Author's Accepted Manuscript
The antidepressant-like pharmacological profile of Yuanzhi-1, a novel serotonin, norepinephrine and dopamine reuptake inhibitor Zeng-liang Jin, Nana Gao, Xiao-rong Li, Yu Tang, Jie Xiong, Hong-xia Chen, Rui Xue, Yun-Feng Li
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S0924-977X(15)00006-1 http://dx.doi.org/10.1016/j.euroneuro.2015.01.005 NEUPSY10963
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European Neuropsychopharmacology
Received date: 14 August 2014 Revised date: 11 December 2014 Accepted date: 9 January 2015 Cite this article as: Zeng-liang Jin, Nana Gao, Xiao-rong Li, Yu Tang, Jie Xiong, Hong-xia Chen, Rui Xue, Yun-Feng Li, The antidepressant-like pharmacological profile of Yuanzhi-1, a novel serotonin, norepinephrine and dopamine reuptake inhibitor, European Neuropsychopharmacology, http://dx.doi.org/10.1016/j.euroneuro.2015.01.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The antidepressant-like pharmacological profile of Yuanzhi-1, a novel serotonin, norepinephrine and dopamine reuptake inhibitor Zeng-liang Jina,b,1, Nana Gaoa,1, Xiao-rong Lia, Yu Tanga,Jie Xiongb,Hong-xia Chenb, Rui Xueb, Yun-Feng Lib* a
Department of Pharmacology, School of Basic Medical Sciences,
Capital Medical University, Beijing, 100069, P. R. China b
Beijing Institute of Pharmacology and Toxicology, Beijing, 100850, P.
R. China Yunfeng Li, Beijing Institute of Pharmacology and Toxicology, Beijing, 100850, P. R. China Tel : +86 10 83911519 Fax : +86 10 83911519 E-mail address : [email protected] (Y.-F. Li), 1
These authors contributed equally to this research.
The category : Regular Research Word Count of the abstract :247 Word Count of the manuscript :6007 The number of references :39 The number of figures :8 The number of tables :4
Abstract Triple
reuptake
inhibitors
that
block
dopamine
transporters
(DATs),
norepinephrine transporters (NETs), and serotonin transporters (SERTs) are being developed as a new class of antidepressants that might have better efficacy and fewer side effects than traditional antidepressants. In this study, we performed in vitro binding and uptake assays as well as in vivo behavioural tests to assess the pharmacological properties and antidepressant-like efficacy of Yuanzhi-1. In vitro, Yuanzhi-1 had a high affinity for SERTs, NETs, and DATs prepared from rat brain tissue (Ki=3.95, 4.52 and 0.87 nM, respectively) and recombinant cells (Ki=2.87, 6.86 and 1.03 nM, respectively). Moreover, Yuanzhi-1 potently inhibited the uptake of serotonin (5-hydroxytryptamine; 5-HT), norepinephrine (NE) and dopamine (DA) into rat brain synaptosomes (Ki=2.12, 4.85 and 1.08 nM, respectively) and recombinant cells (Ki=1.65, 5.32 and 0.68 nM, respectively). In vivo, Yuanzhi-1 decreased immobility in a dose-dependent manner, which was shown among rats via the forced-swim test (FST) and mice via the tail-suspension test (TST). The results observed in the behavioural tests did not appear to result from the stimulation of locomotor activity. Repeated Yuanzhi-1 treatment (2.5, 5 or 10 mg/kg) significantly reversed depression-like behaviours in chronically stressed rats, including reduced sucrose preference, decreased locomotor activity, and prolonged time to begin eating. Furthermore, in vivo microdialysis studies showed that 5- and
10-mg/kg administrations of Yuanzhi-1 significantly increased the extracellular concentrations of 5-HT, NE and DA in the frontal cortices of freely moving rats. Therefore, Yuanzhi-1 might represent a novel triple reuptake inhibitor and possess antidepressant-like activity. Key words: Antidepressants; Yuanzhi-1; Triple reuptake inhibitors; Animal models;
1. Introduction Major depressive disorder (MDD) is a common and severe psychiatric disorder with a lifetime prevalence of 10–20% (Licinio and Wong, 2011). MDD is most likely composed of multiple disorders with overlapping symptoms and diverse aetiologies (Belmaker, 2008; Trivedi et al., 2008). Research during the second half of the 20th century was strongly influenced by the discovery that certain agents, particularly including serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine (NE), can alter monoamine metabolism to relieve depressive symptoms. The monoamine deficiency hypothesis posits that depressive symptoms arise from insufficient levels of the monoamine neurotransmitters 5-HT, NE, DA, or some combination thereof (Delgado, 2006). The ability of antidepressants to elevate synaptic levels of biogenic amines such as 5-HT, NE, and DA, has long been a cornerstone of the monoamine deficiency hypothesis of affective illness. Unfortunately, these current therapies for MDD are not satisfactory. Moreover, these drugs can impose a variety of side effects, including cardiac toxicity, blood pressure, sexual dysfunction, weight gain, and sleep disorders (Antai-Otong, 2004; Nutt et al., 2006; Parker, 2005). Therefore, novel antidepressants capable of treating a broad set of symptoms associated with depression, including emotional and physical symptoms, might be more effective. Recently, a growing number of studies suggest that the dopaminergic system might also be an important therapeutic target for the treatment of
depression (Nutt et al., 2006; Bodenlos et al., 2007). A recent study examined the effects of a treatment using duloxetine (an SNRI) in combination with bupropion (a DA and NE reuptake inhibitor) among patients with MDD who had not achieved symptom remission with either treatment alone. The combination of these drugs significantly improved the depressive symptoms of these patients, suggesting that a drug simultaneously targeting serotonergic, noradrenergic, and dopaminergic neurotransmission would more effectively treat depression (Mischoulon et al., 2000 ; Silk et al., 2007; Dutta et al., 2013). In a previous study, we investigated six triterpenoid saponin components derived from Yuanzhi using an evaluation system that combined in vitro high throughput screening with an in vivo behavioural assessment. We found that one of the triterpenoid saponins, Yuanzhi-1 (Jin et al., 2014 Supplementary Fig.1), shows antidepressant-like activity by acting as a triple monoamine reuptake inhibitor. In the present study, we performed a detailed investigation of the pharmacological characteristics of Yuanzhi-1, both in vivo and in vitro. First, we determined the binding profile of Yuanzhi-1 with respect to its affinity for rat and human dopamine transporters (DATs), norepinephrine transporters (NETs), and serotonin transporters (SERTs). Next, we evaluated the inhibitory activity of Yuanzhi-1 regarding the uptake of 5-HT, NE, and DA into rat synaptosomes and recombinant cells. We also investigated the antidepressant-like activity of Yuanzhi-1 in various mouse and rat models. Finally, to characterise the in vivo effect of Yuanzhi-1 on monoaminergic systems, we observed the extracellular
levels of 5-HT, NE, and DA in the prefrontal cortices of freely moving rats treated with Yuanzhi-1 using microanalysis. 2. Experimental Procedures 2.1 Animals Male ICR mice weighing 18–20 g and male Sprague–Dawley rats weighing 180–200 g were used in the current study (Beijing Vital River Laboratory Animal Technology Company, Beijing, China). The animals were group housed under standard conditions: room temperature 22±2ć, humidity 40–60%, 12 h:12 h light/dark cycle (lights on at 8:00 am). Food and water were available Ad libitum. The animals were allowed to adapt for approximately 1 week prior to the experiments. The experiments were performed in compliance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals (NIH publication No. 86-23, revised 1996). 2.2 Materials Yuanzhi-1 was purchased from Shanghai Pureone biotechnology (Shanghai, China). Fluoxetine, paroxetine, desipramine, duloxetine, GBR12909, 5-HT, and NE were purchased from Sigma Co. (St. Louis, MO, USA). [3H]citalopram, [3H]GBR12909, [3H]nisoxetine, [3H]5-HT, [3H]NE, and [3H]DA were purchased from PerkinElmer Life Sciences (NEN, Boston, MA, USA). Dulbecco’s modified Eagle’s medium and foetal bovine serum were purchased from Invitrogen Inc. (Grand Island, NY, USA) or HyClone Corp. (South Logan, UT, USA), respectively. Unless specified below, the compounds were dissolved in
distilled water. 2.3 Binding and uptake assays 2.3.1. Frontal cortex and striatum membrane preparation The frontal cortex and striatum membranes were prepared according to a previously described method (Bymaster et al., 2001; Xue et al., 2013). Briefly, the rats were euthanised, and their brains were rapidly removed. The frontal cortex and striatum were dissected, homogenised in 40 volumes of ice-cold buffer (50 mM Tris–HCl, pH 7.4), and then centrifuged at 40,000 g for 10 min at 41ć. The pellet was suspended and centrifuged a second time. To remove the endogenous monoamines, the final suspensions were incubated at 37ć for 20 min and centrifuged as before. The final pellets were immediately frozen at -80ć. 2.3.2. Cell membrane preparation The membranes were prepared from HEK293 cells stably expressing hSERT, hNET, and hDAT, as described in a previous study (Jin et al., 2012; Xue et al., 2013). Briefly, the cells were harvested via centrifugation at 110 g for 5 min at 41ć. The pellets were homogenised in an assay buffer (50 nM Tris–HCl containing 120 nM NaCl and 5 mM KCl, pH 7.4) and centrifuged twice at 36,000 g for 15 min at 41ć. The final pellet was resuspended in an assay buffer and stored at -80ć until use. 2.3.3. Binding to rat SERTs, NETs, and DATs To assess the binding affinity of Yuanzhi-1 with regard to the SERTs, NETs,
and DATs prepared from rat brain tissues, competitive binding assays were performed as previously described (Millan et al., 2001; Xue et al., 2013) using [3H]citalopram (1.2 nM), [3H]nisoxetine (1.0 nM), and [3H]GBR12909 (1.0 nM) as radioligands and 10 µM fluoxetine, 10 µM desipramine, and 10 µM GBR12909 as non-specific ligands. The parameters are specified in Table 1. The binding assays were performed in duplicate across three independent experiments. 2.3.4. Binding to hSERTs, hNETs, and hDATs The binding affinities for hSERTs, hNETs and hDATs were also determined via competition with [3H]citalopram (3.0 nM), [3H]nisoxetine (2.0 nM), and [3H]GBR12909 (5.0 nM). Non-specific binding was defined using 10 µM of paroxetine, 20 µM of desipramine, and 10 µM of GBR12909, respectively. The binding assays were performed in duplicate in three independent experiments. 2.3.5. The synaptosomal uptake of [3H]5-HT, [3H]NE and [3H]DA As well as the [3H]5-HT, [3H]NE and [3H]DA uptake assays were performed using crude synaptosomes prepared from rat prefrontal cortices as previously described (Xue et al., 2013;Artaiz et al., 2005). Crude synaptosomes were incubated in Krebs bicarbonate solution containing drug solution, and [3H]5-HT (20 nM), [3H]NE (20 nM) or [3H]DA (50 nM) was added and incubated for 10 min at 37ć. Non-specific uptake was determined using 10 µM of fluoxetine, desipramine, or GBR12909. The uptake assays were performed in duplicate across three independent experiments.
2.3.6. Uptake assays in recombinant cells [3H]5-HT, [3H]NE, and [3H]DA uptake assays in HEK-293 cells expressing the corresponding human transporters were determined as previously described, with minor modifications (Xue et al., 2013; Janowsky et al., 1986). Briefly, the medium was removed from the cells, which were then washed with phosphate-buffered saline. One hundred microliters of a Krebs–Ringers–HEPES buffer containing various concentrations of drugs was then added to the 96-well plate, and [3H]5-HT(50 nM), [3H]NE (80 nM) or [3H]DA (50 nM) was added and incubated for 10 min at 37ć. Non-specific uptake was determined using 10 µM of fluoxetine, desipramine, or GBR12909. The uptake assays were performed in duplicate across three independent experiments. 2.4 Behavioural assays 2.4.1 Tail suspension test (TST) for mice The TST was performed using mice as previously described (Chen et al., 2013; Steru et al., 1985) but with minor modifications. A total of 70 naive mice were randomly assigned to seven treatment groups (N=10 per group): control group, 20 mg/kg duloxetine, 2.5 mg/kg Yuanzhi-1, 5 mg/kg Yuanzhi-1, 10 mg/kg Yuanzhi-1, 20 mg/kg Yuanzhi-1and 40 mg/kg Yuanzhi-1. All drugs were orally administered, once a day at 8:00 am for 1 week. Twenty-four hours after the 1-week treatment period, the mice were suspended for 6 min at the top of the apparatus using adhesive tape placed approximately 1 cm from the tip of their
tails. The duration of immobility was measured during the last 4 min of their suspension. The mice were considered immobile when they hung passively without moving. 2.4.2 Forced swimming test (FST) for mice FST was performed on the mice following the protocol of Porsolt et al. (Porsolt et al., 1977a), with minor modifications. A total of 70 naive mice were randomly assigned to seven treatment groups (N=10 per group): control group, 20 mg/kg duloxetine, 2.5 mg/kg Yuanzhi-1, 5 mg/kg Yuanzhi-1, 10 mg/kg Yuanzhi-1, 20 mg/kg Yuanzhi-1and 40 mg/kg Yuanzhi-1.All drugs were orally administered once a day at 8:00 am for 1 week. Twenty-four hours after this treatment period, the mice were individually placed in cylindrical containers (diameter=12 cm, height=20 cm, containing 10 cm of water maintained at 25ć) for 6 min. The duration of immobility during the last 4 min of suspension was recorded. The mice were considered immobile when they floated motionless, making only the movements necessary to keep their heads above the water. 2.4.3 Locomotor activity in mice To confirm whether Yuanzhi-1 has an antidepressant-like action, we investigated whether Yuanzhi-1 has a significant effect on the central nervous system by measuring the spontaneous motor activity of mice. A total of 70 naive mice were randomly assigned to seven treatment groups (N=10 per group): control group, 20 mg/kg duloxetine, 2.5 mg/kg Yuanzhi-1, 5 mg/kg Yuanzhi-1, 10 mg/kg Yuanzhi-1, 20 mg/kg Yuanzhi-1and 40 mg/kg Yuanzhi-1.All drugs
were orally administered once a day at 8:00 am for 1 week. The locomotor test was conducted for 60 min, 24 h after this treatment period. In this test, each mouse was placed in the five separated chambers of the autonomous movement instrument (Shandong Medical Academy of Science, China). The total locomotor/ambulation activity of the mice was automatically recorded during the 30-min test. The apparatus was cleaned during the test interval. 2.4.4 Chronic unpredictable mild stress (CMS) model To further evaluate the antidepressant-like effects of Yuanzhi-1, the CMS model was developed as previously described (Chen et al., 2013; Xue et al., 2013; Willner et al., 1987). After a 1-week acclimation period, sixty naive rats were subjected to one 48-h period of sucrose training and several periods of sucrose baseline testing. The rats were randomly divided into six groups based on their sucrose preference in the sucrose baseline test: control (non-stress), stress-vehicle
(distilled
water),
stress-duloxetine
(20
mg/kg),
and
stress-Yuanzhi-1 (2.5 , 5 or 10 mg/kg). The vehicle or drug was administered orally between 8:00 and 9:00 am, 1 h before the stress procedure. Except for the non-stressed control group, the rats were subjected to the variety of stressors described in Table 2. The stressors were applied continuously and randomly. Food and water were freely available to the non-stressed control rats, except for the 14-h period of deprivation prior to each sucrose test. After 4 weeks of stress, the open-field test (on day 29; i.e., after the rats had received the vehicle or drugs for 28 days), the sucrose preference test (on day 33), and the novelty-suppressed
feeding (NSF) test (on day 37; i.e., after the rats had received the vehicle or drugs for 36 days) were performed in the absence of acute drug treatment. 2.4.5.Open-field test in the CMS model The apparatus used in the open-field test was an arena (diameter=122 cm, height=45 cm) with the base equally divided into 16 sectors. Rats were placed in the centre of the arena 24 h after the last drug treatment, and the number of crossings and rearings was recorded within 5 min. 2.4.6 Sucrose preference test in the CMS model The rats were first trained to consume 1% (v/v) sucrose solution for 48 h, without food or water supplies. Three days later, the sucrose baseline test was performed following 14 h of food and water deprivation. The rats were allowed to choose from two bottles for 1 h: one with 1% sucrose solution and the other with water. Their sucrose and water intake was recorded, and their sucrose preference (SP=sucrose intake×100%/[sucrose intake+water intake]) was calculated. Following 4 weeks of exposure to stress, the sucrose preference test was repeated using the same procedure. 2.4.7 NSF test in the CMS model The rats that had fasted for 48 h were placed in the corner of a plastic box (54×28×21 cm) with 12 food pellets placed in the centre (Bodnoff et al., 1988). The time prior to beginning eating (up to 5 min) was recorded. Eating was defined as chewing or biting, not merely sniffing or toying with the food. Moreover, the amount of food consumed in the home cage within 5 min was
immediately evaluated to assess the effect of the drug on normal feeding behaviour. 2.4.8 In vivo microdialysis studies The effect of Yuanzhi-1 on the extracellular monoamine levels in the frontal cortices was evaluated using in vivo microdialysis coupled with high performance liquid chromatography (Carboni and Di Chiara, 1989; Matthews et al., 2005). Briefly, male Sprague–Dawley rats were anaesthetised with 3% halothane using a calibrated vaporiser and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA). Each rat was cannulated in the medial prefrontal cortex (mPFC; anterior 3.2, lateral 0.5, ventral 2.0) relative to the bregma and the skull, according to the atlas of Paxinos et al. (Murone et al., 1997). The probe was secured to the skull with dental cement and stainless steel screws. After surgery, the animals were treated with daily penicillin (10,000 U, s.c.) to prevent infection and were housed in individual test cages. After recovering from the surgery, the animals were subjected to microanalysis: A microanalysis probe (CMA/12, 2.0 mm membrane) was inserted into the guide cannula to replace the dummy cannula. The probe was perfused at 2 ȝl/min with artificial cerebrospinal fluid (147 mM of NaCl, 4 mM of KCl, 2.3 mM of CaCl2, and 1.0 mM of MgCl2, pH 7.4). After at least 2 h of equilibration, dialysate samples were collected every 30 min using tubes filled with chloric acid (20 mM per 10 ȝl/tube to prevent the degradation of monoamines). The steady baseline of each neurotransmitter was established prior to drug treatment by collecting the
first four samples. After the baseline sampling was complete, the animals were orally administered Yuanzhi-1 (2.5, 5, 10 mg/kg) or the vehicle (0.2% Tween 80 and 0.5% methylcellulose dissolved in water) at the end of the sixth sampling. Three hours after Yuanzhi-1 administration, the dialysis samples were collected every 20 min and analysed using high-performance liquid chromatography with electrochemical detection (HPLC-ECD) to determine the levels of NE, 5-HT, and DA. At the end of each experiment, the animals were euthanised, and the probe placement was verified histologically. Data collected from rats with incorrect probe placements were discarded. The HPLC system consisted of a micro-bore reverse-phase column (particle size=5 ȝm, 150×4.6 mm; Model C-18, DIKMA Technologies Ltd., Beijing, China), an Agilent 1100 pump (flow rate=1.0 mL/min; Agilent Technologies, Palo Alto, CA, USA) and a Hewlett-Packard HP 1049A glassy carbon amperometric detector (Agilent Technologies, Palo Alto, CA, USA). The mobile phase consisted of 85 mM of citrate, 100 mM of sodium acetate, 0.9 mM of octyl sodium sulphate, 0.2 mM of EDTA, and 15% methanol, pH 3.7. External standard curves were used to quantify the amounts of NE, 5-HT, and DA in each sample, calculated using the area under curve (AUC). The injection volume was 50 ȝl. The detection limit of the assay was 20 pg per sample. 3. Statistical analyses Unless otherwise specified, statistical analysis was performed using GraphPad Prism (GraphPad Prism 5.0, version 2.0; GraphPad Software Inc., San
Diego, CA).As for data from behavioural assays, Student’s t-test was carried out to compare the difference between two groups (i.e., vehicle vs. duloxetine in TST and FST; control non-stressed rats vs. stressed rats in CMS model); other data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. The transporter binding and monoamine uptake data were analysed using a one-site nonlinear regression of the concentration–effect curve. The
Ki
values
were
calculated
using
the
Cheng–Prusoff
equation:
Ki=IC50/([L/KD]+1) (Cheng and Prusoff, 1973). For the data from the CMS behavioural assays, Student’s t-test was performed to compare between-group differences. For the microdialysis experiments, a two-way repeated-measures ANOVA, followed by Tukey's test, was used to compare the percentage increase from baseline among groups. Time and treatment were treated as the independent factors, and time was the repeated factor. 4. Results 4.1 The binding affinities of Yuanzhi-1 to rat monoamine transporters In this study, radioligand binding assays were conducted to determine the affinity of Yuanzhi-1 for rat SERTs, NETs, and DATs (the Ki values are shown in Table 3). We found that Yuanzhi-1 showed a high affinity for the rat monoamine transporters, including SERTs, NETs and DATs (Table 3). Our results showed that Yuanzhi-1 was 4 times more potent than fluoxetine in binding to rat SERTs (Ki values for Yuanzhi-1: 3.95±0.21; Fluoxetine: 15.67±0.43 nM, Table 1; Fig. 2A). To evaluate the binding capacity of Yuanzhi-1 to DATs, we found that the
affinity of Yuanzhi-1 to rat DATs was 30 times higher than nomifensine (the Ki values for Yuanzhi-1 and nomifensine were 0.87±0.14 and 30.24±2.24 nM, respectively; Table 3; Fig. 2C). Similarly, the affinity of Yuanzhi-1 to rat NETs was twice as strong as that of desipramine, a known specific inhibitor of NETs. The Ki values for Yuanzhi-1 and desipramine were 4.52±0.82 and 7.78±0.54 nM, respectively (Table 3; Fig. 2B). Comparing the affinity of Yuanzhi-1 with the three tested monoamine transporters, we found that Yuanzhi-1 binds to DATs with the highest affinity, followed by SERTs and NETs. 4.2 The binding of Yuanzhi-1 to human monoamine transporters To study whether Yuanzhi-1 has similar affinities to monoamine transporters from humans as those from rats, we used HEK293 cells transfected with SERTs, NETs or DATs and tested the capacity of Yuanzhi-1 to compete with specific inhibitors of these transporters. As Table 1 displays, Yuanzhi-1 showed high affinity to human monoamine transporters, consistent with our results in rats. Yuanzhi-1 and fluoxetine competed in a dose-dependent manner with the binding of the SERT from cells transfected with hSERT. The Ki values for Yuanzhi-1 and fluoxetine were 2.78±0.58 and 9.42±0.65 nM, respectively (Table 3; Fig. 2D). The binding of [3H]-GBR12909 to hDAT was competed by Yuanzhi-1 and nomifensine with Ki values of 1.03±0.21 and 16.63±3.04 nM, respectively (Table 3; Fig. 2F). Yuanzhi-1 and desipramine competed with the binding of the NET radiolabeled [3H]-nisoxetine to the membranes from cells transfected with hNET, with Ki values of 6.86±0.43 and 11.41±1.04 nM, respectively (Table 3;
Fig. 2E). Similarly to the rat monoamine transporter, the affinity of Yuanzhi-1 for hDAT was higher than that for hSERT and hDAT. 4.3 Yuanzhi-1 inhibits monoamine uptake in rat synaptosomes Our results showed that Yuanzhi-1 inhibited all three transporters (SERTs, DATs and NETs) in a dose-dependent manner (Table 4;Fig.3). The Ki values for the uptake of [3H]-5-HT into rat frontal cortex synaptosomes were 2.12±0.32 and 78.89±3.54 nM for Yuanzhi-1 and fluoxetine, respectively (Table 4; Fig. 3A). Yuanzhi-1 and desipramine inhibited the uptake of [3H]-NE into the synaptosomes, with Ki values of 4.85±0.52 and 6.54±0.84 nM, respectively (Table 4; Fig. 3B). Yuanzhi-1 and nomifensine inhibited [3H]-DA uptake in rat striatal synaptosomes, with Ki values of 1.08±0.04 and 42.14±3.31 nM, respectively (Table 4; Fig. 3C). Similar to its binding profile, Yuanzhi-1 more potently inhibited these transporters than their corresponding specific inhibitors. Furthermore, Yuanzhi-1 had a greater inhibition effect on DATs than SERTs and NETs (Table 4). 4.4 Yuanzhi-1 inhibits the uptake activities of human monoamine transporters HEK cells were transfected with hSERT, hDAT or hNET and treated with Yuanzhi-1 at varying doses. Consistent with the results from the rat synaptosomes, we found that Yuanzhi-1 also significantly inhibited the activities of the NETs, SERTs and DATs in these transfected cell lines. The Ki values for the Yuanzhi-1 inhibiting the uptake of [3H]-5-HT, [3H]-5-NE, and [3H]-DA into
the cells expressing the corresponding human recombinant transporters were 1.65±0.14, 5.32±0.53 nM and 0.68±0.09 nM, respectively (Table 4; Fig. 3). Again, these data demonstrate that Yuanzhi-1 inhibits the transport activities of DATs with more potency than SERTs and NETs. 4.5 The effect of repeated treatment with Yuanzhi-1 on the immobility time in the FST, TST and locomotor activity. To investigate whether Yuanzhi-1 can produce chronic changes to the depression-related behaviours observed in the FST and TST, we treated mice with different doses via continuous oral administration for 7 days. Compared with vehicle, acute administration of duloxetine at 20 mg/kg significantly reduced the immobility time in TST. (Fig. 4A, Student’s t-test, t=2.298, p=0.0338), indicating the behavioral tests were performed under normal condition and reliable. A one-way ANOVA revealed a significant difference (F[6, 63]=7.722, p!-! ! - 6 # 2 0 & & 0) &%0 6 2& 6 % % # 2
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Figure legend: Fig.1.The outline of design for chronic unpredictable stress and behavioral tests. Fig.2. Yuanzhi-1 competes for the binding of radioligands specific to the rSERT (A), rNET(B), rDAT (C), hSERT (D), hNET(E), hDAT (F). For each transporter bioassay, a known comparator was used [fluoxetine(5-HT reuptake inhibitor); desipramine (NE reuptake inhibitor); and nomifensine (DA reuptake inhibitor)]. The IC50 value was generated from each of these curves and usedto generate the Ki values. Each data point depicted represents the mean±S.E.M. of three independent experiments performed in triplicate. The Ki values for Yuanzhi-1 and the comparators are shown in Table 2. Fig.3.Functional activity of Yuanzhi-1 demonstrates inhibition of radiolabeled uptake of serotonin norepinephrine and dopamine in rat synaptosomes(A-C) and in cloned human transporters expressing in
HEK293 cell lines(D-F). For each uptake transporter bioassay, a known comparator was used [fluoxetine (5-HT reuptake inhibitor); desipramine (NE reuptake inhibitor); nomifensine (DA reuptake inhibitor)]. Each data point depicted represents the mean ±S.E.M. of three independent experiments performed in triplicate. The IC50 values for Yuanzhi-1 are shown in Table 3. Fig.4. The sub-chronic effect of Yuanzhi-1 (dose range: 2.5-40 mg/kg) and DLX (20 mg/kg) on the time of immobility during the total 6-min testing period in the tail suspension test (A) , forced swimming test (B) and locomotor activity (C)in mice. Results are expressed as mean ± SEM (n = 8-10/group). The drugs were administered via the oral route 60 min prior to testing. For statistical significance, *p
The antidepressant-like pharmacological profile of Yuanzhi-1, a novel serotonin, norepinephrine and dopamine reuptake inhibitor Zeng-liang Jin, Nana Gao, Xiao-rong Li, Yu Tang, Jie Xiong, Hong-xia Chen, Rui Xue, Yun-Feng Li
www.elsevier.com/locate/euroneuro
PII: DOI: Reference:
S0924-977X(15)00006-1 http://dx.doi.org/10.1016/j.euroneuro.2015.01.005 NEUPSY10963
To appear in:
European Neuropsychopharmacology
Received date: 14 August 2014 Revised date: 11 December 2014 Accepted date: 9 January 2015 Cite this article as: Zeng-liang Jin, Nana Gao, Xiao-rong Li, Yu Tang, Jie Xiong, Hong-xia Chen, Rui Xue, Yun-Feng Li, The antidepressant-like pharmacological profile of Yuanzhi-1, a novel serotonin, norepinephrine and dopamine reuptake inhibitor, European Neuropsychopharmacology, http://dx.doi.org/10.1016/j.euroneuro.2015.01.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
The antidepressant-like pharmacological profile of Yuanzhi-1, a novel serotonin, norepinephrine and dopamine reuptake inhibitor Zeng-liang Jina,b,1, Nana Gaoa,1, Xiao-rong Lia, Yu Tanga,Jie Xiongb,Hong-xia Chenb, Rui Xueb, Yun-Feng Lib* a
Department of Pharmacology, School of Basic Medical Sciences,
Capital Medical University, Beijing, 100069, P. R. China b
Beijing Institute of Pharmacology and Toxicology, Beijing, 100850, P.
R. China Yunfeng Li, Beijing Institute of Pharmacology and Toxicology, Beijing, 100850, P. R. China Tel : +86 10 83911519 Fax : +86 10 83911519 E-mail address : [email protected] (Y.-F. Li), 1
These authors contributed equally to this research.
The category : Regular Research Word Count of the abstract :247 Word Count of the manuscript :6007 The number of references :39 The number of figures :8 The number of tables :4
Abstract Triple
reuptake
inhibitors
that
block
dopamine
transporters
(DATs),
norepinephrine transporters (NETs), and serotonin transporters (SERTs) are being developed as a new class of antidepressants that might have better efficacy and fewer side effects than traditional antidepressants. In this study, we performed in vitro binding and uptake assays as well as in vivo behavioural tests to assess the pharmacological properties and antidepressant-like efficacy of Yuanzhi-1. In vitro, Yuanzhi-1 had a high affinity for SERTs, NETs, and DATs prepared from rat brain tissue (Ki=3.95, 4.52 and 0.87 nM, respectively) and recombinant cells (Ki=2.87, 6.86 and 1.03 nM, respectively). Moreover, Yuanzhi-1 potently inhibited the uptake of serotonin (5-hydroxytryptamine; 5-HT), norepinephrine (NE) and dopamine (DA) into rat brain synaptosomes (Ki=2.12, 4.85 and 1.08 nM, respectively) and recombinant cells (Ki=1.65, 5.32 and 0.68 nM, respectively). In vivo, Yuanzhi-1 decreased immobility in a dose-dependent manner, which was shown among rats via the forced-swim test (FST) and mice via the tail-suspension test (TST). The results observed in the behavioural tests did not appear to result from the stimulation of locomotor activity. Repeated Yuanzhi-1 treatment (2.5, 5 or 10 mg/kg) significantly reversed depression-like behaviours in chronically stressed rats, including reduced sucrose preference, decreased locomotor activity, and prolonged time to begin eating. Furthermore, in vivo microdialysis studies showed that 5- and
10-mg/kg administrations of Yuanzhi-1 significantly increased the extracellular concentrations of 5-HT, NE and DA in the frontal cortices of freely moving rats. Therefore, Yuanzhi-1 might represent a novel triple reuptake inhibitor and possess antidepressant-like activity. Key words: Antidepressants; Yuanzhi-1; Triple reuptake inhibitors; Animal models;
1. Introduction Major depressive disorder (MDD) is a common and severe psychiatric disorder with a lifetime prevalence of 10–20% (Licinio and Wong, 2011). MDD is most likely composed of multiple disorders with overlapping symptoms and diverse aetiologies (Belmaker, 2008; Trivedi et al., 2008). Research during the second half of the 20th century was strongly influenced by the discovery that certain agents, particularly including serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine (NE), can alter monoamine metabolism to relieve depressive symptoms. The monoamine deficiency hypothesis posits that depressive symptoms arise from insufficient levels of the monoamine neurotransmitters 5-HT, NE, DA, or some combination thereof (Delgado, 2006). The ability of antidepressants to elevate synaptic levels of biogenic amines such as 5-HT, NE, and DA, has long been a cornerstone of the monoamine deficiency hypothesis of affective illness. Unfortunately, these current therapies for MDD are not satisfactory. Moreover, these drugs can impose a variety of side effects, including cardiac toxicity, blood pressure, sexual dysfunction, weight gain, and sleep disorders (Antai-Otong, 2004; Nutt et al., 2006; Parker, 2005). Therefore, novel antidepressants capable of treating a broad set of symptoms associated with depression, including emotional and physical symptoms, might be more effective. Recently, a growing number of studies suggest that the dopaminergic system might also be an important therapeutic target for the treatment of
depression (Nutt et al., 2006; Bodenlos et al., 2007). A recent study examined the effects of a treatment using duloxetine (an SNRI) in combination with bupropion (a DA and NE reuptake inhibitor) among patients with MDD who had not achieved symptom remission with either treatment alone. The combination of these drugs significantly improved the depressive symptoms of these patients, suggesting that a drug simultaneously targeting serotonergic, noradrenergic, and dopaminergic neurotransmission would more effectively treat depression (Mischoulon et al., 2000 ; Silk et al., 2007; Dutta et al., 2013). In a previous study, we investigated six triterpenoid saponin components derived from Yuanzhi using an evaluation system that combined in vitro high throughput screening with an in vivo behavioural assessment. We found that one of the triterpenoid saponins, Yuanzhi-1 (Jin et al., 2014 Supplementary Fig.1), shows antidepressant-like activity by acting as a triple monoamine reuptake inhibitor. In the present study, we performed a detailed investigation of the pharmacological characteristics of Yuanzhi-1, both in vivo and in vitro. First, we determined the binding profile of Yuanzhi-1 with respect to its affinity for rat and human dopamine transporters (DATs), norepinephrine transporters (NETs), and serotonin transporters (SERTs). Next, we evaluated the inhibitory activity of Yuanzhi-1 regarding the uptake of 5-HT, NE, and DA into rat synaptosomes and recombinant cells. We also investigated the antidepressant-like activity of Yuanzhi-1 in various mouse and rat models. Finally, to characterise the in vivo effect of Yuanzhi-1 on monoaminergic systems, we observed the extracellular
levels of 5-HT, NE, and DA in the prefrontal cortices of freely moving rats treated with Yuanzhi-1 using microanalysis. 2. Experimental Procedures 2.1 Animals Male ICR mice weighing 18–20 g and male Sprague–Dawley rats weighing 180–200 g were used in the current study (Beijing Vital River Laboratory Animal Technology Company, Beijing, China). The animals were group housed under standard conditions: room temperature 22±2ć, humidity 40–60%, 12 h:12 h light/dark cycle (lights on at 8:00 am). Food and water were available Ad libitum. The animals were allowed to adapt for approximately 1 week prior to the experiments. The experiments were performed in compliance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals (NIH publication No. 86-23, revised 1996). 2.2 Materials Yuanzhi-1 was purchased from Shanghai Pureone biotechnology (Shanghai, China). Fluoxetine, paroxetine, desipramine, duloxetine, GBR12909, 5-HT, and NE were purchased from Sigma Co. (St. Louis, MO, USA). [3H]citalopram, [3H]GBR12909, [3H]nisoxetine, [3H]5-HT, [3H]NE, and [3H]DA were purchased from PerkinElmer Life Sciences (NEN, Boston, MA, USA). Dulbecco’s modified Eagle’s medium and foetal bovine serum were purchased from Invitrogen Inc. (Grand Island, NY, USA) or HyClone Corp. (South Logan, UT, USA), respectively. Unless specified below, the compounds were dissolved in
distilled water. 2.3 Binding and uptake assays 2.3.1. Frontal cortex and striatum membrane preparation The frontal cortex and striatum membranes were prepared according to a previously described method (Bymaster et al., 2001; Xue et al., 2013). Briefly, the rats were euthanised, and their brains were rapidly removed. The frontal cortex and striatum were dissected, homogenised in 40 volumes of ice-cold buffer (50 mM Tris–HCl, pH 7.4), and then centrifuged at 40,000 g for 10 min at 41ć. The pellet was suspended and centrifuged a second time. To remove the endogenous monoamines, the final suspensions were incubated at 37ć for 20 min and centrifuged as before. The final pellets were immediately frozen at -80ć. 2.3.2. Cell membrane preparation The membranes were prepared from HEK293 cells stably expressing hSERT, hNET, and hDAT, as described in a previous study (Jin et al., 2012; Xue et al., 2013). Briefly, the cells were harvested via centrifugation at 110 g for 5 min at 41ć. The pellets were homogenised in an assay buffer (50 nM Tris–HCl containing 120 nM NaCl and 5 mM KCl, pH 7.4) and centrifuged twice at 36,000 g for 15 min at 41ć. The final pellet was resuspended in an assay buffer and stored at -80ć until use. 2.3.3. Binding to rat SERTs, NETs, and DATs To assess the binding affinity of Yuanzhi-1 with regard to the SERTs, NETs,
and DATs prepared from rat brain tissues, competitive binding assays were performed as previously described (Millan et al., 2001; Xue et al., 2013) using [3H]citalopram (1.2 nM), [3H]nisoxetine (1.0 nM), and [3H]GBR12909 (1.0 nM) as radioligands and 10 µM fluoxetine, 10 µM desipramine, and 10 µM GBR12909 as non-specific ligands. The parameters are specified in Table 1. The binding assays were performed in duplicate across three independent experiments. 2.3.4. Binding to hSERTs, hNETs, and hDATs The binding affinities for hSERTs, hNETs and hDATs were also determined via competition with [3H]citalopram (3.0 nM), [3H]nisoxetine (2.0 nM), and [3H]GBR12909 (5.0 nM). Non-specific binding was defined using 10 µM of paroxetine, 20 µM of desipramine, and 10 µM of GBR12909, respectively. The binding assays were performed in duplicate in three independent experiments. 2.3.5. The synaptosomal uptake of [3H]5-HT, [3H]NE and [3H]DA As well as the [3H]5-HT, [3H]NE and [3H]DA uptake assays were performed using crude synaptosomes prepared from rat prefrontal cortices as previously described (Xue et al., 2013;Artaiz et al., 2005). Crude synaptosomes were incubated in Krebs bicarbonate solution containing drug solution, and [3H]5-HT (20 nM), [3H]NE (20 nM) or [3H]DA (50 nM) was added and incubated for 10 min at 37ć. Non-specific uptake was determined using 10 µM of fluoxetine, desipramine, or GBR12909. The uptake assays were performed in duplicate across three independent experiments.
2.3.6. Uptake assays in recombinant cells [3H]5-HT, [3H]NE, and [3H]DA uptake assays in HEK-293 cells expressing the corresponding human transporters were determined as previously described, with minor modifications (Xue et al., 2013; Janowsky et al., 1986). Briefly, the medium was removed from the cells, which were then washed with phosphate-buffered saline. One hundred microliters of a Krebs–Ringers–HEPES buffer containing various concentrations of drugs was then added to the 96-well plate, and [3H]5-HT(50 nM), [3H]NE (80 nM) or [3H]DA (50 nM) was added and incubated for 10 min at 37ć. Non-specific uptake was determined using 10 µM of fluoxetine, desipramine, or GBR12909. The uptake assays were performed in duplicate across three independent experiments. 2.4 Behavioural assays 2.4.1 Tail suspension test (TST) for mice The TST was performed using mice as previously described (Chen et al., 2013; Steru et al., 1985) but with minor modifications. A total of 70 naive mice were randomly assigned to seven treatment groups (N=10 per group): control group, 20 mg/kg duloxetine, 2.5 mg/kg Yuanzhi-1, 5 mg/kg Yuanzhi-1, 10 mg/kg Yuanzhi-1, 20 mg/kg Yuanzhi-1and 40 mg/kg Yuanzhi-1. All drugs were orally administered, once a day at 8:00 am for 1 week. Twenty-four hours after the 1-week treatment period, the mice were suspended for 6 min at the top of the apparatus using adhesive tape placed approximately 1 cm from the tip of their
tails. The duration of immobility was measured during the last 4 min of their suspension. The mice were considered immobile when they hung passively without moving. 2.4.2 Forced swimming test (FST) for mice FST was performed on the mice following the protocol of Porsolt et al. (Porsolt et al., 1977a), with minor modifications. A total of 70 naive mice were randomly assigned to seven treatment groups (N=10 per group): control group, 20 mg/kg duloxetine, 2.5 mg/kg Yuanzhi-1, 5 mg/kg Yuanzhi-1, 10 mg/kg Yuanzhi-1, 20 mg/kg Yuanzhi-1and 40 mg/kg Yuanzhi-1.All drugs were orally administered once a day at 8:00 am for 1 week. Twenty-four hours after this treatment period, the mice were individually placed in cylindrical containers (diameter=12 cm, height=20 cm, containing 10 cm of water maintained at 25ć) for 6 min. The duration of immobility during the last 4 min of suspension was recorded. The mice were considered immobile when they floated motionless, making only the movements necessary to keep their heads above the water. 2.4.3 Locomotor activity in mice To confirm whether Yuanzhi-1 has an antidepressant-like action, we investigated whether Yuanzhi-1 has a significant effect on the central nervous system by measuring the spontaneous motor activity of mice. A total of 70 naive mice were randomly assigned to seven treatment groups (N=10 per group): control group, 20 mg/kg duloxetine, 2.5 mg/kg Yuanzhi-1, 5 mg/kg Yuanzhi-1, 10 mg/kg Yuanzhi-1, 20 mg/kg Yuanzhi-1and 40 mg/kg Yuanzhi-1.All drugs
were orally administered once a day at 8:00 am for 1 week. The locomotor test was conducted for 60 min, 24 h after this treatment period. In this test, each mouse was placed in the five separated chambers of the autonomous movement instrument (Shandong Medical Academy of Science, China). The total locomotor/ambulation activity of the mice was automatically recorded during the 30-min test. The apparatus was cleaned during the test interval. 2.4.4 Chronic unpredictable mild stress (CMS) model To further evaluate the antidepressant-like effects of Yuanzhi-1, the CMS model was developed as previously described (Chen et al., 2013; Xue et al., 2013; Willner et al., 1987). After a 1-week acclimation period, sixty naive rats were subjected to one 48-h period of sucrose training and several periods of sucrose baseline testing. The rats were randomly divided into six groups based on their sucrose preference in the sucrose baseline test: control (non-stress), stress-vehicle
(distilled
water),
stress-duloxetine
(20
mg/kg),
and
stress-Yuanzhi-1 (2.5 , 5 or 10 mg/kg). The vehicle or drug was administered orally between 8:00 and 9:00 am, 1 h before the stress procedure. Except for the non-stressed control group, the rats were subjected to the variety of stressors described in Table 2. The stressors were applied continuously and randomly. Food and water were freely available to the non-stressed control rats, except for the 14-h period of deprivation prior to each sucrose test. After 4 weeks of stress, the open-field test (on day 29; i.e., after the rats had received the vehicle or drugs for 28 days), the sucrose preference test (on day 33), and the novelty-suppressed
feeding (NSF) test (on day 37; i.e., after the rats had received the vehicle or drugs for 36 days) were performed in the absence of acute drug treatment. 2.4.5.Open-field test in the CMS model The apparatus used in the open-field test was an arena (diameter=122 cm, height=45 cm) with the base equally divided into 16 sectors. Rats were placed in the centre of the arena 24 h after the last drug treatment, and the number of crossings and rearings was recorded within 5 min. 2.4.6 Sucrose preference test in the CMS model The rats were first trained to consume 1% (v/v) sucrose solution for 48 h, without food or water supplies. Three days later, the sucrose baseline test was performed following 14 h of food and water deprivation. The rats were allowed to choose from two bottles for 1 h: one with 1% sucrose solution and the other with water. Their sucrose and water intake was recorded, and their sucrose preference (SP=sucrose intake×100%/[sucrose intake+water intake]) was calculated. Following 4 weeks of exposure to stress, the sucrose preference test was repeated using the same procedure. 2.4.7 NSF test in the CMS model The rats that had fasted for 48 h were placed in the corner of a plastic box (54×28×21 cm) with 12 food pellets placed in the centre (Bodnoff et al., 1988). The time prior to beginning eating (up to 5 min) was recorded. Eating was defined as chewing or biting, not merely sniffing or toying with the food. Moreover, the amount of food consumed in the home cage within 5 min was
immediately evaluated to assess the effect of the drug on normal feeding behaviour. 2.4.8 In vivo microdialysis studies The effect of Yuanzhi-1 on the extracellular monoamine levels in the frontal cortices was evaluated using in vivo microdialysis coupled with high performance liquid chromatography (Carboni and Di Chiara, 1989; Matthews et al., 2005). Briefly, male Sprague–Dawley rats were anaesthetised with 3% halothane using a calibrated vaporiser and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA). Each rat was cannulated in the medial prefrontal cortex (mPFC; anterior 3.2, lateral 0.5, ventral 2.0) relative to the bregma and the skull, according to the atlas of Paxinos et al. (Murone et al., 1997). The probe was secured to the skull with dental cement and stainless steel screws. After surgery, the animals were treated with daily penicillin (10,000 U, s.c.) to prevent infection and were housed in individual test cages. After recovering from the surgery, the animals were subjected to microanalysis: A microanalysis probe (CMA/12, 2.0 mm membrane) was inserted into the guide cannula to replace the dummy cannula. The probe was perfused at 2 ȝl/min with artificial cerebrospinal fluid (147 mM of NaCl, 4 mM of KCl, 2.3 mM of CaCl2, and 1.0 mM of MgCl2, pH 7.4). After at least 2 h of equilibration, dialysate samples were collected every 30 min using tubes filled with chloric acid (20 mM per 10 ȝl/tube to prevent the degradation of monoamines). The steady baseline of each neurotransmitter was established prior to drug treatment by collecting the
first four samples. After the baseline sampling was complete, the animals were orally administered Yuanzhi-1 (2.5, 5, 10 mg/kg) or the vehicle (0.2% Tween 80 and 0.5% methylcellulose dissolved in water) at the end of the sixth sampling. Three hours after Yuanzhi-1 administration, the dialysis samples were collected every 20 min and analysed using high-performance liquid chromatography with electrochemical detection (HPLC-ECD) to determine the levels of NE, 5-HT, and DA. At the end of each experiment, the animals were euthanised, and the probe placement was verified histologically. Data collected from rats with incorrect probe placements were discarded. The HPLC system consisted of a micro-bore reverse-phase column (particle size=5 ȝm, 150×4.6 mm; Model C-18, DIKMA Technologies Ltd., Beijing, China), an Agilent 1100 pump (flow rate=1.0 mL/min; Agilent Technologies, Palo Alto, CA, USA) and a Hewlett-Packard HP 1049A glassy carbon amperometric detector (Agilent Technologies, Palo Alto, CA, USA). The mobile phase consisted of 85 mM of citrate, 100 mM of sodium acetate, 0.9 mM of octyl sodium sulphate, 0.2 mM of EDTA, and 15% methanol, pH 3.7. External standard curves were used to quantify the amounts of NE, 5-HT, and DA in each sample, calculated using the area under curve (AUC). The injection volume was 50 ȝl. The detection limit of the assay was 20 pg per sample. 3. Statistical analyses Unless otherwise specified, statistical analysis was performed using GraphPad Prism (GraphPad Prism 5.0, version 2.0; GraphPad Software Inc., San
Diego, CA).As for data from behavioural assays, Student’s t-test was carried out to compare the difference between two groups (i.e., vehicle vs. duloxetine in TST and FST; control non-stressed rats vs. stressed rats in CMS model); other data were analyzed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. The transporter binding and monoamine uptake data were analysed using a one-site nonlinear regression of the concentration–effect curve. The
Ki
values
were
calculated
using
the
Cheng–Prusoff
equation:
Ki=IC50/([L/KD]+1) (Cheng and Prusoff, 1973). For the data from the CMS behavioural assays, Student’s t-test was performed to compare between-group differences. For the microdialysis experiments, a two-way repeated-measures ANOVA, followed by Tukey's test, was used to compare the percentage increase from baseline among groups. Time and treatment were treated as the independent factors, and time was the repeated factor. 4. Results 4.1 The binding affinities of Yuanzhi-1 to rat monoamine transporters In this study, radioligand binding assays were conducted to determine the affinity of Yuanzhi-1 for rat SERTs, NETs, and DATs (the Ki values are shown in Table 3). We found that Yuanzhi-1 showed a high affinity for the rat monoamine transporters, including SERTs, NETs and DATs (Table 3). Our results showed that Yuanzhi-1 was 4 times more potent than fluoxetine in binding to rat SERTs (Ki values for Yuanzhi-1: 3.95±0.21; Fluoxetine: 15.67±0.43 nM, Table 1; Fig. 2A). To evaluate the binding capacity of Yuanzhi-1 to DATs, we found that the
affinity of Yuanzhi-1 to rat DATs was 30 times higher than nomifensine (the Ki values for Yuanzhi-1 and nomifensine were 0.87±0.14 and 30.24±2.24 nM, respectively; Table 3; Fig. 2C). Similarly, the affinity of Yuanzhi-1 to rat NETs was twice as strong as that of desipramine, a known specific inhibitor of NETs. The Ki values for Yuanzhi-1 and desipramine were 4.52±0.82 and 7.78±0.54 nM, respectively (Table 3; Fig. 2B). Comparing the affinity of Yuanzhi-1 with the three tested monoamine transporters, we found that Yuanzhi-1 binds to DATs with the highest affinity, followed by SERTs and NETs. 4.2 The binding of Yuanzhi-1 to human monoamine transporters To study whether Yuanzhi-1 has similar affinities to monoamine transporters from humans as those from rats, we used HEK293 cells transfected with SERTs, NETs or DATs and tested the capacity of Yuanzhi-1 to compete with specific inhibitors of these transporters. As Table 1 displays, Yuanzhi-1 showed high affinity to human monoamine transporters, consistent with our results in rats. Yuanzhi-1 and fluoxetine competed in a dose-dependent manner with the binding of the SERT from cells transfected with hSERT. The Ki values for Yuanzhi-1 and fluoxetine were 2.78±0.58 and 9.42±0.65 nM, respectively (Table 3; Fig. 2D). The binding of [3H]-GBR12909 to hDAT was competed by Yuanzhi-1 and nomifensine with Ki values of 1.03±0.21 and 16.63±3.04 nM, respectively (Table 3; Fig. 2F). Yuanzhi-1 and desipramine competed with the binding of the NET radiolabeled [3H]-nisoxetine to the membranes from cells transfected with hNET, with Ki values of 6.86±0.43 and 11.41±1.04 nM, respectively (Table 3;
Fig. 2E). Similarly to the rat monoamine transporter, the affinity of Yuanzhi-1 for hDAT was higher than that for hSERT and hDAT. 4.3 Yuanzhi-1 inhibits monoamine uptake in rat synaptosomes Our results showed that Yuanzhi-1 inhibited all three transporters (SERTs, DATs and NETs) in a dose-dependent manner (Table 4;Fig.3). The Ki values for the uptake of [3H]-5-HT into rat frontal cortex synaptosomes were 2.12±0.32 and 78.89±3.54 nM for Yuanzhi-1 and fluoxetine, respectively (Table 4; Fig. 3A). Yuanzhi-1 and desipramine inhibited the uptake of [3H]-NE into the synaptosomes, with Ki values of 4.85±0.52 and 6.54±0.84 nM, respectively (Table 4; Fig. 3B). Yuanzhi-1 and nomifensine inhibited [3H]-DA uptake in rat striatal synaptosomes, with Ki values of 1.08±0.04 and 42.14±3.31 nM, respectively (Table 4; Fig. 3C). Similar to its binding profile, Yuanzhi-1 more potently inhibited these transporters than their corresponding specific inhibitors. Furthermore, Yuanzhi-1 had a greater inhibition effect on DATs than SERTs and NETs (Table 4). 4.4 Yuanzhi-1 inhibits the uptake activities of human monoamine transporters HEK cells were transfected with hSERT, hDAT or hNET and treated with Yuanzhi-1 at varying doses. Consistent with the results from the rat synaptosomes, we found that Yuanzhi-1 also significantly inhibited the activities of the NETs, SERTs and DATs in these transfected cell lines. The Ki values for the Yuanzhi-1 inhibiting the uptake of [3H]-5-HT, [3H]-5-NE, and [3H]-DA into
the cells expressing the corresponding human recombinant transporters were 1.65±0.14, 5.32±0.53 nM and 0.68±0.09 nM, respectively (Table 4; Fig. 3). Again, these data demonstrate that Yuanzhi-1 inhibits the transport activities of DATs with more potency than SERTs and NETs. 4.5 The effect of repeated treatment with Yuanzhi-1 on the immobility time in the FST, TST and locomotor activity. To investigate whether Yuanzhi-1 can produce chronic changes to the depression-related behaviours observed in the FST and TST, we treated mice with different doses via continuous oral administration for 7 days. Compared with vehicle, acute administration of duloxetine at 20 mg/kg significantly reduced the immobility time in TST. (Fig. 4A, Student’s t-test, t=2.298, p=0.0338), indicating the behavioral tests were performed under normal condition and reliable. A one-way ANOVA revealed a significant difference (F[6, 63]=7.722, p!-! ! - 6 # 2 0 & & 0) &%0 6 2& 6 % % # 2
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Figure legend: Fig.1.The outline of design for chronic unpredictable stress and behavioral tests. Fig.2. Yuanzhi-1 competes for the binding of radioligands specific to the rSERT (A), rNET(B), rDAT (C), hSERT (D), hNET(E), hDAT (F). For each transporter bioassay, a known comparator was used [fluoxetine(5-HT reuptake inhibitor); desipramine (NE reuptake inhibitor); and nomifensine (DA reuptake inhibitor)]. The IC50 value was generated from each of these curves and usedto generate the Ki values. Each data point depicted represents the mean±S.E.M. of three independent experiments performed in triplicate. The Ki values for Yuanzhi-1 and the comparators are shown in Table 2. Fig.3.Functional activity of Yuanzhi-1 demonstrates inhibition of radiolabeled uptake of serotonin norepinephrine and dopamine in rat synaptosomes(A-C) and in cloned human transporters expressing in
HEK293 cell lines(D-F). For each uptake transporter bioassay, a known comparator was used [fluoxetine (5-HT reuptake inhibitor); desipramine (NE reuptake inhibitor); nomifensine (DA reuptake inhibitor)]. Each data point depicted represents the mean ±S.E.M. of three independent experiments performed in triplicate. The IC50 values for Yuanzhi-1 are shown in Table 3. Fig.4. The sub-chronic effect of Yuanzhi-1 (dose range: 2.5-40 mg/kg) and DLX (20 mg/kg) on the time of immobility during the total 6-min testing period in the tail suspension test (A) , forced swimming test (B) and locomotor activity (C)in mice. Results are expressed as mean ± SEM (n = 8-10/group). The drugs were administered via the oral route 60 min prior to testing. For statistical significance, *p
