Acta Neuropsychiatrica 2015 All rights reserved DOI: 10.1017/neu.2015.14
© Scandinavian College of Neuropsychopharmacology 2015 ACTA NEUROPSYCHIATRICA
Effect of NMDAR antagonists in the tetrabenazine test for antidepressants: comparison with the tail suspension test Skolnick P, Kos T, Czekaj J, Popik P. Effect of NMDAR antagonists in the tetrabenazine test for antidepressants: comparison with the tail suspension test.
Objective: The N-methyl-D-aspartate receptor (NMDAR) antagonist ketamine, produces rapid and enduring antidepressant effect in patients with treatment-resistant depression. Similar dramatic effects have not been observed in clinical trials with other NMDAR antagonists indicating ketamine may possess unique pharmacological properties. Tetrabenazine induces ptosis (a drooping of the eyelids), and the reversal of this effect, attributed to a sympathomimetic action, has been used to detect firstgeneration antidepressants, as well as ketamine. Because the actions of other NMDAR antagonists have not been reported in this measure, we examined whether reversal of tetrabenazine-induced ptosis was unique to ketamine, or a class effect of NMDAR antagonists. Methods: The effects of ketamine and other NMDAR antagonists to reverse tetrabenazine-induced ptosis were examined and compared with their antidepressant-like effects in the tail suspension test (TST) in mice. Results: All the NMDAR antagonists tested produced a partial reversal of tetrabenazine-induced ptosis and, as expected, reduced immobility in the TST. Ketamine, memantine, MK-801 and AZD6765 were all about half as potent in reversing tetrabenazine-induced ptosis compared to reducing immobility in the TST, while an NR2B antagonist (Ro 25-6981) and a glycine partial agonist (ACPC) were equipotent in both tests. Conclusion: The ability to reverse tetrabenazine-induced ptosis is a class effect of NMDAR antagonists. These findings are consistent with the hypothesis that the inability of memantine, AZD6765 (lanicemine) and MK-0657 to reproduce the rapid and robust antidepressant effects of ketamine in the clinic result from insufficient dosing rather than a difference in mechanism of action among these NMDAR antagonists.
Phil Skolnick1, Tomasz Kos2, Janusz Czekaj3, Piotr Popik2,3 1 Division of Pharmacotherapies & Medical Consequences of Drug Abuse, NIDA, NIH, Bethesda, MD, USA; 2Behavioral Neuroscience and Drug Development Institute of Pharmacology, Polish Academy of Sciences, Krako´w, Poland; and 3Faculty of Health Sciences, Collegium Medicum, Jagiellonian University, Krako´w, Poland
Keywords: antidepressant; NMDAR antagonist; ptosis; tail suspension test; tetrabenazine Piotr Popik, Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland. Tel: + 48 + 12 6623375; Fax: + 48 + 12 6374500; E-mail: [email protected] Accepted for publication February 09, 2015 First published online April 10, 2015
Significance outcomes
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All of the N-methyl-D-aspartate receptor (NMDAR) antagonists tested (ketamine, memantine, MK-801, AZD6765, Ro 25-6981 and ACPC) partially reversed tetrabenazine-induced ptosis and reduced immobility in the tail-suspension test. The ability to reverse tetrabenazine-induced ptosis is attributable to a sympathomimetic action that appears an effect common to NMDAR antagonists. Since we found no differences between ketamine and other NMDAR antagonists tested, we hypothesise that the failure of memantine and possibly other NMDAR antagonists (the channel blocker lanicemine and an NR2B antagonist, MK-0657) to manifest robust, ketamine-like antidepressant efficacy in the clinic results from inadequate target engagement (insufficient dosing) rather than a fundamental difference in mechanism of action.
NMDAR antagonists and tetrabenazine-induced ptosis Limitations
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The pharmacokinetic profile (including Tmax and potential metabolites) of the compounds in this mouse strain are not known. Since behavioural testing was performed at fixed time points, the relative potencies of the compounds in these measures are not optimised. The use of a subjective rating scale to determine the potency of compounds to reverse tetrabenazineinduced ptosis may reduce the reliability of the estimated minimum effective dose (MED) values of NMDAR antagonists. There are alternative, and perhaps equally plausible hypotheses to account for the unique antidepressant profile of ketamine compared with other NMDAR antagonists. These hypotheses range from a transient blockade of NMDAR defined by the unique pharmacokinetic profile of ketamine to its ability to produce a burst of glutamate release, which has been hypothesised to be necessary for the rapid and robust effects observed in treatment-resistant depression. Several of these hypotheses are amenable to further testing in preclinical studies. Clinical studies will be required to critically test the hypothesis that inadequate dosing is responsible for the inability of NMDAR antagonists like memantine, lanicemine and MK-0657 to mimic the robust antidepressant effects of ketamine.
Introduction
Numerous clinical trials have demonstrated that a single intravenous infusion of the NMDAR antagonist ketamine produces a rapid, robust and relatively long-lived antidepressant effect in patients with treatment-resistant depression (1). There are multiple mechanisms that can produce NMDAR blockade, and ketamine acts at the NMDAR-associated ion channel (2,3). However, these dramatic clinical effects have not been replicated using other clinically available NMDA channel blockers, including memantine (4) and AZD6765 (lanicemine) (5,6), despite the remarkably similar electrophysiological properties of these compounds at NMDAR (3). Multiple explanations can be invoked to explain the inability of these compounds to replicate the antidepressant effects of ketamine, ranging from inadequate dosing (4) to pharmacokinetic differences among these agents (7) that could result in dramatically different intracellular effects following NMDAR blockade. All pharmacological classes of NMDAR antagonists produce antidepressant-like effects in ‘behavioural despair’ models, including the forced swim (8) and tail suspension (9) tests (for a recent review see (10)). These tests, while lacking substantial construct and face validity, have high predictive validities (11,12). In addition, ketamine, like classical tricyclic antidepressants, was reported to mimic the effect of imipramine in reversing tetrabenazine-induced ptosis (13). This test relies on the ability of tetrabenazine to deplete monoamines from nerve endings causing the eyelids to droop. Monoamine reuptake inhibitors (such as desmethylimipramine, amitriptyline and imipramine) are particularly effective in reversing this ptosis (13). This study by Sofia and Harakal (13) was prompted by the sympathomimetic actions of ketamine
manifested in both animals and humans (14), and published a decade before ketamine was identified as an NMDAR antagonist (2). Given the apparent lack of concordance between clinical and preclinical findings with NMDAR antagonists (10), the purpose of the present study was to compare the effects of ketamine to other NMDAR antagonists in the tetrabenazineinduced ptosis test. Parallel studies were conducted in the tail suspension test (TST) to minimise potential confounds (such as strain differences) when comparing data among laboratories (10). Materials and methods Animals
Male C57Bl/6J mice (obtained from Institute of Pharmacology breeding facility, originating from the Jackson Laboratories and purchased from Charles-River, Germany) weighing ∼24 g, were ∼7 to 8 weeks old at the start of the experiment. The mice were housed in standard laboratory cages under standard colony A/C-controlled conditions: room temperature of 21 ± 2°C, humidity of 40% to 50% and a 12-h light/dark cycle (lights on at 06:00). The mice had ad libitum access to the standard lab chow and tap water. Behavioural testing was performed during the light phase of the light/dark cycle. All mice were used only once, and each separate vehicle-controlled experiment was carried out on a separate cohort of animals. Drugs
HCl salts of imipramine, memantine and AZD6765 (lanicemine); (Sigma-Aldrich, Poznan, Poland) dizocilpine maleate (MK-801; Abcam Biochemicals, Cambridge, UK), ketamine [10% aqueous solution 229
Skolnick et al. (115.34 mg/ml) Biowet, Pulawy, Poland], Ro 25-6981 [(aR,bS)-a-(4-hydroxyphenyl)-b-methyl-4(phenylmethyl)-1-piperidinepropanol hydrochloride] (Abcam Biochemicals, Cambridge, UK) and ACPC (1-aminocyclopropanecarboxylic acid, kindly donated by Dr. M-L Maccecchini) were dissolved in sterile water. Tetrabenazine (Sequoia Research Products, Pangbourne, UK) was dissolved in 1N acetic acid followed by adjustment to pH 6 with NaOH 10% and supplemented with distilled water to the appropriate volume. Physiological saline was used as a vehicle; all solutions were prepared in the volume of 10 ml/kg, right before the administration. The doses selection for the TST was based on previous reports showing a significant reduction of immobility for imipramine (≥2 mg/kg) (15), ketamine (≥50 mg/kg) (16,17), memantine (≥2.5 mg/kg) (18), MK-801 (0.01 mg/kg) (19), Ro 25-6981 (10 mg/kg) (20) and ACPC (in the mouse forced swim test; ≥100 mg/kg) (21). The dose selection for tetrabenazine-induced ptosis and its reversal by ketamine (20 mg/kg) and imipramine (1.25 mg/kg) was based on the initial report of Sofia and Harakal (13). To our knowledge, the effects of MK-801, memantine, Ro 25-6981, AZD6765 and ACPC on tetrabenazine-induced ptosis have not been reported.
ANOVA followed by nonparametric Dunn’s multiple comparisons post-hoc test. This was done to identify the MED, the lowest dose of test compound producing a statistically significant effect. Data are presented as median ± interquartile range. TST. Vehicle and various doses of tested compounds were administered IP, 45 min before the test. Immobility in the TST was measured as previously described (9,23). Mice were suspended 65 cm above the table top using a paper adhesive tape; the tape was placed ∼1 cm from the tip of the tail. Mice were suspended for 6 min and an observer blind to the treatment conditions recorded the duration of immobility, defined as a complete lack of movement. Statistical analyses were performed using GraphPad Prism 5.0 for Windows. The α value was set at p < 0.05. The given set of treatments (each having its own vehicle control) was subjected to one-way ANOVA followed by Dunnett’s multiple comparisons post-hoc test. This was done to identify the MED in the TST. Data are presented as mean ± SEM. In this set of experiments we did not measure drug-induced locomotor activity. The measurement of animal motility is often used as a control for the specificity of the antidepressant-like effects of compounds, which was not a goal of the present study.
Behavioural procedures
In both behavioural tests, we investigated the effects of seven different NMDAR antagonists, each used at two to five doses. This was done on separate cohorts of mice within separate small experiments. Thus, every experiment (a set of treatments) contained its own vehicle control. Care was taken to have no more treatment groups than six per a separate vehicle control. Tetrabenazine-induced ptosis and its reversal. Groups of mice were administered various doses of test compounds or saline (vehicle) intraperitoneally (IP). Thirty minutes later, the animals were injected with tetrabenazine (IP, 32 mg/kg), and then placed individually into 1 l glass beakers (13). Thirty minutes following tetrabenazine injection (i.e. 60 min following test compound), each mouse was evaluated for the presence of palpebral ptosis by an observer ‘blinded’ to the treatment conditions. The degree of ptosis was rated according to the following rating scale: eyes open (0 points), eyes half closed (1 point), eyes completely closed (2 points) (22). Statistical analyses were performed using GraphPad Prism 5.0 for Windows. The α value was set at p < 0.05. Due to the lack of normal distribution, the given set of treatments (each having its own vehicle control) was subjected to one-way Kruskal–Wallis 230
Results
Tetrabenazine produced complete ptosis in all mice by 30 min post injection. Imipramine, employed as a positive control, produced a dose-dependent reduction in tetrabenazine-induced ptosis, with a MED of 2.5 mg/kg (which was also the lowest dose tested) and a complete reversal at 10 mg/kg (Fig. 1, upper panel). Imipramine also produced a dose-dependent reduction in immobility in the TST, with a MED of 2.5 mg/kg (Fig. 1, lower panel). While all NMDAR antagonists produced statistically significant reductions in tetrabenazine-induced ptosis at one or more doses, none of these compounds fully reversed the ptosis in all animals. The NMDA channel blockers (ketamine, memantine, MK-801, AZD6765) were all less potent (based on MED) in reversing tetrabenazine-induced ptosis than reducing immobility in the TST. For ketamine, the MEDs for attenuating tetrabenazine-induced ptosis and reducing immobility were 50 and 20 mg/kg respectively; for memantine these doses were 15 and 10 mg/kg, respectively, for MK-801 these doses were 0.2 and 0.1 mg/kg, respectively and for AZD6765 these doses were 18 and 4.5 mg/kg, respectively. In contrast, the MEDs of Ro 25-6981 (5 mg/kg) and ACPC (200 mg/kg) in the reversal of tetrabenazine induced ptosis and the TST were identical.
NMDAR antagonists and tetrabenazine-induced ptosis Reversal of Tetrabenazine Ptosis
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IM VE I2 H IM .5 IM I 5 I K 10 KEET 5 KET 2 T 0 50 M VE M EM H EM M 75 E . M M 5 E 1 M M1 0 EM 5 M K2 M 801 V 0 K- 0 EH M 80 .0 K- 1 5 R 80 0. o 1 1 25 0. R -69 2 o R 2 81 VE o 5 2 H 25 -6 .5 -6 98 98 1 1 5 1 AC 0 V ACPC EH ACPC 100 PC 20 0 AZ 4 AZ D V 00 6 E AZD6 765 H D 765 9 67 1 65 8 36
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V IM EH I2 .5 IM I1 KE 0 T KE 20 T M 5 EM 0 M 10 EM 2 M K- V 0 80 E M 1 H K- 0. 0 M 801 5 K- 0 80 .1 1 R 0. o R 25 V 2 o 25 69 EH -6 8 98 1 1 5 10 AC V P EH AC C 1 P 00 AC C 2 PC 00 40 AZ 0 D 67 VE H 6 AZ 5 4 AZ D67 .5 D 65 67 9 65 18
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Fig. 1. Tetrabenazine-induced ptosis and its reversal (upper panel) and anti-immobility effects in the tail-suspension tests (lower panel) of imipramine (IMI) and N-methyl-D-aspartate receptor (NMDAR) antagonists ketamine (KET), memantine (MEM), dizocilpine (MK-801), Ro 25-6981, ACPC and AZD6765. Testing for each compound was done on a separate group of mice and was controlled with a separate vehicle group. Upper panel: For tetrabenazine-induced ptosis, experiment 1 involving imipramine and ketamine, experiment 2 involving memantine, experiment 3 involving MK-801, experiment 4 involving Ro 25-6981, experiment 5 involving ACPC and experiment 6 involving AZD6765, yielded the following Kruskal–Wallis ANOVA values: 57.55 (df = 6); p < 0.0001; 26.68 (df = 5); p < 0.0001; 10.09, (df = 3), p < 0.05; 33.20 (df = 3), p < 0.0001; 26.22 (df = 3), p < 0.0001, 26.70 (df = 3), p < 0.0001, respectively. Symbols: *p < 0.05–p < 0.0001 versus respective vehicle (the precise p-value is not given for the clarity of figure). In experiment 1, the number of mice for vehicle, imipramine at 2.5 mg/kg, imipramine at 5 mg/kg, imipramine 10 mg/kg, ketamine at 5 mg/kg, ketamine at 20 mg/kg and ketamine at 50 mg/kg were 14, 10, 10, 10, 11, 10 and 10, respectively. In experiment 2, the number of mice for vehicle, memantine at 5, 7.5, 10, 15 and 20 mg/kg, were 16, 10, 8, 10, 8 and 10, respectively. In experiment 3, the number of mice for vehicle, MK-801 at 0.05, 0.1 and 0.2 mg/kg were 9, 10, 10 and 10, respectively. In experiment 4, the number of mice for vehicle, Ro 25-6981 at 2.5, 5 and 10 mg/kg was 17, 10, 10 and 10, respectively. In experiment 5, the number of mice for vehicle, ACPC at 100, 200 and 400 mg/kg was 20, 12, 10 and 10, respectively. In experiment 6, the number of mice for vehicle, AZD6765 at 9, 18 and 36 mg/kg was 10 in each group. Lower panel: For the tail suspension tests, experiment 1 involving imipramine, ketamine and memantine, experiment 2 involving MK-801, experiment 3 involving Ro 25-6981, experiment 4 involving ACPC and experiment 5 involving AZD6765, yielded the following one-way ANOVA values: F(6,55) = 16.35, p < 0.0001; F(3,33) = 44.77, p < 0.0001; F(2,21) = 28.37, p < 0.0001, F(3,36) = 31.02, p < 0.0001 and F(3,36) = 28.45, p < 0.0001, respectively. Symbols: *p < 0.05–p < 0.0001 versus respective vehicle (the precise p-value is not given for the clarity of figure). In experiment 1, the number of mice for vehicle, imipramine at 2.5 mg/kg, imipramine at 10 mg/kg, ketamine at 20 mg/kg, ketamine at 50 mg/kg, memantine at 10 mg/kg and memantine at 20 mg/kg, was 8, 10, 8, 10, 8, 10 and 8, respectively. In experiment 2, the number of mice for vehicle, MK-801 at 0.05, 0.1 and 0.2 mg/kg was 13, 8, 8 and 8, respectively. In experiment 3, the number of mice for vehicle, Ro 25-6981 at 5 and 10 mg/kg was 8 in each group. In experiment 4, the number of mice for vehicle, ACPC at 100, 200 and 400 mg/kg was 10 in each group. In experiment 5, the number of mice for vehicle, AZD6765 at 4.5, 9 and 18 mg/kg was 10 in each group.
Discussion
Multiple intracellular events have been linked to the rapid and sustained effects of ketamine following
NMDAR blockade. These include α-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor activation, increased brain-derived neurotrophic factor (BDNF) formation and activation of 231
Skolnick et al. tropomyosin-related kinase B (TrKB), activation of mammalian target of rapamycin (mTOR), vascular endothelial growth factor (VEGF) and their associated cascades (24–27). However, there appear to be significant differences among NMDAR antagonists with respect to reported duration of action and sensitivity to AMPA receptor blockade in both behavioural despair tests (16,23,28–31) and models like chronic mild stress (32,33), which appear to possess better face and construct validity. An emerging body of clinical work indicates the rapid and robust antidepressant effects of ketamine are not shared by either other NMDA channel blockers [e.g. memantine (4) and AZD6765 (lanicemine) (5,6)] or the NR2B antagonist MK-0657 (34). Ketamine was reported to reverse tetrabenazine-induced ptosis (13), an action shared by tricyclic antidepressants, and indicative of a sympathomimetic action. To our knowledge, the ability of other NMDAR antagonists to reverse tetrabenazine-induced ptosis had not been examined. Thus, a series of NMDAR antagonists were tested in order to determine if this action was shared with ketamine. Since NMDAR antagonists are active in behavioural despair tests (reviewed in (10)), these compounds were evaluated in parallel using the TST to eliminate experimental variables that can make direct comparisons across laboratories problematic. In this regard, the MED of ketamine in the TST (20 mg/kg) merits additional comment. While there is general agreement that ketamine is active in behavioural despair measures, some investigators report activity in the 3–10 mg/kg range, whereas other studies require substantially higher doses (>20 mg/kg) (reviewed in Pilc et al. (10)). It has been speculated that differences in both the potency and duration of action may be attributable to strain differences in the metabolism of ketamine (10). The sympathomimetic properties of ketamine (35) led Sofia and Harakal (13) to examine its ability to reverse tetrabenazine-induced ptosis, an early preclinical screen for biogenic amine-based antidepressants (36). Similarly, the sympathomimetic properties of MK-801 were recognised early in its development (37), before its identification as a highly selective NMDAR antagonist (38). Like ketamine, these effects of MK-801 also appear to arise from a centrally mediated sympathomimetic action (39). Consistent with the effects of other NMDA channel blockers, doserelated increases in blood pressure have also been noted following AZD6765 administration (6). Hypertension is one of the side effects associated with mild to moderate memantine overdose (40). The present findings, demonstrating that all NMDAR antagonists tested partially reverse tetrabenazineinduced ptosis, indicate this is a class effect, which may be mediated via a centrally mediated 232
sympathomimetic action by dint of NMDAR receptor blockade (39). At face value, the present findings do not provide insights that may help explain potential differences between ketamine and other NMDAR antagonists reported in both preclinical and clinical studies. Nonetheless, the ability of memantine to produce both an antidepressant-like effect in behavioural despair procedures (Fig. 1a and (18,41–43)) and partially reverse tetrabenazineinduced ptosis, suggest these doses are sufficient to block NMDAR. If a central sympathomimetic action is a class effect produced by NMDAR antagonists, then the failure to observe an antidepressant effect with memantine – either in a randomised clinical trial (4) or an antidepressant-like signal after many years of clinical use – indicates that the doses used in this randomised clinical trial and routinely in the treatment of Alzheimer’s disease (starting at 5 mg and titrating to 20 mg/day) may, despite claims to the contrary (44), be insufficient to block NMDAR. Similarly, the failure to observe an antidepressant signal with the NR2B antagonist MK-0657 could be due to the lack of target engagement. Thus, no symptoms of either hypertension or dissociative-like effects were reported in the study using divided doses of 4 to 8 mg/day (34). In contrast, hypertension was reported in an earlier study (45), administering a single oral dose of 7 mg/kg of MK-0657 to patients with Parkinson’s disease. Since ketamine (1) produces feelings of depersonalisation and even psychosis (46), it cannot be excluded that these unwanted side-effects, manifestations of target activation (NMDAR blockade), are inextricably linked to its efficacy for treating affective disorder (47). Psychotomimetic-like symptoms have also been reported following administration of both dizocilpine (MK-801 (48)); and memantine (49), consistent with the hypothesis that this is a class effect of NMDAR antagonists. Also consistent with this hypothesis, Luckenbaugh et al. (50) recently reported that ketamine-induced increases in dissociation and psychotomimesis at 40 min post infusion were highly correlated (r = − 0.41, p = 0.01) with reductions in the Hamilton Depression rating scale at 7 days post infusion. Thus, if NMDAR antagonists are dosed to produce target engagement (e.g. manifested as dissociative-like reactions and/or hypertension), then this class of compounds, including memantine, lanicemine (5,6) and MK-0657 should produce efficacies comparable to ketamine (47) in treatment-resistant depression. Additional preclinical studies (for example, determining if a glutamate burst and subsequent AMPA receptor activation are observed following administration of all NMDAR antagonists) could further refine this hypothesis by comparing ketamine to other NMDAR antagonists that do not
NMDAR antagonists and tetrabenazine-induced ptosis appear to produce the same robust antidepressant effects in the clinic (10). Acknowledgements
This work was funded by the Statutory Activity of Institute of Pharmacology, Polish Academy of Sciences. The authors thank Dr. A. Bespalov for the generous gift of tetrabenazine. P.S. and P.P. designed the study and wrote the manuscript. T.K. carried out the experiments, analysed the data and wrote parts of the manuscript. J.C. provided important intellectual content, assisted with the interpretation of results and approved the final version. Conflicts of Interest
None. Ethical Standards
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© Scandinavian College of Neuropsychopharmacology 2015 ACTA NEUROPSYCHIATRICA
Effect of NMDAR antagonists in the tetrabenazine test for antidepressants: comparison with the tail suspension test Skolnick P, Kos T, Czekaj J, Popik P. Effect of NMDAR antagonists in the tetrabenazine test for antidepressants: comparison with the tail suspension test.
Objective: The N-methyl-D-aspartate receptor (NMDAR) antagonist ketamine, produces rapid and enduring antidepressant effect in patients with treatment-resistant depression. Similar dramatic effects have not been observed in clinical trials with other NMDAR antagonists indicating ketamine may possess unique pharmacological properties. Tetrabenazine induces ptosis (a drooping of the eyelids), and the reversal of this effect, attributed to a sympathomimetic action, has been used to detect firstgeneration antidepressants, as well as ketamine. Because the actions of other NMDAR antagonists have not been reported in this measure, we examined whether reversal of tetrabenazine-induced ptosis was unique to ketamine, or a class effect of NMDAR antagonists. Methods: The effects of ketamine and other NMDAR antagonists to reverse tetrabenazine-induced ptosis were examined and compared with their antidepressant-like effects in the tail suspension test (TST) in mice. Results: All the NMDAR antagonists tested produced a partial reversal of tetrabenazine-induced ptosis and, as expected, reduced immobility in the TST. Ketamine, memantine, MK-801 and AZD6765 were all about half as potent in reversing tetrabenazine-induced ptosis compared to reducing immobility in the TST, while an NR2B antagonist (Ro 25-6981) and a glycine partial agonist (ACPC) were equipotent in both tests. Conclusion: The ability to reverse tetrabenazine-induced ptosis is a class effect of NMDAR antagonists. These findings are consistent with the hypothesis that the inability of memantine, AZD6765 (lanicemine) and MK-0657 to reproduce the rapid and robust antidepressant effects of ketamine in the clinic result from insufficient dosing rather than a difference in mechanism of action among these NMDAR antagonists.
Phil Skolnick1, Tomasz Kos2, Janusz Czekaj3, Piotr Popik2,3 1 Division of Pharmacotherapies & Medical Consequences of Drug Abuse, NIDA, NIH, Bethesda, MD, USA; 2Behavioral Neuroscience and Drug Development Institute of Pharmacology, Polish Academy of Sciences, Krako´w, Poland; and 3Faculty of Health Sciences, Collegium Medicum, Jagiellonian University, Krako´w, Poland
Keywords: antidepressant; NMDAR antagonist; ptosis; tail suspension test; tetrabenazine Piotr Popik, Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland. Tel: + 48 + 12 6623375; Fax: + 48 + 12 6374500; E-mail: [email protected] Accepted for publication February 09, 2015 First published online April 10, 2015
Significance outcomes
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All of the N-methyl-D-aspartate receptor (NMDAR) antagonists tested (ketamine, memantine, MK-801, AZD6765, Ro 25-6981 and ACPC) partially reversed tetrabenazine-induced ptosis and reduced immobility in the tail-suspension test. The ability to reverse tetrabenazine-induced ptosis is attributable to a sympathomimetic action that appears an effect common to NMDAR antagonists. Since we found no differences between ketamine and other NMDAR antagonists tested, we hypothesise that the failure of memantine and possibly other NMDAR antagonists (the channel blocker lanicemine and an NR2B antagonist, MK-0657) to manifest robust, ketamine-like antidepressant efficacy in the clinic results from inadequate target engagement (insufficient dosing) rather than a fundamental difference in mechanism of action.
NMDAR antagonists and tetrabenazine-induced ptosis Limitations
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The pharmacokinetic profile (including Tmax and potential metabolites) of the compounds in this mouse strain are not known. Since behavioural testing was performed at fixed time points, the relative potencies of the compounds in these measures are not optimised. The use of a subjective rating scale to determine the potency of compounds to reverse tetrabenazineinduced ptosis may reduce the reliability of the estimated minimum effective dose (MED) values of NMDAR antagonists. There are alternative, and perhaps equally plausible hypotheses to account for the unique antidepressant profile of ketamine compared with other NMDAR antagonists. These hypotheses range from a transient blockade of NMDAR defined by the unique pharmacokinetic profile of ketamine to its ability to produce a burst of glutamate release, which has been hypothesised to be necessary for the rapid and robust effects observed in treatment-resistant depression. Several of these hypotheses are amenable to further testing in preclinical studies. Clinical studies will be required to critically test the hypothesis that inadequate dosing is responsible for the inability of NMDAR antagonists like memantine, lanicemine and MK-0657 to mimic the robust antidepressant effects of ketamine.
Introduction
Numerous clinical trials have demonstrated that a single intravenous infusion of the NMDAR antagonist ketamine produces a rapid, robust and relatively long-lived antidepressant effect in patients with treatment-resistant depression (1). There are multiple mechanisms that can produce NMDAR blockade, and ketamine acts at the NMDAR-associated ion channel (2,3). However, these dramatic clinical effects have not been replicated using other clinically available NMDA channel blockers, including memantine (4) and AZD6765 (lanicemine) (5,6), despite the remarkably similar electrophysiological properties of these compounds at NMDAR (3). Multiple explanations can be invoked to explain the inability of these compounds to replicate the antidepressant effects of ketamine, ranging from inadequate dosing (4) to pharmacokinetic differences among these agents (7) that could result in dramatically different intracellular effects following NMDAR blockade. All pharmacological classes of NMDAR antagonists produce antidepressant-like effects in ‘behavioural despair’ models, including the forced swim (8) and tail suspension (9) tests (for a recent review see (10)). These tests, while lacking substantial construct and face validity, have high predictive validities (11,12). In addition, ketamine, like classical tricyclic antidepressants, was reported to mimic the effect of imipramine in reversing tetrabenazine-induced ptosis (13). This test relies on the ability of tetrabenazine to deplete monoamines from nerve endings causing the eyelids to droop. Monoamine reuptake inhibitors (such as desmethylimipramine, amitriptyline and imipramine) are particularly effective in reversing this ptosis (13). This study by Sofia and Harakal (13) was prompted by the sympathomimetic actions of ketamine
manifested in both animals and humans (14), and published a decade before ketamine was identified as an NMDAR antagonist (2). Given the apparent lack of concordance between clinical and preclinical findings with NMDAR antagonists (10), the purpose of the present study was to compare the effects of ketamine to other NMDAR antagonists in the tetrabenazineinduced ptosis test. Parallel studies were conducted in the tail suspension test (TST) to minimise potential confounds (such as strain differences) when comparing data among laboratories (10). Materials and methods Animals
Male C57Bl/6J mice (obtained from Institute of Pharmacology breeding facility, originating from the Jackson Laboratories and purchased from Charles-River, Germany) weighing ∼24 g, were ∼7 to 8 weeks old at the start of the experiment. The mice were housed in standard laboratory cages under standard colony A/C-controlled conditions: room temperature of 21 ± 2°C, humidity of 40% to 50% and a 12-h light/dark cycle (lights on at 06:00). The mice had ad libitum access to the standard lab chow and tap water. Behavioural testing was performed during the light phase of the light/dark cycle. All mice were used only once, and each separate vehicle-controlled experiment was carried out on a separate cohort of animals. Drugs
HCl salts of imipramine, memantine and AZD6765 (lanicemine); (Sigma-Aldrich, Poznan, Poland) dizocilpine maleate (MK-801; Abcam Biochemicals, Cambridge, UK), ketamine [10% aqueous solution 229
Skolnick et al. (115.34 mg/ml) Biowet, Pulawy, Poland], Ro 25-6981 [(aR,bS)-a-(4-hydroxyphenyl)-b-methyl-4(phenylmethyl)-1-piperidinepropanol hydrochloride] (Abcam Biochemicals, Cambridge, UK) and ACPC (1-aminocyclopropanecarboxylic acid, kindly donated by Dr. M-L Maccecchini) were dissolved in sterile water. Tetrabenazine (Sequoia Research Products, Pangbourne, UK) was dissolved in 1N acetic acid followed by adjustment to pH 6 with NaOH 10% and supplemented with distilled water to the appropriate volume. Physiological saline was used as a vehicle; all solutions were prepared in the volume of 10 ml/kg, right before the administration. The doses selection for the TST was based on previous reports showing a significant reduction of immobility for imipramine (≥2 mg/kg) (15), ketamine (≥50 mg/kg) (16,17), memantine (≥2.5 mg/kg) (18), MK-801 (0.01 mg/kg) (19), Ro 25-6981 (10 mg/kg) (20) and ACPC (in the mouse forced swim test; ≥100 mg/kg) (21). The dose selection for tetrabenazine-induced ptosis and its reversal by ketamine (20 mg/kg) and imipramine (1.25 mg/kg) was based on the initial report of Sofia and Harakal (13). To our knowledge, the effects of MK-801, memantine, Ro 25-6981, AZD6765 and ACPC on tetrabenazine-induced ptosis have not been reported.
ANOVA followed by nonparametric Dunn’s multiple comparisons post-hoc test. This was done to identify the MED, the lowest dose of test compound producing a statistically significant effect. Data are presented as median ± interquartile range. TST. Vehicle and various doses of tested compounds were administered IP, 45 min before the test. Immobility in the TST was measured as previously described (9,23). Mice were suspended 65 cm above the table top using a paper adhesive tape; the tape was placed ∼1 cm from the tip of the tail. Mice were suspended for 6 min and an observer blind to the treatment conditions recorded the duration of immobility, defined as a complete lack of movement. Statistical analyses were performed using GraphPad Prism 5.0 for Windows. The α value was set at p < 0.05. The given set of treatments (each having its own vehicle control) was subjected to one-way ANOVA followed by Dunnett’s multiple comparisons post-hoc test. This was done to identify the MED in the TST. Data are presented as mean ± SEM. In this set of experiments we did not measure drug-induced locomotor activity. The measurement of animal motility is often used as a control for the specificity of the antidepressant-like effects of compounds, which was not a goal of the present study.
Behavioural procedures
In both behavioural tests, we investigated the effects of seven different NMDAR antagonists, each used at two to five doses. This was done on separate cohorts of mice within separate small experiments. Thus, every experiment (a set of treatments) contained its own vehicle control. Care was taken to have no more treatment groups than six per a separate vehicle control. Tetrabenazine-induced ptosis and its reversal. Groups of mice were administered various doses of test compounds or saline (vehicle) intraperitoneally (IP). Thirty minutes later, the animals were injected with tetrabenazine (IP, 32 mg/kg), and then placed individually into 1 l glass beakers (13). Thirty minutes following tetrabenazine injection (i.e. 60 min following test compound), each mouse was evaluated for the presence of palpebral ptosis by an observer ‘blinded’ to the treatment conditions. The degree of ptosis was rated according to the following rating scale: eyes open (0 points), eyes half closed (1 point), eyes completely closed (2 points) (22). Statistical analyses were performed using GraphPad Prism 5.0 for Windows. The α value was set at p < 0.05. Due to the lack of normal distribution, the given set of treatments (each having its own vehicle control) was subjected to one-way Kruskal–Wallis 230
Results
Tetrabenazine produced complete ptosis in all mice by 30 min post injection. Imipramine, employed as a positive control, produced a dose-dependent reduction in tetrabenazine-induced ptosis, with a MED of 2.5 mg/kg (which was also the lowest dose tested) and a complete reversal at 10 mg/kg (Fig. 1, upper panel). Imipramine also produced a dose-dependent reduction in immobility in the TST, with a MED of 2.5 mg/kg (Fig. 1, lower panel). While all NMDAR antagonists produced statistically significant reductions in tetrabenazine-induced ptosis at one or more doses, none of these compounds fully reversed the ptosis in all animals. The NMDA channel blockers (ketamine, memantine, MK-801, AZD6765) were all less potent (based on MED) in reversing tetrabenazine-induced ptosis than reducing immobility in the TST. For ketamine, the MEDs for attenuating tetrabenazine-induced ptosis and reducing immobility were 50 and 20 mg/kg respectively; for memantine these doses were 15 and 10 mg/kg, respectively, for MK-801 these doses were 0.2 and 0.1 mg/kg, respectively and for AZD6765 these doses were 18 and 4.5 mg/kg, respectively. In contrast, the MEDs of Ro 25-6981 (5 mg/kg) and ACPC (200 mg/kg) in the reversal of tetrabenazine induced ptosis and the TST were identical.
NMDAR antagonists and tetrabenazine-induced ptosis Reversal of Tetrabenazine Ptosis
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IM VE I2 H IM .5 IM I 5 I K 10 KEET 5 KET 2 T 0 50 M VE M EM H EM M 75 E . M M 5 E 1 M M1 0 EM 5 M K2 M 801 V 0 K- 0 EH M 80 .0 K- 1 5 R 80 0. o 1 1 25 0. R -69 2 o R 2 81 VE o 5 2 H 25 -6 .5 -6 98 98 1 1 5 1 AC 0 V ACPC EH ACPC 100 PC 20 0 AZ 4 AZ D V 00 6 E AZD6 765 H D 765 9 67 1 65 8 36
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V IM EH I2 .5 IM I1 KE 0 T KE 20 T M 5 EM 0 M 10 EM 2 M K- V 0 80 E M 1 H K- 0. 0 M 801 5 K- 0 80 .1 1 R 0. o R 25 V 2 o 25 69 EH -6 8 98 1 1 5 10 AC V P EH AC C 1 P 00 AC C 2 PC 00 40 AZ 0 D 67 VE H 6 AZ 5 4 AZ D67 .5 D 65 67 9 65 18
0
Fig. 1. Tetrabenazine-induced ptosis and its reversal (upper panel) and anti-immobility effects in the tail-suspension tests (lower panel) of imipramine (IMI) and N-methyl-D-aspartate receptor (NMDAR) antagonists ketamine (KET), memantine (MEM), dizocilpine (MK-801), Ro 25-6981, ACPC and AZD6765. Testing for each compound was done on a separate group of mice and was controlled with a separate vehicle group. Upper panel: For tetrabenazine-induced ptosis, experiment 1 involving imipramine and ketamine, experiment 2 involving memantine, experiment 3 involving MK-801, experiment 4 involving Ro 25-6981, experiment 5 involving ACPC and experiment 6 involving AZD6765, yielded the following Kruskal–Wallis ANOVA values: 57.55 (df = 6); p < 0.0001; 26.68 (df = 5); p < 0.0001; 10.09, (df = 3), p < 0.05; 33.20 (df = 3), p < 0.0001; 26.22 (df = 3), p < 0.0001, 26.70 (df = 3), p < 0.0001, respectively. Symbols: *p < 0.05–p < 0.0001 versus respective vehicle (the precise p-value is not given for the clarity of figure). In experiment 1, the number of mice for vehicle, imipramine at 2.5 mg/kg, imipramine at 5 mg/kg, imipramine 10 mg/kg, ketamine at 5 mg/kg, ketamine at 20 mg/kg and ketamine at 50 mg/kg were 14, 10, 10, 10, 11, 10 and 10, respectively. In experiment 2, the number of mice for vehicle, memantine at 5, 7.5, 10, 15 and 20 mg/kg, were 16, 10, 8, 10, 8 and 10, respectively. In experiment 3, the number of mice for vehicle, MK-801 at 0.05, 0.1 and 0.2 mg/kg were 9, 10, 10 and 10, respectively. In experiment 4, the number of mice for vehicle, Ro 25-6981 at 2.5, 5 and 10 mg/kg was 17, 10, 10 and 10, respectively. In experiment 5, the number of mice for vehicle, ACPC at 100, 200 and 400 mg/kg was 20, 12, 10 and 10, respectively. In experiment 6, the number of mice for vehicle, AZD6765 at 9, 18 and 36 mg/kg was 10 in each group. Lower panel: For the tail suspension tests, experiment 1 involving imipramine, ketamine and memantine, experiment 2 involving MK-801, experiment 3 involving Ro 25-6981, experiment 4 involving ACPC and experiment 5 involving AZD6765, yielded the following one-way ANOVA values: F(6,55) = 16.35, p < 0.0001; F(3,33) = 44.77, p < 0.0001; F(2,21) = 28.37, p < 0.0001, F(3,36) = 31.02, p < 0.0001 and F(3,36) = 28.45, p < 0.0001, respectively. Symbols: *p < 0.05–p < 0.0001 versus respective vehicle (the precise p-value is not given for the clarity of figure). In experiment 1, the number of mice for vehicle, imipramine at 2.5 mg/kg, imipramine at 10 mg/kg, ketamine at 20 mg/kg, ketamine at 50 mg/kg, memantine at 10 mg/kg and memantine at 20 mg/kg, was 8, 10, 8, 10, 8, 10 and 8, respectively. In experiment 2, the number of mice for vehicle, MK-801 at 0.05, 0.1 and 0.2 mg/kg was 13, 8, 8 and 8, respectively. In experiment 3, the number of mice for vehicle, Ro 25-6981 at 5 and 10 mg/kg was 8 in each group. In experiment 4, the number of mice for vehicle, ACPC at 100, 200 and 400 mg/kg was 10 in each group. In experiment 5, the number of mice for vehicle, AZD6765 at 4.5, 9 and 18 mg/kg was 10 in each group.
Discussion
Multiple intracellular events have been linked to the rapid and sustained effects of ketamine following
NMDAR blockade. These include α-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor activation, increased brain-derived neurotrophic factor (BDNF) formation and activation of 231
Skolnick et al. tropomyosin-related kinase B (TrKB), activation of mammalian target of rapamycin (mTOR), vascular endothelial growth factor (VEGF) and their associated cascades (24–27). However, there appear to be significant differences among NMDAR antagonists with respect to reported duration of action and sensitivity to AMPA receptor blockade in both behavioural despair tests (16,23,28–31) and models like chronic mild stress (32,33), which appear to possess better face and construct validity. An emerging body of clinical work indicates the rapid and robust antidepressant effects of ketamine are not shared by either other NMDA channel blockers [e.g. memantine (4) and AZD6765 (lanicemine) (5,6)] or the NR2B antagonist MK-0657 (34). Ketamine was reported to reverse tetrabenazine-induced ptosis (13), an action shared by tricyclic antidepressants, and indicative of a sympathomimetic action. To our knowledge, the ability of other NMDAR antagonists to reverse tetrabenazine-induced ptosis had not been examined. Thus, a series of NMDAR antagonists were tested in order to determine if this action was shared with ketamine. Since NMDAR antagonists are active in behavioural despair tests (reviewed in (10)), these compounds were evaluated in parallel using the TST to eliminate experimental variables that can make direct comparisons across laboratories problematic. In this regard, the MED of ketamine in the TST (20 mg/kg) merits additional comment. While there is general agreement that ketamine is active in behavioural despair measures, some investigators report activity in the 3–10 mg/kg range, whereas other studies require substantially higher doses (>20 mg/kg) (reviewed in Pilc et al. (10)). It has been speculated that differences in both the potency and duration of action may be attributable to strain differences in the metabolism of ketamine (10). The sympathomimetic properties of ketamine (35) led Sofia and Harakal (13) to examine its ability to reverse tetrabenazine-induced ptosis, an early preclinical screen for biogenic amine-based antidepressants (36). Similarly, the sympathomimetic properties of MK-801 were recognised early in its development (37), before its identification as a highly selective NMDAR antagonist (38). Like ketamine, these effects of MK-801 also appear to arise from a centrally mediated sympathomimetic action (39). Consistent with the effects of other NMDA channel blockers, doserelated increases in blood pressure have also been noted following AZD6765 administration (6). Hypertension is one of the side effects associated with mild to moderate memantine overdose (40). The present findings, demonstrating that all NMDAR antagonists tested partially reverse tetrabenazineinduced ptosis, indicate this is a class effect, which may be mediated via a centrally mediated 232
sympathomimetic action by dint of NMDAR receptor blockade (39). At face value, the present findings do not provide insights that may help explain potential differences between ketamine and other NMDAR antagonists reported in both preclinical and clinical studies. Nonetheless, the ability of memantine to produce both an antidepressant-like effect in behavioural despair procedures (Fig. 1a and (18,41–43)) and partially reverse tetrabenazineinduced ptosis, suggest these doses are sufficient to block NMDAR. If a central sympathomimetic action is a class effect produced by NMDAR antagonists, then the failure to observe an antidepressant effect with memantine – either in a randomised clinical trial (4) or an antidepressant-like signal after many years of clinical use – indicates that the doses used in this randomised clinical trial and routinely in the treatment of Alzheimer’s disease (starting at 5 mg and titrating to 20 mg/day) may, despite claims to the contrary (44), be insufficient to block NMDAR. Similarly, the failure to observe an antidepressant signal with the NR2B antagonist MK-0657 could be due to the lack of target engagement. Thus, no symptoms of either hypertension or dissociative-like effects were reported in the study using divided doses of 4 to 8 mg/day (34). In contrast, hypertension was reported in an earlier study (45), administering a single oral dose of 7 mg/kg of MK-0657 to patients with Parkinson’s disease. Since ketamine (1) produces feelings of depersonalisation and even psychosis (46), it cannot be excluded that these unwanted side-effects, manifestations of target activation (NMDAR blockade), are inextricably linked to its efficacy for treating affective disorder (47). Psychotomimetic-like symptoms have also been reported following administration of both dizocilpine (MK-801 (48)); and memantine (49), consistent with the hypothesis that this is a class effect of NMDAR antagonists. Also consistent with this hypothesis, Luckenbaugh et al. (50) recently reported that ketamine-induced increases in dissociation and psychotomimesis at 40 min post infusion were highly correlated (r = − 0.41, p = 0.01) with reductions in the Hamilton Depression rating scale at 7 days post infusion. Thus, if NMDAR antagonists are dosed to produce target engagement (e.g. manifested as dissociative-like reactions and/or hypertension), then this class of compounds, including memantine, lanicemine (5,6) and MK-0657 should produce efficacies comparable to ketamine (47) in treatment-resistant depression. Additional preclinical studies (for example, determining if a glutamate burst and subsequent AMPA receptor activation are observed following administration of all NMDAR antagonists) could further refine this hypothesis by comparing ketamine to other NMDAR antagonists that do not
NMDAR antagonists and tetrabenazine-induced ptosis appear to produce the same robust antidepressant effects in the clinic (10). Acknowledgements
This work was funded by the Statutory Activity of Institute of Pharmacology, Polish Academy of Sciences. The authors thank Dr. A. Bespalov for the generous gift of tetrabenazine. P.S. and P.P. designed the study and wrote the manuscript. T.K. carried out the experiments, analysed the data and wrote parts of the manuscript. J.C. provided important intellectual content, assisted with the interpretation of results and approved the final version. Conflicts of Interest
None. Ethical Standards
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