Evaluation of Dimebon in cellular model of Huntington's disease

Background Dimebon is an antihistamine compound with a long history of clinical use in Russia. Recently, Dimebon has been proposed to be useful for treating neurodegenerative disorders. It has demonstrated efficacy in phase II Alzheimer's disease (AD) and Huntington's disease (HD) clinical trials. The mechanisms responsible for the beneficial actions of Dimebon in AD and HD remain unclear. It has been suggested that Dimebon may act by blocking NMDA receptors or voltage-gated Ca2+ channels and by preventing mitochondrial permeability pore transition. Results We evaluated the effects of Dimebon in experiments with primary striatal neuronal cultures (MSN) from wild type (WT) mice and YAC128 HD transgenic mice. We found that Dimebon acts as an inhibitor of NMDA receptors (IC50 = 10 μM) and voltage-gated calcium channels (IC50 = 50 μM) in WT and YAC128 MSN. We further found that application of 50 μM Dimebon stabilized glutamate-induced Ca2+ signals in YAC128 MSN and protected cultured YAC128 MSN from glutamate-induced apoptosis. Lower concentrations of Dimebon (5 μM and 10 μM) did not stabilize glutamate-induced Ca2+ signals and did not exert neuroprotective effects in experiments with YAC128 MSN. Evaluation of Dimebon against a set of biochemical targets indicated that Dimebon inhibits α-Adrenergic receptors (α1A, α1B, α1D, and α2A), Histamine H1 and H2 receptors and Serotonin 5-HT2c, 5-HT5A, 5-HT6 receptors with high affinity. Dimebon also had significant effect on a number of additional receptors. Conclusion Our results suggest that Ca2+ and mitochondria stabilizing effects may, in part, be responsible for beneficial clinical effects of Dimebon. However, the high concentrations of Dimebon required to achieve Ca2+ stabilizing and neuroprotective effects in our in vitro studies (50 μM) indicate that properties of Dimebon as cognitive enhancer are most likely due to potent inhibition of H1 histamine receptors. It is also possible that Dimebon acts on novel high affinity targets not present in cultured MSN preparation. Unbiased evaluation of Dimebon against a set of biochemical targets indicated that Dimebon efficiently inhibited a number of additional receptors. Potential interactions with these receptors need to be considered in interpretation of results obtained with Dimebon in clinical trials.


Conclusion:
Our results suggest that Ca 2+ and mitochondria stabilizing effects may, in part, be responsible for beneficial clinical effects of Dimebon. However, the high concentrations of Dimebon required to achieve Ca 2+ stabilizing and neuroprotective effects in our in vitro studies (50 μM) indicate that properties of Dimebon as cognitive enhancer are most likely due to potent inhibition of H1 histamine receptors. It is also possible that Dimebon acts on novel high affinity targets not present in cultured MSN preparation. Unbiased evaluation of Dimebon against a set of biochemical targets indicated that Dimebon efficiently inhibited a number of additional receptors. Potential interactions with these receptors need to be considered in interpretation of results obtained with Dimebon in clinical trials.

Background
Huntington disease (HD) is an inherited, incurable, autosomal dominant disease caused by the expansion of CAG trinucleotide repeats in the first exon of the huntingtin gene [1,2]. It is characterized by progressive neurodegeneration resulting in motor abnormalities including chorea and psychiatric disturbance with gradual dementia. HD is fatal and causes death within 15-20 years of the onset of the symptoms. The mutant huntingtin (Htt) with expanded polyglutamine (polyQ) is widely expressed in the brain and peripheral tissues but causes selective and most prominent loss of medium spiny neurons in striatum (MSN) which leads to the major clinical abnormalities that characterize the disease. The exact cause of neuronal loss in HD remains unknown [3]. Recent evidence indicates that dysregulation of glutamate and Ca 2+ (calcium) signaling in MSN play an important role in HD pathogenesis [4]. The "Ca 2+ hypothesis of HD" suggests that Ca 2+ signaling inhibitors may have a therapeutic value for treatment of HD [4]. Abnormal neuronal Ca 2+ signaling has also been proposed to play an important role in Alzheimer's disease [5,6].
Dimebon is a drug that has been developed and used as an antihistamine in Russia since 1983. Recently, Dimebon has been proposed to be useful for treating neurodegenerative disorders [7]. Dimebon demonstrated significant positive effects in six-month randomized, double-blinded, placebo-controlled phase II trial of 183 patients with mild to moderate Alzheimer's disease (AD) conducted by Medivation [8]. The phase III trial of Dimebon in AD will soon be initiated. Dimebon also demonstrated efficacy in phase 2 trial of patients with Huntington's disease (HD) conducted by Medivation and Huntington Study Group (DIMOND). Despite extremely encouraging results in clinical trials, the mechanisms responsible for beneficial actions of Dimebon in AD and HD remain poorly understood. Previous reports suggested that Dimebon may act as an inhibitor of NMDA receptors [9], voltage-gated Ca 2+ channels [10] or as a blocker of mitochondrial permeability transition pore [11]. These potential targets indicated that Dimebon may act by stabilizing neuronal Ca 2+ signaling, which may explain clinical benefits observed in HD and AD trials. In the present study, we evaluated the ability of Dimebon to inhibit NMDA receptors and voltage-gated Ca 2+ channels in cultured MSN from wild type mice and from the YAC128 HD transgenic mice model. We also evaluated neuroprotective effects of Dimebon in previously developed glutamate-toxicity assay with cultured YAC128 MSN [12]. We concluded that Ca 2+ and the mitochondria stabilizing effects of Dimebon may only in part be responsible for beneficial effects in human clinical trials and additional mechanisms of Dimebon's actions need to be uncovered to explain its beneficial clinical actions in HD and AD trials.

Calcium imaging experiments
Ca 2+ imaging experiments with 13-14 DIV (days in vitro) MSN cultures were performed as previously described [12,[14][15][16], using a DeltaRAM illuminator, an IC-300 camera, and IMAGEMASTER PRO software (all from PTI). The MSN were loaded with 5 μM fura-2 AM (Molecular Probes) for 45 min at 37°C in artificial cerebrospinal fluid (ACSF, containing the following: 140 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 2 mM CaCl 2 , 10 mM Hepes, pH7.3). Coverslips were mounted onto a recording/perfusion chamber (RC-26G, Warner Instruments) and positioned on the movable stage of an Olympus (Melville) IX-70 inverted microscope. The cells were maintained in ACSF at 37°C during experiments (PH1 heater, Warner Instruments). Images at 340 and 380 nm excitation wavelengths were acquired every 6 s and shown as 340/380 image ratios. Baseline (1-3 min) measurements were obtained before first pulse of glutamate. The 20 μM glutamate solution was dissolved in ACSF and 1-min pulses of 37°C glutamate solution (SH-27B in-line solution heater, Warner Instruments) were applied by using a valve controller (VC-6, Warner Instruments) driven by a squarepulse electrical wave-form generator (Model 148A, Wavetek). 10 μM or 50 μM Dimebon was dissolved in ACSF or 20 μM glutamate solution for the Dimebon application.

Electrophysiology for NMDAR and voltage-gated Ca 2+ channels
Whole-cell patch-clamp recordings of NMDAR activity were performed with cultured MSN from WT and YAC128 mice at DIV9-10 as we previously described [15]. Medium spiny neurons were distinguished based on morphological identification and membrane capacitance ranging from 4-10 pF. A multi-barrel perfusion system was employed to achieve a rapid exchange of extracellular solutions as we previously described [15]. All drugs were prepared according to the specifications of the manufacturers and applied with a gravity-fed "sewer pipe" capillary array. Whole-cell currents were recorded using Axopatch 200B amplifiers (Axon Instruments). Data were filtered at 2 kHz and digitized at 5 Hz using a Digidata 1200 DAC unit (Axon Instruments). The online acquisition was done using pCLAMP software (Version 8, Axon Instruments). Cultured MSN were held at V H = -60 mV membrane potential. Standard extracellular solutions contained 140 mM NaCl, 5 mM KCl, 2.0 mM CaCl 2 , 10 mM HEPES (pH 7.4; 310 mOsm). The pipette solution contained 135 mM CsMeSO4, 10 mM HEPES, 5 mM 1,2bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 3 mM MgATP, 1 mM MgCl 2 , 0.3 mM GTP-tris. In all experiments, 50 μM glycine was added to both control and NMDA-containing extracellular solutions. 10 μM CNQX and 0.1 μM TTX were added to the extracellular recording solution immediately before each experiment to block AMPA/kainate-type glutamate receptors and voltage-gated sodium currents, respectively.

In vitro HD assay
The in vitro HD assay with wild type and YAC128 MSN cultures was conduced as previously described [12,13]. Dimebon was added to the 14 DIV MSN at the final concentration of 5 μM, 10 μM or 50 μM. After 30 minutes incubation with Dimebon, the MSN were exposed for 7 h to 250 μM glutamate in Neurobasal-A added to the culture medium. During exposure to glutamate, the cells were maintained in a cell culture incubator (humidified 5% CO 2 , 37°C). Immediately after the treatment with glutamate, neurons were fixed for 30 min in 4% paraformaldehyde plus 4% sucrose in PBS (pH7.4), permeabilized for 5 min in 0.25% Triton-X-100, and stained by using the DeadEnd fluorometric TUNEL System (Promega). Nuclei were counterstained with 5 μM propidium iodine (PI) (Molecular Probes). Coverslips were extensively washed with PBS and mounted in Mowiol 4-88 (Polysciences). For quantification, six to eight randomly chosen microscopic fields containing 100-300 MSN each were cell-counted for YAC128 and wild type cultures. The number of TUNEL-positive neuronal nuclei was calculated as a fraction of PI-positive neuronal nuclei in each microscopic field. The fractions of TUNEL-positive nuclei determined for each microscopic field were averaged and the results are presented as means ± SE (n = number of fields counted).

Evaluation against biochemical targets
The activity of Dimebon against selected set of biochemical targets was performed by MDS Pharma Services http:/ /discovery.mdsps.com/. The Dimebon was provided to MDS Pharma as dry powder, dissolved in DMSO and tested in 10 μM concentrations. The standard Lead Profiling + CYP450 screen was performed according to MDS Pharma specifications with additional custom-chosen targets added to the screen as indicated.

Statistical analysis
All experiments were repeated at least three times. Data were evaluated for statistical significance by analysis using SigmaPlot t-test or One-Way ANOVA. Statistical difference was considered to be significant if p < 0.05.

Dimebon inhibits glutamate-induced Ca 2+ increase in YAC128 MSN
To test the postulated "Ca 2+ stabilizing" effects of Dimebon, in the first series of experiments, we compared Ca 2+ responses induced by glutamate application to wild type (WT) and YAC128 MSN at 13-14 DIV. In our previous studies, we found that repetitive pulses of 20 μM glutamate resulted in a bigger elevation of cytosolic Ca 2+ levels in the YAC128 MSN compared with that in WT MSN [12,14,15]. To test effects of Dimebon on glutamateinduced Ca 2+ signals, we applied 20 pulses of 20 μM glutamate (each pulse 1 min in duration, followed by a 1 min washout) in the presence of 10 μM or 50 μM Dimebon. Control experiments were performed in the absence of Dimebon. The intracellular Ca 2+ concentration in the experiments was continuously monitored by Fura-2 imaging and the 340/380 ratio was used to quantitatively determine the concentration of the intracellular Ca 2+ ([Ca 2+ ] i ). The increase in Ca 2+ was calculated as a difference between basal values of Ca 2+ prior to glutamate application and at completion of "20 glutamate pulses" protocol in the same cell. On average, the increase in Ca 2+ was 0.250 ± 0.029 for WT MSN and 0.403 ± 0.046 for YAC128 MSN (Figs 2A, B, G). Thus, in agreement with our previous findings [12,14,15], the increase in Ca 2+ was significantly higher in YAC128 MSN than in WT MSN. In the presence of 10 μM Dimebon, the increase in Ca 2+ was 0.236 ± 0.021 for WT MSN and 0.461 ± 0.034 for YAC128 MSN (Figs 2C, D, G). Thus, incubation with 10 μM Dimebon had no significant effect on the glutamate-induced Ca 2+ increase in WT or YAC128 MSN. In the presence of 50 μM Dimebon, the increase in Ca 2+ was 0.290 ± 0.027 for WT MSN and 0.234 ± 0.022 for YAC128 MSN (Figs 2E,   F, G). Thus, 50 μM Dimebon significantly reduced the glutamate-induced Ca 2+ increase of YAC128 MSN without affecting the Ca 2+ signals in WT MSN. These results indicate that Dimebon exerts "Ca 2+ stabilizing" effects in YAC128 MSN at 50 μM but not at 10 μM concentration.

NMDAR inhibition by Dimebon
Previous reports suggested that Dimebon may act as an inhibitor of NMDA receptors [9]. In the next series of experiments, we evaluated the ability of Dimebon to block NMDA-activated currents in DIV9-10 WT and YAC128 MSN cultures. The MSN were voltage-clamped at -60 mV and the currents were evoked by local and rapid application of 100 μM NMDA using multi-barrel perfusion system ("sewer pipe"). Consistent with previous findings [15], we found that NMDA induced much larger currents in YAC128 MSNs than in the WT littermate cultures ( Fig 3A). Application of 1 μM, 10 μM, or 50 μM of Dimebon caused a significant reduction in the size of NMDA-induced currents in both WT and YAC128 MSN (Fig 3A). The inhibitory effects of Dimebon were reversible and the size of NMDA currents quickly recovered following a washout of Dimebon (data not shown). To compare results from different experiments, we normalized the peak amplitude of NMDA-evoked currents to the amplitude recorded in the absence of Dimebon in the same cell and averaged normalized data from multiple experiments (from at least 4 independent cultures established from YAC128 and WT). Analysis of obtained results suggested that Dimebon blocks NMDAR-currents in WT and YAC128 MSN with IC50 = 10 μM (Fig 3B). The YAC128 MSN have increased contribution of NR2B subtype of NMDAR when compared to WT MSN [15,18]. Our results suggest that Dimebon does not display selectivity for NR2B subtype of NMDAR, as both WT and YAC128 MSN currents were inhibited with similar potency ( Fig  3B). In general, our results are consistent with previous studies of Dimebon's inhibitory effects on NMDAR [9].

Voltage-gated calcium channel inhibition by Dimebon
High voltage-activated Ca 2+ channels have been proposed to be another target of Dimebon [10]. In the next series of experiments, we evaluated effects of Dimebon on high voltage-activated Ca 2+ currents recorded in wild type (WT) and YAC128 MSN cultures at DIV9-10. Whole-cell patchclamp recordings of currents in cultured MSN were performed according to the published procedures [17] using 10 mM Ba 2+ as a current carrier. The MSN were voltage clamped at -80 mV, and test pulses to 0 mV were applied at 10 s intervals. Consecutive application of 1 μM, 10 μM, and 50 μM of Dimebon resulted in a progressive reduction in the size of depolarization-evoked currents ( Fig  4A). To compare results from different experiments, we normalized the peak size of voltage-gated Ca 2+ currents to the amplitude recorded in the absence of Dimebon in the Effects of Dimebon on glutamate-induced Ca 2+ signals   (Fig 4B). Dimebon demonstrated similar potency in WT and YAC128 MSN cultures (Fig 4B). In general, our results are consistent with previous studies of Dimebon's inhibitory effects on high voltage-activated Ca 2+ channels [10].   of YAC128 and WT MSN is highly significant and constitutes a quantitative basis for the in vitro HD assay we have previously described [12,13,15]. The neuroprotective effects of Dimebon were evaluated at 5 μM, 10 μM and 50 μM concentrations using the in vitro HD assay (Table 1, Fig 5). In these experiments, Dimebon was added 30 minutes prior to the exposure of MSN cultures to glutamate. We found that 5 μM and 10 μM Dimebon had no significant effects on the glutamate-induced apoptosis of YAC128 MSN (Table 1, Figs 5A, B). At 50 μM concentration, Dimebon showed significant protective effects in the in vitro HD assay (Table 1, Fig 5C).

Discussion
Dimebon demonstrated significant positive effects in phase II AD clinical trial conducted by Medivation in Russia [8]. It has also demonstrated efficacy in a phase 2 trial of patients with Huntington's disease (HD) conducted by Medivation and Huntington Study Group (DIMOND). Despite extremely encouraging results in clinical trials, the mechanisms responsible for the beneficial actions of Dimebon in AD and HD remain poorly understood. Here, we evaluated neuroprotective effects of Dimebon in a previously developed cellular model of HD [12]. We also tested the ability of Dimebon to function as a "Ca 2+ signaling stabilizer" and an inhibitor of NMDAR and voltagegated Ca 2+ channels in Ca 2+ imaging and electrophysiological experiments with wild type and YAC128 MSN cultures. Consistent with the previous reports [9,10], we found that Dimebon indeed inhibits NMDAR and voltage-gated Ca 2+ channels. The IC50 for inhibitory actions of Dimebon was equal to 10 μM for NMDAR and 50 μM for voltage-gated Ca 2+ channels in our experiments (Figs 3B and 4B). At 50 μM concentration, Dimebon also exerted Ca 2+ stabilizing and neuroprotective effects in YAC128 MSN preparation (Figs 2G and 5C, Table 1). At the concentration of 10 μM, Dimebon was not effective in these in vitro assays (Figs 2G and 5B, Table 1). Similar efficacy of Dimebon was observed in experiments with the Drosophila model of HD [19]. Consistent with our find-ings, neuroprotective effects of Dimebon in the Drosophila model of HD were observed in the 50 -100 μM concentration range [19]. The protective effects of Dimebon in the in vitro HD assay are in quantitative agreement with the "Ca 2+ stabilizing" effects of Dimebon ( Fig 2G). Thus, neuroprotective effects of Dimebon observed in our experiments (Table 1, Fig 5C) most likely can be explained by "Ca 2+ stabilizing" effects of Dimebon resulting from inhibition of NMDAR (Fig 3) and voltage-gated Ca 2+ channels (Fig 4). It is also possible that Dimebon exerts additional beneficial actions at the level of neuronal mitochondria [11]. The concentration of Dimebon required to inhibit mitochondrial permeability pore transition in isolated mitochondria was in the range of 50 μM [11], the same concentration range as the neuroprotective effects observed in our experiments with YAC128 MSN cultures ( Fig 5C, Table 1).
Using an identical experimental approach, in previous studies we demonstrated that clinically relevant NMDAR antagonist memantine (Namenda) was protective in the YAC128 MSN glutamate toxicity assay at 10 μM concentration [13]. Thus, Dimebon is 5-fold less effective than memantine when tested in the in vitro HD model. We also previously demonstrated that clinically relevant putative mitochondrial permeability pore inhibitors Nortriptyline, Desipramine, Trifluoperazine, and Maprotiline [20] were also protective in YAC128 MSN toxicity assay at 2 μM concentration [12]. The Dimebon was 25-fold less effective than these compounds when tested in the in vitro HD model.
Unbiased evaluation of Dimebon against a set of biochemical targets indicated that Dimebon efficiently inhibits α-Adrenergic receptors (α 1A , α 1B , α 1D , and α 2A ), Histamine H 1 and H 2 receptors and Serotonin 5-HT 2c , 5-HT 5A , 5-HT 6 receptors with high affinity (Fig 6). Dimebon also had significant effect on Dopamine D 1 , D 2S , D 3 receptors, Imidazoline I 2 receptor, Serotonin 5-HT 2 and 5-HT 2B receptors (Fig 6). Interactions with these receptors need to be taken into consideration in interpretation of results obtained with Dimebon in HD and AD clinical trials. The fraction of apoptotic (TUNEL-positive) neurons is shown for wild type (WT) and YAC128 MSN without glutamate application and following application of 250 μM glutamate for 7 h. The experiments were conduced in the presence of 5, 10 and 50 μM Dimebon as indicated. a The number in parenthesis is the untreated control cells in the same batch of MSN culture. *P < 0.05, compared with control cells and show protective effect.

Conclusion
From our results, we concluded that the "Ca 2+ stabilizing" effects of Dimebon may, in part, be responsible for the clinical benefits observed in HD and AD trials. However, 50 μM concentration of Dimebon that is required to achieve "Ca 2+ stabilizing" and neuroprotective effects in our experiments is not likely to be achieved in human tri-als. In AD trials of Dimebon, the patients received 20 mg pills [8], which should not lead to concentrations higher than 0.6 μM assuming an ideal absorption and bloodbrain-barrier brain permeability profile. Thus, most beneficial effects of Dimebon are likely to be due to its properties as a cognitive enhancer based on its ability to inhibit H1 histamine receptors with IC50 = 3.4 nM [7]. It is also Significant biochemical targets of Dimebon Figure 6 Significant biochemical targets of Dimebon. Significant biochemical targets of Dimebon are shown. The Cat numbers refer to the MDS Pharma Services assay specification. The receptors and species (human or rat) are listed. Dimebon was tested at 10 μM concentration. The % inhibition is shown for each receptor. Significant targets are defined as % inhibition > 50%.