Molecular mechanisms governing different

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British Journal of Pharmacology

DOI:10.1111/bph.12971 www.brjpharmacol.org

RESEARCH PAPER

Correspondence

Molecular mechanisms governing different pharmacokinetics of ginsenosides and potential for ginsenoside-perpetrated herb–drug interactions on OATP1B3

Professor Chuan Li, Laboratory for DMPK Research of Herbal Medicines, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 501 Haike Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China. E-mail: [email protected] ---------------------------------------------------------------†

Visiting graduate student from Tianjin University of Traditional Chinese Medicine. ----------------------------------------------------------------

Received 31 January 2014

Revised 20 September 2014

Accepted 30 September 2014

Rongrong Jiang1,2, Jiajia Dong1, Xiuxue Li1, Feifei Du1, Weiwei Jia1†, Fang Xu1, Fengqing Wang1, Junling Yang1, Wei Niu1 and Chuan Li1,2,3 1

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China, University of Chinese Academy of Sciences, Shanghai, China, and 3Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China 2

BACKGROUND AND PURPOSE Ginsenosides are bioactive saponins derived from Panax notoginseng roots (Sanqi) and ginseng. Here, the molecular mechanisms governing differential pharmacokinetics of 20(S)-protopanaxatriol-type ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 and 20(S)-protopanaxadiol-type ginsenosides Rb1, Rc and Rd were elucidated.

EXPERIMENTAL APPROACH Interactions of ginsenosides with human and rat hepatobiliary transporters were characterized at the cellular and vesicular levels. A rifampin-based inhibition study in rats evaluated the in vivo role of organic anion-transporting polypeptide (Oatp)1b2. Plasma protein binding was assessed by equilibrium dialysis. Drug–drug interaction indices were calculated to estimate potential for clinically relevant ginsenoside-mediated interactions due to inhibition of human OATP1Bs.

KEY RESULTS All the ginsenosides were bound to human OATP1B3 and rat Oatp1b2 but only the 20(S)-protopanaxatriol-type ginsenosides were transported. Human multidrug resistance-associated protein (MRP)2/breast cancer resistance protein (BCRP)/bile salt export pump (BSEP)/multidrug resistance protein-1 and rat Mrp2/Bcrp/Bsep also mediated the transport of the 20(S)-protopanaxatriol-type ginsenosides. Glomerular-filtration-based renal excretion of the 20(S)-protopanaxatriol-type ginsenosides was greater than that of the 20(S)-protopanaxadiol-type counterparts due to differences in plasma protein binding. Rifampin-impaired hepatobiliary excretion of the 20(S)-protopanaxatriol-type ginsenosides was effectively compensated by the renal excretion in rats. The 20(S)-protopanaxadiol-type ginsenosides were potent inhibitors of OATP1B3.

CONCLUSION AND IMPLICATIONS Differences in hepatobiliary and in renal excretory clearances caused markedly different systemic exposure and different elimination kinetics between the two types of ginsenosides. Caution should be exercised with the long-circulating 20(S)-protopanaxadiol-type ginsenosides as they could induce hepatobiliary herb–drug interactions, particularly when patients receive long-term therapies with high-dose i.v. Sanqi or ginseng extracts. © 2014 The British Pharmacological Society

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Abbreviations ABC, ATP-binding cassette; BCRP/Bcrp, breast cancer resistance protein; BSEP/Bsep, bile salt export pump; CLB, hepatobiliary excretory clearance; CLint, intrinsic clearance; CLR, renal excretory clearance; CLtot,p, total plasma clearance; Cum.Ae-B, cumulative amount excreted into bile; Cum.Ae-U, cumulative amount excreted into urine; DDI, drug–drug interaction; E1S, oestrone-3-sulfate; E217βG, oestradiol-17β-D-glucuronide; fe-B, fractional biliary excretion; fe-U, fractional urinary excretion; fu, fraction of plasma protein-unbound compound; IC50, half maximal inhibitory concentration; Km, Michaelis constant; Ki, inhibition constant; MDR/Mdr, multidrug resistance protein; MRP/Mrp, multidrug resistance-associated protein; OATP/Oatp, organic anion-transporting polypeptide; SLC, solute carrier; t1/2, elimination half-life; Vmax, maximum transport velocity

Tables of Links TARGETS

LIGANDS

Transporters

Oestrone-3-sulfate

BCRP, breast cancer resistance protein, ABCG2

Rifampin

BSEP, bile salt export pump, ABCB11

MTX, methotrexate

MDR1, multidrug resistance protein, ABCB1

TCA, taurocholic acid

MRP2, multidrug resistance-associated protein, ABCC2 OATP1B1, organic anion-transporting polypeptide, SLCO1B1 OATP1B3, organic anion-transporting polypeptide, SLCO1B3 These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http:// www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (Alexander et al., 2013).

Introduction The root of Panax notoginseng (Sanqi in Chinese) is a clinically important cardiovascular herb. It is extensively used both alone and in combination with other herbs, such as the root of Salvia miltiorrhiza (Danshen), for patients with coronary artery disease (Ng, 2006; Jia et al., 2012; Shang et al., 2013). Ginsenosides are pharmacologically relevant to Sanqi therapies because they have multiple bioactivities, including vasodilatation, hypolipidaemic effects, anti-inflammatory and antioxidant properties (Lü et al., 2009; Karmazyn et al., 2011; Sun et al., 2013). Ginsenosides (including notoginsenoside R1) are dammarane triterpene saponins and are classified by structure into two types: 20(S)-protopanaxatriol-type (ppttype) and 20(S)-protopanaxadiol-type (ppd-type). Despite their extensive clinical use, Sanqi therapies remain to be optimized. In this context, pharmacokinetic (PK) information of ginsenosides is crucial to the link between the individual ginsenosides’ bioactivities and the Sanqi therapies. Although therapeutic advantages may exist in the Sanqibased combination therapy, PK herb–herb interactions, including ginsenoside-based interactions, should be addressed. Because the use of medicinal herbal products is prevalent among patients who are taking synthetic drugs, PK studies that help to address the possible interactions between ginsenosides and synthetic drugs are also necessary. Like Sanqi, herbs of other Panax species, such as P. quinquefolius roots (American ginseng) and P. ginseng roots (Asian ginseng), have their pharmacological properties, which are also ascribed to ginsenosides. Coronary artery disease is the most common cause of heart failure, which is a devastating condition with limited options for treatment (Tamargo and López-Sendón, 2011). Recently, Guo et al. 1060


(2011) and Moey et al. (2012) found that a ginsenoside extract from American ginseng could prevent and reverse cardiac hypertrophy, myocardial remodelling and heart failure in vitro and in vivo. However, these researchers were unable to determine which of the ginsenosides present accounted for the observed salutary effects. Dysfunctional blood vessel formation is a major problem in heart failure and therapeutic angiogenesis may be a suitable treatment option (Taimeh et al., 2013). Intriguingly, the ppt-type ginsenosides Rg1 and Re may be useful as non-peptide-based angiotherapeutic agents for tissue regeneration (Leung et al., 2006; 2007a; 2011). However, the coexisting ppd-type ginsenoside Rb1 has antiangiogenic properties and the ratio of the concentrations of the two types of ginsenosides can alter angiogenic properties (Sengupta et al., 2004; Leung et al., 2007b). Although there is a predominance of ppt-type ginsenosides in Sanqi (Li et al., 2005), it is unclear whether dosing a patient with the herbal extract via conventional oral and intravenous routes can enhance angiogenesis in the failing heart. To this end, a key step in moving forward is to determine the levels and durations of systemic exposure to ginsenosides and their intestinal absorption and heart disposition and to investigate the factors governing the pharmacokinetics that may affect their antihypertrophic, anti-remodelling, cardioprotective and angiogenic properties. Recent PK studies of ginsenosides from aqueous Sanqi extracts in rat and human subjects indicated that both the ppt-type ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 and ppd-type ginsenosides Rb1 and Rd were present in the blood stream after dosing (Liu et al., 2009; Hu et al., 2013). However, the two types of ginsenosides had markedly different elimination kinetics and hence significantly different levels and durations of systemic exposure despite their

Biliary and renal excretions of ginsenosides

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Figure 1 Chemical structures of ginsenosides. Ara, α-L-arabinopyranosyl; Glc, β-D-glucopyranosyl; Rha, α-L-rhamnopyranosyl and Xyl, β-D-xylopyranosyl.

similar chemical structures (Figure 1). The ppt-type ginsenosides were primarily eliminated via rapid hepatobiliary excretion, but the ppd-type ginsenosides were slowly excreted into the bile (Liu et al., 2009). Transporter-mediated hepatobiliary excretion is a major determinant of plasma pharmacokinetics of many drugs and is therefore a likely target for possible PK drug–drug interactions. The current study was designed to elucidate the molecular mechanisms underlying the relevant elimination processes, which caused different pharmacokinetics of the ginsenosides. The data were analysed to derive further information about possible herb–drug and herb–herb interactions associated with the ginsenosides.

Methods A detailed description of experimental procedures and materials is provided in the Supporting Information Appendix S1, which is available online.

Cellular transport assays HEK293 cells were grown in a DMEM fortified with 10% fetal calf serum in a humidified incubator at 37°C and 5% CO2. The human organic anion-transporting polypeptide (OATP)1B1, OATP1B3 and rat Oatp1b2 expression plasmids and the empty vector were introduced separately into the HEK293 cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) transfection reagent according to the manufacturer’s protocol. Before use, the cells transfected with human OATP1B1, human OATP1B3 or rat Oatp1b2 plasmid were functionally characterized by measuring the protein express-

ing of transporters (Supporting Information Appendix S2) and uptake of oestradiol-17β-D-glucuronide (E217βG). Transport studies were carried out in 24-well poly-D-lysine-coated plates with the transfected cells 48 h after transfection. After pre-incubation, Krebs-Henseleit buffer was replaced by the same buffer fortified with the test compound or the prototypical substrate to initiate transport. The incubation was stopped at the designated time by aspirating the buffer from the well. After washing three times with ice-cold KrebsHenseleit buffer, the cells were lysed and the resulting cellular lysates were analysed by LC-MS/MS. The transport rate of test compound in pmol·min−1 per mg protein was calculated using the following equation:

Transport = (CL × VL ) T WL

(1)

where CL represents the concentration of test compound in the cellular lysate (μM), VL is the volume of the lysate (μL), T is the incubation time (10 min) and WL is the measured cellular protein amount of each well (mg). Differential uptake between the transfected cells (TC) and the mock cells (MC) was defined as a net transport ratio (TransportTC/TransportMC ratio) and net transport ratio greater than three suggested a positive result. Kinetics of human OATP1B3- and rat Oatp1b2-mediated cellular uptakes of ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 were assessed with respect to Km and maximum velocity (Vmax) and the incubation time was set at 5 min to ensure that the assessment was performed under linear uptake conditions (Supporting Information Appendix S3). The inhibitory effect of rifampin on the activity of human OATP1B3 or rat Oatp1b2 that mediated transport of ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 was further measured in terms of inhibition constant (Ki). British Journal of Pharmacology (2015) 172 1059–1073

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The inhibitory effects of ginsenosides on the activity of human OATP1B3- and OATP1B1-mediated transport of E217βG were measured in terms of half maximal inhibitory concentration (IC50) and rifampin was used as positive control.

Vesicular transport assays Inside-out membrane vesicles expressing human multidrug resistance-associated protein (MRP)2, breast cancer resistance protein (BCRP), bile salt export pump (BSEP) or multidrug resistance protein (MDR)1 transporters or rat Mrp2, Bcrp or Bsep transporter were tested with the ginsenosides using a rapid filtration method according to a modified version of the manufacturer’s protocol. ATP was used as energy source to transport the test compounds across vesicle membrane and AMP was used for negative control. The quantity of compounds trapped inside the vesicles was measured by LC-MS/MS. The transport rate of test compound in pmol·min−1 per mg protein was calculated using the following equation:

Transport = (CV × VV ) T WV

(2)

where CV represents the concentration of the compound in the vesicular lysate supernatant (μM), VV is the volume of the lysate (μL), T is the incubation time (10 min) and WV is the amount of vesicle protein amount per well (0.05 mg). Positive results for ATP-dependent transport were defined as a net transport ratio (TransportATP/TransportAMP ratio) greater than three. Kinetics of human MRP2-, BCRP-, BSEP- and MDR1and rat Mrp2-, Bcrp- and Bsep-mediated vesicular transports of ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 were assessed with respect to Km and Vmax. The inhibitory effects of 100 μM ginsenosides on the activity of human MRP2, BCRP and BSEP that mediated transport of E217βG, methotrexate (MTX) and taurocholate acid (TCA), respectively, were also assessed.

Rat studies All animal care and experimental procedures complied with the Guidance for Ethical Treatment of Laboratory Animals (The Ministry of Science and Technology of China, 2006; www.most.gov.cn/fggw/zfwj/zfwj2006) and were approved by the Institutional Animal Care and Use Committee at Shanghai Institute of Materia Medica (Shanghai, China). All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath et al., 2010). A total of 243 animals were used in the experiments described here. Male Sprague Dawley rats (230–300 g) were obtained from the Sino-British SIPPR/BK Laboratory Animal Co., Ltd. (Shanghai, China). The femoral arteries of rats were cannulated for blood sampling and other rats underwent bile duct cannulation for bile collection (Chen et al., 2013; Cheng et al., 2013). Three rat studies were performed with the rodents treated with or without rifampin (i.v., 20 mg·kg−1). For each rat study, rats were randomly assigned to nine groups to receive a bolus i.v. dose of ginsenosides Rg1, Re, Rb1 (using rifampin-free rats only), Rc (using rifampin-free rats only), Rd (using rifampin-free rats only) or notoginsenoside R1 at 2.5 μmol·kg−1. In the first rat study, serial blood samples (60 μL; 0, 5, 15, 30 min, 1, 2, 4, 6, 8, 10 and 24 h) were 1062


collected after dosing and heparinized. The plasma fractions were prepared by centrifugation. In the second rat study, bile samples were collected from rats between 0–2, 2–6, 6–12 and 12–24 h after dosing and were weighed. A sodium taurocholate solution (1.5 mL·h−1; pH 7.4) was infused into the duodenum during the day of bile collection. In the third rat study, urine samples were collected at 0–8 and 8–24 h after dosing and were weighed. The rats were housed singly in metabolic cages and the collection tubes containing their urine were frozen at −15°C during sample collection. All rat samples were stored at −70°C until analysis. Each rat study was repeated twice.

Determination of plasma protein binding Equilibrium dialysis was used to assess the fraction of plasma protein-unbound compound (fu) (Guo et al., 2006). The test compounds were individually added into blank plasma to generate nominal concentrations of 5 μM for ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 and 50 μM for ginsenosides Rb1, Rc and Rd. The fu value was calculated using the following equation:

f u = Cd Cp × 100%

(3)

where Cd represents the concentration in the dialysate (μM) after completion of dialysis and Cp is the corresponding concentration in the plasma (μM).

LC-MS/MS-based bioanalytical assays Validated LC-MS/MS-based bioanalytical methods were used to measure test compounds [including the ginsenosides, rifampin, oestrone-3-sulfate (E1S), E217βG, TCA and MTX] in a variety of biomatrices. A TSQ Quantum mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) was interfaced via an electrospray ionization probe with a LC (Agilent Technologies, Waldbronn, Germany).

Data processing GraFit software (version 5.0; Erithacus Software, Surrey, UK) was used to determine the Km, Vmax, Ki and IC50 values. The calculation of Ki value was based on the following equation:

v = Vmax ⋅ S [ Km (1 + I Ki ) + S ]

(4)

where v represents the difference in the transport of compound transported by transfected cells and the mock cells pmol·min−1 per mg protein, I and S are the concentration of inhibitor (μM) and substrate (μM) respectively. The IC50 for inhibition of transport activity obtained from a plot of percentage activity remaining (relative to control) versus log10 inhibitor concentration. Plasma PK parameters were estimated by non-compartmental analysis using Thermo Kinetica software package (version 5.0; InnaPhase, Philadelphia, PA, USA). The hepatobiliary excretory clearance (CLB) or the renal excretory clearance (CLR) was calculated by dividing the cumulative amount excreted into bile (Cum.Ae-B,0–t) or urine (Cum.Ae-U,0–t), respectively, by the plasma AUC0–t. The drug– drug interaction index (DDI index) was calculated using the following equation (Giacomini et al., 2010; Wang et al., 2013):

DDI index = unbound Cmax ⋅ R IC50

(5)

where unbound Cmax represents the maximal plasma unbound concentration after therapeutic dosing (μM), IC50


represents the half maximal inhibitory concentration (μM) and R is the accumulative factor. R was calculated according to the following equation:

R = 1 (1 − e− kτ )

(6)

where k represents the elimination rate constant (0.693/t1/2) and τ is the dosing interval (h). All data are expressed as the mean ± SD. Statistical analysis was performed using IBM SPSS Statistics Software (version 19.0; IBM, Somers, NY, USA). Data reported in the current study were assumed to describe a normal standard distribution and comparisons between two groups were performed by means of Student’s unpaired t-test. A value of P ≤ 0.05 was considered to be the minimum level of statistical significance.

Materials Ginsenosides Rg1, Re, Rb1, Rc and Rd and notoginsenoside R1 were obtained from Tauto Biotech (Shanghai, China) and their purity exceeded 98%. Rifampin, E1S, E217βG, TCA, MTX, poly-D-lysine hydrobromide (70 000–150 000 Da) and ATP were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used for in vitro studies. Rifampin for injection (Huapont Pharmaceutical, Chongqing, China; with a China Food and Drug Administration ratification number of GuoYaoZhunZiH20041320) that was used in the animal studies was freezedried solid and was available in a sterile parenteral dosage form for i.v. injection. HEK293 cells were obtained from American Type Culture Collection (Manassas, VA, USA). Human OATP1B1 (NM_006446) and OATP1B3 (NM_019844) cDNA clones (Thermo Scientific, Waltham, MA, USA) were subcloned into pcDNA3.1 expression plasmid by Invitrogen Life Technologies (Shanghai, China). The open reading frame of rat Oatp1b2 (NM_031650) was synthesized and subcloned into pcDNA3.1 expression plasmid by Invitrogen Life Technologies. Inside-out membrane vesicle suspensions that expressed human MRP2, MDR1, BCRP, BSEP or rat Mrp2, Bcrp or Bsep were obtained from Genomembrane (Kanazawa, Japan). Inside-out membrane vesicle suspensions that expressed rat Mdr1a or rat Mdr1b were obtained from BD Gentest (Woburn, MA, USA).

Results In vitro interactions of ginsenosides with human hepatobiliary transporters The ppt-type ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 were found to be substrates of human OATP1B3, rather than those of human OATP1B1; the relevant net transport ratios are shown in Table 1. Ginsenosides Rb1, Rc and Rd were not transported by OATP1B3 and OATP1B1 (Table 1). The OATP1B3-mediated uptakes of ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 were saturable with Km, Vmax and intrinsic clearance (CLint) values, as shown in Table 2 and were competitively inhibited by rifampin (Table 2 and Supporting Information Appendix S4). Although the OATP1B transporters did not transport ginsenosides Rb1, Rc and Rd, these ppd-type ginsenosides inhibited the uptake of E217βG in a concentration-dependent manner with IC50 values of less than 1 μM for OATP1B3 and, for OATP1B1, of between 1 and

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5 μM (Table 3). The ppt-type ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 exhibited weak inhibitory properties towards the OATP1B transporters (IC50 values > 39 μM; Table 3). In addition to the preceding solute carrier (SLC) transporters, human hepatic efflux transporters also exhibited in vitro transport activities for the ppt-type ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 with net transport ratios for MRP2, BCRP, BSEP and MDR1 shown in Table 1. However, these ATP-binding cassette (ABC) transporters exhibited little or no activity towards the transports of the ppd-type ginsenosides Rb1, Rc and Rd (Table 1). The Km Vmax and CLint values for the MRP2-, BCRP-, BSEP- and MDR1-mediated transports of the ppt-type ginsenosides are shown in Table 2. The ppdtype ginsenosides did not exhibit any significant inhibitory activity towards these ABC transporters. This was indicated by 100 μM ginsenosides producing only 7.3–21.3% inhibition of transport activities of MRP2 (E217βG), BCRP (MTX) and BSEP (TCA).

In vitro transports of ginsenosides by rat hepatobiliary transporters The rat hepatic uptake transporter Oatp1b2 (Slco1b2) is the closest orthologue of human OATP1B1 and OATP1B3 (Cattori et al., 2000; Kullak-Ublick et al., 2001). Rat Oatp1b2 also preferentially transported the ppt-type ginsenoside Rg1, ginsenoside Re and notoginsenoside R1, but it did not have any significant transport activity for the ppd-type ginsenosides Rb1, Rc and Rd (Table 1). The Km values of the ppt-type ginsenosides for rat Oatp1b2 were slightly greater than those for human OATP1B3 (Table 2). The Oatp1b2-mediated uptake of the ppt-type ginsenosides was also inhibited competitively by rifampin (Table 2 and Supporting Information Appendix S4). The rat ABC transporters Mrp2, Bcrp and Bsep exhibited transport activities for the ppt-type ginsenosides (Table 1), with Km Vmax and CLint values shown in Table 2. These ABC transporters did not exhibit any significant activity towards the transports of the ppd-type ginsenosides Rb1, Rc and Rd (Table 1).

Rifampin-induced disruption of Oatp1b2 altered the levels of rat systemic exposure to ppt-type ginsenosides but their elimination kinetics were still significantly different from those of ppd-type ginsenosides Rifampin is an OATP/Oatp inhibitor and substrate (Vavricka et al., 2002; Zaher et al., 2008). As shown in Supporting Information Appendix S4, the plasma level of rifampin declined with a mean t1/2 of 3.3 h in rats after an i.v. dose of 20 mg·kg−1. Rifampin was moderately bound to rat plasma protein (fu, 23.1%). Accordingly, the unbound plasma concentrations of rifampin (1.1–9.8 μM) during 0–8 h after dosing exceeded its Ki values for inhibition of rat Oatp1b2-mediated transports of the ppt-type ginsenosides (0.7–0.9 μM). There were significant differences between the ppt-type ginsenosides (ginsenoside Rg1, ginsenoside Re and notoginsenoside R1) and the ppd-type ginsenosides (ginsenosides Rb1, Rc and Rd) in the systemic exposure and elimination kinetics after i.v. dosing with the individual compounds at 2.5 μmol·kg−1 (Figures 2 and 3 and Table 4). This was shown British Journal of Pharmacology (2015) 172 1059–1073

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British Journal of Pharmacology (2015) 172 1059–1073 6.81 ± 0.8 3.12 ± 0.09 5.91 ± 0.17

12.1 ± 1.4 (MTX) 4.22 ± 0.12 (TCA) 4.67 ± 0.57 (VER)

BCRP

BSEP

4.12 ± 1.11

1.21 ± 0.37

21.9 ± 3.9 (TCA)

Ntcp

5.91 ± 0.27 1.52 ± 0.12 1.22 ± 0.07

43.0 ± 7.6 (MTX) 10.5 ± 0.7 (TCA) 4.23 ± 0.06 (VER) 3.60 ± 0.04 (VER)

Bcrp

Bsep

Mdr1a

Mdr1b

10.2 ± 2.00

1.28 ± 0.04

1.32 ± 0.07

3.93 ± 0.14

4.01 ± 0.79

1.28 ± 0.06

1.52 ± 0.09

4.32 ± 0.67

3.31 ± 0.24

19.8 ± 4.1

0.836 ± 0.023

5.50 ± 0.23

0.614 ± 0.005

22.0 ± 1.3

5.31 ± 0.15

6.72 ± 0.22

23.9 ± 3.0

1.02 ± 0.16

37.2 ± 4.1

1.29 ± 0.06

Notoginsenoside R1 (50 μM)

1.11 ± 0.13

0.912 ± 0.012

0.923 ± 0.105

1.03 ± 0.07

1.21 ± 0.29

0.813 ± 0.106

0.912 ± 0.123

1.11 ± 0.10

1.49 ± 0.07

1.82 ± 0.08

1.51 ± 0.14

1.20 ± 0.20

0.933 ± 0.266

0.921 ± 0.135

1.04 ± 0.12

Ginsenoside Rb1 (50 μM)

Ppd-type

0.922 ± 0.065

1.20 ± 0.01

1.22 ± 0.16

1.41 ± 0.08

1.39 ± 0.06

1.11 ± 0.12

1.02 ± 0.03

1.03 ± 0.01

1.40 ± 0.23

0.842 ± 0.050

0.833 ± 0.074

0.921 ± 0.087

1.14 ± 0.06

0.892 ± 0.044

1.02 ± 0.14

Ginsenoside Rc (50 μM)

1.03 ± 0.04

1.41 ± 0.09

1.03 ± 0.00

1.21 ± 0.01

1.11 ± 0.04

1.03 ± 0.26

1.17 ± 0.05

0.916 ± 0.092

1.24 ± 0.23

0.907 ± 0.078

1.04 ± 0.15

0.911 ± 0.075

1.03 ± 0.19

0.922 ± 0.006

0.914 ± 0.077

Ginsenoside Rd (50 μM)

E217βG, oestradiol-17β-D-glucuronide; E1S, oestrone-3-sulfate; MTX, methotrexate; TCA, taurocholic acid; VER, verapamil. The functions of human MDR1, rat Mdr1a and rat Mdr1b were verified with veripamil by ATPase assay according to the manufacturers’ protocols and using the ratio of vesicular transport in the absence of sodium vanadate to that in the presence of sodium vanadate. Values represent the means ± SDs (n = 3). For those with net transport ratios greater than three, the differences between TransportTC and TransportMC or between TransportATP and TransportAMP were statistically significant (P < 0.05).

12.5 ± 0.12 4.03 ± 0.32

13.1 ± 3.2 (E217βG)

Mrp2

Rat hepatic ABC transporters

9.92 ± 1.19

10.9 ± 1.44

63.0 ± 7.4 (E217βG) 0.927 ± 0.201

1.21 ± 0.02

1.13 ± 0.00

7.61 ± 1.1 (E217βG)

Oatp1b2

5.99 ± 0.56

5.13 ± 0.45

Oatp1a1

Rat hepatic SLC transporters

MDR1

10.9 ± 1.8

10.2 ± 0.9 (E217βG)

MRP2

16.8 ± 2.3

88.6 ± 5.2 (E1S)

OATP2B1

Human hepatic ABC transporters

107 ± 15 0.844 ± 0.021

15.1 ± 0.8 0.918 ± 0.036

73.2 ± 10.9 (E217βG)

OATP1B3

1.20 ± 0.04

Ginsenoside Re (50 μM)

1.11 ± 0.02

Ginsenoside Rg1 (50 μM)

97.7 ± 7.1 (E217βG)

Positive control (10 μM)

OATP1B1

Human hepatic SLC transporters

Hepatic transporter

Ppt-type

Net transport ratios of in vitro transports of ginsenosides by various human and rat hepatic transporters

Table 1

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Table 2 Comparison of kinetic parameters for transports of ginsenosides by human and rat hepatic transporters

Compound

Km (μM)

Vmax (pmol·min−1 per mg protein)

CLint (μL·min−1 per mg protein)

Ki of rifampin (μM)

Human OATP1B3 Positive control (E217βG)

12.5 ± 3.7

9.74 ± 0.72

0.779

—

Ginsenoside Rg1

45.4 ± 29.1

12.3 ± 6.2

0.270

0.725 ± 0.039

Ginsenoside Re

14.3 ± 4.2

21.5 ± 1.8

1.51

0.647 ± 0.043

Notoginsenoside R1

73.4 ± 33.6

12.3 ± 4.9

0.168

0.851 ± 0.062

Human MRP2 Positive control (E217βG)

18.2 ± 3.9

30.1 ± 3.0

1.65

—

Ginsenoside Rg1

93.5 ± 21.5

190 ± 21

2.03

—

Ginsenoside Re

89.3 ± 21.8

39.9 ± 4.5

0.447

—

Notoginsenoside R1

108 ± 22

119 ± 12

1.10

—

Positive control (E1S)

37.8 ± 15.4

130 ± 19

3.44

—

Ginsenoside Rg1

221 ± 110

38.1 ± 9.3

0.172

—

Ginsenoside Re

175 ± 69

33.9 ± 7.7

0.194

—

Notoginsenoside R1

208 ± 98

24.4 ± 6.9

0.117

— —

Human BCRP

Human BSEP Positive control (TCA)

8.09 ± 4.22

26.5 ± 4.5

3.28

Ginsenoside Rg1

113 ± 52

33.4 ± 7.3

0.296

—

Ginsenoside Re

106 ± 30

25.5 ± 3.1

0.241

—

Notoginsenoside R1

74.6 ± 34.9

23.1 ± 4.04

0.310

—

Human MDR1 Positive control (VER)

12.2 ± 1.0

45.3 ± 3.1

3.71

—

Ginsenoside Rg1

42.4 ± 7.9

113 ± 8

2.67

—

Ginsenoside Re

9.69 ± 7.05

4.09 ± 0.76

0.422

—

Notoginsenoside R1

38.4 ± 16.1

47.4 ± 6.4

1.23

— —

Rat Oatp1b2 Positive control (E217βG)

20.7 ± 2.5

184 ± 7

8.89

Ginsenoside Rg1

72.6 ± 20.4

62.8 ± 7.6

0.864

0.681 ± 0.041

Ginsenoside Re

64.8 ± 24.0

131 ± 70

2.01

0.866 ± 0.047

Notoginsenoside R1

122 ± 37

41.7 ± 16.4

0.342

0.713 ± 0.031

Rat Mrp2 Positive control (E217βG)

22.2 ± 6.2

36.3 ± 4.1

1.64

—

Ginsenoside Rg1

19.7 ± 5.8

32.6 ± 2.5

1.65

—

Ginsenoside Re

68.6 ± 20.6

54.1 ± 7.4

0.789

—

Notoginsenoside R1

42.9 ± 11.2

36.9 ± 3.0

0.860

— —

Rat Bcrp Positive control (E1S)

19.2 ± 9.1

166 ± 28

8.65

Ginsenoside Rg1

172 ± 68

41.8 ± 7.1

0.243

—

Ginsenoside Re

101 ± 73

20.7 ± 6.2

0.205

—

Notoginsenoside R1

248 ± 83

78.5 ± 17.4

0.317

— —

Rat Bsep Positive control (TCA)

23.1 ± 11.9

134 ± 26

5.80

Ginsenoside Rg1

126 ± 43

94.3 ± 13.6

0.748

—

Ginsenoside Re

181 ± 33

61.1 ± 12.9

0.338

—

Notoginsenoside R1

149 ± 76

34.2 ± 11.1

0.230

—

The positive control compounds used were E217βG, oestradiol-17β-D-glucuronide; E1S, oestrone-3-sulfate; TCA, taurocholic acid; VER, verapamil. The function of human MDR1 was verified with verapamil by ATPase assay according to the manufacturer’s protocol and using the ratio of vesicular transport in the absence of sodium vanadate to that in the presence of sodium vanadate. The Vmax for stimulation of human MDR1 ATPase activity by verapamil is in pmol Pi·min−1 per mg protein. Values represent the means ± SDs (n = 3, except for the values for human OATP1B3- and rat Oatp1b2-mediated transports of the ppt-type ginsenosides for which n = 9).


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Table 3 Comparative IC50 values for ginsenosides on human OATP1B3 and OATP1B1 activities (mediating transport of E217βG) and associated DDI indices

Compound

IC50 (μM)

Unbound Cmax (μM)

t1/2 (h)

R

DDI index

OATP1B3 Rifampin

1.63 ± 0.25

1.84–3.45

1.00–4.40

1.00–1.02

1.13–2.16

Ginsenoside Rg1

43.1 ± 3.0

0.789–5.00

1.27–2.01

1.00

0.0183–0.116

Ginsenoside Re

39.4 ± 3.5

0.210–0.226

0.59–0.88

1.00

0.00533–0.00574

Notoginsenoside R1

332 ± 29

0.812

1.41

1.00

0.00245

Ginsenoside Rb1

0.467 ± 0.051

0.085–0.242

40.1–48.0

2.95–3.41

0.540–1.77

Ginsenoside Rc

0.208 ± 0.030

—

—

—

—

Ginsenoside Rd

0.235 ± 0.022

0.005

36.6

2.74

0.0583

OATP1B1 Rifampin

3.79 ± 0.28

1.84–3.45

1.00–4.40

1.00–1.02

0.485–0.928

Ginsenoside Rg1

70.4 ± 9.9

0.789–5.00

1.27–2.01

1.00

0.0112–0.0710

Ginsenoside Re

133 ± 15

0.210–0.226

0.59–0.88

1.00

0.00158–0.00170

Notoginsenoside R1

359 ± 101

0.812

1.41

1.00

0.00226

Ginsenoside Rb1

4.60 ± 0.75

0.085–0.242

40.1–48.0

2.95–3.41

0.0545–0.179

Ginsenoside Rc

2.72 ± 0.26

—

—

—

—

Ginsenoside Rd

1.42 ± 0.12

0.005

36.6

2.74

0.00965

The clinical Cmax and t1/2 values of rifampin at the therapeutic dose of 600 mg per day (p.o. administration) were from reported human data (Acocella, 1978; Loos et al., 1985). The clinical Cmax and t1/2 values of ginsenosides were from our PK data of human subjects receiving XueShuanTong (a ginsenoside injection prepared from Sanqi; details pending publication elsewhere) and from the reported human PK data for ShenMai injection and ShengMai injection by Li et al. (2011). The drug–drug interaction index (DDI index = unbound Cmax·R/IC50) were calculated for the transporter-ginsenoside couples. R is the accumulative factor that is calculated using the equation R = 1/(1 − e−kτ), where k is the elimination rate constant (0.693/t1/2) and τ is the dosing interval (24 h). A DDI index value greater than 0.1 indicates the potential for DDIs and the need for an in vivo DDI study. Values represent the means ± SDs (n = 6, except for the values for rifampin for which n = 3).

by the plasma AUC0–∞ and t1/2 values of the latter, which were 105–368 times and 26–88 times, respectively, greater than those of the former. The total plasma clearance (CLtot,p) values of the ppd-type ginsenosides were only 0.3–1.1% of those of the ppt-type ginsenosides. To determine the role of the rapid hepatobiliary excretion of the ppt-type ginsenosides in the preceding differences relative to their ppd-type counterparts, rats were treated with rifampin. The rifampin treatment led to slowed biliary excretion of the ppt-type ginsenosides (Figure 3 and Table 4). This was indicated by the CLB values of the ppt-type ginsenosides in the rifampin-treated rats, which were only 3.1–10.6% of those in the control rats. The plasma AUC0–∞ of the ppt-type ginsenosides in the rifampin-treated rats were 1.6–2.9-fold higher than those in control rats and the t1/2 values were 1.7–2.3-fold greater (P < 0.01 for all). The increased levels of systemic exposure (in plasma AUC0–∞) to these ppt-type ginsenosides induced with rifampin were associated with increased levels of tissue exposure (1.7–2.5-fold greater for the heart, 1.4–2.6-fold greater for the lung, 1.2– 1.7-fold greater for the brain and 1.6–2.6-fold greater for the kidneys relative to the respective control organ tissues; P < 0.05 for all), except for the liver levels (only 37.5–39.9% of those in the control rats; P, 0.0005–0.02) (Supporting Information Appendix S5). However, even after the rifampininduced increases, the levels of systemic exposure to ppt-type 1066


ginsenosides, as assessed by plasma AUC0–∞, were still significantly lower than those of ppd-type ginsenosides (Figures 2 and 3 and Table 4). The rifampin treatment did not considerably change the elimination kinetics of the ppt-type ginsenosides. This was indicated by their increased t1/2 values (0.5– 0.6 h), which were still significantly shorter than those of the ppd-type ginsenosides (8.3–20.6 h).

Plasma protein binding and renal excretion of ginsenosides Dramatic differences in CLR were also observed between the ppt-type ginsenosides (ginsenoside Rg1, ginsenoside Re and notoginsenoside R1) and the ppd-type ginsenosides (ginsenosides Rb1, Rc, and Rd) in rats (Figure 3 and Table 4). This was most clearly indicated by the CLR values, which were more than 150-fold greater for the former group than for the latter. Despite these differences, the renal excretion of all these ginsenosides appeared to primarily involve passive glomerular filtration, rather than active tubular secretion. This was indicated by the CLR values of all the ginsenosides, none of which exceeded the products of the reported average glomerular filtration rate of Sprague Dawley rats (624 mL·h−1·kg−1; Yu et al., 2007) and the measured plasma fu values (Table 5). The rifampin treatment did not significantly


BJP

Discussion and conclusions

Figure 2 Plasma concentrations of the ppt-type ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 and the ppd-type ginsenosides Rb1, Rc and Rd over time after a bolus i.v. dose of individual compound at 2.5 μmol·kg−1 in normal rats (solid lines for both types of ginsenosides) and rifampin-treated rats (dashed lines for the ppt-type ginsenosides only). The data represent means and SDs from three independent experiments where each rat group was assayed in triplicate.

affect the CLR values of the ppt-type ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 (Figure 3 and Table 4). This was because rifampin did not affect the glomerular-filtrationbased renal excretion, for example by changing the plasma protein binding of the ppt-type ginsenosides (Figure 3). However, the fractions of administered ginsenosides that were excreted into urine in the rifampin-treated rats (fe-U, 75.6%, 81.1% and 75.8% for ginsenoside Rg1, ginsenoside Re and notoginsenoside R1, respectively) were significantly higher than the fe-U values for the control rats (36.9%, 21.8% and 41.9%, respectively) (P < 0.01 for all).

Both ppd-type and ppt-type ginsenosides present in Sanqi exhibit cardiovascular properties. However, PK studies in rats and humans indicated that the two types of ginsenosides had markedly different elimination kinetics and hence significantly different levels and durations of systemic exposure after dosing with a Sanqi extract (Liu et al., 2009; Hu et al., 2013). These PK differences of ginsenosides are expected to affect the outcomes of Sanqi therapies. Studying the elimination of ginsenosides at the molecular level facilitates better understanding of these PK differences. The difference in elimination kinetics, observed in rats, involves a significant discrepancy in CLB between the two types of ginsenosides. Such a CLB discrepancy most probably also occurs in humans. Hepatobiliary excretion of various endogenous and xenobiotic compounds is often accomplished in two steps: (i) SLC transporter-mediated cellular uptake from blood through the sinusoidal membrane to the hepatocytes and (ii) ABC transporter-mediated efflux from the hepatocytes into bile across the canalicular membrane (Pfeifer et al., 2014). In the current study, the ppt-type ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 were found to be substrates of the human hepatobiliary SLC transporter OATP1B3 (SLCO1B3) and the human hepatobiliary ABC transporters MRP2 (ABCC2), BCRP (ABCG2), BSEP (ABCB11) and MDR1 (ABCB1). Although the ppd-type ginsenosides Rb1, Rc and Rd were also bound to OATP1B3, they were not transported. These ppd-type ginsenosides were also not transported by the preceding ABC transporters. Based on this and on their poor membrane permeability (Liu et al., 2009), it was concluded that the circulating ppd-type ginsenosides in the bloodstream were very slowly transported into bile via human hepatocytes. This suggested that there was a significant CLB discrepancy between the two types of ginsenosides in humans. Similarly, although all the ginsenosides were bound to the rat SLC transporter Oatp1b2 (Slco1b2), only the ppt-type ginsenosides were transported. The ppt-type ginsenosides were also substrates of the rat ABC transporters Mrp2 (Abcc2), Bcrp (Abcg2) and Bsep (Abcb11). Despite their poor membrane permeability, multiple ABC transporters working in concert resulted in short half-lives of the ppt-type ginsenosides in the livers of rats (t1/2, 0.2–0.4 h; Supporting Information Appendix S5) and prevented their accumulation in the organs. In contrast, the ppd-type ginsenosides Rb1, Rc and Rd were not the substrates of the rat ABC transporters. The liver t1/2 values of these ppd-type ginsenosides were long (10.8–16.4 h; Supporting Information Appendix S5). A similar scenario is expected in humans, including the interactions of the human ABC transporters with the ginsenosides and the differences in liver disposition between the two types of the ginsenosides. Therefore, rats were used to delineate and extrapolate the in vivo relevance of the human hepatobiliary transporters to the levels of systemic exposure to the ginsenosides. Neither the ppt-type ginsenosides nor the ppd-types ginsenosides are significantly eliminated via metabolism by rat enterohepatic enzymes (Liu et al., 2009). The markedly different elimination kinetics between the two types of ginsenosides in rats mainly resulted from the differences in both transporter-mediated CLB and glomerular-filtration-based CLR. For the ppt-type ginsenosides, the two excretion British Journal of Pharmacology (2015) 172 1059–1073

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CLB (mL–1·kg·h–1)

No rifampin With rifampin 14 7 0

0 No rifampin With rifampin

6 3

900

0

0

100

No rifampin With rifampin

3

fu (%)

1

1.5

300

0

0.5

50

0

Notoginsenoside R1

Ginsenoside Re

Ginsenoside Rg1

Ginsenoside Rd

Ginsenoside Rc

Ginsenoside Rb1

Notoginsenoside R1

0 Ginsenoside Re

0

Ginsenoside Rd

600

No rifampin With rifampin

Ginsenoside Rc

700

1800

Ginsenoside Rb1

1400

Ginsenoside Rg1

CLR (mL–1·kg·h–1)

AUC0-24h (µM·h)

BJP

Figure 3 Different levels of systemic exposure (AUC), hepatobiliary excretory clearance (CLB), renal excretory clearance (CLR) and plasma protein-unbound fraction (fU) of the ppt-type ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 and the ppd-type ginsenosides Rb1, Rc and Rd after a bolus i.v. dose of individual compound at 2.5 μmol·kg−1 in normal rats (No rifampin) and rifampin-treated rats. The data represent means and SDs from three independent experiments where each rat group was assayed in triplicate.

pathways worked in concert but competed with each other; their roles in the elimination of the compounds depended on the sizes of the CLB (810, 1208 and 403 mL·h−1·kg−1 for ginsenoside Rg1, ginsenoside Re and notoginsenoside R1, respectively) and CLR (434, 251 and 300 mL·h−1·kg−1 respectively) (Table 4). The rifampin-induced disruption of rat hepatobiliary excretion resulted in 1.8-, 2.9- and 1.6-fold increases in plasma AUC0–∞ levels of ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 respectively. However, the elevated exposure levels were still significantly lower than the exposure levels of the ppd-type ginsenosides (Figures 2 and 3 and Table 4). Although rifampin could impair the hepatobiliary excretion with respect to CLB, it did not change the CLR of the ppt-type ginsenosides. This is because rifampin did not change the plasma protein binding of these ginsenosides and in turn the associated glomerular filtration. The impairment of hepatobiliary excretion reduced the total clearance (CLtot,p). This, in turn, increased plasma concentrations of the ppt-type ginsenosides and their concentration-dependent rates of renal excretion. The effect of rifampin could also be viewed from another angle. The impairment of hepatobiliary excretion considerably reduced competition with the alternate pathway renal excretion for eliminating the ppt-type ginsenosides. Under the given i.v. dose of the ppt-type ginsenoside 2.5 μmol·kg−1, this led to the renal excretion having longer time to eliminate these ginsenosides than it did when the hepatobiliary excretion worked normally. This is indicated by the rifampin-affected t1/2 (0.5, 0.5 and 0.6 h, for ginsenoside Rg1, ginsenoside Re and notoginsenoside R1, respectively), which were longer than those in the rats not treated with rifampin (0.2, 0.2 and 0.3 h, respectively; P < 0.001). Taken together, both the increased rate of renal excretion (due to the increased plasma concentrations) and the prolonged time for renal excretion (due to the decreased competition from the 1068


hepatobiliary excretion) resulted in the increased urinary Cum.Ae-U,0–t and fe-U of the ppt-type ginsenosides in the rats treated with rifampin (Table 4). Because of the compensation by renal excretion, the drug-induced impairment of hepatobiliary excretion did not alter considerably the differential elimination kinetics between the two types of ginsenosides in rats. Similar mechanisms governing differential Pharmacokinetics of ginsenosides is expected in humans. In rats, the difference in CLB mainly resulted from the ppt-type ginsenosides that were transported by Oatp1b2. The ppd-type ginsenosides were not transported. In humans, the difference in CLB mainly resulted from the ppt-type ginsenosides that were transported by OATP1B3. The ppd-type ginsenosides were not transported. In rats, the difference in CLR resulted from the ppd-type ginsenosides extensively bound to plasma protein (fu values, 0.4–0.9%). The fu values of the ppt-type ginsenosides were 62.3–77.1%. In humans, the difference in CLR resulted from the ppd-type ginsenosides extensively bound to plasma protein (0.4–0.9%). The fu values of the ppt-type ginsenosides were 66.8–78.1%. Like rats, metabolic elimination of unchanged ginsenosides is negligible in humans (Hu et al., 2013). The use of medicinal herbal products is prevalent among patients who are taking synthetic drugs. All substances from the administered drugs and herbs can interact with each other (Fugh-Berman, 2000; Tachjian et al., 2010; Shi and Klotz, 2012). In the practice of Chinese traditional medicine, herb combination therapy (‘fangji peiwu’ in Chinese) is often used to therapeutic advantage either because the beneficial effects are synergistic or additive or because therapeutic effects can be achieved with fewer herb-specific side effects by using submaximal doses of herbs in concert. However, herb combination therapy can give rise to PK herb–herb interactions. PK herb–drug and herb–herb interactions are relevant


BJP

Table 4 PK parameters of ginsenosides after i.v. administration at 2.5 μmol·kg−1 in rats treated with and without rifampin (20 mg·kg−1)

Ppt-type

PK parameter

Ginsenoside Rg1

Ginsenoside Re

Notoginsenoside R1

(−) Rifampin

(−) Rifampin

(−) Rifampin

(+) Rifampin

(+) Rifampin

(+) Rifampin

Plasma data C5min (μM)

6.80 ± 1.98

8.56 ± 1.12*

6.63 ± 2.40

11.3 ± 2.34***

7.07 ± 1.30

8.23 ± 0.79*

AUC0–∞ (h·μM)

2.38 ± 0.66

4.30 ± 0.91***

2.77 ± 1.63

8.11 ± 2.88***

3.19 ± 0.63

5.19 ± 0.73***

t1/2 (h)

0.235 ± 0.017

0.450 ± 0.047***

0.236 ± 0.071

0.537 ± 0.083***

0.316 ± 0.049

0.552 ± 0.063***

−1

VSS (mL·kg )

332 ± 108

316 ± 87

287 ± 82

216 ± 50

308 ± 29

286 ± 27

CLtot,p (mL·h−1·kg−1)

1148 ± 399

624 ± 163**

1238 ± 684

320 ± 88**

786 ± 172

471 ± 89***

Cum.Ae-B,0–24h (nmol·kg−1)

1680 ± 261

325 ± 130***

2319 ± 277

248 ± 65***

1281 ± 211

139 ± 71***

fe-B (%)

67.9 ± 9.7

12.7 ± 5.0***

94.6 ± 11.7

10.5 ± 3.3***

52.3 ± 8.7

5.69 ± 2.80***

CLB (mL·h−1·kg−1)

810 ± 117

85.7 ± 42.6***

1208 ± 587

37.6 ± 18.1***

403 ± 70

25.2 ± 13.1***

932 ± 301

1808 ± 497***

535 ± 236

1928 ± 316***

954 ± 225

1775 ± 369***

Bile data

Urine data Cum.Ae-U,0–24h (nmol·kg−1) fe-U (%)

36.9 ± 12.7

75.6 ± 16.0***

21.8 ± 9.2

81.1 ± 17.5***

41.9 ± 12.1

75.8 ± 22.4**

CLR (mL·h−1·kg−1)

434 ± 93

447 ± 71

251 ± 87

277 ± 63

300 ± 72

322 ± 72

Ppd-type Ginsenoside Rb1

Ginsenoside Rc

Ginsenoside Rd

(−) Rifampin

(−) Rifampin

(−) Rifampin

C5min (μM)

61.5 ± 9.4

69.1 ± 25.9

66.9 ± 16.3

AUC0–∞ (h·μM)

773 ± 210

875 ± 311

336 ± 116

t1/2 (h)

19.2 ± 2.4

20.6 ± 4.7

8.31 ± 2.64

VSS (mL·kg−1)

92.7 ± 20.4

97.7 ± 56.0

80.4 ± 20.2

CLtot,p (mL·h−1·kg−1)

3.54 ± 1.17

3.28 ± 1.31

8.34 ± 3.11

Cum.Ae-B,0–24h (nmol·kg−1)

540 ± 241

738 ± 195

1165 ± 248

fe-B (%)

21.6 ± 9.6

29.1 ± 8.3

45.0 ± 9.4

CLB (mL·h−1·kg−1)

1.29 ± 0.54

1.71 ± 0.39

4.54 ± 1.68

554 ± 255

509 ± 106

422 ± 101

PK parameter Plasma data

Bile data

Urine data Cum.Ae-U,0–24h (nmol·kg−1) fe-U (%)

21.3 ± 9.0

20.0 ± 4.7

16.2 ± 4.4

CLR (mL·h−1·kg−1)

1.45 ± 1.00

1.22 ± 0.42

1.59 ± 0.42

(+) Rifampin, rat group treated with rifampin. (-) Rifampin, rat group not treated with rifampin. C5min, concentration at 5 min after dosing; AUC0–∞, area under concentration-time curve from zero to infinity; t1/2, elimination half-life; VSS, distribution volume at steady state; CLtot,p, total plasma clearance; fe-B, fractional biliary excretion; Cum.Ae-B,0–24h, cumulative amount excreted into bile; CLB, hepatobiliary excretory clearance; fe-U, fractional urinary excretion; Cum.Ae-U,0–24h, cumulative amount excreted into urine; CLR, renal excretory clearance. The data represent means ± SDs from three independent experiments where each rat group was performed in triplicate (total n = 9). *P < 0.05, **P < 0.01, ***P < 0.001; significantly different from the values in rats not treated with rifampin).

because they can be a source of variability in drug and herb responses. This may complicate the dosing of long-term medications. Because of their function and broad substrate specificity, OATP1B transporters are very likely to be targets for drug interactions in the liver (Kallioloski and Niemi, 2009; König et al., 2013).

In the current study, the propensity of the ginsenosides to cause OATP1B-related herb–drug and herb–herb interactions was determined. Notably, the ppd-type ginsenosides Rb1, Rc and Rd showed rather high affinity for OATP1B3 (IC50 values, 0.2–0.5 μM for inhibition of E217βG transport) and high affinity for OATP1B1 (1.4–4.6 μM for inhibition of E217βG British Journal of Pharmacology (2015) 172 1059–1073

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Table 5 Fractions of plasma protein-unbound ginsenosides

fu (%) Human plasma Compound

Rat plasma

Male

Female

Male

64.8 ± 4.2

Ppt-type ginsenoside Ginsenoside Rg1

78.1 ± 2.6

71.5 ± 5.8

Ginsenoside Re

77.9 ± 5.9

66.8 ± 9.2

77.1 ± 2.7

Notoginsenoside R1

69.4 ± 2.3

77.1 ± 5.0

62.3 ± 5.7

Ppd-type ginsenoside Ginsenoside Rb1

0.891 ± 0.110

0.921 ± 0.019

0.912 ± 0.089

Ginsenoside Rc

0.612 ± 0.012

0.597 ± 0.012

0.611 ± 0.011

Ginsenoside Rd

0.392 ± 0.022

0.390 ± 0.032

0.402 ± 0.024

The fu values were measured with drug-free human plasma and rat plasma fortified with the ppt-type ginsenosides at 5 μM and the ppd-type ginsenosides at 50 μM individually. The blank human plasma was obtained from a human study with freeze-dried XueShuanTong for injection (ChiCTR-ONRC-13003905; http://www.chictr.org/en/proj/show.aspx?proj=5603). Values represent the means ± SDs (n = 3).

transport). The affinity of the ppt-type ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 for OATP1B transporters was lower than that of the ppd-type ginsenosides. Their IC50 values for OATP1B3 and OATP1B1 (both for inhibition of E217βG transport) were 39.4–332.0 and 70.4–358.6 μM respectively. To assess the clinical relevance of these findings, the knowledge on plasma concentrations of ginsenosides in humans are essential, particularly the unbound plasma concentrations. The ppd-type ginsenosides had extremely high plasma protein binding (fu values, 0.4–0.9%). The fu values of the ppt-type ginsenosides were 66.8–78.1%. Ginsenosides had poor intestinal absorption, their unbound plasma concentrations after p.o. dosing with Sanqi extract and ginseng preparations in humans were in the low nanomolar range (Ji et al., 2004; Hu et al., 2013), which was much lower than the IC50 values. Oral ginsenosides had little propensity to act as inhibitory agents in OATP1B-based interactions. In China, an herbal injection prepared from Sanqi (freeze-dried XueShuanTong for injection) and those containing red Asian ginseng (including ShenMai injection, ShengMai injection and ShenFu injection) are extensively used, which are sterile, non-pyrogenic parenteral dosage forms for i.v. injection. Unlike the situation with the oral dosage forms, the plasma concentrations of the ginsenosides are quite high after i.v. administration of these herbal injections in human. Based on our own human data and clinical reports by others, the range of Cmax of ginsenoside Rb1 was 26.3 ± 2.6 μM after i.v. administration of a therapeutic dose of XueShuanTong (details pending publication elsewhere), 14.9 ± 10.6 μM after i.v. administration of a therapeutic dose of ShenMai injection and 9.5 ± 8.1 μM after i.v. administration of a therapeutic dose of ShengMai injection (Li et al., 2011). Although ginsenoside Rb1 was extensively bound to human plasma protein, the unbound Cmax ranged from 0.1 to 0.2 μM after a single i.v. dose of the herbal injections. In addition, a long t1/2 of 40.1– 48.0 h in humans indicated that multiple doses of the herbal injections would lead to accumulation of this ppd-type gin1070


senoside in plasma and the unbound Cmax could increase to 0.3–0.8 μM. Accordingly, the ginsenoside Rb1-based DDI indices were calculated as 0.54–1.77 for OATP1B3 and 0.05– 0.18 for OATP1B1 (Table 3). ShenMai injection and ShengMai injection also contained comparable amounts of ginsenosides Rc and Rd to ginsenoside Rb1 (Supporting Information Appendix S6). It is worth mentioning that all these ppd-type ginsenosides from the herbal injections probably have additive inhibitory effect on OATP1B transporters. Ginsenoside Rg1 represented the most abundant ppt-type ginsenosides in freeze-dried XueShuanTong for injection. The ginsenoside Rg1-based DDI indices were 0.02–0.12 for OATP1B3 and 0.01– 0.07 for OATP1B1 (Table 3). Clinically important drugs, including telmisartan and paclitaxel, have been identified as substrates of OATP1B3 (Smith et al., 2005; Ishiguro et al., 2006). The clinically relevant herb–drug interactions may be caused by inhibition of OATP1B3-mediated uptake of these drugs in the liver by the ppd-type ginsenosides from the herbal injections. The ppt-type ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 were found to be weak in vitro inhibitors of the hepatic efflux transporters MRP2, BCRP and BSEP with the IC50 values > 100 μM. Therefore, the ppt-type ginsenosides tended not to alter the liver concentrations of other drugs and herbal compounds. Multiple ABC transporters appeared to mediate the efflux of the ppt-type ginsenosides from the hepatocytes into bile. Although the individual contributions of these transporters to the overall hepatobiliary efflux has not been characterized, alteration of the efflux of the ppt-type ginsenosides via drug interaction might involve affecting the concerted action of multiple transporters, rather than affecting function of a single transporter. The ppd-type ginsenosides did not interact with the ABC transporters either as substrates or as inhibitors. Saponins are the bioactive constituents of many medicinal herbs (Francis et al., 2002; Sparg et al., 2004). They are amphiphilic glycosides containing one or more sugar chains


linked to a non-polar triterpene or steroid aglycone skeleton. Rapid and extensive hepatobiliary excretion represents an important route of elimination for many saponins and can affect their oral bioavailability and Pharmacokinetics (Yu et al., 2012). Several saponins have been found to interact with the human OATP1B transporters. Ismair et al. (2003) reported that glycyrrhizin inhibited OATP1B1-mediated uptake of [3H]E1S into Xenopus laevis oocytes (Ki, 15.9 μM) and OATP1B3-mediated uptake of [35S]bromosulfophthalein (12.5 μM). Recently, Wu et al. (2012) assessed the effects of multiple herbal compounds, including several saponins, on the function of OATP1B1. Here, the ppd-type ginsenosides Rb1, Rc and Rd were found to have high inhibitory activities towards OATP1B3. Besides triterpene saponins, the steroid saponin dioscin was also found to be a substrate of OATP1B3 (Km, 2 μM) rather than that of OATP1B1 (Zhang et al., 2013). So far, a limited number of saponin compounds have been investigated with respect to their interactions with OATP1B transporters. Multiple hepatobiliary ABC transporters were found to have transport activities for the ppt-type ginsenosides. However, there are gaps regarding the manner in which the ABC transporters interact with other medicinal saponins. It is necessary to examine more medicinal saponins. The most important findings from the current study include the following. (i) Both the types of ginsenosides were bound to human OATP1B3/rat Oatp1b2 transporters, but only the ppt-type ginsenosides were transported. This explained their markedly different CLB values. (ii) Multiple ABC transporters mediated the efflux of the ppt-type ginsenosides from the hepatocytes into bile. This led to their short half-lives in the rat livers and a similar scenario is expected in humans. (iii) Unlike the ppt-type ginsenosides, the ppd-type counterparts were extensively bound to plasma protein. This caused their significant differences in glomerular-filtrationbased CLR. (iv) The hepatobiliary excretion and renal excretion of the ppt-type ginsenosides worked in concert and competed with each other in normal rats. (v) The ppd-type ginsenosides had high inhibitory potency towards OATP1B3. Caution should be exercised with these long circulating saponins which could induce herb-drug interactions, especially when patients receive long-term therapies with high-dose i.v. Sanqi or ginseng extracts. The results reported here have implications for translation of many promising findings regarding ginsenosides from the laboratory to the clinical settings and for safety assessment of Sanqi and ginseng therapies.

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brane Co., Ltd., Kanazawa, Japan) for kindly providing the mRNA level data of transporter-expressing vesicles (Supporting Information Appendix S2). We are also grateful for Dr Min Tan for technical assistance on western blotting analysis.

Author contributions C. L. and R. J. participated in research design, performed the data analysis and wrote or contributed to the writing of the manuscript. R. J., J. D., X. L., F. D., W. J., F. X., F. W., J. Y. and W. N. conducted the experiments.

Conflict of interest There are no competing interests to declare.

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Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:

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http://dx.doi.org/10.1111/bph.12971 Appendix S1 Supplemental methods. Appendix S2 Protein expression of OATP1B1, OATP1B3 and Oatp1b2 in transfected HEK293 cells and mRNA levels of MRP2, BCRP, BSEP, MDR1, Mrp2, Bcrp and Bsep in membrane vesicles. Appendix S3 Time courses for accumulation of the ppt-type ginsenosides in HEK293 cells expressing human OATP1B3 or rat Oatp1b2. Appendix S4 Rifampin inhibition of human OATP1B3- and rat Oatp1b2-mediated uptake of the ppt-type ginsenosides, plasma pharmacokinetics of rifampin in rats and rifampinmediated drug–drug interactions. Appendix S5 Rat tissue distribution of ginsenosides. Appendix S6 Content levels of ginsenosides in herbal injections.


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Molecular mechanisms governing different pharmacokinetics of ginsenosides and potential for ginsenoside-perpetrated herb-drug interactions on OATP1B3 Rongrong Jiang, Jiajia Dong, Xiuxue Li, Feifei Du, Weiwei Jia, Fang Xu, Fengqing Wang, Junling Yang, Wei Niu, Chuan Li

British Journal of Pharmacology (DOI:10.1111/bph.12971) - Supporting Information Appendix S1 Supplemental methods Cell culture and transfection HEK293 cells were grown in a Dulbecoo’s modified Eagle’s medium fortified with 10% fetal calf serum in a humidified incubator at 37°C and 5% CO2. Cells were harvested at 90% confluence and then seeded in poly-D-lysine-coated 24-well plates at a density of 2×105 cells/well 24 h prior to transfection. Human OATP1B1 (GeneBank accession no., NM_006446) and OATP1B3 (NM_019844) cDNA clones, purchased from Thermo Scientific (Waltham, MA, USA), were subcloned into pcDNA3.1 expression plasmid by Invitrogen Life Technologies (Shanghai, China). Plasmid expressing human OATP2B1 (NM_007256.2) was obtained from Origene (Rockville, MD). The open reading frames of rat hepatic transporters of Oatp1a1 (NM_0177111), Oatp1b2 (NM_031650), and Ntcp cDNAs (NM_017047) were synthesized and subcloned into pcDNA3.1 expression plasmid by Invitrogen Life Technologies. The expression plasmid and the empty vector were introduced separately into the HEK293 cells with Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. This produced transporter-expressing cells (TC) and mock cells (MC), respectively. Before use, the cells transfected with human OATP1B1, human OATP1B3 or rat Oatp1b2 plasmid were functionally characterized by measuring uptake of oestradiol-17β-D-glucuronide (E217βG). Those transfected with human OATP2B1, rat Oatp1a1 or rat Ntcp plasmid were functionally characterized by measuring uptake of oestrone-3-sulfate (E1S), E217βG or taurocholate acid (TCA), respectively.

Western blotting analysis The transfected cells and the mock cells were homogenized in homogenate buffer (250 mM sucrose, 10 mM HEPES, and 10 mM Tris-base, pH 7.4) with a protease inhibitor cocktail (1:100, v/v; Sigma, Oakville, ON, Canada) to provide crude membrane fractions of the cells. The homogenate was first centrifuged at 3,000g for 10 min at 4°C, and then the resultant supernatant was centrifuged at 33,000g for 30 min at 4°C. The formed pellet (membrane fraction) was resuspended in buffer (50 mM mannitol, 20 mM HEPES, and 20 mM Tris-base, pH 7.5) with the protease inhibitor cocktail. Immunoblotting was conducted with SDS-polyacrylamide gel electrophoresis (10% gel) and proteins were electrophoretically transferred to poly vinylidene fluoride membranes. The primary monoclonal antibody against human OATP1B1 (Abcam, Cambridge, MA, USA; dilution 1:1000) and primary polyclonal antibody against human OATP1B3 (Abcam, Cambridge, MA, USA; dilution 1:250) were used for the immunodetection of human OATP1B1 and OATP1B3. The primary monoclonal antibody against rat Oatp1b2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; dilution 1:500) were used for the immunodetection of Oatp1b2. Blots were incubated with horseradish peroxidase-anti mouse or rabbit IgG (Bio-Rad, Richmond, CA, USA) for 1 h and signals were detected using the Immun-Star Chemiluminescent Protein Detection System according to the manufacturer's instruction (Bio-rad, Hercules, CA, USA).

Transport studies with human OATP1B1- and OATP1B3- and rat Oatp1b2-expressing HEK293 cells Before the transport studies, the transfected HEK293 cells and mock cells were cultured with Dulbecoo’s modified Eagle’s medium for 48 h. The cells were then washed and preincubated at 37°C for 5 min with 250 µL of a prewarmed Krebs-Henseleit buffer (containing 142 mM NaCl, 23.8 mM NaHCO3, 4.83 mM KCl, 0.96 mM KH2PO4, 1.20 mM MgSO4, -1-

12.5 mM HEPES, 5 mM glucose and 1.53 mM CaCl2; adjusted to pH 7.4 with 1 M NaOH). After aspiration of the preincubation buffer from the cell-coated wells, 200 µL of the Krebs-Henseleit buffer fortified with the test compound or the prototypical substrate were added to initiate the transport, which were maintained by incubation for 10 min for transporter identification. The transport was terminated by aspirating the buffer from the wells. After washing three times with 400 µL of ice-cold Krebs-Henseleit buffer, the cells were lysed in water (150 μL) by freeze/thaw-enhanced ultrasonic treatment. The resulting cellular lysates (100 μL) were precipitated with 300 μL of methanol. After centrifugation at 21,100 g for 10 min, the supernatants were analyzed by LC-MS/MS. The total protein concentration of lysate was measured using a method first described by Bradford (1976). The transport rate of test compound in pmol·min−1 per mg protein was calculated using the following equation: Transport = (CL × VL)/T/ WL

(1)

where CL represents the concentration of test compound in the cellular lysate (μM), VL is the volume of the lysate (μL), T is the incubation time (10 min) and WL is the measured cellular protein amount of each well (mg). Differential uptake between the transfected cells (TC) and the mock cells (MC) was defined as a net transport ratio (TransportTC/TransportMC ratio) and net transport ratio greater than three suggested a positive result. Kinetics of human OATP1B3- and rat Oatp1b2-mediated cellular uptakes of ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 were assessed with respect to Michaelis constant (Km) and maximum velocity (Vmax) and the incubation time was set at 5 min to ensure that the assessment was performed under linear uptake conditions (Supporting Information Appendix S3). Compound uptake during the linear phase (first 5 min) was assessed at concentrations over ranges of 3.1–200

μM. The inhibitory effect of rifampin on the activity of human OATP1B3 or rat Oatp1b2 that mediated transport of ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 was further measured in terms of inhibition constant (Ki). The inhibitory effects of ginsenosides on the ability of human OATP1B3 and OATP1B1 to mediate transport of oestradiol-17β-D-glucuronide (E217βG) were measured in terms of half maximal inhibitory concentration (IC50) and rifampin was used as positive control. The difference in the amounts of compound transported by transfected cells and the mock cells served as the transporter-dependent uptake for the kinetic and inhibition studies. All experiments were run in triplicates.

Transport studies with human MRP2-, BCRP-, BSEP- and MDR1-expressing membrane vesicles and rat Mrp2-, Bcrp- and Bsep-expressing membrane vesicles A transport study was performed to identify the transporters that mediated the ATP-dependent cellular efflux of the test compound. A rapid filtration method was used with membrane vesicles expressing one of human MRP2, BCRP, BSEP and MDR1 and rat Mrp2, Bcrp and Bsep according to the manufacturer’s protocol. In brief, a buffer [containing 50 mM 3-(N-morpholino) propanesulfonic acid, 70 mM KCl and 7.5 mM MgCl2; adjusted to pH 7.0 with 1.7 M Tris-base] was used as transport medium, except for human MRP2 and rat Mrp2 using a buffer further containing 2 mM glutathione. The buffer was also used to dissolve the test compound and ATP (or AMP for the negative control), which was preincubated at 37°C for 5 min. The suspension of membrane vesicles was mixed with an equal volume of the buffer and preincubated at 37°C for 5 min. The transport was started by combining 20 µL of the membrane vesicle mixture with 30 µL of the test compound/ATP solution (or the test compound/AMP solution), and the resulting concentration of test compound was 20 µM in all cases. After incubation for 10 min, the transport was terminated by addition of 200 µL of an ice-cold buffer [containing 40 mM 3-(N-morpholino)propanesulfonic acid and 70 mM KCl; adjusted to pH 7.0 with 1.7 M Tris-base] followed by immediate transfer of the mixture into a Millipore MultiScreen-FB filtration plate (0.65 µm; Billerica, MA, USA; pre-wet with the ice-cold buffer). A Millipore MultiScreen vacuum manifold was used with a filtration plate and the membrane vesicles were washed five times with 200 µL of the ice-cold terminating buffer. Filters that retained membrane vesicles were transferred to 1.5 mL polypropylene tubes for lysis with 200 µL of 80% methanol. After centrifugation at 21,100 g for 10 min, the supernatants were analyzed by LC-MS/MS. The transport rate of test compound in pmol·min−1 per mg protein was calculated using the following equation: Transport = (CV × VV)/T/WV

(2) -2-

where CV represents the concentration of the compound in the vesicular lysate supernatant (μM), VV is the volume of the lysate (μL), T is the incubation time (10 min) and WV is the amount of vesicle protein amount per well (0.05 mg). Positive results for ATP-dependent transport were defined as a net transport ratio (TransportATP/TransportAMP ratio) greater than three. A kinetic study was performed using the transporter-expressing membrane vesicles. The transport of compounds during the linear phase (first 5 min) was assessed at concentrations over ranges of 5–300 μM. The inhibitory effects of 100 μM ginsenosides on the activities of human MRP2, BCRP, and BSEP that mediated transport of E217βG, MTX, and TCA, respectively, were also assessed. The difference between the amounts of transport in the presence of ATP and in its absence was defined as transporter-dependent uptake for the kinetic and inhibition studies. All experiments were run in triplicates.

Laboratory animals Male Sprague-Dawley rats (230–300 g) were obtained from Sino-British SIPPR/BK Laboratory Animal Co., Ltd. (Shanghai, China). The use and treatment of rats were in compliance with the Guidance for Ethical Treatment of Laboratory Animals (The Ministry of Science and Technology of China, 2006, www.most.gov.cn/fggw/zfwj/zfwj2006). Animal studies were implemented according to the protocols approved by the Institutional Animal Care and Use Committee at Shanghai Institute of Materia Medica (Shanghai, China). China). All studies involving animals are reported in accordance with the ARRIVE guidelines for reporting experiments involving animals (Kilkenny et al., 2010; McGrath et al., 2010). All the rats were housed in cages (48 × 29 × 18 cm3) in a room with unidirectional airflow, controlled temperature (20–25°C), relative humidity (40–70%) and a 12 h light/dark cycle. Filtered tap water was available ad libitum. The animals were given commercial rat chow ad libitum except during the night before dosing. The rats were allowed to acclimate to the facilities and environment for 5 days prior to experimental use. The femoral arteries of rats were cannulated for blood sampling, and other rats underwent bile duct cannulation for bile collection (Chen et al., 2013; Cheng et al., 2013). After the aseptic surgery, the rats were housed singly and allowed to regain their preoperative body weights before use. All used rats were euthanatized with CO2 gas.

Rat studies To determine the in vivo functional importance of Oatp1b2 in pharmacokinetics and disposition of ginsenosides, four rat studies were performed with the rodents treated or not treated with rifampin (i.v., 20 mg·kg−1). In the first rat study, rats were randomly assigned to nine groups. After a bolus i.v. dose of ginsenosides Rg1, Re, Rb1 (rifampin-free rats only), Rc (rifampin-free rats only), Rd (rifampin-free rats only) or notoginsenoside R1 at 2.5 μmol·kg−1, serial blood samples (60 μL; 0, 5, 15, 30 min, 1, 2, 4, 6, 8, 10 and 24 h) were collected and heparinised. The plasma fractions were prepared by centrifugation. In the second rat study, rats were randomly assigned to nine groups to receive a bolus i.v. dose of ginsenosides Rg1, Re, Rb1 (rifampin-free rats only), Rc (rifampin-free rats only), Rd (rifampin-free rats only) or notoginsenoside R1 at 2.5 μmol·kg−1. Bile samples were collected from rats between 0–2, 2–6, 6–12 and 12–24 h after dosing and were weighed. A sodium taurocholate solution (1.5 mL·h−1 during day time; pH 7.4) was infused into the duodenum during bile collection. In the third rat study, rats were assigned randomly into nine groups to receive a bolus i.v. dose of ginsenosides Rg1, Re, Rb1 (rifampin-free rats only), Rc (rifampin-free rats only), Rd (rifampin-free rats only) or notoginsenoside R1 at 2.5 μmol·kg−1. The rats were housed singly in metabolic cages and the collection tubes containing their urine were frozen at −15°C during sample collection. Urine samples were collected at 0–8 and 8–24 h after dosing and were weighed. Each rat study was repeated twice. In the last study, rats were randomly assigned to nine groups to receive a bolus i.v. dose of ginsenosides Rg1, Re, Rb1 (rifampin-free rats only), Rc (rifampin-free rats only), Rd (rifampin-free rats only) or notoginsenoside R1 at 2.5 μmol·kg−1. The rats under isoflurane anesthesia were killed by bleeding at the abdominal aorta at 5 and 30 min and at 1, 4 and 8 h (three rats per sampling time) after dosing. Selected tissues including the heart, lungs, brain, liver and kidneys were excised, rinsed in ice-cold saline, blotted, weighed and homogenized in four volumes of ice-cold saline. All rat samples were stored at −70°C until analysis.

Determination of plasma protein binding -3-

Equilibrium dialysis was used to assess the fraction of plasma protein-unbound compound (fu) (Guo et al., 2006). The test compounds were individually added into blank human and rat plasma to generate nominal concentrations of 5 μM for ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 and rifampin and 50 μM for ginsenosides Rb1, Rc and Rd. The resulting plasma samples were used for 24 h equilibrium dialysis using Sepctra/Por 2 RC dialysis membranes (molecular weight cutoff, 12–14 kDa; Rancho Dominguez, CA, USA) and phosphate buffered saline (pH 7.4) as dialysate at 37°C. After completion of the dialysis, both the plasma and the dialysate were sampled (100 μL), mixed with methanol (300 and 100 μL, respectively) and centrifuged for LC-MS/MS-based analysis. The fu value was calculated using the following equation: fu = Cd / Cp × 100%

(3)

where Cd represents the concentration in the dialysate (μM) after completion of dialysis and Cp is the corresponding concentration in the plasma (μM).

LC-MS/MS-based bioanalytical assays Validated bioanalytical methods were used to measure test compounds in biomatrices using a TSQ Quantum mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) interfaced via an electrospray ionization probe with a liquid chromatography (Agilent Technologies, Waldbronn, Germany). Chromatographic separations for measurement of the ginsenosides were achieved on an Agilent Eclipse Plus 5-μm C18 column (50 × 2.1 mm i.d.; Santa Clara, CA, USA) and those for measurement of rifampin, E1S, E217βG, TCA and MTX were achieved on a Phenomenex Gemini 5-μm C18 column (50 × 2.0 mm i.d.; Torrance, CA, USA). Mobile phases for measurement of ginsenosides were methanol/water (1:99, v/v, containing 100 μM lithium acetate and 1 mM formic acid; solvent A) and methanol/water (99:1, v/v, containing 100 μM lithium acetate and 1 mM formic acid; solvent B). Mobile phase for measurement of rifampin, E1S, E217βG, TCA and MTX were methanol/water (1:99, v/v, containing 0.7 mM ammonium formate; solvent A) and methanol/water (99:1, v/v, containing 0.7 mM ammonium formate; solvent B). To simplify the assay development, a pulse gradient elution technique was used for the compound measurement (Wang et al., 2007). In brief, the start proportions for measurement of ginsenosides, for measurement of rifampin, E1S, E217βG and TCA and for measurement of MTX were 5% B, 50% B and 30% B, respectively, and the elution proportion segments were 3.9 min, 1.0 min and 1.0 min, respectively. The elution proportions were the same for all the analytes, i.e. 100% B, and the column equilibrium segment were normalized to 3.5 min. MS/MS was performed in the positive or negative ion mode using the precursor-to-product ion pairs m/z 1115→349, 1085→319, 953→773, 807→627, 953→773, 939→319, 824→151, 349→269, 447→271, 514→124 and 455→308 of ginsenosides Rb1, Rc, Rd, Rg1, Re, notoginsenoside R1, rifampin, E1S, E217βG, TCA and MTX, respectively. The LC effluent flow was introduced into the electrospray ionization probe at a flow rate of 0.35 mL·min−1 and the data acquisition period was 2.0–4.5 min. The other effluent flows (0–2 and 5–6.5) were diverted to waste. Sample clean-up was achieved using methanol as precipitant at a methanol-to-sample volume ratio of three. After centrifugation at 21,100g for 10 min, the supernatant (5 μL) was applied to LC-MS/MS-based analysis. Matrix-matched calibration curves were constructed for the test compounds using weighted (1/X) linear regressions of the compound response (peak area; Y) against the corresponding nominal concentrations of compound (X, μM), which showed good linearity (r2 > 0.99).

Data processing GraFit software (version 5.0; Erithacus Software, Surrey, UK) was used to determine the Km and Vmax values by nonlinear regression analysis of initial transport rates as a function of substrate concentration. To determine the Ki and IC50, the data was fitted to an appropriate equation by nonlinear regression analysis with simple weighting using the GraFit software. The calculation of Ki value was based on the following equation: v = Vmax·S/[Km(1 + I/Ki) + S]

(4)

where v represents the difference in the transport of compound transported by transfected cells and the mock cells pmol·min−1 per mg protein, I and S are the concentration of inhibitor (μM) and substrate (μM), respectively. The IC50 for inhibition of transport activity obtained from a plot of percentage activity remaining (relative to control) versus log10 inhibitor

-4-

concentration. Plasma pharmacokinetic parameters were estimated by noncompartmental analysis using Thermo Kinetica software package (version 5.0; InnaPhase, Philadelphia, PA, USA). The area under concentration-time curve (AUC0-t) up to the last measured point in time was calculated using the trapezoidal rule. The value of AUC0-∞ was generated by extrapolating AUC0-t to infinity using the elimination rate constant k and the last measured concentration Ct. The total plasma clearance (CLtot,p) was estimated by dividing the dose by the AUC0-∞. The distribution volume at steady state (VSS) was estimated by multiplying the CLtot,p by the mean residence time (MRT). The hepatobiliary excretory clearance (CLB) or the renal excretory clearance (CLR) was calculated by dividing the biliary or urinary cumulative amount excreted (Cum.Ae,0-t), respectively, by the plasma AUC0-t. The fraction of administered ginsenoside excreted into bile (fe-B) or urine (fe-U) was obtained by dividing the biliary or urinary Cum.Ae,0-t by the dose. The drug-drug interaction (DDI) index was estimated as the ratio of the maximal plasma unbound concentration after therapeutic dosing of ginsenosides divided by the in vitro OATP1B-specific IC50 (Giacomini et al., 2010; Wang et al., 2013). The accumulative factor (R) was calculated using the following equation: R = 1/(1− e−kτ)

(5)

where k represents the elimination rate constant (0.693/t1/2) and τ is the dosing interval (h). All data are expressed as the mean ± standard deviation (SD). Statistical analysis was performed using IBM SPSS Statistics Software (version 19.0; IBM, Somers, NY, USA). A value of P ≤ 0.05 was considered to be the minimum level of statistical significance.

Chemicals and Materials Ginsenosides Rg1, Re, Rb1, Rc and Rd and notoginsenoside R1 were obtained from the National Institutes for Food and Drug Control (Beijing, China). The purity of these test compounds exceeded 98%. Rifampin (rifampicin; ≥97%), oestrone-3-sulfate (E1S; potassium salt; ≥98%), oestradiol-17β-D-glucuronide (E217βG; sodium salt; ≥98%), taurocholic acid (TCA; sodium salt hydrate; ≥95%), methotrexate (MTX; ≥99%), poly-D-lysine hydrobromide (70,000–150,000 Da) and adenosine 5′-triphosphate (ATP; disodium salt hydrate, ≥99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used for in vitro studies. Rifampin that was used in animal studies was freeze-dried solid available as a sterile parenteral dosage form for i.v. injection and manufactured by Huapont Pharm. (Chongqing, China) with a China Food and Drug Administration ratification number of GuoYaoZhunZi-H20041320. HPLC-grade organic solvents were purchased from Sinopharm Chemical Reagent (Shanghai, China). HPLC-grade water was prepared with a Millipore Direct-Q 3 UV water purification system (Bedford, MA, USA). HEK-293 cells were obtained from American Type Culture Collection (Manassas, VA, USA). Inside-out membrane vesicle suspensions that expressed human MRP2, MDR1, BCRP, BSEP and rat Mrp2, Bcrp and Bsep were obtained from Genomembrane (Kanazawa, Japan). Inside-out membrane vesicle suspensions that expressed rat Mdr1a or rat Mdr1b were obtained from BD Gentest (Woburn, MA, USA).

References Bradford MM. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254. Chen F, Li L, Xu F, Sun Y, Du F-F, Ma X-T et al. (2013). Systemic and cerebral exposure to and pharmacokinetics of flavonols and terpene lactones after dosing standardized Ginkgo biloba leaf extracts to rats via different routes of administration. Br J Pharmacol 170: 440–457. Cheng C, Liu X-W, Du F-F, Li M-J, Xu F, Wang F-Q et al. (2013). Sensitive assay for measurement of volatile borneol, isoborneol, and the metabolite camphor in rat pharmacokinetic study of Borneolum (Bingpian) and Borneolum syntheticum (synthetic Bingpian). Acta Pharmacol Sin 34: 1337–1348. Giacomini KM, Huang S-M, Tweedie DJ, Benet LZ, Brouwer KLR, Chu X-Y et al. (2010). Membrane transporters in drug development. Nat Rev Drug Discov 9:215–236.

-5-

Guo B, Li C, Wang G-J, Chen L-S (2006). Rapid and direct measurement of free concentrations of highly protein-bound fluoxetine and its metabolite norfluoxetine in plasma. Rapid Commun Mass Spectrom 20: 39–47. Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG (2010). Animal research: Reporting in vivo experiments: the ARRIVE guidelines. Br J Pharmacol 160: 1577–1579. McGrath J, Drummond G, McLachlan E, Kilkenny C, Wainwright C (2010). Guidelines for reporting experiments involving animals: the ARRIVE guidelines. Br J Pharmacol 160: 1573–1576. Wang L, Pan X-L, Sweet DH (2013). The anthraquinone drug rhein potently interferes with organic anion transporter-mediated renal elimination. Biochem Pharmacol 86: 991–996. Wang L, Sun Y, Du F, Niu W, Lu T, Kan J et al. (2007). 'LC-electrolyte effects' improve the bioanalytical performance of liquid chromatography/tandem mass spectrometric assays in supporting pharmacokinetic study for drug discovery. Rapid Commun Mass Spectrom 21: 2573–2584.

-6-


British Journal of Pharmacology (DOI:10.1111/bph.12971) - Supporting Information Appendix S2 Protein expression of OATP1B1, OATP1B3 and Oatp1b2 in transfected HEK293 cells and mRNA levels of MRP2, BCRP, BSEP, MDR1, Mrp2, Bcrp and Bsep in membrane vesicles Human OATP1B1-expressing HEK293 cells

Human OATP1B3-expressing HEK293 cells

Mock OATP1B1

Rat Oatp1b2-expressing HEK293 cells Mock

Mock OATP1B3

Oatp1b2

130 kDa

94 kDa

94 kDa 94 kDa

66 kDa

66 kDa

Actin

Actin

Actin

Figure 1 The protein expression of human OATP1B1, human OATP1B3 and rat Oatp1b2 in transfected HEK293 cells and mock cells. The western blotting analysis is described in Supplemental Methods.

‐ 1 ‐

Human MRP2-exprssing vesicle (Lot. 0911H )

M

A

B

C

M

Human BSEP-exprssing vesicle (Lot. DUDHG26 )

M

Rat Mrp2-exprssing vesicle (Lot. AKARG03 )

M

A

B

C

A

B

Human BCRP-exprssing vesicle (Lot. DMU9G03)

A

B

C

Human MDR1-exprssing vesicle (Lot. DMIJG23)

C

M

A

B

C

Rat Bcrp-exprssing vesicle (Lot. ASIBG22 )

M

A

B

C

Rat Bsep-exprssing vesicle (Lot. 1007Y )

M

A

B

C

Figure 2 The mRNA levels of human MRP2, human BCRP, human BSEP, human MDR1, rat Mrp2, rat Bcrp and rat Bsep in membrane vesicles obtained from Genomembrane (Kanazawa, Japan). M, A, B and C represent the standard DNA maker, the mixture of three vials with the same batch number, positive control (the same vesicle products that prepared before and were already functionally verified) and negative control (Cat. No. GM0003), respectively. These mRNA data were kindly provided by Genomembrane.

‐ 2 ‐


British Journal of Pharmacology (DOI:10.1111/bph.12971) - Supporting Information Appendix S3 Time courses for accumulation of the ppt-type ginsenosides in HEK293 cells expressing human OATP1B3 or rat Oatp1b2

120

Ginsenoside Rg1

Transport amount (pmol·mg protein-1)

60

0 0

5

10

15

20

10

15

20

15

20

Ginsenoside Re 240

120

0 0

5

Notoginsenoside R1 240

120

0 0 Figure 1

5

10

Time (min)

Time courses for accumulation of ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 in HEK293 cells expressing pcDNA vector (circle), human OATP1B3 (square) or rat Oatp1b2 (triangle). The initial concentrations of ginsenosides used for incubation were 50 µM. Data represent the means and standard errors and each time point was performed in triplicate.

‐ 1 ‐


British Journal of Pharmacology (DOI:10.1111/bph.12971) - Supporting Information Appendix S4 Rifampin inhibition of human OATP1B3- and rat Oatp1b2-mediated uptakes of the ppt-type ginsenosides, plasma pharmacokinetics of rifampin in rats and rifampin-perpetrated drug-drug interactions

Human OATP1B3

20

Transport rat (pmol·min−1 per mg protein)

22

Ginsenoside Rg1 Ki=0.73 ± 0.04 μM

18

Ginsenoside Re Ki=0.65 ± 0.04 μM

11

10

9

0

0 0

0.25

0.50

Notoginsenoside R1 Ki=0.85 ± 0.06 μM

0 0

0.25

0.50

0

0.25

0.50

Rat Oatp1b2 60

Ginsenoside Rg1

100

60

Ginsenoside Re

Ki=0.68 ± 0.04 μM

Notoginsenoside R1 Ki=0.71 ± 0.03 μM

Ki=0.87 ± 0.05 μM

30

30

50

0

0 0

0.35

0.70

0 0

0.40

0.80

0

0.35

0.70

Rate/[Substrate] (μL·min−1 per mg protein)

Figure 1 Eadie-Hofstee plots for rifampin inhibition of human OATP1B3- and rat Oatp1b2-mediated uptakes of the ppt-type ginsenosides. The rifampin concentrations for the inhibition of ginsenosides were 0 (red ○), 0.5 (blue ●), 1 (blue □), 2 (blue ■) and 4 µM (blue Δ).

‐ 1 ‐

Plasma concentration of rifampin (μM)

100

60

10 1

45

Ki

0.1 0.01

30

0.001

15

0

12

24

12

18

24

0 0

6

Time (h) Figure 2 Total concentrations (bound + unbound; black line) and unbound concentrations (red line) of rifampin in plasma over time after a bolus i.v. dose at 20 mg·kg−1 in rats. The unbound plasma concentrations of rifampin, during 0–8 h after dosing, exceeded its Ki values for inhibition of rat Oatp1b2-mediated transports of the ppt-type ginsenosides (0.7–0.9 μM).

Table 1 Plasma PK parameters of rifampin after a bolus i.v. dose at 20 mg·kg−1 in rats PK parameters

Rifampin

C5min (μM)

42.2 ± 4.1

AUC0-t (h·μM)

138 ± 29

AUC0-∞(h·μM)

139 ± 29

t1/2 (h)

3.33 ± 0.37

MRT (h)

4.58 ± 0.5 −1

VSS (mL·kg ) -1

821 ± 31 −1

CLtot,p (mL·h ·kg )

182 ± 40

fU (%)

23.1 ± 1.0

Rifampin-perpetrated drug-drug interactions Effects of rifampin on drug metabolism and transport are broad and of established clinical significance (Niemi et al., 2003). Rifampin induces intestinal and hepatic CYP3A4-mediated metabolism of drugs (including midazolam, triazolam, simvastatin, antimycotics, itraconazole, cyclosporin and most dihydropyridine calcium channel antagonists) and also induces hepatic CYP2C-mediated metabolism of drugs [including (S)-warfarin and the sulfonylurea antidiabetic drugs]. In addition, rifampin induces intestinal MDR1 and can reduce the plasma concentrations of drugs that are substrates of MDR1 (including veripamil and digoxin). Recent research has demonstrated that induction of the P450 enzymes and P-gp by rifampin is mediated by activation of the nuclear PXR (Chen and Raymond, 2006). Because rifampin is an OATP/Oatp-inhibitor and substrate (Zaher et al., 2008; Vavricka et al., 2002), this compound was used in the current study to estimate the role of Oatp1b2 in regulation of rat systemic exposure to the ppt-type ginsenosides Rg1 and Re and notoginsenoside R1 via impairment of hepatobiliary excretion of these saponin compounds. The ppt-type ginsenosides are very poorly oxidized by P450 enzymes (Liu et al., 2009). Therefore, induction of P450 enzymes by

‐ 2 ‐

rifampin is not considered to have significant effect on the systemic exposure to the ginsenosides in rats, particularly when rats were treated with a single i.v. dose of rifampin. Although the ppt-type ginsenosides Rg1 and Re and notoginsenoside R1 were found to be substrates of human MDR1, but its rat ortholog appeared not to transport these ginsenosides. Therefore, induction of Mdr1 by rifampin probably has no effect on the systemic exposure to the ginsenosides in rats. It was also found that both the human BSEP and rat Bsep could be inhibited by rifampin. However, rifampin treatment of rats was found to result in decreased liver concentrations of ginsenosides (Supplemental Data 4). This suggested that rifampin-based inhibition of Oatp1b2 had more significant effect than rifampin-based inhibition of Bsep. In addition, reduced function of Bsep by rifampin in the liver could be compensated by function of hepatic Mrp2 and Bcrp, which were not affected by rifampin. Taken together, despite its broad effects on drug metabolism and transport, rifampin was a useful tool compound for estimation of the role of Oatp1b2 in regulation of rat systemic exposure to the ppt-type ginsenosides.

References Chen J, Raymond K. (2006) Roles of rifampicin in drug-drug interactions: underlying molecular mechanisms involving the nuclear pregnane X receptor. Ann Clin Microbiol Antimicrob 5: 3. Liu H-F, Yang J-L, Du F-F, Gao X-M, Ma X-T, Huang Y-H (2009). Absorption and disposition of ginsenosides after oral administration of Panax notoginseng extract to rats. Drug Metab Dispos 37: 2290–2298. Niemi M, Backman JT, Fromm MF, Neuvonen PJ, Kivistӧ KT. (2003). Pharmacokinetic interactions with rifampicin: clinical relevance. Clin Pharmacokinet 42: 819–850. Vavricka SR, Montfoort JV, Ha HR, Meier PJ, Fattinger K (2002). Interactions of rifamycin SV and rifampicin with organic anion uptake systems of human liver. Hepatology 36: 164–172. Zaher H, zu Schwabedissen HEM, Tirona RG, Cox ML, Obert LA, Agrawal N et al. (2008). Targeted disruption of murine organic anion-transporting polypeptide 1b2 (Oatp1b2/Slco1b2) significantly alters disposition of prototypical drug substrates pravastatin and rifampin. Mol Pharmacol 74: 320–329.

‐ 3 ‐


British Journal of Pharmacology (DOI:10.1111/bph.12971) - Supporting Information Appendix S5 Rat tissue distribution of ginsenosides Table 1 Tissue distribution of ginsenosides after i.v. administration at 2.5 µmoL∙kg−1 in rats treated with and without rifampin at 20 mg∙kg−1 Ppt-type

Ppd-type

Ginsenoside Rg1

Ginsenoside Re

Notoginsenoside R1

Ginsenoside Rb1

Ginsenoside Rc

Ginsenoside Rd

(−) Rif

(+) Rif

(−) Rif

(+) Rif

(−) Rif

(+) Rif

(−) Rif

(−) Rif

(−) Rif

C5min (μM)

6.30 ± 0.80

10.7 ± 1.4*

4.38 ± 0.48

5.28 ± 0.30*

6.33 ± 1.21

8.27 ± 0.44*

64.8 ± 6.8

57.0 ± 1.8

29.0 ± 1.2

AUC0-t (h·μM)

1.29 ± 0.20

3.65 ± 0.32*

0.938 ± 0.210

2.79 ± 0.17*

2.85 ± 0.34

4.40 ± 0.42*

237 ± 58

221 ± 4

92.3 ± 5.9

AUC0-∞(h·μM)

1.30 ± 0.21

3.66 ± 0.33*

0.942 ± 0.212

2.80 ± 0.17*

2.86 ± 0.34

4.41 ± 0.43*

590 ± 215

517 ± 32

136 ± 14

t1/2 (h)

0.121 ± 0.027

0.412 ± 0.094*

0.273 ± 0.154

0.549 ± 0.031*

0.483 ± 0.015

0.820 ± 0.251*

9.00 ± 4.38

10.3 ± 0.5

4.88 ± 0.47

MRT (h)

0.178 ± 0.040

0.469 ± 0.112*

0.198 ± 0.086

0.562 ± 0.043*

0.455 ± 0.097

0.686 ± 0.169*

12.9 ± 6.2

14.6 ± 0.8

6.94 ± 0.71

C5(15)min (μM)

1.02 ± 0.15

1.24 ± 0.04

0.723 ± 0.103

0.667 ± 0.103

0.824 ± 0.199

1.32 ± 0.11*

5.11 ± 0.47

3.99 ± 0.37

2.42 ± 0.36

AUC0-t (h·μM)

0.361 ± 0.019

0.572 ± 0.143

0.147 ± 0.010

0.389 ± 0.029*

0.352 ± 0.026

0.581 ± 0.112*

23.6 ± 2.6

19.0 ± 0.3

12.4 ± 0.8

AUC0-∞(h·μM)

0.370 ± 0.013

0.611 ± 0.129*

0.167 ± 0.034

0.413 ± 0.036*

0.391 ± 0.008

0.670 ± 0.124*

48.1 ± 5.5

33.4 ± 2.9

27.6 ± 4.9

t1/2 (h)

0.172 ± 0.081

0.361 ± 0.067*

0.172 ± 0.040

1.017 ± 0.101*

0.298 ± 0.064

0.312 ± 0.119

8.00 ± 0.90

6.67 ± 0.80

9.23 ± 1.48

MRT (h)

0.251 ± 0.103

0.481 ± 0.052*

0.216 ± 0.033

1.112 ± 0.193*

0.427 ± 0.094

0.452 ± 0.183

11.7 ± 1.1

9.55 ± 1.09

13.3 ± 2.1

Parameters Plasma data

Heart data

‐ 1 ‐

Liver data C5(15)min (μM)

8.44 ± 1.40

1.51 ± 0.27*

4.28 ± 0.28

1.12 ± 0.10*

7.38 ± 1.02

1.32 ± 0.09*

5.43 ± 0.46

4.53 ± 0.31

3.48 ± 0.33

AUC0-t (h·μM)

2.03 ± 0.15

0.660 ± 0.021*

1.77 ± 0.09

0.675 ± 0.105*

2.60 ± 0.30

0.797 ± 0.062*

24.1 ± 2.1

20.9 ± 1.5

17.2 ± 0.8

AUC0-∞(h·μM)

2.08 ± 0.15

0.781 ± 0.039*

1.86 ± 0.08

0.742 ± 0.121*

2.91 ± 0.37

1.10 ± 0.11*

62.6 ± 12.6

70.6 ± 22.2

42.6 ± 13.4

t1/2 (h)

0.182 ± 0.013

0.339 ± 0.028*

0.225 ± 0.016

0.593 ± 0.057*

0.431 ± 0.213

0.533 ± 0.068

11.9 ± 3.1

16.4 ± 8.0

10.8 ± 5.5

MRT (h)

0.251 ± 0.020

0.491 ± 0.052*

0.325 ± 0.023

0.861 ± 0.089*

0.532 ± 0.195

0.770 ± 0.101

17.1 ± 4.4

23.6 ± 11.4

15.6 ± 7.8

C5(15)min (μM)

7.62 ± 0.32

9.32 ± 0.35*

9.58 ± 1.89

17.0 ± 1.3*

14.6 ± 2.2

23.1 ± 6.1

6.17 ± 0.22

4.88 ± 1.15

4.08 ± 0.48

AUC0-t (h·μM)

6.94 ± 0.24

13.2 ± 1.5*

4.98 ± 0.47

14.0 ± 1.4*

13.6 ± 2.0

21.9 ± 2.6*

24.4 ± 2.9

24.2 ± 0.5

16.9 ± 4.1

AUC0-∞(h·μM)

10.4 ± 2.3

21.8 ± 3.2*

6.81 ± 1.19

17.7 ± 0.7*

17.2 ± 4.1

26.7 ± 3.2*

69.5 ± 11.3

105 ± 24

39.6 ± 10.6

t1/2 (h)

5.69 ± 3.41

4.67 ± 3.05

7.14 ± 2.55

4.19 ± 0.53

5.80 ± 2.14

3.69 ± 0.22

16.6 ± 7.8

20.9 ± 5.4

8.14 ± 3.12

MRT (h)

7.01 ± 4.25

6.41 ± 3.16

7.86 ± 3.32

4.69 ± 0.92

6.31 ± 1.91

4.34 ± 0.26

19.3 ± 6.3

30.2 ± 7.7

11.8 ± 4.4

C5(15)min (μM)

1.67 ± 0.31

1.92 ± 0.16

1.64 ± 0.53

1.92 ± 0.30

2.01 ± 0.54

2.87 ± 0.15

5.21 ± 0.24

4.57 ± 0.49

3.53 ± 0.44

AUC0-t (h·μM)

0.701 ± 0.020

1.12 ± 0.07*

0.417 ± 0.106

1.056 ± 0.268*

1.20 ± 0.24*

1.74 ± 0.14*

30.4 ± 1.8

25.8 ± 0.3

17.5 ± 0.5

AUC0-∞(h·μM)

0.712 ± 0.019

1.12 ± 0.07*

0.433 ± 0.105

1.11 ± 0.24*

1.24 ± 0.25*

1.78 ± 0.14*

136 ± 57

81.2 ± 15.3

40.9 ± 10.5

t1/2 (h)

2.11 ± 0.63

1.09 ± 0.18*

0.386 ± 0.296

0.618 ± 0.274

0.769 ± 0.061

0.763 ± 0.063

21.7 ± 9.7

14.4 ± 3.5

9.90 ± 3.40

MRT (h)

0.897 ± 0.047

0.770 ± 0.061*

0.503 ± 0.397

0.749 ± 0.273

0.807 ± 0.158

0.869 ± 0.085

31.2 ± 14.0

20.9 ± 4.9

14.3 ± 4.8

C5(15)min (μM)

0.063 ± 0.017

0.081 ± 0.017*

0.050 ± 0.001

0.113 ± 0.067

0.05 ± 0.01

0.058 ± 0.005

0.345 ± 0.078

0.330 ± 0.014

0.303 ± 0.055

AUC0-t (h·μM)

0.025 ± 0.003

0.038 ± 0.010*

0.078 ± 0.012

0.126 ± 0.021*

0.08 ± 0.01

0.108 ± 0.014*

1.17 ± 0.11

1.30 ± 0.17

0.92 ± 0.32

AUC0-∞(h·μM)

0.028 ± 0.005

0.047 ± 0.010*

0.132 ± 0.011

0.189 ± 0.020*

0.15 ± 0.01

0.184 ± 0.011*

2.02 ± 0.37

2.47 ± 0.52

1.12 ± 0.55

t1/2 (h)

0.299 ± 0.080

0.372 ± 0.151

3.007 ± 0.484

3.05 ± 2.79

3.90 ± 0.33

4.01 ± 2.18

6.00 ± 2.45

7.07 ± 1.54

2.95 ± 1.19

MRT (h)

0.430 ± 0.117

0.538 ± 0.223

4.422 ± 0.690

3.93 ± 3.35

5.61 ± 0.54

5.96 ± 3.66

9.10 ± 3.66

10.4 ± 2.47

4.37 ± 1.62

Kidney data

Lung data

Brain data

Three rats per group. (−) Rif, rats free of rifampin treatment. (+) Rif, rats treated with Rifampin. *, P