Comparative Metabolism of Cinobufagin in Liver Microsomes from Mouse, Rat, Dog, Minipig, Monkey, and Human

Abstract

ABSTRACT:Cinobufagin (CB), a major bioactive component of the traditional Chinese medicine Chansu, has been reported to have potent antitumor activity. In this study, in vitro metabolism of CB among species was compared with respect to metabolic profiles, enzymes involved, and catalytic efficiency by using liver microsomes from human (HLM), mouse (MLM), rat (RLM), dog (DLM), minipig (PLM), and monkey (CyLM). Significant species differences in CB metabolism were revealed. In particular, species-specific deacetylation and epimerization combined with hydroxylation existed in RLM, whereas hydroxylation was a major pathway in HLM, MLM, DLM, PLM, and CyLM. Two monohydroxylated metabolites of CB in human and animal species were identified as 1␣-hydroxylcinobufagin and 5␤-hydroxylcinobufagin by using liquid chromatography-mass spectrometry and two-dimensional NMR techniques. CYP3A4 was identified as the main isoform involved in CB hydroxylation in HLM on the basis of the chemical inhibition studies and screen assays with recombinant human cytochrome P450s. Furthermore, ketoconazole, a specific inhibitor of CYP3A, strongly inhibited CB hydroxylation in MLM, DLM, PLM, and CyLM, indicating that CYP3A was responsible for CB hydroxylation in these animal species. The apparent substrate affinity and catalytic efficiency for 1␣-and 5␤-hydroxylation of CB in liver microsomes from various species were also determined. PLM appears to have K m and total intrinsic clearance value (V max /K m ) similar to those for HLM, and the total microsomal intrinsic clearance values for CB obeyed the following order: mouse > dog > monkey > human > minipig. These findings provide vital information to better understand the metabolic behaviors of CB among various species.

Characterization of Phase I Metabolism of Resibufogenin and Evaluation of the Metabolic Effects on Its Antitumor Activity and Toxicity

“…2). These findings agree well with our previous metabolic investigation of cinobufagin, which is a structural analog of RB (Ma et al, 2007;Ning et al, 2010;Ma et al, 2011). The significant differences in metabolic behaviors between rat and human suggested that rat could not serve as a preferred surrogate model for in vivo study of RB, and the in vivo pharmacological or toxicological data obtained in rat models previously may be misleading.…”

“…By monitoring the apoptosis-associated protein, this investigation indicated that the anticancer bioactivities of 5-HRB were partially mediated through the activation of cytochrome c/caspase-dependent apoptotic signaling pathways for A549 cells. However, attention should be paid to the discrepancy between the present findings and the reported structurecytotoxic activity relationship of bufadienolides, which demonstrated that 5-hydroxylation reduced anticancer activity significantly (Kamano et al, 1998;Ma et al, 2011). The discrepancy makes apparent that the metabolic effect on bufadienolides may change case by case, making it meaningful to investigate the biotransformation of bufadienolides, which have great potential as a chemotherapeutic agents in oncology.…”

“…Recently, the metabolic characteristic of RB in rats was illustrated by isolation and identification of metabolites excreted in bile (Zhu et al, 2013). Consistent with the metabolic fate of cinobufagin (a RB analog) (Ning et al, 2010;Ma et al, 2011), it has been suggested that 3-O-epimerization of RB is a rapid and important metabolic reaction in rats (Chen et al, 2012). However, the metabolic behaviors of RB in humans as well as the metabolic effects on its bioactivities and toxicity have not been well characterized.…”

Bioactivation of herbal constituents: simple alerts in the complex system

“…A number of reports have demonstrated that drugmetabolizing enzymes display significant species difference with regards to isoforms composition, expression and catalytic activities of drug-metabolizing enzymes [121][122][123]. CYP3A, the most important CYP isoform involved in the metabolism of xenobiotics, shows different substrate specificities in different species, resulting in inaccurate extrapolation from animal to human.…”

Identification of the metabolites of TCM prescription Sinisan, found in miniature pig urine via intragastric administration

“…Miniature pig has been reported to possess similar kinetic parameters in the liver microsomes to human [9]. Thus it would be a better choice to serve as a surrogate model for drug metabolism.…”