Application of a high-resolution in vitro human MDR1-MDCK assay and in vivo studies in preclinical species to improve prediction of CNS drug penetration.

P-glycoprotein (P-gp, MDR1) is expressed at the blood-brain barrier (BBB) and restricts penetration of its substrates into the central nervous system (CNS). In vitro MDR1 assays are frequently used to predict the in vivo relevance of MDR1-mediated efflux at the BBB. It has been well established that drug candidates with high MDR1 efflux ratios (ERs) display poor CNS penetration. Following a comparison of MDR1 transporter function between the MDR1-MDCKI cell line from National Institutes of Health (NIH) and our internal MDR1-MDCKII cell line, the former was found to provide better predictions of in vivo brain penetration than our in-house MDR1-MDCKII cell line. In particular, the NIH MDR1 assay has an improved sensitivity to differentiate the compounds with ERs of <3 in our internal cell line and is able to reduce the risk of false negatives. A better correlation between NIH MDR1 ERs and brain penetration in rat and non-human primate (NHP) was demonstrated. Additionally, a comparison of brain penetration time course of MDR1 substrates and an MDR1 non-substrate in NHP demonstrated that MDR1 interaction can delay the time to equilibrium of drug concentration in the brain with plasma. It is recommended to select highly permeable compounds without MDR1 interaction for rapid brain penetration to produce the maximal pharmacological effect in the CNS with a quicker onset.

Azetidine-based selective glycine transporter-1 (GlyT1) inhibitors with memory enhancing properties.

A strategy to conformationally restrain a series of GlyT1 inhibitors identified potent analogs that exhibited slowly interconverting rotational isomers. Further studies to address this concern led to a series of azetidine-based inhibitors. Compound 26 was able to elevate CSF glycine levels in vivo and demonstrated potency comparable to Bitopertin in an in vivo rat receptor occupancy study. Compound 26 was subsequently shown to enhance memory in a Novel Object Recognition (NOR) behavioral study after a single dose of 0.03 mg/kg, and in a contextual fear conditioning (cFC) study after four QD doses of 0.01-0.03 mg/kg.

Design and Synthesis of Novel and Selective Glycine Transporter-1 (GlyT1) Inhibitors with Memory Enhancing Properties.

We report here the identification and optimization of a novel series of potent GlyT1 inhibitors. A ligand design campaign that utilized known GlyT1 inhibitors as starting points led to the identification of a novel series of pyrrolo[3,4- c]pyrazoles amides (21-50) with good in vitro potency. Subsequent optimization of physicochemical and in vitro ADME properties produced several compounds with promising pharmacokinetic profiles. In vivo inhibition of GlyT1 was demonstrated for select compounds within this series by measuring the elevation of glycine in the cerebrospinal fluid (CSF) of rats after a single oral dose of 10 mg/kg. Ultimately, an optimized lead, compound 46, demonstrated in vivo efficacy in a rat novel object recognition (NOR) assay after oral dosing at 0.1, 1, and 3 mg/kg.