Identification and characterization of in vitro and in vivo metabolites of steroidal alkaloid veratramine

Abstract

Veratramine, a steroidal alkaloid originating from Veratrum nigrum L., has demonstrated distinct anti-tumor and anti-hypertension effects, however, its metabolism has rarely been explored. The objective of the current study was to provide a comprehensive investigation of its metabolic pathways. The in vitro metabolic profiles of veratramine were evaluated by incubating it with liver microsomes and cytosols. The in vivo metabolic profiles in plasma, bile, urine and feces were monitored by UPLC-MS/MS after oral (20 mg/kg) and i.v. (50 µg/kg) administration in rats. Meanwhile, related P450s inhibitors and recombinant P450s and SULTs were used to identify the isozymes responsible for its metabolism. Eleven metabolites of veratramine, including seven hydroxylated, two sulfated and two glucuronidated metabolites, were characterized. Unlike most alkaloids, the major reactive sites of veratramine were on ring A and B instead of on the amine moiety. CYP2D6 was the major isozyme mediating hydroxylation, and substrate inhibition was observed with a Vmax , Ki and Clint of 2.05 ± 0.53 nmol/min/mg, 33.08 ± 10.13 µ m and 13.58 ± 1.27 µL/min/mg. SULT2A1, with Km , Vmax and Clint values of 19.37 ± 0.87 µ m, 1.51 ± 0.02 nmol/min/mg and 78.19 ± 8.57 µL/min/mg, was identified as the major isozyme contributing to its sulfation. In conclusion, CYP2D6 and SULT2A1 mediating hydroxylation and sulfation were identified as the major biotransformation for veratramine.

Gender-Dependent Pharmacokinetics of Veratramine in Rats: In Vivo and In Vitro Evidence

“…Since the contribution of CYP2C11 for the formation of 7-hydroxylveratramine could not be thoroughly ruled out, the malespecific CYP2C11 may also partly mediate the significant gender-dependent formation of 7-hydroxyl-veratramine. SULT2A1 was the major isozyme mediating the sulfation of veratramine in humans (35). Based on the similarity in the sulfation of veratramine in male and female rat liver cytosols with the existence of rat SULT2A (67)(68)(69), it could be inferred that SULT2A was responsible for the sulfation of veratramine in rats.…”

“…1) and veratramine-3-O-sulfate ( Fig. 1) (35). During our preliminary pharmacokinetic investigations in rats after oral administration of 30 mg/kg of veratramine, it was surprisingly noticed that male rats easily suffered from toxic reactions, such as severe trembling, with a fatality rate of nearly 50%, whereas female rats were all alive without any toxic response.…”

“…Our previous study manifested that CYP2D and CYP3A mainly mediated the formation of 7-hydroxl-veratramine in rats (35). The enzyme activity of CYP2D in male rats was higher than that in female rats, especially for the male dominant CYP2D1 (60)(61)(62).…”

“…Due to the gender differences in drug metabolism being notoriously species dependent, the limitation of the current findings should be noted. Our previous study demonstrated that different enzymes mediated the formation of 7-hydroxylveratramine from veratramine in rats (CYP2D and CYP3A) and humans (only CYP2D6) (35). It was previously suggested that CYP2D in rats had gender differences with more predominant expression and activity in males (60,61), whereas no gender difference for CYP2D6 has been identified in humans.…”

“…The results of the bioanalytical method validation of veratramine including precision, accuracy, recovery, matrix effect, and stability are shown in Supplemental 1, which indicated that the assay was acceptable for the determination of veratramine in vitro and in vivo. Since the authentic standards of 7-hydroxyl-veratramine and veratramine-3-Osulfate were unavailable, the signal strength conversion factor was used to adjust the standard curve of veratramine for the quantification of 7-hydroxyl-veratramine and veratramine-3-O-sulfate as previously described (35,40,41). In brief, rat liver cytosolic incubations of veratramine with a series of concentrations were performed in the absence and presence of PAPS.…”