of indiplon in vivo; [38]), N-despropyl-indiplon, and [18F]fluoropropyl- derivatives was not fundamentally improved. It has been N-desacetyl-indiplon likely. Thus, the primary radiometabo- shown that enzymatic N-demethylation and N-deacteylation lites resulting from either the esterase-catalysed deacetyla- 28 Current Radiopharmaceuticals, 2009, Vol. 2, No. 1 Deuther-Conrad et al.

Germany, and [N-methyl-3H]flunitrazepam (specific activity: 3,600 GBq/mmol) was purchased from GE Healthcare. All chromatographic measurements were performed at room temperature (RT).

TLC was performed on silica gel 60F254 plates with ethyl acetate as eluent, and spots were visualised using a UV lamp (wavelength: 366 nm). Retention values of FPI and the tosy- late precursor (see 2.3) were 0.45 and 0.40, respectively. Radio-TLCs were recorded using a Fuji BAS 2000 scanner and evaluated with a standard image analysis software Fig. (3). Ex vivo digital autoradiographs of sagittal mice brain sec- (AIDA 2.31; Raytest). tions after intravenous application of [18F]FPI. Brain sections (12 m) were obtained at 20 (A), 60 (B), and 120 min (C), respectively, Analytical radio-HPLC was performed on a Merck-Hita- post intravenous injection of 16 MBq [18F]FPI (specific activity ~ chi La Chrome device equipped with a binary pump, a UV 250 GBq/mol EOS) on a cryostat microtome and exposed to 18F- detector (wavelength: 228 and 254 or 344 nm), and a radio- sensitive film plates for 19 h at ambient temperature. activity detector (GINA; Raytest). HPLC column: Multisorb RP 18-5 (250 x 4 mm; Chromatographie Servise); elution -1 tion of [18F]fluoro-indiplon, [18F]-2-fluoroacetaldehyde [23], (isocratic; 1 ml min ): 37% MeCN/20 mM ammonium ace- or the cytochrome P450-catalysed dealkylation of [18F]FPI, tate; recording: D-7000 HPLC System Manager 3.1. [18F]-3-fluoropropionaldehyde, are comparable. These re- 4.3. Radiosynthesis of [18F]FPI sults indicate that (i) attempts to introduce 18F via N-alkyl- acetamide groups should take into consideration potential Radiosynthesis and radiochemical characterization of N- 18 pharmacokinetic restrictions and (ii) due to their BBB per- (3-[ F]fluoro-propyl)N-{3-[3-(thiophene-2-carbonyl)-pyra- 18 meability, the pharmacodynamics of particular metabolites zolo[1,5-a]pyrimidin-7-yl)-phenyl}-acetamide, [ F]FPI, are of indiplon should be investigated with the utmost accuracy. described in detail in Fischer et al. [39]. Briefly, the tosylate precursor N-{3-[3-(thiophene-2-carbonyl)-pyrazolo[1,5-a] In summary, [18F]FPI undergoes a fast and extensive he- pyrimidin-7-yl)-phenyl}-N-[4-(-4-sulfonyl)-propyl]- 18 patic metabolism in NMRI mice with no detectable specific acetamide was reacted with [ F]/K2CO3/kryptofix 2.2.2 in accumulation of [18F]FPI in the brain and radiometabolites DMF (140°C, 10 min; labelling efficiencies 55-65%). [18F] which are able to penetrate the blood-brain barrier at a sig- FPI was separated with a C18 Sep-Pack cartridge, purified nificant level. Despite being promising in vitro, the current in by isocratic (37% MeCN/20 mM ammonium acetate; 0.75- vivo data obtained in NMRI mice exclude [18F]FPI from fur- 1.5 ml min-1) semi-preparative radio-HPLC (Kromasil 100-5 ther PET tracer development. C18, 250 x 10 mm; Eka Chemicals), and analysed by radio- 18 4. EXPERIMENTAL TLC and -HPLC as described in 2.2. The [ F]FPI-contai- ning fractions were combined, highly diluted with water, 4.1. Animals adsorbed on an Sep-Pak Plus C18 cartridge and almost quan- titatively desorbed with . Before injection into SPRD rats and NMRI mice were purchased from the animals, diethyl ether was removed under reduced pressure University of Leipzig, Germany (Medizinisch-Experimentel- and [18F]FPI was solved in sterile saline at ~1 MBq/ml. By les Zentrum). All experiments involving animals were per- this procedure, less than 8% of the radiotracer remained ad- formed in accordance with the International Guiding Princi- sorbed on syringe material. ples for Biomedical Research Involving Animals by the WHO and the national regulations for animal research, and 4.4. In Vitro Evaluation of [18F]FPI had the approval of the Saxon Ministry of Science and the Fine Arts. Animals used in ex vivo, in vivo, or in vitro ex- 4.4.1. Preparation of Rat Cerebellum periments were anaesthetised with CO2/O2 (70%:30%). In- Animals were decapitated, the brains rapidly dissected travenous injections were performed into the tail vein. In out, and the cerebella homogenised in ice-cold TRIS-HCl vivo and ex vivo studies were performed on female (n=31) (50 mM, pH 7.4) using a glass-PTFE Potter-Elvehjem tissue NMRI mice weighing 28 ± 4 g (8 to 10 weeks old). The mice homogenizer (type Potter S; B. Braun Biotech International) were housed under standard conditions (24°C, humidity 55 ± for 1 min at 1,200 rpm. The homogenate was sedimented at 5%, controlled 12 h light-dark cycle) with free access to 20,000 g for 15 min at 4°C. The pellet was washed two times standard food and drinking water. For in vitro studies, mem- by resuspension, homogenisation, and centrifugation as de- brane preparations and cryostat slices of the rat brains were scribed above, and the final pellet was resuspended at 100 obtained from 10 to 12 weeks old female SPRD rats. Isolated mg wet weight/mL ice-cold TRIS-HCl (50 mM, pH 7.4) and brain hemispheres of juvenile female pigs (mixed German stored at -25°C. domestic breed, 36-40 kg) were obtained from the Institute of Molecular Cell Biology, University of Jena, Germany. 4.4.2. Determination of Equilibrium Dissociation Con- stant of [18F]FPI 4.2. Chemicals and Analytical Methods The equilibrium dissociation constant KD of FPI was de- All chemical reagents used were of highest commercially termined by homologous competition studies with [18F]FPI available quality. Zolpidem was obtained from TOCRIS, and FPI according to a recently published protocol [27]. GABAA Receptor Specific Pyrazolopyrimidines as Potential Imaging Agents Current Radiopharmaceuticals, 2009, Vol. 2, No. 1 29

Fig. (4). Representative chromatograms of [18F]FPI and radiometabolites in the brain and plasma at 15 (A and C, resp.) and 30 min (B and D, resp.), and urine at 30 min (E) post intravenous injection of ~ 50 - 200 MBq [18F]FPI (specific activity ~ 250 GBq/mol EOS) in mice. The labelled radioactive peak (*) corresponds to [18F]FPI (retention time: 26.4 min). The recovery of tissue radioactivity after the extraction with acetonitrile was  85% at 15 and 30 min. The curves represent one of four individual experiments.

Briefly, the cerebellar membranes were thawed, diluted at 1 totally applied radioactivity). The binding parameter IC50 mg wet weight/15 ml incubation buffer (50 mM TRIS-HCl, was estimated by non-linear curve fitting, and KD was calcu- pH 7.4 at 21°C), and centrifuged. Washing was repeated lated using the equation KD = IC50 - [radioligand] [40]. twice and the diluted suspension finally homogenised by a 4.4.3. In Vitro Autoradiography 23-gauge needle. Membrane aliquots (~100 g protein/vial) were incubated with [18F]FPI (~0.02 nM working concentra- To compare the distribution of specific binding sites of 18 tion) and 8 to 10 concentrations of FPI (0.01 nM to 0.3 M) various GABAA receptor radioligands, the binding of [ F] in incubation buffer at room temperature (RT) for 60 min. At FPI and [3H]flunitrazepam was studied in rats and pig brain the end of the incubation, the samples were filtered rapidly slices according to recently published protocols [23,27]. In through polyethyleneimine presoaked Whatman GF/B glass brief, anaesthetised rats were decapitated, the brains rapidly fibre filters using a Brandel cell harvester (Biomedical Re- removed on ice, and the separated hemispheres frozen in search and Development Labs) and washed three times (4 isopentane and stored at -25°C until cut into sagittal sections. mL each) with ice-cold incubation buffer. Filter bound ra- Cryostat sections (12 m and 20 m for rat and pig brains, dioactivity was counted decay corrected in a -counter. Two respectively) were mounted on microscope glass slides, air independent experiments were performed, each in triplicate. dried, and stored at -25°C. For the autoradiography, slices Nonspecific binding of [18F]FPI (~5% of total binding) was were thawed at RT, dried, preincubated with incubation determined by 10 M zolpidem. Specific binding was calcu- buffer at RT for 10 min followed by incubation with 0.8 nM lated by subtracting nonspecific from total binding (~10 % of [18F]FPI (specific activity ~ 120 GBq/mol EOS) or 1 nM 30 Current Radiopharmaceuticals, 2009, Vol. 2, No. 1 Deuther-Conrad et al.

[3H]flunitrazepam at RT for 60 min. Nonspecific binding 228, 254 or 344 nm), and a radioactivity detector: NaI(TI)- was determined by 10 M zolpidem. The incubation was counter with supplement FAP 100K/HV (bte, Germany). terminated by washing the slices twice (2 min each) in ice- HPLC columns: Multisorb RP 8C-5 (20 x 4 mm) and cold incubation buffer followed by a short rinse with ice-cold RP18C-7 (250 x 4 mm) (Chromatographie Servise); solvent ultra pure water. Sections were air dried, placed in X-ray A: 5% MeCN/20 mM ammonium acetate, solvent B: 80% cassettes, apposed to 18F- or 3H-sensitive imaging plates MeCN/20 mM ammonium acetate; elution (gradient; 1 ml (Fuji Film, Japan), and exposed at RT for 60 min or four min-1): 0-10 min 100% A, 10-40 min steady change to 100% weeks, respectively. B; recording and handling: HP ChemStation data system WIN NT; retention time of [18F]FPI: 26.4 min. To determine 4.5. In Vivo and Ex Vivo Evaluation of [18F]FPI the percentage of [18F]FBI and radiometabolites, the frac- 4.5.1. Biodistribution Studies in Mice tions eluated from the radio-HPLC (0.25 to 0.5 mL) were 18 collected (Foxy Jr.; Knauer, Germany) and the radioactivity 200 l saline containing 200 to 300 kBq [ F]FPI (~ 1 of each fraction was measured in a calibrated -counter pmol/animal and 20 ng/kg at a specific activity of 250 (Wallac WIZARD 3”; Perkin Elmer). GBq/mol at EOS) was injected into the tail vein of awake mice. No pharmacological side effects were observed at the ABBREVIATIONS injected tracer concentrations. Under anaesthesia (see 2.1 Cb = Cerebellar cortex Animals), blood samples were taken from the retro-orbital plexus at 5 (n=6), 15 (n=7), 30 (n=5), 60 (n=7), and 120 Ci = Inferior colliculus (n=6) min post injection (p.i.), and the animals were sacri- Co = Cortex ficed by cervical dislocation. The brain and the organs of interest were rapidly removed, weighed, and radioactivity Cs = Superior colliculus was measured in a calibrated -counter (Wallac WIZARD 3”; Perkin Elmer). The 18F uptake was calculated individu- DG = Dentate gyrus ally as percentage injected dose per gram tissue (%ID/g), and Hip = Hippocampus the data are given as mean values ± standard deviation (S.D.). OB = Olfactory bulb 4.5.2. Ex Vivo Autoradiography Str = Striatum To investigate the in vivo distribution of radiotracer in the Th = Thalamus brain, sagittal brain sections of NMRI mice were qualita- VP = Ventral pallidum tively analysed by ex vivo digital autoradiography. Three NMRI mice received a tail-vein injection of ~16 MBq REFERENCES [18F]FPI solved in 200 L saline. The animals were anaes-  [1] Macdonald, R. L.; Olsen, R. W. GABAA receptor channels. Annu. thetised at 20, 60, and 120 min p.i., respectively, decapitated, Rev. 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Received: July 01, 2008 Revised: November 03, 2008 Accepted: November 07, 2008