Donepezil in the treatment of ischemic stroke: Review and future perspective
Arnavaz Hajizadeh Barfejania,b,⁎,1, Mahbod Jafarvandc,1, Seyed Mohammad Seyedsaadatd, Roozbeh Tarighati Rasekhie
aDepartment of Pharmacy, Mayo Clinic, Rochester, MN, 55902, USA
bSchool of Medicine, Royal College of Surgeons in Ireland, Dublin 2, Ireland
cDepartment of Surgery, Harlem Hospital Center, affiliated to Columbia University, NYC, NY 10037, USA
dDepartment of Radiology, Mayo Clinic, Jacksonville, FL 32224, USA
eDepartment of Radiology and Imaging Sciences, Emory University School of Medicine, Atlanta, GA 30322, USA

A R T I C L E I N F O

Keywords: Donepezil Ischemic stroke
Vascular dementia Neuroprotection
A B S T R A C T
Ischemic stroke remains the leading cause of morbidity and the second most common cause of mortality worldwide. Over the past decade, endovascular thrombectomy (EVT) drastically changed the care of patients with ischemic stroke due to large vessel occlusion. Nevertheless, despite revascularization, many patients do not achieve a good functional outcome. Moreover, not all patients with ischemic stroke are eligible for EVT.
During ischemia, a cascade of ischemic and inflammatory changes lead to permanent damage. As such, ad- junct therapies that can protect neurons during acute ischemic phase prior to revascularization have the po- tential of enhancing functional recovery. Donepezil, an acetylcholinesterase inhibitor, improves cognition and global function in patients with Alzheimer’s and Vascular dementia via modulation of acetylcholine receptors and downstream inflammatory response. Preclinical studies demonstrated the potential neuroprotective effects of donepezil in ischemic stroke. However, only a handful of clinical studies investigated this drug’s safety and efficacy in stroke patients. In this review, we summarize the current evidence for the utility, or lack thereof, donepezil in treating and rehabilitating patients with ischemic stroke.

1.Introduction
Stroke is the leading cause of permanent disability in adults and the second most common cause of mortality worldwide [1]. Stroke also poses a significant economic burden on societies. The estimated cost of post-stroke care in the United States is more than 4500 dollars per patient per month [2] with an estimated annual cost of 40 billion dollars [3]. Stroke can be ischemic or hemorrhagic, with 87% of pa- tients suffer ischemic form. An acute ischemic stroke (AIS) occurs when there is a disruption of blood flow to brain tissue, leading to neuronal injury and cell death. Depending on the affected area of the brain, AIS patients may suffer from various neurological deficits.
The cornerstone of AIS treatment remains the restoration of blood flow to the ischemic area. In select group patients who present within 4.5 h of the last known well, thrombolysis with tissue plasminogen activator (tPA) is the only US Food and Drug Administration (FDA) approved medication that improves the functional outcome [4]. Over

the past decade, endovascular thrombectomy (EVT) showed promising results in patients with AIS and large vessel occlusion, extending the AIS treatment time-window up to 24 h [5]. Despite advances in treat- ment, many stroke patients don’t achieve good functional outcomes. In two recent trials, less than half of the patients who underwent EVT gained functional independence in 3 months [6].
1.1.Pathogenesis of ischemia
Ischemia causes a cascade of pathological processes that leads to acute and chronic brain damage. With the lack of blood flow, glucose and oxygen can’t be delivered to neurons, leading to failure of pro- duction of high energy phosphate compounds. Energy failure adversely affects maintaining the ionic gradient across the cell membranes, re- sulting in an influx of calcium. Increased entry of calcium into the cell initiates a cascade of nuclear and cytoplasmic events, including acti- vation of proteolytic enzymes, reactive oxygen species (ROS) formation,

⁎ Corresponding author at: Department of Pharmacy, Mayo Clinic, Rochester, MN, 55902, USA.
E-mail address: [email protected] (A.H. Barfejani). 1 These authors contributed equally to this work.

https://doi.org/10.1016/j.lfs.2020.118575

Received 10AvailableonlineJuly122020;OctoberReceived2020 in revised form 1 October 2020; Accepted 4 October 2020
0024-3205/ ©2020 Elsevier Inc. All rights reserved.

lipid peroxidation, DNA fragmentation, and activation of apoptotic pathways, which together lead to inflammatory response and cell death [7]. In addition, excitotoxicity due to glutamate release can further trigger inflammation and exacerbate neuronal injury [8]. An array of neurotransmitters receptors, including glutamatergic, purinergic, and cholinergic, are overexpressed in microglia and contribute to down- stream processes [9]. N-methyl-D-aspartate (NMDA) receptors, for ex- ample, are upregulated in ischemic core and penumbra, and glutamate surge results in downstream inflammatory responses [10].
Reducing inflammation in the acute phase of ischemic stroke at- tenuates brain damage and improves functional outcome [11]. Acet- ylcholine receptors (AChR) show neuroprotective properties that have been attributed to the modulation of immune cells by the cholinergic anti-inflammatory pathways via activation of α7-nAChR [12]. These receptors are commonly expressed on the surface of neurons, glial, and endothelial cells [13]. Activation of these receptors attenuates the production of inflammatory cytokines [14] and enhances neuronal re- sistance to ischemic insult [15].
1.2.Donepezil
Donepezil (( ± )-2-[(1-benzylpiperidin-4-yl) methyl]-5, 6-di- methoxy-indan-1-one monohydrochloride), is a reversible non- competitive acetylcholine esterase inhibitor that improves cognition and global function in patients with Alzheimer’s [16] and Vascular dementia [17]. Donepezil is transported across the Blood-Brain Barrier by an organic cation transporter and increases acetylcholine in synaptic clefts. It is generally well-tolerated, and its side effects are mild, tran- sient, and cholinergic, as expected [18].
Like many other neuroprotective agents, donepezil has been shown to improve outcomes in preclinical studies of cerebral ischemia but failed to exert the same promising results in randomized clinical trials. There are a few explanations for discrepancies between basic and clinical stroke research of neuroprotective agents [19]:
1.Neuroprotective agents have been examined in animal models of ischemia but not studied in combination with reperfusion in clinical trials
2.The efficacy of agents are determined by infarct volumes in animal models but by long-term functional outcomes in clinical trials
3.Animal models are performed in young rodents, whereas stroke patients tend to be older men and women with multiple comorbid conditions
4.Neuroprotective agents might be administered in sub-therapeutic dose in clinical trials to prevent potential adverse events
5.Publication bias in reporting of animal stroke studies leads to sig- nificant overestimation of efficacy [20]
With the recent advances in AIS treatment, we decided to review the evidence behind Donepezil’s efficacy in ischemic stroke and its potential use in conjunction with reperfusion therapy.
2.Method
We searched PubMed and Web of Science databases to find studies between January 1996 and April 2019, which used donepezil to treat AIS in human or animal stroke models. Keywords and synonyms in- cluding “donepezil”, “Aricept” and “acute ischemic stroke”, as well as “stroke,” “outcomes,” “functional recovery,” “brain Infarction,” “cere- bral infarction” were searched and original articles in the English lan- guage were included. The reference lists of included articles were also reviewed for additional relevant studies. Preclinical studies quality were assessed according to a ten-item checklist comprising: (1) pub- lication in peer-reviewed journal; (2) statement of control of tempera- ture; (3) randomization to control or treatment; (4) blinded ischemia induction; (5) blinded outcome assessment; (6) avoidance of anesthetics
with marked neuroprotective properties; (7) use of animals with hy- pertension or diabetes; (8) sample size calculation; (9) statement of compliance with regulatory requirements; and (10) statement regarding possible conflicts of interest [21].
2.1.Preclinical studies
Multiple in vivo and in vitro studies showed neuroprotective effects of Donepezil. As expected, most studies are performed in Alzheimer’s dementia models, and many of the proposed neuroprotective mechan- isms are independent of cholinesterase inhibition. We have found six preclinical studies investigating the effects of donepezil in ischemic conditions, of which four were focal or global ischemic stroke models in rodents. (Table 1).
In oxygen-glucose deprived neuronal culture, Akosufo et al. de- monstrated that Donepezil could inhibit the fast inflow of Na+ and Ca2+, reduce the release of glutamate, and therefore protects cortical neurons against depolarization-induced neurotoxicity [22]. In a more recent study, he and his colleagues showed that donepezil’s protective effects against ischemic damage and Aβ toxicity persist in the presence of nicotinic and muscarinic AChR antagonists, suggesting that its neu- roprotective effect arises via other mechanisms than acet- ylcholinesterase inhibition. In addition, high doses of donepezil only slightly inhibited the binding of ligands to NMDA receptor subtypes in neuronal culture, suggesting that NMDA modulation can’t fully account for its neuroprotective properties, either [23].
Donepezil also increases neurotrophins such as nerve growth factor [24] and brain-derived neurotrophic factor [25]. Chronic treatment of old rats with donepezil increased dendritic spine numbers of neurons in the prefrontal cortex, hippocampus, and nucleus accumbens. The re- sultant increase in neural connectivity may be beneficial in neurode- generative disorders [26]. Donepezil is shown to decrease the amount of Aβ fibrils via activation of phosphatidylinositol 3-kinase (PI3K)-Akt [27] by inhibiting Glycogen synthase kinase (GSK)-3β pathway [28]. GSK-3β plays a critical role in oxidative stress-induced neuronal cell death mechanisms, and its inhibition confers neuroprotection [29]. In addition, stimulation of α7-nAChR by donepezil reduces glutamate toxicity via down-regulation of NMDA receptors [30].
Fujiki et al. studied the impact of donepezil pretreatment in Middle Cerebral Artery Occlusion (MCAo) in rats. Young male Wistar rats were treated with 12 mg/kg of donepezil 2 h prior to induction of ischemia. Neurological outcome and infarct volume were assessed 24 h after MCAo. Donepezil significantly reduced infarct volume by more than 30% with no significant improvement in functional outcome [31]. In contrast, chronic treatment with donepezil starting 24 h after MCAo in mice did not affect infarct volume but significantly improved neuro- logic deficit score and use of the impaired right forelimb on day seven after ischemia [32]. Post-stroke functional recovery was attributed to neurogenesis in the subventricular zone. In particular, donepezil acti- vated choline acetyltransferase (ChAT) + neurons in the subventricular zone, which controls neural stem cell proliferation by indirect activa- tion of fibroblast growth factor receptor (FGFR) [33]. Lack of infarct size reduction by donepezil in this study is in agreement with the clinical findings, where any potential benefit of a neuroprotective agent in infarct volume reduction is limited to the first few hours after AIS, before the evolution of infarct.
Transient occlusion of bilateral carotid arteries in rodents causes neuronal damage in hippocampal CA1 neurons [34] and is commonly used as a model to assess the effect of pharmacological neuroprotection on memory. Wang and colleagues demonstrated that treatment with 3 mg/kg of donepezil one day after transient forebrain ischemia in mice significantly reduced neuronal loss in the hippocampal CA1 region. The treatment group animals performed better in the water maze test than controls; however, the difference was not significant. During cerebral ischemia, the influx of Ca2+ results in the activation of the Ca2+-de- pendent protease calpain and its downstream cyclin-dependent kinase 5

(CDK5) [35]. Under pathological conditions, CDK5 dysfunction is im- plicated in neuronal injury [36]. Wang et al. found that the ability of donepezil to improve learning and memory might be related to de- creased expression of calpain I-CDK5 pathway and reduced oxidative damage. A 21-day course treatment with 5 mg/kg donepezil in gerbils significantly inhibited delayed CA1 neuronal death in a transient bi- lateral carotid occlusion. Calcium/calmodulin (CaM)-dependent pro- tein kinase II (CaMKII)/cAMP-responsive element-binding protein (CREB) signaling pathway is involved in synaptic plasticity and memory. Donepezil treatment maintains CaMKII/CREB level in the hippocampus after ischemia, playing a role in observed memory im- provement [37]. Interestingly, It has been shown that CDK5 down- regulation induces neural plasticity and enhancement of brain-derived neurotrophic factor (BDNF)/CREB pathway, further bridging the two mechanisms associated with neuroprotective effects of donepezil in learning and memory [38]. Verma and Sharma investigated the po- tential impact of donepezil in vascular dementia. Female Wistar rats were ovariectomized and fed high-fat diets for 4-weeks. 1 mg/kg/d of oral donepezil was administered between day 14 and day 28, and an- imals were assessed on day 32 with a battery of tests. Donepezil im- proves vascular function, learning, memory ability, and decreased neuronal cell death and oxidative stress in ovariectomized dyslipidemic rats [39].
2.2.Anti-inflammatory effects
Inflammatory response to neuronal cell death after ischemia leads to the formation of ROS and other proinflammatory cytokines, which in turn activates microglia [40]. Activation of microglia results in induc- tion of adhesion molecules (Integrin and selectins) and further release of proinflammatory cytokine in cerebral endothelial vasculature, causing blood-brain barrier (BBB) disruption [41]. Microglia and other immune cells express AChRs. Stimulation of AChR inhibits the release of cytokines such as tumor necrosis factor (TNF), interleukin (IL)-1beta, IL-6, and IL-18 [42]. High dose donepezil has been shown to reduce inflammation via stimulation of microglial α7-nAChRs [43]. In a murine model of multiple sclerosis, 2 mg/kg/d donepezil prevents BBB disruption, inhibits matrix metalloproteinase-2 (MMP-2) and MMP-9 production, modulates NGF and pro-NGF, hence exerting anti-in- flammatory effects [44]. Similar effects have been shown in animal models of Alzheimer’s disease, manifested as amelioration of tauopathy and synaptic loss [45].
2.3.Clinical studies
We identified 14 original investigations in which donepezil effect was assessed in stroke and vascular dementia. Nine of the studies in- cluded control group (four on stroke and five on vascular dementia). Only one study assessed donepezil in acute phase of ischemic stroke. The characteristics of these studies are presented in Table 2.
2.4.Vascular dementia
Three randomized, double-blind, placebo-controlled trial studied the effects of donepezil in patients with vascular dementia. Patients with associated neurodegenerative disorders were excluded from trials. NINDS-AIREN criteria for vascular dementia was used for patient se- lection. The duration of trials was 24 weeks. Alzheimer’s disease Assessment Scale-Cognitive subscale (ADAS-Cog) and the Clinician’s Interview-Based Impression of Change–Plus version (CIBIC-plus) were used for primary outcome assessment.
Black and colleagues included 603 patients in their study (5 mg/d (n = 198), 10 mg/d (n = 206), or placebo (n = 199)). Patients with stroke within the past 3 months were excluded from the study. The mean age of patients was 73.9 and 55.2% were men. Both donepezil groups demonstrated significant improvement in ADAS-Cog score
(mean change from baseline: donepezil 5 mg/d, -1.90; P = 0.001; donepezil 10 mg/d, -2.33; P < 0.001). On the other hand, im- provement in CIBIC-plus was only significant for patients on 5 mg/d group (p = 0.014). Clinical Dementia Rating Scale–Sum of Boxes (CDR–SB) was used as a secondary outcome in the study, and was only significant in 10 mg/d treatment group (p = 0.022), but not in 5 mg/d [46]. In a separate but similar RCT, Wilkinson et al. studied 616 pa- tients with possible and probable vascular dementia, treated with pla- cebo (n = 193), donepezil 5 mg/day (n = 208), or donepezil 10 mg/
day (after 5 mg/day for the first 28 days) (n = 215) for 24 weeks. Patients who had a stroke within the prior 28 days of enrollment were excluded. 76% of patients carried a diagnosis of probable vascular de- mentia and 24% had possible vascular dementia. The average of pa- tients was 75 and 59.9% were men. Like the Black et al. study, both groups showed significant improvement in ADAS-Cog score (donepezil 5 mg, -1.65 [p = 0.003]; 10 mg, -2.09 [p = 0.0002]). In contrast to Black et al, Both donepezil groups showed significant improvement in CIBIC-plus score. The AD Functional Assessment and Change Scale (ADFACS) was used to assess global function. Patients treated with 10 mg/d donepezil showed significant improvement in ADFACS com- paring to control. A trend toward improvement was noted in 5 mg/d group; however, it was not significantly different [47]. By combining the data from both RCTs, Roman and colleagues showed that donepezil significantly improves global function, cognition and ability to perform IADL in vascular dementia patients [48]. The findings of another study from the same group was presented on 10th International Conference on Alzheimer's Disease and related disorders. Treatment with 5 mg/d donepezil for 24 weeks led to marked improvement of mean V-ADAS- cog score (-1·16 points [95% CI -1·98 to -0·33]). However, the same study also showed a significant increased risk of death: 1·7% (11 of 648) assigned to donepezil versus 0% (0 of 326) assigned to placebo (OR 4·57 [95% CI 1·30–16·08]). The findings of this study have not published [49]. In a 30 week extension open-label trial, Wilkinson et al. showed a mean ADAS-cog score reduction of 0.6–1.15 between baseline and week 54. 14.4% discontinued the medication due to adverse reactions (nausea and diarrhea were the most common side effects). Patients who were initiated on donepezil during the extension phase did not perform as good as patients who were initiated on treatment during initial phase [50]. In a single-arm, open label trial, patients with Binswanger type vascular dementia showed significant improvement in cognitive func- tion on the Seoul Neuropsychological Screening Battery-Dementia version (SNSB-D) comparing to baseline. No significant difference was noted in activities of daily living score [51].
2.5.Stroke
We identified nine studies evaluating the potential benefit of do- nepezil in patients with stroke. In the only prospective randomized, double-blind, placebo-controlled trial, 20 patients were randomized to either donepezil (5 mg/d for 2 weeks, followed by 10 mg/d for 4 weeks) or placebo. During the last 2 weeks of study, all patients underwent constraint-induced therapy (CIT) for upper-limb dysfunction. Patients with more than a year history of unilateral ischemic or hemorrhagic stroke, leading to upper extremity paresis were included in study. Multiple motor function assessment was done and none of them showed significant differences between the two group [52]. Recently, Lin et al. studied the effect of 3-month course of 5 mg/d donepezil on gait per- formance of patients with acute cerebral infarction with hemiparesis. In this prospective cohort study, Donepezil significantly improved Wis- consin Gait Scale (WGS) score after three months of treatment com- paring to control group [53]. Whyte and colleagues investigated the effects of donepezil and galantamine in a 12-week open-label study of patients with ischemic stroke. Forty patients of at least 60 years of age who had a stroke within the prior 28 days, resulting in cognitive im- pairment assigned to either donepezil (maximum 10 mg/day) or ga- lantamine (maximum 24 mg/day). The mean age of patients was 69.5

Fig. 1. Protective mechanisms of donepezil in ischemic stroke.

and 61.5% were male. The mean interval between time of stroke and initiation of medication was 8.3 days. Outcome was assessed using the Functional Independence Measure–motor subscale (FIM-motor). After 12 weeks, patients treated with donepezil, but not galantamine, showed marked improvement in FIM-motor score in comparison to historical control (p < 0.0001). Change in apathy was noted to be associated with functional recovery [54]. The same group is currently running a phase-3 RCT to determine the efficacy of donepezil after stroke to im- prove functional recovery [55]. Berthier and colleagues studied the effects of donepezil on post hemispheric stroke recovery. In an open- label, single arm study, 10 patients with chronic post stroke aphasia received 5 mg/d of donepezil for 4 weeks, followed by a 10 mg/d dose for 12 weeks and a 4 weeks washout period. The mean interval between stroke and trial initiation was 4.4 years. The average age of the patients was 56 and 90% of them were men. Aphasia Quotient (AQ) of the Western Aphasia Battery (WAB) was used as primary outcome and measured at baseline, week 4, 16 and 20. Higher score represents better global language function. Baseline AQ score was 55.1 ± 17.6. There was a significant improvement of AQ score on week 4 (mean change ± SD: 6.3 ± 3.9) and week 16 (9.5 ± 3.8). There was a significant worsening of AQ score during washout period (-1.6 ± 2.0) [56]. In a case study, a 64-year-old woman with right hemispheric ischemic stroke with residual left hemiplegia, left hemi- sensory loss and conduction aphasia at age 59, received donepezil 5 mg/d for 4 weeks, followed by 10 mg/d for 12 weeks and a 4 week washout period. Baseline and posttreatment assessment were done using the Fugl-Meyer scale and the Rivermead Mobility Index. After two weeks of treatment, the patient developed allodynia in left hemibody. On week 8, she regained voluntary movements of the left leg and shoulder, but left hand remained paralyzed. On week 16, her Fugl- Meyer scale score increased from 10 to 34 and Rivermead Mobility Index score from 6 to 13 [57]. In a case-control study, Chen et al. di- vided sixty acute stroke patients with aphasia to donepezil 5 mg/d and placebo groups. After 12 weeks of treatment, patients in the treatment group showed significant improvement in AQ score compared to pla- cebo group (34.14 ± 17.70 Vs. 20.69 ± 17.26, p = 0.004) [58]. In a prospective, multicenter, single arm Phase II clinical trial to investigate the efficacy and safety profile of donepezil, 33 patients with possible
stroke within the past 24 h started on donepezil 5 mg/d for 30 days, followed by 10 mg/day for 60 days. Three patients (9%) discontinued donepezil because of adverse effects and 3 participants (9%) required a reduction to 5 mg/day after increasing the dose to 10 mg/d. In com- parison with historical data, patients showed significant improvement in multiple secondary cognitive outcomes [59]. Chang and colleagues further investigated the mechanisms underlying cognitive improvement on donepezil in patients with right hemispheric stroke. Patients either received 5 mg/d donepezil or placebo for four weeks. The treatment group showed marked improvement in MMSE compared to control. fMRI demonstrated increase activity of bilateral prefrontal, inferior frontal and left inferior parietal cortices in donepezil, pointing toward increased recruitment of frontoparietal networks as a potential me- chanism of donepezil effects [60] (Fig. 1).

3. Discussion

Donepezil has been proven to exert protection against cerebral ischemia in a number of preclinical studies. Although pretreatment with donepezil led to decreased infarct volume in rats, the reduction in infarct size was not translated to improved outcome in early post-stroke phase. On the other hand, chronic treatment with donepezil led to improved functional outcome without significant change in infarct size. In transient forebrain ischemia, donepezil displayed neuroprotection in CA1 neurons which translated to enhanced learning and memory. A wide variety of mechanisms are proposed for donepezil neuroprotec- tion; neurogenesis, stimulation of α7-nAChR leading to down-regula- tion of NMDA receptors and excitotoxicity, decrease in expression of CI- CDK5 pathway and ROS formation, GSK-3β pathway inhibition, acti- vation of CaMKII/CREB pathway, among many others. Nevertheless, the preclinical studies suffered from many short coming of rodent models of ischemic neuroprotection. First, none of the studies used aged animals with vascular risk factors, making their findings at best sub- optimal for the clinical practice. Second, in the majority of animal studies, it is not clear whether or not the induction of ischemia or as- sessment of outcome was performed in a blinded manner, making the results prone to bias. Third, the basis of sample size estimation is not explained in any of the studies. Fourth, the heterogeneity of ischemic

models, dosage, duration of therapy, and outcome assessment methods further limits the clinical translation of the findings.
Clinical studies of donepezil in vascular dementia showed sig- nificant improvement in cognitive assessment in 24 weeks. There was more significant ADAS-cog amelioration seen with 10 mg/d compared to 5 mg/d, which was not replicated in CIBIC-plus score [61]. Aguilar and colleagues showed marked improvement with 5 mg/d donepezil in V-ADAS-cog score, at the expense of a significant increase in mortality. The results of this trial were never published, raising the concern for publication bias and potential other unpublished studies with equivocal results. One other limitation of vascular dementia trials is their reliance on the cognitive scoring methods, designed for Alzheimer's dementia and other neurodegenerative disorders. Although sharing some clinical characteristics, these diseases are different pathophysiological entities, and each requires its validated assessment score.
The clinical evidence behind the potential utility of donepezil in ischemic stroke is even less robust. The only prospective randomized, double-blind, placebo-controlled, parallel-group trial investigated the effect of donepezil, initiated one year after the stroke and only showed a trend toward improvement. The remaining studies lacked adequate sample size, randomization, control group, or included patients with different stroke subtypes further compromising the quality of their findings. Nevertheless, there might be a possible role for donepezil in the recovery of cognitive and motor symptoms due to ischemic stroke. In general, donepezil is well tolerated with minimal adverse effect profile. Future randomized, double-blind, placebo-controlled trials that assess the efficacy of donepezil as an adjunct treatment to EVT and tPA are needed. Given the modest effect, rigorous patient selection criteria to ensure the homogeneity of data and capturing beneficial effects is required. For the time being, current evidence is insufficient to support widespread use of donepezil in patients with ischemic stroke.
Funding
No funding was used for publication of this study. Declaration of competing interest
On behalf of all authors, the corresponding author states that there is no conflict of interest.
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