Neuropharmacology 81 (2014) 116e125

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Neuropharmacology

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Antidepressant-like and -like effects of YL-IPA08, a potent ligand for the (18 kDa)

Li-Ming Zhang a, 1, Nan Zhao a, 1, Wen-Zhi Guo b, Zeng-Liang Jin a, Zhi-Kun Qiu a, c, Hong-Xia Chen a, Rui Xue a, You-Zhi Zhang a, Ri-Fang Yang d, Yun-Feng Li a, * a Department of New Drug Evaluation, Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Beijing, PR China b Department of Anesthesiology, General Hospital of Beijing Military Command, Beijing 100007, China c Department of Pharmacology, School of Pharmaceutical Sciences, Southern Medical University, 1023 Shatainan Road, Guangzhou, PR China d Department of Medicinal Chemistry, Beijing Institute of Pharmacology and Toxicology, Beijing, PR China article info abstract

Article history: It has been demonstrated that the translocator protein (18 kDa) (TSPO) plays an important role in stress- Received 8 June 2013 response and stress-related disorders, such as anxiety and depression, by affecting the production of Received in revised form , supporting the potential use of selective TSPO ligands as or anxiolytic 4 September 2013 drugs. N-ethyl-N-(2-pyridinylmethyl)- 2-(3,4-ichlorophenyl)- 7-methylimidazo [1,2-a] pyridine-3- Accepted 9 September 2013 acetamide hydrochloride (YL-IPA08), a novel TSPO ligand that has been synthesized at our institute, Available online 22 September 2013 exerted a high affinity for TSPO in a crude mitochondrial fraction prepared from rat cerebellum but exhibited only a negligible affinity for the central receptor. As expected, YL-IPA08 in- Keywords: The translocator protein 18KD cubation with the cultured rat astrocyte cells increased the pregnenolone and progesterone concen- fi YL-IPA08 tration from the cultured medium. Moreover, YL-IPA08 produced signi cant antidepressant-like and Anxiolytic anxiolytic-like effects in a series of mouse and rat behavior models. In addition, the antidepressant- Antidepressant like behavior of YL-IPA08 was totally blocked by the TSPO antagonist PK11195 in a tail suspension Neurosteroids test, and the anxiolytic effect was blocked by PK11195 but not by a CBR antagonist in the elevated plus- maze test. Furthermore, compared with the CBR agonist , YL-IPA08 had no myorelaxant effects and did not affect the motor coordination, memory or hexobarbitone-induced sleep in mice. Overall, these results indicate that YL-IPA08 is a more potent and selective TSPO ligand, which exerts antidepressant-like and anxiolytic-like effects on behaviors that are mediated by TSPO but does not cause the side effects that are typically associated with conventional . Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction serotonin reuptake inhibitors (SSRIs), which have broad spectrum anxiolytic efficacy in generalized anxiety disorder, panic, social Depression and anxiety disorders are serious and burdensome anxiety disorder, Posttraumatic stress disorder (PTSD) and antide- psychiatric illnesses that frequently turn into chronic clinical con- pressant efficacy in major depression (Mula, 2013; Zonda et al., ditions. Until now, the underlying pathophysiological mechanism 2011; Baldwin, 2000). However, SSRIs have weaknesses, including of depression and anxiety have remained obscure. First-line treat- a delayed onset of action, a partial response with residual symp- ment of both anxiety disorders and depression comprises selective toms, non-response, and undesirable side effects (e.g., loss of sexual drive, gastrointestinal effects, changes in body weight) (Haddad, 1998; Nelson and Philbrick, 2012; Sheeler et al., 2012). Tricyclic Abbreviations: Allo, ; BZD, Benzodiazepine; CBR, central are efficacious in some anxiety disorders and benzodiazepine receptors; CNS, central nervous system; Cort, Corticosterone; CSF, depression but are associated with a greater burden of adverse cerebrospinal fluid; FST, forced swimming test; GABA, g-aminobutyricacid; GC, glucocorticoid; PBR, peripheral benzodiazepine receptor; PTSD, Posttraumatic effects compared to SSRIs and are usually used only after a poor stress disorder; SBSSs, selective brain steroidogenic stimulants; SSRIs, selective response or no response to SSRI treatment (Baldwin et al., 2005; serotonin reuptake inhibitors; TSPO, translocator protein (18 kDa); TST, tail sus- Anderson et al., 2008). In addition, benzodiazepines (BZDs) (e.g., pension test. diazepam) have proved to be effective in anxiety disorders (panic * þ þ Corresponding author. Tel.: 86 10 66874606; fax: 86 10 68211656. disorders, GAD and SAD) (Baldwin et al., 2005) but are not used in E-mail addresses: [email protected], [email protected] (Y.-F. Li). 1 Li-Ming Zhang and Nan Zhao contributed equally to this study. depression (Anderson et al., 2008). However, BZDs also produce

0028-3908/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2013.09.016 L.-M. Zhang et al. / Neuropharmacology 81 (2014) 116e125 117

future (Taliani et al., 2009; Verleye et al., 2005; Rupprecht et al., 2010). Given all of the factors mentioned above, in search for a new TSPO ligand for the therapeutic treatment of stress-related disor- ders, we designed and synthesized a series of compounds that have novel structures. YL-IPA08 [N-ethyl-N-(2-pyridinylmethyl)-2-(3,4- ichlorophenyl)-7-methylimidazo [1,2-a] pyridine-3-acetamide hy- drochloride, Fig.1] was screened as the final candidate because of its high TSPO affinity and strong antidepressant and anxiolytic-like effects in the absence of the tolerance and withdrawal liabilities. In the present study, we performed detailed investigation of the Fig. 1. Chemical structure of YL-IPA08. pharmacological characteristics of YL-IPA08 in vivo and in vitro. First, we determined the binding and selection profile of YL-IPA08, undesirable side effects (motor coordination deficits and memory i.e., its affinity for rat TSPO and CBR. Then, we measured its effects impairment, consecutive abuse liability, and withdrawal symptoms on production in cultured rat astrocyte and its following chronic administration), which limit their usefulness. antidepressant-like and anxiolytic-like behaviors in various mouse Accordingly, considerable effort has been invested in the search for and rat models. To confirm the role of TSPO in the behavioral action better drugs for more effective treatment of depression and anxiety. of YL-IPA08, we evaluated the effects of TSPO antagonist on the During the last decade, the down-regulation of neurosteroid behavioral effects of YL-IPA08. Finally, we investigated its potential biosynthesis has been implicated as a possible contributor to the to cause side effects (including myorelaxant effects, motor coordi- development of anxiety and depressive disorders (Pinna et al., nation, memory impairment or the potentiation of hexobarbitone- 2006). In clinical studies, decreases in the serum, plasma, and ce- induced sleep) that are typically associated with conventional BZDs. rebrospinal fluid (CSF) content of neuroactive steroids, including the progesterone metabolite, allopregnanolone (Allo), which is a 2. Materials and methods potent positive allosteric modulator of the action of g-amino- butyricacid (GABA) at GABAA receptors (Uzunova et al., 1998; 2.1. Animals Romeo et al., 1998; Rasmusson et al., 2006; Nin et al., 2011), are associated with several psychiatric disorders such as depression, Both male ICR mice (18 2 g) and male SpragueeDawley rats anxiety, PTSD, schizophrenia, and impulsive aggression. In the so- (180 10 g) were purchased from the Beijing Vital Laboratory cially isolated mouse, it was also found that corticolimbic Allo levels Animal Technology Company (Beijing, China). The animals were become markedly decreased in association with the development maintained under standard conditions of controlled temperature of anxiety-like behaviors, resistance to sedation, and extreme (23 1 C), humidity (45%), and lighting (12 h/d). The experiments aggression. Importantly, neuroactive steroids had been shown to were conducted according to the National Institute of Health Guide elicit anxiolytic and antidepressant properties in a variety of animal for the Care and Use of Laboratory Animals (NIH Publications No. models (Brinton, 2013; Eser et al., 2006, 2008; Pinna and 80-23, revised 1996). The experimental procedures were approved Rasmusson, 2012). by the institutional committee on animal care and use, and all ef- Recently, the role of the 18 kDa translocator protein (TSPO), forts were made to minimize animal suffering and reduce the which represents the starting point and an important rate-limiting number of animals used for the experiments. step in neurosteroidogenesis, has received increased attention in the pathophysiology of stress-response and stress-related disor- 2.2. Drugs and reagents ders (Beurdeley-Thomas et al., 2000; Pinna and Rasmusson, 2012). TSPO is a five trans-membrane domain protein that is located Desipramine (DMI), (DLX), PK11195, and Flumazenil Ò mainly in the outer mitochondrial membrane in peripheral tissues were purchased from Sigma-Aldrich (SIGMA , St. Louis, MO, USA). and the central nervous system (CNS). In the CNS, TSPO is mainly Diazepam (DZP) was purchased from Wako Pure Chemical In- located in the glial cells and mediates the translocation of choles- dustries Ltd. (Osaka, Japan). YL-IPA08 (Purity 99%) and AC-5216 (a terol from the outer to the inner mitochondrial membrane, which is selective TSPO ligand that has entered phase II clinical research, the rate-limiting step in the synthesis of steroids and neurosteroids Purity 99%) were both synthesized by the Department of Me- and is one of the most well-characterized functions of this protein. dicinal Chemistry at our institute. In the mitochondria, cholesterol is converted to pregnenolone by P450scc, which is located in the inner mitochondrial membrane. 2.3. Binding assays Pregnenolone then moves to the cytosol, where it is processed by several enzymes in a cascade of neurosteroidogenesis (Pinna et al., 2.3.1. TSPO-binding assay 2006; Papadopoulos et al., 2006a, b.). In humans, there is a decrease TSPO-binding assays were performed using crude mitochon- in platelet and lymphocyte TSPO levels in patients who have drial fractions that were prepared separately from rat cerebellum, a generalized anxiety disorder (Rocca et al., 1998). Certain TSPO li- rich TSPO-dense area in the primate brain. The rats were decapi- gands, such as AC-5216 (Emapunil, XBD173) and , have tated, and the cerebellums were dissected, homogenized in 10 been shown to enhance neurosteroidogenesis in the brain and to volumes of ice-cold HS buffer (10 mM HEPES buffer pH 7.4 con- exert anxiolytic/antidepressant activity in some rodent models taining 320 mM sucrose), and then centrifuged at 770 rpm for (Kita et al., 2004; Rupprecht et al., 2009, 2010). The anxiolytic/an- 10 min at 4 C, and the supernatant was centrifuged at 9000g for tidepressant effects of TSPO agents were related their ability to 15 min at 4 C. Then, the obtained pellet was resuspended in HS increase neurosteroid biosynthesis, as confirmed by studies in buffer (pH 7.4) and centrifuged at 13000g for 10 min. The crude which key enzyme blockers for neurosteroid biosynthesis, mitochondrial pellet was resuspended in 50 mM HEPES buffer (pH including finansteride and trilostane (Schüle et al., 2011; Kita and 7.4) and stored at 80 C until required. Furukawa, 2008), were used. Thus, TSPO ligands might represent Each crude mitochondrial preparation (50 mg) was incubated promising candidates for antidepressant and anxiolytic drugs in the with [3H]-PK11195 (final concentration 0.5 mM) and various 118 L.-M. Zhang et al. / Neuropharmacology 81 (2014) 116e125 concentrations of the test compounds in a total volume of 200 mL 2.5. Antidepressant-like effects for 60 min at 37 C, and the reaction was terminated by rapid filtration through 0.3% polyethylenimine pretreated Whatman 2.5.1. Forced swimming test in mice GF/C glass fiber filters, using a cell harvester (MB-48, Brandel). The traditional forced swimming test (FST) in mice was per- The reaction was terminated by rapid filtration through a GF/B formed following the protocol of Porsolt et al. (1978), with minor glass filter that was presoaked with 0.3% polyethyleneimine. The modifications. All of the mice received a single drug administration. filters were immediately washed with ice-cold 50 mM TriseHCl Sixty minutes after oral administration (p.o.), the mice were indi- buffer (pH 7.6), and the filter-bound radioactivity was quantified vidually placed in cylindrical containers (diameter 12 cm, height using a liquid scintillation analyzer (Tri Carb 2700 TR, Packard). 20 cm, containing 10 cm of water maintained at 25 C). The dura- Nonspecific binding was determined in the presence of 10 mM tion of immobility during the last 4 min of the total 6 min was AC-5216. All of the assays were performed in duplicate except for recorded. The mice were considered to be immobile when they the total binding and nonspecific binding, which were in floated motionless, making only the movement necessary to keep quadruplicate. their heads above the water.

2.3.2. CBR-binding assay 2.5.2. FST in rats CBR-binding assays were performed using crude mitochondrial The test was performed as described previously (Porsolt et al., fractions that were prepared separately from rat whole brain. The 1978). The procedure constituted two sessions, the pretest ses- whole brain from Male SD rats was homogenized in 10 volumes of sion and the test session, using the same apparatus and conditions ice-cold Tris-citrate buffer (50 mM buffer, pH 7.1) with a micro- (diameter 20 cm, height 40 cm, containing 25 cm of water main- homogenizer. Then, the homogenate was centrifuged at 40000g tained at 28 C). During the pretest session, the rats were forced to and 4 C for 10 min, and the resulting supernatant was centrifuged swim for 15 min; 24 h later, the rats were placed in the same at 40000g and 4 C for 10 min. Next, the obtained pellet was apparatus for 5 min, which was designated as a test session. The resuspended in TriseHCl buffer (pH 7.4) and centrifuged at 4000g duration of immobility (defined as described above) during this for 10 min. The pellet was resuspended in 50 nM TriseHCl buffer 5 min was recorded. Desipramine (DMI) or YL-IPA08 was admin- (pH 7.4) and frozen at 80 C until required. istered orally 1 h before the test session. The receptor preparations (100 mg) were incubated with [3H]- Ro151788 (final concentration 10 nM) and various concentrations 2.5.3. Tail suspension test in mice of the test compounds in a total volume of 20 mL for 20 min at 37 C. The tail suspension test (TST) in mice was performed as previ- The reaction was terminated by rapid filtration through a GF/B glass ously described (Steru et al., 1985), with minor modifications. The filter that was presoaked with 0.3% polyethyleneimine. The filters mice were suspended on the top of the apparatus using adhesive were immediately washed with ice-cold potassium phosphate tape placed approximately 1 cm from the tip of the tail. The dura- buffer (200 mM KCl, 20 mM KOH, 20 mM KH2PO4, pH 7.4), and the tion of immobility during the last 4 min of the total 6 min was filter-bound radioactivity was quantified using a liquid scintillation measured. The mice were judged to be immobile when they hung analyzer (Tris Carb 2700 TR, Packard). Non-specific binding was passively without moving. Duloxetine was used as a positive anti- determined in the presence of 10 mM flumazenil. All of the assays depressant drug and was p.o. 60 min before the test. were performed in duplicate. Also, the specific binding was deter- To investigate the role of TSPO in the behavioral effects of YL- mined by subtracting the non-specific binding from the total IPA08, the mice were treated with PK11195 (a potent TSPO binding. antagonist ligand, 3 mg/kg, i.p.) 30 min after YL-IPA08 (0.1 mg/kg, The specific binding was determined by subtracting the non- p.o.), and 30 min thereafter, the animals were challenged with the specific from the total binding. The IC50 values were determined TST. according to a nonlinear least-square curve-fitting method using GraphPad Prism (GraphPad Prism 5.0, version 2.0; GraphPad Soft- 2.6. Anxiolytic-like effects ware Inc., San Diego, CA). 2.6.1. Novelty suppressed feeding 2.4. Evaluation of in vitro pregnenolone or progesterone production This test was performed as described previously (Santarelli from YL-IPA08-treated astrocyte line et al., 2003) with a minor modification. After 24 h of food depri- vation, the mice were individually placed in a corner of the white, The rat astrocyte line CRL-2005TM (generation 6) was pur- plastic, open chamber (35 28 16 cm); the floor was covered by a chased from ATCC (American type culture collection). The source 1-cm thick layer of wooden bedding; four pellets of food (regular was Sprague-Dawley rat brain (diencephalon), and the cell type chow) were placed in the center of the floor. The latency to begin to was astrocyte (type 1 phenotype). The cells were cultured in chew the pellets was recorded for up to 5 min. Diazepam (DZP, DMEM, which was supplemented with 10% FBS at 37 C in hu- 2 mg/kg,) was i.p. 30 min before the test, and YL-IPA08 (0.1, 0.3, 1, midified air with 5% CO2. 3 mg/kg) was orally administered 1 h before the test session. The cells were seeded in 96-well plates at a density of approx- imately 10 000 cells/well in a final volume of 100 mL for 12 h until 2.6.2. Vogel-type conflict test in rats the cells adhered. For the correct assessment of PROG released into For this test, the method of Vogel and colleagues with a minor the cell medium and to exclude the interference of pregnenolone or modification was used in male SD rats. For the test operant, behavior progesterone presence in the FBS-containing culture medium, the boxes (50 30 20 cm) with a stainless steel grid floor (Columbus cells were washed twice with DMEM without serum and were then Co., Ltd) were used. A water bottle with a metal drinking tube was treated with different dosages of YL-IPA08（dissolved in DMEM, fitted outside the box so that only the drinking tube extended into with a final concentration of 0.5, 1, 2 mM）or AC-5216 (dissolved in the box. Electric shocks (0.3 mA, 2 s) were administered to each rat DMEM, with a final concentration 0.5, 1, 2 mM) for 2 h. Then, the by automatically switching the connections to the drinking tube and pregnenolone or progesterone levels that were secreted into the the grid floor from the drinkometer to an electric stimulator. After cell medium were determined by ELISA, under the conditions rec- 48 h of water deprivation, the rats were individually placed in the ommended by the supplier (Abnova, RB, USA). test chamber. The number of shocks that each rat received after L.-M. Zhang et al. / Neuropharmacology 81 (2014) 116e125 119 every 20 licks was recorded for 3 min. Diazepam (DZP, 2 mg/kg,) was Table 1 3 i.p. 30 min before the test, and YL-IPA08 (1, 3, 6,10 mg/kg) was orally Effects of YL-IPA08 and reference compounds on [ H]-PK11195 binding to the TSPO or [3H]-Flumazenil binding to the CBR. The data represent the mean SEM. The IC administered 1 h before the test session. 50 values were calculated from three independent experiments on different days. Each concentration was run in triplicate. 2.6.3. Elevated plus-maze test Compounds IC (nM) In this test, the mouse was placed in the center of the maze 50 (40 cm above the floor), facing an enclosed arm. The number of TSPO CBR entries into (with all four paws) and the time spent in both the open YL-IPA08 0.23 0.04 706.18 10.18 and enclosed arms (30 5 cm and 30 5 15 cm high, respec- AC-5216 0.65 0.02 67.4 6.82 tively) were recorded for 5 min, as described previously (Masood Flumazenil 742.41 12.6 9.91 0.48 et al., 2008). The percentages of entries and time spent in the open arms were calculated as open-arm entries, and the time was divided by total arm entries and total time, respectively. Diazepam 2.7.4. Subhypnotic pentobarbital-induced sleep test （DZP, 2 mg/kg) was i.p. 30 min before the test, and YL-IPA08 (0.03, Observers were blind to the drug treatment. The mice were 0.1, 0.3, 1, 3 mg/kg) was p.o. 60 min before the test. treated with diazepam (DZP, 2 mg/kg, i.p.) or YL-IPA08 (0.3e30 mg/ To investigate the role of TSPO in the behavioral effects of YL- kg, p.o.) and,1 h later, received an injection of pentobarbital (25 mg/ IPA08, the mice were treated with PK11195（1 mg/kg, i.p) 30 min kg, i.p). Following the pentobarbital injection, and 30 min or 60 min after YL-IPA08 (0.3 mg/kg, p.o.), and 30 min thereafter, the animals later, each mouse was observed with regard to onset of sleep, with were challenged with the elevated plus-maze test. To exclude the the criterion for sleep onset being placement on its back and loss of possible involvement of CBR in the behavioral effects of YL-IPA08, a righting reflex over 5 min. The percentage of sleep onset was the mice were treated with Flumazenil (a CBR antagonist, 10 mg/ calculated as follows: sleep onset ¼ No. of falling asleep/total no. kg, i.p) 30 min after YL-IPA08 (0.3 mg/kg, p.o.), and 30 min there- after, the animals were challenged with the elevated plus-maze 2.8. Statistical analysis test. All of the data were expressed as the mean SEM (binding 2.7. Evaluation of undesirable effects assays, FST, TST, novelty suppressed feeding, vogel-type conflict test, elevated plus-maze test, step-down passive avoidance test) or 2.7.1. Traction test incidence rate (traction test, rota-rod test, subhypnotic The apparatus was a horizontal stainless-steel bar (30 cm long, pentobarbital-induced sleep test). Comparisons between experi- 2 mm in diameter) that was set at a height of 15 cm above the mental and control groups were analyzed by one-way or two-way bench. The mice were suspended from the horizontal bar by their ANOVA followed by Dunnett’s t-test or Fisher’s exact probability forepaws. Failure to cling to the bar with their hindpaws within 5 s test, as indicated in the Results section. For all of the tests, differ- at least twice in three consecutive trials was regarded as positive ences with P < 0.05 were considered to be significant. for a muscle relaxation effect. This traction test was conducted 30 min and 60 min after diazepam (DZP, 2 mg/kg, i.p.) treatment or 3. Results oral administration of YL-IPA08 (0.3e100 mg/kg). 3.1. In vitro binding assays 2.7.2. Rota-rod test The method proposed by Dunham and Miya (1957) was used. In the TSPO-binding assay, YL-IPA08 showed a high affinity for The mice were placed on the horizontal rotating bar (diameter TSPO in the crude mitochondrial fraction prepared from rat cere- 2.5 cm, 12 rpm) of the rota-rod apparatus for 30 min or 60 min after bellum. As a parallel comparison, YL-IPA08 potency for binding to treatment with Diazepam（DZP, 2 mg/kg, i.p.) or YL-IPA08 (0.3e TSPO was higher than that of AC-5216. In the CBR-binding assay, 100 mg/kg, p.o.), respectively. The number of falls during a 5-min YL-IPA08 showed negligible affinity for CBR, while AC-5216 showed session was recorded. weak inhibition at CBR. Flumazenil, a well-known classic CBR antagonist, showed high affinity for CBR but very low affinity for 2.7.3. Step-down passive avoidance test TSPO. These results indicated that YL-IPA08 was more potent and The apparatus was a white acrylic open-topped box more selective to TSPO binding than AC-5216. The results are (30 30 50 cm3) with a grid floor (2-mm in diameter, with a 0.5- shown in Table 1. cm interval). A wooden platform (4 4 4 cm) was fixed at the corner of the grid floor. Electric shocks were delivered via the grid floor by an electric stimulator. Experiments constituted a training 3.2. YL-IPA08-stimulated pregnenolone and progesterone session and a retention session, which were separated by a 24-h production in rat astrocyte cells in vitro interval. In the training session, 30 min or 60 min after the treat- fi ment with Diazepam（DZP, 2 mg/kg, i.p.) or YL-IPA08 (0.3e30 mg/ After incubation with astrocyte cells for 2 h, AC-5216 signi cantly kg, p.o.), the mice were placed on the platform. When a mouse increased (in a concentration-dependent way) the level of pregnen- ’ < stepped down from the platform and placed all of its paws on the olone and progesterone in the supernatant (Dunnett s test, P 0.001 grid floor, electric shocks (38 V, 50 Hz) were delivered for 15 s vs. vehicle). Similarly, astrocyte cells were incubated with increasing fi through the grid floor. The training trials were repeated until the concentrations of YL-IPA08, and a signi cant increase in pregneno- mouse stayed on the platform for 60 s. After the training session, lone and progesterone production was also obtained (Fig. 2). the mice were returned to their home cages. At 24 h after the training session, the mice were placed on the platform again 3.3. Antidepressant-like effects of YL-IPA08 without an electric shock, and 30 min or 60 min after treatment with Diazepam (DZP, 2 mg/kg, i.p.) or YL-IPA08 (0.3e30 mg/kg, As shown in Fig. 3a and b, acute treatment with DMI (40 mg/kg, p.o.), the step-down latency was measured with a cut-off time of p.o.), a tricyclic antidepressant, significantly decreased the immo- 300 s. bility time in the FST in the rats and mice (P < 0.01, in rats; P < 0.05, 120 L.-M. Zhang et al. / Neuropharmacology 81 (2014) 116e125

when given orally at doses of 3e6 mg/kg, significantly increased the number of shocks that the rats received (Dunnett’s test, P < 0.05 vs. vehicle). Diazepam at a dose of 2 mg/kg also produced a significant increase in the number of shocks (P < 0.01 vs. vehicle) (Fig. 3f). The elevated plus-maze test is a well-established model for detecting both anxiolytic- and anxiogenic-like behavior; in this test, the proven anxiolytic diazepam at 2 mg/kg increased the percentage of both the time spent and the entries into in the open arms (P < 0.01 for the percentages of entries and time). A similar effect was produced by YL-IPA08 (0.1e1mg/kg),which increased the percentages of both the time spent and the entries into the open arms; the result was significant at a dose of 0.3 mg/ kg for the former and 0.1e1 mg/kg for the latter, compared with the corresponding vehicle. Neither YL-IPA08 nor diazepam, at the doses used, altered the total arm entries or the total time spent in arm exploration (Fig. 3g).Inaddition,inthismodel,we also found that the anxiolytic effect of YL-IPA08 (0.3 mg/kg, p.o.) was antagonized by PK11195 (1 mg/kg, i.p.), a TSPO antagonist, but not by Flumazenil (10 mg/kg, i.p.), a CBR antagonist, whereas the anxiolytic effect of diazepam (2 mg/kg, i.p.) was reversed by Flumazenil but not by PK11195. In addition, either PK11195 or Flumazenil did not affect the total arm entries or the total time Fig. 2. Stimulation of pregnenolone (PREG, a) and progesterone (PROG, b) production spentinarmexploration(Fig. 3h, i). These results indicated that from AC-5216 or YL-IPA08-treated rat astrocyte cells. The astrocyte cells were incu- the anxiolytic effect of YL-IPA08 was mediated by TSPO activa- fi bated for 2 h at 37 C in a humidi ed atmosphere of 5% CO2/95% air, with salt medium tion but not by CBR, which is consistent with its TSPO binding in the presence of an increasing concentration of YL-IPA08 (0.5, 1, 2 mM). After incu- selection. bation, the amount of pregnenolone or progesterone secreted into the salt medium was quantified by an ELISA test, as described in the Methods section. The values are the mean SEM of at least three independent experiments, performed in triplicate. 3.5. Profile of undesirable effects *P < 0.05, **P < 0.01 and ***P < 0.001 compared with vehicle control (one-way ANOVA followed by Dunnett’s t-test). 3.5.1. Traction test The muscle-relaxing effects of test compounds were assessed in in mice). Further post-hoc analysis revealed that YL-IPA08 at doses mice by studying their effect in the traction test. Neither vehicle nor of 1, 3, 10 mg/kg produced a marked reduction on the immobility YL-IPA08 at very large ranges of the doses tested (0.3, 3, and 30 mg/ time in the FST (Dunnett’s test, P < 0.05 vs. vehicle for the dose of kg) impaired the traction performance or the motor coordination of 10 mg/kg; P < 0.01 vs. vehicle for the dose of 1, 3 mg/kg). the mice at any of the time points that were examined (30 and Fig. 3c illustrates the acute effects of YL-IPA08 on the immobility 60 min after drug administration), which suggests that YL-IPA08 time in the TST in mice. Compared with vehicle, p.o. administration had no muscle relaxant activity. In contrast, diazepam (2 mg/kg, of the positive drug duloxetine at 10 mg/kg significantly decreased i.p.) caused traction impairment in a number of mice, indicating its the immobility time (Dunnett’s t-test, P < 0.05 vs. vehicle), which muscle-relaxing effect (P < 0.05 vs. vehicle) (Table 2). indicated that the behavioral tests were performed under normal conditions and were reliable. Further post-hoc analysis revealed 3.5.2. Rota-rod test that YL-IPA08 at doses of 0.1, 0.3 mg/kg produced a marked The motor coordination effects of YL-IPA08 were evaluated by reduction on the immobility time in the TST (Dunnett’s test, means of the rota-rod test. Diazepam (2 mg/kg, i.p.) affects the P < 0.01 vs. vehicle for the dose of 0.1 mg/kg; P < 0.05 vs. vehicle for motor coordination in a number of mice (P < 0.05 vs. vehicle). Both the dose of 0.3 mg/kg). In another set of experiments, the YL-IPA08 vehicle and YL-IPA08 at the very large ranges of doses tested (0.3, 3, (0.1 mg/kg)-induced decrease in the immobility time was antago- and 30 mg/kg) did not impair the motor coordination of the mice at nized by the TSPO antagonist PK11195 (3 mg/kg, i.p, P < 0.05 vs. YL- any of the time points examined (30 and 60 min after drug IPA08-treated-mice), which itself had no effect on the immobility administration) (Table 2). time (Fig. 3d). 3.5.3. Step-down passive avoidance test 3.4. Anxiolytic-like effects of YL-IPA08 It is well known that BZDs induce anterograde amnesia; test compounds were administered to the mice before the training In the mouse novelty suppressed feeding test, which is a widely session in which they acquired a passive avoidance response. A used test to assess the effects of anti-anxiety agents, DZP (2 mg/kg, retention test was carried out 24 h after the training session. YL- i.p., P < 0.05 vs. Vehicle) produced anxiolytic-like effects, as evi- IPA08, even at a very high dose relative to its effective dose on denced by the increased latency to feed. A similar result was behaviors (30 mg/kg, p.o.), had no significant decrease in the step- observed after acute YL-IPA08 treatment (Dunnett’s test, P < 0.05 down latency in the retention test at any of the doses that were vs. vehicle for the dose of 0.1 mg/kg; P < 0.01 vs. vehicle for the dose tested. Diazepam, on the other hand, significantly decreased the of 1, 3 mg/kg) (Fig. 3e). step-down latency in the retention test at doses of 2 mg/kg, i.p. The Vogel-type conflict test in rats is another paradigm that is (P < 0.01 vs. vehicle) (Fig. 4). widely used for anxiolytic testing; in this test, the anxiolytic effect is indicated by an increase in the number of shocks that the rats 3.5.4. Sub-hypnotic pentobarbital-induced sleep test receive as a result of inhibition of the conflict between the desire BZDs are widely used sedatives and hypnotics. In this test, to drink water and the aversion to an electric shock. YL-IPA08, Diazepam (2 mg/kg), the positive control used in this study, L.-M. Zhang et al. / Neuropharmacology 81 (2014) 116e125 121 significantly potentiated pentobarbital hypnosis (P < 0.01 vs. 4. Discussion vehicle). YL-IPA08, at a very high dose relative to its effective dose on behaviors (30, 60, and 100 mg/kg), did not affect the rate of sleep In the present study, we evaluated the pharmacological profile onset induced by subhypnotic dosage of pentobarbital, which was of YL-IPA08, a new potent TSPO ligand, in both receptor binding and also consistent with its TSPO binding selection (Table 2). behavioral studies. Our results demonstrate that YL-IPA08 has a

Fig. 3. Antidepressant-like and anxiolytic-like effects of YL-IPA08 on behavior in rats and mice. Treatment with YL-IPA08 decreased immobility in the rats forced swim (FST, a), mice FST (b), and tail-suspension tests (TST, c), which is indicative of antidepressant-like behavior. (d) Antagonism by PK11195 of the antidepressant effects of YL-IPA08 in the TST. The mice were pre-treated with PK11195 (a potent TSPO antagonist ligand, 3 mg/kg, i.p.) 30 min after YL-IPA08 (0.1 mg/kg, p.o.), and 30 min thereafter, the animals were challenged with the tail suspension test. (e) Treatment with YL-IPA08 increased the latency to feed in the mouse novelty suppressed feeding (NSF) test, increased the number of shocks in the Vogel drinking conflict test in rats (f), and increased the percentages of entries into and the time spent in the open arms in the elevated plus-maze test (g). These findings suggest that there are anxiolytic-like effects of the drug treatment on the behavior. (h) Effects of Flumazenil on the anxiolytic-like effects of YL-IPA08 in the EPM. The mice were pre-treated with Flumazenil (a potent CBR antagonist, 10 mg/kg, i.p.) 30 min after the YL-IPA08 (0.1 mg/kg, p.o.) treatment, and 30 min thereafter, the animals were challenged with the EPM test. (i) Antagonism by PK11195 on the anxiolytic-like effects of YL-IPA08 in the EPM. The mice were pre-treated with PK11195 (a potent TSPO antagonist, 1 mg/kg, i.p) 30 min after YL-IPA08 (0.3 mg/kg, p.o.) treatment, and 30 min thereafter, the animals were challenged with the EPM test. The data are expressed as the mean SEM (n ¼ 10). *P < 0.05, **P < 0.01, compared with the Vehicle control group (ANOVA followed by Dunnett’s t-test); #P < 0.05, ##P < 0.05 compared with YL-IPA08 alone-treated group (Wilcoxon’s rank- sum test). 122 L.-M. Zhang et al. / Neuropharmacology 81 (2014) 116e125

Fig. 3. (continued). more potent and selective affinity for TSPO. Furthermore, YL-IPA08 antidepressant-like potential of YL-IPA08 was evaluated using increased the concentrations of neurosteroids in the conditioned behavioral despair paradigms. It is well-accepted that behavioral medium from cultured rat astrocytes. Additionally, YL-IPA08 pro- despair paradigms, including TST in mice and FST in mice and rats, duced TSPO-mediated antidepressant-like and anxiolytic effects, are well-established measures for the evaluation of antidepressant without causing the undesirable effects that are associated with drugs; both are highly reliable and have the highest rates of pre- conventional BZDs. dictive validity for antidepressants. In these two models, YL-IPA08 In the in vitro binding assay, it was found that AC-5216 showed a showed antidepressant-like effects with a maximum effect com- high affinity for the TSPO from rat cerebellum. In a parallel com- parable to that of desipramine or duloxetine. In addition, the dose parison study, YL-IPA08 showed a relatively higher affinity to TSPO of desipramine or duloxetine were determined based on previous (IC50 ¼ 0.23 nM), which appears to be more potent than AC-5216 studies in which these compounds, given alone at the indicated (IC50 ¼ 0.65 nM), and a more negligible binding to CBR than AC- doses, showed good antidepressant-like effects. In support of our 5216 (706.18 vs. 67.4 nM), which indicates that YL-IPA08 should findings, other TSPO ligands, such as AC-5216, Ro5-4864 and be a more potent and selective TSPO ligand. These binding results FGIN1-27, have been reported to produce antidepressant-like ef- are also demonstrated by evaluating the behavioral effects and the fects in the mouse and rat forced swimming test (Schüle et al., 2011; undesirable effects. Kita et al., 2004; Gavioli et al., 2003). Additionally, the Depression, as well as anxiety, is often thought to be associated antidepressant-like effects of YL-IPA08 were totally blocked by with stress. Because TSPOs have been shown to be involved in PK11195. These results, together with those of the present study, stress responses, we first investigated the ability of YL-IPA08 to demonstrate that YL-IPA08-induced antidepressant-like effects produce antidepressant-like effects. In the present study, the were mediated by the TSPO, and TSPO ligands have the potential to

Table 2 Effects of YL-IPA08 in the traction test, the rota-rod test and on pentobarbital-induced sleep in mice. The data are expressed as the mean SEM (n ¼ 10). **P < 0.01 compared with Vehicle (Fisher’s exact probability test). *P < 0.05, **P < 0.01 compared with the Vehicle (Fisher’s exact probability test).

Compound Dose Traction test Rota-rod test Subhypnotic pentobarbital-induced sleep test (mg/kg) Muscle-relaxation/number of mice tested Number of falls/number of mice tested Sleep onset

30 min 60 min 30 min 60 min 30 min 60 min

Vehicle e 0/10 0/10 0/10 1/10 2/20 1/20 DZP 2 8/10* 2/10 5/10* 0/10 10/10** 10/10** YL-IPA08 0.3 0/10 0/10 0/10 0/10 ee 3 0/10 0/10 0/10 0/10 ee 30 0/10 0/10 1/10 1/10 2/20 1/20 60 eeee2/10 1/10 100 eeee1/10 0/10 L.-M. Zhang et al. / Neuropharmacology 81 (2014) 116e125 123

traction test, memory impairment in the passive avoidance test and prolonged hexobarbitone-induced sleep, at doses close to those that produced anxiolytic effects. In contrast, unlike diazepam, YL- IPA08 did not induce myorelaxant effects, memory impairment or potentiation of hexobarbitone-induced sleep even at doses of up to 100 mg/kg (p.o.). The results of the present study suggest that YL- IPA08, even when used across a very wide dose range, induces neither inhibition nor excitation of general CNS functions, which might be due to its high selectivity for the TSPO but not CBR. Taken together, the in vivo results observed in this study indicate that the antidepressant and anxiolytic effects of YL-IPA08 at the doses tested are mediated by the TSPO but not by the CBR, which Fig. 4. Effects of YL-IPA08 on the passive avoidance response in the step-down passive warrants a further pharmacological characterization of this avoidance test in mice. Diazepam (DZP, 2 mg/kg,) was given i.p. 30 min before the test, compound. and YL-IPA08 (0.3e30 mg/kg) was orally administered 1 h before the test session. The Neurosteroids also serve as neuromodulators at several other ¼ < data are expressed as the mean SEM (n 10). **P 0.01 compared with the Vehicle neurotransmitter receptors, such as glutamate and acetylcholine (ANOVA followed by Dunnett’s t-test). receptors, and influence emotion, memory/learning and stress re- sponses. Altered steroid production could contribute to the devel- produce antidepressant-like effects based on a novel mechanism opment and maintenance of psychiatric disorders, including that is distinct from existing antidepressants. anxiety states and depression (Nothdurfter et al., 2012; Eser et al., Our results from behavioral studies also demonstrated that YL- 2006, 2008; Costa et al., 2012). Some neurosteroids have been IPA08 induced anxiolytic effects in several animal models. A found to produce anxiolytic effects in several models of anxiety considerable amount of data have been published demonstrating (e.g., Romeo et al.,1993) and to induce antidepressant-like effects in that Vogel-type conflict test procedures reliably fulfill the criteria the forced swimming test in mice (Khisti et al., 2000; Schüle et al., established for animal models of anxiety. This procedure is capable 2011). In agreement with this idea, it has been shown that part of of reliably detecting almost all known classes of (Serra the action mechanism exerted by other conventional anxiolytic et al., 1999; Basso et al., 2011). By decreasing the shock level drugs is to act on specific enzymes of the neurosteroidogenic (thus increasing the number of shocks received by the control an- pathway and to normalize the levels of neurosteroids that posi- imals), it is also possible to detect the effects of anxiogenic com- tively modulate GABAAR activity (Griffin and Mellon, 1999; Guidotti pounds. In this model, a single treatment of YL-IPA08 as well as et al., 2001). Neurosteroids have also been reported to exert pro- diazepam produced an anti-conflict effect significantly. The NSF tective effects in numerous experimental models that mimic a va- test is a paradigm that is sensitive to treatment with acute BZDs riety of pathogenic aspects of brain dysfunction, such as (such as diazepam) or after 2e3 weeks of antidepressant treatment Alzheimer’s disease, stroke, and traumatic brain injury (Chen and (e.g., SSRIs) (Bodnoff et al., 1988; Dulawa and Hen, 2005), which Guilarte, 2008; Brinton, 2013). As discussed above, it is possible reduce the feeding latency that is increased by a novel environ- that newly synthesized neurosteroids are involved in the phar- ment. In this study, a single administration with YL-IPA08 signifi- macological effects of YL-IPA08. We, thus, studied the level of cantly decreased the latency to eat, which first suggested that the endogenous neurosteroids after the administration of YL-IPA08, to acute treatment YL-IPA08, a selective TSPO ligand, produced an substantiate this hypothesis. The treatment of YL-IPA08 with anxiolytic effect in this model. astrocyte cells significantly enhanced pregnenolone and proges- To further investigate the anxiolytic effects of YL-IPA08, the terone production. This finding is consistent with previous obser- elevated plus-maze test was used. In this model, YL-IPA08 vations, which demonstrate that treatment with selective TSPO increased both the entries and time spent in the open arms, ligands increased pregnenolone and progesterone production in which suggests that YL-IPA08 possesses an anxiolytic activity. steroidogenic cell lines such as Y-10 adrenocortical and C6 glioma Furthermore, YL-IPA08, at the same dose, did not influence the total cells (Guarneri et al., 1992; Romeo et al., 1992; Primofiore et al., number of arm entries. The findings of this test are consistent with 2004). a previous report that showed that a 21-day repeated treatment It is important to point out that several preclinical studies with 40-chlorodiazepam (Ro-5e4864), a potent TSPO ligand, re- observed that SSRIs such as fluoxetine or norfluoxetine were able to duces anxiolytic-like effects in the mouse elevated plus-maze test reverse the decrease of brain neurosteroid levels (such as Allo) and (Rägo et al., 1992). In this animal model, we also found that the YL- to correct behavioral deficits expressed by socially isolated mice IPA08-induced anxiolytic effects were antagonized by the TSPO even at dosages that are 50-fold lower than those required to cause antagonist PK11195 but not by the CBR antagonist flumazenil, an effective 5-HT reuptake inhibition (Pinna et al., 2003, 2004, which indicates that the anxiolytic effects of YL-IPA08 are mediated 2009; Pinna, 2010). On the basis of these considerations, these by TSPO. In contrast to YL-IPA08, diazepam-induced anxiolytic ef- drugs, which were originally termed ‘SSRI’ antidepressants, were fects were completely antagonized by flumazenil but not by suggested changing to the more appropriate term ‘selective brain PK11195. All of these results are consistent with the TSPO/CBR steroidogenic stimulants’ (SBSSs), which more accurately defines binding selection results above. In addition, it has been reported the pharmacological mechanisms (Pinna et al., 2006, 2009, 2012). that PK11195, at doses of 0.1e10 mg/kg (i.p.), had no anti-anxiety Many studies suggest the novel SBSSs, devoid of SSRI activity, but effects in any of the in vivo tests used but antagonized the effects that are potent neurosteroidogenic agents should be developed for of AC-5216. In addition, PK11195 at an i.v. dose of 4.2 mmol/kg has the treatment of psychiatric disorders that result from the down- no anti-anxiety effects and blocks the effects of FGIN1e27, a TSPO regulation of neurosteroid expression, including major depression, ligand, but not those of diazepam in rats (Romeo et al., 1993). These anxiety and in the prevention of PTSD. results are consistent with our present study. It is also worth noting that Allo is potent (nM affinity) positive BZDs cause several adverse effects through their action on the endogenous allosteric modulator of GABA action that acts at the CBR, although they have a pronounced anxiolytic effect. In the majority of synaptic and extrasynaptic GABAA receptor subtypes present study, diazepam produced myorelaxant effects in the (Bellelli and Lambert, 2005; Puia et al., 2003). In the socially 124 L.-M. Zhang et al. / Neuropharmacology 81 (2014) 116e125 isolated mouse model of behavioral deficits that resemble symp- Baldwin, D.S., Anderson, I.M., Nutt, D.J., Bandelow, B., Bond, A., Davidson, J.R., den toms of human anxiety disorders and PTSD, Allo levels decrease in Boer, J.A., Fineberg, N.A., Knapp, M., Scott, J., Wittchen, H.U., 2005. 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