BACE, a β-secretase, is an attractive potential disease-modifying therapeutic strategy for Alzheimer's disease (AD) as it results directly in the decrease of amyloid precursor protein (APP) processing through the β-secretase pathway and a lowering of CNS amyloid-β (Aβ) levels. The interaction of the β-secretase and α-secretase pathway-mediated processing of APP in the rhesus monkey (nonhuman primate; NHP) CNS is not understood. We hypothesized that CNS inhibition of BACE would result in decreased newly generated Aβ and soluble APPβ (sAPPβ), with increased newly generated sAPPα. A stable isotope labeling kinetics experiment in NHPs was performed with a (13)C6-leucine infusion protocol to evaluate effects of BACE inhibition on CNS APP processing by measuring the kinetics of sAPPα, sAPPβ, and Aβ in CSF. Each NHP received a low, medium, or high dose of MBI-5 (BACE inhibitor) or vehicle in a four-way crossover design. CSF sAPPα, sAPPβ, and Aβ were measured by ELISA and newly incorporated label following immunoprecipitation and liquid chromatography-mass spectrometry. Concentrations, kinetics, and amount of newly generated APP fragments were calculated. sAPPβ and sAPPα kinetics were similar, but both significantly slower than Aβ. BACE inhibition resulted in decreased labeled sAPPβ and Aβ in CSF, without observable changes in labeled CSF sAPPα. ELISA concentrations of sAPPβ and Aβ both decreased and sAPPα increased. sAPPα increased by ELISA, with no difference by labeled sAPPα kinetics indicating increases in product may be due to APP shunting from the β-secretase to the α-secretase pathway. These results provide a quantitative understanding of pharmacodynamic effects of BACE inhibition on NHP CNS, which can inform about target development.

Inhibition of Beta-site APP-cleaving enzyme 1 (BACE1), the β-secretase in the central nervous system (CNS), is an attractive potential disease modifying therapeutic strategy for Alzheimer's disease (AD) as it decreases amyloid precursor protein (APP) processing through the amyloidogenic pathway and CNS β-amyloid peptide levels. The interaction of the β-secretase (amyloidogenic) and α-secretase (non-amyloidogenic) pathway-mediated processing of APP in the rhesus monkey, non-human primate (NHP) CNS is not understood. We hypothesized that CNS inhibition of BACE1 would result in decreased newly-generated amyloid β peptide (Aβ) and sAPPβ, with increased sAPPα. A Stable Isotope Labeling Kinetic (SILK) experiment in NHPs was performed with a 13 C 6 -Leucine infusion protocol to evaluate the effects of a BACE1 inhibitor on CNS processing of APP by measuring the production rates and clearance rates of sAPPα, sAPPβ, and Aβ in cerebrospinal fluid (CSF). Each NHP received vehicle, low dose (10 mg/kg), medium dose (30 mg/kg), and a high dose (125 mg/kg) of the BACE1 inhibitor MBI-5 in a 4-way crossover design. CSF sAPPα, sAPPβ, and Aβ were measured by both ELISA and newly-incorporated label following immunopurification and liquid-chromatography-mass spectrometry. Concentrations, production rates, clearance rates, and total amount of newly-generated APP fragments were calculated. Models were developed to further explore the data and results. Vehicle-treated NHP Aβ production and clearance rates were similar to human measures. sAPPβ and sAPPα production and clearance rates were similar to each other, but much slower than Aβ. The newly-synthesized sAPPα to sAPPβ ratio in NHP CSF is approximately 3:1. Inhibition of BACE1 by MBI-5 resulted in a dose-dependent decrease in newly-labeled CSF sAPPβ and Aβ, without an observable change in newly-labeled CSF sAPPα, while the concentrations of sAPPβ and Aβ both decreased and sAPPα increased as measured by ELISA. While effects on the estimated, newly-synthesized Aβ and sAPPβ levels were directionally consistent to those measured by ELISA, the magnitude of AUC reductions in the newly-synthesized APP fragments, captured during steady state plasma 13 C leucine and peak MBI-5 levels, was greater. These results of BACE1 inhibition in the primate CNS suggest a common pool of APP for both the α-secretase and β-secretase pathways.

Alzheimer's disease (AD) is characterized neuropathologically by the presence of Aß-peptide containing plaques along with neurofibrillary tangles in the brain. Aß generation depends on the proteolytic cleavage of the amyloid precursor protein (APP) by b -secretase; this protease is a prime therapeutic target for the treatment of AD. A BACE inhibitor compound should prevent or reduce the upsurge of Aß accumulation and in theory slow or stop the progression of AD. An animal model was established to allow chronic, simultaneous sampling of cerebral spinal fluid (CSF) and plasma in conscious non-human primates. A dynamic colony of cisterna magna ported (CMP) rhesus monkeys is maintained and used extensively to evaluate the potential of multi-mechanistic agents to alter CSF and plasma biomarkers, with the ability to concurrently measure CSF and plasma drug concentrations. In this study, we compared the effects of the Merck-BACE inhibitor at 30mpk PO gavage, in a crossover fashion with vehicle, n = 6. Baseline CSF and plasma samples were taken prior to single drug or vehicle administration followed by sampling at multiple post dose timepoints over 5 days. Samples were assayed for CSF Aβ40, CSF Aβ42, sAPPa, sAPPb and plasma Aβ40. Post dosing, the compound showed a significant peak reduction relative to both vehicle and baseline values, these reductions were observed as follows: CSF Aβ40 58-65% at 9-13hr; CSF Aβ42 58-67% at 7-13hr; sAPPb 58-72% at 16-25hr; Plasma Aβ40 ∼63% at 9hr, with a peak increase in sAPPa of 60 % at 13-16hr. sAPPa/β ratio had an average increase of ∼180% at 13-25hr. The pharmacokinetic profile of this Merck-BACE inhibitor compound in Rhesus monkeys reveals a Tmax in plasma and CSF of 7hr and 6hr, with a Cmax of 4mM and 0.3mM respectively. These results suggest that the oral administration of this novel BACE inhibitor decreases Aβ significantly in the central compartment in this CMP non-human primate model.

Conscious coronary sinus-cannulated dogs were used to assess the hemodynamic effects and local cardiac norepinephrine (NE) and histamine (HA) release of 4 mechanistically diverse agents either clinically approved or representing a potential novel mechanism for the promotion of wakefulness or attention. Dosing regimens were based on reported or concurrently determined wake-promoting activities in canine models. The central nervous system stimulant, d-amphetamine [0.1 mg x kg(-1) x 10 min intravenous (IV)], significantly elevated mean arterial pressure (+30%) and increased coronary sinus and peripheral venous NE concentrations, indicative of cardiac neurotransmitter release. The selective NE reuptake inhibitor atomoxetine (2.0 mg x kg(-1) x 10 min(-1) IV) and modafinil (30.0 mg x kg(-1) x 10 min(-1) IV) also significantly elevated mean arterial pressure (+15% and +30%, respectively), but with no effect on coronary sinus or peripheral NE concentration, suggesting central mechanisms underlying the hemodynamic effects. The preclinical demonstrations of pressor effects with d-amphetamine, atomoxetine, and modafinil are consistent with clinically reported hemodynamic effects with these agents. The quinazolinone HA receptor subtype H3 inverse agonist 5r (0.3 mg x kg(-1) x 10 min(-1) IV) displayed no effect on hemodynamics or on coronary sinus or peripheral NE and HA concentrations. These data suggest the potential for therapeutic effect with the latter mechanism in the absence of peripheral cardiac neurotransmitter release or obvious changes in cardiovascular function.