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

Abstract

Resibufogenin (RB), one of the major active compounds of the traditional Chinese medicine Chansu, has displayed great potential as a chemotherapeutic agent in oncology. However, it is a digoxinlike compound that also exhibits extremely cardiotoxic effects. The present study aimed to characterize the metabolic behaviors of RB in humans as well as to evaluate the metabolic effects on its bioactivity and toxicity. The phase I metabolic profile in human liver microsomes was characterized systemically, and the major metabolite was identified as marinobufagenin (5b-hydroxylresibufogenin, 5-HRB) by liquid chromatography-mass spectrometry and nuclear magnetic imaging techniques. Both cytochrome P450 (P450) reaction phenotyping and inhibition assays using P450-selective chemical inhibitors demonstrated that CYP3A4 was mainly involved in RB 5b-hydroxylation with much higher selectivity than CYP3A5. Kinetic characterization demonstrated that RB 5b-hydroxylation in both human liver microsomes and human recombinant CYP3A4 obeyed biphasic kinetics and displayed similar apparent kinetic parameters. Furthermore, 5-HRB could significantly induce cell growth inhibition and apoptosis in A549 and H1299 by facilitating apoptosome assembly and caspase activation. Meanwhile, 5-HRB displayed very weak cytotoxicity of human embryonic lung fibroblasts, and in mice there was a greater tolerance to acute toxicity. In summary, CYP3A4 dominantly mediated 5b-hydroxylation and was found to be a major metabolic pathway of RB in the human liver, whereas its major metabolite (5-HRB) displayed better druglikeness than its parent compound RB. Our findings lay a solid foundation for RB metabolism studies in humans and encourage further research on the bioactive metabolite of RB.

Metabolic Profile of 3-Acetyl-11-Keto-β-Boswellic Acid and 11-Keto-β-Boswellic Acid in Human Preparations In Vitro, Species Differences, and Bioactivity Variation

“…A series of selective inhibitors for different P450 isoforms were used to explore the potential contribution of CYP3A to the metabolism of KBA (10 μM) in HLM (0.003 mg of protein). The selective inhibitors and their concentrations were as follows: α-naphthoflavone (1 μM) for CYP1A2, 8-methoxypsoralen (2.5 μM) for CYP2A6, TEPA (50 μM) for CYP2B6, montelukast (2 μM) for CYP2C8, sulfaphenazole (1 μM) for CYP2C9, omeprazole (20 μM) for CYP2C19, quinidine (10 μM) for CYP2D6, clomethiazole (10 μM) for CYP2E1, ketoconazole (1 μM) for CYP3A, CYP3cide (1 μM, a potent and selective inhibitor for CYP3A4) for CYP3A4, and verapamil (20 μM) for CYP3A (43)(44)(45).…”

“…To obtain the metabolism kinetic parameters of KBA hydroxylation in HLM, CYP3A4, and CYP3A5, a general operating procedure was performed (42,43). In brief, KBA (0-50 μM) was incubated with pooled HLM (0.03 mg of protein/mL), CYP3A4 (0.005 mg of protein/mL), and CYP3A5 (0.005 mg of protein/mL) at 37°C for 30 min, respectively.…”

“…For estimating the hydrolysis kinetic parameters of AKBA, a general and standard operating procedure was performed (42,43). In brief, AKBA (1-100 μM) was incubated with different enzyme sources including HLM, HIM, and CE2 for 30 min, respectively.…”

Gomisin A is a Novel Isoform-Specific Probe for the Selective Sensing of Human Cytochrome P450 3A4 in Liver Microsomes and Living Cells

“…However, 8-hydroxylation schizandrin undergoes subsequent rapid demethylation by CYP3A4, which might affect the accurate quantification of the total intrinsic metabolic clearance of CYP3A4. More interestingly, the substrates appeared to be selectively metabolized by CYP3A4 through hydroxylation reactions, such as GA 8-hydroxylation, schizandrin 8-hydroxylation [18], bufalin 5β-hydroxylation [14], and resibufogenin 5β-hydroxylation [35]. In contrast, the dealkylation reactions of substrates are generally mediated by multiple CYP enzymes other than CYP3A4.…”

Identification of Antitumor Constituents in Toad Venom by Spectrum-Effect Relationship Analysis and Investigation on Its Pharmacologic Mechanism

“…Here, we noticed that the coefficient of some components were negative, such as 19 peak, which was resibufogenin. However, according to known references [ 27 ], resibufogenin also shows some antitumor effects on A549 cells with IC50 of about 25 nM [ 28 ], which means that resibufogenin also has some degree of antitumor activity. To explain this result, we analyzed the relationship between the peak areas of 6, 7, 8, 11, 13, 15, and 16, the most significant peaks according to our analysis, and peak 19 with CCA.…”