Organic anion and cation transporters are possibly involved in renal excretion of entecavir in rats

Life Sciences 89 (2011) 1–6

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Life Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i f e s c i e

Organic anion and cation transporters are possibly involved in renal excretion of entecavir in rats Chen Yanxiao a,1, Xu Ruijuan a,1, Yang Jin a,⁎, Chen Lei a, Wang Qian a, Yin Xuefen a, Tang Hong a, Zhang Xueying a, Andrew K. Davey b, Wang Jiping b a b

Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing 210009, China Sansom Institute, School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, SA 5000, Australia

a r t i c l e

i n f o

Article history: Received 9 November 2010 Accepted 21 March 2011 Keywords: Entecavir Renal excretion Organic anion and cation transporters MRP2 P-glycoprotein

a b s t r a c t Aims: The purpose of the present study was to investigate the roles of transporters in the renal excretion of entecavir. Main methods: We analyzed the effect of probenecid, cimetidine, sulfobromophthalein sodium (BSP), verapamil, inhibitors of organic anion transporter (OAT), organic cation transporter (OCT), multidrug resistance-associated protein 2 (MRP2) and P-glycoprotein respectively, on the excretion of entecavir. The area under plasma concentration–time curve (AUC), body clearance, and renal clearance of entecavir was examined in each group. Key findings: After intravenous coadministration with entecavir in conscious rats, cimetidine, probenecid, BSP and verapamil significantly increased the AUC of entecavir by 40.07%, 48.78%, 37.49%, and 54.58%, and reduced the body clearance by 27.14%, 31.69%, 29.79%, and 42.17%, respectively. Then the effects of these inhibitors on the renal clearance of entecavir in unconscious rats were studied. Coadministration of cimetidine and probenecid increased the steady plasma concentration of entecavir by 127.61% and 169.46%, reduced the renal clearance by 50.47% and 67.76%, and decreased the excretion ratio by 44.81% and 64.16% compared to initial values. However, the effects of BSP and verapamil were slight. Cimetidine and probenecid also increased the concentration of entecavir in kidney from 34.00 ± 0.80 ng/mL to 55.19 ± 4.92 ng/mL and 49.92 ± 1.53 ng/mL, while the concentration of entecavir in kidney from BSP and verapamil groups was 30.96 ± 0.81 ng/mL and 35.72 ± 7.30 ng/mL, respectively. Significance: These results suggest that cimetidine and probenecid inhibit the renal excretion of entecavir in rats, which indicates the most likely involvement of organic anion and cation transporters in the renal excretion of entecavir. © 2011 Elsevier Inc. All rights reserved.

Introduction Entecavir (Fig.1) is a synthetic guanosine nucleoside analog widely used in the treatment of chronic hepatitis B (HBV) infection. Entecavir is phosphorylated to the triphosphate form and inhibits all the three replication steps of the HBV. Previous studies in human have indicated that entecavir is well absorbed orally, undergoes minimal metabolism and is eliminated primarily in the urine (62%–73%) (Matthews, 2006; Rivkin, 2007; Scott and Keating, 2009; Shaw and Locarnini, 2004). As entecavir's renal clearance exceeds the glomerular filtration rate (GFR), tubular secretion should positively contribute to its renal clearance (Matthews, 2006). It is well known that renal tubular secretion of drugs includes several different transport systems (Lee and Kim, 2004), such as organic anion transporter (OAT), organic ⁎ Corresponding author. Tel./fax: + 86 25 83271386. E-mail address: [email protected] (Y. Jin). 1 These authors contributed equally to this work. 0024-3205/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2011.03.018

cation transporter (OCT), multidrug resistance-associated protein (MRP) family and P-glycoprotein. However, the tubular excretion mechanism of entecavir remains unclear. Entecavir is a hydrophilic, weak alkaline and its structure is similar to that of other nucleoside antiviral drugs such as acyclovir, ganciclovir, lamivudine, AM188 (Wang et al., 2004), and adefovir. Wada et al., (2000) reported that the uptake of acyclovir in kidney is probenecid-sensitive and mediated by rat OAT1 (rOAT1). Coadministration of probenecid and cimetidine decreased the renal clearance of acyclovir (De Bony et al., 2002). Similarly, Takeda et al., 2002 reported that acyclovir and ganciclovir are substrates of OCT1 and OAT1. Lamivudine is a substrate of human OCT1 (hOCT1), hOCT2, and hOCT3 (Minuesa et al., 2009) and cimetidine inhibits its renal secretion through inhibition of the organic cation transport system (Takubo et al., 2000). Renal secretion of the antiviral nucleoside analog, AM188, is inhibited by probenecid and cimetidine in the isolated perfused rat kidney (Wang et al., 2004). OAT3 and, particularly, OAT1 mediate the transport of adefovir in the kidney (Cihlar et al., 1999;

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C. Yanxiao et al. / Life Sciences 89 (2011) 1–6

OH H2 N

N HN

N

N

CH 2

OH

O Fig. 1. Structure of entecavir.

Servais et al., 2006; Uwai et al., 2007). MRP2, 4 and 5 have a role in the transport of adefovir (Servais et al., 2006). It is therefore reasonable to hypothesize that these transporters contribute to the transport of entecavir. In order to determine the role of transporters in the tubular secretion of entecavir, the effects of probenecid, cimetidine, sulfobromophthalein sodium (BSP) and verapamil on the clearance and renal excretion of entecavir in rats were investigated. Materials and methods Chemicals Entecavir (Department of Medical Chemistry, China Pharmaceutical University, Jiangsu, China); inulin (Bomei Bio-Technology Co., Ltd, Anhui, China); probenecid, cimetidine and verapamil (China Pharmaceutical Biological Products Analysis Institute, Jiangsu, China); BSP (Acros Organics); urethane (chemical reagent grade, Shanghai Qingxi Chemical Technology Co., Ltd, Shanghai, China); isotonic saline (Nanjing Xiaoying Pharmaceutical Group Co. Ltd, Jiangsu, China); methanol and acetonitrile (high-performance liquid chromatography grade, Merck Co., USA); formic acid and ammonia water (analytical reagent grade, Nanjing Chemical Reagent Co., Ltd, China) were used for this work. Chemicals were dissolved in isotonic saline for intravenous injection. Animals Male Sprague–Dawley rats (200 ± 20 g) were purchased from Yangzhou University. Rats were housed under controlled lighting and temperature, with a commercial food diet and water freely available. All animals were fasted overnight with free access to water before surgery. All procedures involving animals were carried out according to the guidance of the Animal Ethics Committee in China Pharmaceutical University. Animal experiments Effects of inhibitors on the body clearance of entecavir administered as a bolus injection in conscious rats Probenecid (20 mg/kg), cimetidine (50 mg/kg), BSP (20 mg/kg), verapamil (1.5 mg/kg) or an equivalent volume of normal saline (1.0 mL/100 g) were intravenously administered to rats. Ten minutes later, entecavir (10 μg/kg) was administered to rats by tail vein injection (t = 0 min). Blood samples were collected through the orbital venous plexus at 0, 2, 10, 30, 60, 90 and 120 min. Plasma samples were obtained by centrifugation at 9600 ×g for 3 min and stored at − 20 °C until analysis. Effects of inhibitors on the renal excretion of entecavir at steady state in unconscious rats Rats were anesthetized by intraperitoneal injection of 20% (w/v) urethane solution (7 mL/kg). The rats were then placed on their backs,

the body temperature maintained at 37 °C with a heat lamp, and the right jugular vein and left femoral artery cannulated with polyethylene tubes for drug administration and blood sampling, respectively. Then, 5% (w/v) mannitol solution was infused (0.1 mL/min) into the jugular vein to stabilize the urinary flow rate, and the right and left ureters were catheterized for urine collection. After the surgery, rats received a loading dose of inulin (30 mg/kg) and entecavir (1.7 μg), followed by a constant-rate infusion of 5% mannitol solution to administer entecavir (0.5 μg/h) and inulin (15 mg/h) at a rate of 6 mL/h using an infusion pump till the end of the experiment. After 30 min infusion, when the concentration of entecavir reached steady state, urine was collected into pre-weighed tubes for 10 min, and blood samples were taken at the midpoint of the urine collection periods. Then, cimetidine (50 mg/kg), probenecid (20 mg/kg), BSP (20 mg/kg) and verapamil (1.5 mg/kg) or saline (1.0 mL/100 g) were administered intravenously. Twenty minutes later, urine samples were collected at 10 min intervals for 40 min and blood samples were taken at the midpoint of the urine collection periods. The volume of urine samples was measured gravimetrically, with specific gravity assumed to be 1.0. At the end of the experiment, both kidneys of each rat were removed, washed with cold saline, blotted dry, weighed and homogenized with an equivalent volume of saline. Plasma samples were obtained by centrifugation at 9600 ×g for 3 min. All plasma, urine and homogenate samples were stored at −20 °C until analysis. Drug analysis Concentrations of entecavir in plasma, urine and kidney homogenate were determined by LC/MS after solid-phase extraction. A portion (200 μL) of plasma or diluted urine (20 fold) or homogenate sample and 120 μL of 10% (v/v) perchloric acid containing 10 μL of ganciclovir (0.5 μg/mL) as internal standards were mixed and centrifuged at 20627 ×g for 5 min. The supernatant was loaded into a solid-phase extraction column (Waters Oasis MCX) preactivated by methanol and distilled water and then the column was washed with 1 mL of 2% (v/v) formic acid and methanol. Finally, 0.5 mL of 4% ammonia in methanol was added and the eluate was dried under a stream of nitrogen gas at 40 °C, the residue was reconstituted in 0.02% formic acid and centrifuged at 20627 ×g for 5 min before LC/MS analysis. The conditions were as follows: column, Lichrospher C18 column (2.0 × 150 mm); column temperature: 35 °C; mobile phase, 0.02% formic acid (A)-methanol (B) and B%: 0–1.00 min: 13%; 1.01–4.00 min: 13%–35%; 4.01–9.00 min: 13%; and flow rate: 0.2 mL/min. Concentrations of inulin in plasma and urine were determined by HPLC. Diluted plasma (2 fold) or urine (20 fold) sample (100 μL) and 100 μL 10% (v/v) perchloric acid containing 10 μL of paracetamol as internal standard were mixed and centrifuged at 20627 ×g for 5 min, then the supernatant was boiled for 10 min, cooled for 5 min on ice, centrifuged at 20627 ×g for 5 min and separated on a reversed-phase column (C18, 4.6 × 250 mm). The mobile phase consisted of 10% (v/v) acetonitrile in distilled water and the flow rate was 1.0 mL/min. Inulin was detected by UV at 280 nm and the column temperature was 30 °C. Data analysis The area under plasma concentration–time curve (AUC) and half life (t1/2) was calculated based on moment methods. The body clearance (CLtol) of entecavir after a bolus injection in conscious rats was calculated using the following formula: CLtol =

Div AUC

where Div represents the dose of intraveneous injection.

C. Yanxiao et al. / Life Sciences 89 (2011) 1–6 Table 1 Effects of cimetidine, probenecid, BSP and verapamil on the clearance of entecavir in rats following an intravenous bolus injection. Parameters

Control

Cimetidine

Probenecid

BSP

Verapamil

t1/2 (h) 0.41±0.05 0.42±0.01 0.41±0.04 0.37±0.04 0.37±0.03 AUC 2.62±0.17 3.69±0.39* 3.90±0.17** 3.61±0.22* 4.05±0.29** (ng h/mL) CL (mL/min) 13.13±1.03 9.57±0.86* 8.97±0.41** 9.22±0.57** 7.59±0.99** All data are expressed as mean ± SE (n = 4). *Significantly different from control (*, p b 0.05, **, p b 0.01).

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the CLr by 50.47% and 67.76%, and decreased the excretion ratio by 44.81% and 64.16%. However, BSP and verapamil had no apparent influence on these parameters. The GFR in all the groups was consistent. The concentration of entecavir in the kidney at the end of the experiment was also determined, and the concentration of entecavir in kidney homogenates is shown in Table 3. Cimetidine and probenecid increased the concentration of entecavir in the kidney homogenates. Discussion

The GFR, renal excretion (CLr) and excretion ratio (ER) of entecavir at steady state in unconscious rats were calculated as follows: CLr =

Ku × U Cpss

GFR = CLr;

inulin

CLr ER = GFR where Ku and U represent the urine flow rate and urine concentration, respectively; and Cpss represents the plasma concentration at midpoint; and CLr, inulin represents the renal clearance of inulin. Statistical analysis All data were expressed as mean ± standard errors (SE) respectively. Statistical analysis was assessed by one-way ANOVA followed by post hoc Dunnett's test, and statistical significance was assumed when p value was lower than 0.05. Results Effects of inhibitors on the clearance of entecavir administered as a bolus injection in conscious rats The effects of cimetidine, probenecid, BSP and verapamil on the pharmacokinetics of entecavir are presented in Table 1. Coadministration of cimetidine, probenecid, BSP and verapamil significantly increased the AUC of entecavir by 40.07%, 48.78%, 37.49%, and 54.58%, and reduced the body clearance by 27.14%, 31.69%, 29.79%, and 42.17%, respectively. However, the half life of entecavir was not significantly affected by the inhibitors. Effects of inhibitors on the renal excretion of entecavir at steady state in unconscious rats The effects of cimetidine, probenecid, BSP and verapamil on the renal excretion of entecavir are summarized in Table 2 and Fig. 2. The concentration of entecavir in plasma and renal clearance was kept at a steady state during the infusion. Coadministration of cimetidine and probenecid increased the Cpss by 127.61% and 169.46%, reduced

In order to explore the role of renal tubular transporters in the renal excretion of entecavir, the effects of inhibitors of OAT, OCT, MRP2 and P-glycoprotein on the body clearance in rats were initially screened. After a single dose of entecavir in conscious rats, coadministration of cimetidine, probenecid, BSP and verapamil significantly increased the AUC and reduced the body clearance of entecavir compared to the control group. All of these drugs inhibited the elimination of entecavir, indicating that these transporters are involved in the elimination of entecavir in rats. Entecavir is mainly excreted by the kidneys but these results do not exclude the possibility of an effect on hepatic/biliary clearance. To further clarify the role of renal transporters on the clearance of entecavir, the effects of cimetidine, probenecid, BSP and verapamil on the renal excretion of entecavir at steady state were investigated. During the experiment, the GFR was not affected by these inhibitors, and the renal clearance of entecavir was greater than the GFR, suggesting that the renal excretion of entecavir includes glomerular filtration and tubular secretion, which is consistent with the behavior of entecavir in human. After 55 min infusion, the renal clearance and excretion ratio of entecavir in the cimetidine group and probenecid group were notably reduced, compared to the initial values while these parameters remained stable in the control group, BSP group and verapamil group. Cimetidine and probenecid inhibited the renal excretion of entecavir, the inhibitory potency of probenecid was greater than that of cimetidine. We also detected the concentration of entecavir in kidney homogenates after the infusion and found that the concentration of entecavir in cimetidine group and probenecid group was 62.3% and 46.8% higher than the control group, respectively. This result indicated that probenecid and cimetidine added the accumulation of entecavir in kidney and reduced its excretion. From the results it is apparent that entecavir excretion in the rat kidney is sensitive to cimetidine and probenecid. Cimetidine is a well-known substrate and inhibitor of OCT. It is reported to inhibit the uptake of organic cation N1-methylnicotinamide (NMN) in brush-border membrane vesicles of donor human and rat kidneys and the inhibition constant (Ki) in rats is 140 μM (Ott et al., 1991; Ullrich and Rumrich, 1992). It has also been found that cimetidine is transported by human OAT3(hOAT3) (Cha et al., 2001) and rOAT3 (Tahara et al., 2005). In addition to hOAT3, cimetidine was also observed to inhibit organic anion uptake mediated by hOAT1 and hOAT4

Table 2 Effects of cimetidine, probenecid, BSP and verapamil on the renal excretion of entecavir at steady state in rats. Group

Control + cimetidine + probenecid + BSP + verapamil

Cpss (ng/mL)

GFR (mL/min)

CLr (mL/min)

ER (CLr/GFR)

Initial

55 min

Initial

55 min

Initial

55 min

Initial

55 min

3.50 ± 0.22 2.39 ± 0.28 2.02 ± 0.35 2.37 ± 0.22 3.65 ± 0.53

3.12 ± 0.36 5.45 ± 0.60** 5.44 ± 0.49** 2.74 ± 0.10 3.91 ± 0.34

1.20 ± 0.23 1.29 ± 0.14 1.54 ± 0.18 2.08 ± 0.41 2.12 ± 0.35

0.94 ± 0.20 1.24 ± 0.19 1.35 ± 0.20 2.10 ± 0.67 1.77 ± 0.33

4.42 ± 0.51 4.89 ± 0.66 5.16 ± 1.03 4.87 ± 0.33 3.82 ± 1.08

4.70 ± 0.81 2.42 ± 0.39** 1.66 ± 0.27** 3.93 ± 0.98 4.64 ± 0.87

4.35 ± 0.49 3.95 ± 0.42 3.42 ± 0.58 2.92 ± 0.74 1.85 ± 0.56

5.43 ± 0.85 2.18 ± 0.14** 1.23 ± 0.03** 3.18 ± 1.26 2.27 ± 0.64

All data are expressed as mean ± SE (n = 4). *Significantly different from initial steady state values (*, p b 0.05, **, p b 0.01).

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Cpss of entecavir (ng/mL)

A

7.00

35min

**

** **

** *

4.00 3.00 2.00 1.00

3.50

GFR of entecavir (mL/min)

3.00

control

cimetidine

before

25min

45min

55min

probenecid

BSP

verapamil

probenecid

BSP

verapamil

BSP

verapamil

BSP

verapamil

35min

2.50 2.00 1.50 1.00 0.50 0.00

CLr of entecavir (mL/min)

55min

5.00

B

7.00 6.00

control

cimetidine

before

25min

45min

55min

35min

5.00 4.00

*

*

*

3.00

**

** ** **

2.00 1.00 0.00

D

25min

6.00

0.00

C

before 45min

9.00

control

cimetidine

before

25min

45min

55min

probenecid

35min

ER of entecavir

7.50 6.00 4.50 3.00

**

0.00

** **

1.50 control

cimetidine

**

**

probenecid

Fig. 2. Effects of cimetidine (50 mg/kg), probenecid (20 mg/kg), BSP (20 mg/kg) and verapamil (1.5 mg/kg) on the renal excretion of entecavir at steady state in rats. (A) Effects of cimetidine, probenecid, BSP and verapamil on the Cpss of entecavir at steady state. The concentration of entecavir in plasma was kept at a steady state during the infusion. Coadministration of cimetidine and probenecid increased the Cpss by 127.61% and 169.46%, while BSP and verapamil had no apparent influence on Cpss. (B) Effects of cimetidine, probenecid, BSP and verapamil on the GFR of entecavir at steady state. The glomerular filtration rate in each group had no significant difference. (C) Effects of cimetidine, probenecid, BSP and verapamil on the CLr of entecavir at steady state. Coadministration of cimetidine and probenecid reduced the CLr by 50.47% and 67.76%. However, CLr in BSP and verapamil group didn't change significantly. (D) Effects of cimetidine, probenecid, BSP and verapamil on the ER of entecavir at steady state. Coadministration of cimetidine and probenecid decreased the excretion ratio by 44.81% and 64.16%, while BSP and verapamil failed to change ER of entecavir. Each point represents the mean ± SE (n = 4). *, significantly different from initial steady state values (*, p b 0.05, **, p b 0.01).

(Khamdang et al., 2004). According to these findings, it is reasonable to assume that the inhibitory effect of cimetidine on the renal excretion of entecavir in rats is mediated by OAT or/and OCT.

Probenecid is commonly used to inhibit OAT in experiments and is reported to dose-dependently inhibit the uptake of organic anion para-aminohippuric acid (PAH) by rOAT1(Ki value: 14.1 μM)

C. Yanxiao et al. / Life Sciences 89 (2011) 1–6

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Table 3 Effects of cimetidine, probenecid, BSP and verapamil on the concentration of entecavir in kidney homogenates after 55 min-infusion. Group

Control

+ cimetidine

+ probenecid

+ BSP

+ verapamil

C (ng/mL)

34.00 ± 0.80

55.19 ± 4.92**

49.92 ± 1.53*

30.96 ± 0.81

35.72 ± 7.30

All data are expressed as mean ± SE (n = 4). *Significantly different from control (*, p b 0.05, **, p b 0.01).

(Khamdang et al., 2004). Interestingly, probenecid also inhibits the transport of some organic cation drugs. For example, probenecid inhibits the renal transport of the organic cation cimetidine (Brandle and Greven, 1992; Gisclon et al., 1989). Inotsume et al., 1990 reported that probenecid inhibits the renal tubular secretion of famotidine, which exists partly in a cationic form under physiological conditions. So the inhibitory effect of probenecid on the renal excretion of entecavir might act through the inhibition of organic anion transport system or/and organic cation transport system. Furthermore, Masereeuw et al., 2000 found the various inhibitory effects of probenecid on the transport of organic anion and cation in cells is likely to, at least in part, be a result of membrane disordering due to its lipophilic character rather than competition for the transporter. Probenecid is also an inhibitor of MRP2, 4, 5 and 6 (Lee and Kim, 2004) and the Ki value of a typical MRP2 substrate in isolated canalicular membrane vesicles was 44.6 μM (Horikawa et al., 2002). MRP2, the multidrug resistance protein isoform characterized by its apical localization in polarized cells and substrate multi-specificity, has been identified in rat and human kidney (Schaub et al., 1997, 1999). In order to determine whether MRP2 is involved in the excretion of entecavir, we examined the effect of BSP, a well-known MRP2 substrate, on the excretion of entecavir. The Ki value of BSP and its metabolite sulfobromophthalein–glutathione (BSP-SG) for MRP2 are 2.18 μM and 0.0461 μM respectively (Horikawa et al., 2002). BSP reduced the AUC and body clearance of entecavir significantly, but it had no significant effect on the renal excretion of entecavir. P-glycoprotein is a well-known transporter related to drug–drug interactions in the clinical situation and it is present in the apicial membrane of many secretory cell types in organs such as the kidney, liver and intestine (Ito et al., 1993; Okamura et al., 1993; Rebbeor JF, 1998; Zhou, 2008). The substrates of P-glycoprotein vary greatly in structure and functionality, ranging from small molecules including organic cations to macromolecules such as proteins (Wang et al., 2003). Verapamil, a P-glycoprotein inhibitor [half-maximal inhibition constant (K0.5) =1 μM] was used to investigate the role of P-glycoprotein in the elimination of entecavir in rats. Verapamil reduced the whole body clearance of entecavir, but it did not affect the renal excretion of entecavir. As both MRPs and P-glycoprotein are expressed in liver and intestine (Lee and Kim, 2004; Sankatsing et al., 2004) they play an important role in the absorption and elimination of drugs at these sites as well as having a role in renal excretion. Within the current study there was some evidence that entecavir clearance was affected by verapamil and BSP following a bolus intravenous injection but this was not apparent at steady state and there is no evidence of an effect on renal clearance. It is therefore possible that the observed results in the i.v. bolus study are explained by BSP and verapamil having more pronounced effects on biliary and/or intestinal clearance of entecavir than on renal clearance as hypothesized. However, as renal clearance of entecavir is its predominant route of clearance, the Cpss during an intravenous infusion is likely to be relatively insensitive to altered transporter activity in the liver/intestine. Certainly further research is required to clarify the role of MRPs and P-glycoprotein in the clearance of entecavir. In addition to OAT, OCT, MRPs and P-glycoprotein, there are other transport systems expressed on the renal tubular membrane, such as the nucleoside transport system. The nucleoside transport system is divided into two types based on the transport

mechanism: equilibrative bidirectional nucleoside transporter family (ENT) driven by chemical gradients, and inwardly directed concentrative nucleoside transporter family (CNT) driven by the sodium electrochemical gradient (Baldwin et al., 2004; Cass et al., 1998; Gray et al., 2004). Nucleoside transporters contribute to the uptake and disposition of many nucleoside analogs used in the treatment of cancers and viral diseases. It is reported that CNT1, 2 and ENT2 are involved in the transport of nucleoside antiviral drug zidovudine in the kidney (Ritzel et al., 1997, 1998; Yao et al., 2001). Fukuchi et al., 2010 reported that ENT1 is an uptake transporter of ribavirin in human hepatocytes, rat intestine and human epithelial LS180 cells (Takaai et al., 2008). So it is reasonable to suspect if there are interactions between entecavir and nucleoside transporters, which have not yet been clarified. In conclusion, these results suggest that cimetidine and probenecid inhibit the renal excretion of entecavir in rats, which indicates the most likely involvement of organic anion and cation transporters in the renal excretion of entecavir. To date, the research in the field of transport systems is still limited by the overlapping of substrates, inducers, inhibitors and the lack of specific biotechnology. The present study is a preliminary experiment about the possible involvement of renal transporters in the renal excretion of entecavir; our hypothesis needs to be verified by further studies of drug transport in knockout rat and OCT/OAT transfected cells, and it will be useful for the further study about the drug–drug interactions based on renal tubular transporters between entecavir and other drugs which are also mainly eliminated in urine. Conflict of interest statement There are no competing interests in this paper.

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