In Vitro and in Vivo Metabolism of Verproside in Rats

Verproside, a catalpol derivative iridoid glycoside isolated from Pseudolysimachion rotundum var. subintegrum, is a biologically active compound with anti-inflammatory, antinociceptic, antioxidant, and anti-asthmatic properties. Twenty-one metabolites were identified in bile and urine samples obtained after intravenous administration of verproside in rats using liquid chromatography-quadrupole Orbitrap mass spectrometry. Verproside was metabolized by O-methylation, glucuronidation, sulfation, and hydrolysis to verproside glucuronides (M1 and M2), verproside sulfates (M3 and M4), picroside II (M5), M5 glucuronide (M7), M5 sulfate (M9), isovanilloylcatalpol (M6), M6 glucuronide (M8), M6 sulfate (M10), 3,4-dihydroxybenzoic acid (M11), M11 glucuronide (M12), M11 sulfates (M13 and M14), 3-methyoxy-4-hydroxybenzoic acid (M15), M15 glucuronides (M17 and M18), M15 sulfate (M20), 3-hydroxy-4-methoxybenzoic acid (M16), M16 glucuronide (M19), and M16 sulfate (M21). Incubation of verproside with rat hepatocytes resulted in thirteen metabolites (M1–M11, M13, and M14). Verproside sulfate, M4 was a major metabolite in rat hepatocytes. After intravenous administration of verproside, the drug was recovered in bile (0.77% of dose) and urine (4.48% of dose), and O-methylation of verproside to picroside II (M5) and isovanilloylcatalpol (M6) followed by glucuronidation and sulfation was identified as major metabolic pathways compared to glucuronidation and sulfation of verproside in rats.

The pharmacokinetics of verproside were evaluated in rats after intravenous (dose range 2-10 mg/kg) and oral (dose range 20-100 mg/kg) administration [7,8]. Short half-life (12.2-16.6 min), high systemic clearance (56.7-86.2 mL/min/kg), and low renal clearance (2.7-4.1 mL/min/kg) of verproside after intravenous administration and very low oral bioavailability of verproside (less than 0.5%) in male Sprague-Dawley rats suggest that verproside may be extensively metabolized [8]. Isovanilloylcatalpol was identified as a metabolite of verproside after intravenous administration, but was not detected after oral administration to rats in our previous study [8]. Therefore, it is necessary to elucidate the other metabolic pathways of verproside in rats, aside from the formation of isovanilloylcatalpol, for characterization of its pharmacodynamics and toxicity.
In this work the in vivo metabolites of verproside in the bile and urine samples obtained after intravenous administration of verproside, and the predominant metabolites formed from in vitro incubation of verproside and its possible metabolites from rat liver S9 fractions and hepatocytes were elucidated using liquid chromatography-high resolution quadrupole Orbitrap mass spectrometry (LC-HRMS).

Results and Discussion
LC-HRMS analysis of the bile and urine samples obtained after intravenous injection of verproside to rats resulted in twenty-one metabolites (M1-M21), along with unchanged verproside (Figures 1 and 2). The accurate mass of deprotonated molecular ions ([M−H] − ) and the retention times (t R ) for verproside and its twenty-one metabolites, M1-M21, are shown in Table 1 Table 1). After many trials with different columns and mobile phase combinations, verproside and its twenty-one metabolites were well separated on a Halo C18 column using a gradient elution of methanol and 1 mM ammonium formate (pH 3.1).

Figure 1.
Extracted ion chromatograms of verproside and its possible metabolites in the bile samples obtained for 6 hours after intravenous administration of verproside at a dose of 10 mg/kg to a Sprague Dawley rat using 5 ppm mass accuracy.  The identification of twenty-one metabolite peaks was achieved using the accurate mass and the prominent and informative product ions from the product scan spectra (Table 1, Figures 3 and 4). The mass deviation between experimental and theoretical m/z ratio for each metabolite was less than 5 ppm, indicating good correlation between the theoretical mass calculated from the molecular elemental composition and the experimental mass obtained after full scan MS analysis. The identity of some metabolites of verproside in the bile and urine samples was ascertained by comparing their retention times and accurate masses with those of the authentic standards. Because no authentic standards were available for the metabolites of sulfates and glucuronides, sulfates and glucuronides were also identified by treating the bile and urine samples with -glucuronidase or sulfatase and by in vitro metabolism of verproside and its authentic metabolite standards including isovanilloylcatalpol, picroside II, 3,4-dihydroxybenzoic acid, 3-methoxy-4-hydroxybenzoic acid and 4-methoxy-3-hydroxybenzoic acid with rat liver S9 fractions in the presence of uridine 5'-diphosphoglucuronic acid (UDPGA) and 3'-phosphoadenosine-5'-phosphosulfate (PAPS), respectively.    (Figure 3). M2 was identified as the major metabolite after incubation of verproside with rat liver S9 fraction in the presence of UDPGA and rat hepatocytes. Treatment of the bile and urine samples with -glucuronidase resulted in the increase of the peak area for verproside and the disappearance of M1 and M2 peaks. From these results, M1 and M2 were tentatively identified as verproside glucuronides, but the exact site for glucuronidation could not be determined. The amount of M2 was more than M1 in the bile and urine samples.  Figure 3). M3 and M4 were produced after incubation of verproside with rat liver S9 fraction in the presence of PAPS and with rat hepatocytes. After sulfatase treatment of the bile and urine samples, the peak area of verproside was increased but M3 and M4 peaks were decreased. These results indicate that M3 and M4 might be verproside sulfates, but the position of sulfation was not accurately identified. M4 was identified as a major metabolite after incubation of verproside in rat hepatocytes, and the amount of M4 was more than that of M3 in the bile and urine samples.  (Figure 3). While M7 was produced after incubation of picroside II (M5) with rat liver S9 fraction in the presence of UDPGA, M8 was produced from isovanilloylcatalpol. -glucuronidase treatment of the bile and urine samples increased the peak areas of M5 (picroside II) and M6 (isovanilloylcatalpol), but M7 and M8 peaks were not detected. These results indicate that M7 and M8 may be M5 (picroside II) glucuronide and M6 (isovanilloylcatalpol) glucuronide, respectively, but the position of glucuronidation was not accurately identified. These results support the results of Li et al. [10] that identified picroside II glucuronide in the bile samples obtained after intravenous administration of picroside II in rats.

In Vivo Metabolism of Verproside in the Rats
Four male Sprague-Dawley rats (230 ± 10 g, Samtako Co., Osan, Korea) were anaesthetized via intraperitoneal injection of zoletil (50 mg/kg) and the bile duct was catheterized using PE-10 tubing. The cannulated rat was kept in Ballman cage individually. Bile and urine samples were collected for 24 h after intravenous administration of verproside dissolved in sterile water to the tail vein at a dose of 10 mg/kg. Bile and urine samples were kept in −20 C until analysis. The bile and urine samples (50 μL) were mixed with 50 μL of methanol and centrifuged. The supernatant (40 μL) was diluted with 160 μL of water. An aliquot (5 μL) was analyzed by the LC-HRMS method to identify verproside and its possible metabolites.
To characterize the nature of glucuronidation and sulfation, the bile and urine samples (25 μL) were incubated with -glucuronidase (18.75 unit) or sulfatase (0.97 unit) at 37 C for 1 h and were analyzed as described above.

In Vitro Metabolism of Verproside and Its Possible Metabolites in Rat Hepatocytes and Liver S9 Fractions
Cryopreserved rat hepatocytes were recovered with a cryopreserved hepatocyte purification kit and viable hepatocytes were resuspended in Krebs-Henseleit buffer at a final concentration of 1.6 × 10 6 cells/mL. 100 μL of rat hepatocyte suspensions (1.6 × 10 5 cells) and 100 μL of 100 μM verproside were added into a 96 well plate and the mixture was incubated for 120 min at 37 C in a CO 2 incubator. 40 μL of the incubation mixture was transferred to a 1.5 mL eppendorf tube and mixed with 40 μL of methanol followed by centrifugation at 10,000 ×g for 5 min. The supernatant (40 μL) was diluted with 60 μL of water and an aliquot (5 μL) was injected onto the LC-HRMS system.

LC-HRMS Analysis of Verproside and Metabolites
To separate and identify the structures of verproside and its metabolites, a Q Exactive Orbitrap mass spectrometer (Thermo Scientific, San Jose, CA, USA) coupled with an Accela UPLC system was used. KGaA, Darmstadt, Germany) were tried using the gradient elution of methanol and ammonium formate buffer (1 mM, pH 3.1) as the mobile phase. Best separation of metabolites was obtained with a Halo C18 column using a gradient elution of 5% methanol in ammonium formate (1 mM, pH 3.1) (mobile phase A) and methanol (mobile phase B) at a flow rate of 0.5 mL/min: 5% mobile phase B for 3 min, 5% to 25% mobile phase B in 6 min, 25% to 90% mobile phase B in 0.4 min, 90% mobile phase B for 4.5 min, 90% to 13% mobile phase B in 0.4 min, 13% mobile phase B for 4 min. The column and the autosampler were maintained at 40 C and 6 C, respectively.
Accurate mass measurements for verproside and its metabolites were performed by electrospray ionization in the negative mode with the following electrospray source settings: ion transfer capillary