IN VITRO DETECTION OF (S)-NAPROXEN AND IBUPROFEN BINDING TO PLAQUES IN THE ALZHEIMER’S BRAIN USING THE POSITRON EMISSION TOMOGRAPHY MOLECULAR IMAGING PROBE 2-(1-{6-[(2- [ 18 F]FLUOROETHYL)(METHYL)AMINO]-2-NAPHTHYL}ETHYLIDENE) MALONONITRILE E. D. AGDEPPA, a V. KEPE, a A. PETRIC ˆ , b N. SATYAMURTHY, a J. LIU, a S.-C. HUANG, a G. W. SMALL, c G. M. COLE d,e AND J. R. BARRIO a * a Division of Nuclear Medicine, Department of Molecular and Medical Pharmacology, Laboratory of Structural Biology and Molecular Medi- cine, The David Geffen School of Medicine at UCLA, Box 956948, Los Angeles, CA 90095, USA b Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia c Department of Psychiatry and Biobehavioral Sciences, Neuropsychi- atric Institute, The David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA d The Greater Los Angeles Veterans Affairs Healthcare System, Sepulveda Geriatric Research, Education, and Clinic Center, Sepulveda, CA 91343, USA e Departments of Medicine and Neurology, The David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA Abstract—Epidemiological studies have suggested that the chronic use of non-steroidal anti-inflammatory drugs (NSAIDs) reduces the relative risk of Alzheimer’s disease (AD). The possible neuroprotection by NSAIDs in AD is gen- erally attributed to anti-inflammatory activity. An additional mode of drug action may involve anti-aggregation of -amy- loid (A) peptides by commonly used NSAIDs. We utilized in vitro competition assays, autoradiography, and fluorescence microscopy with AD brain specimens to demonstrate con- centration-dependent decreases in the binding of the in vivo molecular imaging probe, 2-(1-{6-[(2-[ 18 F]fluoroethyl)(methyl) amino]-2-naphthyl}ethylidene)malononitrile ([ 18 F]FDDNP), against (S)-naproxen and (R)- and (S)-ibuprofen (but not di- clofenac) to A fibrils and ex vivo A senile plaques. Con- versely, in vitro amyloid dyes Congo Red and Thioflavine T were demonstrated in the same experiments not to bind to the FDDNP binding site. FDDNP and the NSAIDs that share the same binding site also exhibit anti-aggregation effects on A peptides, suggesting that the shared binding site on A fibrils and plaques may be a site of anti-aggregation drug action. Our results indicate for the first time the binding of select NSAIDs to plaques, specifically to the binding site of the molecular imaging probe [ 18 F]FDDNP. Our understanding of the molecular requirements of FDDNP binding may help in the optimization of the A anti-aggregation potency of exper- imental drugs. [ 18 F]FDDNP has been used to image plaques in vivo with positron emission tomography (PET), and inves- tigations into the influence of A anti-aggregation on the risk-reduction effects of NSAIDs on AD could utilize [ 18 F]FDDNP and PET in determining the occupancy rate of NSAIDs and experimental drugs in plaques in the living brain of AD patients. © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: anti-aggregation, -amyloid fibrils, competitive binding, digital autoradiography, fluorescence microscopy, non-steroidal anti-inflammatory drugs. Epidemiological studies have implicated the chronic use of non-steroidal anti-inflammatory drugs (NSAIDs) in reduc- ing the relative risk (Breitner et al., 1995; Stewart et al., 1997; in t’ Veld et al., 2001) or delaying the onset of Alzheimer’s disease (AD) (Rogers et al., 1993; Breitner et al., 1995). It is believed that chronic inflammatory response in AD contributes to neurodegeneration (McGeer and Mc- Geer, 1995; Halliday et al., 2000), but it is not clear if the possible neuroprotective effect of NSAIDs in AD is solely due to the suppression of chronic inflammatory response of microglia and astrocytes and reduced neuronal expres- sion of cyclo-oxygenase enzymes (Halliday et al., 2000), or is due to mitigation of other pathological mechanisms in AD (McGeer and McGeer, 1995). Recently, NSAIDs have been suggested to exert an anti-aggregation effect on -amyloid (A) peptide (Thomas et al., 2001). Aggregates, or fibrils, of A(1– 40/42) peptides are major constituents of one of the hallmark lesions in AD, A senile plaques (SPs). These lesions are considered central in the pathogenesis of AD (Selkoe, 1994; Teplow, 1998), suggesting the ther- apeutic paradigm involving the use of small-molecule drugs to inhibit pathological fibrillogenesis of A peptides (Findeis, 2000). We report herein the previously unrealized in vitro NSAID binding to SPs and A fibrils using 2-(1-{6- [(2-[ 18 F]fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)- malononitrile ([ 18 F]FDDNP; 1, Scheme I). [ 18 F]FDDNP is a molecular imaging probe developed in our laboratories and previously used in the in vivo detection of SPs and neuro- fibrillary tangles in the living brain of AD patients using positron emission tomography (PET) (Shoghi-Jadid et al., 2002). *Corresponding author. Tel: +1-310-825-4167; fax: +1-310-825- 4517. E-mail address: jbarrio@mednet.ucla.edu (J. R. Barrio). Abbreviations: A, -amyloid; AD, Alzheimer’s disease; ANOVA, anal- ysis of variance; CR, Congo Red; [ 18 F]FDDNP, 2-(1-{6-[(2-[F]fluor- oethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrile; NSAIDs, non-steroidal anti-inflammatory drugs; PBS, phosphate-buffered saline; PET, positron emission tomography; SPs, senile plaques; TT, thioflavine T. Neuroscience 117 (2003) 723–730 0306-4522/03$30.00+0.00 © 2003 IBRO. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0306-4522(02)00907-7 723