br Secretase modulators a loss of

γ-Secretase modulators: a loss of pharmacology? A lack of translation from in vitro to in vivo pharmacology is an unresolved issue among most GSMs. R-flurbiprofen was tested in clinical trials, however, it did not achieve statistical significance on either of its primary endpoints – cognition or activities of daily living. R-flurbiprofen is a weak GSM with an IC50 for Aβ reduction at approximately 300 μM [100]. Due to its poor Octopamine HCl penetration, it was unlikely to have lowered brain Aβ42 levels in the clinical studies. Treatment of monkeys at 100 mg/kg of ibuprofen or humans with 800 mg single dose did not reveal any changes of Aβ in plasma; CSF Aβ from ibuprofen dosed monkeys did not show any changes [111]. Among 100 CHF5074 treated subjects with MCI, apoEε4 carrier improved on several cognitive measures over the initial three months of treatment [106,107]. During the open label extension period of more than one and half year, apoEε4 carriers maintained their improved cognition and even score better on verbal memory and tests of attention and executive function, compared to baseline performance. Non-carriers’ cognitive abilities have remained stable for almost two years [106,107]. The clinical outcome from apoEε4 carriers versus non-carriers could be explained by findings that apoEε4 carriers usually have more neuroinflammation compared to non-carriers [5], thus it was easier to achieve any anti-neuroinflammatory benefit of CHF5074. However, the IC50 for Aβ42 was at 3.6 μM in cultured cells, indicating that CHF5074 is a weak GSM [103,104]. Another class of non-NSAID derivative GSMs include Eisai’s E-2012 [112,113] and NeuroGenetics’ Compound 4 (Cpd 4) [114]. While Cpd4 directly interacts with PS and Pen2, the binding protein for E-2012 is not clear. When brain exposure was over 100-fold of IC50, Cpd 4 inhibited all three Aβ peptides (Aβ38, 40 and 42) in animals under chronic treatment [114], which is similar to Eisai’s E-2012 that inhibits both Aβ40 and Aβ42 [112,113]. In addition, a number of GSMs were reported by Merck [[115], [116], [117]], and among them, one GSM showed 70% Aβ42 reduction when brain GSM exposure reached 7.8 μM (∼400-fold of IC50) [117]. More potent GSMs with IC50 at sub-μM have been reported [[118], [119], [120]]. GSM-10h is a NSAID-derived GSM with an in vitro IC50 of 0.8 μM. In a transgenic mouse line expressing mutant APP and PS1, GSM-10h brain and plasma levels at 6 hpd reached 54.7 μM and 32.9 μM, respectively, which were 40–70 fold of IC50, but brain Aβ42 was reduced by about 20% [121]. There was a concomitant >30% increase in Aβ38, with no effect on Aβ40 [122]. In rats, GSM-10h caused a dose-dependent decrease in the level of Aβ42, but not Aβ40, in brain, CSF, and plasma [123]. An analogue of GSM-10h, GSM-1, carries an in vitro IC50 at 0.35 μM, causes a dose dependent decrease of Aβ42 and an increase of Aβ38 in mice expressing Swedish mutant APP [80,124]. Among the GSMs with IC50 at sub-μM, EVP-A, EVP-B and JNJ-40418677 show similar potency. EVP-A and EVP-B (EnVivo, later Forum Pharmaceuticals) showed in vitro IC50 for reduction of Aβ40 and Aβ42 at 0.24 μM and 0.14 μM, respectively. In rats, a brain concentration of 2.7 μM EVP-A produced no reduction of Aβ, while a brain concentration of 10 μM (40–70 fold above the IC50) of EVP-B produced a 20–30% reduction of brain Aβ [125]. A better compound EVP-0015962 showed a similar IC50 of 0.12 μM in stable human cells, a 4-fold higher IC50 of 0.49 μM in neuronal cells, and no effect on Notch processing. Dose dependent reduction of Aβ42 was observed in rat models. Brain exposures at 2.8 μM and 8.3 μM (5- and 17-fold of IC50) led to a 22% and 38% reduction in brain Aβ42 respectively. Chronic dosing at 20 and 60 mg/kg/day in APP transgenic mice for 6 months led to a lowering of brain plaque load of 81% and 95% respectively [126,127]. A GSM with a similar potency to the EnVivo compounds, JNJ-40418677, selectively inhibited Aβ42 production with IC50s in neuroblastoma cells and primary rat cortical neuronal cultures of 0.20 μM and 0.18 μM, respectively [128]. A lack of effect of JNJ-40418677 on α- and β-secretase was confirmed by visualizing unchanged APP CTFα and CTFβ. In cell-free APP and Notch assays in vitro, JNJ-40418677 did not affect the AICD generation at 100 μM and NICD at 10 μM. Although the difference in its effect on AICD and NICD generation is not clear, a 50-fold selection for Aβ42 inhibition over NICD inhibition was achieved [128]. In mice, four hours after a single oral dose, both brain and plasma GSM exposures achieved 17 μM (85-fold of IC50), with a brain/plasma ratio of 1. Between 2 and 24 h, Aβ42 levels were significantly reduced, and total Aβ levels were not changed in the brain. Chronic dosing of JNJ-40418677 in Tg2576 mice for 7-months at doses of 20, 60 and 120 mg/kg/day led to corresponding dose dependent effect. When brain exposure was at 2-fold of IC50 (0.42 μM), no effect on Aβ levels was found. When the brain exposure reached 12-fold of IC50 (2.4 μM) or higher, a significant reduction of Aβ42 was observed, and all three Aβ peptides, Aβ38, 40, and 42, were reduced. The Aβ reduction correlated with a significant reduction in the numbers of plaques that contained Aβ38, Aβ40 and Aβ42 [128]. This is similar to “Notch sparing” GSI, BMS-708163, that reduces levels of CSF Aβ38, 40 and 42 [129]. Therefore, chronic dosing of JNJ-40418677 in animals led to a complete inhibition of all Aβ peptides, a feature like that of GSI.