In vitro and in vivo characterization of PA01, a novel promising triple reuptake inhibitor

Physiology & Behavior 138 (2015) 141–149

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In vitro and in vivo characterization of PA01, a novel promising triple reuptake inhibitor Jian Hou a,b, Yanli Xing a,b, Daiying Zuo a, Yingliang Wu a,⁎, Jingwei Tian b,c,⁎⁎, Qingguo Meng b,c, Mina Yang b a b c

School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016, PR China State key laboratory of Long-acting and Targeting Drug Delivery Technologies (luye Pharma Group Ltd.), Yantai, Shandong 264003, PR China School of Pharmacy, Yantai University, Yantai, Shandong 264005, PR China

H I G H L I G H T S • PA01 potently bound to the human 5-HT, NE, and DA transporters. • PA01 potently inhibited the reuptake of 5-HT, NE, and DA into recombinant cells. • PA01 displayed more potent antidepressant activity than desvenlafaxine succinate.

a r t i c l e

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Article history: Received 12 April 2014 Received in revised form 14 June 2014 Accepted 9 October 2014 Available online 20 October 2014 Keywords: Antidepressant activity PA01 Triple reuptake inhibitors Forced swimming test Tail suspension test

a b s t r a c t Triple reuptake inhibitors (TRIs) that inhibit the reuptake of serotonin (5-HT), norepinephrine (NE), and dopamine (DA) are being developed as a new class of antidepressants, which is hypothesized to produce more rapid onset and better efficacy than conventional antidepressants in part due to the addition of the DA component. 4[2-(dimethylamino)-1-(1-hydroxycyclohexyl)-ethyl]-phenyl benzoate hydrochloride (PA01), a novel compound, potently bound to the human 5-HT, NE, and DA transporters (Ki = 105, 644, and 813 nM, respectively), and inhibited the reuptake of 5-HT, NE, and DA into recombinant cells (IC50 = 341, 427, and 753 nM, respectively). In vivo, PA01 dose-dependently decreased immobility time in the forced swimming test (FST) in rats, and the tail suspension test (TST) in mice with higher efficacy than desvenlafaxine succinate (DVS), and showed no stimulatory effect on the spontaneous locomotor activity. The anti-immobility effect of PA01 in the TST was significantly prevented by the pretreatment of mice with DL-p-chlorophenylalanine (pCPA, 300 mg/kg, an inhibitor of serotonin synthesis), SCH23390 (0.05 mg/kg, s.c., dopamine D 1 receptor antagonist), and sulpiride (50 mg/kg, i.p., dopamine D2 receptor antagonist). PA01 significantly increased head-twitch response induced by 5-hydroxytryptophan (80 mg/kg, i.p., a metabolic precursor to serotonin) in rats, potentiated yohimbine (25 mg/kg, s.c., a α2-adrenoceptor antagonist) toxicity, and antagonized high dose apomorphine-induced hypothermia in mice. Taken together, these in vitro and in vivo results indicated that PA01 is a novel triple reuptake inhibitor, and exerts an excellent antidepressant activity in the behavioral despair animal models of depression, with more potent antidepressant activity than DVS at the same dose. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Depression is one of the most common mental disorders with high morbidity and mortality, representing a major social and economic burden [1,2]. Recent projections for direct and indirect economic costs of depression are projected to be near the top of the list for the year 2030 [3]. Unfortunately, although the antidepressants have been developed and used for several decades, there are still many shortcomings.

⁎ Corresponding author Yingliang Wu: Tel./fax: +86 24 23986278. ⁎⁎ Correspondence may also be addressed to Jingwei Tian: Tel./fax: +86 535 3808268. E-mail addresses: [email protected] (Y. Wu), [email protected] (J. Tian).

http://dx.doi.org/10.1016/j.physbeh.2014.10.007 0031-9384/© 2014 Elsevier Inc. All rights reserved.

The current prescribed antidepressants mainly include tricyclic antidepressants (TCAs), monoamine oxidase inhibitors (MAOIs), selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), norepinephrine reuptake inhibitors (NARIs), serotonin antagonist and reuptake inhibitor (SARIs), and noradrenergic and specific serotonergic antidepressants (NaSSAs). All these antidepressants have serious drawbacks such as limitations in efficacy, slow onset, and severe side effects [4–7]. Therefore, it is urgent to explore more promising antidepressants for the clinical needs of depressed patients. The dopaminergic system plays an important role in the pathophysiology of depression [8–11]. A recent study examined effects of treatment with duloxetine, a 5-HT/NE reuptake inhibitor, in combination with the

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bupropion, a DA/NE reuptake inhibitor, in a small percentage of patients diagnosed with a major depressive disorder who had not achieved symptom remission with either treatment alone. The combination of these drugs resulted in a significant improvement in depressive symptoms in this group of patients, suggesting that a drug targeting serotonergic, noradrenergic, and dopaminergic neurotransmission simultaneously would likely be more effective in reducing depression [12,13]. Furthermore, bupropion, combined with selective 5-HT-reuptake inhibitors, reduced the sexual side effects associated with this class of drugs [14]. Given these findings, the TRI strategy represents a promising approach to developing a superior antidepressant with better efficacy and fewer side effects. 4-[2-(Dimethylamino)-1-(1-hydroxycyclohexyl) ethyl] phenyl benzoate hydrochloride (PA01, Fig. 1), a novel compound, was synthesized based on the structure of O-desmethylvenlafaxine (ODV) by the Department of Chemistry. In the present study, we performed a detailed investigation of the pharmacological characteristics of PA01 in vitro and in vivo. First, we determined the binding affinity of PA01 to human 5-HT, NE, and DA transporters (hSERT, hNET, and hDAT), and the inhibitory activity on the reuptake of 5-HT, NE, and DA into recombinant CHO cells; then, we evaluated antidepressant activity of PA01 in the FST in rats and the TST in mice; finally, to further validate our in vitro test results, we investigated the effects of PA01 on serotonergic, noradrenergic, and dopaminergic systems in vivo. 2. Materials and methods 2.1. Animals Male Sprague-Dawley rats (180–220 g) and male Kunming mice (18–22 g) were obtained from Shandong Luye Pharmaceutical Company (Yantai, PR China). The animals were housed in a temperature and humidity controlled facility (22.0 ± 2.0 °C, 50 ± 10% relative humidity) with a 12-h light/dark cycle and free access to food and water. All experiments in this study were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23) revised 1996. 2.2. Drugs and reagents PA01 (molecular formula: C23H29NO3·HCl, molecular weight: 403.94, purity N 99.3%) and desvenlafaxine succinate (DVS) were provided by State Key Laboratory of Long-acting and targeting Drug Delivery Technologies (Yantai, PR China). DOV21947 was provided by Shanghai Institute

of Pharmaceutical Industry. GBR12909 dihydrochloride, imipramine hydrochloride, desipramine hydrochloride, fluoxetine hydrochloride, 5-hydroxytryptophan (5-HTP), DL-p-chlorophenylalanine (pCPA), yohimbine hydrochloride, apomorphine hydrochloride (APO), (R)-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5tetrahydro-1H-3-benzazepine hydrochloride (SCH23390), sulpiride, pargyline, and tropolone were purchased from Sigma-Aldrich (St. Louis, MO, USA). [3H]imipramine, [3H]nisoxetine, [3H]WIN35428, hSERT, hNET, hDAT, [3H]5-HT, [3H]NE, [3H]DA, and Microscint 20 scintillation fluid were purchased from Perkin-Elimer Life Sciences (NEN, Boston, MA). Transfected CHO cells were provided by Shanghai Institute of Materia Medica, Chinese Academy of Science.

2.3. Experimental designs Fig. 2 displays an overview of the study designs. We previously reported that DVS is effective at 7.5 mg/kg on FST in rats [15]. In this study, we selected 8 mg/kg to study the efficacy of DVS in rats. If the conversion of the rat dose (8 mg/kg) to the corresponding mouse equivalent doses were to be based on body surface area, as recommended by FDA Guidance for Industry, “Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers” (July 2005), the dose was about 16 mg/kg in mice. When these doses of PA01 at 1, 2, 4, 8, and 16 mg/kg in rats were converted into mice doses, they were about 2, 4, 8, 16, and 32 mg/kg in mice. The previous time–response relationship indicated that PA01 produced the maximum anti-immobility effect occurring 60 min post administration on both FST at 8 mg/kg in rats, and TST at 16 mg/kg in mice (unpublished data). Therefore, in this study, we selected the endpoint at 60 min post administration of PA01.

2.4. In vitro experiments 2.4.1. Binding to human transporters The competitive binding assays of PA01 to hSERT, hNET, and hDAT were performed as previously described [15,16]. [ 3 H]imipramine (1.5 nM), [ 3 H]nisoxetine (0.5 nM), and [ 3H]WIN35428 (2.5 nM) were used as radioligands for hSERT, hNET, and hDAT, respectively. Test compounds (PA01, DVS, and DOV21947) were tested in triplicate at 6 different concentrations, spanning 3 orders of magnitude. Non-specific binding was defined with imipramine (10 μM), desipramine (10 μM) and GBR12909 (10 μM) for hSERT, hNET, and hDAT, respectively. The specified experiment conditions are listed in Table 1.

ODV

PA01 Fig. 1. Chemical structure of ODV and PA01.

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Fig. 2. An overview of the experimental design.

2.4.2. Reuptake assays on human transporters The reuptake assays were performed on CHO cells stably expressing hSERT, hNET, and hDAT as previously described with minor modifications [17]. Cells were seeded in opaque 24 well-plates in RPMI-1640 culture medium (plus 10% FCS, 1.25 mg/kg geneticin, 10,000 units/ml penicillin, 10,000 units/ml streptomycin, 29.2 mg/ml L-glutamine; Invitrogen, Gibco, USA), and reuptake assays were run once cells reached 80–90% confluence. Removing the culture medium, cells were rinsed twice with phosphate-buffered saline (PBS) and incubated with test compounds (PA01, DVS, and DOV21947) or no drug at 37 °C for 10 min in HBSS containing 10 μM of pargyline, ascorbate, and tropolone before adding [3H]5-HT, [3H]NE, and [3H]DA. Following the addition of the radiolabeled neurotransmitter (20 μl, approximately 20,000 cpm), cells were gently mixed and then incubated at 37 °C for 5 min. Reactions were terminated by rapid aspiration of the reuptake buffer and rapidly rinsed twice with ice-cold HBSS. The cell layer was incubated with a Microscint 20 scintillation buffer (500 μl/well) for at least 2 h under gentle shaking. Radioactivity incorporated in the cells was determined for each well by liquid scintillation counting using a multifunctional liquid scintillation counter (LS-6500, Beckman). Non-specific reuptake was defined with fluoxetine (10 μM), desipramine (10 μM), and GBR12909 (10 μM) for hSERT, hNET, and hDAT experiments, respectively. Experiments were performed in triplicate in three independent experiments. The specified experimental conditions are listed in Table 1.

2.5. In vivo experiments 2.5.1. Forced swimming test (FST) in rats The FST is widely used to screen novel compounds for potential antidepressant activity [18]. As previously described [19], seventy

naive rats were randomly assigned to seven groups (n = 10/group). The procedure consisted of a pre-test and a test session separated by 24 h, using the same cylinder and conditions (height, 36 cm; internal diameter, 29 cm; containing 24 cm of water at 24 ± 1 °C). During the pre-test session, rats (adapted to the test room for at least 1 h) were individually placed in the cylinder for 15 min. Following this initial exposure, the rats were dried with towels and transferred to a drying cage situated under a warming lamp. The following day, the rats were transferred to the test room and reacclimated for no less than 1 h. Rats were administered intragastrically (i.g.) with DVS (8 mg/kg), PA01 (1, 2, 4, 8, and 16 mg/kg) or vehicle (0.5% sodium carboxymethyl cellulose) 60 min before testing and then placed in the test chambers. The duration of immobility was recorded during the last 5 min of the 6 min test period. The rat was considered immobile when floating in the water without struggling and making only those movements necessary to keep its head above water. The operator's attributing FST scores were blind to the treatments.

2.5.2. Tail suspension test (TST) in mice The TST in mice seems to be a corroboration of the FST, with possible sensitivity to a broader range of antidepressants [20]. As previously described [21], seventy naive mice were randomly assigned to seven groups (n = 10/group), and were administered intragastrically with DVS (16 mg/kg), PA01 (2, 4, 8, 16, and 32 mg/kg) or vehicle 60 min before testing. Mice were then individually suspended by their tails 35 cm above the tabletop by adhesive tape placed approximately 1 cm from the tip of the tail. The duration of immobility was recorded during the last 5 min of the 6 min test period. A mouse was considered immobile only when hanging passively and completely motionless. The operator's attributing TST scores were blind to the treatments during the experiment.

Table 1 Specified experiment condition in binding and uptake assays. Assays

Source

[3H]Ligand

Non-specific ligand (μM)

Reaction condition

hSERT binding hNET binding hDAT bindng 5-HT uptake NE uptake DA uptake

CHO CHO CHO CHO CHO CHO

Imipramine (1.5 nM) Nisoxetine (0.5 nM) WIN 35428 (2.5 nM) 5-HT (10 μM) NE (10 μM) DA (10 μM)

Imipramine (10) Desipramine (10) GBR 12909 (10) Fluoxetine (10) Desipramine (10) GBR 12909 (10)

25 °C, 30 min 25 °C, 30 min 4 °C, 60 min 37 °C, 5 min 37 °C, 5 min 37 °C, 5 min

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2.5.3. Spontaneous locomotor activity In order to assess the possible effects of PA01 on spontaneous locomotor activity, sixty naive rats and sixty naive mice were randomly assigned to six groups (n = 10/group), respectively. Rats and mice were transferred into the test room at least 2 h prior to drug administration (the doses and time corresponding to the FST and the TST). Fiftyfive minutes after oral administration of DVS, PA01 or vehicle, animals were placed individually into the corner of each test chamber (50 × 50 × 45 cm for rats, 25 × 25 × 30 cm for mice) for a 5 min acclimation period. Sixty minutes after the administration of drugs, spontaneous locomotor activity was recorded for 30 min using a tracking and computerized analysis system (BehaviorSys 2.20, Shanghai Mobile Datum Information Technology Co., Ltd., China). After subcutaneous (s.c.) injection of APO (0.25 or 0.5 mg/kg), spontaneous locomotor activity was measured immediately. The area was wiped clean with 75% alcohol solution between the tests of each animal. 2.5.4. Investigation of the serotonergic system in the antidepressant activity of PA01 2.5.4.1. 5-HTP-induced head-twitch response in rats. To reveal whether the serotonergic system is involved in the antidepressant activity of PA01, 5-HTP-induced head-twitch response was performed as previously described [22], with minor modifications. Fifty naive rats were randomly assigned to five groups (n = 10/group), rats were administered intragastrically with DVS (8 mg/kg), PA01 (4, 8, and 16 mg/kg) or vehicle 40 min before intraperitoneal (i.p.) injection of 5-HTP (80 mg/kg). Immediately after receiving 5-HTP, rats were placed individually in a plastic cage and the number of head twitches was recorded from 20 to 40 min after the injection of 5-HTP (Fig. 3). 2.5.4.2. The depletion of 5-HT in mouse TST. To further study the possible serotonergic mechanism in the antidepressant activity of PA01, One hundred naive mice were randomly assigned to ten groups (n = 10/group). Mice were pretreated with pCPA (300 mg/kg, i.p., an inhibitor of serotonin synthesis) or 0.9% saline once a day for three consecutive days, respectively. On the fourth day, mice received oral administration of DVS (16 mg/kg), PA01 (8, 16, and 32 mg/kg) or vehicle 60 min prior to the TST testing. The procedure and dose of pCPA were based on previous studies [23].

mice were randomly assigned to five groups (n = 10/group), and were treated with DVS (16 mg/kg, i.g.), PA01 (8, 16, and 32 mg/kg, i.g.) or vehicle orally 30 min prior to APO (16 mg/kg, s.c). The rectal temperature of the mice was measured using a commercially available electronic thermometer (Dierte Ltd., China), which was dipped in glycerine, and inserted about 1 cm in the gently hand-restrained mouse. Rectal temperature was measured at three time points: T0 min, prior to administering any drugs, as initial body temperature; T30 min, 30 min after administration of drugs, namely, just before the injection of APO, to study the effect of the drugs on basal body temperature; T60 min, 30 min after the APO injection, in order to assess the effect of the drugs on APOinduced hypothermia. During the entire experiment, the ambient room temperature was strictly maintained at 22 ± 1 °C. 2.5.6. Investigation of the dopaminergic system in the antidepressant activity of PA01 We also illustrated the influence of the dopaminergic system in the antidepressant activity of PA01. As previously described [26], sixty naive mice were randomly assigned to six groups (n = 10/group), and were pretreated with SCH23390 (0.05 mg/kg, s.c., a dopamine D1 receptor antagonist), Sulpiride (50 mg/kg, i.p., a dopamine D2 receptor antagonist) or saline. After 30 min they received PA01 (32 mg/kg, i.g.) or vehicle and were tested in the TST 60 min later. Spontaneous locomotor activity was determined to investigate whether the administration of PA01 with SCH23390 or sulpiride impairs motor abilities. 2.6. Statistical analysis In vitro experiments, the transporter binding and monoamine uptake results were analyzed using one-site nonlinear regression of concentration-effect curve by GraphPad Prism (version 5.0). The ki values were calculated from the Cheng–Prusoff equation: Ki = IC50 / [(L / KD) + 1] [27]. In vivo experiments, results are expressed as mean ± standard error (S.E.M.) and analyzed by SPSS statistical software (version 17.0). A chi-square test was used to analyze the data in the yohimbine toxicity potentiation test. One or two-way analysis of variance (ANOVA) followed by Tukey's test was used to compare the differences among groups. A value of P b 0.05 was considered statistically significant. 3. Results

2.5.5. Investigation of the noradrenergic system in the antidepressant activity of PA01

3.1. Results in in vitro experiments

2.5.5.1. Yohimbine toxicity potentiation test in mice. To evaluate a possible contribution of the noradrenergic system in the antidepressant activity of PA01, a test of yohimbine toxicity potentiation in mice was performed [24]. Seventy-five naive mice were randomly assigned to five groups (n = 15/group), and were treated with DVS (16 mg/kg), PA01 (8, 16, and 32 mg/kg) or vehicle orally 60 min before yohimbine (25 mg/kg, s.c., a α2-adrenoceptor antagonist). The number of dead mice was recorded during a 24 h period after the injection of yohimbine.

3.1.1. Binding of PA01 to human monoamine transporters PA01 potently inhibited the binding of [3H]imipramine to hSERT, 3 [ H]nisoxetine to hNET, and [3H]WIN35428 to hDAT, with Ki values of 105 ± 13, 644 ± 137, and 813 ± 107 nM, respectively (Table 2). DOV21947 displayed higher affinity for hSERT, hNET, and hDAT than PA01, with Ki values of 27.4 ± 4.2, 221 ± 29, and 149 ± 33 nM, respectively. The Ki value of PA01 to hSERT is significantly less than DVS (7.84 ± 0.6 nM).

2.5.5.2. Antagonism of APO-induced hypothermia in mice. To further investigate the possible effect of PA01 on the noradrenergic system, a high dose of APO-induced hypothermia test was performed [25]. Fifty naive

3.1.2. Inhibition of PA01 on [3H]monoamines uptake PA01 concentration dependently and potently inhibited the uptake of [3H]5-HT, [3H]NE, and [3H]DA into human recombinant cells, with

Fig. 3. An outline of the design of the 5-HTP induced head-twitch test.

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Table 2 The Ki or IC50 values (nM) for binding to human transporters and inhibitory activity on the reuptake of monoamines into the recombinant cells. Compound

PA01 DVS DOV21947

Binding assay (Ki, nM)

Reuptake assay (IC50, nM)

hSERT

hNET

hDAT

hSERT

hNET

hDAT

105 ± 13 7.84 ± 0.6 27.4 ± 4.2

644 ± 137 1250 ± 197 221 ± 29

813 ± 107 8549 ± 757 149 ± 33

341 ± 36 56.5 ± 6.3 71.8 ± 9.2

427 ± 28 583 ± 39 168 ± 18

753 ± 97 N10,000 217 ± 31

Results are expressed as mean ± S.E.M., the Ki or IC50 values were calculated from three independent experiments. Each concentration was run in triplicate.

IC50 values of 341 ± 36, 427 ± 28, and 753 ± 97 nM, respectively (Table 2). The IC50 values for DOV21947 at inhibiting uptake of [3H] monoamines were 71.8 ± 9.2, 168 ± 18, and 217 ± 31 nM, respectively. DVS inhibited the uptake of [3H]5-HT and [3H]NE into recombinant cells with IC50 values 56.5 ± 6.3 and 583 ± 39 nM, respectively.

3.2. Results in in vivo experiments 3.2.1. Effect of PA01 in the FST in rats As shown in Fig. 4, one-way ANOVA showed statistically significant differences in the immobility time among the groups in FST [F (6, 63) = 9.01, P b 0.01]. Post hoc analysis indicated that PA01 (4, 8, and 16 mg/kg) produced a significant reduction in immobility time by 28%, 35%, and 43% as compared with the vehicle control group, respectively (P b 0.05 or P b 0.01). Moreover, PA01 (8 mg/kg) exhibited more potency than DVS at the same dose (P b 0.05).

3.2.2. Effect of PA01 in the TST in mice As shown in Fig. 5, one-way ANOVA showed statistically significant differences in the immobility time among the groups in TST [F (6, 63) = 7.04, P b 0.01]. Post hoc analysis indicated that PA01 (8, 16, and 32 mg/kg) produced a significant reduction in the immobility time by 33%, 41%, and 54% as compared with the vehicle control group, respectively (P b 0.05 or P b 0.01). Moreover, PA01 (16 mg/kg) exhibited more potency than DVS at the same dose (P b 0.05).

Fig. 4. Effect of PA01 on the amount of immobility time in rats during the FST. Rats received oral administration of PA01, DVS, or vehicle 60 min prior to the FST. Values are expressed as mean ± S.E.M. (n = 10/group). One-way ANOVA followed by Tukey's test: ⁎p b 0.05, ⁎⁎p b 0.01 as compared with the vehicle control group; #P b 0.05 as compared with the DVS group.

3.2.3. Effect of PA01 on spontaneous locomotor activity As shown in Fig. 6, one-way ANOVA showed statistically significant differences in the immobility time among the groups in rats [F (5, 54) = 22.64, P b 0.01] and in mice [F (5, 54) = 4.01, P b 0.01]. Post hoc analysis revealed that PA01 did not significant increase the spontaneous locomotor activity in rats and mice as compared with the vehicle control group, respectively; by contrast, the positive drug APO (0.25 or 0.5 mg/kg, s.c.) significantly stimulated spontaneous locomotor activity in rats and mice (P b 0.01). 3.2.4. Involvement of the serotonergic system 3.2.4.1. Effect of PA01 on 5-HTP-induced head-twitch response. As shown in Fig. 7, one-way ANOVA showed statistically significant differences in the cumulative number of 5-HTP-induced head twitches [F (4, 45) = 12.00, P b 0.01]. Post hoc analysis revealed that PA01 (4, 8, and 16 mg/kg) significantly increased the cumulative number of head twitches in rats as compared with the vehicle control group (P b 0.01 or P b 0.05). 3.2.4.2. Effect of PA01 on the immobility time of pretreatment with pCPA in the TST. As shown in Fig. 8, one-way ANOVA showed statistically significant differences in the immobility time among the groups in TST [F (9, 90) = 7.35, P b 0.01]. Post hoc analysis showed that PA01 (8, 16, and 32 mg/kg) treatment induced a significant reduction in the immobility time (P b 0.05 or P b 0.01). pCPA alone did not affect the immobility time. However, pretreatment with pCPA (300 mg/kg, i.p.) significantly attenuated the anti-immobility action of PA01 at doses of 8 and

Fig. 5. Effect of PA01 on the amount of immobility time in mice during the TST. Mice received oral administration of PA01, DVS, or vehicle 60 min prior to the TST. Values are expressed as mean A0S.E.M. (n = 10/group). One-way ANOVA followed by Tukey's test: ⁎p b 0.05, ⁎⁎p b 0.01 as compared with the vehicle control group; #P b 0.05 as compared with the DVS group.

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Fig. 6. Effect of PA01 on spontaneous locomotor activity in rats (A) and mice (B). Rats or mice received oral administration of PA01, APO, DVS or vehicle, and locomotor activity was measured at 60 min after administration for the next 30 min. Values are expressed as mean ± S.E.M. (n = 10/group). One-way ANOVA followed by Tukey's test: ⁎⁎p b 0.01 as compared with the vehicle control group.

16 mg/kg (P b 0.05 or P b 0.01). PA01 at a dose of 32 mg/kg remained active (P b 0.05), despite pCPA pretreatment. 3.2.5. Involvement of the noradrenergic system 3.2.5.1. Effect of PA01 on APO-induced hypothermia. As shown in Fig. 9, one-way ANOVA showed that the temperature variation among groups was not observed at T0 min [F (4, 45) = 0.07, P = 0.99] or T30 min [F (4, 45) = 0.27, P = 0.89]. The maximal change in temperature was found at 30 min after APO (16 mg/kg, s.c.) injection [F (4, 45) = 5.38, P b 0.01]. Post hoc analysis showed that PA01 significantly antagonized the hypothermic response induced by APO in mice (P b 0.01). 3.2.5.2. Effect of PA01 on the yohimbine toxicity potentiation test. As shown in Table 3, the mortality among groups showed statistical differences (Chi-square = 8.73, df = 4, P b 0.05). Pretreatment with PA01 significantly potentiated yohimbine-induced lethality in a dose-dependent

Fig. 7. Effect of PA01 on the number of 5-HTP-induced head response in rats. Rats orally administrated PA01, DVS, or vehicle 40 min prior to injection of 5-HTP (80 mg/kg). Values are expressed as means ± S.E.M. (n = 10/group). One-way ANOVA followed by Tukey's test: ⁎p b 0.05, ⁎⁎p b 0.01 as compared with the vehicle control group.

manner (P b 0.05). The mortality of PA01 at 8, 16, and 32 mg/kg groups were 46.7%, 66.7%, and 80%, respectively. The mortality of the DVS (16 mg/kg) group was 60%. 3.2.6. Effect of dopaminergic receptor antagonists on the action of PA01 in mice TST SCH23390 (0.05 mg/kg, s.c.) per se did not have any effect on the immobility time in TST. SCH23390 pretreatment, however, was able to reverse the anti-immobility effect of PA01 (Fig. 10A). A two-way ANOVA showed significant differences for PA01 treatment [F (1, 36) = 27.54, P b 0.01], SCH23390 pretreatment [F (1, 36) = 13.58, P b 0.01] and PA01 × SCH23390 interaction [F (1, 36) = 11.32, P b 0.01] on immobility time in the TST. Post hoc analyses indicated that the anti-immobility effect of PA01 (32 mg/kg) was reversed by pretreatment with SCH23390. Moreover, sulpiride (50 mg/kg, i.p.) per se did not have any effect on the immobility time in TST. Sulpiride pretreatment, however, was also able to reverse the anti-immobility effect of PA01 (Fig. 10B). A two-way ANOVA showed significant differences for PA01 treatment [F (1, 36) = 15.30, P b 0.01], sulpiride pretreatment [F (1, 36) = 8.93, P b 0.01] and PA01 × sulpiride interaction [F (1, 36) = 7.81, P b 0.01].

Fig. 8. Effect of PA01 on the immobility time of pretreatment with pCPA in the TST. Mice were pretreated with pCPA (300 mg/kg, i.p.) once a day for three consecutive days. On the fourth day, drugs or vehicle were administered 60 min prior to testing. Values are expressed as means ± S.E.M. (n = 10/group). One-way followed by Tukey's test: ⁎p b 0.05, ⁎⁎p b 0.01 as compared with the vehicle control group; #p b 0.05, ##p b 0.01 compared with the same group pretreated with saline.

J. Hou et al. / Physiology & Behavior 138 (2015) 141–149

Fig. 9. Effect of PA01 on APO-induced hypothermia in mice. Drugs or vehicle were administered 30 min prior to the injection of APO (16 mg/kg, s.c.). Rectal temperature of mice was measured at 0, 30 and 60 min after administration of PA01. Values are expressed as mean ± S.E.M. (n = 10/group). One-way ANOVA followed by Tukey's test: ⁎p b 0.05, ⁎⁎p b 0.01 as compared with the vehicle control group.

Post hoc analyses showed that the anti-immobility effect of PA01 (32 mg/kg) was also reversed by pretreatment with sulpiride. As shown in Table 4, PA01 (32 mg/kg) administered alone or in combination with SCH23390 (0.05 mg/kg, s.c.) did not significantly cause changes in the spontaneous locomotor activity of mice. A twoway ANOVA revealed no significant effects of PA01 treatment [F (1, 36) = 1.37, P = 0.25], SCH23390 pretreatment [F (1, 36) = 0.05, P = 0.83] and PA01 × SCH23390 interaction [F (1, 36) = 0.10, P = 0.76]. Similarly, sulpiride (50 mg/kg, i.p.) alone or in combination with PA01 (32 mg/kg) did not significantly alter the spontaneous locomotor activity of mice. A two-way ANOVA showed no significant effects of PA01 treatment [F (1, 36) = 1.17, P = 0.29], sulpiride pretreatment [F (1, 36) = 0.32, P = 0.56] and PA01 × sulpiride interaction [F (1, 36) = 0.33, P = 0.51]. 4. Discussion In this study, we first characterized the in vitro and in vivo pharmacological profile of PA01, a structurally novel compound, and provided convincing evidence that PA01 was a novel triple reuptake inhibitor with significant antidepressant properties. Compared with DVS, a commonly prescribed SNRI, PA01 displayed more potent inhibitory activity on NE and DA uptake in vitro, and higher antidepressant efficacy through the modulation of monoaminergic systems in vivo. In the in vitro study, we evaluated and compared the binding profile of PA01 with DVS and DOV21947 at human monoamine transporters.

Table 3 Effect of PA01 on yohimbine-induced lethality in mice. Groups

Dose (mg/kg)

Yohimbine (mg/kg)

Total

Died

Lethality (%)

Vehicle DVS PA01 PA01 PA01

– 16 8 16 32

25 25 25 25 25

15 15 15 15 15

5 9⁎ 7 10⁎ 12⁎

33.3 60.0⁎ 46.7 66.7⁎ 80.0⁎

Drugs or vehicle were administered 60 min prior to the injection of yohimbine (25 mg/kg, s.c.). ⁎P b 0.05 compared with vehicle control group (Chi-square test).

147

PA01 displayed high affinity for hSERT, hNET, and hDAT. In comparison with DVS, PA01 showed about 2-fold and 10-fold higher affinity for hNET and hDAT, respectively, whereas about 13-fold lower affinity for hSERT. Compared with DOV21947, PA01 displayed lower affinity for three monoamine transporters. Consistent with the results from binding assays, the data from functional studies for the transporters showed a similar pattern in the capacity of PA01 to inhibit the monoamine reuptake using recombinant CHO cells. DVS had no effect on DA uptake, whereas PA01 exerted a more potent inhibitory effect on [3H]DA uptake. The study showed that PA01 is most potent in inhibiting 5-HT uptake, followed by NE and DA uptake (Table 2). These in vitro data indicate that PA01 is a novel triple reuptake inhibitor. In recent years, both preclinical studies and clinical trials indicate that triple reuptake inhibitors may produce a faster onset of action, or provide greater efficacy and fewer side effects than traditional antidepressants [28–30]. We believe that such a “broad spectrum” antidepressant could represent a positive breakthrough in the treatment of depression. The optimum preclinical profile for a TRI, defined as maximum antidepressant efficacy and minimum side effects, cannot be accurately predicted based on the in vitro method alone. Therefore, we used a behavioral screening approach to supplement the data for identifying TRIs with favorable in vivo profiles. In tests predictive of antidepressant activity in humans, the FST in rats and the tail suspension test in mice were used. These tests are predictive of clinically effective antidepressants [31]. In this study, PA01 significantly decreased the immobility time in FST at 4, 8, and 16 mg/kg (Fig. 4) and TST at 8, 16, and 32 mg/kg (Fig. 5). Moreover, PA01 exhibited more potency than DVS at the same dose. In the FST and TST, false positive results can be obtained for compounds that stimulate spontaneous locomotor activity [18]. Therefore, spontaneous locomotor activity was studied to exclude this stimulation effect of PA01 itself. The results showed that PA01 did not increase the spontaneous locomotor activity in rats at 4, 8, and 16 mg/kg and in mice at 8, 16, and 32 mg/kg. These findings proved that PA01induced decrease in the immobility time in the despair tests was not relevant to a psychostimulant effect, but rather an antidepressant activity. It is well known that monoamine neurotransmitters play important roles in the pathophysiology and treatment of depression [32,33]. To further validate our in vitro test results, we studied the effects of PA01 on serotonergic, noradrenergic, and dopaminergic systems in vivo. The serotonergic system plays a major role in the etiology of depression, and drugs acting on the serotonergic system have been largely implicated in the treatment of depressive disorders [32,34]. 5-HTP (a precursor to serotonin)-induced head-twitch response was commonly used to investigate the effects of antidepressant drugs on serotonergic function [35]. It was reported that pCPA pretreatment induced a significant reduction of 5-HT (over 60%) in the whole brain, whereas NE and DA levels were not affected [36]. In the present study, PA01 significantly enhanced the number of 5-HTP-induced head twitches. Meanwhile, the anti-immobility effect of PA01 in mouse TST was significantly attenuated by pretreatment of pCPA. Thus, these results indicated that the serotonergic system was implicated in the antidepressant activity of PA01. The hypofunction of the noradrenergic system has also been involved in the pathogenesis of depression [32]. Yohimbine can produce excessive NE releasing by its antagonistic action on pre-synaptic α2-adrenoceptor [24], and APO acts on the presynaptic D2 receptors situated on noradrenergic nerve terminals to inhibit the release of noradrenaline [25]. Antagonism of a high dose of APO (16 mg/kg) induced hypothermia is proposed as an improved screening test for antidepressants inhibiting norepinephrine reuptake [37]. These two models are commonly used for the evaluation of a noradrenergic effect of antidepressant drugs [24,38]. In the present study, pretreatment with PA01 (16 and 32 mg/kg) significantly enhanced the mouse lethality induced by yohimbine as compared with the vehicle control group. Moreover, PA01 (8, 16, and 32 mg/kg) antagonized the hypothermia induced by APO. These results suggested that the noradrenergic system participated in the antidepressant activity of PA01.

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Fig. 10. Effect of pretreatment of mice with SCH23390 (0.05 mg/kg, s.c., Fig. 10A) or Sulpiride (50 mg/kg, i.p., Fig. 10B) on the ant-immobility effect of PA01 in the TST. Values are expressed as mean ± S.E.M. (n = 10/group). Two-way ANOVA followed by Tukey's test: ⁎p b 0.05, ⁎⁎p b 0.01 as compared with the vehicle control group; #p b 0.05, ##p b 0.01 versus the same group pretreated with saline.

In parallel with the serotonergic and noradrenergic systems, the dopaminergic system is also suggested to be implicated in the regulation of mood, and may be an important therapeutic target [8,9]. In fact, there are currently several lines of evidence regarding the efficacy of antidepressants with dopaminergic effects in the treatment of depression [39–41]. It was reported that dopamine D1 and D2 receptors might display a significant role in the mechanism of action of SSRIs [42] and dopamine re-uptake inhibitors such as bupropion and nomifensine [43]. In this study, the anti-immobility effect of PA01 was significantly blocked by pre-treatment with SCH23390 or sulpiride, respectively. PA01 alone or in combination with dopamine receptor antagonists (SCH23390 and sulpiride) had no significant effects on the spontaneous locomotor activity. These results demonstrated that the dopaminergic system was also involved in the antidepressant activity of PA01. In summary, these results indicate that the antidepressant activity of PA01 is likely mediated by an interaction with the serotonergic, noradrenergic, and dopaminergic systems. 5. Conclusion The present study indicates that PA01 is a novel potent triple reuptake inhibitor that is highly bound to all three monoamine transporters (SERT, NET, and DAT), and displays excellent antidepressant activity in FST and TST. PA01 may be a promising antidepressant with a broad prospect. However, further preclinical studies should be investigated as a possible agent for the treatment of depression. Acknowledgments This work was supported through funds provided by the “Major New Drugs” special projects of the Ministry of Science and Technology of PR

Table 4 Effect of the administration of PA01 alone or in combination with SCH23390 (0.05 mg/kg, s.c.) or sulpiride (50 mg/kg, i.p.) on spontaneous locomotor activity of mice. Groups

Dose (mg/kg)

n

Mean activity (cm)

Vehicle PA01 SCH23390 PA01 + SCH23390 sulpiride PA01 + sulpiride

– 32 0.05 32 + 0.05 50 32 + 50

10 10 10 10 10 10

964 1299 986 1179 966 1068

± ± ± ± ± ±

211 227 188 270 198 215

Results are expressed as mean ± S.E.M. (n = 10/group). Results were analyzed by two-way ANOVA followed by Tukey's test.

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