The Study On Glucuronidation Metabolism Of Desacetylcinobufagin(DACB) In Vitro

Objective:In this paper, we studied the glucuronidation metabolism of Desacetylcinobufagin(DACB) that is one of the important active components in Chan’su. Our aim is to revel the conjugation position of DACB metabolites, the major UGT enzyme involved in the metabolism, the kinetic models and parameters of various metabolites in human liver microsomes, human intestine microsomes, liver microsomes from different experimental animals and the recombinant UGTs, the metabolism differences among seven species and verify the possibility of DACB with various hydroxyl groups used as a probe substrate.Methods:(1) In vitro metabolism experiments:investigation of the glucuronidation metabolism of DACB in different liver microsomes which from different species including human (HLM, HIM), pig (PLM), dog (DLM), cynomolgus monkey (MLM), SD rat (RtLM), mouse (MsLM), rabbit (RLM), guinea pig (TLM) and 14 recombinant UGT isoforms. (2) Preparing the metabolites of DACB by using the in vitro incubation method, isolated and purified the metabolites with the phytochemistry method, identified the metabolites of glucuronidation of DACB by spectral methods including two-dimensional NMR such as heteronuclear single quantum correlationk, heteronuclear multiple-bond correlation spectroscopy, and nuclear overhauser effect spectroscopy. (3) Determining the expression level of UGT 1 A3 and UGT1A4 in twelve human liver microsomes was performed by Western Blotting to identify the metabolic rate of DACB in twelve human liver microsomes. Then, correlation analysis of the expression level and the metabolic rate was carried out.Results:The structures of two monoglucuronide metabolites (DACB-16-O-β-D-glucuronide and DACB-3-O-β-D-glucuronide) of DACB were determined. UGT1A4 was the only recombinant UGT isoform for the formation of M-1; UGT1A3 was the major recombinant UGT isoform for the formation of M-2, although UGT1A1 made less contribution M-2 formation. Michaelis-Menten kinetic model were shown in the glucuronidation of DACB to generate M-1 in HLM and UGT1A4, substrate inhibition kinetics model were shown in the glucuronidation of DACB to generate M-2 in HLM and UGT1A3, Michaelis-Menten kinetic model were shown in UGT1A1, and glucuronidation of these components in different UGT isoforms. We also systematically analyzed various kinetic parameters (Km, Vmax, and CLint) for glucuronidation of DACB. Regioselective glucuronidation showed considerable species differences, DACB-3-O-Glucuronidation was observed in liver microsomes from dog, cynomolgus monkey, rabbit and guinea pig. And species screening combined with the kinetic analysis indicated that cynomolgus monkey was the model animal for the study of formation of M-1 in vivo; DACB-16-O-Glucuronidation was observed in liver microsomes from pig, dog, cynomolgus monkey and rabbit. And species screening combined with the kinetic analysis indicated that rabbit was the model animal for the study of formation of M-2 in vivo. Western Blotting used to determine the expression level of UGT isoforms in twelve HLMs, exhibited specificity of UGT1A3 and UGT1A4 for metabolism of DACB. UGT isoforms screening, kinetic analysis, correlation analysis and inhibition test were all the evidences that DACB can be the probe substrate to detect the activity of UGT1A3 and UGT1A4.Conclusions:In this paper, we firstly determined the DACB metabolism in vitro by using different organs microsomes of human, different experiment animal liver micorsomes and 14 recombinant UGTs. The kinetic models and paprameters were determined, which would provid the useful information for chan’su and DACB. Meantime, DACB could be used as the specific probe of UGTA3 and UGT1A4, according to the various probe reactions towards UGTA3 and UGT1A4.