In vitro metabolism of the anti-inflammatory clerodane diterpenoid polyandric acid A and its hydrolysis product by human liver microsomes and recombinant cytochrome P450 and UDP-glucuronosyltransferase enzymes

Abstract

1. The metabolism of the anti-inflammatory diterpenoid polyandric acid A (PAA), a constituent of the Australian Aboriginal medicinal plant Dodonaea polyandra, and its de-esterified alcohol metabolite, hydrolysed polyandric acid A (PAAH) was studied in vitro using human liver microsomes (HLM) and recombinant UDP-glucuronosyltransferase (UGT) and cytochrome P450 (CYP) enzymes.

2. Hydrolysis of PAA to yield PAAH occurred upon incubation with HLM. Further incubations of PAAH with HLM in the presence of UGT and CYP cofactors resulted in significant depletion, with UGT-mediated depletion as the major pathway.

3. Reaction phenotyping utilising selective enzyme inhibitors and recombinant human UGT and CYP enzymes revealed UGT2B7 and UGT1A1, and CYP2C9 and CYP3A4 as the major enzymes involved in the metabolism of PAAH.

4. Analysis of incubations of PAAH with UDP-glucuronic acid-supplemented HLM and recombinant enzymes by UPLC/MS/MS identified three glucuronide metabolites. The metabolites were further characterised by β-glucuronidase and mild alkaline hydrolysis. The acyl glucuronide of PAAH was shown to be the major metabolite.

5. This study demonstrates the in vitro metabolism of PAA and PAAH and represents the first systematic study of the metabolism of an active constituent of an Australian Aboriginal medicinal plant.

Keywords:

• Clerodane diterpenoid
• cytochrome P450
• esterases
• glucuronidation
• in vitro–in vivo extrapolation
• polyandric acid A
• reaction phenotyping
• UDP-glucuronosyltransferase

Acknowledgements

The authors wish to acknowledge the Chuulangun Aboriginal Corporation, on behalf of the Kuuku I'yu Northern Kaanju people, for sharing their cultural knowledge and facilitating access to raw plant materials from their land, as well as Kushari Burns, Nuy Chau, Dr. Pramod Nair, Dr. Andrew Rowland and Dr. Chi Ndi for technical assistance with this work.

Declaration of interest

The authors have no declarations of interest to report. This work was partially supported by the National Health and Medical Research Council Development Grant 1017556.