Phosphodiesterase 10A (PDE10A) is a cAMP/cGMP phosphodiesterase highly expressed in medium spiny neurons (MSNs) in the striatum. We evaluated the in vivo pharmacological profile of a potent and selective PDE10A inhibitor, TAK-063 (1-[2-fluoro-4-(1H-pyrazol-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazol-5-yl)-pyridazin-4(1H)-one). TAK-063 at 0.3 and 1 mg/kg p.o., increased cAMP and cGMP levels in the rodent striatum and upregulated phosphorylation levels of key substrates of cAMP- and cGMP-dependent protein kinases. TAK-063 at 0.3 and 1 mg/kg p.o., strongly suppressed MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine]–induced hyperlocomotion, which is often used as a predictive model for antipsychotic-like activity in rodents. Upregulation of striatal cAMP/cGMP levels and the antipsychotic-like effect of TAK-063 were not attenuated after 15 days of pretreatment with TAK-063 in mice. The potential side effect profile of TAK-063 was assessed in rats using the clinical antipsychotics haloperidol, olanzapine, and aripiprazole as controls. TAK-063 did not affect plasma prolactin or glucose levels at doses up to 3 mg/kg p.o. At 3 mg/kg p.o., TAK-063 elicited a weak cataleptic response compared with haloperidol and olanzapine. Evaluation of pathway-specific markers (substance P mRNA for the direct pathway and enkephalin mRNA for the indirect pathway) revealed that TAK-063 activated both the direct and indirect pathways of MSNs. These findings suggest that TAK-063 represents a promising drug for the treatment of schizophrenia with potential for superior safety and tolerability profiles.
The basal ganglia are a series of interconnected subcortical nuclei that integrate widespread cortical inputs with dopaminergic signaling to plan and execute relevant motor and cognitive patterns while suppressing unwanted or irrelevant patterns (Albin et al., 1989; Graybiel, 2000; McHaffie et al., 2005). Dysfunction in this circuit has been implicated in various central nervous system (CNS) disorders and diseases, including schizophrenia, Parkinson disease, Huntington disease, and addiction (Albin et al., 1989; Chesselet and Delfs, 1996; Hyman et al., 2006; Perez-Costas et al., 2010). Schizophrenia is a devastating neuropsychiatric syndrome that typically strikes in late adolescence or early adulthood (van Os and Kapur, 2009). Positive symptoms, including delusions and hallucinations, are the most apparent manifestation of the disorder. These emerge episodically and usually trigger the first hospitalization in early adulthood. Chronic aspects of the disorder include negative symptoms, such as social withdrawal, flattened affect, and anhedonia as well as pervasive cognitive deficits. The latter have been closely linked to a poor functional outcome and long-term prognosis (Green et al., 2004; Harvey et al., 2004). Current antipsychotics are known to have clinically significant effects on positive symptoms that are mediated by dopamine D2 receptor antagonism, but their efficacy is considerably limited for other aspects of the disease, such as cognitive and negative symptoms. Typical antipsychotics are known to cause extrapyramidal symptoms (EPS) by excessive dopamine D2 receptor antagonism in the striatum, and hyperprolactinemia by dopamine D2 receptor antagonism in the pituitary gland (Krebs et al., 2006). Although atypical antipsychotics, such as olanzapine and risperidone, produce a lower incidence of EPS than typical antipsychotics, these drugs are still associated with hyperprolactinemia and serious metabolic side effects, including hyperglycemia, weight gain, diabetes, and an abnormal lipid profile (Krebs et al., 2006). Thus, novel drugs with potent efficacy against more symptoms of schizophrenia and a better safety profile would be of considerable therapeutic value.
Inhibition of cyclic nucleotide phosphodiesterase (PDE) may provide a new therapeutic approach for the treatment of CNS disorders (Menniti et al., 2006, 2007; Reneerkens et al., 2009). The PDE superfamily of enzymes is encoded by 21 genes and subdivided into 11 distinct families according to their structural and functional properties (Bender and Beavo, 2006). Identification of the entire PDE gene family and a greater understanding of the complex expression patterns of the different PDEs have renewed interest in the therapeutic potential of this important class of enzymes. Among PDE families, PDE10A is emerging as a promising target for CNS disorders, such as schizophrenia and Huntington’s disease (Giampà et al., 2009, 2010; Kehler and Nielsen, 2011). PDE10A mRNA expression was found to be particularly high in certain brain regions and testis in mice, rat, and humans (Fujishige et al., 1999; Soderling et al., 1999; Seeger et al., 2003; Lakics et al., 2010). Subsequent studies in multiple mammalian species indicated that PDE10A protein is highly expressed in the GABA-containing medium spiny neurons (MSNs) in the mammalian striatum and substantia nigra, with restricted distribution in the periphery (Seeger et al., 2003; Coskran et al., 2006). MSNs are the principal input site for information integration in the basal ganglia of the mammalian brain, and they give rise principally to two pathways: a direct (striatonigral) pathway, which expresses dopamine D1 receptors, and an indirect (striatopallidal) pathway, which expresses dopamine D2 receptors (Graybiel, 1990, 2000). These pathways have competing effects on the striatal output. As PDE10A is expressed in both pathways, PDE10A inhibition and resulting elevation of striatal cyclic nucleotide levels would potentially have the effect of D2 receptor antagonism, the standard treatment of psychosis, along with D1 receptor agonism, which may minimize EPS liabilities. The results of preclinical studies of several PDE10A inhibitors suggest that PDE10A inhibition may be a novel therapeutic approach for the treatment of schizophrenia (Schmidt et al., 2008; Grauer et al., 2009; Smith et al., 2013).
We recently discovered the novel PDE10A inhibitor, TAK-063 (1-[2-fluoro-4-(1H-pyrazol-1-yl)phenyl]-5-methoxy-3-(1-phenyl-1H-pyrazol-5-yl)-pyridazin-4(1H)-one), and confirmed that TAK-063 can specifically bind to PDE10A under physiologic conditions (Kunitomo et al., 2014; Harada A, Suzuki K, Kamiguchi N, Miyamoto M, Tohyama K, Nakashima K, Taniguchi T, and Kimura H, manuscript in preparation). In this study, we characterized the in vivo efficacy of TAK-063 as a novel treatment of schizophrenia. TAK-063 activates striatal signaling pathways, has antipsychotic-like activity in rodent models, and has the potential to demonstrate a superior side-effect profile in the clinic. These findings suggest that TAK-063 is a promising drug for the treatment of schizophrenia.
Materials and Methods
Male imprinting control region (ICR) mice and Sprague-Dawley (SD) rats were supplied by CLEA Japan Inc. (Tokyo, Japan) and Charles River Laboratories Japan Inc. (Yokohama, Japan), respectively. Animals were housed in a light-controlled room (12-hour light/dark cycle with lights on from 7:00 AM). Animals used in the experiments were 6–8 weeks old and had completed an acclimation period of at least 1 week. The Experimental Animal Care and Use Committee of Takeda Pharmaceutical Company Limited approved the care and use of animals and the experimental protocols used in this research.
TAK-063 was synthesized by Takeda Pharmaceutical Company Limited (Fujisawa, Japan) (Kunitomo et al., 2014). TAK-063 and haloperidol (Sigma-Aldrich, St. Louis, MO) were suspended in 0.5% (w/v) methylcellulose in distilled water. The doses of TAK-063 were calculated as the free base. Olanzapine was extracted from Zyprexa (Eli Lilly and Company, Indianapolis, IN) at KNC Laboratories Co. Ltd. (Kobe, Japan), and then dissolved in 1.5% (v/v) lactic acid. The pH of this solution was then adjusted to neutral using 1 M NaOH. Aripiprazole was purchased from AK Scientific Inc. (Union City, CA), and suspended in a 1% (v/v) solution of Tween 80 in distilled water. These compounds were administered orally. (+)-MK-801 hydrogen maleate (MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine]; Sigma-Aldrich) was dissolved in saline and administered subcutaneously. Rolipram (Sigma-Aldrich) was suspended in a 0.5% (w/v) solution of methylcellulose in saline and administered intraperitoneally. To evaluate the activation of the direct and indirect pathways in MSNs, haloperidol was suspended in a 0.5% (w/v) solution of methylcellulose in saline. SKF82958 hydrobromide [(±)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide; Sigma-Aldrich] was dissolved in two drops of Tween 80 and then diluted in saline. These compounds were administered intraperitoneally. All compounds were dosed at 20 ml/kg body weight in mice and 2 m/kg body weight in rats.
Measurement of Cyclic Nucleotides and Phosphorylated Protein Levels in Various Brain Regions.
Male ICR mice and SD rats were sacrificed using an MMW-05 focused microwave irradiation system (Muromachi Kikai Co. Ltd., Tokyo, Japan) 60 minutes after oral administration of TAK-063. Brain tissues were isolated, sonicated in 0.5 N HCl, and clarified by centrifugation. Cyclic nucleotide concentrations in the supernatants were measured using enzyme immunoassay kits (Cayman Chemical Company, Ann Arbor, MI). To assess the phosphorylation of cAMP response element–binding protein (pCREB) and of AMPA [(±)-α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid] receptor subunit GluR1 at Ser845 (pGluR1), microwaved brain tissues were sonicated in extraction buffer (Invitrogen, Carlsbad, CA) containing the protease inhibitor cocktail (Sigma-Aldrich) and 0.5 mM p-amidinophenylmethanesulfonyl fluoride hydrochloride (Sigma-Aldrich). After centrifugation, protein concentrations in the supernatant were determined using the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA). Each sample (5 μg of protein) was subjected to electrophoresis through polyacrylamide gradient gels, followed by Western blotting to nitrocellulose membranes (Bio-Rad Laboratories Inc., Hercules, CA). The blots were incubated with antibodies to pCREB (Cell Signaling Technology Inc., Danvers, MA), total CREB (tCREB; Cell Signaling Technology), pGluR1 (PhosphoSolutions, Aurora, CO), total GluR1 (Millipore, Temecula, CA), or β-actin (Sigma-Aldrich). Individual protein bands were visualized with horseradish peroxidase–conjugated secondary antibodies (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) followed by treatment with ECL Western Blotting Detection Reagents (GE Healthcare UK Ltd., Buckinghamshire, UK) and exposure to Hyperfilm ECL (GE Healthcare). Protein levels were calculated from densitometric analysis using a Bio-Rad GS-800 calibrated scanning densitometer. Densities of phosphorylated protein bands for each sample were normalized to the density of the corresponding total protein bands. These ratios are expressed as the relative change divided by the average of the vehicle-treated samples. Amounts of pCREB and tCREB in the various brain regions were measured using the CREB [pS133] human ELISA kit and the CREB [total] human ELISA kit (Life Technologies, Carlsbad, CA), respectively.
MK-801–Induced Hyperlocomotion in Rodents.
Locomotion was measured using a SUPERMEX spontaneous motor analyzer (Muromachi Kikai) for rats and an MDC system (Brain Science Idea Co. Ltd., Osaka, Japan) for mice. Animals (male ICR mice or SD rats) were placed in locomotor chambers (length × width × height: 24 × 37 × 30 cm for rats and 36 × 22 × 13.5 cm for mice) for more than 60 minutes for habituation. Thereafter, animals were removed from each chamber and treated with either vehicle or test compounds and then quickly returned to the chamber. After an appropriate pretreatment time, animals were again removed from the chambers and treated with either vehicle (saline) or MK-801 (0.3 mg/kg s.c., as a salt) and then quickly transferred to the test chamber. Activity counts were recorded in successive 1-minute bins and then cumulative counts during 120 minutes after psychostimulant administration was calculated. A 60-minute pretreatment time was used in the mouse study. Pretreatment times in rats varied according the pharmacokinetic profile (i.e., Tmax) of each agent, resulting in a 90-minute pretreatment time for TAK-063, a 30-minute pretreatment time for olanzapine, and a 60-minute pretreatment time for haloperidol and aripiprazole.
Measurement of Plasma Prolactin Levels.
Male SD rats were orally administered either vehicle or test compounds after a habituation period of >30 minutes. Ninety minutes after administration, blood was collected from tail vein into a 1.5-ml Eppendorf tube containing 25 μl of EDTA. Blood was immediately mixed with EDTA, placed on ice, and then centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatants were collected in another tube as plasma, and were stored in a deep-freezer until use. The prolactin concentrations in the plasma samples were measured using an ELISA kit (Bertin Pharma, Montigny le Bretonneux, France).
Measurement of Plasma Glucose Levels.
Male SD rats were fasted overnight and were decapitated 150, 60, 60, and 90 minutes after administration of TAK-063, haloperidol, olanzapine, and aripiprazole, respectively. Trunk blood was collected into 50-ml centrifuge tubes. Plasma glucose levels were measured using a model 7180 Clinical Analyzer (Hitachi High-Technologies Inc., Tokyo, Japan).
The degree of cataleptic response in rats to antipsychotics such as haloperidol and ziprasidone was reported to increase as a function of both dose and time (Schmidt et al., 2008). The catalepsy-like behavior of male SD rats was measured 2 and 4 hours after administration of test compounds in a blind manner, with exceptions noted. Forelimbs were placed on a horizontal metal bar at a 13-cm height and the length of time during which both forelimbs remained on the bar was determined with a stopwatch (Seiko Holdings Corporation, Tokyo, Japan). Animals not responding within 90 seconds were removed from the apparatus and assigned a latency of 90 seconds (cut-off value). The average of three trials was recorded as the duration of the cataleptic response.
Real-Time Quantitative Polymerase Chain Reaction Expression Analysis.
Total RNA from different brain regions was extracted using Isogen (Nippon Gene Co. Ltd., Toyama, Japan) and the RNeasy kit (Qiagen, Hilden, Germany) following the manufacturer’s instruction. Real-time quantitative polymerase chain reaction was performed using an ABI PRISM 7900HT sequence detection system (Life Technologies) and TaqMan reagents (Eurogentec, Seraing, Belgium). RNA quantities were normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) TaqMan probes according to the manufacturer’s instruction. The following primers were used for mouse PDE10A analysis: forward primer, 5′-AAGAGACAGCAATGTGGATTTCAG-3′; reverse primer, 5′-TCTTCAACCTTCACGTTCAGCTT-3′; TaqMan probe (MGB probe), 5′-CCCGGCGCCTAGCAAGAGCAC-3′. The following primers were used for mouse PDE4D analysis: forward primer, 5′-GACGTGGCCTATCACAACAACA-3′; reverse primer, 5′-GGTGTAGAGAGCAGCACGTGAGT-3′; TaqMan probe (MGB probe), 5′-CCATGCTGCAGACGTCGTCCAGTC-3′. The following primers were used for enkephalin analysis: forward primer, 5′-GGACTGCGCTAAATGCAGCTA-3′; reverse primer, 5′-GTGTGCATGCCAGGAAGTTG-3′; TaqMan probe (MGB probe), 5′-CGCCTGGTACGTCCCGGCG-3′. The following primers were used for substance P analysis: forward primer, 5′-CGCAAAATCCAACATGAAAATC-3′; reverse primer, 5′-GCAAACAGTTGAGTGGAAACGA-3′; TaqMan probe (MGB probe), 5′-CGTGGCGGTGGCGGTCTTTTT-3′. The following primers were used for rat GAPDH analysis: forward primer, 5′-TGCCAAGTATGATGACATCAAGAAG-3′; reverse primer, 5′-AGCCCAGGATGCCCTTTAGT-3′; TaqMan probe (MGB probe), 5′-TGGTGAAGCAGGCGGCCGAG-3′. The primers used for mouse GAPDH analysis were from ABI (TaqMan Rodent GAPDH Control Reagents, VIC probe).
The statistical significance of differences between two groups was assessed by the Student’s t test (for homogenous data) or the Aspin-Welch test (for nonhomogeneous data). Differences yielding P values ≤0.05 were considered significant. In the experiments that examined the effects of multiple doses of test compounds, statistical significance was determined using a one-tailed Williams’ test (for homogenous data) or a one-tailed Shirley-Williams’ test (for nonhomogeneous data). One-tailed differences yielding P values ≤0.025 were considered significant.
Effect of TAK-063 on Striatal Cyclic Nucleotide Levels and Their Downstream Signaling.
PDE10A inhibition and the resulting elevation of cAMP and cGMP levels are known to activate cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) (Kehler and Nielsen, 2011). Activated PKA and PKG increase the phosphorylation of key substrates, such as CREB and AMPA receptor GluR1 subunit (Lonze and Ginty, 2002; Wang et al., 2005; Serulle et al., 2007). To evaluate in vivo PDE10A inhibition by TAK-063, the effects of TAK-063 on cAMP and cGMP levels and phosphorylation levels of CREB and GluR1 were assessed. Oral administration of TAK-063 increased cAMP and cGMP levels in the mouse and rat striatum in a dose-dependent manner (Fig. 1, A and B). The minimum effective dose (MED) of TAK-063 for the upregulation of cAMP and cGMP in the mouse striatum was 0.3 mg/kg p.o. (P ≤ 0.025). The phosphorylation of CREB and GluR1 in striatal extracts was evaluated by Western blotting. TAK-063 increased phosphorylation levels of CREB and GluR1 in the mouse striatum in a dose-dependent manner, in the same dose range of TAK-063 that upregulated striatal cAMP and cGMP levels (P ≤ 0.025; Fig. 1, A, C, and D). These results suggest that TAK-063 suppressed PDE10A activity and activated downstream cAMP and cGMP signals in the striatum.
Striatal-Selective Upregulation of cAMP and CREB Phosphorylation by TAK-063.
We investigated the upregulation of cAMP and pCREB by TAK-063 in various brain regions, such as the frontal cortex, striatum, thalamus, brainstem, hippocampus, and cerebellum, in mice. A quantitative polymerase chain reaction expression analysis revealed that PDE10A mRNA was expressed mainly in the striatal complex within these brain regions (Fig. 2A). TAK-063 at 0.3 mg/kg p.o., selectively increased cAMP (P ≤ 0.01; Fig. 2B) and pCREB levels (P = 0.12; Fig. 2C) in the striatal complex, where the PDE10A mRNA expression level was high. Rolipram (10 mg/kg i.p.), a selective PDE4 inhibitor, increased cAMP levels in all brain regions tested, probably owing to the broader expression pattern of PDE4 (Supplemental Fig. 1; Lakics et al., 2010; Johansson et al., 2012). These results indicate that TAK-063 selectively inhibits PDE10A in vivo under these conditions.
Effects of TAK-063 on MK-801–Induced Hyperactivity in Rodents.
Hyperlocomotion induced by MK-801, an NMDA receptor antagonist, is commonly used as a model for acute psychosis on the basis of the NMDA hypofunction hypothesis of schizophrenia (O’Neill and Shaw, 1999). TAK-063 produced dose-dependent suppression of MK-801–induced hyperlocomotion in mice with an MED of 0.3 mg/kg p.o. (P ≤ 0.025; Fig. 3A). In rats, TAK-063 produced dose-dependent suppression of MK-801–induced hyperlocomotion with an MED of 0.1 mg/kg p.o. (P ≤ 0.025; Fig. 3B). Current clinically-used antipsychotics such as haloperidol, olanzapine, and aripiprazole also showed dose-dependent suppression of MK-801–induced hyperlocomotion. Their MEDs were 3, 30, and 100 mg/kg, respectively (P ≤ 0.025; Fig. 3B). These data suggest that TAK-063 has a potent antipsychotic-like effect in rodents.
Effects of TAK-063 on Striatal Cyclic Nucleotide Levels and MK-801–Induced Hyperactivity after Repeated Administration.
To assess whether the pharmacological effects of TAK-063 are altered after chronic treatment, we examined striatal molecular marker responses and antipsychotic-like effects of TAK-063 after repeated administration in mice. After 15 days of pretreatment with TAK-063 (0.5 mg/kg per day), TAK-063 at 0.5 mg/kg p.o., significantly upregulated striatal cAMP and cGMP levels (P ≤ 0.05 for cAMP, P ≤ 0.01 for cGMP; Fig. 4A) and produced a suppression of MK-801–induced hyperlocomotion in mice (P ≤ 0.025; Fig. 4B). These effects did not differ from those seen after administration of a single dose (Fig. 4, A and B). These results suggest that the antipsychotic-like effects of TAK-063 are maintained after repeated administration.
Evaluation of Potential Clinical Side Effects.
The potential of TAK-063 to produce well known side effects of current clinical antipsychotics, such as hyperprolactinemia, hyperglycemia, and EPS, was evaluated in rats. TAK-063 did not affect plasma prolactin levels in male rats at doses up to 3 mg/kg p.o., whereas current antipsychotics such as haloperidol, olanzapine, and aripiprazole significantly increased plasma prolactin levels. The MEDs of haloperidol, olanzapine, and aripiprazole for upregulation of plasma prolactin levels were 0.3, 3, and 30 mg/kg p.o., respectively (P ≤ 0.025; Fig. 5). It has been reported that the acute effects of antipsychotics on rodent plasma glucose levels are well correlated with clinically observed effects (Assié et al., 2008). Oral administration of olanzapine significantly increased rat plasma glucose levels in a dose-dependent manner, with an MED of 10 mg/kg p.o. (P ≤ 0.025; Fig. 6). In contrast, TAK-063, haloperidol, and aripiprazole did not affect plasma glucose levels (Fig. 6).
Next, the potential of these compounds to produce EPS was assessed by evaluating their cataleptogenic activities 2 and 4 hours after administration in rats. All compounds tested produced a cataleptic response in a time-dependent manner; the MEDs for TAK-063, haloperidol, olanzapine, and aripiprazole were 3, 0.3, 10, 30 mg/kg p.o., respectively (4 hours after administration, P ≤ 0.025; Fig. 7). Suppression of MK-801–induced hyperlocomotion and the potential to produce side effects in rats are summarized in Table 1. These results suggest that TAK-063 administration carries lower risk of the side effects assessed compared with administration of current antipsychotics.
Activation of the Direct and Indirect Pathways by PDE10A Inhibition.
It has been reported that PDE10A is expressed both in the direct (dopamine D1 receptor–dependent, facilitate activity) and in the indirect (dopamine D2 receptor–dependent, tonically inhibitory) striatal pathways of the striatum complex (Sano et al., 2008). To evaluate activation of the direct and the indirect pathways by a D2 antagonist (haloperidol), a D1 agonist (SKF82958), and a PDE10A inhibitor (TAK-063), we measured the expression of mRNA encoding substance P and enkephalin in the rat striatum. Substance P and enkephalin are markers of the direct and indirect pathways, respectively. RNAs encoding these neuropeptides are known to be upregulated via CREB response elements in the promoter regions of the respective genes (Gerfen et al., 1990; Simpson and McGinty, 1995). SKF82958 (2 mg/kg i.p.) selectively increased the substance P mRNA level (P ≤ 0.05; Fig. 8A), and haloperidol (1 mg/kg i.p.) selectively increased the enkephalin mRNA level in the rat striatum (P ≤ 0.01; Fig. 8A). TAK-063 (1 mg/kg p.o.) increased both enkephalin and substance P mRNA levels in the rat striatum (P ≤ 0.01 for substance P and P ≤ 0.05 for enkephalin). The indirect and direct pathways are thought to have competing effects on striatal output (Graybiel, 1990, 2000). The effect of D1 direct pathway activation on the D2 indirect pathway–mediated cataleptic response was evaluated using SKF82958 and haloperidol. Pretreatment with SKF82958 (2 mg/kg i.p.) significantly decreased the magnitude of the haloperidol (1 mg/kg i.p.)-induced cataleptic response in rats (P ≤ 0.05; Fig. 8B). These results suggest that the reduced cataleptic response seen in rats after TAK-063 is attributable to modulation of both the direct and indirect pathways by TAK-063.
We previously reported the discovery of a novel PDE10A inhibitor, TAK-063 (Kunitomo et al., 2014), that displays potent PDE10A inhibitory activity (hPDE10A IC50 = 0.30 nM) and selectivity (>15,000-fold) over other PDE families (PDE1–PDE9 and PDE11) in in vitro assays using recombinant human enzymes. TAK-063 also selectively binds to native PDE10A in the rodent brain (Harada A, Suzuki K, Kamiguchi N, Miyamoto M, Tohyama K, Nakashima K, Taniguchi T, and Kimura H, manuscript in preparation). In the present study, we described data validating the functional PDE10A selectivity of TAK-063. TAK-063 selectively (i.e., only in the striatum) upregulated cAMP level and phosphorylation levels of their downstream signaling molecules CREB and GluR1. This striatal-specific signal activation is consistent with the striatal-specific expression of PDE10A and its inhibition by TAK-063 in the mouse brain.
The antipsychotic-like effects of TAK-063 were evaluated through inhibition of MK-801–induced hyperlocomotion in rodents that is often used as a predictive assay for antipsychotic-like activity. In this model, pharmacological effects were observed at 0.3 mg/kg p.o., a dose consistent with doses shown to activate striatal signaling pathways in rodents. Furthermore, a dose of 0.3 mg/kg p.o., of TAK-063 has been estimated to produce a striatal PDE10A occupancy level of 26% in rats (Harada A, Suzuki K, Kamiguchi N, Miyamoto M, Tohyama K, Nakashima K, Taniguchi T, and Kimura H, manuscript in preparation). Positron emission tomography occupancy studies could be useful as a translational approach between preclinical and clinical studies of TAK-063. We developed a clinical positron emission tomography radioligand targeting PDE10A, [11C]T-773 (1-[2-fluoro-4-(tetrahydro-2H-pyran-4-yl)phenyl]-5-[11C]methoxy-3-(1-phenyl-1H-pyrazol-5-yl)pyridazin-4(1H)-one), and successfully estimated striatal PDE10A occupancy by TAK-063 using nonradiolabeled T-773 in rats (Harada et al., 2014; Harada A, Suzuki K, Kamiguchi N, Miyamoto M, Tohyama K, Nakashima K, Taniguchi T, and Kimura H, manuscript in preparation). Combined with the results of the present study, investigation of the dose-occupancy relationship in humans will provide further characterization of the preclinical and clinical aspects of the pharmacological profile of TAK-063.
Activation of the indirect striatal pathway by blockade of the dopamine D2 receptor is the hypothesized mechanism underlying the effect of current antipsychotics. Antipsychotic effects similar to those of current antipsychotics could be expected from TAK-063 through its upregulation of cAMP and concomitant activation of the indirect pathway. This hypothesis could be explored in future clinical studies.
Plasma glucose and prolactin levels and catalepsy were evaluated in rats dosed with TAK-063 as measures of potential adverse effects in the clinic. TAK-063, haloperidol, and aripiprazole did not affect plasma glucose levels, even at doses higher than those that produce antipsychotic-like effects, whereas olanzapine significantly increased plasma glucose levels in rats. These effects of current antipsychotics correlate well with clinical observations (Krebs et al., 2006). In addition, a dose of 3 mg/kg of TAK-063, at which the striatal PDE10A occupancy level was 77% as determined by nonlinear regression analysis (Harada A, Suzuki K, Kamiguchi N, Miyamoto M, Tohyama K, Nakashima K, Taniguchi T, and Kimura H, manuscript in preparation), did not increase prolactin release in rats. TAK-063 showed a lower potential for producing a cataleptic response than haloperidol, olanzapine, and aripiprazole.
Given the unique pharmacological profile of TAK-063 relative to clinically-used antipsychotics and the high levels of PDE10A in MSNs, we examined the striatal pathways modulated by TAK-063. We confirmed that TAK-063 induces the expression of substance P (a marker of the direct striatal pathway) and enkephalin (a marker of the indirect striatal pathway) in rats. We also confirmed that activation of the direct pathway by SKF82958 (D1 agonist) inhibited the indirect pathway–mediated cataleptic response to haloperidol (D2 antagonist) in rats. The modulation of both the direct (D1-dependent, facilitate activity) and indirect (D2-dependent, tonically inhibitory) striatal pathways by PDE10A inhibitors (Siuciak et al., 2006) probably relates to their reduced cataleptic versus antipsychotic profile in animal models. Altogether these results suggest that the striatum-selective expression of PDE10A and the activation of both striatal pathways by PDE10A inhibition contribute to a superior side-effect profile of TAK-063. Clinical studies, however, are critical to validating these predictions on the basis of preclinical findings.
In the present study, our results demonstrated that TAK-063 is a potent and selective inhibitor of PDE10A in vivo. Additionally, the compound showed potent activity in rodent models of schizophrenic-like symptoms but was less potent than clinically-used antipsychotics in their metabolic and cataleptic effects. PDE10A inhibitors have been reported to show efficacy in preclinical assays that test cognitive performance and possible models of negative symptoms in rodents (Grauer et al., 2009; Smith et al., 2013). Because cognitive and negative symptoms are not adequately treated by current antipsychotics, it is worthwhile to see the potential of TAK-063 in these domains.
In summary, these results suggest that TAK-063 could be a promising drug in the treatment of schizophrenia, for which this compound is currently in clinical development (ClinicalTrials.gov Identifiers: NCT01879722 and NCT01892189).
The authors thank Naomi Kamiguchi for performing the pharmacokinetic analysis of test compounds, and Takahiko Taniguchi for providing a chemical compound.
Participated in research design: Suzuki, Kimura.
Conducted experiments: Suzuki, Harada, Shiraishi.
Performed data analysis: Suzuki, Harada, Shiraishi, Kimura.
Wrote or contributed to the writing of the manuscript: Suzuki, Kimura.
- (±)-α-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid
- central nervous system
- cAMP response element–binding protein
- extrapyramidal symptoms
- glyceraldehyde-3-phosphate dehydrogenase
- imprinting control region
- minimum effective dose
- medium spiny neuron
- (±)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide
- Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics