NT-0796

Although the authors describe having conducted 'Cerep panel of 121 in vitro binding assays to assess its [NT-0796] selectivity across a broad range of biological receptors', no data has been provided for these tests. Also, there were similar Cerep panel results reported for the hydrolyzed active carboxylate form NT-19795. Same holds true for the CYP inhibition, hERG blockade patch clamp, AMES, phospholipidosis, and phototoxicity assays. In my opinion, at least the in vitro Cerep panel and BYP inhibition studies should also be conducted with the active NT-19795 compound itself. Another point - The authors mention that they synthesized and tested the (S-) eutomer of NT-0796 (Cmpd 33 in Table 5), but then ascribed the 375-fold lower potency in the PBMC assay to a 0.1% contamination with the active (R-) enantiomer. My question is two-fold: 1) Is NT-0796 susceptible to racemization either in vitro or in vivo? and 2) If not, then would it be possible to utilize purified (S-) eutomer as a Neg Ctrl compound to be tested in parallel with the active prodrug (R-) NT-0796?

NT-0796 is an isopropyl ester pro-drug undergoing carboxylesterase-mediated cleavage to give its carboxylic acid product NDT-19795, which is a potent NLRP3 inhibitor (PBMC IC50 = 66 nM, WB IC50=4.7 μM). NT-0796 was designed to significantly increase the cell permeability of the negatively charged NDT-19795. This is reflected in the PBMC assay, where the compound was dosed as the ester, the potency increased by more than 200-fold (IC50=0.32 nM). A mouse in vivo model (IV dosing) was used to determine the blood/brain concentration ratio of 0.79 which further indicated its good CNS penetration. As expected, the carboxylic acid NDT-19795 was shown to be poorly CNS permeable. NT-0796 is a selective NLRP3 inflammasome inhibitor, which affects the release of cytokines IL-1β but not IL-6 or TNFα at concentration up to 2 uM. At 3 nM, NLRP3-driven IL-1β release was inhibited by >90%. However, NT-0796 does not show an effect on IL-1β produced by the NLRC4 inflammasome at the same concentration. In terms of drug-like properties, at 30 μM concentration NT-0796 showed no inhibition against CYP isoforms (1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4). IC50 of hERG patch clamp is >30 μM, indicating a low risk on blockage of hERG potassium channels. Cerep multitarget binding panel (121 targets) indicates that only one potential off-target receptor (5HT2B). Further in vitro safety assessments including phototoxicity, Ames mutagenicity, phospholipidosis, and steatosis all showed NT-0796 as safe and progressible. NT-0796 is currently in Ph. Ib/IIa clinical trial for cardiovascular risk and neuroinflammation.