Discovery of Potent, Orally Bioavailable, Tricyclic NLRP3 Inhibitors.

NLRP3 is a molecular sensor recognizing a wide range of danger signals. Its activation leads to the assembly of an inflammasome that allows for activation of caspase-1 and subsequent maturation of IL-1ß and IL-18, as well as cleavage of Gasdermin-d and pyroptotic cell death. The NLRP3 inflammasome has been implicated in a plethora of diseases including gout, type 2 diabetes, atherosclerosis, Alzheimer's disease, and cancer. In this publication, we describe the discovery of a novel, tricyclic, NLRP3-binding scaffold by high-throughput screening. The hit (1) could be optimized into an advanced compound NP3-562 demonstrating excellent potency in human whole blood and full inhibition of IL-1ß release in a mouse acute peritonitis model at 30 mg/kg po dose. An X-ray structure of NP3-562 bound to the NLRP3 NACHT domain revealed a unique binding mode as compared to the known sulfonylurea-based inhibitors. In addition, NP3-562 shows also a good overall development profile.

Non-clinical characterization of the disposition of EMA401, a novel small molecule angiotensin II type 2 receptor (AT2R) antagonist.

EMA401, (the S-enantiomer of 5-(benzyloxy)-2-(2,2-diphenylacetyl)-6-methoxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), also known as Olodanrigan, is an orally active selective angiotensin II type 2 receptor (AT2 R) antagonist that is in Phase IIb clinical development as a novel analgesic for the relief of chronic pain. The main purpose of the present work was to investigate the disposition of a single 14 C- labeled EMA401 in non-clinical studies. The in vitro metabolism studies of EMA401 were undertaken to understand the hepatic biotransformation pathways in animal species used in toxicology studies and how they compare to human. Furthermore, investigation of EMA401's PK was carried out in vivo in rats. The study demonstrates the rapid absorption and distribution of drug-related material mainly to the tissues associated with absorption and elimination (GI tract, liver, and kidney). EMA401was then readily eliminated metabolically via the bile (95% of dose) predominantly in the form of the direct acylglucuronide (40% of dose), which was further hydrolysed by the intestinal flora to the active parent drug. Other metabolic pathways such as dealkylations and hydroxylation were also involved in the elimination of EMA401 to a lesser extent. EMA401 was metabolically unstable in hepatocytes of all species investigated and the key metabolites produced in the in vitro model were also detected in vivo. Independent of the dosing route, the S-enantiomer EMA401 showed a good in vivo chiral stability. Overall, the present study provides the first full characterization of the disposition of EMA401 in preclinical species.

Further Assessment of the Relay Hepatocyte Assay for Determination of Intrinsic Clearance of Slowly Metabolised Compounds Using Radioactivity Monitoring and LC-MS Methods.

BACKGROUND AND OBJECTIVES: Low-clearance drugs are widely used by industry mostly because of their often longer half-life, allowing for lower or less frequent dosing. Nevertheless, prediction of human clearance for these molecules from in vitro models presents a great challenge for pharmaceutical scientists. The objective of this study was to further characterise the predictive accuracy of the relay hepatocyte assay using 14C and 3H labelled proprietary compounds with a low extraction ratio and the known clearance mechanism in rats. Highly permeable compounds cleared by metabolism as well as rate limitation by transport were included in this study. METHODS: Blood clearance was determined from concentration-time profiles following intravenous dosing to rats. In vitro clearance was determined from the single concentration parent depletion-time profiles throughout the incubation period of up to 20 h (five relays) using radioactivity monitoring in tandem with mass spectrometry. A new approach was proposed to correct concentrations for loss and dilution during the relay steps. Clearance was predicted with a standard well-stirred model for the liver and predicted values were then compared with observed data to evaluate method accuracy. RESULTS: The results showed that intrinsic clearance values predicted using the relay hepatocyte assay from either radioactivity or mass spectrometry concentration data were comparable. A significant difference in prediction accuracy between the permeable compounds cleared by hepatic metabolism (about 2-fold) and the compound that was the hepatic uptake substrate (5- to 6-fold of actual) was demonstrated. CONCLUSIONS: The relay method is effective in predicting in vivo clearance for the compounds that are cleared via hepatic metabolism but tends to be notably underpredictive for drugs that rely on uptake transport. Consistent with the overall trend toward underprediction of hepatic clearance from the in vitro models prevalently used in the pharmaceutical industry, all values predicted from the hepatocyte relay method were lower than observed.

In Vitro Monitoring of the Mitochondrial Beta-Oxidation Flux of Palmitic Acid and Investigation of Its Pharmacological Alteration by Therapeutics.

BACKGROUND AND OBJECTIVE: The present study was designed to validate the functional assay that enables rapid screening of therapeutic candidates for their effect on mitochondrial fatty acid oxidation. METHODS: The two whole-cell systems (tissue homogenates and hepatocytes) have been evaluated to monitor the total beta-oxidation flux of physiologically important 3H-palmitic acid by measurement of tritiated water enrichment in incubations using UPLC coupled on-line to radioactivity monitoring and mass spectrometry. RESULTS: Our results with several known inhibitors of fatty acid oxidation showed that this simple assay could correctly predict a potential in alteration of mitochondrial function by drug candidates. Since the beta-oxidation of palmitic acid takes place almost exclusively in mitochondria of human hepatocytes, this model can be also utilized to distinguish between the mitochondrial and peroxisomal routes of this essential metabolic pathway in some cases. CONCLUSIONS: The present work offers a new in vitro screen of changes in mitochondrial beta-oxidation by xenobiotics as well as a model to study the mechanism of this pathway.

Quantitative characterization of a polystyrene/poly(alpha-methylstyrene) blend by MALDI mass spectrometry and size-exclusion chromatography.

The use of coupled size-exclusion chromatography and MALDI mass spectrometry for quantitative measurement of the composition of technical polymer blends at the molecular level is described. The method is illustrated with a model binary blend consisting of polystyrene and poly(alpha-methylstyrene), both polymers having similar molecular weights and polydispersities. The proposed MALDI data treatment allows determination of the chemical composition of the blend, the molecular weights of the constituents, and the distributions of the homopolymers.

Polymer characterization by combining liquid chromatography with MALDI and ESI mass spectrometry.

High-performance polymers are complex mixtures of materials of different size and chemical composition and with different end groups and architecture. To determine the molecular heterogeneity of such systems, hyphenation of several techniques is required. The value of coupling mass spectrometry (MS) with separation techniques has already been recognized - such methods have proved to be among the most powerful for molecular characterization of complex polymer systems. The review focuses on matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) MS coupled with liquid chromatography (LC). Such hyphenation has been used for most polymer analysis by mass spectrometry coupled with separation techniques. The advantages and/or limitations of these techniques for polymer characterization are discussed. Future prospects are briefly outlined.