An Investigation on Glucuronidation Metabolite Identification, Isozyme Contribution, and Species Differences of GL-V9 In Vitro and In Vivo

Abstract

GL-V9 is a prominent derivative of wogonin with a wide therapeutic spectrum and potent anti-tumor activity. The metabolism characteristics of GL-V9 remain unclear. This study aimed to clarify the metabolic pathway of GL-V9 and investigate the generation of its glucuronidation metabolites in vitro and in vivo. HPLC-UV-TripleTOF was used to identify metabolites. The main metabolite that we found was chemically synthesized and the synthetic metabolite was utilized as standard substance for the subsequent metabolism studies of GL-V9, including enzyme kinetics in liver microsomes of five different species and reaction phenotyping metabolism using 12 recombinant human UDP-glucuronosyltransferase (UGT) isoforms. Results indicated that the glucuronidation reaction occurred at C5-OH group, and 5-O-glucuronide GL-V9 is the only glucuronide metabolite and major phase II metabolite of GL-V9. Among 12 recombinant human UGTs, rUGT1A9 showed the strongest catalytic capacity for the glucuronidation reaction of GL-V9. rUGT1A7 and rUGT1A8 were also involved in the glucuronidation metabolism. K_m of rUGT1A7-1A9 was 3.25 ± 0.29, 13.92 ± 1.05, and 4.72 ± 0.28 μM, respectively. In conclusion, 5-O-glucuronide GL-V9 is the dominant phase II metabolite of GL-V9 in vivo and in vitro, whose formation rate and efficiency are closely related to isoform-specific metabolism profiles and the distribution of UGTs in different tissues of different species.

Keywords: GL-V9; enzyme kinetics; glucuronidation; human recombinant UGTs.

Figure 1

HPLC-UV-TripleTOF analysis of GL-V9 and its metabolites in HLMs system: (A) Total ion current chromatogram; (B) extracted ion current chromatogram; (C) spectrum of 5-O-glucorunide GL-V9; (D) UV chromatogram). The structure of 5-O-glucorunide GL-V9 and predominant fragmentation pattern was shown in C, and the fragment drawn in red is the chemical modification in GL-V9, while the fragment in blue is the glucuronic acid part.

Figure 2

HPLC-UV-TripleTOF analysis of GL-V9 and metabolites in rat plasma: (A) Total ion current chromatogram; (B) extracted ion current chromatogram; (C) spectrum of 5-O-glucorunide GL-V9; (D) UV chromatogram). The structure of 5-O-glucorunide GL-V9 and predominant fragmentation pattern was shown in (C), and the fragment drawn in red is the chemical modification in GL-V9, while the fragment in blue is the glucuronic acid part.

Figure 3

HPLC-UV-TripleTOF analysis of chemically synthesized and biosynthesized 5-O-glucuronide GL-V9 in rat plasma: (A) UV channel chromatogram of rat plasma, the peak area of 5-O-glucuronide GL-V9 was 2.3215 × 10⁵; (B) UV channel chromatograph of rat plasma with chemically synthesized 5-O-glucuronide GL-V9 standard solution, the peak area of 5-O-glucuronide GL-V9 was 6.5219 × 10⁵; (C) TripleTOF Mass spectrum for 5-O-glucuronide GL-V9).

Figure 4

Glucuronidation of GL-V9 by twelve recombinant human UGTs. Three concentrations of GL-V9 were selected to incubate with 12 commercially available UGTs (0.05 mg/mL) at 37 °C for 1 h. Glucuronides of GL-V9 in incubation system were quantified through UPLC-MS/MS. The formation rates were calculated and described as pmol/min/mg protein. Each bar is the average of three detection values, and the error bars are standard deviations of the mean (n = 3).

Figure 5

Eadie-Hofstee plots for the formation of 5-O-glucuronide GL-V9 by MLMs (A), DLMs (B), RLMs (C), MiceLMs (D), HLMs (E), and enzyme kinetics comparisons between these liver microsomes (F). Serial concentrations of GL-V9 were incubated with liver microsomes from five different species (monkey, beagle dog, rat, mouse, and human). Glucuronides of GL-V9 in incubation system were quantified using UPLC-MS/MS. The formation rates were calculated and described as pmol/min/mg protein. Kinetic profiles were fitted into Eadie-Hofstee plots to determine enzyme-mediated reaction types. All the data were expressed as the average of three determinations and the error bar represents the standard deviation of the mean (mean ± SD, n = 3). *** p < 0.001 indicating significantly different.

Figure 6

Eadie-Hofstee plots for the formation of 5-O-glucuronide GL-V9 by rUGT1A7 (A), rUGT1A8 (B), rUGT1A9 (C) and enzyme kinetics comparisons between these isoenzymes (D). Serial concentrations of GL-V9 were incubated with rUGT1A7, 1A8 or 1A9 for 1 h at 37 °C. Glucuronides of GL-V9 in incubation system were quantified using UPLC-MS/MS. The formation rates were calculated and described as pmol/min/mg protein. Kinetic profiles were fitted into Eadie-Hofstee plots and Michaelis-Menten plot to determine enzyme-mediated reaction types. All the data were expressed as the average of three determinations and the error bar represents the standard deviation of the mean (mean ± SD, n = 3). *** p < 0.001 indicating significantly different.

Figure 7

Mean concentration-time profiles of GL-V9 or 5-O-glucuronide GL-V9 in rat plasma after oral administration of GL-V9 (gavage, 50 mg/kg). Concentrations of GL-V9 or 5-O-glucuronide GL-V9 in rat plasma were quantified using UPLC-MS/MS. The concentration data of GL-V9 were reported by Xing et al. [38]. All the data were expressed as the average of six determinations and the error bar represents the standard deviation of the mean (mean ± SD, n = 6).

Scheme 1

Synthetic route of 5-O-glucuronide GL-V9.