Nuclear Medicine and Biology 42 (2015) 146–154

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Characterization of the binding properties of T-773 as a PET radioligand for phosphodiesterase 10A Akina Harada a, Kazunori Suzuki a, Shotaro Miura a, Tomoaki Hasui a, Naomi Kamiguchi b, Tsuyoshi Ishii c, Takahiko Taniguchi a, Takanobu Kuroita a, Akihiro Takano d, Vladimir Stepanov d, Christer Halldin d, Haruhide Kimura a,⁎ a

CNS Drug Discovery Unit, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Fujisawa, Japan Drug Metabolism and Pharmacokinetics Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Fujisawa, Japan c Biomolecular Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Fujisawa, Japan d Center for Psychiatric Research, Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden b

a r t i c l e

i n f o

Article history: Received 1 August 2014 Received in revised form 2 September 2014 Accepted 4 September 2014 Keywords: PDE10A Autoradiography PET T-773 C-11

a b s t r a c t Introduction: Phosphodiesterase 10A (PDE10A) is a dual-substrate PDE that hydrolyzes both cAMP and cGMP and is selectively expressed in striatal medium spiny neurons. Recent studies have suggested that PDE10A inhibition is a novel approach for the treatment of disorders such as schizophrenia and Huntington's disease. A positron emission tomography (PET) occupancy study can provide useful information for the development of PDE10A inhibitors. We discovered T-773 as a candidate PET radioligand for PDE10A and investigated its properties by in vitro autoradiography and a PET study in a monkey. Methods: Profiling of T-773 as a PET radioligand for PDE10A was conducted by in vitro enzyme inhibitory assay, in vitro autoradiography, and PET study in a monkey. Results: T-773 showed a high binding affinity and selectivity for human recombinant PDE10A2 in vitro; the IC50 value in an enzyme inhibitory assay was 0.77 nmol/L, and selectivity over other PDEs was more than 2500fold. In autoradiography studies using mouse, rat, monkey, or human brain sections, radiolabeled T-773 selectively accumulated in the striatum. This selective accumulation was not observed in the brain sections of Pde10a-KO mice. The binding of [3H]T-773 to PDE10A in rat brain sections was competitively inhibited by MP-10, a selective PDE10A inhibitor. In rat brain sections, [3H]T-773 bound to a single high affinity site of PDE10A with Kd values of 12.2 ± 2.2 and 4.7 ± 1.2 nmol/L in the caudate-putamen and nucleus accumbens, respectively. In a monkey PET study, [11C]T-773 showed good brain penetration and striatum-selective accumulation. Conclusion: These results suggest that [11C]T-773 is a potential PET radioligand for PDE10A. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Phosphodiesterase 10A (PDE10A) is a dual-substrate PDE that hydrolyzes both cAMP and cGMP and is selectively expressed in medium spiny neurons (MSNs) in the striatum of mammalian brains [1–3]. The striatal outputs mediated by MSNs are mainly divided into two pathways, with the dopamine D2 receptor expressing the indirect pathway and the D1 receptor expressing the direct pathway [4,5]. Activation of the indirect pathway by D2 antagonism is a principal mechanism of action of most antipsychotic drugs [6]; however, excessive blockade of D2 receptors in this pathway is known to cause unwanted side effects such as extrapyramidal symptoms (EPS) [7]. Activation of the direct pathway is expected to counteract excessive activation of the indirect pathway and reduce these side effects. In line with this idea, PDE10A inhibition ⁎ Corresponding author at: CNS Drug Discovery Unit, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan. Tel.: +81 466 321859; fax: +81 466 324422. E-mail address: [email protected] (H. Kimura). http://dx.doi.org/10.1016/j.nucmedbio.2014.09.005 0969-8051/© 2014 Elsevier Inc. All rights reserved.

has shown lower risks of EPS through the activation of both the direct and indirect pathways in pre-clinical studies [8–10]. In addition to EPS, some current antipsychotics lead to hyperprolactinemia due to their D2 antagonism in the pituitary gland [11]. PDE10A inhibitors can avoid hyperprolactinemia because of low PDE10A expression in the pituitary gland. Moreover, a PDE10A inhibitor is considered effective for Huntington's disease (HD) by improving cAMP signaling and protecting striatal MSNs against neurodegeneration [12–14]. Thus, PDE10A inhibitors are strongly desired as therapeutic drugs for those central nervous system (CNS) disorders. Biological markers (biomarkers) can provide beneficial information such as delivery of drugs to the intended targets, alteration of proposed pathophysiological mechanisms, and prediction of clinical outcomes [15]. Accordingly, clinical biomarkers are valuable for the confirmation of proof-of-concept during drug development. Recently, positron emission tomography (PET) imaging has been used as a clinical marker. PET is a highly sensitive non-invasive imaging modality, which can visualize the biological actions of small molecules in living animals including human beings. As a translational tool, PET provides useful information

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such as biodistribution of drugs, functional or morphological changes, and target occupancy by drugs [16,17]. Thus, clinical PET studies can accelerate drug development especially in CNS. PDE10A is a promising drug target for several disorders; thus, development of a PET radioligand for PDE10A is needed. Several candidate PET radioligands for PDE10A have been reported in a preclinical setting [18–28], and some fluorine-18 labeled radioligands have been assessed in humans [22,24,26,28]. The aim of this study was to develop a carbon11 labeled PDE10A PET radioligand potential for human examination. Here we report the profile of a novel PDE10A PET radioligand T-773 by evaluating its target specificity and binding properties using in vitro autoradiography and monkey PET imaging.

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trapping the cartridge was rinsed with 8 mL of sterile water, the product was then eluted with 1 mL of 99.4% ethanol and collected into sterile vial pre-filled with 10–11 mL of phosphate buffered saline. As the final step the formulation was passed through a 0.22 μm sterile filter (Millipore) for sterilization purposes. The QC analysis was performed using HPLC system equipped with Ascentis RP-Amide column (150 × 4.6 mm, 3 μm) (Supelco) and acetonitrile/aq. phosphoric acid 0.05 mol/L 460:540 v/v as mobile phase at a flow of 1 mL/min equipped with UV absorbance and radio detectors. Identification of the product was achieved by comparing retention time of the radioactive peak with retention time of a known cold standard of T-773. 2.3. In vitro PDE inhibition assay

2. Materials and methods 2.1. Animals Seven-week-old male Sprague–Dawley rats were purchased from Charles River Laboratories Japan, Inc (Kanagawa, Japan). After acclimation for 1 week, the 8-week-old rats were used for experiments. Pde10a wild-type (WT) and knockout (KO) mice (129/SvEv-C57BL/6) were purchased from Taconic Farms, Inc. (Hudson, NY), and were used for experiments after at least 1 week of acclimation. The animals were housed in a light controlled room (12-h light/dark cycle with lights on at 07:00 h). Food and water were provided ad libitum. The care and use of these animals and the experimental protocols used in this research were approved by the Experimental Animal Care and Use Committee of Takeda Pharmaceutical Company, Limited. A rhesus monkey used for PET study weighing 5000 g was supplied by the Swedish Institute of Infectious Disease Control (Solna, Sweden). The experimental protocol used in the PET study was approved by the Animal Ethics Committee at Karolinska Institutet (Stockholm, Sweden) and was consistent with Good Laboratory Practice, applicable regulatory requirements, and the TAKEDA policy on Bioethics. 2.2. Radioligands and chemicals T-773 [1-[2-fluoro-4-(tetrahydro-2H-pyran-4-yl)phenyl]-5-methoxy3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one] and MP-10 succinate [2-[4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)-phenoxymethy]quinoline succinate] were synthesized by Medicinal Chemistry Research Laboratories, Takeda Pharmaceutical Company Limited (Kanagawa, Japan). MP-10 has been reported as a selective PDE10A inhibitor developed by Pfizer Inc. (New York City, NY) [29]. [3H]T-773 (37.0 MBq/mL in ethanol) was radiosynthesized by Quotient Bioresearch (Radiochemicals) Limited (Cambridgeshire, UK). The specific radioactivity and radiochemical purity of the radioligand were 555 GBq/mmol and 99.9%, respectively. Other reagents and materials were purchased from Sigma-Aldrich (St. Louis, MO) unless specified otherwise. [ 11C]T-773 was synthesized from the desmethylated precursor, desmethyl-T-773, via Carbon-11 methylation using 11C-methyl triflate ([ 11C]MeOTf) produced from 11C-methane as described elsewhere [30,31]. In short, [ 11C]methyl triflate was trapped at room temperature into a 5 mL glass vial with 1-[4-(3,3-difluoroazetidin-1-yl)-2fluorophenyl]-5-hydroxy-3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-4 (1H)-one (0.2–0.4 mg), acetone (400 μL) and sodium hydroxide (0.5 mol/L, 3 μL), and lowed to react for 60 seconds at room temperature. The product, [ 11C]T-773, was purified by injecting reaction mixture onto Ascentis RP-Amide reverse-phase column (250 × 10 mm, 5 μm) (Supelco), and eluting the column with mobile phase consisting of acetonitrile/aq. triethylamine 0.1% 440:560 v/v at a flow of 6 mL/min. The carbon-11 labeled compound-containing fraction from HPLC was collected into a vial containing 50 mL of sterile water and 70–100 mg of sodium ascorbate. The resulting solution was then pushed through an Oasis HLB 1 cc cartridge (Waters), previously conditioned by 5 mL of 99.4% ethanol and 5 mL of sterile water (in that order). After product

COS-7 or Sf9 cells were transfected with full-length human PDE10A2 or His-tagged full-length human PDE2A3 cDNA respectively, and cell lysates containing human PDE enzymes were recovered. PDE2A3 were purified by His-tag affinity chromatography using Ni–NTA Superflow Cartridges (Qiagen, Hilden, Germany) and Sephacryl S300 (GE Healthcare UK Ltd., Buckinghamshire, UK) and then stored at − 70 °C until use. Human PDE1A, PDE3A, PDE4D2, PDE5A1, PDE7B, PDE8A1, PDE9A2, and PDE11A4 were purchased from BPS Bioscience (San Diego, CA). PDE6AB was purchased from Scottish Biomedical (Glasgow, UK). Inhibitory activities of T-773 against recombinant PDEs were evaluated by the scintillation proximity assay (GE Healthcare UK Ltd.). Each PDE enzyme was incubated with various concentrations of T-773 in 30 μL of assay buffer (for PDE1A: 50 mmol/L Tris-HCl, 8.3 mmol/L MgCl2, 0.2 mmol/L CaCl2, 30 nmol/L calmodulin, 0.1% BSA, pH 7.5; for other PDEs: 50 mmol/L HEPES-NaOH, 8.3 mmol/L MgCl2, 1.7 mmol/L EGTA, 0.1% BSA, pH 7.4) containing 1.3% DMSO in 96-well half-area plates (Corning Incorporated, Corning, NY) for 30 min at room temperature. After addition of 10 μL of [3H]cAMP (for PDE3A, PDE4D2, PDE7B, and PDE8A1) or [ 3H]cGMP (for PDE1A, PDE2A3, PDE5A1, PDE6AB, PDE9A2, PDE10A2, and PDE11A4), the mixtures were further incubated for 60 min at room temperature. The total assay volume was 40 μL, and the concentration of DMSO was 1% in these mixtures. Tritium-labeled cyclic nucleotides were purchased from GE Healthcare UK Ltd. Then, 20 μL of 20 mg/mL yttrium SPA beads containing zinc sulphate were added to terminate the reactions. After being settled for more than 120 min at room temperature, radioactivity was measured using a scintillation counter (PerkinElmer Inc., Waltham, MA), and inhibition rates and half-maximal inhibitory concentrations (IC50 values) were calculated. The inhibition rate was calculated on the basis of 0% control wells without compound and 100% control wells with 10 μmol/L of papaverine for PDE10A2 or without enzyme for other PDEs. 2.4. Real-time quantitative polymerase chain reaction (RT-PCR) analysis Rat and monkey total RNA were extracted from brain tissue using Isogen (Nippon Gene Co., Ltd., Tokyo, Japan) and the RNeasy kit (Qiagen) following the manufacturer's instruction. Human total RNA samples were purchased from Clontech Laboratories, Inc. (Madison, WI). RT-PCR was performed using an ABI PRISM 7900HT sequence detection system (Life Technologies, Carlsbad, CA) with qPCR Mastermix Plus (Eurogentec, Seraing, Belgium). RNAs were normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA transcripts according to the manufacturer's instruction. Primers used for rat PDE10A analysis were as follows: forward primer, 5′-CGTGTT CAGACGTCGGCTATT-3′; reverse primer, 5′-TCTGGCCTGGTAGCAAAT GG-3′; TaqMan probe (MGB probe), 5′-CATGGCTCCGCCTGACCCCC-3′. Primers used for rat DRD2 analysis were as follows: forward primer, 5′-GCAGCAGTCGAGCTTTCAGA-3′; reverse primer, 5′-CGCCTGTTCA CTGGGAAACT-3′; TaqMan probe (MGB probe), 5′-CCTGAAGACAC CACTCAAGGGCAACTGT-3′. The primer set used for rat GAPDH analysis was TaqMan Rodent GAPDH Control Reagents (Life Technologies). Primers used for human PDE10A analysis were as follows: forward

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primer, 5′-GATGTACCAGACCGGATCACTAAA-3′; reverse primer, 5′-AA CGGGCCACAGTTTTGTCA-3′; TaqMan probe (MGB probe), 5′-CACAT AGAGACCGTGTAATTGGTTTGATGATGAC-3′. Primers used for human DRD2 analysis were as follows: forward primer, 5′-GACCAGAACGA GTGCATCATTG-3′; reverse primer, 5′-GGGCACGTAGAAGGAGACGAT-3′; TaqMan probe (MGB probe), 5′-CAACCCGGCCTTCGTGGTCTACTCC-3′. Primers used for human GAPDH analysis were as follows: forward primer, 5′-CATCCATGACAACTTTGGTATCGT-3′; reverse primer, 5′-CAGTCTTCT GGGTGGCAGTGA-3′; TaqMan probe (VIC probe), 5′-AAGGACTCATGA CCACAGTCCATGC-3′. 2.5. Western blotting analysis Male Sprague–Dawley rats and male Pde10a WT and KO mice were sacrificed by decapitation. Brain tissues were dissected and sonicated in extraction buffer (Life Technologies) containing protease inhibitor cocktail (Sigma-Aldrich, St.Louis, MO) and 0.5 mmol/L 4-amidinophenylmethanesulfonyl fluoride hydrochloride (SigmaAldrich). After centrifugation, protein concentrations in the supernatant were determined using the BCA protein assay kit (Pierce Chemical Co., Rockford, IL). Each sample (10 μg protein) was subjected to electrophoresis with 4%–20% polyacrylamide gradient gels (Cosmo Bio Co., Ltd., Tokyo, Japan), followed by western blotting with nitrocellulose membranes (Bio-Rad Laboratories Inc., Hercules, CA). The blots were incubated with a primary antibody to PDE10A (FabGennix Inc., Frisco, TX) or beta-actin (Sigma-Aldrich). Individual protein bands were visualized with horseradish peroxidaseconjugated secondary antibodies (for mice, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD; for rats, Cell Signaling Technology Inc., Danvers, MA) followed by treatment with ECL Western blotting detection reagents (GE Healthcare UK Ltd.) and exposure to Hyperfilm ECL (GE Healthcare UK Ltd.). 2.6. In vitro autoradiography (ARG) using rodent brain sections 2.6.1. Preparation of tissue slices Male Sprague–Dawley rats and male Pde10a WT and KO mice were sacrificed by decapitation. The brains were rapidly removed and were slowly frozen by immersion into an isopentane–dry ice bath. The frozen brains were stored in a deep freezer. Twenty-micrometer-thick sagittal or coronal sections were cut on a cryostat (Leica Microsystems, Wetzlar, Germany) and were thaw-mounted onto glass slides. The rat coronal sections were prepared from between the bregma 1.7 mm and 0.2 mm, and the rat sagittal sections were prepared from within 1.9–3.4 mm lateral to the midline (coordinates taken from the Paxinos and Watson rat brain atlas [32]). The mouse sagittal sections were prepared from within 1.2–1.4 mm lateral to the midline (coordinates taken from the Franklin and Paxinos mouse brain atlas [33]). One hundredmicrometer-thick monkey brain and human brain hemisphere slices were prepared. Postmortem human brain tissue, provided by the brain bank of the Psychiatry Section, Department of Clinical Neuroscience, Karolinska Institutet, was obtained from deceased subjects without CNS diseases. 2.6.2. In vitro ARG in rats and Pde10a KO mouse brain sections Sagittal sections prepared from a rat brain or Pde10a WT and KO mouse brains (3 mice in each genotype) on glass slides were warmed to room temperature. The sections were pre-incubated twice for 5 min at room temperature in pre-incubation buffer (50 mmol/L TrisHCl pH 7.5, 1.7 mmol/L EDTA, 6 mmol/L MgCl2, 120 mmol/L NaCl, and 0.1% BSA). The sections were then incubated for 60 min at room temperature in binding buffer (pre-incubation buffer containing 0.03% Triton X-100) with 20 nmol/L [ 3H]T-773. Simultaneously, a blocking study was conducted with adjacent sections by the addition of an excess amount of PDE10A inhibitors to the radioligand containing buffer (1 μmol/L of MP-10 or 10 μmol/L of T-773 for rat sagittal sections and

1 μmol/L MP-10 for mice sagittal sections). The sections were rinsed twice for 1 min at 4 °C in pre-incubation buffer and then rapidly rinsed in ice-cold distilled water. The sections were dried under a stream of cool air and were exposed to imaging plates BAS IP TR 2040E (GE Healthcare UK Ltd.) for 7 days. The imaging plates were analyzed using an image analyzer FLA-7000 (Fujifilm, Tokyo, Japan) and image analyzing software ImageGauge 4.0 (Fujifilm). In an autoradiographic study of Pde10a WT and KO mice, regions of interest (ROIs) were placed at the caudate putamen (CPu) in each section. [ 3H]T-773 radioactivities in the ROIs were analyzed and represented as photostimulated luminescence (PSL) values. The background was subtracted from the PSL values of each ROI, and then the PSL values in the striatum were averaged for each group. The PSL values in the absence and presence of an excess amount of MP-10 in WT mice brain sections were represented as total binding and non-specific binding (NSB), respectively. 2.7. Saturation binding experiments in rat brain sections Coronal brain sections prepared from rat brains (4 rats) on glass slides were warmed to room temperature. The sections were preincubated twice for 5 min at room temperature in pre-incubation buffer. The sections were then treated with 0.3125, 0.625, 1.25, 2.5, 5, 10, 20, and 40 nmol/L [ 3H]T-773 in binding buffer for 60 min at room temperature. NSB was determined in the presence of 1 μmol/L MP-10. The sections were rinsed, dried using the same procedure described above, and then exposed to an imaging plate for 7 days. Autoradiograms were read using FLA-7000, and the images were analyzed using ImageGauge 4.0. ROIs were placed at CPu in each section, and [ 3H]T-773 radioactivities in the ROIs were represented as PSL values. The background PSL value was subtracted from the PSL values of each ROI, and then the PSL values of the left and right CPu were averaged in each section. The PSL values in the presence and absence of an excess amount of MP-10 were represented as total binding and NSB, respectively. The saturation binding curve was fit by non-linear regression using GraphPad Prism 5.01 (GraphPad Software, Inc., La Jolla, CA), and the dissociation constant (Kd) was calculated using the same software. 2.8. In vitro ARG in monkey and human brain sections using [11C]T-773 The horizontal sections obtained from the monkey or human brain were pre-incubated for 20 min at room temperature in buffer (50 mmol/L Tris-HCl pH 7.4 containing 50 mmol/L sodium chloride) and then supplied with 30–50 MBq of [ 11C]T-773. After incubation, the sections were washed twice for 5 min in the same cold buffer, dried on warm plates, and placed on phosphoimaging plates for 45 min. The plates were digitized using an image analyzer BAS-5000 (Fujifilm). 2.9. Baseline PET imaging in monkeys using [ 11C]T-773 Anesthesia in a rhesus monkey (body weight 5000 g) was induced by intramuscular injection of ketamine hydrochloride and maintained by inhalation of a mixture of O 2 , air, and sevoflurane (2%–8%) in the PET unit. The monkey was intubated, and ventilation was controlled using a respirator. The head of the monkey was immobilized using a fixation device [34]. The PET experiment was performed using a Siemens high-resolution research tomograph PET scanner (Siemens, Munich, Germany). The PET scan was conducted for 123 min after injection of 162 MBq (specific radioactivity: 1144 GBq/μmol and injected mass: 0.06 μg) of [ 11C]T-773. PET images were summed from 9 to 123 min. Previously acquired T1weighted magnetic resonance images were used for anatomical identification in corresponding PET images. The mean ratio between the striatum and the cerebellum was calculated from 87 min to 123 min. Venous blood samples were taken at 30 min and 90 min for the metabolite analysis with HPLC system.

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Fig. 1. Phosphodiesterase 10A (PDE10A) expression in rat and human brains. PDE10A mRNA expression levels were evaluated by quantitative real-time quantitative polymerase chain reaction (RT-PCR) in rat (A) and human brain tissues (B). Western blotting analysis showed that a PDE10A immunoreactive band was selectively observed in the striatum of the rat brain (C). Beta-actin (β-actin) was used as a loading control. The mRNA expression level of PDE10A was compared with that of dopamine D2 receptor (DRD2) in the striatal complex of rat (D) and human brain tissues (E). Each mRNA expression level was corrected by the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression level. Fcx, frontal cortex; Str, striatum; NAc, nucleus accumbens; Thal, thalamus; Bs, brainstem; Hipp, hippocampus; Cb, cerebellum; Cn, caudate nucleus; Pu, putamen; Ob, olfactory bulb.

3. Results 3.1. Expression patterns and levels of PDE10A in mammalian brains The expression pattern and level of target molecules are key information in predicting the feasibility of PET imaging. Restricted expression is advantageous for quantitative PET studies because a region with low expression levels can be used as a reference site [35]. In addition, the expression level is important in predicting the sensitivity of detection in PET imaging. Quantitative RT-PCR analysis revealed that PDE10A mRNA was highly expressed in the striatum (Str), including the caudate nucleus (Cn) and putamen (Pu), and in the nucleus accumbens (NAc) of both rat and human brains (Fig. 1A,B). The expression levels in the frontal cortex (Fcx), thalamus (Thal), brainstem (Bs), hippocampus (Hipp), and cerebellum (Cb) were more than 40-fold lower than the expression level in Str. Western blotting analysis showed that the PDE10A protein was also highly expressed in the Str of rat brains (Fig. 1C). In this study, obvious PDE10A immunoreactive bands were detected only in Str and whole brain tissue samples, and the expression

ratio (Str/whole brain) was 15.8. Accordingly, PDE10A protein expression levels in the other regions were at least 15-fold lower than the expression level in Str. These results were consistent with those of the previous reports of PDE10A expression [1–3]. This restricted distribution of PDE10A in the brain is favorable for clinical PET studies. Next, we compared the mRNA expression levels of PDE10A with those of dopamine D2 receptor. In the Str and NAc of both rat and human brains, the mRNA expression levels of PDE10A were comparable to those of D2 receptor (Fig. 1D,E). Thus, PDE10A would be sufficiently expressed to be visualized using the PET technique. 3.2. In vitro PDE10A inhibitory activity of T-773 High binding affinity and high selectivity are required in a PET radioligand to sensitively and selectively visualize the target molecule in vivo by PET imaging [16]. We discovered T-773 to be a small molecule with PDE10A inhibitory activity (Fig. 2A). The PDE superfamily of enzymes are encoded by 21 genes and subdivided into 11 distinct families according to structural and functional properties [36]. We evaluated the

Fig. 2. Chemical structure and phosphodiesterase (PDE) inhibitory activities of T-773 in vitro. Chemical structure of T-773 (A). Human recombinant PDE10A2 inhibitory activity of T-773 and its selectivity over the other 10 PDE family proteins were evaluated by the scintillation proximity assay (B). T-773 exhibited potent and selective inhibitory activity against recombinant human PDE10A2. Data were represented as mean ± SD.

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Table 1 Inhibitory activities of T-773 for recombinant PDE family proteins. PDEs

IC50 (nmol/L)

PDE10A2 PDE1A PDE2A3 PDE3A PDE4D2 PDE5A1 PDE6AB PDE7B PDE8A1 PDE9A2 PDE11A4

0.77 N10000 N10000 N10000 N10000 9100 2200 N10000 N10000 N10000 N10000

Each IC50 value was rounded to two significant figures.

PDE10A2 inhibitory activity of T-773 and its selectivity over other recombinant human PDE family enzymes, including PDE1A, PDE2A3, PDE3A, PDE4D2, PDE5A1, PDE6AB, PDE7B, PDE8A1, PDE9A2, and PDE11A4 (Fig. 2B). The IC50 value of T-773 for PDE10A2 was

0.77 nmol/L, and the minimum IC50 value among the other 10 PDE families was 2200 nmol/L for PDE6AB. Therefore, the PDE family selectivity of T-773 for recombinant PDE10A2 was more than 2500-fold in this experiment (Table 1). 3.3. In vitro ARG using [ 3H]T-773 in Pde10a WT and KO mouse brain sections To confirm the specificity of T-773 for native PDE10A, we performed in vitro ARG using [ 3H]T-773 and brain sagittal sections of Pde10a WT and KO mice. The chemical structure of [ 3H]T-773 is shown in Fig. 3A. Western blotting analysis confirmed the complete deletion of PDE10A protein in the striatum of Pde10a KO mice (Fig. 3B). In WT mouse brain sagittal sections, [ 3H]T-773 selectively accumulated in the caudate putamen (CPu), globus pallidus (GP), nucleus accumbens (NAc), and substantia nigra (SN; Fig. 3C). The striatal MSNs are divided into two output pathways as described above, and each MSN projects to distinct brain regions; direct and indirect pathway MSNs are projecting to SN and GP, respectively [2]. Indeed, high expression of the PDE10A protein was observed not only in Str but also in SN and GP [2,3]. Thus, [3H]T-773

Fig. 3. In vitro autoradiography (ARG) using [3H]T-773 and Pde10a WT or KO mouse brain sections. Chemical structure of [3H]T-773 (A). Phosphodiesterase 10A (PDE10A) protein expression in the striatum of WT and KO mice was analyzed by western blotting (B). Beta-actin (β-actin) was used as a loading control. [3H]T-773 selectively accumulated in the caudate putamen (CPu; white arrow), globus pallidus (GP; black arrow), nucleus accumbens (NAc; white arrow head), and substantia nigra (SN; black arrow head) of WT mouse brain sections (C). The selective accumulation of [3H]T-773 in these areas was not observed in Pde10a KO mouse brain sections (D). In vitro ARG in the presence of 1 μmol/L of MP-10 using WT mouse brain slices was conducted to measure non-specific binding (E). In vitro ARG in the presence of 1 μmol/L of MP-10 using KO mouse brain slices was conducted (F). [3H]T-773 binding in the CPu of each group was presented as percent of total binding in the CPu of WT mouse sections (G).

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Fig. 4. Saturation binding of [3H]T-773 in rat brain sections. Regions of interest (ROIs) were placed at the bilateral caudate putamen (CPu; arrows) and nucleus accumbens (NAc; arrowheads) in the autoradiograms (A). Non-specific binding (NSB) was determined in the presence of an excess amount of MP-10. Total binding and NSB in each ROI were represented as PSL values (/mm2), and the saturation binding curves of CPu (B) and NAc (C) were analyzed by non-linear regression. Data were represented as mean ± SEM. Kd values in CPu and NAc were estimated at 12.2 ± 2.2 nmol/L and 4.7 ± 1.2 nmol/L, respectively.

selectively accumulated in PDE10A high expression areas. This selective accumulation of [ 3H]T-773 was not observed in Pde10a KO mouse brain sections (Fig. 3D). NSB was measured by in vitro ARG study using WT mouse brain slices in the presence of an excess amount (1 μmol/L) of MP-10 (Fig. 3E). [3H]T-773 binding in the CPu of each group was calculated as percent of total binding in WT mouse sections (Fig. 3G). NSB

was only 4.4%, and was almost equal to binding amount of [ 3H]T-773 in KO mouse sections in the absence (4.5%, Fig. 4D) and presence (3.7%, Fig. 4F) of 1 μmol/L of MP-10. These results suggest that [ 3H]T773 selectively binds to native PDE10A in the brain. 3.4. Saturation binding analysis with [ 3H]T-773 in rat brain sections To assess the binding affinity of T-773 to native PDE10A, we measured the Kd values of [ 3H]T-773 in the CPu and NAc of rat brain sections using the saturation binding assay. A representative autoradiogram of a rat brain coronal section is shown in Fig. 4A. Selective and saturable bindings of [ 3H]T-773 were observed with Kd values of 12.2 ± 2.2 nmol/L for CPu and 4.7 ± 1.2 nmol/L for NAc (Fig. 4B,C), indicating that [ 3H]T-773 binds to a single high affinity site of PDE10A in the rat brain. 3.5. [11C]T-773 production The total synthesis time was 31 ± 3 min from the time that the activity was delivered from cyclotron with average radioactivity yield of 2828 ± 351 MBq (n = 6) and radiochemical purity in excess of 99.5%. Mean specific radioactivity was 1643 ± 377 GBq/μmol (n = 6) at 15 min past end of synthesis. Decay-corrected radioactive yield was approximately 80% relative to the amount of [ 11C]Methyl triflate produced. 3.6. In vitro ARG using [11C]T-773 in primate brain sections

Fig. 5. In vitro autoradiography (ARG) using [11C]T-773 in monkey and human brain horizontal sections. Chemical structure of [11C]T-773 (A). Intense [11C]T-773 radioactivity was selectively detected in the striatum of monkey (B) and human (C) brain sections (black arrows).

Next, we performed in vitro ARG using primate brain sections. Taking availability for PET imaging into consideration, we used T773 labeled with a short-lived radionuclide, carbon-11. The substitution of carbon-12 by carbon-11 would not change the biochemical and pharmacological properties of a compound [16]. The chemical structure of [ 11C]T-773 is shown in Fig. 5A. [ 11C]T-773 selectively accumulated in the striatum of monkey and human brain horizontal sections (Fig. 5B,C), in a manner similar to the previously reported expression patterns of PDE10A protein in monkey and human brain

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Fig. 6. Competitive binding of [3H]T-773 with phosphodiesterase 10A (PDE10A) inhibitors in rat brain sagittal sections. [3H]T-773 was highly accumulated in the caudate putamen (CPu; white arrow), globus pallidus (GP; black arrow), nucleus accumbens (NAc; white arrow head), substantia nigra (SN; black arrow head), and the connecting pathway between the striatal complex and SN (gray arrow) of rat brain sagittal sections (A). In vitro autoradiography (ARG) in the presence of an excess amount of PDE10A inhibitors, MP-10 (B) and T-773 (C), was performed with adjacent sections.

sections [3]. These results suggest that T-773 selectively binds to monkey and human native PDE10A. 3.7. Competitive binding of [3H]T-773 with PDE10A inhibitors in rat brain sections To measure target occupancy by a PET study, specific binding of a PET radioligand should be competitively inhibited by a candidate drug. We investigated whether binding of [ 3H]T-773 to PDE10A could be blocked by various PDE10A inhibitors, such as MP-10 and nonradiolabeled T-773, by in vitro ARG in rat brain sagittal sections. [ 3H]T773 selectively accumulated in CPu, GP, NAc, and SN, and radioactivity was also observed in the connecting pathway between the striatal complex and SN (Fig. 6A). This selective accumulation of [ 3H]T-773 was abolished in the presence of an excess amount of both MP-10 and T773 (Fig. 6B,C). Thus, PDE10A occupancy by PDE10A inhibitors could be measured using T-773 as a tracer. 3.8. PET measurement in a rhesus monkey Baseline PET imaging in the monkey brain was conducted using [11C] T-773. After intravenous administration of [ 11C]T-773, its high uptake was observed in the striatum (Fig. 7A–C). Anatomical identification

was conducted using previously acquired MR images (Fig. 7D–F). This restricted distribution was consistent with the result of in vitro ARG using [11C]T-773 in monkey brain sections (Fig. 7B) and the PDE10A expression pattern in the monkey brain [3]. The mean ratio between the striatum and the cerebellum calculated from 87 min to 123 min was 5.1. Thus, the selective binding of systemically administered [ 11C]T773 to native PDE10A could be visualized by PET imaging in the monkey brain. The fraction of unchanged [ 11C]T-773 was 56% and 45% at 30 min and 90 min, respectively. The HPLC chromatogram did not show any more lipophilic radiometabolites than [ 11C]T-773 itself. It is therefore unlikely that these radiometabolites should enter the brain. 4. Discussion To obtain insight into the required profile of a PDE10A radioligand, we firstly measured PDE10A expression levels and patterns in mammalian brains. The mRNA expression level of PDE10A was comparable to that of dopamine D2 receptor in the striatal complex of both rat and human brains. D2 receptor is a well-established target for preclinical and clinical PET imaging in CNS [37–39]; therefore, PDE10A is likely to be a feasible molecule for PET imaging. In accordance with previous reports, PDE10A showed restricted expression in rat and human brains; high expression in the striatum, and low expression in Fcx, Hipp, and

Fig. 7. Baseline positron emission tomography (PET) study using [11C]T-773 in monkeys. Horizontal (A), sagittal (B), and coronal (C) baseline PET images were summed from 9 to 123 min after intravenous injection of [11C]T-773 to an anesthetized monkey. The corresponding magnetic resonance (MR) images are shown (D–F). A high intensity signal of [11C]T-773 was observed in the striatum (white arrows).

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Cb [1–3]. This restricted distribution of PDE10A in the brain would be favorable for the evaluation of target engagement of drugs in a clinical PET study. In addition, such restricted expression will allow for a PET occupancy study because a region with a low expression level for the target molecule could serve as a reference region in quantitative analysis [34]. T-773 showed potent inhibitory activity (IC50 value of 0.77 nmol/L) and high selectivity for recombinant human PDE10A (more than 2500-fold over other recombinant human PDE families) in vitro. In ARG experiments, radiolabeled T-773 selectively accumulated in PDE10A high expression areas in the brains of multiple mammals including the mouse, rat, monkey, and human. In rat brain sections, [ 3H] T-773 also accumulated in the connecting pathway between the striatum and SN. This pathway was also observed in the previous immunohistochemical study of PDE10A using rat brain sagittal sections and was reported to correspond to the striatonigral MSN [2]. The selective accumulation of [ 3H]T-773 in brain sections was not observed in Pde10a KO mice. Thus, T-773 would specifically bind to native PDE10A under physiological conditions. The saturation binding assay using rat brain sections demonstrated that [ 3 H]T-773 binds to a single high affinity site of native PDE10A in CPu and NAc with Kd values of 12.2 and 4.7 nmol/L, respectively. In this experiment, an excess amount of MP-10 was used to determine NSB of [ 3H]T-773 because MP-10 competed with [ 3 H]T-773 in the blocking study, and NSB was linear over the range of concentrations used. The obtained K d values are almost comparable to those of [ 3H]raclopride in a previous autoradiographic study of the striatum of rat brains (2.1–3.0 nmol/L) [40]. [ 11C]raclopride is a well-established PET radiotracer for D2 receptor imaging [38,41]; thus, visualization of PDE10A using [ 11C]T773 in a monkey PET study is a reasonable result. The PDE10Aspecific binding of [ 3H]T-773 was competitively inhibited by PDE10A inhibitors with different chemical structures in rat brain sagittal sections. Therefore, PDE10A occupancy of PDE10A inhibitors could be measured using T-773 as a tracer, although an in vivo blocking study using various PDE10A inhibitors is necessary to develop T-773 as a PET radioligand for PDE10A. Blood–Brain barrier penetration is also important for PET radioligands for PDE10A. This property is known to depend on the capacity for passive entry versus susceptibility to rejection by efflux pumps such as multidrug resistance protein 1 (MDR1) [42,43]. PET radioligands with moderate lipophilicity, indicated by logD values in the approximate range 2.0–3.5, have been considered to show good brain entry in vivo [42,44]. T-773 exhibited optimal lipophilicity with a logD value of 2.13. In addition, the membrane permeability test using human MDR1-expressing cells showed that the excretion/ absorption efflux ratio of T-773 was 0.6, suggesting that T-773 was not a substrate for MDR1. From these results, we expected T-773 to have good brain permeability. As expected, in a baseline monkey PET study using [ 11C]T-773, high uptake of [ 11C]T-773 was observed in the striatum compared with other regions of the brain. This result is consistent with that of in vitro ARG using [ 11C]T-773 in monkey brain sections and previously reported PDE10A expression patterns in the monkey brain [3]. Thus, [ 11C]T-773 can selectively visualize PDE10A using the PET imaging technique. In PET studies, a brain-permeable radioactive metabolite is known to complicate quantitative analysis. In fact, high accumulation of radioactivity in the striatum was observed in recent human PET studies using 18 F-JNJ42259152, a radiotracer for PDE10A, although the possible influence of a brain-penetrating radioactive metabolite was pointed out [22–24]. The present results of venous blood samples indicate that the radiometabolites of [ 11C] T-773 are not likely to enter the brain. Further PET study will use arterial blood sampling for a more detailed investigation. In the present study, we showed that T-773 selectively bound to native PDE10A in multiple mammals including humans. The in vivo selective binding of [11C]T-773 to PDE10A was successfully visualized by PET imaging in the monkey brain. As a next step, in vivo blocking studies

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using various PDE10A inhibitors and careful evaluation of radioactive metabolites should be conducted. In summary, [ 11C]T-773 could be a promising PET radioligand for the evaluation of PDE10A occupancy by PDE10A inhibitors in humans.

Acknowledgements This work was sponsored by Takeda Pharmaceutical Company Limited. We wish to express our sincere thanks to Nicholas Hird, Teruaki Okuda, and Keisuke Hirai for their support and helpful discussions.

References [1] Fujishige K, Kotera J, Michibata H, Yuasa K, Takebayashi S, Okumura K, et al. Cloning and characterization of a novel human phosphodiesterase that hydrolyzes both cAMP and cGMP (PDE10A). J Biol Chem 1999;274:18438–45. [2] Seeger TF, Bartlett B, Coskran TM, Culp JS, James LC, Krull DL, et al. Immunohistochemical localization of PDE10A in the rat brain. Brain Res 2003;985:113–26. [3] Coskran TM, Morton D, Menniti FS, Adamowicz WO, Kleiman RJ, Ryan AM, et al. Immunohistochemical localization of phosphodiesterase 10A in multiple mammalian species. J Histochem Cytochem 2006;54:1205–13. [4] Graybiel AM. Neurotransmitters and neuromodulators in the basal ganglia. Trends Neurosci 1990;13:244–54. [5] Graybiel AM. The basal ganglia. Curr Biol 2000;10:R509–11. [6] Karam CS, Ballon JS, Bivens NM, Freyberg Z, Girgis RR, Lizardi-Ortiz JE, et al. Signaling pathways in schizophrenia: emerging targets and therapeutic strategies. Trends Pharmacol Sci 2010;31:381–90. [7] Krebs M, Leopold K, Hinzpeter A, Schaefer M. Current schizophrenia drugs: efficacy and side effects. Expert Opin Pharmacother 2006;7:1005–16. [8] Siuciak JA, Chapin DS, Harms JF, Lebel LA, McCarthy SA, Chambers L, et al. Inhibition of the striatum-enriched phosphodiesterase PDE10A: a novel approach to the treatment of psychosis. Neuropharmacology 2006;51:386–96. [9] Menniti FS, Chappie TA, Humphrey JM, Schmidt CJ. Phosphodiesterase 10A inhibitors: a novel approach to the treatment of the symptoms of schizophrenia. Curr Opin Investig Drugs 2007;8:54–9. [10] Kehler J, Nielsen J. PDE10A inhibitors: novel therapeutic drugs for schizophrenia. Curr Pharm Des 2011;17:137–50. [11] Rourke C, Starr KR, Reavill C, Fenwick S, Deadman K, Jones DN. Effects of the atypical antipsychotics olanzapine and risperidone on plasma prolactin levels in male rats: a comparison with clinical data. Psychopharmacology 2006;184:107–14. [12] Giampà C, Patassini S, Borreca A, Laurenti D, Marullo F, Bernardi G, et al. Phosphodiesterase 10 inhibition reduces striatal excitotoxicity in the quinolinic acid model of Huntington's disease. Neurobiol Dis 2009;34:450–6. [13] Giampà C, Laurenti D, Anzilotti S, Bernardi G, Menniti FS, Fusco FR. Inhibition of the striatal specific phosphodiesterase PDE10A ameliorates striatal and cortical pathology in R6/2 mouse model of Huntington's disease. PLoS One 2010;5:e13417. [14] Kleiman RJ, Kimmel LH, Bove SE, Lanz TA, Harms JF, Romegialli A, et al. Chronic suppression of phosphodiesterase 10A alters striatal expression of genes responsible for neurotransmitter synthesis, neurotransmission, and signaling pathways implicated in Huntington's disease. J Pharmacol Exp Ther 2011;336:64–76. [15] Frank R, Hargreaves R. Clinical biomarkers in drug discovery and development. Nat Rev Drug Discov 2003;2:566–80. [16] Halldin C, Gulyás B, Farde L. PET studies with carbon-11 radioligands in neuropsychopharmacological drug development. Curr Pharm Des 2001;7:1907–29. [17] Lee CM, Farde L. Using positron emission tomography to facilitate CNS drug development. Trends Pharmacol Sci 2006;27:310–6. [18] Celen S, Koole M, De Angelis M, Sannen I, Chitneni SK, Alcazar J, et al. Preclinical evaluation of 18F-JNJ41510417 as a radioligand for PET imaging of phosphodiesterase10A in the brain. J Nucl Med 2010;51:1584–91. [19] Tu Z, Fan J, Li S, Jones LA, Cui J, Padakanti PK, et al. Radiosynthesis and in vivo evaluation of [11C]MP-10 as a PET probe for imaging PDE10A in rodent and non-human primate brain. Bioorg Med Chem 2011;19:1666–73. [20] Plisson C, Salinas C, Weinzimmer D, Labaree D, Lin SF, Ding YS, et al. Radiosynthesis and in vivo evaluation of [(11)C]MP-10 as a positron emission tomography radioligand for phosphodiesterase 10A. Nucl Med Biol 2011;38:875–84. [21] Andrés JI, De Angelis M, Alcázar J, Iturrino L, Langlois X, Dedeurwaerdere S, et al. Synthesis, in vivo occupancy, and radiolabeling of potent phosphodiesterase subtype-10 inhibitors as candidates for positron emission tomography imaging. J Med Chem 2011;54:5820–35. [22] Van Laere K, Ahmad RU, Hudyana H, Celen S, Dubois K, Schmidt ME, et al. Human biodistribution and dosimetry of 18F-JNJ42259152, a radioligand for phosphodiesterase 10A imaging. Eur J Nucl Med Mol Imaging 2013;40:254–61. [23] Celen S, Koole M, Ooms M, De Angelis M, Sannen I, Cornelis J, et al. Preclinical evaluation of [(18)F]JNJ42259152 as a PET tracer for PDE10A. NeuroImage 2013;82: 13–22. [24] Van Laere K, Ahmad RU, Hudyana H, Dubois K, Schmidt ME, Celen S, et al. Quantification of 18F-JNJ-42259152, a novel phosphodiesterase 10A PET tracer: kinetic modeling and test-retest study in human brain. J Nucl Med 2013;54:1285–93. [25] Hwang DR, Hu E, Rumfelt S, Easwaramoorthy B, Castrillon J, Davis C, et al. Initial characterization of a PDE10A selective positron emission tomography tracer [11C] AMG 7980 in non-human primates. Nucl Med Biol 2014;41:343–9.

154

A. Harada et al. / Nuclear Medicine and Biology 42 (2015) 146–154

[26] Plisson C, Weinzimmer D, Jakobsen S, Natesan S, Salinas C, Lin SF, et al. Phosphodiesterase 10A PET radioligand development program: from pig to human. J Nucl Med 2014;55:595–601. [27] Fan J, Zhang X, Li J, Jin H, Padakanti PK, Jones LA, et al. Radiosyntheses and in vivo evaluation of carbon-11 PET tracers for PDE10A in the brain of rodent and nonhuman primate. Bioorg Med Chem 2014;22:2648–54. [28] Barret O, Thomae D, Tavares A, Alagille D, Papin C, Waterhouse R, et al. In vivo assessment and dosimetry of 2 novel PDE10A PET radiotracers in humans: 18FMNI-659 and 18F-MNI-654. J Nucl Med 2014;55:1297–304. [29] Verhoest PR, Chapin DS, Corman M, Fonseca K, Harms JF, Hou X, et al. Discovery of a novel class of phosphodiesterase 10A inhibitors and identification of clinical candidate 2-[4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)-phenoxymethyl]quinoline (PF-2545920) for the treatment of schizophrenia. J Med Chem 2009; 52:5188–96. [30] Andersson J, Truong P, Halldin C. In-target produced [11C]methane: increased specific radioactivity. Appl Radiat Isot 2009;67:106–10. [31] Stepanov V, Miura S, Takano A, Kimura H, Taniguchi T, Halldin C. Development of a series of novel carbon-11 labeled PDE10A inhibitors. J Labelled Comp Radiopharm 2013:S312 [Korea]. [32] Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 4th ed. San Diego: Academic Press; 1998. [33] Paxinos G, Franklin KBJ. The mouse brain in stereotaxic coordinates. 2nd ed. San Diego: Academic Press; 2001.

[34] Karlsson P, Farde L, Halldin C, Swahn CG, Sedvall G, Foged C, et al. PET examination of [11C]NNC 687 and [11C]NNC 756 as new radioligands for the D1-dopamine receptor. Psychopharmacology 1993;113:149–56. [35] Waarde A. Measuring receptor occupancy with PET. Curr Pharm Des 2000;6:1593–610. [36] Bender AT, Beavo JA. Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use. Pharmacol Rev 2006;58:488–520. [37] Takano A. The application of PET technique for the development and evaluation of novel antipsychotics. Curr Pharm Des 2010;16:371–7. [38] Farde L, Wiesel FA, Halldin C, Sedvall G. Central D2-dopamine receptor occupancy in schizophrenic patients treated with antipsychotic drugs. Arch Gen Psychiatry 1988;45:71–6. [39] Nord M, Farde L. Antipsychotic occupancy of dopamine receptors in schizophrenia. CNS Neurosci Ther 2011;17:97–103. [40] Minuzzi L, Cumming P. Agonist binding fraction of dopamine D2/3 receptors in rat brain: a quantitative autoradiographic study. Neurochem Int 2010;56:747–52. [41] Arakawa R, Ito H, Takano A, Okumura M, Takahashi H, Takano H, et al. Dopamine D2 receptor occupancy by perospirone: a positron emission tomography study in patients with schizophrenia and healthy subjects. Psychopharmacology 2010;209:285–90. [42] Pike VW. PET radiotracers: crossing the blood-brain barrier and surviving metabolism. Trends Pharmacol Sci 2009;30:431–40. [43] Raub TJ. P-glycoprotein recognition of substrates and circumvention through rational drug design. Mol Pharm 2006;3:3–25. [44] Waterhouse RN. Determination of lipophilicity and its use as a predictor of blood-brain barrier penetration of molecular imaging agents. Mol Imaging Biol 2003;5:376–89.