CLINICAL TRIALS SECTION EDITOR: IRA SHOULSON, MD. Efficacy of Donepezil in Early-Stage Alzheimer Disease

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1 CLINICAL TRIALS SECTION EDITOR: IRA SHOULSON, MD Efficacy of Donepezil in Early-Stage Alzheimer Disease A Randomized Placebo-Controlled Trial Ben Seltzer, MD; Parvaneh Zolnouni, MD; Margarita Nunez, MD; Robert Goldman, PhD; Dinesh Kumar, MA; John Ieni, PhD; Sharon Richardson, PhD; for the Donepezil 402 Study Group Objective: To evaluate the efficacy of donepezil in patients with early-stage Alzheimer disease. Design: Multicenter, randomized, double-blind, 24- week, placebo-controlled study that enrolled patients with early-stage Alzheimer disease. Patients were randomized in an approximately 2:1 ratio to donepezil, 5 mg/d, for the first 6 weeks, with a forced escalation to 10 mg/d thereafter (n=96), or placebo (n=57). The primary efficacy measure was the modified Alzheimer Disease Assessment Scale cognitive subscale. Secondary efficacy measures included the Mini-Mental State Examination, the Computerized Memory Battery Test, the Clinical Dementia Rating Scale Sum of the Boxes, the Patient Global Assessment Scale, and the Apathy Scale. Results: Improvements favoring donepezil on the Alzheimer Disease Assessment Scale cognitive subscale were found at weeks 12 and 24 and at the end point (last observation carried forward); treatment differences were 1.9 (P=.03), 2.3 (P=.008), and 2.3 (P=.001) points, respectively. Improvements favoring donepezil on the Mini- Mental State Examination were found at weeks 6, 12, and 24 and at the end point (last observation carried forward); treatment differences were 1.4 (P =.02), 1.2 (P=.04), 1.4 (P=.03), and 1.8 (P=.002) points, respectively. Donepezil-treated patients showed greater mean improvement compared with placebo-treated patients on the following Computerized Memory Battery Test subscales: facial recognition (P=.007 in the intent-to-treat population and P=.04 in the fully evaluable population), first and last name total acquisition (P=.02), and name-face association delayed recall (P=.04). Donepezil was safe and well tolerated in this population; serious adverse events occurred in similar numbers of donepezil- and placebo-treated patients. Conclusion: These data suggest significant treatment benefits of donepezil in early-stage Alzheimer disease, supporting the initiation of therapy early in the disease course to improve daily cognitive functioning. Arch Neurol. 2004;61: Author Affiliations are listed at the end of this article. Group Information: A list of the members of the Donepezil 402 Study Group appears on page Financial Disclosure: Dr Seltzer has received research support from and is on the speakers bureau of Eisai Inc and Pfizer Inc; Dr Goldman is an employee of Pfizer Inc; and Mr Kumar and Drs Ieni and Richardson are employees of Eisai Inc. PIVOTAL CLINICAL TRIALS OF cholinesterase inhibitors such as donepezil have focused on patients with mild to moderate Alzheimer disease (AD) and have demonstrated the treatment benefits of cholinesterase inhibitors in this patient population. 1,2 The diagnosis of AD is often delayed until 2 to 3.5 years after the initial complaints of cognitive impairment, 3 by which time patients have well-entrenched cognitive symptoms. Little prospective research has been conducted to evaluate the benefit of initiating treatment in the earliest phase of the disease, when cognitive symptoms are only mild and patients activities of daily living are preserved. Yet, improving symptoms and delaying progression of the disease at this stage might be expected to be particularly beneficial because these individuals have the highest level of functioning to preserve. To our knowledge, this is the first clinical trial to prospectively evaluate donepezil s effects on patients with early-stage AD. These patients do meet the criteria for probable AD and have crossed the threshold from mild cognitive impairment. Standardized psychometric tests, including the modified Alzheimer Disease Assessment Scale cognitive subscale (ADAS-cog) and the Mini-Mental State Examination (MMSE), were used to evaluate donepezil s treatment effects. The Computerized Memory Battery Test (CMBT) 4 initially developed for age-associated memory impairment was used to assess donepezil s effects on critical cognitive tasks of everyday life. 1852

2 METHODS PATIENTS Generally healthy ambulatory patients, aged 50 to 92 years, diagnosed as having probable AD 5,6 within the past 12 months were enrolled. Inclusion criteria included a modified Hachinski Ischemia Scale 7 score of 4 or less; a global Clinical Dementia Rating Scale (CDR) 8 score of 0.5 or 1.0; an MMSE 9 score of 21 to 26 (inclusive); and only mild impairment in activities of daily living, defined by a summed score of 2 to 4 on the 3 functional domains (home and hobbies, community affairs, and personal care) of the CDR, with no more than 1 functional domain with a score of 2 or more. Patients were excluded if the decline in memory was possibly attributable to a psychiatric or neurologic disorder (eg, stroke or Parkinson disease) or to cognitive deficits following head trauma. Previous treatment with cholinesterase inhibitors, whether approved or in development, was not permitted. The study was conducted in accordance with the principles stated in the Declaration of Helsinki, and was approved by institutional review boards at each site. Written informed consent was obtained from patients and their caregivers before screening. STUDY DESIGN This 24-week, randomized, double-blind, placebo-controlled study was conducted at 17 sites in the United States. Patients were randomized in an approximately 2:1 ratio to donepezil, 5 mg/d, for the first 6 weeks, with a forced escalation to 10 mg/d thereafter, or placebo. Patients unable to tolerate 10 mg of donepezil were discontinued from the study. Assessments were conducted at screening, at baseline, and at 6-week intervals thereafter through week 24 (final study visit) or at unscheduled termination visits. OUTCOME MEASURES The primary efficacy measure was the least squares means change from baseline to end point on the modified ADAS-cog 10 total score. Secondary efficacy measures included change from baseline to end point on the MMSE, the CDR Sum of the Boxes, the CMBT, 11 and the Apathy Scale. 12 Patients were asked to rate their impression of change using the Patient Global Assessment Scale at the end point visit. SAFETY EVALUATIONS Safety assessments were conducted at screening and at the final visit. Adverse events (AEs) were elicited at each visit by questioning the patient throughout the study by telephone and through direct observation by the patient treatment team. DATA ANALYSIS Efficacy analyses were performed on the intent-to-treat and fully evaluable (FE) populations, whereas safety analyses were performed on the safety population. Study end point was the final visit observation (ie, if a patient was missing a week 24 observation, then the last observed value was carried forward and used as the end point visit observation). The intent-to-treat population included all patients randomized to treatment regardless of follow-up evaluations or compliance with treatments. The FE population comprised patients who completed 24 weeks of treatment with 80% or more compliance at the week 24 visit and at 2 other clinic visits, with no significant protocol violations. Completed 70 (73%) Donepezil (n = 96) Withdrawn 26 (27%) Reasons for Withdrawal Adverse Event 15 (16%) Medication Noncompliance 1 (1%) Protocol Violation 3 (3%) Unavailable for Follow-up 4 (4%) Subject Withdrew Consent 3 (3%) Screening (N = 309) Screen Failures (n = 156) Randomization (n = 153) Statistical analyses of efficacy variables were performed on the change from baseline scores at weeks 6, 12, 18, and 24 and at the end point(last observation carried forward). The overall treatment effect was assessed using type III sums of squares performed at the 5% level to determine statistical significance. Ordinal variables were analyzed using the Cochran-Mantel-Haenszel 2 test with MODRIDITS scores. Treatment differences in the incidence rates for AEs were tested using the Fisher exact test. RESULTS Completed 46 (81%) Placebo (n = 57) PATIENT DISPOSITION AND DEMOGRAPHIC CHARACTERISTICS A total of 153 patients were randomized to treatment, 96 receiving donepezil and 57 placebo (Figure 1). Patient characteristics are summarized in Table 1. The mean±sd time to onset of dementia symptoms, as determined by interview of patient and caregiver, was 2.8±1.9 years before enrollment. The treatment groups were similar with respect to scores on screening and baseline neuropsychologic and cognitive tests (Table 1). The proportions of patients with comorbid illnesses and concomitant medication use were similar in the 2 groups. PRIMARY EFFICACY MEASURE Withdrawn 11 (19%) Reasons for Withdrawal Adverse Event 5 (9%) Medication Noncompliance 1 (2%) Protocol Violation 1 (2%) Unavailable for Follow-up 0 Subject Withdrew Consent 4 (7%) Figure 1. Patient enrollment and completion through the study. The asterisk indicates that percentages sum to more than 19% because of rounding. Improvements favored donepezil on the modified ADAScog 13-item scores as early as week 12 (P=.03) (Figure 2). The drug-placebo difference was approximately 2.3 points at week 24 (P=.008) and at the end point (P=.001). Improvement of 4 points or more was seen in 16% of placebotreated patients and in 37% of donepezil-treated patients; improvement of 7 points or more was noted in 7% of the placebo- and 10% of the donepezil-treated patients. In addition, improvements favored donepezil in the FE population at the end point (P=.02) (data not shown). 1853

3 Table 1. Demographic Characteristics of Patients Randomized to Study Treatments* Characteristic (n = 96) (n = 57) Age, y 73.3 ± 9.6 (50-90) 75.1 ± 8.8 (52-92) Female sex 48 (50) 34 (60) Onset of 2.9 ± ± 1.8 cognitive symptoms, y Screening CDR score Global (31) 19 (33) (64) 38 (67) Memory Box (21) 15 (26) (69) 40 (70) Score at screening MMSE 24.1 ± 1.7 (20-27) 24.3 ± 1.3 (22-27) Modified ADAS-cog 21.0 ± 7.9 ( ) 21.3 ± 6.8 ( ) total (13 items) Hachinski 0.6 ± 0.7 (0-4) 0.7 ± 0.8 (0-3) Ischemia Scale HAM-D total 2.9 ± 2.8 (0-12) 2.2 ± 2.7 (0-11) Abbreviations: ADAS-cog, Alzheimer Disease Assessment Scale cognitive subscale; CDR, Clinical Dementia Rating Scale; HAM-D, Hamilton Depression Rating Scale; MMSE, Mini-Mental State Examination. *Data are given as number (percentage) of each group unless otherwise indicated. Data are given as mean±sd (range). Data are given as mean±sd. Change From Baseline on ADAS-cog Scores Improvement Baseline Decline End Point Study Week (LOCF) Figure 2. Mean±SE change from baseline through 24 weeks of treatment on the Alzheimer Disease Assessment Scale cognitive subscale (ADAS-cog) total score for the donepezil and placebo groups. The difference between groups was as follows: P=.03 at week 12, P=.008 at week 24, and P=.001 at the end point. LOCF indicates last observation carried forward. At least 70% of the patients with early-stage AD receiving donepezil did not experience cognitive worsening vs 47% of the placebo patients, during the 24 weeks of treatment. SECONDARY EFFICACY MEASURES Change From Baseline on MMSE Scores End Point Study Week (LOCF) Improvements in MMSE scores favoring donepezil were observed as early as week 6 (P=.02), and were sustained through week 24 (P=.03) (Figure 3). The drugplacebo difference at the end point was 1.8 points in favor of donepezil (P =.002). In the FE population, improvements favored donepezil in MMSE scores at the end point (P=.04) (data not shown). Improvements favoring donepezil were seen on CMBT tasks testing verbal and visual memory. Donepezil improved performance of the intent-to-treat population on name-face association delayed recall (mean±sd scores: donepezil group, 0.1±0.1; placebo group, 0.1±0.1; P=.04) and facial recognition (First Miss) (mean±sd scores: donepezil group, 0.8±0.6; placebo group, 1.6±0.8; P=.007). In addition, donepezil improved performance on first and last name total acquisition (mean±sd scores: donepezil group, 0.7±0.2; placebo group, 0.2±0.3; P=.02) and facial recognition (First Miss) (mean±sd scores: donepezil group, 0.1±0.8; placebo group, 2.3±0.9; P=.04) in the FE population. No significant improvements were seen on other CMBT tasks (P.07). On the Apathy Scale, the donepezil group tended to score higher than the placebo group; however, the difference was not significant. No significant differences on CDR Sum of the Boxes or Patient Global Assessment Scale scores were observed between treatment groups. SAFETY Improvement Baseline Decline Figure 3. Mean±SE change from baseline through 24 weeks of treatment on the Mini-Mental State Examination (MMSE) score for the donepezil and placebo groups. The difference between groups was as follows: P=.02 at week 6, P=.04 at week 12, P=.03 at week 24, and P=.002 at the end point. LOCF indicates last observation carried forward. Donepezil was well tolerated in this study; 15 (16%) of the 96 patients receiving donepezil withdrew because of AEs compared with 5 (9%) of the 57 patients receiving placebo. The incidence rates of common AEs are presented in Table 2. Most AEs were mild to moderate in severity and transient. Serious AEs were experienced by 5 (5%) donepezil-treated patients and by 3 (5%) placebo-treated patients. Serious AEs in donepezil-treated patients included colon cancer, dizziness, fall and cerebral hemorrhage, syncope, and right-sided carotid stenosis. Only 1 serious AE (fall and cerebral hemorrhage) for 1 donepeziltreated patient was considered possibly related to treatment. Serious AEs in placebo-treated patients included pancreatic cancer, bradycardia, and prostate cancer. 1854

4 Table 2. Adverse Events Occurring in 5% or More of Patients Receiving Donepezil and at Least Twice the Rate of Placebo Adverse Event (n = 96)* COMMENT (n = 57)* P Value Any 67 (70) 37 (65) NA Diarrhea 19 (20) 5 (9).11 Nausea 10 (10) 2 (4).21 Asthenia 9 (9) 1 (2).09 Abnormal dreams 9 (9) 0.03 Dizziness 8 (8) 1 (2).16 Injury 6 (6) 0.08 Insomnia 5 (5) 0.16 Abbreviation: NA, data not applicable. *Data are given as number (percentage) of each group. This trial demonstrates that donepezil treatment resulted in significant improvements in cognitive function over 24 weeks in patients with early-stage AD. The modified ADAS-cog and MMSE scores improved significantly during the treatment period in the donepezil group. In contrast, the placebo group failed to improve on both assessments. The rate of cognitive decline seems to vary during the course of AD. 13,14 The present study found, on average, little or no decline in the ADAS-cog or MMSE scores of the patients with early-stage AD in the placebo group during the 24-week period. This is in contrast to earlier clinical studies enrolling patients with mild to moderate AD, who showed significant decline (1-1.8 points in ADAScog scores) within this time frame. 2,15 Thus, data from this study suggest that treatment response of patients with early-stage AD may be different from the response of patients with advanced stages of AD. Significant differences in cognitive function favoring donepezil were also observed for the following components of the CMBT: facial recognition (First Miss), nameface association delayed recall, and first and last name total acquisition. These tests assess verbal and visual memory, which are impaired in the early stages of AD. 16 Given the minimal functional impairment of patients at enrollment, the lack of a drug-placebo difference on the CDR Sum of the Boxes was not unexpected. The CDR Sum of the Boxes may have limited ability to detect functional differences over a 6-month period in minimally impaired patients and, thus, may not be the best assessment tool for this population. A similar argument can be made for the Apathy Scale and Patient Global Assessment Scale scores. Patients enrolled in the trial had only mild apathy at study enrollment, consistent with the notion that apathy is a symptom with a later onset. 17 Thus, the absence of drug-placebo differences may not be an unexpected outcome. The lack of significant improvement in Patient Global Assessment Scale scores may reflect a limited sensitivity of the measure in this population because the reliability of the selfassessment by the patients may not adequately reflect the actual treatment benefits. Donepezil 402 Study Group Bernardo Arias, MD, Gulf Coast Education and Research Center, Port Charlotte, Fla; Mahan Chehrenama, DO, Innovative Clinical Research Center, Alexandria, Va; Anita Chopra, MD, University of Medicine and Dentistry New Jersey School of Osteopathic Medicine Center for Aging, Stratford; Larry S. Eisner, MD, Baumel-Eisner Neuromedical Institute, Ft Lauderdale, Fla; Mildred V. Farmer, MD, Meridien Research, St Petersburg, Fla; James Ferguson, MD, Pharmacology Research Clinic, Salt Lake City, Utah; Barry Gordon, MD, PhD, The Johns Hopkins University School of Medicine, Baltimore, Md; Louis C. Kirby II, MD, Pivotal Research, Peoria, Ariz; Melinda Waldrop, MD, Clinical Research Services, Tennessee Christian Medical Center, Madison; Craig McCarthy, MD, Pivotal Research, Scottsdale, Ariz; Margarita Nunez, MD, ICSL Clinical Studies, St Petersburg, Fla; Peter Ripley, MD, ICSL Clinical Studies, South Yarmouth, Mass; Beth E. Safirstein, MD, Baumel-Eisner Neuromedical Institute, Miami Beach, Fla; Ben Seltzer, MD, Tulane University School of Medicine, New Orleans, La; Steven Sevush, MD, University of Miami Memory Disorder Center, Miami, Fla; Alex Shepherd, MD, Air Force Village II Freedom House Research Clinic, San Antonio, Tex; Parvaneh Zolnouni, MD, California Clinical Trials Medical Group, Beverly Hills. Donepezil was safe in this population; serious AEs occurred in similar numbers of donepezil- and placebotreated patients. Good tolerability was demonstrated overall. Discontinuations due to AEs were higher in the donepezil than in the placebo group, but similar to those in donepezil trials of patients with mild to moderate AD. 2,18 The use of cholinesterase inhibitors such as donepezil has been shown to impact long-term outcomes, such as maintaining patients at a higher level of functioning 19 and keeping them in the community longer. 20,21 Thus, initiating treatment early may preserve cognitive function and maintain patient independence longer, thereby reducing the pharmacoeconomic impact of the disease. While clinical studies such as this one provide further evidence for the symptomatic benefits of donepezil treatment, imaging studies suggest additional neuroprotective effects with donepezil, compared with placebo, causing significantly smaller decreases in hippocampal volumes in patients with AD. 22 Suggested mechanisms of action include an antagonist effect on N-methyl-Daspartate receptor sites, 23 induction of neuroprotective factors by stimulation of nicotinic acetylcholine receptors, 24 and modulation of gene expression. 24,25 The robust effect of donepezil on cognitive performance provides further evidence of the benefit of early initiation of donepezil therapy in the treatment of AD. Longer-term studies are required to further evaluate the potential benefits to the patient, family, caregivers, and society by early initiation of donepezil therapy. Accepted for Publication: May 12, Author Affiliations: Department of Psychiatry and Neurology, Tulane University School of Medicine, New Orleans, La (Dr Seltzer); California Clinical Trials Medical 1855

5 Group, Beverly Hills (Dr Zolnouni); ICSL Clinical Studies, St Petersburg, Fla (Dr Nunez); Pfizer Inc, New York, NY (Dr Goldman); and Eisai Inc, Teaneck, NJ (Mr Kumar and Drs Ieni and Richardson). Correspondence: Ben Seltzer, MD, Department of Psychiatry and Neurology, Tulane University School of Medicine, 1430 Tulane Ave, New Orleans, LA Author Contributions: Study concept and design: Ieni and Richardson. Acquisition of data: Seltzer, Zolnouni, Nunez, and Richardson. Analysis and interpretation of data: Seltzer, Goldman, Kumar, Ieni, and Richardson. Drafting of the manuscript: Seltzer, Goldman, and Richardson. Critical revision of the manuscript for important intellectual content: Seltzer, Zolnouni, Nunez, Goldman, Kumar, Ieni, and Richardson. Statistical analysis: Kumar. Obtained funding: Goldman and Ieni. Administrative, technical, and material support: Seltzer and Goldman. Study supervision: Seltzer, Ieni, and Richardson. Funding/Support: This study was supported by Eisai Inc and Pfizer Inc. Acknowledgment: We thank Yvonne Noble, RN, clinical study manager, Eisai Inc, for her commitment and hard work; and Teresa Griesing, PhD, Pfizer Inc, for her role in the early phases of this study. REFERENCES 1. Rogers SL, Doody RS, Mohs RC, Friedhoff LT; Donepezil Study Group. Donepezil improves cognition and global function in Alzheimer disease: a 15-week, double-blind, placebo-controlled study. Arch Intern Med. 1998;158: Rogers SL, Farlow MR, Doody RS, Mohs R, Friedhoff LT; Donepezil Study Group. A 24-week, double-blind, placebo-controlled trial of donepezil in patients with Alzheimer s disease. Neurology. 1998;50: Knopman D, Donohue JA, Gutterman EM. Patterns of care in the early stages of Alzheimer s disease: impediments to timely diagnosis. J Am Geriatr Soc. 2000; 48: Larrabee GJ, Crook TI. A computerized battery for assessment of everyday memory. In: West R, Sinnott J, eds. Every Day Memory and Aging. New York: Springer- Verlag NY Inc; 1992: American Psychiatric Association. Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision. Washington, DC: American Psychiatric Press; McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical diagnosis of Alzheimer s disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer s Disease. Neurology. 1984;34: Rosen WG, Terry RD, Fuld PA, Katzman R, Peck A. Pathological verification of ischemic score in differentiation of dementias. Ann Neurol. 1980;7: Hughes CP, Berg L, Danziger WL, Coben LA, Martin RL. A new clinical scale for the staging of dementia. Br J Psychiatry. 1982;140: Folstein MF, Folstein SE, McHugh PR. Mini-Mental State : a practical method for grading the cognitive state of patients for the clinician. J Psychiatr Res. 1975; 12: Mohs RC, Knopman D, Petersen RC, et al. Development of cognitive instruments for use in clinical trials of antidementia drugs: additions to the Alzheimer s Disease Assessment Scale that broaden its scope: the Alzheimer s Disease Cooperative Study. Alzheimer Dis Assoc Disord. 1997;11(suppl 2):S13-S Crook TH, Youngjohn J, Larrabee G, Salama M. Aging and everyday memory: a cross cultural study. Neuropsychology. 1992;6: Starkstein SE, Mayberg HS, Preziosi TJ, Andrezejewski P, Leiguarda R, Robinson RG. Reliability, validity, and clinical correlates of apathy in Parkinson s disease. J Neuropsychiatry Clin Neurosci. 1992;4: Morris JC, Storandt M, Miller JP, et al. Mild cognitive impairment represents earlystage Alzheimer s disease. Arch Neurol. 2001;58: Galasko D. An integrated approach to the management of Alzheimer s disease: assessing cognition, function and behaviour [abstract]. Eur J Neurol. 1998; 5(suppl 4):S9-S Burns A, Rossor M, Hecker J, et al. The effects of donepezil in Alzheimer s disease: results from a multinational trial. Dement Geriatr Cogn Disord. 1999; 10: Almkvist O, Basun H, Backman L, et al. Mild cognitive impairment: an early stage of Alzheimer s disease? J Neural Transm Suppl. 1998;54: Gilley DW, Wilson RS, Bennett DA, Bernard BA, Fox JH. Predictors of behavioral disturbance in Alzheimer s disease. J Gerontol. 1991;46:P362-P Doody RS, Geldmacher DS, Gordon B, Perdomo CA, Pratt RD; Donepezil Study Group. Open-label, multicenter, phase 3 extension study of the safety and efficacy of donepezil in patients with Alzheimer s disease. Arch Neurol. 2001;58: Mohs RC, Doody RS, Morris JC, et al. A 1-year, placebo-controlled preservation of function survival study of donepezil in AD patients [published erratum appears in Neurology. 2001;57:1942]. Neurology. 2001;57: Lopez OL, Becker JT, Wisniewski S, Saxton J, Kaufer DI, DeKosky ST. Cholinesterase inhibitor treatment alters the natural history of Alzheimer s disease. J Neurol Neurosurg Psychiatry. 2002;72: Geldmacher DS, Provenzano G, McRae T, Mastey V, Ieni JR. Donepezil is associated with delayed nursing home placement in patients with Alzheimer s disease. J Am Geriatr Soc. 2003;51: Krishnan KR, Charles HC, Doraiswamy PM, et al. Randomized, placebocontrolled trial of the effects of donepezil on neuronal markers and hippocampal volumes in Alzheimer s disease. Am J Psychiatry. 2003;160: Wang XD, Chen XQ, Yang HH, Hu GY. Comparison of the effects of cholinesterase inhibitors on [ 3 H]MK-801 binding in rat cerebral cortex. Neurosci Lett. 1999; 272: Takada Y, Yonezawa A, Kume T, et al. Nicotinic acetylcholine receptor-mediated neuroprotection by donepezil against glutamate neurotoxicity in rat cortical neurons. J Pharmacol Exp Ther. 2003;306: Akasofu S, Kosasa T, Kimura M, Kubota A. Protective effect of donepezil in a primary culture of rat cortical neurons exposed to oxygen-glucose deprivation. Eur J Pharmacol. 2003;472: