MIST-relevant metabolism of ganaxolone
This cracking paper just out on the complex MIST-relevant metabolism of ganaxolone is our paper pick for March. It highlights the limitations of in vitro and animal in vivo studies, which, in this case, led to the initial false belief that other biotransformations were responsible for clearance of the drug.
Ganaxalone, a neuroactive steroid approved for use by the FDA for treating seizures associated with cyclin-dependent kinase-like 5 (CDKL5) deficiency disorder, is subject to complex metabolism with 59 metabolites identified (Fitch et al., 2023). It is a synthetic C-3 methyl analog of allopregnanolone. The major routes of ganaxalone metabolism involve multiple steps, comprising hydroxylation at the 16a-hydroxy position, stereoselective reduction of the 20-ketone to give the corresponding 20a-hydroxysterol, and sulfation of the 3a-hydroxy group. The latter reaction yielded an unstable tertiary sulfate, which undergoes elimination to form a double bond in the A ring. A combination of these pathways, together with oxidation of the 3b-methyl substituent to a carboxylic acid and sulfation at the 20a position, led to two major circulating metabolites in plasma, M2 and M17.
In vitro and animal in vivo studies led to the initial belief that alternative biotransformations were responsible for clearance of the drug. The major human liver microsomal (HLM) metabolite was identified as 16α-hydroxy-ganaxalone mainly through the action of CYP3A4, whilst in human hepatocytes the corresponding glucuronide M11 was also observed as a major metabolite along with a minor metabolite M5. A later key finding was the high conversion of this dehydrated metabolite to M2 in hepatocytes, but not in HLMs.
Key intermediates of M2 and M17, M5, M6 and M53, were observed at low levels in human hepatocyte and human S9 incubations of GNX, but their levels were overlooked in the presence of the much more dominant M1 and M11.
None of the preclinical species yielded detectable amounts of M2 or M17, except trace levels in the female rat. However, findings in the human 14C radiolabel human mass balance study pointed to an alternative situation afoot, with a very slow rate of excretion and only 72.7% ± 9.6% of the administered radioactivity recovered by the end of the study at 720 h.
Subsequently, M2 and M17 were shown to be disproportionate human metabolites which persisted in circulation, highlighting the limitations of traditional animal studies and human in vitro systems in predicting major circulating metabolites in man. Unfortunately, there was an added complication in that minimal structural information could be gleaned from MS/MS, and NMR was needed to elucidate the structure of M2 and other metabolites.
Multiple enzymes are involved in ganaxalone’s biotransformation including CYP3A4, AKR1, SULTs and UGTs. Interestingly it is proposed that the species-specific nature of one sulfation step is the reason that preclinical species fail to produce appreciable amounts of the M5 or M5-derived intermediate metabolites. The involvement of extrahepatic sulfotransferase(s) also point to the disconnect between hepatic in vitro systems and the in vivo situation.
Although M2 demonstrated no functional activity at the GABAA receptor, either as a modulator or as a direct activator, safety studies were needed. The additional evaluation of M17 as a MIST metabolite was seen as less of a concern.
Hypha are proud to have played a part in solving a piece of the jigsaw.
William L. Fitch, Steven Smith, Michael Saporito, Gregory Busse, Mingbao Zhang, Julie Ren, Michael E. Fitzsimmons, Ping Yi, Stephen English, Adam Carter and Thomas A. Baillie
Drug Metabolism and Disposition June 1, 2023, 51 (6) 753-763; DOI: https://doi.org/10.1124/dmd.122.001218