Multiple UDP-Glucuronosyltransferase and Sulfotransferase Enzymes are Responsible for the Metabolism of Verproside in Human Liver Preparations

Abstract

Verproside, an active iridoid glycoside component of Veronica species, such as Pseudolysimachion rotundum var. subintegrum and Veronica anagallis-aquatica, possesses anti-asthma, anti-inflammatory, anti-nociceptive, antioxidant, and cytostatic activities. Verproside is metabolized into nine metabolites in human hepatocytes: verproside glucuronides (M1, M2) via glucuronidation, verproside sulfate (M3) via sulfation, picroside II (M4) and isovanilloylcatalpol (M5) via O-methylation, M4 glucuronide (M6) and M4 sulfate (M8) via further glucuronidation and sulfation of M4, and M5 glucuronide (M7) and M5 sulfate (M9) via further glucuronidation and sulfation of M5. Drug-metabolizing enzymes responsible for verproside metabolism, including sulfotransferase (SULT) and UDP-glucuronosyltransferase (UGT), were characterized. The formation of verproside glucuronides (M1, M2), isovanilloylcatalpol glucuronide (M7), and picroside II glucuronide (M6) was catalyzed by commonly expressed UGT1A1 and UGT1A9 and gastrointestinal-specific UGT1A7, UGT1A8, and UGT1A10, consistent with the higher intrinsic clearance values for the formation of M1, M2, M6, and M7 in human intestinal microsomes compared with those in liver microsomes. The formation of verproside sulfate (M3) and M5 sulfate (M9) from verproside and isovanilloylcatalpol (M5), respectively, was catalyzed by SULT1A1. Metabolism of picroside II (M4) into M4 sulfate (M8) was catalyzed by SULT1A1, SULT1E1, SULT1A2, SULT1A3, and SULT1C4. Based on these results, the pharmacokinetics of verproside may be affected by the co-administration of relevant UGT and SULT inhibitors or inducers.

Keywords: UDP-glucuronosyltransferase; in vitro metabolism; sulfotransferase; verproside.

Figure 1

Chromatograms of the extracted ions of verproside and its metabolites in the liquid chromatography-mass spectrometry (LC-MS)analysis of the assays using human hepatocytes. The extracted ion chromatograms were reconstructed based on deprotonated molecular ions: m/z 497.12939 for verproside, 673.16138 for M1 and M2 (verproside glucuronides), 577.08594 for M3 (verproside sulfate), 511.14484 for M4 (picroside II) and M5 (isovanilloylcatalpol), 687.17657 for M6 (picroside II glucuronide) and M7 (isovanilloylcatalpol glucuronide), and 591.10150 for M8 (picroside II sulfate) and M9 (isovanilloylcatalpol sulfate).

Figure 2

Possible in vitro metabolic pathways of verproside in human hepatocytes.

Figure 3

Formation of (A) verproside glucuronides (M1, M2) from 500 μM verproside; (B) picroside II glucuronide (M6) from 100 μM picroside II (M4); and (C) isovanilloylcatalpol glucuronide (M7) from 100 μM isovanilloylcatalpol (M5) in supersomes expressing recombinant human UDP-glucuronosyltransferase (UGT)1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17. ND: <0.67 pmol/min/mg protein for verposide. ND: <2.5 pmol/min/mg protein for picroside II and isovanilloylcatalpol. The data represent mean ± S.D. (n = 3).

Figure 4

Michaelis–Menten plots for the formation of verproside glucuronide M1 from verproside in (A) human liver microsomes; (B) human intestinal microsomes, and supersomes expressing recombinant human (C) UGT1A1; (D) UGT1A8; (E) UGT1A9; and (F) UGT1A10 enzymes. An Eadie–Hofstee plot is provided in the inset. The solid line is the curve fit line obtained using the Enzyme Kinetics program.

Figure 5

Michaelis-Menten plots for the formation of verproside glucuronide M2 from verproside in (A) human liver microsomes; (B) human intestinal microsomes, and supersomes expressing recombinant human (C) UGT1A1; (D) UGT1A8; (E) UGT1A9; and (F) UGT1A10 enzymes. An Eadie-Hofstee plot is provided in the inset.

Figure 6

Michaelis–Menten plots for the formation of isovanilloylcatalpol glucuronide (M7) from isovanilloylcatalpol in (A) human liver microsomes, (B) human intestinal microsomes; and supersomes expressing recombinant human (C) UGT1A1; (D) UGT1A7; (E) UGT1A8; (F) UGT1A9; and (G) UGT1A10 enzymes. An Eadie–Hofstee plot is provided in the inset.

Figure 7

Formation of (A) verproside sulfate (M3) from 1.2 μM verproside; (B) isovanilloylcatalpol sulfate (M9) from 25 μM isovanilloylcatalpol (M5); and (C) picroside II sulfate (M8) from 25 μM picroside II (M4) in supersomes expressing recombinant human sulfotransferase (SULT)1A1*1, SULT1A1*2, SULT1A2, SULT1A3, SULT1B1, SULT1C2, SULT1C4, SULT1E1, and SULT2A1. Data represent the mean ± S.D. (n = 3). ND: <0.5 pmol.

Figure 8

Michaelis-Menten plots of the formation of verproside sulfate (M3) from verproside in (A) human liver S9 fractions and (B) supersomes expressing recombinant human SULT1A1*1 and (C) SULT1A1*2. An Eadie–Hofstee plot is provided in the inset.

Figure 9

Michaelis–Menten plots of the formation of isovanilloylcatalpol sulfate (M9) from isovanilloylcatalpol (M5) in (A) human liver S9 fractions and (B) supersomes expressing recombinant human SULT1A1*1 and (C) SULT1A1*2. An Eadie–Hofstee plot is provided in the inset.

Figure 10

Michaelis–Menten plots of the formation of picroside II sulfate (M8) from picroside II (M4) in (A) human liver S9 fractions and (B) supersomes expressing recombinant human SULT1A1*1; (C) SULT1A1*2; (D) SULT1A2, (E) SULT1A3; (F) SULT1C4; and (G) SULT1E1. An Eadie–Hofstee plot is provided in the inset.