Tetrahydrofurofuranoid Lignans, Eudesmin, Fargesin, Epimagnolin A, Magnolin, and Yangambin Inhibit UDP-Glucuronosyltransferase 1A1 and 1A3 Activities in Human Liver Microsomes

Eudesmin, fargesin, epimagnolin A, magnolin, and yangambin are tetrahydrofurofuranoid lignans with various pharmacological activities found in Magnoliae Flos. The inhibition potencies of eudesmin, fargesin, epimagnolin A, magnolin, and yangambin on six major human uridine 5′-diphospho-glucuronosyltransferase (UGT) activities in human liver microsomes were evaluated using liquid chromatography–tandem mass spectrometry and cocktail substrates. Eudesmin, fargesin, epimagnolin A, magnolin, and yangambin inhibited UGT1A1 and UGT1A3 activities, but showed negligible inhibition of UGT1A4, UGT16, UGT1A9, and UGT2B7 activities at 200 μM in pooled human liver microsomes. Moreover, eudesmin, fargesin, epimagnolin A, magnolin, and yangambin noncompetitively inhibited UGT1A1-catalyzed SN38 glucuronidation with Ki values of 25.7, 25.3, 3.6, 26.0, and 17.1 μM, respectively, based on kinetic analysis of UGT1A1 inhibition in pooled human liver microsomes. Conversely, the aforementioned tetrahydrofurofuranoid lignans competitively inhibited UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation with 39.8, 24.3, 15.1, 37.6, and 66.8 μM, respectively in pooled human liver microsomes. These in vitro results suggest the necessity of evaluating whether the five tetrahydrofurofuranoid lignans can cause drug–drug interactions with UGT1A1 and UGT1A3 substrates in vivo.

Aschantin, a bioactive tetrahydrofurofuranoid lignan found in M. biondii and Hernandia nymphaeifolia, exhibited the time-dependent inhibition of CYP2C8 (Ki: 10.2 μM and kinact: 0.056 min −1 ), CYP2C9 (Ki: 3.7 μM and kinact: 0.044 min −1 ), CYP2C19 (Ki: 5.8 μM and The ethanol extract of the dried flower buds of M. fargesii (encoded as NDC-052 contained eudesmin, fargesin, epimagnolin A, magnolin, and yangambin as 4.1, 3.4, 11.9, 21.5, and 9.1%, respectively, as determined by the LC-APCI-MS/MS method [18]) has been developed as an effective alternative or complement to standard asthma therapy based on their biological activities [19]. Consequently, taking NDC-052 (600 mg/day for 8 weeks per oral) in adult asthmatic patients was safe and tolerated [19]. The add-on therapy of NDC-052 (600 mg/day for 8 weeks per oral) with inhaled corticosteroids in asthmatic patients had a beneficial effect on asthma control [19]. The pharmacokinetics of herbal constituents in rats have been investigated in addition to their biological activities. Eudesmin, yangambin, epimagnolin A, fargesin, and magnolin were identified in rat plasma following oral administration (5.5-22 mg/kg) of NDC-052 to rats [18]. The area under the plasma concentration curve (AUC) and maximum plasma concentration (C max ) values of eudesmin, yangambin, and epimagnolin linearly increased with dose increase [18]. However, AUC and C max values of fargesin and magnolin were not increased with dose proportionality, suggesting the nonlinear pharmacokinetic properties of fargesin and magnolin [18]. Additionally, the AUC and C max values of magnolin in rats were 7568 ± 1085 ng·h/mL and 2493 ± 513 ng/mL, respectively, when administered NDC-052 (22.2 mg/kg containing 4 mg/kg of magnolin) [20] and had similar results with the previous report [18]. However, the AUC and C max values of magnolin in rats were 3630 ± 581 ng·h/mL and 1340 ± 113 ng/mL, respectively, when administered magnolin alone (4 mg/kg) [21]. These results suggested the possibility of drug interactions among the constituents of herbal drugs.
Therefore, this study aimed to investigate the in vitro inhibitory potentials and inhibition kinetics of eudesmin, fargesin, epimagnolin A, magnolin, and yangambin, that are major tetrahydrofurofuranoid lignans contained in NDC-052 and also found in rat plasma samples [18], on UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, and UGT2B7 activities using pooled human liver microsomes to provide the underlying drug interaction potentials of eudesmin, fargesin, epimagnolin A, magnolin, and yangambin.

Data Analysis
IC 50 values (i.e., the inhibitor concentrations required for 50% inhibition of the control activity) of eudesmin, fargesin, epimagnolin A, magnolin, and yangambin on UGT activities were estimated using SigmaPlot (version 12.0; Systat Software, San Jose, CA, USA). The apparent kinetic inhibition constant (K i ) values of eudesmin, fargesin, epimagnolin A, magnolin, and yangambin and the mode of inhibition were calculated from Dixon plot transformation [31].

Inhibitory Effect of Eudesmin on Human Uridine 5 -diphospho-glucuronosyltransferase (UGT) Isoforms
The inhibitory effects of five tetrahydrofurofuranoid lignans on six major human UGT isoforms were measured in ultrapooled human liver microsomes and the IC 50 values calcu-Pharmaceutics 2021, 13, 187 5 of 13 lated from the concentration dependent inhibition curves of UGT activities are summarized in Table 1. The UGT1A1 and UGT1A3 activities were inhibited by the presence of eudesmin  Table 1).

Inhibitory Effect of Eudesmin on Human Uridine 5′-diphospho-glucuronosyltransferase (UGT) Isoforms
The inhibitory effects of five tetrahydrofurofuranoid lignans on six major human UGT isoforms were measured in ultrapooled human liver microsomes and the IC50 values calculated from the concentration dependent inhibition curves of UGT activities are summarized in Table 1.
The UGT1A1 and UGT1A3 activities were inhibited by the presence of eudesmin  Table 1).

Inhibitory Effect of Fargesin on Human UGT Isoforms
The UGT1A1 and UGT1A3 activities were inhibited by the presence of fargesin (0.1-200 μM) in a concentration dependent manner with IC50 values of 24.5 and 21.5 μM, respectively, when measured by the formation of SN-38 glucuronide and chenodeoxycholic acid 24-acyl-glucuronide, respectively. The metabolic activities of UGT1A4, UGT1A6, UGT1A9, and UGT2B7 were slightly inhibited by the presence of fargesin with IC50 values of 182.7, 193.9, 110.9, and 94.7 μM, respectively ( Figure 3, Table 1).

Inhibitory Effect of Fargesin on Human UGT Isoforms
The UGT1A1 and UGT1A3 activities were inhibited by the presence of fargesin

Inhibitory Effect of Epimagnolin A on Human UGT Isoforms
The UGT1A1 and UGT1A3 activities were inhibited by the presence of epimagnolin A (0.1-200 μM) in a concentration dependent manner with IC50 values of 7.5 and 26.6 μM, respectively, when measured by the formation of SN-38 glucuronide and chenodeoxycholic acid 24-acyl-glucuronide. The metabolic activities of UGT1A4, UGT1A6, UGT1A9, and UGT2B7 were negligibly inhibited by epimagnolin A up to 200 μM tested ( Figure 4, Table 1).

Inhibitory Effect of Epimagnolin A on Human UGT Isoforms
The UGT1A1 and UGT1A3 activities were inhibited by the presence of epimagnolin A (0.1-200 µM) in a concentration dependent manner with IC 50 values of 7.5 and 26.6 µM, respectively, when measured by the formation of SN-38 glucuronide and chenodeoxycholic acid 24-acyl-glucuronide. The metabolic activities of UGT1A4, UGT1A6, UGT1A9, and UGT2B7 were negligibly inhibited by epimagnolin A up to 200 µM tested ( Figure 4, Table 1).

Inhibitory Effect of Magnolin on Human UGT Isoforms
The UGT1A1 and UGT1A3 activities were inhibited by the presence of magnolin (0.1-200 µM) in a concentration dependent manner with IC 50 values of 21.3 and 22.9 µM, respectively, when measured by the formation of SN-38 glucuronide and chenodeoxycholic acid 24-acyl-glucuronide. The UGT1A9 activity was slightly inhibited by the presence of magnolin with IC 50 values of 145.7 µM, when measured by the formation of mycophenolic acid glucuronide. However, the UGT1A4, UGT1A6, and UGT2B7 activities were not inhibited by the presence of magnolin up to 200 µM tested ( Figure 5, Table 1).

Inhibitory Effect of Yangambin on Human UGT Isoforms
The UGT1A1 and UGT1A3 activities were inhibited by the presence of yangambin (0.1-200 µM) in a concentration dependent manner with IC 50 values of 29.7 and 56.5 µM, respectively, when measured by the formation of SN-38 glucuronide and chenodeoxycholic acid 24-acyl-glucuronide. The metabolic activities of UGT1A4, UGT1A6, UGT1A9, and UGT2B7 were negligibly inhibited by yangambin up to 200 µM tested ( Figure 6, Table 1).

Inhibitory Effect of Magnolin on Human UGT Isoforms
The UGT1A1 and UGT1A3 activities were inhibited by the presence of magnolin (0.1-200 μM) in a concentration dependent manner with IC50 values of 21.3 and 22.9 μM, respectively, when measured by the formation of SN-38 glucuronide and chenodeoxycholic acid 24-acyl-glucuronide. The UGT1A9 activity was slightly inhibited by the presence of magnolin with IC50 values of 145.7 μM, when measured by the formation of mycophenolic acid glucuronide. However, the UGT1A4, UGT1A6, and UGT2B7 activities were not inhibited by the presence of magnolin up to 200 μM tested ( Figure 5, Table 1).

Inhibitory Effect of Magnolin on Human UGT Isoforms
The UGT1A1 and UGT1A3 activities were inhibited by the presence of magnolin (0.1-200 μM) in a concentration dependent manner with IC50 values of 21.3 and 22.9 μM, respectively, when measured by the formation of SN-38 glucuronide and chenodeoxycholic acid 24-acyl-glucuronide. The UGT1A9 activity was slightly inhibited by the presence of magnolin with IC50 values of 145.7 μM, when measured by the formation of mycophenolic acid glucuronide. However, the UGT1A4, UGT1A6, and UGT2B7 activities were not inhibited by the presence of magnolin up to 200 μM tested ( Figure 5, Table 1).

Inhibitory Effect of Yangambin on Human UGT Isoforms
The UGT1A1 and UGT1A3 activities were inhibited by the presence of yangambin (0.1-200 μM) in a concentration dependent manner with IC50 values of 29.7 and 56.5 μM, respectively, when measured by the formation of SN-38 glucuronide and chenodeoxycholic acid 24-acyl-glucuronide. The metabolic activities of UGT1A4, UGT1A6, UGT1A9, and UGT2B7 were negligibly inhibited by yangambin up to 200 μM tested ( Figure 6, Table 1).
As expected, NDC-052 inhibited UGT1A1-and UGT1A3-mediated glucuronidation in a concentration dependent manner with IC 50 values of 38.1 and 65.0 µg/mL, respectively. When the IC 50 values were calculated as the concentration of individual lignans by using the content of five lignans in NDC-052, 38.1 and 65.0 µg/mL of NDC-052 contained 4.0 and 6.9 µM of eudesmin, 3.5 and 6.0 µM of fargesin, 10.9 and 18.6 µM of epimagnolin A, 19.7 and 33.6 µM of magnolin, and 7.8 and 13.2 µM of yangambin, respectively. These concentrations were lower than the IC 50 values of individual lignans in Table 1, which suggested that the inhibitory effect of NDC-052 on the metabolic activities of UGT1A1 and UGT1A3 may be caused by the coexistence of lignans rather than the inhibition by single lignan. However, we also should note that the total content of five lignans accounts for 50% of NDC-052 and unveiled components such as terpenoids, alkaloids, and flavonoids may serve as inhibitors of UGT enzymes [42].
The efficacy of NDC-052 has been evaluated for the treatment of asthma in guinea pig chronic asthma model at a repeated oral dose (50 mg/kg/day for 8 weeks) as well as in adult asthmatic patients (600 mg/day for 8 weeks) [19]. Although the plasma and gastric concentrations of individual eudesmin, fargesin, epimagnolin A, magnolin, and yangambin in asthmatic patients were not reported, the sum of the plasma and gastric concentrations for these five lignans may reach or exceed K i values to UGT1A1 and UGT1A3 in the above efficacy model considering the C max of the previous study [18] and the expected gastric concentration of five lignans after repeated oral administration. Therefore, these five lignans may potentiate coordinately the likelihood of UGT1A1-and UGT1A3-mediated drug interaction with co-administration of victim drugs.
In a previous study, the AUC of magnolin was linearly increased with an increase in the intravenous dose (0.5-2 mg/kg) and in the oral dose (1-4 mg/kg) [21]. In addition, magnolin was mainly eliminated by metabolism [21]. Three major metabolites (i.e., Odesmethylmagnolin, i.e., M1 and M2, and hydroxymagnolin, M4) were formed by CYP2C8, CYP2C9, CYP2C19, and CYP3A4 in the in vitro studies of magnolin metabolism [43]. However, the AUC of magnolin was not proportionally increased with increasing oral dose of NDC-052 (5.5-22 mg/kg containing 1-4 mg/kg magnolin) and about two-fold higher than that following oral administration of magnolin alone [18,20]. Taken together, fargesin may be involved in the two-fold increase of magnolin AUC by inhibiting the CYP2C8, CYP2C9, CYP2C19, and CYP3A4-mediated magnolin metabolism when orally administered as NDC-052 to the same dose of single magnolin. For the elucidation of the coordinated drug interaction among the similar structure possessing components such as these tetrahydrofurofuranoid lignans in the NDC-052, the CYPs and UGTs-mediated metabolism of these tetrahydrofurofuranoid lignans awaits further investigation. In this regard, this study would provide the molecular mechanism for understanding the drug interactions among the structurally related herbal components and the concomitantly administered victim drugs.