A pharmacokinetic evaluation and metabolite identification of the GHB receptor antagonist NCS-382 in mouse informs novel therapeutic strategies for the treatment of GHB intoxication

Abstract

Gamma-aminobutyric acid (GABA) is an endogenous inhibitory neurotransmitter and precursor of gamma-hydroxybutyric acid (GHB). NCS-382 (6,7,8,9-tetrahydro-5-hydroxy-5H-benzo-cyclohept-6-ylideneacetic acid), a known GHB receptor antagonist, has shown significant efficacy in a murine model of succinic semialdehyde dehydrogenase deficiency (SSADHD), a heritable neurological disorder featuring chronic elevation of GHB that blocks the final step of GABA degradation. NCS-382 exposures and elimination pathways remain unknown; therefore, the goal of the present work was to obtain in vivo pharmacokinetic data in a murine model and to identify the NCS-382 metabolites formed by mouse and human. NCS-382 single-dose mouse pharmacokinetics were established following an intraperitoneal injection (100, 300, and 500 mg/kg body weight) and metabolite identification was conducted using HPLC-MS/MS. Kinetic enzyme assays employed mouse and human liver microsomes. Upon gaining an understanding of the NCS-382 clearance mechanisms, a chemical inhibitor was used to increase NCS-382 brain exposure in a pharmacokinetic/pharmacodynamic study. Two major metabolic pathways of NCS-382 were identified as dehydrogenation and glucuronidation. The Km for the dehydrogenation pathway was determined in mouse (Km = 29.5 ± 10.0 μmol/L) and human (Km = 12.7 ± 4.8 μmol/L) liver microsomes. Comparable parameters for glucuronidation were >100 μmol/L in both species. Inhibition of NCS-382 glucuronidation, in vivo, by diclofenac resulted in increased NCS-382 brain concentrations and protective effects in gamma-butyrolactone-treated mice. These initial evaluations of NCS-382 pharmacokinetics and metabolism inform the development of NCS-382 as a potential therapy for conditions of GHB elevation (including acute intoxication & SSADHD).