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 1α-hydroxylcinobufagin and 5β-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 1α- and 5β-hydroxylation of CB in liver microsomes from various species were also determined. PLM appears to have Km and total intrinsic clearance value (Vmax/Km) 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.
Footnotes
This work was supported in part by the National Basic Research Program of China [Grant 2009CB522808]; National Key Technology R&D Program in the 11th Five Year Plan of China [Grant 2008ZX10002-019]; National Natural Science Foundation of China [Grants 30701088, 30630075, 30973590, 81001473]; and the Foundation of Education Department of Liaoning Province [Grant LS2010059].
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.036830.
↵ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
-
ABBREVIATIONS:
- TCM
- traditional chinese medicine
- CB
- cinobufagin
- HLM
- human liver microsomes
- MS
- mass spectrometry
- ABT
- 1-aminobenzotriazole
- DAD
- diode array detector
- LC
- liquid chromatography
- P450
- cytochrome P450
- UFLC
- ultrafast liquid chromatography
- ESI
- electrospray ionization
- MLM
- Swiss-Hauschka mouse liver microsome(s)
- DLM
- beagle dog liver microsome(s)
- PLM
- minipig liver microsome(s)
- CyLM
- cynomolgus monkey liver microsome(s)
- DCB
- 16-deacetylcinobufagin
- RLM
- Sprague-Dawley rat liver microsomes
- 1-HCB
- 1α-hydroxylcinobufagin
- 5-HCB
- 5β-hydroxylcinobufagin.
- Received October 19, 2010.
- Accepted January 4, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
DMD articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|