CNS amyloid-ß, soluble APP-α and -ß kinetics during BACE inhibition.

BACE, a ß-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 ß-secretase pathway and a lowering of CNS amyloid-ß (Aß) levels. The interaction of the ß-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 Aß and soluble APPß (sAPPß), 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, sAPPß, and Aß 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, sAPPß, and Aß 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. sAPPß and sAPPα kinetics were similar, but both significantly slower than Aß. BACE inhibition resulted in decreased labeled sAPPß and Aß in CSF, without observable changes in labeled CSF sAPPα. ELISA concentrations of sAPPß and Aß 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 ß-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.

Acute gamma-secretase inhibition of nonhuman primate CNS shifts amyloid precursor protein (APP) metabolism from amyloid-beta production to alternative APP fragments without amyloid-beta rebound.

The accumulation of amyloid beta (Abeta) in Alzheimer's disease is caused by an imbalance of production and clearance, which leads to increased soluble Abeta species and extracellular plaque formation in the brain. Multiple Abeta-lowering therapies are currently in development: an important goal is to characterize the molecular mechanisms of action and effects on physiological processing of Abeta, as well as other amyloid precursor protein (APP) metabolites, in models which approximate human Abeta physiology. To this end, we report the translation of the human in vivo stable-isotope-labeling kinetics (SILK) method to a rhesus monkey cisterna magna ported (CMP) nonhuman primate model, and use the model to test the mechanisms of action of a gamma-secretase inhibitor (GSI). A major concern of inhibiting the enzymes which produce Abeta (beta- and gamma-secretase) is that precursors of Abeta may accumulate and cause a rapid increase in Abeta production when enzyme inhibition discontinues. In this study, the GSI MK-0752 was administered to conscious CMP rhesus monkeys in conjunction with in vivo stable-isotope-labeling, and dose-dependently reduced newly generated CNS Abeta. In contrast to systemic Abeta metabolism, CNS Abeta production was not increased after the GSI was cleared. These results indicate that most of the CNS APP was metabolized to products other than Abeta, including C-terminal truncated forms of Abeta: 1-14, 1-15 and 1-16; this demonstrates an alternative degradation pathway for CNS amyloid precursor protein during gamma-secretase inhibition.

Laser Doppler imaging of reactive hyperemia exposes blood flow deficits in a rat model of experimental limb ischemia.

The primary pathophysiology of peripheral artery occlusive disease is associated with impaired perfusion to the lower extremities. The lack of effective pharmacologic agents to treat this disease emphasizes the need for well-characterized animal models that can be used to evaluate the efficacy of emerging therapies. A major limitation with the current animal models of peripheral artery occlusive disease is that the variety of surgical methods employed to reduce peripheral blood flow produce differences in the severity and time course of the resting and reserve blood flow deficits. Furthermore, the methods used to evaluate the restoration of peripheral flow are often not suitable for serial measurements. This study used laser Doppler imaging to serially evaluate resting blood flow and the development of a functional collateral circulation after the induction of hind limb ischemia in the rat. Reserve blood flow was assessed by measuring hyperemic blood flow in the hind paw after temporary arterial occlusion. The magnitude of the hyperemic response was found to be dependent upon both the duration of arterial occlusion and the measurement time after release of the occluder. After ligation of the common iliac artery, but at a time when resting blood flow was reestablished, hyperemic tests unmasked a sustained deficit in reserve blood flow capacity that persisted for at least 14 days. Therefore, the use of a noninvasive vascular occluder and laser Doppler imaging represents a sensitive and consistent technique to measure peripheral blood flow status to assess the development of functional collateral vessels. These findings will enhance the ability to effectively study pharmacologic therapies aimed at promoting the growth and development of collateral vessels.

Effect of odanacatib on bone turnover markers, bone density and geometry of the spine and hip of ovariectomized monkeys: a head-to-head comparison with alendronate.

Odanacatib (ODN) is a selective and reversible Cathepsin K (CatK) inhibitor currently being developed as a once weekly treatment for osteoporosis. Here, effects of ODN compared to alendronate (ALN) on bone turnover, DXA-based areal bone mineral density (aBMD), QCT-based volumetric BMD (vBMD) and geometric parameters were studied in ovariectomized (OVX) rhesus monkeys. Treatment was initiated 10 days after ovariectomy and continued for 20 months. The study consisted of four groups: L-ODN (2 mg/kg, daily p.o.), H-ODN (8/4 mg/kg daily p.o.), ALN (15 µg/kg, twice weekly, s.c.), and VEH (vehicle, daily, p.o.). L-ODN and ALN doses were selected to approximate the clinical exposures of the ODN 50-mg and ALN 70-mg once-weekly, respectively. L-ODN and ALN effectively reduced bone resorption markers uNTx and sCTx compared to VEH. There was no additional efficacy with these markers achieved with H-ODN. Conversely, ODN displayed inversely dose-dependent reduction of bone formation markers, sP1NP and sBSAP, and L-ODN reduced formation to a lesser degree than ALN. At month 18 post-OVX, L-ODN showed robust increases in lumbar spine aBMD (11.4%, p<0.001), spine trabecular vBMD (13.7%, p<0.001), femoral neck (FN) integral (int) vBMD (9.0%, p<0.001) and sub-trochanteric proximal femur (SubTrPF) int vBMD, (6.4%, p<0.001) compared to baseline. L-ODN significantly increased FN cortical thickness (Ct.Th) and cortical bone mineral content (Ct.BMC) by 22.5% (p<0.001) and 21.8% (p<0.001), respectively, and SubTrPF Ct.Th and Ct.BMC by 10.9% (p<0.001) and 11.3% (p<0.001) respectively. Compared to ALN, L-ODN significantly increased FN Ct. BMC by 8.7% (p<0.05), and SubTrPF Ct.Th by 7.6% (p<0.05) and Ct.BMC by 6.2% (p<0.05). H-ODN showed no additional efficacy compared to L-ODN in OVX-monkeys in prevention mode. Taken together, the results from this study have demonstrated that administration of ODN at levels which approximate clinical exposure in OVX-monkeys had comparable efficacy to ALN in DXA-based aBMD and QCT-based vBMD. However, FN cortical mineral content clearly demonstrated superior efficacy of ODN versus ALN in this model of estrogen-deficient non-human primates.

High-resolution peripheral quantitative computed tomography and finite element analysis of bone strength at the distal radius in ovariectomized adult rhesus monkey demonstrate efficacy of odanacatib and differentiation from alendronate.

Translational evaluation of disease progression and treatment response is critical to the development of therapies for osteoporosis. In this study, longitudinal in-vivo monitoring of odanacatib (ODN) treatment efficacy was compared to alendronate (ALN) in ovariectomized (OVX) non-human primates (NHPs) using high-resolution peripheral computed tomography (HR-pQCT). Treatment effects were evaluated using several determinants of bone strength, density and quality, including volumetric bone mineral density (vBMD), three-dimensional structure, finite element analysis (FEA) estimated peak force and biomechanical properties at the ultradistal (UD) radius at baseline, 3, 6, 9, 12, and 18 months of dosing in three treatment groups: vehicle (VEH), low ODN (2 mg/kg/day, L-ODN), and ALN (30 µg/kg/week). Biomechanical axial compression tests were performed at the end of the study. Bone strength estimates using FEA were validated by ex-vivo mechanical compression testing experiments. After 18months of dosing, L-ODN demonstrated significant increases from baseline in integral vBMD (13.5%), cortical thickness (24.4%), total bone volume fraction BV/TV (13.5%), FEA-estimated peak force (26.6%) and peak stress (17.1%), respectively. Increases from baseline for L-ODN at 18 months were significantly higher than that for ALN in DXA-based aBMD (7.6%), cortical thickness (22.9%), integral vBMD (12.2%), total BV/TV (10.1%), FEA peak force (17.7%) and FEA peak stress (11.5%), respectively. These results demonstrate a superior efficacy of ODN treatment compared to ALN at the UD radii in ovariectomized NHPs.