Leonard N. Boggs

Functional gamma‐secretase inhibitors reduce beta‐amyloid peptide levels in brain

Converging lines of evidence implicate the beta‐amyloid peptide (Aβ) as causative in Alzheimers disease. We describe a novel class of compounds that reduce Aβ production by functionally inhibiting γ‐secretase, the activity responsible for the carboxy‐terminal cleavage required for Aβ production. These molecules are active in both 293 HEK cells and neuronal cultures, and exert their effect upon Aβ production without affecting protein secretion, most notably in the secreted forms of the amyloid precursor protein (APP). Oral administration of one of these compounds, N‐[N‐(3,5‐difluorophenacetyl)‐l‐alanyl]‐S‐phenylglycine t‐butyl ester, to mice transgenic for human APPV717F reduces brain levels of Aβ in a dose‐dependent manner within 3 h. These studies represent the first demonstration of a reduction of brain Aβin vivo. Development of such novel functional γ‐secretase inhibitors will enable a clinical examination of the Aβ hypothesis that Aβ peptide drives the neuropathology observed in Alzheimers disease.

Neurotoxicity of Human Amylin in Rat Primary Hippocampal Cultures: Similarity to Alzheimer's Disease Amyloid-β Neurotoxicity

Amylin, a 37‐amino‐acid amyloidogenic peptide, bears biophysical similarities to the amyloid‐β peptide (Aβ) deposited in Alzheimers disease. Using embryonic rat hippocampal cultures we tested whether amylin induces neurotoxicity similar to that previously observed with Aβ(1–40). Treatment with human amylin (1–37) resulted in prominent toxicity as assessed by phasecontrast microscopy and quantification of lactate dehydrogenase in the medium. Amylin‐induced neurotoxicity was morphologically similar to that induced by Aβ(1–40). In contrast, the nonamyloidogenic rat amylin showed negligible neurotoxicity despite having 95% sequence similarity to human amylin. Only full‐length human amylin was toxic; various amylin peptide fragments including amino acid residues 20–29 were nontoxic at similar concentrations. These studies suggest that unrelated amyloidogenic peptides like human amylin and Aβ can adopt a similar neurotoxic conformation in vitro. Similar conformation‐dependent neurotoxicity may drive the prominent neurite degeneration around compacted but not diffuse deposits of Aβ in Alzheimers disease.

Clusterin (Apo J) Protects Against In Vitro Amyloid-β(1–40) Neurotoxicity

Abstract: Clusterin is a secreted glycoprotein that is markedly induced in many disease states and after tissue injury. In the CNS, clusterin expression is elevated in neuropathological conditions such as Alzheimers disease (AD), where it is found associated with amyloid‐β (Aβ) plaques. Clusterin also coprecipitates with Aβ from CSF, suggesting a physiological interaction with Aβ. Given this interaction with Aβ, the goal of this study was to determine whether clusterin could modulate Aβ neurotoxicity. A mammalian recombinant source of human clusterin was obtained by stable transfection of hamster kidney fibroblasts with pADHC‐9, a full‐length human cDNA clone for clusterin. Recombinant clusterin obtained from this cell line, as well as a commercial source of native clusterin purified from serum, afforded dose‐dependent neuroprotection against Aβ(1–40) when tested in primary rat mixed hippocampal cultures. Clusterin afforded substoichiometric neuroprotection against several lots of Aβ(1–40) but not against H2O2 or kainic acid excitotoxicity. These results suggest that the elevated expression of clusterin found in AD brain may have effects on subsequent amyloid‐β plaque pathology.

The Potent BACE1 Inhibitor LY2886721 Elicits Robust Central Aβ Pharmacodynamic Responses in Mice, Dogs, and Humans

BACE1 is a key protease controlling the formation of amyloid β, a peptide hypothesized to play a significant role in the pathogenesis of Alzheimers disease (AD). Therefore, the development of potent and selective inhibitors of BACE1 has been a focus of many drug discovery efforts in academia and industry. Herein, we report the nonclinical and early clinical development of LY2886721, a BACE1 active site inhibitor that reached phase 2 clinical trials in AD. LY2886721 has high selectivity against key off-target proteases, which efficiently translates in vitro activity into robust in vivo amyloid β lowering in nonclinical animal models. Similar potent and persistent amyloid β lowering was observed in plasma and lumbar CSF when single and multiple doses of LY2886721 were administered to healthy human subjects. Collectively, these data add support for BACE1 inhibition as an effective means of amyloid lowering and as an attractive target for potential disease modification therapy in AD.

Regulation of Cytokine Secretion and Amyloid Precursor Protein Processing by Proinflammatory Amyloid Beta (Aβ)

Abstract: Neurodegenerative processes in Alzheimers disease (AD) are thought to be driven, in part, by the deposition of amyloid beta (Aβ), a 39‐43‐aminoacid peptide product resulting from an alternative cleavage of amyloid precursor protein (APP). In addition to its neurotoxic properties, Aβ may influence neuropathology by stimulating glial cell cytokine and acute phase protein secretion in affected areas of the brain (e.g., cortex, hippocampus). Using an in vitro human astrocyte model (U‐373 MG astrocytoma cells), the effects of Aβ treatment on acute phase protein (APP and alpha‐1‐antichymot rypsin [α1‐ACT]) and interleukin‐8 (IL‐8) were examined. U‐373 MG cells secreted increased levels of α1‐ACT and neurotrophic/neuroprotective alpha‐cleaved APP (αAPP) after exposure to interleukin‐1β (IL‐1β) for 24 hours. Aβ treatment resulted in a similar, but modest increase in α1‐ACT secretion, a two‐ to threefold stimulation of IL‐8 production, and, conversely, a profound reduction in the levels of secreted αAPPs. Aβ inhibited αAPP secretion by U‐373 MG cells in a concentration‐ and conformation‐dependent manner. Moreover, the reduction in αAPP secretion was accompanied by an increase in cell‐associated APP. Another proinflammatory amyloidogenic peptide, human amylin, similarly affected APP processing in U‐373 astrocytoma cells. These data suggest that Aβ may contribute to Alzheimers‐associated neuropathology by lowering the production of neuroprotective/neurotrophic αAPPs. Moreover, the concomitant increase in cell‐associated APP may provide increased substrate for the generation of amyloidogenic peptides within astrocytes.

Biochemical and kinetic characterization of BACE1: investigation into the putative species-specificity for β- and β′-cleavage sites by human and murine BACE1

β‐amyloid peptides (Aβ) are produced by a sequential cleavage of amyloid precursor protein (APP) by β‐ and γ‐secretases. The lack of Aβ production in beta‐APP cleaving enzyme (BACE1)–/– mice suggests that BACE1 is the principal β‐secretase in mammalian neurons. Transfection of human APP and BACE1 into neurons derived from wild‐type and BACE1–/– mice supports cleavage of APP at the canonical β‐secretase site. However, these studies also revealed an alternative BACE1 cleavage site in APP, designated as β′, resulting in Aβ peptides starting at Glu11. The apparent inability of human BACE1 to make this β′‐cleavage in murine APP, and vice versa, led to the hypothesis that this alternative cleavage was species‐specific. In contrast, the results from human BACE1 transgenic mice demonstrated that the human BACE1 is able to cleave the endogenous murine APP at the β′‐cleavage site. To address this discrepancy, we designed fluorescent resonance energy transfer peptide substrates containing the β‐ and β′‐cleavage sites within human and murine APP to compare: (i) the enzymatic efficiency; (ii) binding kinetics of a BACE1 active site inhibitor LY2039911; and (iii) the pharmacological profiles for human and murine recombinant BACE1. Both BACE1 orthologs were able to cleave APP at the β‐ and β′‐sites, although with different efficiencies. Moreover, the inhibitory potency of LY2039911 toward recombinant human and native BACE1 from mouse or guinea pig was indistinguishable. In summary, we have demonstrated, for the first time, that recombinant BACE1 can recognize and cleave APP peptide substrates at the postulated β′‐cleavage site. It does not appear to be a significant species specificity to this cleavage.

COMBINATION THERAPY WITH A PLAQUE-SPECIFIC ABETA ANTIBODY AND A BACE INHIBITOR RESULTS IN DRAMATIC PLAQUE REDUCTION IN A DOSE-DEPENDENT MANNER IN AGED PDAPP TRANSGENIC MICE

lowed. Results:High antibody responses were found in both species. In the rabbit, 277.16 118.4 mg/ml plasmaweremeasured after 5 immunizations with no significant differences between the dose groups. Antibody levels declined slightly to 246.66 109.5 mg/ml after a resting period of two months. Low levels of IFNg and IL-17 secreting splenocytes were found in the ELISPOTassays from rabbit splenocytes. Rhesusmonkeys reachedmean antibody levels of 114.2 6 41.67 mg/ml plasma after five immunizations. The isotype profile was high on IgG4 antibodies, indicative of a Th2 immune response. After three immunizations, no IL-17 or IFNg producing cells were found in ELISPOT assays from PBMCs. Conclusions: DNA Ab42 immunization leads to high antibody titers in large mammals, and is likely to produce high antibody levels and a safe (Th2 biased) immune response in humans as well. supported by NIA/NIH P30AG12300-21, Zale Foundation, Rudman Foundation, AWARE, Presbyterian Village North, Freiberger, and Denker Family Funds

Proof-of-concept pharmacodynamic assessment of a prototypic BACE1 inhibitor at steady-state using IV infusion dosing in the PDAPP transgenic mouse model of Alzheimer's disease

Background: BACE1 is a key protease involved in Abeta generation but has proven challenging as a drug target. Many early BACE1 inhibitors which were optimized for potency at the enzyme, failed to achieve sufficient brain penetration to demonstrate central pharmacodynamic (PD) effects in vivo. Methods: Young PDAPP transgenic mice (n 1⁄4 10/group) were surgically implanted with a central catheter and dosed IV over a period of 22 hrs with vehicle or BACE1 inhibitor at 4.3, 8.6 or 14.4 mg/ml at 60 ul/hr rate. Doses were determined by prior pharmacokinetic measurements using a single IV bolus and calculated to reach steady-state levels exceeding their cellular IC50. Plasma and brain Ab (1-X) levels were measured using human-specific total Ab (1-X) ELISAs. The proximal biomarkers of BACE1 cleavage, C99 and sAPPbeta, were also measured in brain homogenates using appropriately configured sandwich ELISAs. Compound exposure was measured in plasma and remaining brain using LC/MS. Results: Steady-state IV infusion of this small molecule BACE1 inhibitor in PDAPP transgenic mice resulted in dose-dependent decreases in Ab, C99 and sAPPbeta measured in brain. Plasma Ab levels were not measurable beyond the lowest dose due to the robust effect of treatment. The reductions in brain biomarkers were dose-dependent and a change of similar magnitude occurred in all three in both brain regions (w71-85% decrease in the high dose group). The exposures measured in the plasma and brains increased with dose and were consistent with the dose-dependent efficacy observed. There were no adverse reactions to these doses in this study. Conclusions: IV administration of a prototypic BACE1 small molecule inhibitor to steady state produces robust reduction of brain Abeta levels by a mechanism of action consistent with BACE1 inhibition in vivo. Robust BACE1 inhibition can be maintained for 22 hours without significant adverse events offering hope for targeting this protease for the treatment of AD.

A CORRELATIONAL ANALYSIS OF EXPOSURE AND PHARMACODYNAMIC EFFECTS OF THE BACE1 INHIBITOR LY3202626 IN PDAPP MICE FOLLOWING ACUTE ORAL DOSING

Background: LY3202626 is a potent, freely CNS-penetrant small molecule BACE1 inhibitor in development for the treatment of Alzheimer’s disease (AD). Herein, we demonstrate strong pharmacokinetic / pharmacodynamic (PK/PD) relationships in PDAPP mice between central LY3202626 exposure and central (hippocampal and cortical brain tissue) BACE1 inhibition as determined by quantifying markers of amyloid precursor protein (APP) metabolism. Methods: In a dose response study, young female PDAPP mice (n1⁄46/group) were orally administered 0, 0.3, 1.0, or 3.0 mg LY3202626/kg and were sacrificed at 3 hr post-dose. In a separate time course study, young female PDAPP mice were sacrificed at 3, 6, 9, or 12 hours following a 3 mg LY3202626/kg oral dose. In all studies, LY3202626 concentrations were determined in plasma and brain samples by LC/MS/MS and concentrations of sAPPbeta, C99 and Abeta 1-X were determined in hippocampus and cortex using ELISA methodology. Results: Oral administration of LY3202626 to PDAPPmice produced dose-dependent reductions in brain Abeta, C99, and sAPPbeta with LY3202626 brain concentrations negatively-correlated with all three PD endpoints (r values > 0.56). Changes in each BACE1 biomarker were similar in the cortical and hippocampal brain regions (all r values > 0.90). In a timecourse study following a dose of 3 mg/kg LY3202626, free brain exposure over the 3-12 hour time-course study correlated well with hippocampal Abeta 1-X changes (r 1⁄4 0.60), indicating that the robust PK/PD relationship was maintained over time. All correlations were significant, with p-values <0.0001. Observed free (unbound) concentrations in brain and plasma across these studies suggest LY3202626 is highly brain penetrant in PDAPP mice. Conclusions:LY3202626 is a potent inhibitor of BACE1. Administration of LY3202626 results in significant changes in central biomarkers of BACE1 activity, and the magnitude of these changes correlate well with observed.

A COMPARISON OF IN VIVO POTENCY IN TWO BACE INHIBITORS AS DETERMINED IN HUMANS AND DOGS

P1-365 A COMPARISON OF IN VIVO POTENCY IN TWO BACE INHIBITORS AS DETERMINED IN HUMANS AND DOGS Brian A. Willis, Patrick C. May, Scott A. Monk, Stephen L. Lowe, Stuart Friedrich, Dustin J. Mergott, Leonard Boggs, Hakop Gevorkyan, Stan Jhee, Robert A. Dean, Masako Nakano, Celedon R. Gonzales, Eli Lilly and Co., Indianapolis, Indiana, United States; Eli Lilly and Company, Indianapolis, Indiana, United States; Lilly NUS Center of Clinical Pharmacology, Singapore, Singapore; Eli Lilly and Company, Indianapolis, Indiana, United States; Eli Lilly and Co., Indianapolis, Indiana, United States; Parexel International, Glendale, California, United States; Eli Lilly Japan K.K., Kobe, Japan. Contact e-mail: p.c. may@lilly.com