**1. Introduction**

Catalposide is an active iridoid glycoside of *Veronica* species including *Catalpa ovata* and *Pseudolysimachion lingifolium* [1–3]. Catalposide exhibits various biological effects including anti-inflammatory [4–9], anti-oxidant [10], antinociceptic [8], cytostatic [11], hypolipidemic via peroxisome proliferator-activated receptor-α activation [12], and hepatoprotective activities [13].

Catalposide had a short half-life (19.3 ± 9.5 min), and exhibited high systemic clearance (96.7 ± 44.1 mL/min/kg), and low urinary excretion (9.9 ± 4.1% of the dose) after intravenous administration of 10 mg/kg to male Sprague-Dawley rats [14]. This indicated that catalposide might be extensively metabolized in rats. However, catalposide remained stable after 1 h incubation with rat liver microsomes in the presence of NADPH [14]. Thus, catalposide may be catabolized via non-cytochrome P450 (CYP)-mediated mechanism. Catalposide was the substrate of OAT3, OATP1B1, and OATP1B3 transporters and weakly inhibited their transport activities with IC<sup>50</sup> values of 83, 200, and 235 µM, respectively, suggesting that OAT3, OATP1B1, and OATP1B3 may regulate the pharmacokinetics and drug interactions of catalposide [15].

Pharmacokinetics and metabolism of potential active constituents in herbal drugs is helpful for the determination of dosage regimens and interpretation of pharmacological effects under clinical conditions [16]. It is important to establish the comparative metabolism and drug-metabolizing enzymes of active constituents for a full characterization of its pharmacokinetics, pharmacodynamics, and toxicity. The characterization of drug-metabolizing enzymes such as CYPs, carboxylesterases (CESs), UDP-glucuronosyltransferases (UGTs), and sulfotransferases (SULTs) responsible for the metabolism of a drug may reveal inter-individual variability in drug metabolism and the potential drug interactions [17–19]. However, catalposide metabolism has not been studied in humans and animals. We identified catalposide metabolites and drug-metabolizing enzymes involved to predict its pharmacokinetics and possible drug interactions.

We identified catalposide metabolites formed from in vitro incubations of catalposide with human hepatocytes, intestinal microsomes, and liver S9 fractions using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and characterized the CES, UGT, and SULT enzymes involved in catalposide metabolism using human cDNA-expressed CES, UGT, and SULT supersomes, respectively.

#### **2. Materials and Methods**

#### *2.1. Materials and Reagents*

Catalposide (purity, 98%) was obtained from Aobious Inc. (Gloucester, MA, USA). Alamethicin, 3-phosphoadenosine-5-phosphosulfate (PAPS), and uridine 5′ -diphosphoglucuronic acid (UDPGA) were from Sigma-Aldrich Co. (St. Louis, MO, USA). 4-hydroxybenzoic acid and 4-hydroxybenzoic acid glucuronide were purchased from Toronto Research Chemicals (North York, ON, Canada). Pooled human intestinal microsomes; pooled human liver S9 fractions; human cDNA-expressed UGTs 1A1/3/4/6/7/8/9/10 and 2B4/7/15/17 supersomes; human cDNA-expressed CESs 1b, 1c, and 2 supersomes; cryopreserved human hepatocytes; and hepatocyte purification kits were obtained from Corning Life Sciences (Woburn, MA, USA). Human cDNA-expressed SULT 1A1\*1, 1A1\*2, 1A2, 1A3, 1B1, 1C2, 1C4, 1E1, and 2A1 supersomes were purchased from Cypex Ltd. (Dundee, UK). Methanol (HPLC grade) was from Burdick & Jackson Inc. (SK Chemicals, Ulsan, Korea), and all other chemicals were of the highest quality available. Calibration mixtures for Exactive MS [ProteoMass LTQ/FT-hybrid ESI positive mode Cal Mix (MSCAL5) and negative mode Cal Mix (MSCAL6)] were obtained from Supelco (Bellefonte, PA, USA).

#### *2.2. In Vitro Metabolism of Catalposide in Cryopreserved Human Hepatocytes*

Cryopreserved human hepatocytes were recovered with the aid of a hepatocyte purification kit, and viable cells were resuspended in William's E buffer at a final concentration of 1.28 × 10<sup>6</sup> cells/mL [20]. Human hepatocyte suspensions (62.5 µL, 8.00 × 10<sup>4</sup> cells) and 62.5 µL of 400 µM catalposide in William's E buffer were added to the wells of a 96-well plate and the mixture was incubated for 120 min at 37 ◦C in a CO<sup>2</sup> incubator. Methanol (250 µL) was added to each well and the mixture was centrifuged at 3000× *g* for 10 min. Aliquots of the supernatants (250 µL) were evaporated to dryness using a vacuum evaporator (Genevac Ltd., Ipswich, UK). Each residue was dissolved in 100 µL of 5% methanol and an aliquot (5 µL) was injected into the LC-HRMS system.

### *2.3. In Vitro Metabolism of Catalposide in Human Liver S9 Fractions and Intestinal Microsomes*

Each reaction mixture contained 50 mM potassium phosphate buffer (pH 7.4), 10 mM magnesium chloride, human liver S9 fractions or human intestinal microsomes (100 µg protein), 2 mM UDPGA or 200 µM PAPS, 200 µM catalposide or a possible metabolite, and 1000 µM 4-hydroxybenzoic acid in a volume of 200 µL. Samples lacking UDPGA and PAPS served as controls. The mixtures were incubated at 37 ◦C for 60 min and the reactions were then quenched by adding 500 µL of methanol. The tubes were centrifuged and the supernatants evaporated to dryness using a vacuum concentrator. The residues were dissolved in 100 µL of 5% methanol and 5 µL aliquots were injected into the LC-HRMS system.
