Ming-Chih Crouthamel

First Demonstration of Cerebrospinal Fluid and Plasma Aβ Lowering with Oral Administration of a β-Site Amyloid Precursor Protein-Cleaving Enzyme 1 Inhibitor in Nonhuman Primates

β-Site amyloid precursor protein (APP)-cleaving enzyme (BACE) 1 cleavage of amyloid precursor protein is an essential step in the generation of the potentially neurotoxic and amyloidogenic Aβ42 peptides in Alzheimers disease. Although previous mouse studies have shown brain Aβ lowering after BACE1 inhibition, extension of such studies to nonhuman primates or man was precluded by poor potency, brain penetration, and pharmacokinetics of available inhibitors. In this study, a novel tertiary carbinamine BACE1 inhibitor, tertiary carbinamine (TC)-1, was assessed in a unique cisterna magna ported rhesus monkey model, where the temporal dynamics of Aβ in cerebrospinal fluid (CSF) and plasma could be evaluated. TC-1, a potent inhibitor (IC50 ∼ 0.4 nM), has excellent passive membrane permeability, low susceptibility to P-glycoprotein transport, and lowered brain Aβ levels in a mouse model. Intravenous infusion of TC-1 led to a significant but transient lowering of CSF and plasma Aβ levels in conscious rhesus monkeys because it underwent CYP3A4-mediated metabolism. Oral codosing of TC-1 with ritonavir, a potent CYP3A4 inhibitor, twice daily over 3.5 days in rhesus monkeys led to sustained plasma TC-1 exposure and a significant and sustained reduction in CSF sAPPβ, Aβ40, Aβ42, and plasma Aβ40 levels. CSF Aβ42 lowering showed an EC50 of ∼20 nM with respect to the CSF [TC-1] levels, demonstrating excellent concordance with its potency in a cell-based assay. These results demonstrate the first in vivo proof of concept of CSF Aβ lowering after oral administration of a BACE1 inhibitor in a nonhuman primate.

In Vivo β-Secretase 1 Inhibition Leads to Brain Aβ Lowering and Increased α-Secretase Processing of Amyloid Precursor Protein without Effect on Neuregulin-1

β-Secretase (BACE) cleavage of amyloid precursor protein (APP) is one of the first steps in the production of amyloid β peptide Aβ42, the putative neurotoxic species in Alzheimers disease. Recent studies have shown that BACE1 knockdown leads to hypomyelination, putatively caused by a decline in neuregulin (NRG)-1 processing. In this study, we have tested a potent cell-permeable BACE1 inhibitor (IC50 ∼ 30 nM) by administering it directly into the lateral ventricles of mice, expressing human wild-type (WT)-APP, to determine the consequences of BACE1 inhibition on brain APP and NRG-1 processing. BACE1 inhibition, in vivo, led to a significant dose- and time-dependent lowering of brain Aβ40 and Aβ42. BACE1 inhibition also led to a robust brain secreted (s)APPβ lowering that was accompanied by an increase in brain sAPPα levels. Although an increase in full-length NRG-1 levels was evident in 15-day-old BACE1 homozygous knockout (KO) (–/–) mice, in agreement with previous studies, this effect was also observed in 15-day-old heterozygous (+/–) mice, but it was not evident in 30-day-old and 2-year-old BACE1 KO (–/–) mice. Thus, BACE1 knockdown led to a transient decrease in NRG-1 processing in mice. Pharmacological inhibition of BACE1 in adult mice, which led to significant Aβ lowering, was without any significant effect on brain NRG-1 processing. Taken together, these results suggest that BACE1 is the major β-site cleavage enzyme for APP and that its inhibition can lower brain Aβ and redirect APP processing via the potentially nonamyloidogenic α-secretase pathway, without significantly altering NRG-1 processing.

A presenilin-independent aspartyl protease prefers the γ-42 site cleavage

β‐Amyloid peptides (Aβ40 and Aβ42) are the major constituents of amyloid plaques, which are one of the hallmarks of Alzheimers disease (AD). The Aβ is derived from sequential cleavages of amyloid precursor protein (APP) by β‐ and γ‐secretases. γ‐Secretase consists of at least four proteins where presenilins (PS1 and PS2 or PS) are the catalytic subunit involved in the γ‐site cleavage of APP. Secretion of both Aβ40 and Aβ42 is significantly reduced in PS1 knock‐out cells and completely abolished in cells deficient for both PS1 and PS2. Consequently, both the PS proteins play essential roles in the production of the secretory of Aβ from cells. Recent studies in primary neurons, however, suggest that PSs are not required for intracellular Aβ42 accumulation; thus the intracellular Aβ42 appears to be generated in a PS‐independent manner. Here we present the first biochemical evidence indicating that Aβ, especially Aβ42, can be generated in the absence of PS based on an in vitroγ‐secretase assay employing membranes prepared from PS‐deficient Blastocyst‐derived (BD) cells. This PS‐independent γ‐secretase (PSIG) activity is sensitive to the changes in pH and displays an optimal activity at pH 6.0. Pepstatin A is a potent inhibitor for this proteolytic activity with IC50 of 1.2 nm and 0.4 nm for Aβ40 and Aβ42 generation, respectively. These results indicate that these PS‐independent γ‐site cleavages are mediated by an aspartyl protease. More importantly, the PSIG activity displays a distinct preference in mediating the 42‐site cleavage over the 40‐site cleavage, thereby generating Aβ42 as the predominant product.

P4-305: Allosteric potentiation of the M1 muscarinic receptor provides unprecedented selectivity and a novel therapeutic strategy for the treatment of Alzheimer's disease

practice since 5000 years. Objective: The main goal of the studies reported here is to investigate the cerebro-protective and memory improving potentials of PHYLLSARONE-2H. Methods: -amyloid, scopolamine, ibotenic acid, CO2 and aging induced amnesia, KCN and Carotid artery ligationinduced hypoxia were the experimental models. Basal forebrain lesion induced decrease in cerebral Ach and ChAT activity were assessed, Concentrations of Norepinerphrine, Epinephrine, Dopamine, 5-HT in cerebral cortex, cerebellum, hypothalamus, hippocampus, pons and corpus striatum were measured by HPLC analysis. Neuromorphology, contents of MDA, NO, activities of SOA and CAT were also Measured. Exteroceptive models such as Water maze, Hebb william’s maze, Radial arm maze were used to assess acquistion and retention. Results: Neuromorphology, contents of MDA, NO, activities of SOA and CAT were Measured which indicated that PHYLLSARONE-2H significantly protected the experimental animals from hypoxia, stress, amnesia and neurodegeneration. They inhibited KCN and Carotid artery ligation induced hypoxia, reversed amnesia and neurodegeneration induced by -amyloid, scopolamine, ibotenic acid, CO2 and brain aging, produced normalizing action on discrete regions of brain and controlled alterations in neurotransmitter levels due to neurodegeneration. They also decreased ChAT activity in the parietal cortex and inhibited decrease in Ach levels in both parietal and frontal cortex in amnesic rats.Neurohistochemistry revealed that both PHYLLSARONE-2H profoundly reduced the neurodegeneration in the experimental animals. It also improved acquistion and retention capacities of the animals. Conclusions: PHYLLSARONE-2H, profoundly reduced the neurodegeneration, improved learning and memory in the experimental animals. Hence PHYLLSARONE-2H can be of beneficial in the treatment of dementia of Alzheimer’s type and other neurodegenerative disorders.