The duration of sleep promoting efficacy by dual orexin receptor antagonists is dependent upon receptor occupancy threshold.

BACKGROUND: Drugs targeting insomnia ideally promote sleep throughout the night, maintain normal sleep architecture, and are devoid of residual effects associated with morning sedation. These features of an ideal compound are not only dependent upon pharmacokinetics, receptor binding kinetics, potency and pharmacodynamic activity, but also upon a compound's mechanism of action. RESULTS: Dual orexin receptor antagonists (DORAs) block the arousal-promoting activity of orexin peptides and, as demonstrated in the current work, exhibit an efficacy signal window dependent upon oscillating levels of endogenous orexin neuropeptide. Sleep efficacy of structurally diverse DORAs in rat and dog was achieved at plasma exposures corresponding to orexin 2 receptor (OX2R) occupancies in the range of 65 to 80%. In rats, the time course of OX2R occupancy was dependent upon receptor binding kinetics and was tightly correlated with the timing of active wake reduction. In rhesus monkeys, direct comparison of DORA-22 with GABA-A modulators at similar sleep-inducing doses revealed that diazepam produced next-day residual sleep and both diazepam and eszopiclone induced next-day cognitive deficits. In stark contrast, DORA-22 did not produce residual effects. Furthermore, DORA-22 evoked only minimal changes in quantitative electroencephalogram (qEEG) activity during the normal resting phase in contrast to GABA-A modulators which induced substantial qEEG changes. CONCLUSION: The higher levels of receptor occupancy necessary for DORA efficacy require a plasma concentration profile sufficient to maintain sleep for the duration of the resting period. DORAs, with a half-life exceeding 8 h in humans, are expected to fulfill this requirement as exposures drop to sub-threshold receptor occupancy levels prior to the wake period, potentially avoiding next-day residual effects at therapeutic doses.

Cisterna magna cannulated repeated CSF sampling rat model--effects of a gamma-secretase inhibitor on Aß levels.

Cerebrospinal fluid (CSF) provides a window into central nervous system (CNS) physiology and pathophysiology in human neurodegenerative conditions such as Alzheimer's disease. Changes in CSF bioanalytes also provide a direct readout of target engagement in the CNS following pharmacological interventions in clinical trials. Given the importance of tracking CNS bioanalytes in drug discovery, we have developed a novel cisterna magna cannulated rat model for repeated CSF sampling and used it to assess an amyloid beta (Aß) lowering agent. The surgically implanted cisterna magna cannula was patent over a period of 1-2 weeks and enabled repeated sampling of CSF (volume of ∼30-50µL/sample) from each rat. CSF Aß40 levels showed good intra-animal variability across time points and inter-animal variability within a time point. Peripheral treatment with a gamma-secretase inhibitor (GSI) led to a rapid and robust decline in CSF Aß40 levels that returned to baseline over 24-96h after dosing. Terminal brain, CSF and plasma Aß levels measured at 24h after dosing demonstrated robust Aß lowering and showed excellent correlation across these compartments. These results are the first pharmacological validation of the repeated CSF sampling rat model for Aß lowering agents. This model can have broad applicability in pharmacological evaluation for diverse CNS targets.

A novel pathway for amyloid precursor protein processing.

Amyloid precursor protein (APP) can be proteolytically processed along two pathways, the amyloidogenic that leads to the formation of the 40-42 amino acid long Alzheimer-associated amyloid ß (Aß) peptide and the non-amyloidogenic in which APP is cut in the middle of the Aß domain thus precluding Aß formation. Using immunoprecipitation and mass spectrometry we have shown that Aß is present in cerebrospinal fluid (CSF) as several shorter isoforms in addition to Aß1-40 and Aß1-42. To address the question by which processing pathways these shorter isoforms arise, we have developed a cell model that accurately reflects the Aß isoform pattern in CSF. Using this model, we determined changes in the Aß isoform pattern induced by α-, ß-, and γ-secretase inhibitor treatment. All isoforms longer than and including Aß1-17 were γ-secretase dependent whereas shorter isoforms were γ-secretase independent. These shorter isoforms, including Aß1-14 and Aß1-15, were reduced by treatment with α- and ß-secretase inhibitors, which suggests the existence of a third and previously unknown APP processing pathway involving concerted cleavages of APP by α- and ß-secretase.