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Effect of Phenserine Treatment on Brain Functional Activity and Amyloid in Alzheimer’s Disease Ahmadul Kadir, MD,1 Niels Andreasen, MD, PhD,1,2 Ove Almkvist, PhD,1–3 Anders Wall, PhD,4 Anton Forsberg, MSc,1 Henry Engler, MD,5 Göran Hagman, MSc,2 Marie Lärksäter, BSc,2 Bengt Winblad, MD, PhD,1 Henrik Zetterberg, MD, PhD,6 Kaj Blennow, MD, PhD,6 Bengt Långström, PhD,4,7 and Agneta Nordberg, MD, PhD1,2 Objective: The effects of (⫺)-phenserine (phenserine) and placebo/donepezil treatment on regional cerebral metabolic rate for glucose (rCMRglc) and brain amyloid load were investigated by positron emission tomography in 20 patients with mild Alzheimer’s disease in relation to cerebrospinal fluid (CSF) and plasma biomarkers, and cognitive function. Methods: The first 3 months of the study was a randomized, double-blind, placebo-controlled phase, during which 10 patients received phenserine (30mg/day) and 10 patients the placebo. Three to 6 months was an open-label extension phase, during which the placebo group received donepezil (5mg/day) and the phenserine group remained on phenserine. After 6 months, all patients received phenserine treatment up to 12 months. The patients underwent positron emission tomography examinations to measure rCMRglc (18F-FDG) and amyloid load (11C-PIB) at baseline and after 3 and 6 months of the treatment. Neuropsychological and biomarker data were collected at the three times of positron emission tomography imaging. Results: Statistically significant effects on a composite neuropsychological test score were observed in the phenserine-treated group compared with the placebo and donepezil group at 3 and 6 months, respectively. Values of rCMRglc were significantly increased in several cortical regions after 3 months of phenserine treatment, compared with baseline, and correlated positively with cognitive function and CSF -amyloid 40 (A40). Cortical Pittsburgh Compound B retention correlated negatively with CSF A40 levels and the ratio A/-secretase–cleaved amyloid precursor protein. In CSF, A40 correlated positively with the attention domain of cognition. Interpretation: Phenserine treatment was associated with an improvement in cognition and an increase in rCMRglc. Ann Neurol 2008;63:621– 631 Currently, cholinesterase inhibitors (ChEIs) are clinically used for symptomatic treatment in Alzheimer’s disease (AD). Donepezil, rivastigmine, and galantamine have shown benefits for functional, behavioral, and cognitive measurements.1 The drug studied in this clinical trial, (⫺)-phenserine (phenserine), has a dual mode of action: an acetylcholinesterase inhibitor (AChEI), and an inhibitor of the formation of -amyloid precursor protein (-APP), the source of neurotoxic -amyloid (A) plaque.2 Phenserine interferes with the 5⬘ untranslated region of human APP messenger RNA, decreasing the amount of translation into the actual protein, hence reducing newly synthesized APP. In an experimental study, both (⫺)- and cholinergically inert (⫹)-phenserine have been shown to decrease A levels in cell cultures and mouse brain.3 Other substances, such as monoclonal antibodies and vaccines directed against A, have led to a reduction of plaques in transgenic mice4,5 and nonhuman primates,6 and therefore have been used in the study of patients with AD.7 ChEIs have also been shown to have an effect on APP processing in cell culture, reducing levels of A.8 –10 Although several experimental studies have shown From the 1Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Karolinska University Hospital Huddinge; 2 Department of Geriatric Medicine, Karolinska University Hospital Huddinge; 3Department of Psychology, Stockholm University, Stockholm; 4Uppsala Imanet AB, Imanet, GE Healthcare; 5Department of Nuclear Medicine, Uppsala University Hospital, Uppsala; 6 Institute of Neuroscience and Physiology, Section of Psychiatry and Neurochemistry, Göteborg University, Göteborg; and 7Department of Biochemistry and Organic Chemistry, Uppsala University, Uppsala, Sweden. This article includes supplementary materials available via the Internet at http://www.interscience.wiley.com/jpages/0364-5134/suppmat Received Sep 12, 2007, and in revised form Dec 28. Accepted for publication Jan 2, 2008. Published online Feb 25, 2008, in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ana.21345 Address correspondence to Dr Nordberg, Karolinska Institutet, Department of Neurobiology, Care Sciences and Society, Division of Alzheimer Neurobiology, Karolinska University Hospital Huddinge, Novum Floor-5, S-14186 Stockholm, Sweden. E-mail: [email protected] © 2008 American Neurological Association Published by Wiley-Liss, Inc., through Wiley Subscription Services 621 the efficacy of (⫺)- and (⫹)-phenserine treatment on A metabolism and neurogenesis,3,11,12 there have been no reports regarding effect of (⫺)-phenserine on cortical amyloid plaque in AD patients. Pittsburgh Compound B (11C-PIB) has been used as a promising positron emission tomography (PET) ligand to measure in vivo amyloid plaque.13,14 The primary aim of this explorative study was to use PET to measure the effects of (⫺)-phenserine treatment (30mg daily) and placebo/donepezil (5mg daily) on regional cerebral metabolic rate for glucose (rCMRglc), using 2-(18F)fluoro-2deoxy-D-glucose (18F-FDG), and on amyloid load, using 11C-PIB, in the brains of mild AD patients. The secondary objectives were to measure cerebrospinal fluid (CSF) and plasma biomarkers, to evaluate cognitive function, and to find possible interrelations between PET parameters, CSF and plasma biomarkers, and cognitive function. Subjects and Methods Study Design This was a 12-month study involving 20 patients with mild AD (Supplementary Fig 1 illustrates the study design). The first 3-month period was a double-blind, placebo-controlled, randomized period. The patients were grouped according to the treatment they received: placebo group (n ⫽ 10) and phenserine group (n ⫽ 10). The second 3-month period (3– 6 months) was an open-label extension phase, during which patients in the placebo group received donepezil and patients in the phenserine group remained on phenserine. After 6 months, all patients received phenserine treatment from 6 to 12 months and were clinically followed up with neuropsychological tests. During the dose-escalation periods, in the double-blind phase, the phenserine-treated patients received 5mg twice daily from weeks 1 to 4, 10mg twice daily from weeks 5 to 8, and 15mg twice daily from weeks 9 to 12. In the openlabel phase from week 13, the phenserine-treated patients remained on 15mg twice daily. The placebo-treated patients switched to donepezil at 5mg/day for 3 months; then they had a 1-week washout before being titrated up to a 15mg twice-daily dose of phenserine. Patients Twenty patients with a diagnosis of mild AD (Mini-Mental State Examination [MMSE] score ⱖ 21) were recruited from the Department of Geriatric Medicine, Karolinska University Hospital Huddinge, Stockholm, Sweden. All patients were referred for assessment of dementia because of a memory problem and underwent a thorough clinical investigation including medical history, cognitive screening (MMSE), physical and neurological examination, laboratory blood tests, apolipoprotein E genotyping, neuropsychological assessment, lumbar puncture, and magnetic resonance imaging/computed tomography scans. The diagnosis of AD was made by exclusion of other dementia diseases, in accordance with criteria from the National Institute of Neurological and Communication Disorders and Stroke-Alzheimer’s disease and Related Disorders Association (NINCDS-ADRDA).15 In the study, we used PIB binding data from six agematched (mean ⫾ standard deviation, 67.3 ⫾ 8.8; range, 57–77 years) healthy control subjects to compare with that of patients with mild AD at baseline. Fig 1. Regional cerebral glucose metabolism (rCMRglc, normalized to the pons) in the different cortical brain regions. Data are expressed as mean ⫾ standard error. Posttreatment rCMRglc values were expressed as percentage changes from the baseline (pretreatment) value by using the following formula: %rCMRglc ⫺ 100 ⫽ (rCMRglc (f)/rCMRglc(b) ⫻ 100) ⫺ 100, with f and b indicating the rCMRglc values at follow-up and baseline, respectively. Positive rCMRglc values indicate increased rCMRglc. Solid bars indicate the phenserine group at 3 and 6 months of treatment, respectively. Hatched bars indicate the placebo (A) and donepezil (B) groups. *p ⬍ 0.05 indicates significantly increased in rCMRglc within the group compared with baseline; #p ⬍ 0.05 indicates a significant difference between the groups at 3 months. ctx ⫽ cortex; lt ⫽ left; rt ⫽ right. 622 Annals of Neurology Vol 63 No 5 May 2008 All patients and their responsible caregivers provided written informed consent to participate in the study; it was conducted according to the Declaration of Helsinki and subsequent revisions, and was approved by the Ethics Committee of Karolinska University Hospital Huddinge and the Faculty of Medicine and Radiation Hazard Ethics Committee of Uppsala University Hospital, Uppsala, Sweden. Assessment of Safety Safety was assessed throughout the study. The safety variables were adverse events (AEs), vital signs, physical examination, laboratory safety screen, and electrocardiogram. Positron Emission Tomography Methods PET studies were performed at the PET Center, Uppsala Imanet AB, Imanet GE Healthcare, Uppsala, Sweden, at baseline, and after 3 and 6 months of the study. Radiotracers Production of FDG and PIB was conducted according to standard good manufacturing processes at Uppsala Imanet. Synthesis of PIB was performed by means of a method described previously.13,16 Positron Emission Tomography Scanning The PET scans were performed using Siemens ECAT EXACT HR⫹ scanners (CTI PET-Systems), with an axial field of view of 155mm, providing 63 contiguous 2.46mm slices with 5.6mm transaxial and 5.4mm axial resolution. The patients were scanned after fasting for 4 hours under resting conditions in a dimmed room. The orbitomeatal line was used to center the heads of the patients. The data were acquired in three-dimensional mode. The scanner protocol for transmissions and reconstructions has been described previously.13 The emission scans consisted of 7 or 24 time frames with a progressive increase in frame duration (1 ⫻ 60, 1 ⫻ 140, and 5 ⫻ 300 seconds) and a total duration of 45 minutes for FDG; and 4 ⫻ 30, 9 ⫻ 60, 3 ⫻ 180, and 8 ⫻ 300 seconds with a total duration of 60 minutes for PIB. The average tracer doses administered were 328 ⫾ 34.6 and 210 ⫾ 64.8Mbq for PIB and FDG, respectively (mean ⫾ standard deviation). Regions of Interest A set of standardized regions of interest (ROIs) were defined using the Scanditronix program (IDA, Images Display and Analyses GE 1994). All ROIs were paired for the right and left hemispheres, except for the pons and whole brain. The ROIs placement procedure has been described in detail previously.13,17 In this article, we focused the data analysis on those ROIs that are considered to be most critical in AD, and for presentation of PET data, these regions were grouped into four major areas: (1) frontal cortex (including the ROIs of frontal, anterior cingulate, and frontal association cortices), (2) parietal cortex, (3) parietotemporal cortex, and (iv) temporal cortex (including the ROIs of posterior/anterior lateral, inferior, and medial temporal cortices). The mean cortical was calculated as a composite of the above four regions. Data Management For FDG, venous arterialized blood samples were obtained and parametric maps were generated by means of the Patlak method, using the time course of the tracer in the arterialized venous plasma as an input function.18 The rCMRGlc values were normalized to the pons value (ROI/ref) to allow interindividual and intraindividual comparisons.19 PIB retention was measured by the Patlak reference method,20 assuming irreversible binding of the tracer to the brain tissue. The binding of PIB to the target tissue can be considered as irreversible during the measurement period of 60 minutes. Specific uptake of PIB in the cerebellum is, however, not negligible. The net accumulation rate in the reference region was accounted for by means of a correction based on previous measurements of the accumulation rate constant of PIB in this region.21 The cerebellar cortex was chosen as reference because of its previously reported lack of Congo red– and thioflavin-S–positive plaques.22,23 Among several different PIB evaluation methods, the late scan reference method has shown a large effect size.24 In this study, percentage changes in the CMRglc and PIB retention values were used, defined by the following formula: %PET data ⫺ 100 ⫽ (PET data(f)/PET data(b) ⫻ 100) ⫺ 100, with f and b indicating CMRglc and PIB retention values at the times of follow-up and at baseline, respectively. Cerebrospinal Fluid and Plasma Biomarkers CSF and plasma samples were collected by lumbar puncture and venipuncture, respectively, at baseline and after 3 and 6 months of treatment. All samplings were performed in the morning or 2 to 4 hours after intake of morning medication. A volume of 12ml CSF was collected in polypropylene tubes and gently mixed to avoid gradient effects. CSF samples with more than 500 erythrocytes/l were excluded. The blood samples were collected in tubes containing EDTA as anticoagulant. All samples were centrifuged, aliquoted, and stored in polypropylene tubes at ⫺80°C pending biochemical analyses, without being thawed and refrozen. Levels of -amyloid 1-42 (A42), total-tau (T-tau), and phosphorylated tau (P-tau181) in CSF were analyzed using the INNO-BIA AlzBio3 assay (Innogenetics, Ghent, Belgium), as described previously in detail.25 Levels of CSF ␣- and -secretase–cleaved amyloid precursor protein (␣-sAPP and -sAPP, respectively) were measured using Meso Scale Discovery electrochemiluminescence detection technology (sAPP␣/sAPP multiplex assay; Meso Scale Discovery, Gaithersburg, MD) and a SECTOR Imager 2400 instrument, following the instructions of the manufacturer. The -sAPP assay is based on a -sAPP–specific capture antibody, recombinant -sAPP as standard, and an N-terminal APP-specific SULFO-TAG–labeled antibody as detector. The ␣-sAPP assay is based on 6E10 monoclonal antibody (MAb) as capture antibody, recombinant ␣-sAPP as standard, and an N-terminal, APP-specific, SULFO-TAG– labeled antibody as detector. Levels of CSF A40 were measured by using Meso Scale Discovery human A40 ultrasensitive assays (Meso Scale Discovery) and a SECTOR Imager 2400 instrument, following the instructions of the manufacturer. This assay is based on 6E10 MAb as capture antibody and an A40-specific SULFO-TAG–labeled antibody as detector. Kadir et al: PET Study of Phenserine in AD 623 Plasma A40 levels were determined by using highsensitivity enzyme-linked immunosorbent assay kits (TK40HS; The Genetics Company, Schlieren, Switzerland). In this enzyme-linked immunosorbent assay, MAb W02 (A epitopes 5– 8) is the capture antibody and MAb G2-10 is the detection antibody. The latter has negligible cross-reactivity with A species other than A40.26,27 Plasma A42 concentrations were determined by using the high-sensitivity INNOTEST -AMYLOID(1-42) assay (Innogenetics, Ghent, Belgium). The assay and its characteristics have been described previously in detail.28 Cognitive Function Global cognition was estimated by means of MMSE29 at baseline and at 3, 6, 9, and 12 months of the study. Neuropsychological tests of episodic memory (Word Recall [2and 5-second presentation rate], Word Recognition-d [2and 5-second presentation rate]), attention (Digit Symbol, Trail Making Test-A [time]), and visuospatial ability (Clock Recognition) were performed at baseline and during treatment at months 2, 3, 6, 9, and 12. Further details of the neuropsychological procedure have been described previously.30 To make comparisons between various neuropsychological test results, we Z-transformed all cognitive raw scores (including MMSE score) by using reference data from the Geriatric Clinic, Karolinska University Hospital Huddinge, Stockholm, Sweden.31 Statistical Analysis Data are expressed as mean values and standard error of the mean. The equivalence of groups in demographic variables was checked by means of one-way analysis of variance. Between-group comparisons at specific time points were conducted by means of analysis of covariance. In this model, percentage changes from baseline (for the CMRglc and PIB retention) or absolute value changes from baseline (for cognitive function, as well as CSF and plasma biomarkers) were used as dependent variables, with baseline value as a covariate. For within-group comparisons, Wilcoxon signed rank test was used to compare baseline values versus those at other time points. This nonparametric method was chosen for within-group analysis because of the small sample size (n ⫽ 10 per group) and because we could not assume that all the parameters were normally distributed. Two-tailed Pearson’s correlation coefficients and Spearman’s rank correlations where appropriate were used in correlation analysis, which was then visualized graphically using simple regression plots. All statistical tests were two-tailed at the 0.05 level of significance, and no corrections for multiple comparisons were made because this was an explorative type of study. Results Demographic Data The demographic characteristics of the patients are presented in the Table. The demographic data did not show any significant differences between the groups (all p ⬎ 0.10 except for MMSE score, p ⫽ 0.08, nominally favoring the group initially receiving phenserine). 624 Annals of Neurology Vol 63 No 5 May 2008 Adverse Events Two episodes of nausea, one episode of vomiting, and gastritis commenced during treatment with 15mg phenserine twice daily in three patients, but for two patients, these symptoms gradually resolved when the dose was reduced to 20mg/day. One episode of depression, nasopharyngitis, and headache occurred during treatment with placebo in one patient; one episode of pruritus commenced during treatment with 5mg/day donepezil in one patient. None of these adverse events was severe, and all patients completed the 12-month study. No serious adverse events occurred because of the treatment, and no clinically relevant changes in laboratory values, vital signs, or electrocardiographic readings were observed during the study. Changes in Regional Cerebral Metabolic Rate for Glucose At 3 months, a generalized increase (7 ⫾ 3%; range, 4 –10%) in cortical rCMRglc was observed in the phenserine group compared with baseline. The increase in rCMRglc was significant in the left frontal, right parietal, and the right/left parietotemporal cortices (all p ⬍ 0.05) (Fig 1A), whereas no significant effects were observed in brain regions that are less affected in AD, that is, primary visual, sensory motor, thalamus, putamen, and cerebellum (data not shown). The left parietotemporal cortex showed a significant difference between the phenserine- and placebo-treated groups (F ⫽ 6.28; degrees of freedom [df] ⫽ 1; p ⫽ 0.02). No significant effects were observed in the placebo group compared with baseline (see Fig 1A). The rCMRglc in the cortical regions did not differ from baseline after 6 months of treatment with phenserine or 3 months of treatment with donepezil (see Fig 1B). Changes in Cortical 11C-PIB Retention Because we did not observe any difference in PIB retention between different cortical regions, we aggregated the four studied regions (frontal, parietal, parietotemporal, and temporal) and presented the data as mean cortical PIB retention. At baseline, both groups showed significantly high PIB retention compared with the healthy control group (see Supplementary Fig 2A). After 3 and 6 months of treatment, no significant changes in PIB retention were observed in either group compared with baseline or between the groups at these time points. At 3 months, all placebo-treated patients except one showed results within an estimated test-retest value of ⫾5%.32 In the phenserine group, four patients showed reduced PIB retention compared with baseline (see Supplementary Fig 2B). Supplementary Figure 3 illustrates the parametric image of cortical PIB retention in an AD patient after 3 months of phenserine treatment and showed a 12% reduction in cortical PIB binding compared with baseline. Three patients after 6 months of phenserine treatment and two patients after 3 months of donepezil treatment showed reduced PIB retention compared with the baseline below the test-retest value (see Supplementary Fig 2C). Changes in Global Cognition The phenserine-treated group showed improvement in MMSE Z-score, which reached statistical significance at 9 months ( p ⬍ 0.02; Fig 2A) compared with baseline. After 12 months (Z-score ⫽ 0.4 ⫾ 0.6), no difference was observed in the phenserine-treated group compared with baseline. No significant change in MMSE score was observed in the placebo/donepezil/ phenserine-treated group compared with baseline, the MMSE score at 12 months being comparable with that in the phenserine group (see Fig 2A). Fig 2. Mean (⫾ standard error [SE]) changes in Z-scores from baseline in Mini-Mental State Examination (MMSE) score (A) and in composite of seven neuropsychological tests (B) over time. *p ⬍ 0.02 indicates a significant improvement in the MMSE Z-score in the phenserine-treated group at 9 months compared with baseline (A). #p ⬍ 0.05 indicates a significant difference in the composite neuropsychological Z-score between the groups at the specified treatment intervals (B). Solid squares represent phenserine group; open circles represent 0 to 3 months ⫽ placebo group, 3 to 6 months ⫽ donepezil treatment, and 6 to 12 months ⫽ phenserine treatment. Changes in Neuropsychological Performance Because neuropsychological performance was included as a secondary objective, and because of the limitation of sample size, the outcome of the seven neuropsychological subtests was calculated as mean composite Z-score and presented as changes from baseline (see Fig 2B). At 3months, analysis of covariance of the composite Z-scores yielded significant effect (F ⫽ 5.72; df ⫽ 1; p ⫽ 0.03) between the placebo- and the phenserinetreated group. The significant group effect was found because of improvement in the phenserine-treated group (Z-score ⫽ 0.25 ⫾ 0.13) and decline (Z-score ⫽ ⫺0.16 ⫾ 0.13) in the placebo-treated group after 3 months of treatment compared with baseline. Furthermore, a significant group effect (F ⫽ 7.05; df ⫽ 1; p ⫽ 0.02) was observed between 6 months of treatment with phenserine (improvement, Z-score ⫽ 0.33 ⫾ 0.16) and 3 months of treatment with donepezil (decline, Z-score ⫽ ⫺0.24 ⫾ 0.11) compared with baseline. At 12 months, phenserine-treated patients showed stabilization (Z-score ⫽ ⫺0.09 ⫾ 0.4) compared with baseline (see Fig 2B). After 6 months, when the donepezil-treated group switched to phenserine treatment for another 6 months, they showed a nonsignificant decline (Z-score ⫽ ⫺0.33 ⫾ 0.17) compared with baseline (see Fig 2B). Correlation between Positron Emission Tomography Measures (Regional Cerebral Metabolic Rate for Glucose and Pittsburgh Compound B) and Cognitive Function At baseline, rCMRglc values positively correlated with the composite Z-score of the seven neuropsychological Kadir et al: PET Study of Phenserine in AD 625 right parietal cortex (r ⫽ 0.51; p ⬍ 0.02; see Fig 3B). In addition, at this follow-up point, the changes in rCMRglc correlated positively with changes in plasma A40 levels in the following brain regions: right frontal tests in the following brain regions: the right parietal (r ⫽ 0.50; p ⬍ 0.03), the left parietal (r ⫽ 0.51; p ⬍ 0.03), the right parietotemporal (r ⫽ 0.44; p ⬍ 0.05), and the left parietotemporal cortices (r ⫽ 0.48; p ⬍ 0.04). At 3 months, the changes in rCMRglc in all four cortical regions correlated positively with the composite Z-score. Significant correlations were observed in the right parietotemporal (r ⫽ 0.44; p ⬍ 0.05) and left parietotemporal cortices (r ⫽ 0.45; p ⬍ 0.05). At 6 months, similar positive correlations were observed between the changes in rCMRglc and composite Z-score. Furthermore, significant positive correlations were observed in the left frontal (r ⫽ 0.45; p ⬍ 0.05; see Supplementary Fig 4) and right temporal cortices (r ⫽ 0.44; p ⬍ 0.05). These findings demonstrate that AD patients with a high rCMRglc also showed high cognitive function at the explicit time point. No significant correlations between cortical PIB binding and rCMRglc or cognitive function were observed throughout the study. Changes in Cerebrospinal Fluid and Plasma Biomarkers No statistically significant differences were observed in any of the parameters between the placebo/donepeziland phenserine-treated groups or in the within-group changes from baseline during the study period (see Supplementary Table 1). However, there was a tendency toward increased values of CSF A40 and plasma A40 after 3 months of treatment with phenserine compared with baseline. Correlation between Regional Cerebral Metabolic Rate for Glucose and Cerebrospinal Fluid and Plasma Biomarkers At baseline, rCMRglc values in the right parietal cortex correlated positively with CSF A40 levels (r ⫽ 0.46; p ⬍ 0.04; Fig 3A). At 3 months, changes in rCMRglc in all four cortical regions correlated positively with the changes in CSF A40 concentrations, and a significant positive correlation was observed with changes in the Fig 3. Positive correlations between regional cerebral glucose metabolism (rCMRglc) in the right parietal cortex and cerebrospinal fluid (CSF) -amyloid 40 (A40) concentrations (pg/ml) at baseline (A); changes in rCMRglc in the right parietal cortex and changes in CSF A40 levels at 3 months (B); changes in rCMRglc in the right temporal cortex and changes in CSF A40 levels at 6 months (C). At 3 and 6 months, all values on both axes are changes from individual baseline values. Solid squares at 3 and 6 months represent phenserine group; open circles at 3 months represent placebo group; open circles at 6 months represent donepezil group. (A) r ⫽ 0.46; p ⬍ 0.04. (B) r ⫽ 0.51; p ⬍ 0.02. (C) r ⫽ 0.44; p ⬍ 0.05. 626 Annals of Neurology Vol 63 No 5 May 2008 ‹ (r ⫽ 0.65; p ⬍ 0.003), left frontal (r ⫽ 0.64; p ⬍ 0.003), right parietal (r ⫽ 0.64; p ⬍ 0.003), left parietal (r ⫽ 0.64; p ⬍ 0.003), right parietotemporal (r ⫽ 0.65; p ⬍ 0.002), and left temporal cortices (r ⫽ 0.66; p ⬍ 0.002). At 6 months, positive correlations were observed between changes in cortical rCMRglc and changes in CSF A40 levels, and a significant positive correlation was observed with changes in the right temporal cortex (r ⫽ 0.44; p ⬍ 0.05; see Fig 3C). No significant correlations between changes in cortical rCMRglc versus CSF or plasma A42 concentrations were observed throughout the study. Correlation between Cerebrospinal Fluid and Plasma Biomarkers, and Cognitive Function At baseline, CSF A40 levels correlated positively with the patients’ performance in the domain of attention (composite of Digit Symbol and Trail Making Test-A) Z-score (r ⫽ 0.59; p ⬍ 0.01; Fig 4A). At 3 months, changes in plasma A40 levels correlated positively with changes in the attention Z-score (r ⫽ 0.46; p ⬍ 0.05; see Fig 4B). At 6 months, changes in CSF A40 levels correlated positively with changes in the attention Z-score (r ⫽ 0.58; p ⬍ 0.01; see Fig 4C). In addition, at this time point, changes in plasma A40 levels, only in the phenserine group, correlated positively with attention Z-score (r ⫽ 0.83; p ⬍ 0.004). These findings suggest that an AD patient with a high A40 level in CSF or plasma also had a high attention function. No significant correlation was observed among the changes in CSF A42, plasma A42, and the attention Z-score. Correlation between Pittsburgh Compound B Retention and Cerebrospinal Fluid and Plasma Biomarkers A pattern of negative correlation, although not significant, was observed between mean cortical PIB retention and CSF A40 level at baseline (r ⫽ ⫺0.21; p ⬎ 0.10), and between changes at 3 months (r ⫽ ⫺0.11; p ⬎ 0.10; Fig 5A). However, a significant correlation was found at 6 months (r ⫽ ⫺0.48; p ⬍ 0.03; see Fig 5B). This analysis indicates that patients with lower PIB retention had increased CSF A40 levels. No significant correlations were observed between cortical Š Fig 4. Positive correlations between composite attention Z-score (Digit Symbol and Trail Making Test-A) and cerebrospinal fluid (CSF) -amyloid 40 (A40) concentrations (pg/ ml) at baseline (A); changes in composite attention Z-score and changes in plasma A40 levels at 3 months (B); changes in composite attention Z-score and changes in CSF A40 levels at 6 months (C). At 3 and 6 months, all values are absolute value changes from individual baseline values. Solid squares at 3 and 6 months ⫽ phenserine group; open circles at 3 months ⫽ placebo group; open circles at 6 months ⫽ donepezil group. (A) r ⫽ 0.59; p ⬍ 0.01. (B) r ⫽ 0.46; p ⬍ 0.05. (C) r ⫽ 0.58; p ⬍ 0.01. Kadir et al: PET Study of Phenserine in AD 627 Table. Demography Data Characteristics Total Subjects Placebo/Donepezil Phenserine 20 10 10 5/15 3/7 2/8 Age (yr) 68 ⫾ 2 70 ⫾ 3 66 ⫾ 3 Education (yr) 12 ⫾ 1 12 ⫾ 1 12 ⫾ 1 Duration of disease (yr) 4⫾1 4⫾1 4⫾1 First-degree relative with AD (Y/N) 6/14 2/8 4/6 Total subjects (n) Sex (M/F) Apolipoprotein E ε4 carriers (⫹⫹/⫺⫹/⫺⫺) 6/12/1 3/7/0 3/5/1 MMSE score at baseline 24 ⫾ 1 23 ⫾ 1 25 ⫾ 1 Data are presented as mean ⫾ standard error. AD ⫽ Alzheimer’s disease; MMSE ⫽ Mini-Mental State Examination. Fig 5. Negative correlations between changes in mean cortical Pittsburgh Compound B (PIB) retention and changes in cerebrospinal fluid (CSF) -amyloid 40 (A40) concentrations at 3 (A) and 6 months (B). Negative correlations between changes in mean cortical PIB retention and changes in the CSF total -amyloid (A40 ⫹ A42) and -secretase-cleaved amyloid precursor protein (sAPP) ratio at 3 (C) and 6 months (D). All values are changes from individual baseline values. Solid squares ⫽ phenserine group; open circles at 3 months ⫽ placebo group; open circles at 6 months ⫽ donepezil group. (A) r ⫽ ⫺0.11; p ⬎ 0.10. (B) r ⫽ ⫺0.48; p ⬍ 0.03. (C) r ⫽ ⫺0.45; p ⬍ 0.05. (D) r ⫽ ⫺0.42; p ⬍ 0.06. 628 Annals of Neurology Vol 63 No 5 May 2008 PIB retention and levels of CSF A42 or plasma A40 and A42 throughout the study. Finally, the possible relation between mean cortical PIB retention and the CSF A (A40 ⫹ A42)/sAPP ratio was investigated. At baseline, a pattern of negative correlation was observed between mean cortical PIB retention and the CSF A/-sAPP ratio (r ⫽ ⫺0.24; p ⬎ 0.10). Later, changes in mean cortical PIB retention correlated negatively with changes in the CSF A/-sAPP ratio at 3 months (r ⫽ ⫺0.45; p ⬍ 0.05; see Fig 5C) and at 6 months (r ⫽ ⫺0.42; p ⬍ 0.06; see Fig 5D). The analysis indicates that patients with lower PIB retention had an increased CSF A/-sAPP ratio. Discussion To date, this is the first study in which the new PET amyloid tracer PIB has been used for evaluation of antiamyloid drug treatment in mild AD patients. Moreover, multitracer PET assessment was applied to investigate the relations between biological factors such as rCMRglc and amyloid load, with CSF and plasma biomarkers, and cognitive function of AD patients at regular follow-up intervals. We observed an increase in rCMRglc in cortical regions after 3 months and maintenance after 6 months of treatment with phenserine compared with baseline. This finding is due to the ChEI effect of phenserine treatment. Because the results of 3 months of placebo treatment showed a decrease in rCMRglc in cortical regions, this indicates a possible decrease in synaptic activity during the course of AD, although 3 months is a relatively short time to detect disease deterioration in all patients. No significant effect was observed on rCMRglc when the placebo group switched to donepezil treatment. The lack of effect on rCMRglc might be a result of the low dose of donepezil (5mg) and/or variation in this small number of patients per group. Several ChEIs, such as tacrine,33,34 donepezil,35 rivastigmine,36,37 and galantamine,38 have shown shortterm enhancement or long-term stabilization effects on rCMRglc; they have also shown increases in cholinergic neurotransmitter activity30,33,39 in AD patients. Thus, the increase in rCMRglc observed after short-term treatment with phenserine probably reflected a similar effect as observed with other ChEIs, that is, an increase in cholinergic neuronal signaling and activities, which is highly energy dependent. The neurotoxic peptide A is a major component of the extraneuronal plaques that characterize AD pathologically. Reduction of A levels in the brain is a major focus in the design and development of effective therapies for AD.40 In this study, no significant changes in mean cortical PIB retention were observed after phenserine or donepezil treatment, but at the individual level, changes in PIB retention correlated negatively with the CSF A/-sAPP ratio. However, both in vitro2,11 and in vivo animal41 studies have shown the positive effect of phenserine on reduction of -APP and A. A plausible explanation for the lack of a significant decrease in PIB retention in vivo in AD patients is likely to be related to the dual mode of action of phenserine (ie, AChE inhibition and APP reduction); AChE inhibitors are by nature dose limiting, which prevent achievement of the necessary concentration for optimally inhibiting A production in humans. The results of experimental studies show that phenserine reduces the production and secretion of newly synthesized, rather than already produced, -APP and A in a concentration- and timedependent manner with an inhibitory concentration of 50% (IC50) that is greater than its action on AChE.11 In this study, we found that composite neuropsychological test results correlated positively with rCMRglc, but not with PIB retention. 18F-FDG PET reflects the functional integrity of synapses, rather than the presence or quantity of plaques, which best correlates with cognitive function in patients with AD.42 It has also been reported that, in neuropathological studies, there is a stronger correlation between degree of dementia and neurofibrillary tangle density than amyloid load.43,44 We did not find any significant treatment effect on CSF and plasma biomarkers. However, we found a tendency toward increased CSF and plasma A40 after 3 months of treatment with phenserine compared with baseline. We also observed positive correlations between CSF and plasma A40, and rCMRglc and cognitive subtest attention, demonstrating that the patients with the greatest A40 levels in CSF and plasma also showed the greatest increase in rCMRglc and cognition. To our knowledge, no data are available concerning the direct interrelations among CSF and plasma A, rCMRglc, and cognitive function. Our results suggest that, in AD patients, a high level of A40 in the CSF or plasma is a better diagnostic marker. We also showed that a high level of CSF A40 correlated with low PIB retention in the brain. It should be noted that A42, tau and p-tau did not correlate with imaging and clinical measures. Whether high level of A40 in the CSF or plasma is a feature of improvement in patients with AD will require further testing with larger patient groups. However, the role of A40 in AD pathogenesis is not as well established, but recent experimental45,46 and clinical47 studies have provided evidence for a protective role of A40 in AD. In this study, the phenserine-treated AD patients showed improvement in the composite neuropsychological performance compared with the placebo-treated patients at 3 months. Within the group, the phenserine-treated AD patients showed an improvement for up to 6 months compared with baseline. Af- Kadir et al: PET Study of Phenserine in AD 629 ter 12 months, the performance was comparable with the baseline level. These findings are in agreement with our earlier studies on AD patients treated with the ChEIs galantamine30 and rivastigmine48; however, untreated AD patients showed significant deterioration in performance in similar neuropsychological tests after 12 months.48 On the other hand, once the placebo group switched to active treatment after the end of 3 months, they showed nonsignificant changes (because of the lack of power) compared with baseline data. The global cognition was assessed by MMSE score, and the power of this study was inadequate to draw clinical conclusions on data addressing changes in global cognition. An important limitation of this study is that relatively large numbers of statistical analyses were performed on PET parameters in relation to the CSF and plasma biomarkers, and cognitive function, which were not corrected for multiple comparisons, and this may render some of the results liable to be obtained by chance. Therefore, result patterns were taken into consideration rather than isolated findings. Conversely, this study was designed primarily as a PET investigation, and thus included a small group of mild AD patients. These features make it necessary to be cautious when interpreting the data because small sample size may result in a lack of statistical power, although this might make the significant results even more interesting. In summary, we showed that phenserine (30mg/day) results in a generalized increase in rCMRglc in cortical regions after 3 months of treatment. The changes in cortical rCMRglc were associated with cognitive function. Mean cortical PIB retention correlated negatively with the total CSF A/-sAPP ratio. CSF and plasma A40 concentrations were positively associated with rCMRglc and the attention domain of cognition; conversely, CSF A40 levels were inversely correlated with PIB retention in AD patients. 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