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NEUROPHARMACOLOGY

Ex Vivo Occupancy of -Secretase Inhibitors Correlates with Brain -Amyloid Peptide Reduction in Tg2576 Mice

Research and Development, Neuroscience Drug Discovery, Bristol-Myers Squibb Co., Wallingford, Connecticut

Received May 14, 2007; accepted July 17, 2007.

	Abstract

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Reduction of brain -amyloid peptide (A) synthesis by -secretase inhibitors is a promising approach for the treatment of Alzheimer's disease. However, measurement of central pharmacodynamic effects in the Alzheimer's disease patient will be a challenge. Determination of drug occupancy may facilitate the analysis of efficacy of -secretase inhibitors in a clinical setting. In this study, the relationship of -secretase site occupancy and brain A40 reduction by -secretase inhibitors was examined in Tg2576 mice. [³H](2R,3S)-2-Isobutyl-N¹-((S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-3-propylsuccinamide (IN973) was used as a -secretase radioligand, since it has been shown to bind to -secretase in rat, rhesus, and human brains with high affinity and specificity. We extended these findings by showing that [³H]IN973 bound to -secretase in Tg2576 brains with an affinity, specificity, and regional localization very similar to the other species. To quantify -secretase occupancy by -secretase inhibitors, an ex vivo binding assay was developed using [³H]IN973 and frozen brain sections from drug-treated mice. -Secretase occupancy and brain A40 reduction were found to be highly correlated in animals dosed with either 2-[(1R)-1-[[4-chlorophenyl)-sulfonyl](2,5-difluorophenyl)amino] ethyl]-5-fluoro-benzenepropanoic acid (BMS-299897) or (S)-2-((S)-2-(3,5-difluorophenyl)-2-hydroxyacetamido)-N-((S,Z)-3-methyl-4-oxo-4,5-dihydro-3H-benzo[d][1,2]diazepin-5-yl)propanamide (BMS-433796) over a wide range of doses and times postdose, with the exception of the earliest times postdose. This lag in A40 response to -secretase inhibition is probably related to the delayed clearance of previously produced A40. The excellent correlation between brain A40 and -secretase occupancy suggests that a positron emission tomography ligand for -secretase could be a valuable biomarker to determine whether -secretase inhibitors bind to their target in humans.

Based on the amyloid hypothesis, -secretase inhibitors are being developed as AD therapies (Barten et al., 2006). When compounds are tested in clinical trials, it is important to determine whether the new therapeutic is binding to its target and causing the desired pharmacological effect. Although plaques can be imaged with agents such as Pittsburgh compound B (Rowe et al., 2007), plaque number is not likely to be a good method to measure changes in A synthesis by -secretase inhibitors. In particular, plaques are stable for months in mice (Christie et al., 2001; Dolev and Michaelson, 2004; Jankowsky et al., 2005), and Pittsburgh compound B labeling seems constant in AD patients (Engler et al., 2006). It is likely that soluble and oligomeric forms of A will be cleared first, reductions of which can serve as an earlier indication of efficacy. The -secretase inhibitor BMS-299897 has, in fact, been shown to reduce CSF A in Tg2576 mice with plaques that were resistant to removal (Barten et al., 2005). Since it will not be possible to directly measure the effect of the inhibitors on brain A, two potential biomarkers have been proposed instead: CSF A and -secretase site occupancy. Although CSF A is only partially derived from brain A, the pharmacodynamic response of CSF A to -secretase inhibition is similar to the response of brain A in preclinical studies (Lanz et al., 2003, 2004; Anderson et al., 2005; Barten et al., 2005; Best et al., 2005, 2006; El Mouedden et al., 2006). Although these results are encouraging, it is possible that CSF A in AD patients with diminished CSF A42 levels (Andreasen and Blennow, 2005) will respond differently to -secretase inhibitors. Furthermore, a less invasive procedure may be desirable.

Another potential biomarker for -secretase inhibitors is site occupancy as measured using PET ligands for -secretase. Radioligands that bind specifically to -secretase have been used to explore the binding and enzymological interaction of different classes of -secretase inhibitors (Esler et al., 2000; Seiffert et al., 2000; Churcher et al., 2003; Kornilova et al., 2003; Tian et al., 2003; Clarke et al., 2006) and to localize -secretase binding sites in brain sections from rats, rhesus monkeys, and humans (Yan et al., 2004; Patel et al., 2006). In this study, we used the -secretase inhibitor radioligand [³H]IN973, previously referred to as compound D (Seiffert et al., 2000; Yan et al., 2004; Patel et al., 2006), to characterize the location of -secretase inhibitor binding sites in Tg2576 mice, a transgenic mouse model that overexpresses the Swedish mutant of APP. We then developed an ex vivo binding assay for -secretase inhibitor site occupancy, and we used this assay to measure the relationship of -secretase occupancy and brain A levels after dosing inhibitors in vivo. These results show a strong correlation between site occupancy and brain A in this model, supporting the possibility of developing a PET ligand as a biomarker for -secretase inhibitor clinical studies.

	Materials and Methods

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Animals and Tissue Preparation. Tg2576 mice used in these studies were developed by Karen Hsiao Ashe (University of Minnesota, Minneapolis, MN) (Hsiao et al., 1996) and licensed from the Mayo Foundation for Medical Education and Research (Rochester, MN). Male Tg2576 transgenic mice were bred to normal C57BL6/SJL females at the Bristol-Myers Squibb Co. facility in Wallingford, CT. Mice were housed with a 6:00 AM to 6:00 PM light/dark cycle, and they were allowed free access to food and water. For all studies, brains were removed after CO₂ euthanasia. For membrane binding studies, brains from either male or female mice between the ages of 6 and 9 months were frozen immediately in liquid nitrogen. For the distribution and saturation binding studies, brains were frozen immediately in methyl butane on dry ice, and then they were stored at –70°C until sectioned for ex vivo autoradiography. For studies involving in vivo dosing, both male and female mice between the ages of 3 and 6 months were used, and although no differences in A were observed between them (Barten et al., 2005), only one sex was used in a single study. BMS-299897 (Smith et al., 2000) and BMS-433796 (Prasad et al., 2007) were synthesized in the Process Research and Development and Discovery Chemistry departments of Bristol-Myers Squibb Co., respectively. Animals were dosed by oral gavage with a volume of 6 ml/kg in polyethylene glycol 400 with 1% Tween. After the designated dosing interval, brains were removed, and they were divided in half in the sagittal plane. The left half was cut in half again, frozen immediately on dry ice, and stored at –70°C for the determination of A40. The right half was frozen as described for ex vivo autoradiography. Mice were handled strictly in accordance with the Bristol-Myers Squibb Co. Animal Care and Use Committee guidelines.

Homogenate Binding. Synthesis and labeling of the radioligand ([³H]IN973) were as described previously (Yan et al., 2004). Initial pharmacological characterization of ligand binding was performed using gross homogenates of the Tg2576 mouse frontal cortex. Tissue, which had been stored at –80°C, was thawed in 10 ml of ice-cold homogenization buffer [50 mM HEPES, 0.1% mammalian protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO), pH 7.0], and it was homogenized using a Polytron (Kinematica, Basel, Switzerland) purchased from Brinkmann Instruments (Westbury, NY). The homogenate was diluted to a final volume of 40 ml, and it was centrifuged at 39,000g for 15 min at 4°C using an RC5C centrifuge (Sorvall, Newton, CT). The supernatant was discarded, and the pellet was resuspended in homogenization buffer using a Polytron followed by aspiration through a syringe and 27-gauge needle, and then it was centrifuged. This homogenization procedure was repeated once more before final resuspension. Protein was determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA). Homogenates were diluted to a concentration of 5 mg/ml protein in homogenization buffer.

The binding assay was performed in duplicate in an assay volume of 250 µl consisting of 25 µl of buffer (50 mM HEPES and 0.1% CHAPSO, pH 7.0) or 10 µM BMS-433796 to define nonspecific binding, 25 µl of buffer containing [³H]IN973, and 200 µlof homogenate (200 µg of protein). Incubation was initiated by the addition of the membrane homogenates at 25°C for 1 h. Binding reactions were terminated by filtration through Whatman GF/B filters (Whatman, Maidstone, UK) using a cell harvester (Brandel Inc., Gaithersburg, MD). Unbound radioactivity was removed by rinsing the filters with ice-cold wash buffer (PBS; pH 7.0). Filters were counted using a 2500TR liquid scintillation counter (PerkinElmer Life and Analytical Sciences, Waltham, MA). Saturation data were analyzed by the nonlinear regression analysis program LIGAND (Biosoft, Milltown, NJ). Binding curves were best fit to a one-site model, yielding the equilibrium dissociation constant (K_d) and the maximal number of binding sites (B_max).

Ex Vivo Autoradiography. Brain sections were cut coronally at a thickness of 20 µm on a cryostat, and they were thaw-mounted on Superfrost Plus slides (VWR, West Chester, PA). For saturation binding and occupancy studies, three sections from each of three individual mice at the level of the rostral hippocampus at intervals of approximately 100 µm were used. For distribution studies, sections from the olfactory bulb at the rostral extent through the cerebellum at the caudal extent at intervals of approximately 250 µm were used. Brain sections were warmed to room temperature, dried, incubated for 10 min in 50 mM HEPES buffer, pH 7.4, transferred to the same buffer containing 5 nM [³H]IN973, and then incubated at room temperature for 60 or 10 min for distribution studies or occupancy studies, respectively. For saturation binding studies, sections were incubated with increasing concentrations of [³H]IN973 up to 100 nM in the same buffer. To define nonspecific binding, adjacent sections were incubated in buffer containing [³H]IN973 plus a 100-fold excess of unlabeled -secretase inhibitor (IN973 or BMS-433796 for saturation binding or occupancy, respectively). After the incubation, the slides were washed three times, for 2 min each wash, in ice-cold PBS, pH 7.2, and then they were dipped in ice-cold distilled water and dried with a fan blowing cool air. The slides were placed under tritium-sensitive phosphor storage screens together with ³H microscales (GE Healthcare, Piscataway, NJ), and they were exposed in the dark for 7 days. The screens were then scanned in a Cyclone phosphor scanner (PerkinElmer Life and Analytical Sciences). Images were acquired from the phosphor storage screens using OptiQuant acquisition and analysis software (PerkinElmer Life and Analytical Sciences). Optical densities (expressed as digital light units per square millimeter) over areas of interest (parietal cortex for occupancy studies), and the ³H standards were measured.

Measurement of Brain A. Brain A40 levels were measured following the procedure described previously (Barten et al., 2005). In brief, frozen samples of the forebrain were homogenized in a lysis buffer consisting of 2% CHAPS in PBS with protease inhibitors (Roche Diagnostics, Indianapolis, IN) at a concentration of 50 ml per gram of tissue. Samples were centrifuged at 100,000g for 45 min at 4°C, and the supernatants were diluted 1:2 before determination of A40 levels. Assay plates were coated with the capture antibody TSD-9S3.2 (A40 C terminus-specific). After washing three times with PBS plus 0.05% Tween 20, the plates were blocked with 5% bovine serum albumin before the addition of brain homogenates in triplicate. A40 levels were detected by the addition of the horseradish peroxidase-conjugated monoclonal antibody 26D6 recognizing human A1-12, followed by the addition of Pico Pierce Chemiluminescence reagent (Pierce Chemical, Rockford, IL). The signal was measured in a TopCount chemiluminescence reader (PerkinElmer Life and Analytical Sciences).

View larger version (10K): [in this window] [in a new window]   Fig. 1. Saturation binding analysis of [3H]IN973. A, chemical structure of the radioligand [3H]IN973. B, representative binding isotherm for radioligand binding to brain homogenates from Tg2576 mice. Data from three independent experiments yielded Kd = 2.6 ± 0.2 nM and Bmax = 135 ± 16 fmol/mg protein. C, representative binding isotherm for radioligand binding to frozen sections from Tg2576 mice is shown. Each data point is the mean value from three individual mice based on densitometric analysis of sections from the frontal cortex. Data from three independent experiments yielded Kd = 1.0 ± 0.1 nM and Bmax = 170 ± 2 fmol/mg protein.

	Results

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To further compare [³H]IN973 binding in Tg2576 brains with results from rat and human brains, we determined the regional distribution of [³H]IN973 binding sites. These studies showed that the highest levels of [³H]IN973 binding were observed in the glomerular layer of the olfactory bulb, choroids plexus, and pituitary (Fig. 2, a to f; Table 1). Moderately high levels of binding were also observed in the striatum, lateral septum, frontal cortex, hippocampus, hypothalamus, amygdala, substantia nigra, superior colliculus, central gray, and the molecular layer of the cerebellum. Low to negligible levels of binding were observed in the thalamus, midbrain, brain stem, and white matter. This distribution of binding is very similar to that observed in the rat, rhesus, and human brains (Yan et al., 2004; Patel et al., 2006).

View larger version (94K): [in this window] [in a new window]   Fig. 2. Distribution of binding sites in the Tg2576 mouse brain. Frozen sections (20 µm in thickness) taken at intervals of approximately 250 µm were incubated with [3H]IN973, and they were exposed to phosphor screens together with 3H microscales (red representing the highest level of binding). Pseudocolor images of representative sections at the level of the olfactory bulb (a), striatum (b), thalamus and rostral hippocampus (c), caudal hippocampus (d), midbrain (e), and cerebellum (f) are shown. Densitometric analysis of specific brain regions is shown in Table 1. Scale bar, 0.23 cm.

Effect of BMS-299897 and BMS-433796 on Brain A and [³H]IN973 Binding. -Secretase inhibitors that have been shown to reduce brain A in vivo can be grouped into two major classes: sulfonamides and azepinones (R. Olson and C. Albright, in preparation). We selected a representative -secretase inhibitor from the sulfonamide class, BMS-299897 (Fig. 3A) (Smith et al., 2000; Barten et al., 2005), and the azepinone class, BMS-433796 (Fig. 3B) (Prasad et al., 2007), for this study. To measure the extent of -secretase occupancy by -secretase inhibitors, we developed an ex vivo binding assay. In this assay, frozen brain sections from Tg2576 mice treated orally with -secretase inhibitors were briefly incubated with [³H]IN973, and unbound ligand was removed by washing. The amount of [³H]IN973 bound to sites not already occupied by inhibitors was measured by autoradiography. Nonspecific binding was determined by including 100-fold excess of unlabeled BMS-433796 in parallel binding reactions, and it was subtracted from the total. Examples of brain sections incubated with [³H]IN973 in the presence or absence of a 100-fold excess of unlabeled BMS-433796 (Fig. 3C) show that nonspecific binding is minimal. Occupancy was expressed as the reduction in specific [³H]IN973 binding in drug-treated mice relative to vehicle-treated mice. Control studies were conducted to measure the dissociation of dosed compound during incubation with ligand, which would underestimate the level of occupancy. There was no significant change in the binding of [³H]IN973 between 5 and 10 min of incubation, indicating that BMS-299897 and BMS-433796 were not detectably dissociated during the 10-min incubation used for the occupancy studies (Fig. 3D).

View larger version (21K): [in this window] [in a new window]   Fig. 3. Characterization of binding of -secretase inhibitors. A and B, structures of -secretase inhibitors BMS-299987 (A) and BMS-433796 (B) used in this study. C, two representative brain sections incubated with [3H]IN973 for 5 min in the presence or absence of unlabeled BMS-433796 as examples of nonspecific or specific binding, respectively. D, ex vivo binding of [3H]IN973 for 5, 10, 20, or 30 min to frozen sections taken from brains of Tg2576 mice treated with 30 mg/kg BMS-433796 () or 300 mg/kg BMS-299897 () is shown. Each data point is the mean value from five individual brains compared with vehicle-treated brains based on densitometric analysis of sections from the frontal cortex (**, p < 0.01).

Two dosing paradigms were used to explore the relationship between brain A40 and [³H]IN973 binding in Tg2576 mice. In the first paradigm, the -secretase inhibitors were administered at varying doses, and mice were harvested 3 h postdose for brain A40 and [³H]IN973 binding measurements. The more abundant brain A40 was measured in these studies, since previous studies showed a high correlation of A40 and A42 reductions following dosing with BMS-299897 (Barten et al., 2005). Brain A40 was reduced as a result of administering BMS-299897 and BMS-433796 in a dose-dependent manner (Fig. 4, A and C), with ED₅₀ values of 31.2 and 2.4 mg/kg, respectively. In a second dosing paradigm, the -secretase inhibitors were given at a single dose, and mice were harvested at times ranging from 0.5 to 24 h postdose. Both BMS-299897 and BMS-433796 caused a rapid reduction in brain A40 that partially returned to predose levels by 24 h (Fig. 4, B and D). When the amount of -secretase occupancy in these mice was determined using the ex vivo binding assay described above, the amount of -secretase occupancy and brain A40 reductions were highly correlated for both BMS-299897 and BMS-433796 using both dosing paradigms (Fig. 4E), although brain A40 reductions seemed to lag behind -secretase site occupancy at the earliest times postdose (Fig. 4, B and D). In particular, occupancy of BMS-299897 was at a maximal level of 90% at the 0.5-h time point, whereas A40 reduction did not reach a maximum level of 70% until approximately 3 h postdose. Likewise, occupancy of BMS-433796 reached a maximum of 75% at 0.5 h, whereas A40 did not reach a maximum reduction of 75% until 3 h postdose. At 3 h and later times, the correlation between occupancy and A40 reduction was excellent for both compounds.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Effect of BMS-299897 and BMS-433796 on brain A40 and [3H]IN973 binding. A and C, 3 h after various doses of BMS-299897 (A) or BMS-433796 (C), half of each brain was harvested and assayed for brain A40 (), and the other half for [3H]IN973 binding (). Occupancy is expressed as the mean reduction in specific ligand binding due to the presence of bound drug. Brain A40 is expressed as the mean percentage of A40 level compared with the vehicle control. B and D, at various times after a 100-mg/kg dose of BMS-299897 (B) or 30-mg/kg dose of BMS-433796 (D), brains were harvested and assayed for brain A40 () and occupancy (). Error bars represent standard error for groups of five mice. E, correlation of occupancy and brain A40 reduction for individual animals from the 3- to 24-h time points in the studies above is shown.

	Discussion

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In this study, an ex vivo binding assay was developed using [³H]IN973 and frozen brain sections from mice treated with -secretase inhibitors to quantify -secretase occupancy by -secretase inhibitors. For this purpose, the relationship of -secretase site occupancy and brain A reduction by -secretase inhibitors was examined using Tg2576 mice as a model. [³H]IN973 was used as a -secretase radioligand for these studies, since previous work by others and by us showed that [³H]IN973 bound to -secretase in rat, rhesus, and human brains with high affinity and specificity (Yan et al., 2004; Patel et al., 2006). We extended these findings to -secretase in brains of Tg2576 mice to which [³H]IN973 bound with an affinity specificity, and regional localization very similar to those found in brains of other species. The distribution of binding also correlates well with the distribution of PS-1 expression in the brain (Elder et al., 1996; Moussaoui et al., 1996; Blanchard et al., 1997; Kim et al., 1997; Siman and Salidas, 2004). Levels of binding were unaffected by the presence of plaques in aged Tg2576 mouse brain (data not shown), which represent large deposits of hydrophobic protein with the potential for significant nonspecific binding. Therefore, the presence of plaques in AD brains should not preclude use of a PET ligand for -secretase.

-Secretase inhibitors of two different structural classes were used to test the utility of [³H]IN973 as a ligand to measure ex vivo occupancy. BMS-299897 belongs to the sulfonamide class of -secretase inhibitors, whereas BMS-433796 and [³H]IN973 belong to the azepinone class of -secretase inhibitors (R. Olson and C. Albright, in preparation). All three of these -secretase inhibitors belong to the nontransition state class of molecules that are proposed to bind to an allosteric binding site on the presenilin subunit of -secretase (Kornilova et al., 2003; Tian et al., 2003; Clarke et al., 2006; Morohashi et al., 2006), and that exert their effect through inhibition of the turnover of -secretase processing intermediates (Qi-Takahara et al., 2005; Zhao et al., 2007). Clinical candidates have been developed from this type of -secretase inhibitor, making it likely that a PET ligand of this class will be of clinical value.

The effect of BMS-299897 and BMS-433796 on [³H]IN973 binding to -secretase has been studied using membrane preparations from THP-1 cells that contain high amounts of -secretase (Iben et al., in preparation). [³H]IN973 binding to THP-1 membranes is saturable, with a K_d value of 2.2 nM, similar to the K_d value for -secretase in other systems as described above. Inhibition of [³H]IN973 binding to THP cell homogenates (L. G. Iben, R. E. Olson, L. A. Balanda, S. Jayachandra, B. J. Robertson, V. Hay, J. Corradi, R. Zaczek, C. F. Albright, and J. H. Toyn, manuscript in preparation), as well as to rat brain sections (Yan et al., 2004) and rhesus cortex homogenates (Patel et al., 2006) in the presence of unlabeled -secretase inhibitors indicates that [³H]IN973 is binding specifically to -secretase. Furthermore, previous studies have shown that binding to mouse embryos is greatly reduced in embryos from presenilin-1 knockouts compared with wild-type embryos (Yan et al., 2004). Both BMS-299897 and BMS-433796 cause a concentration-dependent decrease in [³H]IN973 binding, with IC₅₀ values of 12 and 1.2 nM, respectively (Iben et al., manuscript in preparation), very similar to the IC₅₀ values for inhibition of A40 in human embryonic kidney cells overexpressing the Swedish mutation of APP of 7.4 and 0.8 nM, respectively, and for inhibition of A42 of 7.9 and 0.4 nM, respectively (data not shown). In addition, other -secretase inhibitors have shown a similar correspondence of IC₅₀ values (Yan et al., 2004; Patel et al., 2006).

Ex vivo assays can be a powerful method to quantify site occupancy in preclinical studies. For example, Lelas et al. (2004) have recently been able to determine levels of occupancy of a CRF1 antagonist required to elicit desired behavioral effects, while limiting effects on stress-induced activation of the hypothalamic-pituitary-adrenal axis (Lelas et al., 2004). However, ex vivo site occupancy assays can be subject to artifacts. For example, the extent of occupancy can be underestimated if the dosed compound dissociates from the binding site during the incubation with radioligand and subsequent washes. Control experiments measuring the dissociation of dosed compound during incubation with [³H]IN973 showed that the level of occupancy of BMS-299897 or BMS-433796 minimally changed during the 10-min incubation with ligand.

Using the [³H]IN973 ex vivo binding assay and animals dosed with either BMS-299897 or BMS-433796, the relationship between -secretase occupancy and brain A40 was determined. Results from these experiments showed a correlation between -secretase site occupancy and brain A40 levels over a wide range of doses and times, with the exception of the first 3 h when the reduction in brain A40 response lagged -behind secretase occupancy. This lag is probably a result of the time required to clear brain A40 that was produced before -secretase was inhibited. Consistent with this hypothesis, results from pharmacokinetic studies show that the half-life of A40 in the brain of transgenic mice ranges from 38 min to 2.5 h (Cirrito et al., 2003; Lanz et al., 2004; Barten et al., 2005).

The correlation between brain A40 and -secretase occupancy found in this study suggests that a PET ligand for -secretase could be a valuable biomarker to determine whether -secretase inhibitors bind to their target in human clinical trials. The binding potential (B_max/K_d) of IN973 is within a range (>10) that is acceptable for a ligand (R. Innis, personal communication), suggesting that development of a PET ligand for -secretase would be possible. In addition, the high affinity and signal-to-noise ratio, and the specific localization of [³H]IN973, are all qualities of a good PET ligand. Additional effort will probably be needed to identify a compound that fulfills the appropriate lipophilicity and brain penetrance requirements for a successful PET ligand (Laruelle et al., 2003).

	Acknowledgements

 
We thank Yang Hong and Shiang-Yuan Chen for synthesis of the radioligand, and Drs. Carl Bergstrom and Kate McElhone for synthesis of BMS-433796 used in these studies.

	Footnotes

 
A portion of this work was previously presented at the Society for Neuroscience 2005; 12–16 Nov 2005; Washington, DC.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.125492.

ABBREVIATIONS: AD, Alzheimer's disease; A, -amyloid peptide; APP, -amyloid precursor protein; PS, presenilin; CSF, cerebrospinal fluid; IN973, (2R,3S)-2-isobutyl-N¹-((S)-1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)-3-propylsuccinamide; BMS-299897, 2-[(1R)-1-[[(4-chlorophenyl)-sulfonyl](2,5-difluorophenyl)amino]ethyl]-5-fluoro-benzenepropanoic acid; BMS-433796, (S)-2-((S)-2-(3,5-difluorophenyl)-2-hydroxyacetamido)-N-((S,Z)-3-methyl-4-oxo-4,5-dihydro-3H-benzo[d][1,2]diazepin-5-yl)propanamide; PET, positron emission tomography; CHAPS, [(3-cholamidopropyl)dimethylammonio]propanesulfonate; CHAPSO, 3-([3-cholamidopropyl])dimethylammonio])-2-hydroxy-1-propanesulfonate.

Address correspondence to: Dr. Margi Goldstein, Bristol-Myers Squibb Co., Neuroscience Drug Discovery, 5 Research Pkwy., Wallingford, CT 06492. E-mail: margi.goldstein{at}bms.com

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