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NEUROPHARMACOLOGY
in Vivo and in Vitro Using the 
-Secretase Inhibitor, LY-450139
CNS Discovery, Pfizer, Inc., Groton, Connecticut
Received for publication
July 13, 2006
Accepted
August 17, 2006.
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Abstract |
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-secretase inhibitor shown to have efficacy in multiple cellular and animal models. Paradoxically, robust elevations of plasma amyloid-
(A) have been reported in dogs and humans after administration of subefficacious doses. The present study sought to further evaluate A responses to LY-450139 in the guinea pig, a nontransgenic model that has an A sequence identical to that of human. Male guinea pigs were treated with LY-450139 (0.2–60 mg/kg), and brain, cerebrospinal fluid, and plasma A levels were characterized at 1, 3, 6, 9, and 14 h postdose. Low doses significantly elevated plasma A levels at early time points, with return to baseline within hours. Higher doses inhibited A levels in all compartments at early time points, but elevated plasma A levels at later time points. To determine whether this phenomenon occurs under steady-state drug exposure, guinea pigs were implanted with subcutaneous minipumps delivering LY-450139 (0.3–30 mg/kg/day) for 5 days. Plasma A was significantly inhibited at 10–30 mg/kg/day, but significantly elevated at 1 mg/kg/day. To further understand the mechanism of A elevation by LY-450139, H4 cells overexpressing the Swedish mutant of amyloid-precursor protein and a mouse embryonic stem cell-derived neuronal cell line were studied. In both cellular models, elevated levels of secreted A were observed at subefficacious concentrations, whereas dose-responsive inhibition was observed at higher concentrations. These results suggest that LY-450139 modulates the -secretase complex, eliciting A lowering at high concentrations but A elevation at low concentrations.
peptide into dense core plaques in the brains of Alzheimer's disease patients is the ultimate target of multiple disease-modifying drug discovery efforts. One strategy that has entered the clinic is the use of a -secretase inhibitor to reduce central A production. Preclinically, multiple -secretase inhibitors have demonstrated central and peripheral A-lowering activity in transgenic mouse lines overexpressing human mutant amyloid precursor protein (Dovey et al., 2001
; Cirrito et al., 2003; Lanz et al., 2003, 2004; Wong et al., 2004; Barten et al., 2005), as well as nontransgenic species (Anderson et al., 2005; Best et al., 2006; El Mouedden et al., 2006). Whereas acute treatment of old, plaque-bearing mice should have little immediate impact on plaque load (insoluble A), these inhibitors have been shown to inhibit A in CSF (Lanz et al., 2003; Barten et al., 2005) and interstitial fluid (Cirrito et al., 2003) similarly in both plaque-free and plaque-bearing mice. In addition, plasma A has been shown to be reduced similarly by -secretase inhibition in both young and old Tg2576 mice (Lanz et al., 2003; Barten et al., 2005). These findings indicate that despite the presence or absence of insoluble A plaques, these compounds had similar potency in reducing soluble, secreted A in young and old transgenic mice.
The ability of plasma and CSF A to track pharmacological changes in brain A provides a useful method for tracking the efficacy of -secretase inhibitors in the clinic. Because each compartment may have varying degrees of drug exposure and different clearance rates for both drug and A, it is important to understand the dynamics of A in each compartment. Dose-response and time course studies with -secretase inhibitors in transgenic mice have revealed differences in sensitivity, time of maximal efficacy, and rate of A recovery in each of the different compartments. At lower doses or earlier time points, A levels in plasma seemed to be more dynamic than those in CSF or brain (Lanz et al., 2004; Barten et al., 2005). In addition, a low dose of LY-411575 in Tg2576 mice has been shown to elevate plasma A1–40 and A1–42 while having no effect upon CSF or brain A (Lanz et al., 2004). A structurally related -secretase inhibitor, LY-450139, has since been shown to elevate plasma A levels in beagles and in human volunteers either at low doses that had no effect upon CSF A or at higher doses that initially lowered plasma A but later elevated plasma A above baseline (Hyslop et al., 2004; Siemers et al., 2005).
In the present series of experiments, we sought to further characterize the multicompartment efficacy of LY-450139 in guinea pigs, whose A sequence is identical to that of human (Johnstone et al., 1991). To obtain both dose-response and time course information in brain, CSF, and plasma, a six-point dose response was repeated in different cohorts of animals that were evaluated at 1, 3, 6, 9, and 14 h postadministration. At early time points subefficacious doses raised plasma A, whereas higher doses lowered A levels in brain, CSF, and plasma, as expected. At later time points, A levels in animals receiving lower doses had returned to baseline, whereas animals receiving the higher doses exhibited elevated levels of plasma A. To test the hypothesis that this potentiation phenomenon is driven by drug concentration, guinea pigs were implanted with minipumps delivering vehicle or LY-450139 for 5 days to establish steady-state drug levels in plasma. On the final day, brain, CSF, and plasma were collected and analyzed. At high drug concentrations, inhibition of plasma and brain A was observed. A low concentration (1 mg/kg/day) significantly elevated plasma A.
To rule out the possibility that A elevation at low concentrations might be caused by intercompartmental dynamics, two cell lines were dosed with LY-450139, and A was analyzed in media. Both APP overexpressing H4(Swe) cells and embryonic stem cell-derived mixed neuronal cultures showed a similar pattern: dose-responsive A inhibition until a certain low concentration range in which A levels were elevated. IP/MS of H4(Swe) media confirmed ELISA data and showed multiple A fragments responding to concentration-dependent elevation and inhibition.
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Materials and Methods |
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225 to 250 g (n = 6 per group). All animal treatment protocols were approved by Pfizer's Institutional Animal Care and Use Committee and were compliant with Animal Welfare Act Regulations. For acute dosing, animals were administered vehicle (0.5% methylcellulose) or 0.2, 0.6, 2, 6, 20, or 60 mg/kg LY-450139 (see Fig. 1 for structure) by oral gavage. Doses and time points were selected on the basis of pilot experiments (data not shown). For steady-state drug exposure, 7-day minipumps (10 µl/h) were implanted s.c. in male guinea pigs (n = 6 per group). Minipumps were loaded with vehicle (20% DMSO, 20% EtOH, and 60% PEG400) or LY-450139 at concentrations corresponding to a delivery rate of 0.3, 1, 3, 10, or 30 mg/kg/day, and animals were euthanized after 5 days.
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Tissue Isolation and Processing. At specified intervals after dosing, guinea pigs were euthanized with CO2. Once breathing had ceased, a blood sample was obtained via cardiac puncture and stored on ice in EDTA-coated tubes for separation of plasma. The cisterna magna was then exposed by dissection, and a sample of CSF was drawn through a 25-gauge butterfly needle (BD Biosciences, Franklin Lakes, NJ) and frozen on dry ice. Brain hemispheres were harvested into conical tubes and frozen in liquid nitrogen. All tissue was stored at –80°C. Brain tissue was homogenized in 9 volumes of 0.2% diethylamine-50 mM sodium chloride, incubated 3 h on ice, and then centrifuged at 135,000g (1 h, 4°C). Supernatants were applied to 60-mg HLB 96-well plates (Waters, Milford, MA) and concentrated as described previously (Lanz and Schachter, 2006).
A Assay. Half-volume 96-well plates were coated overnight at 4°C with 6E10 (Signet Laboratories, Dedham, MA) diluted (4 µg/ml) in 0.1 M sodium bicarbonate, pH 8.2, and then blocked for 3 h at room temperature with 1% bovine serum albumin in phosphate-buffered saline with 0.05% Tween 20 (Sigma-Aldrich, St. Louis, MO). Standard curves were prepared from stock solutions of species-specific A peptides (Bachem Biosciences, King of Prussia, PA) in blocking buffer. A1–40 peptide was used to prepare the standard curve for A1–X in addition to A1–40. Standards and samples were incubated with capture antibody overnight at 4°C. For detection of A1–X, biotinylated 4G8 (0.2 µg/ml) was incubated for 2 h at room temperature. For detection of A1–40 and A1–42, rabbit polyclonal antibodies directed against each of the fragments (Mehta et al., 1998) were used in place of 4G8 at dilutions of 1:1000 to 1:2000. The signal was amplified with europium-conjugated streptavidin followed by incubation with Delfia enhancement solution (both steps at room temperature for 1 h). Plates were read on a Wallac Victor plate reader (europium-Delfia reagents and equipment from PerkinElmer Life and Analytical Sciences, Boston, MA). Standards were fit to a fourth-order polynomial curve, and sample values were extrapolated with GraphPad Prism 4.0. Each time point was treated as a separate experiment: each was assayed and analyzed separately.
Measurement of Drug Levels in CSF and Plasma
Calibration Standards and QC Samples. Stock solutions of LY-450139 (1 mg/ml) and internal standard (1 mg/ml) were prepared individually in methanol. Standard working solutions of LY-450139 (100 µg/ml) and internal standard (10 µg/ml) were prepared by serial dilution of their stock solutions with methanol-water. Calibration standard samples were prepared by serial dilution with blank guinea pig plasma or ultrafiltered rat plasma to yield concentrations of 1, 2, 5, 10, 20, 50, 100, 500, and 1000 ng/ml. Likewise, QC samples at concentrations of 3.2, 400, and 800 ng/ml were also prepared in each matrix.
Plasma Sample Preparation. Aliquots (100 µl) of plasma sample, calibration standards, and QC samples were pipetted into individual wells of a 96-well plate. All samples were spiked with 10 µl of internal standard (10 µg/ml) followed by 400 µl of acetonitrile. The plate was vortexed, followed by centrifugation at 3000 rpm for 10 min; 300 µl of acetonitrile was drawn into a clean 96-well plate and evaporated to dryness under a stream of nitrogen. Samples were reconstituted in 100 µl of methanol-water and vortexed for approximately 2 min, and 20 µl was injected onto the liquid chromatographtandem mass spectrometer.
CSF Sample Preparation. Aliquots (50 µl) of CSF sample, ultrafiltrate calibration standards, and ultrafiltrate QC samples were pipetted into a 96-well plate containing glass inserts. All samples were spiked with 5 µl of internal standard (10 µg/ml) and vortexed, and 20 µl was injected onto the liquid chromatograph-tandem mass spectrometer.
Liquid Chromatography-Tandem Mass Spectrometry Conditions. An LC-10AD chromatograph (Shimadzu) equipped with a degasser and a CTC Analytics HTS PAL autosampler (Leap Technologies, Carrboro, NC) was used to fractionate 20-µl aliquots of processed samples on a Phenomenex Synergi Polar-RP column (2.0 mm x 50 mm, 4 µm). The mobile phase was a gradient mixture of solvents A (10 mM ammonium acetate, pH 7.0) and B (acetonitrile). Quantitation was achieved by tandem mass spectrometry detection in positive ion modes for analyte and internal standard using a Micromass Quattro Ultima mass spectrometer. Detection of the ions was performed in the multiple reaction-monitoring mode, monitoring the transition of m/z 362.4 
191.1 for LY-450139 and m/z 407.4 153.8 for the internal standard. The analytical data were processed by MassLynx software (version 4.0). Calibration curves were acquired by plotting the peak area ratio of analyte to internal standard against the nominal ratio of concentrations of calibration standards. The results were fitted to linear regression analysis using 1/
2 as the weighting factor.
Whole-Cell Assay
Drug Treatment. H4 cells overexpressing the Swedish mutation of human APP were plated in 96-well plates at 30,000 cells/well grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and penicillin-streptomycin (Invitrogen, Carlsbad, CA). For ELISA analysis, cells were treated in triplicate with vehicle (0.5% DMSO in medium) or LY-450139 at concentrations ranging from 200 pM to 2 µM. For IP/MS analysis, vehicle or a single concentration was administered to every well in a 96-well plate. Cells were treated with drug for 20 h, at which time medium was harvested and frozen. For IP/MS analysis, all wells for a given treatment group were pooled before freezing.
A Assay. Cell medium was assayed at a 1:2 dilution in blocking buffer for A1–X using the ELISA configuration described for in vivo samples.
IP/MS of Conditioned Media. A was immunoprecipitated from conditioned media of vehicle or LY-450139-treated H4(Swe) cells with monoclonal antibodies 6E10 and 4G8. Matrix-assisted time-of-flight mass spectroscopy analysis was performed as described elsewhere (Wood et al., 2005).
Mouse Embryonic Stem Cell-Derived Neurons
Differentiation. Undifferentiated mouse embryonic stem cells were incubated for 5 days in the presence of transferrin, leukemia inhibitory factor, and noggin. Once embryoid bodies formed, cells were dissociated and replated in the presence of basic fibroblast growth factor, sonic hedgehog signal, fibroblast growth factor-8, noggin, laminin, and G418. Medium was replaced after 6 days, and neuronal precursors were allowed to expand for 4 days. The resulting culture contained both cells of neuronal and non-neuronal phenotype.
Drug Treatment. Mouse neuronal precursor cells were adhered to 24-well poly-D-lysine-coated plates at 1.2 x 105 cells/well in a NeuroIII medium (1 µM cAMP, 200 µM ascorbic acid, 100 ng/ml brain-derived neurotrophic factor, and 5 mg/l laminin added to a mixture of 80% Neurobasal medium with B27, L-glutamine, and 20% Dulbecco's modified Eagle's medium F-12/N2) and incubated at 2% O2. At day 8 medium was replaced with 0.5 ml of fresh NeuroIII with 10% fetal bovine serum, and differentiated neurons were treated for 48 h with vehicle (1% DMSO) or LY-450139 at concentrations ranging from 30 pM to 3 µM. Conditioned medium was collected, and viability of neurons was assessed using the 3-(4,5-dimethylthyazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric microassay.
A Assay. AX– 40 levels were quantified by an electrochemiluminescence assay (Origen M-8 analyzer; BioVeris, Gaithersburg, MD). Conditioned medium (100 µl/well) was incubated with biotinylated 4G8 diluted 1:2000 into 100 µl of phosphate-buffered saline with 1% bovine serum albumin, streptavidin M-280 Dynabeads (0.1 mg/ml; BioVeris), and 0.5 µg/ml ruthenylated 8D2 (in-house monoclonal antibody raised against amino acids 33–40 of A).
Statistical Analysis and Data Presentation
At each time point, one-way analysis of variance was used to detect a significant treatment effect on A (GraphPad Prism software). After a significant main effect by ANOVA, individual differences versus vehicle were analyzed with Dunnett's multiple comparison test using p < 0.05 as a statistically significant level. To facilitate comparisons between all studies, data are presented as percentages of the corresponding vehicle control ± S.E.M. Absolute values for the vehicle group are included in the figure legends.
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Results |
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-Secretase in Guinea Pigs by LY-450139. Male guinea pigs were treated orally with vehicle or LY-450139 at doses ranging from 0.2 to 60 mg/kg, and complete dose responses were repeated at multiple time points. As early as 1 h after dosing (Fig. 2), significant inhibition of brain A1–X production was observed at 20 to 60 mg/kg (32–34% reduction, p < 0.05). These doses also significantly lowered plasma A1–X, A1–40 (84–97% reduction, p < 0.01), and A1–42 (63–74% reduction, p < 0.01). Doses of 2 to 6 mg/kg significantly lowered plasma A1–X and 1–40 (63–90% reduction, p < 0.01) but had no effect in CSF or brain at 1 h. The lowest dose, 0.2 mg/kg, significantly elevated plasma A1–X, 1–40, and 1–42 to between 159 and 214% of vehicle (p < 0.01). This dose also elevated CSF A1–42 to 261% of vehicle (p < 0.05). The next higher dose, 0.6 mg/kg, had no effect upon A1–X or A1–40 in any compartment but significantly elevated plasma A1–42 (161% of vehicle, p < 0.01).
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was still observed in plasma, although it only reached statistical significance for A1–40 (183–212% of vehicle, p < 0.05). A1–X and 1–40 were significantly reduced in all compartments at 20–60 mg/kg. As with the 1-h time point, less reduction of A1–42 was observed relative to A1-X and 1–40. At 2 to 6 mg/kg, plasma A1–X and 1–40 still exhibited significant reduction, although the magnitude of effect was less at 3 h versus 1 h, suggesting a very short time of maximal effect at these doses.
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, but rather raised A levels over baseline. A1–X and A1–40 reached 171 and 220% of vehicle, respectively (p < 0.01) at the 6 mg/kg dose. Plasma A1–42 was also significantly elevated at the 6 mg/kg dose (128% of vehicle, p < 0.01). Only the maximum dose, 60 mg/kg, still elicited significant reduction at 6 h, and this efficacy was restricted to plasma.
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was observed at 9 h (Fig. 5). The 2 mg/kg dose, which lowered plasma A at 1–3 h and elevated plasma A at 6 h, had no effect on any compartment by 9 h. The 6 mg/kg dose, which had earlier lowered A in all three compartments, caused an elevation of plasma A1–X and 1–40 at 9 h. By 14 h, A levels in all compartments had returned to baseline (data not shown).
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, predominantly in plasma. Middle doses showed inhibition of plasma A followed by elevation of plasma A, without any significant effect in CSF or brain. The highest two doses showed efficacy in plasma by 1 h, with CSF and brain showing A significant reduction by 3 h. The lower of these two doses eventually caused an elevation of plasma A at 9 h, by which time levels in CSF had returned to baseline.
Dose-Dependent Modulation of A in Guinea Pigs with Steady-State Ly-450139 Infusion. To evaluate whether the A elevations observed in plasma could occur under steady-state conditions, guinea pigs were treated with vehicle or LY-450139 for 5 days through a s.c. minipump. At infusion rates of 10 and 30 mg/kg/day, A1–X (Fig. 6A) was significantly lowered in brain (16–22% reduction, p < 0.05) and plasma (66–73% reduction, p < 0.01). Plasma A1–X was significantly elevated (141% of vehicle) at 1 mg/kg/day (p < 0.01). Plasma A1–40 (Fig. 6B) was similarly lowered at 10 to 30 mg/kg/day (81% reduction, p < 0.01) and was elevated at 1 mg/kg/day (141% of vehicle, p < 0.01). No treatment effect was detected in brain A1–40. Although 10 to 30 mg/kg/day significantly reduced A1–42 by 32 to 35% (p < 0.05), no potentiation was observed at any of the doses tested (Fig. 6C). The only significant change in brain A1–42 was an 18% increase at 30 mg/kg/day (p < 0.05). Whereas CSF A1–42 showed a trend toward elevation at the high doses, treatment effects did not reach significance in any A fragment measured in this compartment.
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Drug levels in CSF and plasma are listed in Table 1. Detectable levels of LY-450139 were measured in plasma at every dose. The 1 mg/kg/day delivery rate resulted in a plasma drug concentration of 40 nM, which caused significant elevation of plasma A. No significant alteration of A levels in any compartment was observed at 116 nM plasma LY-450139, whereas significant reductions of A1–X, 1–40, and 1–42 were detected at plasma drug concentrations of 368 nM and above.
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Concentration-Dependent A Modulation in Cell Culture Models. After the observation of A elevation in plasma at low measurable concentrations of LY-450139, the compound was evaluated in H4 cells overexpressing the "Swedish" mutation of human APP. Cells were treated with LY-450139 at concentrations ranging from 0.6 nM to 2 µM. After exposure to the drug for 20 h, A1–X levels were measured in media (Fig. 7). Across two trials, the IC50 was approximately 60 nM, with near-complete inhibition at higher concentrations. At 6 and 19 nM, A1–X was 30 to 40% elevated. At lower concentrations, A1-X levels were similar to baseline.
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levels was identified, an inhibitory concentration was compared with a potentiating concentration at multiple time points up to 24 h (Fig. 8). With vehicle treatment, cell media continued to accumulate A1–X over a 24-h period. When a high concentration (95 nM) of LY-450139 was present, the rate of A production remained low at every time point. When cells were incubated with 9.5 nM LY-450139, however, the rate of A production was consistently higher than that of vehicle, resulting in elevated A levels at every time point.
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species affected by LY-450139 in this cell culture system, media were pooled from vehicle-treated samples and separately from drug-treated samples for IP/MS analysis (Fig. 9). Fixed concentrations of 15N-substituted synthetic A1–40, 1–42, and 1–43 peptides were included in each set of samples to enable accurate quantitation of these peptides in the sample. At 6 nM LY-450139, A1–40 and A1–42 were elevated 30 to 50%; these changes could be visualized in the spectra by a change in the relative size of these peaks relative to the 15N standards. A11–40 also showed an increase at 6 nM LY-450139. At 60 nM, A1–40 was inhibited by 50%, and A1–42 was inhibited 25%. A11–40, however, showed further elevation beyond the level detected at 6 nM LY-450139. By 600 nM, all A peptides were almost completely reduced; the only major peaks in the spectra were the 15N standards.
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LY-450139 was then dosed in a nonmutant cell culture system, in which APP and A are generated at physiological rates. Mixed neuronal cultures derived from mouse embryonic stem cells were treated with LY-450139 for 48 h (Fig. 10). As in H4(Swe) cells, high drug concentrations depleted the media of AX-40. At 30 and 100 nM LY-450139, A levels were elevated by 60% or more in all three repeats. At drug concentrations of 
10 nM, A levels resembled those of vehicle-treated cells. None of the drug treatments used in these experiments caused a significant change in cell viability as assessed by 3-(4,5-dimethylthyazol-2-yl)-2,5-diphenyltetrazolium bromide activity.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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-secretase inhibitor, LY-450139, differentially modulates guinea pig plasma A in a concentration-dependent manner. At high doses, the compound reduces brain, CSF, and plasma A in a similar fashion within the first 3 h after dosing. At these early time points, low doses of the inhibitor significantly elevate plasma A. Higher doses that reduced plasma A within the first 3 h were shown to significantly elevate plasma A at later time points, presumably as drug exposure waned. After 5 days of steady-state delivery, LY-450139 reduced plasma A at high doses and elevated plasma A at low doses, similar to the pattern seen at early time points after acute dosing. Furthermore, elevation of A at low drug concentrations was observed in two different in vitro model systems. Maintaining steady-state drug levels of LY-450139 both in vitro and in vivo produced sustained inhibition or potentiation of -secretase activity depending on drug concentration.
The acute dosing data in guinea pigs reproduced a similar pattern of reduction versus elevation of plasma A1–X shown previously in beagles (Hyslop et al., 2004) and in healthy human patients (Siemers et al., 2005). In addition, significant elevation of plasma A by a low dose of LY-411575 has been previously demonstrated in Tg2576 mice (Lanz et al., 2004). If potentiation occurred only after a period of inhibition, it is reasonable to hypothesize that substrate may accumulate while drug is present; once the enzyme is no longer inhibited, a transient overproduction of A could occur as the substrate stock is consumed. Our results, however, suggest that an initial inhibition of -secretase activity is not required for LY-450139 to produce elevated A levels. When LY-450139 was maintained at a low concentration (40 nM) in plasma for several days, A levels remained in an elevated state. It is reasonable to assume that after an acute dose, plasma exposure quickly reaches a potentiating concentration, whereas high doses may take several hours before normal clearance mechanisms reduce circulating drug levels to such a concentration. Second, in two different whole-cell models, sustained nanomolar concentrations of LY-450139 also increased levels of multiple A peptides. The potentiation occurred in the earliest time point studied (4 h) and was maintained throughout the whole incubation period with LY-450139. Differences in the IC50 and potentiating concentrations between the two cell systems were observed, possibly due to substrate availability, cell density, and media formulation. However, the detection of this concentration-dependent phenomenon in two very different cell systems using both immunoassay and IP/MS suggests that in vitro potentiation is not merely a model-specific artifact.
Despite the demonstration of reproducible A potentiation in vitro, it is interesting to note that in vivo potentiation in the present work was predominantly restricted to plasma. In CSF, some elevations in A were seen, although few changes were statistically significant because of the high variability in this compartment. Additional variability in this and other compartments in extrapolated vehicle A levels is also observed due to analytical issues: because each time point was run as a separate experiment, each set of samples was extrapolated against a separate standard curve. Absolute A levels therefore exhibit higher variance between assays than within an assay.
Brain A was inhibited by multiple doses across time, but significant potentiation of multiple A peptides was not observed. Because of the gross homogenization of brain tissue, the mixing of intracellular and secreted A pools may account for some differences between this compartment and those of CSF and plasma, which contain only secreted A. In cell culture, potentiation and inhibition was measured in terms of A secreted into the media over several hours. Technical considerations prohibited robust detection of intracellular A at these potentiating concentrations. In vivo, the dynamics of A clearance and transport in and out of the brain are also quite complex, including active transport by receptor for advanced glycation end products, P-glycoprotein, and other proteins (Deane et al., 2003; Cirrito et al., 2005). In a healthy animal, therefore, homeostatic mechanisms may prevent significant accumulation of A in the central nervous system.
The accumulation of secreted A under low, steady-state concentrations of LY-450139 could be due to direct or indirect effects of the compound. Whereas inhibition of an A-degrading enzyme could lead to an increased pool of A, cells have multiple mechanisms for degrading this peptide. Nonspecific effects upon multiple proteases would not be expected at the low drug concentrations that produce potentiation. A peptides have been shown to modulate various intracellular pathways, including synthesis of cholesterol, which can affect A production or transport (Grimm et al., 2005). It is therefore feasible that a subtle change in A levels could feed back into a pathway that influences -cleavage or A secretion. If such a mechanism were in place, however, one might expect that A levels would eventually return to baseline, rather than show continued elevation. In contrast, a mechanism in which both potentiation and inhibition are a result of direct action on -secretase activity, although paradoxical, could be responsible for the sustained effects observed in vivo and in vitro.
In a multisubunit enzyme complex such as -secretase, it is conceivable that more than one binding site is available, and this results in subtle effects on enzymatic activity. The apparent activation of -secretase that results in potentiation could be caused if binding of a first molecule of drug prevents access of the substrate to an unproductive binding mode and so increases the rate of proteolysis. Binding of a second molecule of drug would then result in inhibition. On presenilin alone, at least three sites have been identified whose binding by various substrates affects -secretase cleavage of APP: -secretase substrates bind first to an exosite spanning the N- and C-terminal fragments before entering the active site (Kornilova et al., 2005) and a nucleotide binding site has been shown to selectively modulate APP proteolysis (Fraering et al., 2005).
There is ample literature demonstrating that various genetic modifications of presenilin-1 elevate the cleavage of certain C-terminal A fragments (for review, see German and Eisch, 2004). Gene dosage experiments with other components of the enzyme complex have also been shown to either inhibit or potentiate A production. Neurons and fibroblasts from homozygous nicastrin knockout mice produce no detectable A, whereas the heterozygous knockouts show increased A1–40 and A1–42 production versus wild-type cells (Li et al., 2003). Altered gene dosage of Aph-1b has been shown to affect cleavage of APP, but not that of other -secretase substrates such as ErbB4, neuregulin-2, or p75 in rat cortex (Coolen et al., 2006). Whereas potentiation of A has been shown for more than one peptidomimetic -secretase inhibitors, further work is necessary to determine whether structurally unrelated inhibitors and those with different binding sites modulate A production in a concentration-dependent manner.
Pharmacological manipulations of -secretase have been previously shown to modulate specific -cleavage sites. Select nonsteroidal anti-inflammatory drugs have been shown to increase A1–38 while reducing A1–42 in vitro (Weggen et al., 2001; Beher et al., 2005). In another in vitro model, Qi et al. (2003) demonstrated elevation of A1–42 levels at concentrations that reduced A1–40 with the -secretase inhibitor, difluoroketone. The well-characterized -secretase inhibitor, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester, has elicited modulation in vitro and in vivo: doses that reduced A1–40 and A1–42 simultaneously elevated levels of longer A fragments such as A1–43 and A1–46 (Qi-Takahara et al., 2005; Yagashita et al., 2006). In the present studies, two forms of modulation were observed. Low concentrations of LY-450139 caused A elevations in vivo and in vitro. In addition, greater potency in reducing A1–40 versus A1–42 was observed at multiple doses and in multiple compartments in vivo. The preferential inhibition of A1–40 has also been demonstrated previously using the -secretase inhibitor LY-411575 in Tg2576 mice (Lanz et al., 2004). Furthermore, in the present studies, IP/MS revealed that A11–40 was elevated at a concentration of LY-450139 at which both A1–40 and A1–42 were inhibited. Only at high concentrations (>200 nM) were all detectable A peptides significantly reduced. Whether these forms of modulation occur with -secretase inhibitors from more diverse chemical series remains to be determined.
In the development of a -secretase inhibitor as a therapeutic agent, it is important to understand its pharmacodynamics in measurable compartments. There is ample evidence that -secretase is capable of being modulated to up-regulate select products while down-regulating others. In addition, we and others have observed both elevation and reduction of the same A fragment using a -secretase inhibitor, with the direction of effect being dependent upon drug concentration. In vitro evidence suggests that although high drug concentrations effectively reduce the rate of -secretase activity, a certain range of low concentrations actually increases the rate of -secretase cleavage. This unusual pharmacology is further complicated by the fact that A elevation was not clearly detected in the brain in vivo, despite demonstration of the phenomenon in a mixed culture containing cells with a neuronal phenotype. Whether this discrepancy is due to methodological issues or basic physiology remains to be answered. Even if potentiation is restricted to plasma, characterizing its appearance and disappearance will have an impact on a biomarker strategy dependent upon A endpoints. The fact that elevations in plasma A have already been detected in patients treated with a -secretase inhibitor (Siemers et al., 2005) underscores the importance of more fully understanding the dynamics of -secretase inhibition or modulation, if indeed such a distinction can be made.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: LY-450139, (S)-2-hydroxy-3-methyl-N-[(S)-1-((S)-3-methyl-2-oxo-2,3,4,5,-tetrahydro-1H-benzo[d]azepin-1-carbamoyl)-ethyl]-butyramide; A, amyloid-; CSF, cerebrospinal fluid; APP, amyloid-precursor protein; H4(Sw), H4 cells overexpressing the Swedish mutation of APP; IP/MS, immunoprecipitation/mass spectroscopy; ELISA, enzyme-linked immunosorbent assay; DMSO, dimethyl sulfoxide; QC, quality control.
Address correspondence to: Dr. Thomas A. Lanz, Pfizer, Inc., Eastern Point Rd., MS# 8220-4183, Groton, CT 06340. E-mail: thomas.a.lanz{at}pfizer.com
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References |
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|---|
Anderson JJ, Holtz G, Baskin PP, Turner M, Rowe B, Wang B, Kounnas MZ, Lamb BT, Barten D, Felsenstein K, et al. (2005) Reductions in -amyloid concentrations in vivo by the -secretase inhibitors BMS-289948 and BMS-299897. Biochem Pharmacol 69: 689–698.[CrossRef][Medline]
Barten DM, Guss VL, Corsa JA, Loo AT, Hansel SB, Zheng M, Munoz B, Srinivasan K, Wang B, Robertson BJ, et al. (2005) Dynamics of -amyloid reductions in brain, cerebrospinal fluid and plasma of -amyloid precursor protein transgenic mice treated with a -secretase inhibitor. J Pharmacol Exp Ther 312: 635–643.
Beher D, Clarke EE, Wrigley JD, Martin AC, Nadin A, Churcher I, and Shearman MS (2005) Selected non-steroidal anti-inflammatory drugs and their derivatives target -secretase at a novel site: evidence for an allosteric mechanism. J Biol Chem 279: 43419–43426.
Best JD, Jay MT, Otu F, Churcher I, Reilly M, Morentin-Gutierrez P, Pattison C, Harrison T, Shearman MS, and Atack JR (2006) In vivo characterization of A(40) changes in brain and cerebrospinal fluid using the novel a-secretase inhibitor N-[cis-4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK-560) in the rat. J Pharmacol Exp Ther 317: 786–790.
Cirrito JR, Deane R, Fagan AM, Spinner ML, Parsadanian M, Finn MB, Jiang H, Prior JL, Sagare A, Bales KR, et al. (2005) P-glycoprotein deficiency at the blood-brain barrier increases amyloid- deposition in an Alzheimer disease mouse model. J Clin Investig 115: 3285–3290.[CrossRef][Medline]
Cirrito JR, May PC, O'Dell MA, Taylor JT, Parsadanian M, Cramer JW, Audia JE, Nissen JS, Bales KR, Paul SM, et al. (2003) In vivo assessment of brain interstitial fluid with microdialysis reveals plaque-associated changes in amyloid- metabolism and half-life. J Neurosci 23: 8844–8853.
Coolen MW, van Loo KM, van Bakel NN, Ellenbroek BA, Cools AR, and Martens GJ (2006) Reduced Aph-1b expression causes tissue- and substrate-specific changes in -secretase activity in rats with a complex phenotype. FASEB J 20: 175–177.
Deane R, Du Yan S, Submamaryan RK, LaRue B, Jovanovic S, Hogg E, Welch D, Manness L, Lin C, Yu J, et al. (2003) RAGE mediates amyloid- peptide transport across the blood-brain barrier and accumulation in brain. Nat Med 9: 907–913.[CrossRef][Medline]
Dovey HF, John V, Anderson JP, Chen LZ, de Saint Andrieu P, Fang LY, Freedman SB, Folmer B, Goldbach E, Holsztynska EJ, et al. (2001) Functional -secretase inhibitors reduce -amyloid peptide levels in brain. J Neurochem 76: 173–181.[CrossRef][Medline]
El Mouedden M, Vandermeeren M, Meert T, and Mercken M (2006) Reduction of A levels in the Sprague Dawley rat after oral administration of the functional -secretase inhibitor, DAPT: a novel non-transgenic model for A production inhibitors. Curr Pharm Des 12: 671–676.[CrossRef][Medline]
Fraering PC, Ye W, LaVoie MJ, Ostaszewski BJ, Selkoe DJ, and Wolfe MS (2005) -Secretase substrate selectivity can be modulated directly via interaction with a nucleotide-binding site. J Biol Chem 280: 41987–41996.
German DC and Eisch AJ (2004) Mouse models of Alzheimer's disease: insight into treatment. Rev Neurosci 15: 353–369.[CrossRef][Medline]
Grimm MO, Grimm HS, Patzold AJ, Zinser EG, Halonen R, Duering M, Tschape JA, De Strooper B, Muller U, Shen J, et al. (2005) Regulation of cholesterol and sphingomyelin metabolism by amyloid- and presenilin. Nat Cell Biol 11: 1118–1123.
Hyslop PA, May PC, Audia JE, Calligaro DO, McMillian CL, Garner CO, Cramer JW, Gitter BD, Porter WJ, and Nissen JS (2004) Reduction in A-(1–40) and A-(1–42) in CSF and plasma in the beagle dog following acute oral dosing of the secretase inhibitor, LY450139. Neurobiol Aging 25: S147.
Johnstone EM, Chaney MO, Norris FH, Pascual R, and Little SP (1991) Conservation of the sequence of Alzheimer's disease amyloid peptide in dog, polar bear, and five other mammals by cross-species polymerase chain reaction analysis. Mol Brain Res 10: 299–305.[Medline]
Kornilova AY, Bihel F, Das C, and Wolfe MS (2005) The initial substrate-binding site of -secretase is located on presenilin near the active site. Proc Natl Acad Sci USA 102: 3230–3235.
Lanz TA, Himes CS, Pallante G, Adams L, Yamazaki S, Amore B, and Merchant KM (2003) The -secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester reduces A levels in vivo in plasma and cerebrospinal fluid in young (plaque-free) and aged (plaque-bearing) Tg2576 mice. J Pharmacol Exp Ther 305: 864–871.
Lanz TA, Hosley JD, Adams WJ, and Merchant KM (2004) Studies of A pharmacodynamics in the brain, cerebrospinal fluid, and plasma in young (plaque-free) Tg2576 mice using the -secretase inhibitor N2-[(2S)-2-(3,5-difluorophenyl)-2-hydroxyethanoyl]-N1-[(7S)-5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl]-L-alaninamide (LY-411575). J Pharmacol Exp Ther 309: 49–55.
Lanz TA and Schachter JB (2006) Demonstration of a common artifact in immunosorbent assays of brain extracts: development of a solid-phase extraction protocol to enable measurement of amyloid- from wild type rodent brain. J Neurosci Methods 157: 71–81.[CrossRef][Medline]
Li J, Fici GJ, Mao CA, Myers RL, Shuang R, Donoho GP, Pauley AM, Himes CS, Qin W, Kola I, et al. (2003) Positive and negative regulation of the a-secretase activity by nicastrin in a murine model. J Biol Chem 278: 33445–33449.
Mehta PD, Dalton AJ, Mehta SP, Kim KS, Sersen EA, and Wiskniewski HM (1998) Increased plasma amyloid protein 1–42 levels in Down syndrome. Neurosci Lett 241: 13–16.[CrossRef][Medline]
Qi Y, Morishima-Kawashima M, Sato T, Mitsumori R, and Ihara Y (2003) Distinct mechanisms by mutant presenilin 1 and 2 leading to increased intracellular levels of amyloid -protein 42 in Chinese hamster ovary cells. Biochemistry 42: 1042–1052.[CrossRef][Medline]
Qi-Takahara Y, Morishima-Kawashima M, Tanimura Y, Dolios G, Hirotani N, Horikoshi Y, Kametani F, Maeda M, Saido TC, Wang R, et al. (2005) Longer forms of amyloid protein: implications for the mechanism of intramembrane cleavage by -secretase. J Neurosci 25: 436–445.
Siemers E, Skinner M, Dean RA, Gonzales C, Satterwhite J, Farlow M, Ness D, and May PC (2005) Safety, tolerability, and changes in amyloid concentrations after administration of a -secretase inhibitor in volunteers. Clin Neuropharmacol 28: 126–132.[CrossRef][Medline]
Weggen S, Eriksen JL, Das P, Sagi SA, Wang R, Pietrzik CU, Findlay KA, Smith TE, Murphy MP, Bulter T, et al. (2001) A subset of NSAIDs lower amyloidogenic A42 independently of cyclooxygenase activity. Nature (Lond) 414: 212–216.[CrossRef][Medline]
Wood KM, Du P, Geoghegan KF, and Tate B (2005) In vivo A peptide profiling by immunoprecipitation and mass spectroscopy. In Proceedings of the Annual Meeting of the Society for Neuroscience; 2005 Nov 12–16; Washington, DC.
Wong GT, Manfra D, Poulet FM, Zhang Q, Josien H, Bara T, Engstrom L, Pinzon-Ortiz M, Fine JS, Lee HJ, et al. (2004) Chronic treatment with the -secretase inhibitor LY-411,575 inhibits -amyloid production and alters lymphopoiesis and intestinal cell differentiation. J Biol Chem 279: 12876–12882.
Yagashita S, Morishima-Kawashima M, Tanimura Y, Ishiura S, and Ihara Y (2006) DAPT-induced intracellular accumulations of longer amyloid -proteins: further implications for the mechanism of intramembrane cleavage by -secretase. Biochemistry 45: 3952–3960.[CrossRef][Medline]
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), CSF (
), and plasma (
). A, A
versus 
represents the two different samples. Mean vehicle A
