In vitro toxicological evaluation of NCS-382, a high-affinity antagonist of γ-hydroxybutyrate (GHB) binding

Toxicol In Vitro. 2017 Apr;40:196-202. doi: 10.1016/j.tiv.2017.01.013. Epub 2017 Jan 22.


γ-Hydroxybutyric acid (GHB), a minor metabolite of the inhibitory neurotransmitter GABA, can accumulate to significant concentrations in the heritable disorder of GABA degradation, succinic semialdehyde dehydrogenase (SSADH) deficiency (SSADHD). Moreover, GHB may be employed in therapeutic settings (treatment of narcolepsy), as well as instances of illicit activity, including acquaintance sexual assault and the induction of euphoria. High-affinity binding sites for GHB in the brain have been identified, although the absolute identity of these receptors remains unclear. Pharmacological antagonism of GHB binding may have multiple instances of therapeutic relevance. The high affinity GHB receptor antagonist, NCS-382 (6,7,8,9-tetrahydro-5-hydroxy-5H-benzo-cyclohept-6-ylideneacetic acid) has not been piloted in humans. To address the potential clinical utility of NCS-382, we have piloted initial studies of its toxicology in HepG2 and primary hepatocyte cells. At high dose (0.5mM), NCS-382 showed no capacity for inhibition of microsomal CYPs (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and minimal potential for activation of xenobiotic nuclear receptors. Additional cellular integrity and functional assays (viability, oxidative stress, apoptosis, ATP production) revealed little evidence for cytotoxicity, and a low degree of dysregulation of >370 genes actively engaged in the mediation of cellular toxicity. In vitro testing indicates a low probability of cellular toxicity associated with NCS-382.

Keywords: Apoptosis; Cytochrome P-450 (CYP); Cytotoxicity; Gene expression; HepG2 cells; Microsomal CYPs; Mitochondrial function; PXR; Primary human hepatocytes; Xenobiotic receptor induction.

MeSH terms

  • Benzocycloheptenes / pharmacology*
  • Cell Survival / drug effects
  • Cells, Cultured
  • Cytochrome P-450 Enzyme Inhibitors / pharmacology
  • Cytochrome P-450 Enzyme System / genetics
  • Cytochrome P-450 Enzyme System / metabolism
  • Drug Interactions
  • Gene Expression Regulation, Enzymologic / drug effects
  • Hep G2 Cells
  • Hepatocytes / drug effects
  • Humans
  • Hydroxybutyrates / metabolism*
  • Microsomes, Liver / drug effects
  • Microsomes, Liver / metabolism
  • Receptors, Cell Surface / antagonists & inhibitors*


  • 4-hydroxybutyric acid receptor
  • Benzocycloheptenes
  • Cytochrome P-450 Enzyme Inhibitors
  • Hydroxybutyrates
  • Receptors, Cell Surface
  • NCS 382
  • 4-hydroxybutyric acid
  • Cytochrome P-450 Enzyme System