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 K m for the dehydrogenation pathway was determined in mouse (K m = 29.5 ± 10.0 μmol/L) and human (K m = 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).

Succinic semialdehyde dehydrogenase deficiency, a disorder of GABA metabolism: an update on pharmacological and enzyme‐replacement therapeutic strategies

“…1; Maitre 1997; Vogensen et al 2013), and may be the only known antagonist of GHBRs (Bay et al 2014), whose molecular structure(s) remain undefined. NCS-382 was effective in rescuing aldh5a1 −/− mice from premature lethality and in blocking the motor deficits induced by the GHB prodrug, gamma-butyrolactone (GBL; Ainslie et al 2016; Gupta et al 2002). NCS-382 exists as a racemic mixture ( hydroxyl -carbon; Fig.…”

“…We obtained detailed pharmacokinetic measures following i.p. administration of NCS-382 (100 to 500 mg/kg) in C57/B6 mice (Ainslie et al 2016). The plasma elimination t 1/2 for NCS-382 ranged from 0.25–0.68 h in a dose-dependent fashion.…”

“…Glucuronide formation was linear in both species up to 100 μM. UGT2B7 (uridine 5′-diphosphoglucuronosyltransferase; UDP-glucuronosyltransferase 2B7) was suspected as the primary isoform of glucuronidating enzymes responsible for NCS-382 metabolism, based upon competition studies with the UGT2B7 inhibitor, diclofenac (Ainslie et al 2016). Co-administration of diclofenac (25 mg/kg) improved the efficacy of NCS-382 (300 mg/kg) to block the sedative and motor effects in animals treated with GBL.…”

Pharmacological sensitivity of reflexive and nonreflexive outcomes as a correlate of the sensory and affective responses to visceral pain in mice

“…On the other hand, the surprisingly low ED50 for diclofenac to restore the latency to eat (0.005 mg/kg) suggests that COX-2 expression is extremely relevant at the time we analyzed the drug’s effects, 2 hours after iAA. The AUC after intraperitoneal administration of diclofenac (25 mg/kg) in mice has been calculated as 232 μM × h 42 and assuming a linear pharmacokinetics for diclofenac which has been reported 43 we can estimate an AUC of 46 nM × h for the ED50 0.005 mg/kg, which is higher than the IC50 for the COX-2 inhibition (10 nM) 38 . Thus despite the low doses necessary to restore the appetitive behaviour in AA-treated mice, it seems feasible that diclofenac could in fact be elicit a selective inhibition of the COX-2 enzyme in vivo at such a low doses/exposure.…”

In vitro modeling of experimental succinic semialdehyde dehydrogenase deficiency (SSADHD) using brain-derived neural stem cells

“…The mouse model of SSADH deficiency has provided valuable avenues from which to explore additional preclinical therapeutics targeting SSADHD, including NCS-382 (a GHB receptor antagonist), neuroactive steroids, and agents that inhibit the mechanistic target of rapamycin [ 4 , 8 – 11 ]. The SSADHD mouse has facilitated the collection of outcome data such as survival, electrophysiology, metabolite levels and gene expression [ 3 , 10 , 12 ].…”