New Therapeutic Property of Dimebon As a Neuroprotective Agent

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REVIEW ARTICLE New Therapeutic Property of Dimebon as a Neuroprotective Agent

Aleksey Ustyugov1, Elena Shevtsova1, George E. Barreto2,3, Ghulam Md Ashraf 4, Sergey O. Bachurin1 and Gjumrakch Aliev1,5,6,*

1Institute of Physiologically Active Compounds, Russian Academy of Sciences, Severniy Proezd 1, Cher- nogolovka, 142432, Russia; 2Departamento de Nutrición y Bioquímica, Facultad de Ciencias, Pontificia Uni- versidad Javeriana, Bogotá D.C., Colombia; 3Instituto de Ciencias Biomédicas, Universidad Autónoma de Chile, Santiago, Chile; 4King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Ara- bia; 5GALLY International Biomedical Research Consulting LLC., 7733 Louis Pasteur Drive, #330, San An- tonio, TX, 78229, USA; 6School of Health Science and Healthcare Administration, University of Atlanta, E. Johns Crossing, #175, Johns Creek, GA, 30097, USA

Abstract: Dimebon (or Latrepirdine) was initially used as an anti-histamergic drug but later new therapeutic properties were rediscovered, adding to a growing body of “old” agents with prominent neuroprotective effects. In the present manuscript, we are focusing on our

latest study on Dimebon with regard to brain’s pathological processes using in vivo protei-

A R T I C L E H I S T O R Y nopathy models. In the study, neurodegenerative pathology has been attributed to a group of aggregate-prone proteins: hyperphosphorylated tau, fused in sarcoma and γ-synuclein , which Received: March 13, 2016 Revised: June 08, 2016 are involved in a number of neurological disorders. We have also presented our in vitro Accepted: July 24, 2016 model based on overexpression of an aberrant mutant form of transactive response DNA DOI: 10.2174/0929867323666160804 binding 43 kDa protein in cultured SH-SY5Y neuroblastoma cells. Dimebon treatment fol- 122746 lowed by the activation of autophagy markers resulted in reduced number of inclusion containing cells. The most significant effects of Dimebon appeared to be on the improving cellular energy balance, mitochondria stability by increasing the threshold for nonselective mitochondrial pore opening as well as on increased calcium retention capacity while reducing lipid peroxidation. The therapeutic potential of Dimebon and newly designed analogs show disease modifying properties and could be used to treat neurodegenerative disorders. In addition, new data hint on a possible anti-aging effect and potential application of Dimebon for treatment of anxiety, ischemia and depression. Overall, our findings suggest that the most pronounced effect of Dimebon was observed when treatment was started at the early stages of disease onset and this factor needs to be taken into account while planning future clinical trials. Keywords: Dimebon, Latrepirdine, proteinopathy, neuroprotection, mitochondrial permeability transition.

INTRODUCTION gress towards long term treatment options. In recent years, the steadily increasing number of failures in The drugs for the treatment of majority of neurode- clinical trials led to an alternative strategy based on re- generative disorders (NDDs) such as Alzheimer disease introduction of earlier approved medicines as route for (AD), Parkinson’s disease (PD), amyotrophic lateral modern drug research [1, 2]. Dimebon (or Latrepirdine) sclerosis (ALS), frontotemporal dementia (FTD), etc is one of those “old” drugs which has been quite effec- provide only temporary relief without any visible pro- tive in the treatment of pathological conditions differ-

ing from original targets (Fig. 1) [3-5]. Several decades *Address correspondence to this author at the GALLY International ago it was marketed as a non-competitive anti- Biomedical Research Consulting LLC., 7733 Louis Pasteur Drive, #330, San Antonio, TX, 78229, USA; Tel: +1(440) 263-7461; histamine agent, but unexpectedly showed the ability to E-mail: [email protected] enhance cognition in a small-scale, open-label trial of

mechanistic capabilities of mitochondria which has N opened up new possibilities will make this task even more challenging [10, 11]. The lack of definitive mode N of action is hampering the attempt to understand the Cl remarkable discrepancies in clinical trials and also

Cl eluding the answer to ultimate questions like how, when and whether Dimebon might still be an effective

N drug for AD patients [12, 13]. In addition, our recent studies showed that Aβ induced mitochondrial permeabilization is attenuated by Dimebon [14]. The quest of Fig. (1). Structure of Dimebon [78]. understanding molecular mechanisms/targets that aid mild-to-moderate AD patients [6]. A double blind, pla- Dimebon in boosting AD patients’ cognition proclaims cebo-controlled phase II trial with pre-selected mild-to- the need to explore Dimoben’s action in terms of stabi- moderate AD patients showed significant clinical im- lization of energy balance, modulation of cell clearance provements in cognition assessed by AD cooperative systems, and activation of innate defense mechanism. study-activities of daily living (ADL) as a measure of Earlier studies have shown Dimebon as an inhibitor self care and function, and AD assessment scale on of AchE and BchE [15], blocker of L-type Ca2+ chan- cognitive subscale tests with respect to baseline [7]. nels [16], weak competitive agonist of NDMAR [17], However, the phase III trial on a heterogeneous popula- positive modulator of AMPA receptor [18], and have a tion exhibiting multiple neuropathies (with non-AD high affinity for serotonin 5-HT6 and 5-HT7 receptors symptoms as well) failed to reciprocate the promising [19].. Recent studies have shown that Dimebon lowers findings of phase II trials [8]. Both phase II and III tri- formation and subsequent accumulation of pathological als included the dynamics of deterioration of patients’ proteins [20-25], initiate autophagy [26-29], and stabi- health, recruitment age and use of Dimebon formula- lizes mitochondrial functions by blocking permeability tion, thus making it really tough to understand the re- transition [14, 30-33], thus clearly showing its in- markable distinctions between these two trials. Certain volvement in multiple mechanisms of action (Table 1). discrepancy between the results in clinical trials II and Some new findings also suggest that Dimebon could be III was addressed by Bharadwaj et al [9]. The authors used for potential treatment of ischemia, anxiety and argue that phase III failure could be due to either poor depression [34, 35]. All the above discussed data heav- drug efficacy or lack of trial optimization leading to ily support the re-evaluation of therapeutic significance difficulties in interpreting the results. However, it is of Dimebon, so as to position the newly designed ana- worth mentioning that the results in phase II AD study logs as promising therapeutic agents for NDDs [9, 36]. were reflected by overall improvements in Dimebon group and worsening in placebo group; yet, in phase ROLE OF DIMEBON IN NEUROBLASTOMA III, patients in either group did not significantly dete- CELL LINE SH-SY5Y riorate implying the heterogeneous and diverse patho- A transactive response binding protein 43 kDa logical features in each cohort. Moreover, parameters (TDP-43) is the major component of inclusions in FTD such as Dimebon formulation, age of the recruited pa- and ALS patients [37-40]. The ubiquitin-positive intra- tients (mean age of 68.1 and 74.4 years in phase II and cytoplasmic deposits in nerve and glial cells cause III, respectively), and Baseline Mini Mental State Ex- pathological conditions and inevitable neuronal dam- amination (MMSE) were also altered, thus suggesting age. As a result, we proposed that protection effect that phase III results cannot be interpreted in terms of from Dimebon treatment might be due to its inhibitory drug efficacy. This observation calls for new and alter- effect on formation of deposits. To test this hypothesis, native ways of assaying these findings. Also, the failure we used human neuroblastoma cell line SH-SY5Y with of possible Dimebon protective effects on clinical trials two distinct overexpressing pathological isoforms – a might be due to insufficient knowledge about its TDP-43 mutant lacking nuclear localization signal and mechanism of action. Hence, novel studies are required residues 187–192 or another TDP-43 mutant lacking to establish the effect of Dimebon on N-methyl-D- the C-terminal fragment (162–414) fused with green aspartic acid receptor (NMDAR). In addition, there is a fluorescent protein (GFP) reporter [21]. As a result, need for discovery of affected target proteins as well as Dimebon treatment reduced the formation of TDP-43- Dimebon actions on mitochondria and activation of cell positive deposits in a concentration dependent manner clearance pathways. However, a recent twist in the Dimebon as a Neuroprotective Agent Current Medicinal Chemistry, 2016, Vol. 23, No. 30 3

Table 1. Summary of Dimebon effects recapitulating our current in vitro and in vivo findings.

In vivo models Dimebon treatment

gamma-synucleinopathy Increased average lifespan of transgenic animals Delay in the development of locomotor and coordination pathology Reduced amyloid deposits in spinal cord gray matter Tauopathy Reduced accumulation of hyperphosphorylated tau in spinal cord Slower decline of motor function compared to non-treated animals Autophagic machinery was not responsible for clearance of the intracellular tau deposits Fusopathy Increased mean lifespan compared to non-treated animals Experiment is ongoing Cell models of TDP-43 proteinopathy Concentration-dependent reduction of TDP-43-positive aggregates Fractionated spinal cord tissues of gamma- Decreased levels of aggregated gamma-synuclein in the detergent-insoluble fraction synuclein mice Redistribution of monomeric ubiquitin to the buffer-soluble fraction Mitochondria assays Increased CRC levels in the presence of β-amyloid Inhibition of TBHP induced swelling and lipid peroxidation of rat liver and brain mitochondria

(Fig. 2). Briefly, transfected cells were incubated with of autophagosomal complex [42]. In our experiments in 10 µM Dimebon for 3, 6, and 24 hours and then fixed. non-differentiated SH-SY5Y cells Dimebon (10 µM) The control group (without Dimebon addition) was set triggered a quick increase in p38 and ERK1/2 phos- to 100% and the percentage GTP aggregation was es- phorylation (in the first minute after addition), followed timated. In Dimebon treated groups, overall reduction by a slow reduction during the first hour. This initial of aggregate containing cells was detected after 3 hours spike in p38 and ERK1/2 phosphorylation indicates the (Fig. 3). Citing Dimebon’s anti-histamininc property, initiation of autophagy. On the other hand, p38 and we compared the inhibitory effect of other model anti- ERK1/2 belong to a family of key regulatory kinases histamergic compounds like thioperamide maleate, and activate survival pathways like cell cycle [43], diphenhydramine hydrochloride and promethazine hy- apoptosis [44], development pathways [45], and path- drochloride. All these compounds were found to be ways producing pro-inflammatory cytokines [46]. ineffective, thus implying that the action of Dimebon Thus, it was important to establish the expression of was not due to its anti-histamine properties. However, autophagy related factors, i.e., Atg16 and Atg12-Atg5 we still believe that Dimebon has the ability to prevent complex. The findings of qRT-PCR indicated that formation of deposits/aggregates either by direct inhibi- Atg12-Atg5 complex was transiently up-regulated 1-3 tion of production/accumulation of pathogenic protein h after Dimebon treatment, but maximum expression of aggregates, or by an indirect mechanism of activating Atg16 peaked during the first hour itself. High expres- cell’s innate clearance such as ubiquitin proteasomal sion levels of Atg12-Atg5-Atg16 mRNA and increased pathways and/or autophagy. phosphorylation levels of p38 kinases and ERK 1/2 might be indicative of Dimebon involvement in auto- AUTOPHAGY INDUCING EFFECTS OF DIME- phagosomal response. In yeast, Dimebon was reported BON to facilitate Aβ42-GFP removal via the autophagic The effect of Dimebon on p38 phosphorylation and pathway [26]. In addition, chronic administration of extracellular signal regulated kinase 1/2 (ERK 1l2) as Dimebon has also been attributed to autophagy induc- was used as an index of cell’s capacity to initiate auto- tion and removal of alpha-synuclein in mouse brain phagy [27]. The p38 and ERK1/2 phosphorylation ini- [29]. tiates proliferation acting as a resistance marker to In another study, Dimebon treatment on SH-SY5Y apoptosis by inducing autophagy in response to anti- cells resulted in up-regulation of M protein, which is a tumor agents [41]. However, autophagy-related factors member of immunity related GTPase family and play a (Atg16 and Atg12-Atg5 ) are vital for the development 4 Current Medicinal Chemistry, 2016, Vol. 23, No. 30 Ustyugov et al.

of autophagosomal factors and kinase phosphorylation remained up-regulated during the first hour of Dime- bon treatment and tapered off subsequently with no further changes in activity. Potentially, this might be due to Dimebon stability in aqueous solutions [48] and this feature is attributed and high regarded in later de- rivatives of Dimebon.

CHRONIC DIMEBON TREATMENT SLOWS DOWN Γ-SYNUCLEINOPATHY The trials of Dimebon as a drug for AD and Schizo- phrenic patients yielded rather encouraging results [7, 49]. However, further in vitro and animal studies are required on its possible use as a neuroprotective agent. We for the first time initiated a study to test the effect of Dimebon in a transgenic mouse model having an Fig. (2). Percentage of neuroblastoma SH-SY5Y cells with over-expressed aggregate prone wild type γ-synuclein cytoplasmic inclusions overexpressing TDP-43 mutant pro- in neurons under the control of pan-neuronal Thy-1 tein lacking C-terminal fragment (162–414) fused with green promoter. The striking pathological features exhibited fluorescent protein reporter in the presence (Dim) or absence in these mice included decreased life span, motor defi- (CN) of 10 µM Dimebon. Error bars represent ± SEM. cits, and age-related neuropathology which are all in- Briefly, cells were plated on 10 mm glass slides and trans- dicative of proteinopathy [50]. γ-synuclein aggregated fected with corresponding vector at minimum three separate and formed deposits in neuronal cell bodies and proc- glass slides per group were used in five independent experi- esses with increased levels of astrogliosis. Interest- ments. Values represent mean values of 10 different regions ingly, the spinal motor neurons appeared to be the most examined on the each 10 mm slide roughly covering 10000 affected, suggesting a linkage to motor neuron disease µm2. Mann-Whitney U test resulted in p=0.095 (Reprinted phenotype. These symptoms featured in a number of with permission from [5]). phenotypically different neurological diseases but all sharing core molecular pathology – protein aggregation. Such type of pathology brings together neurological symptoms with drastically distant clinical manifes- tations such as AD, ALS, FTD, PD, and others. Our focus was on studying the effects of Dimebon on formation of pathological protein deposits by exam- ining the disease pattern development and changes in transgenic mice nervous system [20]. Homozygous transgenic animals receiving Dimebon in drinking water at libido were used as an experimental group, whereas controls were on normal drinking water. The dose of 10 µg/ml can be considered to be equivalent to Fig. (3). Comparative average lifespan of gamma-synuclein the commonly used dose of 1.5 mg/kg/day taking into (γ-syn-TG) and Fus (Fus-TG) transgenic mice treated (Dim) account that each animal weighs roughly 30 g and con- or not treated (CN) with Dimebon at 1.5 mg/kg/day starting sumes 5 ml per day. Subsequently, two cohorts were at the ‘pre-symptomatic’ stage. Data are presented in days formed in the experimental group. Treatment of Dime- ±SEM (*p<0.05, Mann-Whitney U-test). The number of bon in the first cohort was started prior to the onset of animals in each group is shown at the bottom of each bar neurological symptoms (from the age of 3 months) and (Reprinted with permission from [5]). was dubbed as ‘pre-symptomatic’. Treatment of Dime- bon in the second cohort of the experimental group was crucial role in autophagy induced by mitochondrial started from the age of 6 months and was dubbed as driven innate immune system defense [47]. These data ‘post-symptomatic’. Animals at the later stages exhib- complements findings on mitochondria stabilization ited impaired motor performance and coordination with [30, 32]. However, it is still intriguing that expression Dimebon as a Neuroprotective Agent Current Medicinal Chemistry, 2016, Vol. 23, No. 30 5 multiple amyloidogenic deposits in the spinal cord gation of autophagosomal membranes, was used to as- [50]. Subsequent analyses of motor and coordination sess autophagy [63]. As number of formed auto- skills showed substantial deferral of locomotor and co- phagosomes is directly related to the amount of LC3-II ordination pathology in ‘pre-symptomatic’ cohort [20] [64], it was possible determine that autophagic machin- with higher average lifespan (Fig. 3) [22]. Moreover, ery did not take part in the clearance of intracellular tau fewer amyloid-like deposits, lower amounts of deter- deposits which were quite abundant in 6 month old gent-insoluble γ-synuclein and reduced levels of astro- control animals. Similar to the γ-synuclein expressing gliosis were detected in spinal cord gray matter of Di- mice model, the Dimebon treated tau-P301S animals mebon-treated animals based on the glial fibrillary demonstrated slower motor function decline compared acidic protein staining [22, 51]. Yet, histological analy- to non-treated controls. Therefore, chronic Dimebon sis of Nissl-stained spinal cord sections did not show treatments offered limited protection in progressing any statistically significant difference in the total num- motor dysfunction and appearance of tau-positive in- ber of surviving motor neurons between control and clusions. However, one limitation of the tau-P301S either of the experimental cohorts. The results from our model is that aggregation of hyperphosphorylated in vivo model system suggested that chronic Dimebon mimic might not resemble the physiological state ob- treatments slowed down but did not reverse pathologi- served in tauopathy patients. cal processes in γ-synuclein overexpressing mice, The clearance of accumulated toxic filaments might which may be attributed to the removal of γ-synuclein also implicate the activation of ubiquitin-proteasome aggregates by the cell clearance systems which may system (UPS) which is an alternative cell clearance become overloaded and halted, thus resulting in the route to autophagy. Analysis of γ-synuclein distribution eventual decline. in fractionated spinal cord tissue extracts demonstrated a lowered level of aggregated γ-synuclein in detergent CHRONIC DIMEBON TREATMENT PAR- TIALLY PREVENTS TAUOPATHY IN VIVO insoluble fraction of Dimebon treated 3 months old mice group as compared to the control group. Subse- The accumulation of extracellular hyperphosphory- quent analysis of buffer and detergent-soluble fractions lated tau builds toxic filaments and inclusions and it is showed higher levels of ubiquitinated proteins in con- regarded as a major feature of AD [52-61]. Aggrega- trols. The presence of monomeric ubiquitin in buffer- tion of hyperphosphorylated tau catches alongside soluble fraction of Dimebon-treated mice is attributed normal tau and other microtubule associated proteins to the activation of UPS [23]. However, further studies like MAP1A/MAP1B and MAP2, thus further tying the are required to establish the level of UPS up-regulation AD knot. A well characterized transgenic mouse model in tau-P301S animals as well as other animals models of tauopathy was used to study the action of Dimebon employed in our studies. in tautopathology. P301s tau expression in neuronal cells of these mice initiates the formation of filamen- ROLE OF CHRONIC DIMEBON TREATMENT tous tau throughout the nervous system, thus resulting ON LIFESPAN OF TRANSGENIC MICE in neuronal cell loss in spinal cord and subsequent mo- Our recent report showed the maximum and average tor dysfunction [62]. The pathology onset in these ani- lifespan of wild-type C57B1/6 female mice was in- mals is rather fast. At the age of 6 months homozygous creased in Dimebon treated group compared to the con- mice developed severe paraparesis and were sacrificed, trol cohort. Also, the general health of aged animals thus making it crucial to start early stage (at 5 weeks) was also improved. According to morphological analy- treatments of Dimebon to overcome the severe impacts sis, cerebral hemispheres and cerebellum in Dimebon- of tau accumulation [25]. The quantitative Western treated animals was better preserved. Particularly, Di- blotting with AT8 anti-tau antibody and immunohisto- mebon-treated mice had less severe loss of neurons and chemical staining of spinal cord sections showed re- pericellular hypostasis compared to age-matched con- duced hyperphosphorylated tau aggregation in Dime- trols [65]. Moreover, the average lifespan analysis from bon treated group as compared to control animals. Yet, other studies revealed that survival of transgenic ani- assessment of cells quantity expressing NeuN marker mals was directly dependent on the stage at which Di- in the thoracic ventral horn did not show statistical dif- mebon treatment was initiated (Fig. 3) [20, 66]. It ference between control and experimental groups. Di- seems apparent that it is critical to start treatment dur- mebon probably acts on a subset of neurons and thus ing the ‘pre-symptomatic’ phase implying that such further analysis is needed. The ration between the type of ‘preconditioning’ could be attributed to en- markers LC3-I and LC3-II, which are involved in elon- hanced survival of animals [67]. 6 Current Medicinal Chemistry, 2016, Vol. 23, No. 30 Ustyugov et al.

The ongoing study on effects of Dimebon on a nisms and/or pathways enhancing survival of neuronal novel animal ALS model proved similar point. In our cells. ALS model, the pathology is induced by the overexpression of aberrant form of human fused in sarcoma DIMEBON AS METABOLIC ENHANCER AND (FUS) protein [68]. FUS belongs to a family of STABILIZER OF MITOCHONDRIAL FUNC- DNA/RNA-binding proteins and are involved in the TIONS regulation of dynamic shuttling between nucleus and One of the significant features of Dimebon is the cytoplasm. In ALS patients, an aberrant form of FUS anti-aging effect in normal animals. It was shown that protein is found in inclusion bodies which is attributed long-term treatment of C57Bl/6 female mice with Di- to motor neuron dysfunction. Our mice model resem- mebon extended average lifespan and decreased the bles ALS-like symptoms as well as phenotypic features aging-determined formation of senile cataract and hair with acute and rapid damage to motor neurons, neu- loss [69]. It was known that mitochondria and oxida- roinflammatory response and subsequent death of mo- tive stress are the key factors which determine the age- tor neurons. The outcome of this pathology lies in a dependent loss or impairment of different organs and shortened lifespan (90-140 days) and fast motor func- functions. And now there are numerous data which tion loss within several days of symptoms onset [68]. have confirmed that mitochondrial dysfunction and The phenotype was a result of a gain of function of 1- abnormal energy metabolism have specific impact on 359 mutant FUS isoform. The key features of the mu- the pathogenesis of several NDDs. It was shown that tant FUS included loss of nuclear localization signal Dimebon could restore the decreased level of CGU in leading to its mislocalization, and removal of RNA aging female B6SJLF2 mice instead of the young con- recognition and binding motif; which in turn hampers trol cohort [70]. Hypometabolism of glucose and mito- the inability to bind target RNA, facilitates irreversible chondrial defects correlate with aging, but significantly aggregation of mutant neuronal cell bodies, and result- pronounced with disease progression in NDD patients ing in selective damage and death of motor neurons. and animal models, and these factors are likely to be Our studies has established that Dimebon treatment key pathologic cellular features of most NDDs. In spo- reduces the number of aggresome containing cells ex- radic forms of NDDs with aging as one of the main risk pressing the mutant 1-359 isoform (unpublished data). factor, a mitochondrial origin of pathophysiology Therefore, we proposed that chronic Dimebon treat- seems to be the most prevalent feature [71]. ment of FUS transgenic mice might attribute to enhanced survival. Similarly to our γ-synuclein mice In a recent study, delayed onset of pathological model, the expression of FUS transgene in neuronal symptoms and significantly increased lifespan ob- tissues relies on Thy-1 promoter causing the expression served in chronic Dimebon treated SOD1-G93A trans- in all neuronal tissues. The experimental group was genic mice (a model of ALS) was attributed to Dime- subdivided into two cohorts due to early onset of pa- bon mediated “preconditioning” of AMP-activated pro- thology in FUS transgenic mice.. The first cohort initi- tein kinase activator which in turn resulted in mTOR- ated Dimebon treatment on 35th day, which was before independent activation of autophagy [67]. Dimebon at the onset of pathology and it was referred to as the sub-nanomolar concentrations (0.1 nM) increases intra- ‘pre-symptomatic’ stage. The second cohort started cellular ATP level and glucose transporter-3 transloca- Dimebon treatment at the time of detection of FUS- tion to the plasma membrane possibly due to activation positive inclusions in the nervous tissue which corre- of the AMP-activated protein kinase [72]. But the di- sponded to the 63rd day. Chronic Dimebon treatments rect influence on mitochondrial functions may be the in drinking water were successfully established in our reason of anti-aging effects of Dimebon for normal previous experiments for other proteinopathy mouse animals and delayed onset of pathological symptoms models [22, 25]. Preliminary findings indicated 29% and significantly increased lifespan in NDD models. and 20% increase in the mean lifespan in ‘pre- Neuroprotection in different neurotoxic cellular symptomatic’ and ‘symptomatic’ Dimebon-treated models was shown for Dimebon. At picomolar levels, groups, respectively, compared to non-treated controls Dimebon inhibits ionomycin induced cell death by en- [66]. Molecular analyses are not yet available, but we hancing the ATP level in primary rat hippocampal cells believe that the major benefit from Dimebon treatment as well as human neuroblastoma cells (SK-N-SH and is attributed to the chronic administration from an early SH-5Y5Y cells) without inducing mitogenesis and pre- stage of pathology and it might involve several mecha- serving mitochondrial protein (as noted by JC-1 fluo- rescence) [32]. At higher Dimebon concentrations mi- Dimebon as a Neuroprotective Agent Current Medicinal Chemistry, 2016, Vol. 23, No. 30 7 tochondria are protected from the toxic effects of Aβ- chondrial-accumulated calcium as well as pro- peptide(1-42) on differentiated neuroblastoma SH- apoptotic factors from the intermembrane space [75]. SY5Y, primary cortical neurons, and protect cerebellar Mitochondrial potential and respiratory activity of granular neurons from the toxic effect of Aβ- isolated mitochondria from rat liver or brain were not peptide(25-35) [15, 32]. Earlier we have shown that affected by Dimebon whereas it calcium-induced depo- one of the main feature of Dimebon is to decrease the larization and swelling were significantly decreased vulnerability of mitochondria towards mitochondrial with elevated calcium retention capacity (CRC) of mi- permeability transition (MPT) induced by calcium ions, tochondria revealing the properties of mitochondrial phosphate, Aβ-peptide and suggest that the neuropro- permeability transition inhibitor [30]. Our data also tective and anti-aging properties of Dimebon are linked suggest that Dimebon contributes to the increased CRC to mitochondria stabilization. The vulnerability to levels in the presence of Aβ (Fig. 4). The cognition MPT-inducing factors increases with aging, and thus enhancing and neuroprotective effects of Dimebon rely compounds like Dimebon with mitochondrial stabiliz- on the increase in mitochondrial CRC, and the calcium ing properties might possess anti-aging effect [73, 74]. addition mode depend on brain mitochondria. No obvi- Accumulation of cytotoxic oligomeric forms of Aβ- ous deviations were observed during the continuous peptide and hyperphosphorylated tau-protein, changes “pump” of calcium ions [76], but the time for MPT in redox status, production of reactive oxygen species induction of after a single addition of calcium ions and and increased intracellular calcium are the hallmarks of CRC during the “bolus mode” was found to be in- AD pathology [53-61]. These factors sensitize mito- creased by Dimebon. This resulted in extra- chondria towards induction of MPT, which represent a mitochondrial calcium decrease, and physiological pro- potential point of convergence in numerous specific tection of active neurons from initiation of apoptotic neurodegenerative and cell death pathways. Induction cascade [14, 30]. Dimebon also inhibits lipid peroxida- of MPT leads to disruption of cellular respiration, ATP tion and swelling of rat brain and liver mitochondria deficit and overproduction of oxygen radicals, destroys which is induced by tert-butylhydroperoxide (TBHP). the proton gradient, and allows the release of mito-

Fig. (4). Dimebon increases calcium retention capacity of isolated brain mitochondria in the presence of a. – amyloid β- peptide(1-42) or b. – β-amyloid(1-40). Rat brain mitochondria (0.2 mg/ml) were suspended in KCl-based medium supple- mented with 5 mM succinate, 1 µM rotenone, 0.15 mM ADP and 1 µg/mL oligomycin. Boluses of Ca2+ (30 nmol/mg protein) were added to the mitochondrial suspensions with or without 1 µM amyloid β-peptide(1-42) or β-peptide(1-40) and 50 µM Dimebon. The data are representative of three independent experiments (Reprinted with permission from [5]). 8 Current Medicinal Chemistry, 2016, Vol. 23, No. 30 Ustyugov et al.

TBHP-induced swelling in the 0.005-0.15 mM was voltage-dependent anion channels, cyclophilin D (pep- found to be effectively blocked, while the inhibitory tidyl prolyl isomerase F), the peripheral benzodi- effect was found to be reduced above 0.2 mM level. On azepine receptor, hexokinase II and several members of the contrary, suppression of THBP-induced lipid per- the Bcl-2 family. In comparison to the MPT inhibitor oxidation was found to be maximal at 0.4 mM [30], cyclosporine A, Dimebon’s action does not depend on while lowered mitochondrial lipid peroxidation was conformational changes in adenine nucleotide translo- found to be induced by calcium and Aβ-peptide(25-35) case and its inhibitory effect is detected only in ener- in response to cyclosporine A [30, 31]. In addition, gized mitochondria [14], but the question about the spontaneous lipid peroxidation was found to be de- mitochondrial molecular targets of Dimebon are still creased in rat brain homogenates, which is a feature of waiting to be solved. an anti-aging component (Fig. 5). CONCLUSION The latest findings on Dimebon have unearthed new targets of central nervous system providing new essential conclusions. Dimebon is a multi-target agent capa- ble of many independent mechanisms of action like inhibition of proteinopathy processes, autophagosomal upregulation, and stabilization of mitochondrial functions in neurodegenerative conditions. Most of the molecular pathways in NDDs are common, and thus Di- mebon and/or its modified analogs have the potential to form an important component in comprehensive treatment of various neuropathological conditions. The fact that Dimebon has the most pronounced effect in initial stage of disease onset should be taken into considera- tion while planning the clinical trials. Recently, a number of novel potential clinical applications of Dimebon have been analyzed. Of particular importance are its efficient application in combination with risperidone Fig. (5). Dimebon decreases spontaneous lipid peroxidation for schizophrenic patients’ treatment, neuroprotective in rat brain mitochondrial homogenates (0.5 mg/ml) incu- properties against ischemic neuronal damage, and very bated with 20 or 50 µM of Dimebon or none (control). Lipid attractive Dimebon effects on delaying ALS progres- peroxidation was estimated as formation of thiobarbituric sion. Hence, further investigations on Dimebon mecha- acid-reactive compounds one of which, malondialdehyde nism of action could radically change our understand- (MDA), is the most commonly reported biomarker of lipid ing of its possible clinical applications. peroxidation. The data are representative of three independent experiments (Reprinted with permission from [5]). LIST OF ABBREVIATIONS AD = Alzheimer disease The cognition stimulating properties of Dimebon might be also linked with its ability to induce migration ALS = Amyotrophic lateral sclerosis of mitochondria to neurites of hippocampal neurons CGU = Cerebral Glucose Utilization and stimulate neurite outgrowth [32] as well as enhance hippocampal neurogenesis by blocking the apoptosis of CRC = Calcium Retention Capacity new hippocampal neurons, thus resulting in a greater ERK1/2 = Extracellular signal-regulated kinase survival of new neurons due to stabilization of mito- 1/2 chondria which in turn increases the threshold to MPT FTD = Frontotemporal Lobar Degeneration induction [77]. Therefore, one potential target of Di- mebon could be the MPT pore complex. Although, the FUS = Fused in Sarcoma MPT pore complex structure is not yet elucidated, sev- MPT = Mitochondrial Permeability Transition eral proteins are known to play an important role and NDDs = Neurodegenerative disorders participate in the MPT pore activity, including adenine nucleotide translocase, the inorganic phosphate carrier, TBHP = Tert-butylhydroperoxide Dimebon as a Neuroprotective Agent Current Medicinal Chemistry, 2016, Vol. 23, No. 30 9

TDP-43 = Transactive response DNA binding placebo-controlled study. Lancet, 2008, 372(9634), 207- 215. protein 43 kDa [8] MacKay, J.; Harnett, S.; Machado, P.: UPS = Ubiquitin-proteasome System http://press.pfizer.com/press-release/pfizer-and-medivation- announce-results-two-phase-3-studies-dimebon- latrepirdine-alzhei. Accessed 11 November 2014., 2010; CONFLICT OF INTEREST Vol. [9] Bharadwaj, P.R.; Bates, K.A.; Porter, T.; Teimouri, E.; The author(s) confirm that this article content has no Perry, G.; Steele, J.W.; Gandy, S.; Groth, D.; Martins, R.N.; conflict of interest. Verdile, G. Latrepirdine: molecular mechanisms underlying potential therapeutic roles in Alzheimer's and other ACKNOWLEDGMENTS neurodegenerative diseases. Transl. Psychiatry, 2013, 3, e332. This work partly supported by the Russian Scien- [10] Solís-Herrera, A.; Ashraf, G.M.; del C A Esparza, M.; tific Foundation ((www.rscf.ru, Российский Научный Arias, R.I.S.; Bachurin, S.O.; Barreto, G.E.; Aliev, G. Biological Activities of QIAPI 1 as a Melanin Precursor Фонд, Grant № 14-23-00160 for 2014-2016: Directed and Its Therapeutic Effects in Wistar Rats Exposed to design, synthesis, and study of biological activity of Arsenic Poisoning. Cent. Nerv. Syst. Agents Med. Chem., multi-target compounds as innovative drugs for treat- 2015, 15(2), 99-108. [11] Herrera, A.S.; Del C A Esparza, M.; Md Ashraf, G.; ment of neurodegenerative diseases). Part of the ex- Zamyatnin, A.A.; Aliev, G. Beyond mitochondria, what perimental work was performed using the equipment of would be the energy source of the cell? Cent. Nerv. Syst. Center for collective use of IPAC RAS (Agreement № Agents Med. Chem., 2015, 15, (1), 32-41. [12] Miller, G., Pharmacology. The puzzling rise and fall of a 14.621.21.0008, ID RFMEFI62114X0008). GEB work dark-horse Alzheimer's drug. Science, 2010, 327(5971), is supported by Pontificia Universidad Javeriana. G. 1309. Aliev work also supported by the GALLY International [13] Bezprozvanny, I. The rise and fall of Dimebon. Drug News Biomedical Research Consulting LLC, San Antonio, Perspect., 2010, 23(8), 518-523. [14] Shevtsova, E.F.; Vinogradova, D.V.; Kireeva, E.G.; Reddy, Texas, USA. Ghulam Md Ashraf gratefully acknowl- V.P.; Aliev, G.; Bachurin, S.O. Dimebon attenuates the edges the facilities provided by King Fahd Medical Abeta-induced mitochondrial permeabilization. Curr. Research Center (KFMRC) and Deanship of Scientific Alzheimer Res., 2014, 11(5), 422-429. [15] Makhaeva, G.F.; Lushchekina, S.V.; Boltneva, N.P.; Research (DSR), King Abdulaziz University, Jeddah, Sokolov, V.B.; Grigoriev, V.V.; Serebryakova, O.G.; Saudi Arabia. Vikhareva, E.A.; Aksinenko, A.Y.; Barreto, G.E.; Aliev, G.; Bachurin, S.O. 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