Alzheimer's disease (AD) patients suffer from an inexorable loss of cognitive function due in part to the degeneration of the basal forebrain cholinergic projection neurons. Restoring signalling through the post-synaptic muscarinic acetylcholine receptors that mediate cholinergic signalling is thus a promising therapeutic strategy for the symptomatic management of AD. Moreover muscarinic receptor agonists reduce Aβ42 in animal models, suggesting that this class of compounds could address the underlying disease pathology. Unfortunately, multiple non-selective muscarinic receptor agonists have been tested in AD patients and were not tolerated due to unwanted peripheral cholinergic stimulation. Improved selectivity for M1, the most prominently expressed post-synaptic muscarinic receptor in the cortex and hippocampus, could address these tolerability issues, however the high conservation of the acetylcholine binding site between receptor subtypes has precluded the discovery of selective agonists. To achieve selectivity for M1 by discovering and exploiting an allosteric ligand binding site. We screened a chemical library for positive allosteric modulators of the M1 receptor and assessed chemical leads for selectivity in vitro and efficacy in rodent AD models in vivo. We identified Benzyl Quinolone Carboxylic Acid (BQCA), a small molecule potentiator selective for M1. In CHO cells expressing recombinant human receptor, BQCA sensitizes M1 to acetylcholine 83.9-fold (inflection point = 449 nM), while having no potentiation, agonist, or antagonist effect on M2, M3, or M4 receptors up to 100 μM. In the mouse contextual fear conditioning model of episodic-like memory, BQCA fully reversed the cognitive impairment caused by the non-selective muscarinic antagonist scopolamine, demonstrating a critical role for M1 in this type of memory. Importantly BQCA does not show signs of unwanted peripheral cholinergic stimulation at doses that produce central physiological responses. In vivo BQCA and other muscarinic agonists can specifically reduce cortical Aβ42 in some experiments. However, this effect is likely complex and indirect, since it does not occur in cultured neurons and is not consistently observed across species and disease models. BQCA is a highly selective pharmacological reagent for understanding the normal physiology of the M1 receptor and its potential as a therapeutic target for AD.
BACE cleavage of Amyloid Precursor Protein (APP) is one of the first steps in the generation of amyloid peptides, including the potentially neurotoxic Aβ42 species. We have previously shown that potent cell-permeable BACE inhibitors can lower brain Aβ levels when introduced directly into the lateral ventricles of mice (Sankaranarayanan et al, JPET, 2007), injected systemically (Sankaranarayanan et al, ICAD, Madrid 2006) or infused intravenously into rhesus monkeys (Simon et al, Salzburg AD/PD 2007). We have further optimized various amyloid-reducing BACE inhibitors and now report oral BACE inhibition in a unique, chronically implanted non-human primate model (Cook et al, submitted). The compound utilized in the present study has an enzymatic IC50 of 0.4 nM and cell culture IC50 of 40 nM, and PK properties suitable for in vivo use. In CSF and plasma samples collected from N = 8 conscious rhesus monkeys treated over a three and one-half day period in a cross-over study design, we observed a significant AUC reduction in CSF Aβ40 (42 %, p<0.001) and CSF Aβ42 (43 %, p<0.002). As anticipated from our previous rhesus infusion studies, CSF sAPPβ levels significantly decreased (41 %, p<0.0002), while sAPPα levels trended upwards (not significant). In the periphery, plasma Aβ40 was dramatically reduced (61 %, p<0.005). BACE inhibition in the monkey exhibits good correlation between sAPPβ and CSF Aβ42 (r=0.78, p<0.0001) as well as between CSF Aβ42 and Aβ40 (r=0.94, p<0.0001), while CSF Aβ40 to Plasma Aβ40 shows increased scatter and variability (r=0.42, p<0.0001). These results are the first reported demonstration of in vivo pharmacological evidence in primates that acute brain Aβ lowering can be achieved after oral administration of a BACE inhibitor.
The cathepsin K (CatK) enzyme is abundantly expressed in osteoclasts, and CatK inhibitors have been developed for the treatment of osteoporosis. In our effort to support discovery and clinical evaluations of a CatK inhibitor, we sought to discover a radioligand to determine target engagement of the enzyme by therapeutic candidates using positron emission tomography (PET). L-235, a potent and selective CatK inhibitor, was labeled with carbon-11. PET imaging studies recording baseline distribution of [11 C]L-235, and chase and blocking studies using the selective CatK inhibitor MK-0674 were performed in juvenile and adult nonhuman primates (NHP) and ovariectomized rabbits. Retention of the PET tracer in regions expected to be osteoclast-rich compared with osteoclast-poor regions was examined. Increased retention of the radioligand was observed in osteoclast-rich regions of juvenile rabbits and NHP but not in the adult monkey or adult ovariectomized rabbit. Target engagement of CatK was observed in blocking studies with MK-0674, and the radioligand retention was shown to be sensitive to the level of MK-0674 exposure. [11 C]L-235 can assess target engagement of CatK in bone only in juvenile animals. [11 C]L-235 may be a useful tool for guiding the discovery of CatK inhibitors.
We report herein the discovery of a fatty acid amide hydrolase (FAAH) positron emission tomography (PET) tracer. Starting from a pyrazole lead, medicinal chemistry efforts directed toward reducing lipophilicity led to the synthesis of a series of imidazole analogues. Compound 6 was chosen for further profiling due to its appropriate physical chemical properties and excellent FAAH inhibition potency across species. [(11)C]-6 (MK-3168) exhibited good brain uptake and FAAH-specific signal in rhesus monkeys and is a suitable PET tracer for imaging FAAH in the brain.
Background: Relatively large disulfide-linked polypeptides can serve as signaling molecules for a diverse array of biological processes and may be studied in animal models to investigate their function in vivo. The aim of this work was to develop an LC-MS/MS assay to measure a model peptide, INSL3, in rat plasma. Results: A dual enrichment strategy incorporating both protein precipitation and solid phase extraction was utilized to isolate INSL3 from rat plasma, followed by targeted LC-MS/MS detection. The method was able to measure full-length INSL3 (6.1 kDa) down to 0.2 ng/ml with acceptable accuracy and precision. Conclusion: The final assay was applied to support an exploratory pharmacokinetic study to evaluate steady-state concentrations of dosed INSL3 in rat plasma.
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