*3.2. Characterization of Human SULT, UGT, and CES Enzymes Involved in Catalposide Metabolism*

A screen using human cDNA-expressed SULTs 1A1\*1, 1A1\*2, 1A2, 1A3, 1B1, 1C2, 1C4, 1E1, and 2A1 to assess the formation of catalposide sulfate (M1) from catalposide identified possible roles for SULTs 1A1\*1, 1A1\*2, 1C4, and 1E1 (Figure 4).

**Figure 4.** Rate of formation of catalposide sulfate (M1) from 150 µM catalposide by human cDNA-expressed SULT enzymes. All data are means ± SD (*n* = 3). ND: <32 pmol/min/mg protein. **500 ND ND ND ND**

**0**

**1000**

The formation of catalposide sulfate from catalposide catalyzed by SULTs 1A1\*1, 1A1\*2, and 1E1 exhibited substrate inhibition kinetics, but the activities of SULT1C4 and pooled human liver S9 fractions fitted the Hill equation (Figure 5). The enzyme kinetic parameters for the formation of catalposide sulfate from catalposide are listed in Table 2. SULT1C4 exhibited a higher affinity for catalposide and more rapid sulfation than did SULT1A1\*1, SULT1A1\*2, and SULT1E1. **SULT isoforms 1A1\*1 1A1\*2 1A2 1A3 1B1 1C2 1C4 1E1 2A1**

**Figure 5.** Michaelis–Menten plots of the sulfation of catalposide to catalposide sulfate (M1) by pooled human liver S9 fractions (**A**) and human cDNA-expressed SULT1A1\*1 (**B**), SULT1A1\*2 (**C**), SULT1C4 (**D**), and SULT1E1 (**E**). Insets: Eadie–Hofstee plots. Each data point represents the average of two determinations.


**Table 2.** Kinetic parameters for the formation of catalposide sulfate (M1) and catalposide glucuronide (M4) from catalposide in pooled human liver S9 fractions, intestinal microsomes, and human cDNA-expressed sulfotransferase (SULT) or UDP-glucuronosyltransferase (UGT) enzymes.

*V*max: pmol/min/mg protein; *Cl*int: µL/min/mg protein; *n*: Hill coefficient.

A screen using human cDNA-expressed UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17 supersomes for the metabolism of 4-hydroxybenzoic acid (M2) to 4-hydroxybenzoic acid glucuronide (M3) identified possible roles of UGT1A6 and UGT1A9 (Figure 6A). The results show that 4-hydroxybenzoic acid glucuronide (M3) was produced on incubation of catalposide with human hepatocytes, liver S9 fractions, and intestinal microsomes.

**Figure 6.** Rates of formation of (**A**) 4-hydroxybenzoic acid glucuronide (M3) from 500 µM 4-hydroxybenzoic acid (M2) and (**B**) catalposide glucuronide (M4) from 400 µM catalposide by human cDNA-expressed UGT enzymes. All data are means ± SD (*n* = 3). ND: <27 pmol/min/mg protein for M3, <0.83 pmol/min/mg protein for M4.

A screen using twelve human cDNA-expressed UGT supersomes, to assess the metabolism of catalposide to catalposide glucuronide (M4), identified possible roles of gastrointestinal tract-specific UGT1A8 and UGT1A10 (Figure 6B). The results show that catalposide glucuronide (M4) was produced after incubation of catalposide with pooled human intestinal microsomes, but not human liver S9 fractions.

Formation of catalposide glucuronide (M4) from catalposide by pooled human intestinal microsomes followed single enzyme kinetics; formation via UGT1A8 and UGT1A10 exhibited Hill equation kinetics (Figure 7, Table 2).

**Figure 7.** Michaelis–Menten plots for glucuronidation of catalposide to catalposide glucuronide (M4) by pooled human intestinal microsomes (**A**) and human cDNA-expressed UGT1A8 (**B**) and UGT1A10 (**C**). Insets: Eadie–Hofstee plots. Each data point represents the average of two determinations.

4-Hydroxybenzoic acid (M2) was formed from catalposide by pooled human liver S9 fractions; intestinal microsomes; and the CES1b, CES1c, and CES2 enzymes (Figure 8). The rate of formation of 4-hydroxybenzoic acid (M2) after incubation of catalposide with pooled human intestinal microsomes was higher than that after incubation with pooled human liver S9 fractions (Figure 8).

**Figure 8.** Rates of formation of 4-hydroxybenzoic acid (M2) from 200 µM and 400 µM catalposide in human liver S9 fractions (Liver S9); human intestinal microsomes (HIM); and human cDNA-expressed CES1b, CES1c, and CES2 enzymes. All data are means ± SD (*n* = 3).

#### **4. Discussion**

μ μ μ μ Catalposide was metabolized to catalposide sulfate (M1), catalposide glucuronide (M4), 4-hydroxybenzoic acid (M2), and M2 glucuronide (M3) by human hepatocytes or intestinal microsomes (Figure 3). On the basis of the kinetics of catalposide sulfate (M1) formation from catalposide catalyzed by human cDNA-expressed SULTs 1A1\*1, 1A1\*2, 1C4, and 1E1 (Figure 4, Table 2), we suggest that SULT1A1, SULT1C4, and SULT1E1 may play major roles in this metabolism. SULT1C4 exhibited higher activity (Clint, 51.0 µL/min/mg protein) in terms of catalposide sulfation than did SULT1A1\*1, SULT1A1\*2, or SULT1E1 (Clint, 10.6~23.8 µL/min/mg protein) (Table 2). SULT1C4 is highly expressed in the fetal lung and kidney, and at lower levels in the fetal heart, adult kidney, ovary, brain, and spinal cord [21–23]. SULT1A1 is the major hepatic SULT (53% of total hepatic SULTs), but is also present in substantial quantities in the small intestine (19% of total SULTs) [23,24]. SULT1E1 is expressed at relatively low levels in the liver (6% of total SULTs) and small intestine (8% of total SULTs), but is the most abundant enzyme in the lung (40% of total SULTs) [23,24]. Catalposide was metabolized to M1 by cytosolic SULTs of both hepatic and extrahepatic tissues. There may be inter-individual variability in catalposide sulfation, given that SULT1A1, SULT1C4, and SULT1E1 polymorphisms are known in humans [25]. SULT1A1, SULT1C4, and SULT1E1 are induced or inhibited by various drugs

and chemicals [26,27]; therefore, co-administration of drugs that inhibit or induce expression of these enzymes may affect catalposide sulfation and thus catalposide pharmacokinetics.

Metabolism of catalposide to catalposide glucuronide (M4) was mediated by human cDNA-expressed UGT1A8 and UGT1A10 enzymes, which are confined to the gastrointestinal tract [28–32] (Figure 7), indicating that catalposide glucuronidation was gastrointestinal tract-specific; glucuronidation was not detected after incubation of catalposide with human hepatocytes and liver S9 fractions. UGT1A10-catalyzed catalposide glucuronidation was more extensive (Clint, 0.3399 µL/min/mg protein) than UGT1A8-catalyzed glucuronidation (Clint, 0.0396 µL/min/mg protein) (Table 2). UGT1A10 is more abundant than UGT1A8 in the small intestine (17.3% vs. 0.8% of total UGT protein) and colon (27.4% vs. 1.5% of total UGT protein) [32]. Thus, UGT1A10 may play the major role in glucuronidation of catalposide and UGT1A8 only a minor role. The human UGT1A8 and UGT1A10 enzymes are inhibited by various drugs [30,32–35]. Therefore, co-administration of drugs that inhibit or induce UGT1A8 and UGT1A10 may affect catalposide glucuronidation.

CES2, the predominant CES of the intestine, was more active in terms of hydrolysis of catalposide to 4-hydroxybenzoic acid (M2) than were the hepato-predominant CES1b and CES1c enzymes [32,36]. Thus, the rate of formation of 4-hydroxybenzoic acid (M2) was higher when catalposide was incubated with pooled human intestinal microsomes than with pooled human liver S9 fractions.

UGT1A6 and UGT1A9 play major roles in the formation of 4-hydroxybenzoic acid glucuronide (M3) from 4-hydroxybenzoic acid (Figure 6A). Abbas et al. [37] found that UGT1A9 played the major role in metabolism of 4-hydroxybenzoic acid to 4-hydroxybenzoic acid glucuronide. UGT1A6 and UGT1A9 are major enzymes of both the liver and intestine; therefore, 4-hydroxybenzoic acid glucuronide (M3) was identified after incubation of catalposide with either human hepatocytes or intestinal microsomes.

#### **5. Conclusions**

Catalposide was metabolized to catalposide sulfate (M1), 4-hydroxybenzoic acid (M2), M2 glucuronide (M3), and catalposide glucuronide (M4) via sulfation, glucuronidation, and hydrolysis, on incubation with human hepatocytes, liver S9 fractions, or intestinal microsomes. SULT1A1, SULT1C4, and SULT1E1 formed catalposide sulfate (M1) from catalposide. Gastrointestine-specific UGT1A8 and UGT1A10 played major roles in formation of catalposide glucuronide (M4). CES2 and CES1 catalyzed hydrolysis of catalposide to 4-hydroxybenzoic acid (M2), which was further metabolized to M2 glucuronide (M3) by UGT1A6 and UGT1A9. These results suggest that SULT1A1, SULT1C4, SULT1E1, UGT1A8, UGT1A10, CES2, and CES1 enzymes may play important roles in the pharmacokinetics and drug–drug interaction of catalposide in humans. The pharmacokinetics of catalposide may be dramatically affected by the co-administration of inhibitors or inducers of UGTs, CESs, or SULTs.

**Author Contributions:** Conceptualization, D.-K.H. and H.S.L.; methodology, D.-K.H., W.-G.C., Y.S., and S.K.; software, D.-K.H. and J.-H.K.; investigation, D.-K.H., W.-G.C., Y.S., and S.K.; data curation, D.-K.H., Y.S., J.-H.K., and H.S.L.; writing—original draft preparation, D.-K.H., J.-H.K., and Y.S.; writing—review and editing, Y.-Y.C., J.Y.L., H.C.K., and H.S.L.; supervision, H.S.L.; project administration, H.S.L.; funding acquisition, H.C.K. and H.S.L.

**Funding:** This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2015M3A9E1028325, NRF-2017M3A9F5028608, and NRF-2017R1A4A1015036).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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