JOURNAL OF NEUROCHEMISTRY
| 2012 | 121 | 277–286
doi: 10.1111/j.1471-4159.2011.07560.x
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*Neurodegeneration Research, R&D China, GlaxoSmithKline, Shanghai, China Drug Metabolism and Pharmacokinetics, R&D China, GlaxoSmithKline, Shanghai, China àMedicinal Chemistry, R&D China, GlaxoSmithKline, Shanghai, China ¶Neurosciences Centre of Excellence for Drug Discovery, GlaxoSmithKline, Harlow, Essex, UK
Abstract c-secretase inhibitors (GSIs) have been developed to reduce amyloid-b (Ab) production for the treatment of Alzheimer’s disease by inhibiting the cleavage of amyloid precursor protein (APP). However, cross-inhibitory activity on the processing of Notch can cause adverse reactions. To avoid these undesirable effects, c-secretase modulators (GSMs) are being developed to selectively reduce toxic Ab production without perturbing Notch signaling. As it is also known that GSIs can cause a paradoxical increase of plasma Ab over the baseline after a transient reduction (known as Ab-rebound), we asked if GSMs would cause a similar rebound and what the potential mechanism might be. Our studies were performed with one GSI (LY-
450139) and two chemically distinct GSMs. Although LY450139 caused Ab-rebound as expected in rat plasma, the two GSMs did not. Inhibition of APP processing by LY-450139 induced an accumulation of c-secretase substrates, a- and b-C-terminal fragments of APP, but neither GSM caused such an accumulation. In conclusion, we discover that GSMs, unlike GSIs, do not cause Ab-rebound, possibly because of the lack of accumulation of b-C-terminal fragments. GSMs may be superior to GSIs in the treatment of Alzheimer’s disease not only by sparing Notch signaling but also by avoiding Ab-rebound. Keywords: c-secretase inhibitor, c-secretase modulator, Alzheimer’s disease, amyloid-b, C-terminal fragment. J. Neurochem. (2012) 121, 277–286.
According to the amyloid cascade hypothesis, amyloid-b (Ab) peptides are the ultimate culprit in the pathogenesis of Alzheimer’s disease (AD) (Sommer 2002). Ab peptides aggregate and form the characteristic senile plaques in the brains of AD patients. They are generated from a parental molecule, amyloid precursor protein (APP), after sequential cleavage at the b- and c-secretase cleavage sites, by b-site APP cleaving enzyme-1 (BACE1) and c-secretase, respectively (Hardy and Selkoe 2002). Deletion of presenilin-1 and
2 Present address: Laboratory for Research of Neurodegenerative Diseases, Center for Human Genetics, KULeuven, Leuven 3000, Belgium. 3 Present address: Takeda Cambridge Ltd., Cambridge CB4 0PA, UK. 4 Present address: Therapeutic Area-Neurodegenerative Diseases, Merck Serono S.A., Geneva 1202, Switzerland. 5 Present address: Neuroscience Product Creation Unit, European Knowledge Centre, Eisai Ltd., Hertfordshire, AL10 9SN, UK. 6 Present address: Roche R&D Center China, Shanghai 201203, China. 7 Present address: New Products and Alliance Development, R&D China, GlaxoSmithKline, Shanghai 201203, China. 8 Present address: Neural Pathways Discovery, GlaxoSmithKline R&D, Singapore 138667, Singapore. None of the compounds disclosed in this manuscript are approved for use in humans. Abbreviations used: Ab, amyloid-b; AD, Alzheimer’s disease; APP, amyloid precursor protein; BACE1, b-site APP cleaving enzyme-1; CTF, C-terminal fragment; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; GSI, c-secretase inhibitor; GSM, c-secretase modulator; LC-MS/MS, liquid chromatography-tandem mass spectrometry; PBS, phosphate-buffered saline; sAPP, soluble form of APP.
Received July 18, 2011; revised manuscript received September 23, 2011; accepted October 25, 2011. Address correspondence and reprint requests to Yasuji Matsuoka, Neural Pathways Discovery, GlaxoSmithKline R&D, 11 Biopolis Way, Singapore 138667, Singapore. E-mail:
[email protected] 1 Present address: Discovery Medicine, R&D China, GlaxoSmithKline, Shanghai 201203, China.
2011 GlaxoSmithKline Journal of Neurochemistry 2011 International Society for Neurochemistry, J. Neurochem. (2012) 121, 277–286
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presenilin-2 completely eliminates c-secretase activity (Herreman et al. 2000). Mutations on presenilin were found among familial AD cases (Mathews et al. 2000). In addition, up-regulation of presenilin has been detected in postmortem brains of sporadic AD cases (Ikeda et al. 2000; Smith et al. 2004). These findings indicate that inhibition of c-secretase is a potential therapeutic target for the treatment of AD. c-Secretase inhibitors (GSIs) have been shown to reduce brain Ab dose-dependently in pre-clinical models (Dovey et al. 2001). The most advanced GSI in drug development was Eli Lilly’s LY-450139 (also known as semagacestat). In phase II clinical trials, LY-450139 inhibited plasma Ab levels transiently followed by a paradoxical increase above the baseline (Siemers et al. 2006; Fleisher et al. 2008). While the total level of Ab in the cerebrospinal fluid was not significantly changed by LY-450139 (Siemers et al. 2005; Siemers et al. 2006, 2007 Fleisher et al. 2008), metabolic labeling studies revealed reduced synthesis consistent with c-secretase inhibition in the CNS (Bateman et al. 2009). However, recent phase III trial results showed no improvements in cognitive functions in AD patients treated with LY450139; these patients actually became worse in their cognitive functions and had higher risk of developing skin cancer (Eli Lilly News Release 2010). The phase III trial of LY-450139 in AD patients was halted. Consistent with outcomes of the phase III trial of LY450139, some pre-clinical studies have suggested that inhibition of c-secretase may impair learning and memory (Saura et al. 2004) and induce skin cancer (Li et al. 2007a,b). cSecretase cleaves many other substrates in addition to APP (Rochette and Murphy 2002; Hemming et al. 2008). It is possible that inhibition of the processing of these other substrates might lead to undesirable reactions. One of these substrates is Notch; inhibition of its cleavage by GSI results in abnormalities in the gastrointestinal tract (Milano et al. 2004; Stanger et al. 2005) and the immune system (Maillard et al. 2003; Wong et al. 2004). It is known that LY-450139 inhibited processing of APP and Notch to a similar extent (Imbimbo et al. 2011). This lack of substrate selectivity could lead to mechanism-associated adverse effects. To avoid undesirable reactions mediated by inhibition of Notch processing, some groups have shifted their focus from GSI to c-secretase modulators (GSMs). Uneven cleavage of APP by c-secretase leads to the production of various Ab fragments, ranging from 37 to 43 amino acid residues. GSIs inhibit cleavage at all c-secretase cleavage sites and production of Ab peptides in all lengths. However, GSMs selectively reduce the production of the longer amyloidogenic Ab peptides (e.g. Ab42) and increase that of the shorter and less amyloidogenic Ab peptides, for example, Ab 37 and 38 (Weggen et al. 2001; Czirr et al. 2008; Peretto and La 2008). As GSMs modulate but do not block c-secretase activity, Notch processing and signaling are largely spared.
Therefore, it is believed that GSMs will be better tolerated than GSIs. In addition, GSIs are also found to induce Ab-rebound. It has been shown that a single dose of LY-450139 in guinea pigs increases plasma Ab levels beyond baseline levels after an transient reduction (Lanz et al. 2006). Similar Ab-rebound in the plasma has been detected with different GSIs in APP transgenic mice (Lanz et al. 2004), dogs (Hyslop et al. 2004), monkeys (Cook et al. 2010) and humans (Siemers et al. 2005, 2007). In this study, we asked if GSMs could cause Abrebound and if there were any differences in the mechanisms of action between GSIs and GSMs that could account for any differences in their effects on Ab-rebound.
Materials and methods In vivo study design and tissue sampling All experimental protocols used in this study were reviewed and approved by Institutional Animal Care and Use Committee at GlaxoSmithKline R&D China. Male Sprague-Dawley rats (200 ± 10 g body weight, Shanghai SLAC Laboratory Animal, Shanghai, China) were maintained under a 12-h light/dark cycle with free access to food and water. Eight rats were assigned in each group. Vehicle, GSI, GSM-A or GSM-B (30 mg/kg) was administrated by oral gavage. GSM-A used in this study is identical to GSM-10h described in an earlier publication (Hall et al. 2010). Although mutant APP over-expressing transgenic mice develop Ab plaque burden and are commonly used in the AD field, there is no clear evidence to support that APP is over-expressed to that extent in sporadic AD patients. The commonly used mutant APP in transgenic models, Swedish mutant APP, is a much more efficient substrate for BACE1, and artificially increases levels of the substrate for c-secretase. Importantly, it has been reported that the effects of GSIs on the processing of wildtype and mutant APP are different (Burton et al. 2008). Therefore, wildtype animals were used in this study and believed to better reflect the effects of GSIs/GSMs in sporadic AD patients. Animals were killed with sodium pentobarbital (40 mg/kg, i.p.). EDTA-treated plasma was prepared (4000 g for 10 min at 4C), and snap-frozen in dry ice. Brains were isolated with the olfactory bulb and cerebellum removed first and then snap-frozen. All samples were stored at )80C until use. Quantification of Ab and APP fragments The frozen hemi-brains were homogenized in ice-cold 50 mM Tris– HCl, pH 8.0, containing 5 M guanidine and protease inhibitor cocktail (EMD Biosciences, Darmstadt, Germany) using BioSpec Mini-Beadbeater-8 homogenizer (BioSpec Products, Bartlesville, OK, USA) for 1.5 min at 4C. One-hundred microliter of brain homogenates were mixed with 900 lL neutralization buffer (50 mM Tris–HCl, pH 7.4, containing 150 mM NaCl, 0.05% Tween 20 and 1% bovine serum albumin). Samples were vortexed and centrifuged at 16 000 g for 20 min at 4C, and the resultant supernatant was used for Ab quantification. Levels of Ab 40 and 42 were quantified using ELISA kits (Wako Pure Chemical, Osaka, Japan) (Suzuki et al. 1994) according to the manufacturer’s instruction.
2011 GlaxoSmithKline Journal of Neurochemistry 2011 International Society for Neurochemistry, J. Neurochem. (2012) 121, 277–286
c-Secretase modulators do not cause Ab-rebound | 279
For western blot, frozen hemi-brains were homogenized in 20 mM Tris–HCl, pH 7.5, containing 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3VO4, 1 lg/ mL leupeptin, and protease inhibitor cocktail. The homogenate was centrifuged at 14 000 g for 10 min at 4C, and the supernatant was mixed with NuPAGE LDS sample buffer (Invitrogen, Carlsbad, CA, USA). Samples were run on a NuPAGE Novex 4–12% Bis–Tris Gel with NuPAGE MES SDS running buffer (Invitrogen), and transferred onto Immobilon PVDF membranes (Millipore, Billerica, MA, USA). After blocking (LI-COR Biosciences, Lincoln, NE, USA), membranes were incubated overnight with rabbit anti-APP N-terminus polyclonal antibody, rabbit anti-APP C-terminus polyclonal antibody, or mouse anti-b-actin monoclonal antibody (clone AC-15) (1, 1, and 0.5 lg IgG/mL, respectively; Sigma, St Louis, MO, USA). After several washes in phosphate-buffered saline (PBS) containing 0.1% Tween 20, membrane was probed with IRDye 800-conjugated secondary antibody (LI-COR Biosciences) for 1 h at 25C, scanned and protein bands were quantified (Odyssey infrared scanner, LI-COR Biosciences). Ab fragments profiling using LC-MS/MS Primary cultured cortical neurons were treated with vehicle, GSI (LY-450139), GSM-A, or GSM-B at 1 lM for 48 h. Purified IgG of mouse anti-Ab antibody clone m3.2 (Morales-Corraliza et al. 2009) were cross-linked with magnetic Dynabeads according to manufacturer’s instructions (Invitrogen). Beads-linked with anti-Ab antibody (0.2 lg IgG) was incubated with 1 mL of culture medium. After incubation on a rocking platform at 4C overnight, immunocomplex was separated using a magnet. Immunoprecipitants were washed twice with PBS, then twice with 25 mM of ammonium bicarbonate. Precipitated Ab fragments were eluted in 100 lL of 0.1% ammonium hydroxide and 20% acetonitrile in water. Supernatant was separated on a Waters Acquity UPLC system (Hertfordshire, UK) and analyzed with a triple quadrupole linear ion trap mass spectrometer (API5000: AB/MDS Sciex, Concord, Canada) equipped with electrospray ionization. Pharmacokinetics Drug concentrations were analyzed using liquid chromatographytandem mass spectrometry (LC-MS/MS). Brains were homogenized in 3-fold PBS (w/v) using PRO200 homogenizer (PRO Scientific, Oxford, CT, USA), and precipitated with 3-fold acetonitrile (v/v). Chromatography was performed on ACQUITY UPLC System at 30C (Waters Corp., Milford, MA, USA) equipped with an ACQUITY UPLC BEH C18 2.1 · 50 mm (1.7 lm) column (Waters). Elution was carried out at a flow-rate of 0.6 mL/min using 1 mM ammonium acetate in 10% acetonitrile and acetonitrile : methanol (4 : 1) as mobile phase with a gradient program. The MS-MS analysis was performed on API 4000 Q TRAP LC/MS/ MS System (Applied Biosystems, Foster City, CA, USA) equipped with electrospray source operating in positive ion mode. Drug concentrations were determined using multiple reaction monitoring scan mode with the following parameter settings; ion spray voltage: 5500, curtain gas: 35, nebulizer gas: 50, auxiliary gas: 70, temperature: 650C, and collision-activated dissociation gas: 8. Linear range of all the analyte was 1.2–1200 ng/mL with lower limit of quantification of 1.2 ng/mL.
In vitro cell culture studies HEK293 cells over-expressing APP with the Swedish mutation (HEK293-APPswe), SH-SY5Y human neuroblastoma cells (American Type Culture Collection, Manassas, VA, USA) and primary cultured rat cortical neurons were used. All cells were maintained at 37C in a humidified chamber containing 5% CO2. HEK293 cells were transfected with plasmid pcDNA3.1-human APPswe or with empty vector (as a negative control) using Lipofectamine (Invitrogen). Stably transfected cells were selected with G418 (Invitrogen), and HEK293-APPswe line was established. HEK293-APPswe cells were seeded in 6-well plates at 4 · 105 cells/well or 96-well plate at 5 · 104 cells/well and grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% (v/v) fetal bovine serum (FBS) (Invitrogen) and 600 lg/mL G418 (GIBCO, Langley, OK). SH-SY5Y cells were seeded in 96-well plates at 5 · 104 cells/well and grown in DMEM/F12 at 1:1 (Invitrogen) with 10% FBS. Primary neurons were prepared from the cerebral cortex of Sprague–Dawley rat embryos on embryonic days 18. Dissected tissues were incubated with 0.25% trypsin-EDTA for 10 min at 37C with occasional swirling. DMEM with 10% FBS and 2 mM GlutaMax (Invitrogen) was used to stop trypsin activity. The digested tissues were then centrifuged at 600 g for 5 min. After the supernatant was discarded, cells were re-suspended with medium and dissociated by passing through a 70-lm mesh filter (BD Biosciences, San Jose, CA, USA). Cortical neurons were seeded in 0.01% poly-L-lysine coated 6-well plates at 2 · 106 cells/well or 24well plates at 5 · 105 cells/well. After overnight incubation, half of the culture medium was replaced by neurobasal medium containing 2 mM GlutaMax and 1· B27 Supplement (Invitrogen). Cells were used for experiments after 5 days in culture. Cells were treated with compounds dissolved in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide was adjusted to 1%. After treatment, culture medium was collected, centrifuged briefly to eliminate debris, and snap-frozen for subsequent Ab quantification using ELISA. Cells were harvested in cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) for Western blot. Cytotoxicity was determined using WST-1 kit (Roche Diagnostics GmbH, Mannheim, Germany). Notch activity was determined using a reporter gene assay as previously described (Eatherton et al. 2009). Briefly, HEK293 cells were transiently transfected using FuGENE6 (Roche Diagnostics) with 5 lg of pGL3 5xGal4-Luc and 750 ng of pcDNA3.2 sp Notch DE-GVP, encoding Gal4/VP16 DNA binding/ transactivation domain and a fusion protein of Notch DE fragment, respectively. Following overnight incubation at 37C, the transfected cells were washed in PBS and frozen in cell freezing medium (Sigma). Frozen cells were thawed and treated with different concentrations of compound or vehicle for 24 h. Then the cultures were harvested and luciferase activity was quantified using Steadylite (PerkinElmer, Waltham, MA, USA) reagents on ViewLux ultraHTS microplate imager (PerkinElmer). Statistical analysis Data were presented as the mean ± standard error of the mean. Differences between treatment and non-treatment groups were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s t-test (Statistica 8; StatSoft, Tulsa, OK, USA). Differences with p < 0.05 were considered as statistically significant changes.
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WinNonLin 5.2 was used for pharmacokinetics and pharmacodynamics modelling and analysis (Pharsight, Mountain View, CA, USA).
15 and 27 h after drug administration when the concentration of LY-450139 was less than 1/100 of Cmax, plasma Ab levels rose above baseline (Ab 40: 142% of vehicle, p < 0.001; Ab 42: 145% of vehicle, p < 0.01). Interestingly, as a result of this rebound, there was no net reduction in Ab40 or 42 levels by LY450139 as represented by the area under the curve (Fig. 2d). Changes of plasma Ab after single oral administration of the two GSMs were also examined (Fig. 2b and c). Both GSM-A and GSM-B significantly reduced the levels of plasma Ab, reaching a peak inhibition between 6 and 9 h. GSM-A reduced Ab 40 by 17% (p < 0.05) and Ab 42 by 61% (p < 0.001) while GSM-B reduced Ab 40 by 46% (p < 0.001) and Ab 42 by 88% (p < 0.001). There was a temporal correlation between drug exposure and Ab reduction. Ab levels returned to baseline as the GSMs were completely cleared from plasma. Unlike GSI, Ab-rebound was not detected for either GSM (Fig. 2b, c and d).
Results In this study, we used two classes of Ab-lowering agents: GSI (LY-450139) and GSM (GSM-A and GSM-B) (Fig. 1). Both GSM-A and GSM-B were >100-fold more active in modulating APP processing than Notch processing while LY-450139 had limited selectivity. In addition, GSM-A and GSM-B represented two different types of GSMs with different effects on APP processing: GSM-A preferentially reduced Ab 42 production to a greater extent than that of Ab 40 while GSM-B was less selective (Fig. 1a). To confirm mechanism of c-secretase modulation, we investigated changes in Ab fragments profile. Ab 1-38, 1-40 and 1-42, were all reduced by LY-450139. In contrast, both GSM-A and GSM-B at 1 lM selectively and markedly increased shorter Ab fragment, Ab 1-38, to >150% of vehicle control when levels of Ab 1-42 were significantly reduced.
GSMs did not induce an accumulation of CTFs in cultured cells To understand if there were any differences between GSI and GSM in their effects on APP processing that might account for their different effects on Ab-rebound, we investigated the accumulation of two c-secretase substrates (a- and b-CTFs) after treatment in HEK293-APPswe (Fig. 3a–f) and primary cultured rat cortical neurons (Fig. 3g–l) with these compounds. HEK293-APPswe cells were treated with LY-450139, GSM-A and GSM-B at 2-fold IC50 for Ab 42, that is, 0.11 lM, 0.58 lM, and 0.32 lM, respectively. After treatment with LY-450139, levels of b- and a-CTFs were rapidly increased in a time-dependent manner (Fig. 3a) to 999% and 213% of vehicle, p < 0.01, respectively, after 4-h incubation; and to 1166% and 365% of vehicle, p < 0.05, respectively,
GSMs did not induce plasma Ab-rebound in rats To test the effects of GSI and GSM on plasma Ab in rats, we orally administered a single dose of LY-450139, GSM-A or GSM-B at 30 mg/kg and collected plasma at different time points up to 45 h after drug administration. As shown in Fig. 2a, LY-450139 significantly reduced Ab 40 and 42 levels in rat plasma; concentration of the compound in the plasma and Ab reduction both peaked around 3 h (Ab 40: 84% reduction, p < 0.001; Ab 42: 31% reduction, p < 0.001). As the compound was cleared from the plasma, magnitude of Ab reduction gradually diminished. Between (a)
pIC50 of compounds HEK293-APPswe cells
(b)
Primary cultured neurons
SH-SY5Y cells
Class
Compounds
Aβ42
Aβ40
Notch
Notch/Aβ42
Aβ42
Aβ40
GSI
LY-450139
7.3
7.2
5.9
20.5
7.0
7.1
6.5
GSM
GSM-A
6.5
4.5
4.5
102.0
6.6
