1. Introduction
Ursodeoxycholate (UDCA), an endogenous hydrophilic bile salt, is predominant in bear bile and has been used as a traditional medicine for the treatment of jaundice. In 1989, the therapeutic efficacy of UDCA was re-demonstrated in the first clinical trials on patients with primary biliary cholangitis. UDCA has been marketed as a therapeutic for cholestasis and preventive drug for liver diseases [
1,
2]. Ursa
® (Daewoong Pharmaceutical Co. Ltd., Seoul, Korea), a single tablet with 100–300 mg of UDCA, has been marketed in Korea since 1961 to cure liver diseases, including cholestasis. Presently, UDCA is the most widely prescribed drug for the treatment of cholestasis and the only medicine approved by the US Food and Drug Administration to treat primary biliary cirrhosis [
2].
The application of UDCA extends to the treatment of non-cholestatic liver diseases, owing to its multiple modes of action. This includes a reduction in the serum levels of toxic hydrophobic bile salts [
3], the stimulation of the hepatobiliary excretion of xenobiotics via phase II and III detoxification processes [
2,
4], antioxidant activity against oxidative stress [
2,
5,
6] and antiapoptotic activity through signaling pathways such as those involving the protein kinase C activator and mitogen-activated protein kinases (MAPKs) [
2,
7,
8,
9].
Studies have shown that UDCA pretreatment also prevents lipid peroxidation, increased cellular concentrations of reduced glutathione (GSH) [
10] and superoxide dismutase and activation of nuclear factor-E2-related factor-2 (Nrf2) in rats [
5,
11,
12]. GSH facilitates the hepatic elimination of xenobiotics by increasing GSH conjugation (phase II metabolism) and, consequently, enhancing the biliary excretion (phase III metabolism or detoxification) of GSH conjugates via canalicular membrane transporters, such as multidrug resistance-associated protein 2 (Mrp2) [
13,
14]. The greater affinity of GSH conjugates for Mrp2, as well as increased GSH conjugation [
13,
14,
15,
16,
17], was proposed as mechanisms underlying the UDCA-mediated increase in the biliary excretion of certain xenobiotics. Mrp2 is involved in the biliary excretion of numerous anionic endobiotics and xenobiotics, as well as their glucuronide, sulfate [
15,
18] and GSH conjugates [
13,
14]. In combination with GSH, Mrp2 mediates the transport of amphipathic, unchanged drugs, such as cisplatin, vinblastine and sulfinpyrazone [
19,
20]. Therefore, Mrp2 is one of the most important canalicular transporters involved in the hepatic excretion of various xenobiotics and their metabolites into the bile.
UDCA also increases the expression of Mrp2, Mrp3 and Mrp4 in rodents overexpressing Nrf2 [
5] and bile salt export pump (Bsep) in rats with 17α-ethinylestradiol (EE)-induced cholestasis [
21]. Therefore, UDCA treatment, which increases GSH conjugation and canalicular expression of efflux pump, may accelerate the hepatic elimination of endo- and xenobiotic Mrp2/Bsep substrates in a coordinated manner.
In this study, we investigated the effects of UDCA treatment, in combination with canalicular transporter expression, hepatic and renal functions and protein binding of probe drug, on biliary probe drug excretion in rats with cholestasis. Methotrexate (MTX) was selected as the probe drug for Mrp2, because it is mainly excreted through the bile and urine in its parent form with limited metabolism, mainly by Mrp2 [
22]. Cholestasis was experimentally induced through a subcutaneous (sc) injection of EE to male rats [
23,
24]. EE-induced cholestasis alters the expression and functions of drug transporters [
25]. For instance, the mRNA expression of Na
+-taurocholate co-transporting polypeptide (Ntcp), organic anion-transporting polypeptide (Oatp) 1a2 was downregulated in rats with EE-induced cholestasis [
26]. Cholestasis reduced the canalicular expression of Bsep and Mrp2 [
21,
27,
28]. Additionally, the internalization of canalicular Bsep and Mrp2 into the vesicular compartment of hepatocytes, followed by the reduction in the canalicular excretion of their substrates has been demonstrated in experimental cholestatic models induced by intravenous injections of estradiol-glucuronide and taurolithocholic acid [
29,
30]. Therefore, the effect of UDCA treatment on the expressional and functional changes of these transporters in cholestasis is worth investigating with respect to the biliary excretion (or detoxification) of their probe substrates.
3. Discussion
The purpose of the present study is to elucidate the association of expressional changes of hepatic transporters by UDCA treatment with the change in the biliary excretion (or detoxification) of MTX in rats with cholestasis. UDCA was orally administered tid at a daily dose of 100 mg/kg for 10 days (UDCA alone for 5 days, followed by co-administration with EE for 5 days). The dose was selected based on the therapeutic dose of UDCA [
33]. UDCA treatment for 10 days restored bile flow and biochemical abnormalities in the livers of rats with EE-induced cholestasis. During this treatment, the concentration of total bile salt in the plasma and bile was inversely changed by the treatment of EE and UDCA, demonstrating that decreased biliary secretion of bile salts is responsible for the increased plasma concentrations of bile salts in rats with cholestasis, consistent with a previous report [
34]. When compared the steady-state plasma concentrations of UDCA and its metabolites TUDCA and GUDCA, UDCA was the main component among UDCA and the two conjugated metabolites. This suggested that UDCA itself contributed largely to the therapeutic effect of UDCA observed in this study. Additionally, the content of UDCA in the plasma increased dramatically from zero to 60% in this study. Altogether, the increased UDCA concentration and increased content of hydrophilic bile acid could play critical roles in facilitating bile flow and hepatic detoxification. Moreover, the steady-state concentration of UDCA observed in this study was comparable to that observed following therapeutic UDCA administration to humans (900 mg/day for 3 weeks) [
35].
The expression of efflux transporters, such as Mrp2 and Bsep, was increased after UDCA treatment; however, the expression of uptake transporters, such as Ntcp, Oatp1a2, Oat1 and Oat3, was decreased by the treatment. Although the underlying mechanisms were not fully elucidated in the present study, transcriptional enhancement of Mrp2 and Bsep, via the enhanced cAMP-dependent protein kinase A (PKA) signaling and farnesoid X receptor (FXR) activation [
36] or increased Nrf2 pathway signaling [
5], might be responsible for the increased expression of the transporters. In addition, a good correlation was found between Mrp2 expression in the liver and the overall hepatobiliary excretion (CL
bile) and canalicular excretion clearance (CL
exc,bile) of MTX, suggesting that Mrp2-mediated canalicular excretion may regulate the elimination process and, thereby, normalize the pharmacokinetics of MTX, a Mrp2 substrate.
In the clinical study, UDCA treatment (900 mg/day for 3 weeks) increases the expression of MRP2 on the bile canalicular membrane in the patients with early-stage primary biliary cholangitis and pregnancy-induced cholestasis [
2,
37]. Breast cancer resistant protein (BCRP) expression was also increased in patients with intrahepatic cholestasis of pregnancy treated with UDCA at a dose of 900 mg/day for 3 weeks [
38]. Another UDCA administration (1 g/day for 3 weeks) to patients with gallstones stimulated the expression of MRP4, BSEP and MDR3 [
39].
Taken together, UDCA treatment may offer benefits by facilitating the hepatobiliary elimination of various xenobiotics and their conjugated metabolites via increased expression of efflux transporters, including MRPs, BSEP, BCRP and MDR. Thus, UDCA could accelerate the phase III detoxification in the cholestasis in humans as well as in rats by controlling the expression and function of efflux transporters.
4. Materials and Methods
4.1. Materials
UDCA was obtained from Daewoong Pharmaceutical (Youngin, Korea), while EE, MTX and taurocholate were purchased from Sigma-Aldrich (St. Louis, MO, USA). GUDCA and TUDCA were supplied by Toronto Research Chemicals (Toronto, ON, Canada).
4.2. Animals
Male rats (Sprague−Dawley, 220−250 g) were obtained from the Samtako Co. (Osan, Korea). All animal care and experimental procedures were approved by the Animal Care and Use Committee of Kyungpook National University (No. 2014-0131-1, 15 January 2015) and were carried out in accordance with the National Institutes of Health guidance for the care and the use of laboratory animals. Rats were housed for 1 week for acclimatization before
4.3. Treatment of Rats with EE and UDCA
Based on the various preliminary experiments with different UDCA treatment regimens, rats were finally divided into three groups: control, EE (cholestasis) and UDCA + EE/UDCA group (
Table 1).
Rats from the control group were administered with a daily sc injection of propylene glycol (PG) at 10 a.m. for 5 consecutive days at a daily dose of 1 mL/kg, followed by an oral administration of 1% (w/v) carboxymethyl cellulose (CMC) suspension in tap water tid (9 a.m., 3 p.m. and 9 p.m.) at 2 mL/kg daily for subsequent 5 days.
Rats from EE group received a daily sc injection of PG solution of EE at 10 a.m. for 5 consecutive days at a dose of 10 mg/mL/kg [
40]. CMC suspension (1%,
w/
v) without UDCA was then orally administered (2 mL/kg daily) tid (9 a.m., 3 p.m. and 9 p.m.) for subsequent 5 days.
Rats from UDCA + EE/UDCA group were orally administered with 1% (w/v) CMC suspension of UDCA tid (9 a.m., 3 p.m. and 9 p.m.) for 5 days at a daily UDCA dose of 100 mg/2 mL/kg, followed by a concomitant administration of EE and UDCA for another 5 days. EE was sc administered as a PG solution daily at 10 a.m. at a dose of 10 mg/mL/kg and UDCA was orally administered tid (9 a.m., 3 p.m. and 9 p.m.) as a 1% (w/v) CMC suspension at a daily dose of 100 mg/2 mL/kg.
The arterial plasma was collected from rats in each group at 12 h after the last treatment. The plasma sample was evaluated for biochemical parameters such as ALT, AST, bilirubin, bilirubin total, blood urea nitrogen (BUN) and serum creatinine level using assay kits from Young-Dong Diagnostics Co. (Seoul, Korea) in Seoul Clinical Laboratories (Yongin, Korea).
The plasma protein binding of MTX was determined for each group using a rapid equilibrium dialysis kit (ThermoFisher Scientific Korea, Seoul, Korea) according to the manufacturer’s protocol. Briefly, 100 μL of plasma withdrawn from each group containing 1 μM MTX was added into the sample chamber of the semipermeable membrane (molecular weight cut-off 8000 Da) and 300 μL of phosphate buffered saline (PBS) was added to the outer buffer chamber. After 4 h incubation on a shaking incubator at 300 rpm at 37 °C, 50 μL aliquots from both sample and buffer chambers were taken and treated with equal volumes of fresh PBS and plasma, respectively, to match the sample matrices for liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis. Aliquots (100 μL) of matrix-matched samples were added to 500 μL of acetonitrile containing 2 ng/mL of propranolol (an internal standard). Samples were vortexed for 10 min and centrifuged at 13,200 rpm for 10 min. An aliquot (1 μL) of the supernatant was injected directly into LC-MS/MS system.
4.4. Isolation of Total RNAs and Real-Time Reverse-Transcription Polymerase Chain Reaction (RT-PCR) Analysis
Liver and kidney samples were collected from rats of each group at 12 h after the last treatment, were snap-frozen in liquid nitrogen and stored at −80 °C. Total RNA was extracted from 100 mg samples of the liver and kidney using RNAzol (Molecular Research Center Inc., Cincinnati, OH, USA). The purity of total RNA was confirmed by the absorption ratio between 260 nm and 280 nm and the concentration of total RNA was measured by UV spectrophotometry.
Real-time reverse transcription polymerase chain reaction was performed with the LightCycler 96 Real-Time PCR System (Roche, Madison, WI, USA). Each PCR program included a pre-incubation period at 95 °C for 10 min, followed by 45 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 10 s and 72 °C for 10 s. Probes used for RT-PCR are shown in
Table 6. A relative quantitation of the mRNA level in the test tissue sample was made by measuring the threshold cycle (
CT) values of target genes and hypoxanthine phosphoribosyltransferase 1 (
Hprt1) house-keeping gene, which was used as an endogenous internal standard (IS) [
41].
4.5. Western Blot Analysis
Total protein samples were prepared by homogenizing 100 mg samples of liver and kidney from each group with one volume of lysis buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholic acid, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris, pH 7.5) for 10 min, followed by centrifugation at 13,200 rpm for 10 min. Protein samples (30–50 μg) were separated by SDS polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA; 4–12% gradient gel) and transferred onto a nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% bovine serum albumin in Tris-buffered saline with Tween 20 (200 mM Tris, 1.37 M NaCl, 0.1% Tween 20, pH 7.6) for 1 h, followed by its overnight incubation at 4 °C with primary antibodies against Oat1 (1:200, Santa Cruz Biotechnology, Dallas, TX, USA), Oat3 (1:100, Santa Cruz Biotechnology), Mrp2 (1:1000, Abcam, San Francisco, CA, USA), Nrf2 (1:1000, Abcam), Ntcp (1:1000, Abcam), Bsep (1:1000, Abcam), and β-actin (1:1000, Santa Cruz Biotechnology). Following incubation, the membrane was rinsed thrice with TBST at 25 °C and treated with horseradish peroxidase-labeled anti-goat or anti-rabbit IgG antibody (Santa Cruz Biotechnology). Protein bands were visualized with an enhanced chemiluminescence system (Santa Cruz Biotechnology). The images were analyzed using an ImageQuant LAS 4000 Mini (GE healthcare Korea, Seoul, Korea).
4.6. Determination of Bile Salts in the Plasma and Bile
Total bile salt concentrations in plasma and bile samples were determined using an enzymatic-fluorometric assay with a slight modification to the method described by Choi et al. [
42]. In brief, 100 μL of bile samples was collected 12 h after the last treatment of rats through the bile duct cannula, followed by the collection of 250 μL of blood samples from the abdominal artery. Aliquots (50 μL) of standard taurocholate solutions (5, 10, 25, 50, 100 and 200 μM), plasma samples and bile samples were added to 950 μL of the reaction buffer containing 1 mM β-nicotinamide adenine dinucleotide (β-NAD), 50 μU 3α-hydroxysteroid dehydrogenase (3α-HSD), 0.385 mM ethylenediaminetetraacetic acid (EDTA) and 760 mM Tris (pH 9.5) and incubated at 37 °C for 30 min. The reaction was stopped by adding 3 mL of ice cold water and the fluorescence of the samples was measured at 340 nm (excitation) and 465 nm (emission).
Concentrations of UDCA and its metabolites, GUDCA and TUDCA, in the plasma samples from the three groups were also measured according to the previously reported method [
43]. Aliquots (50 μL) of the plasma sample were added to 250 μL of acetonitrile containing 0.2 ng/mL of UDCA-d5 (IS). Samples were vortexed for 10 min and centrifuged at 13,200 rpm for 10 min. An aliquot (1 μL) of the supernatant was injected directly into Agilent 6430 Triple Quad LC-MS/MS system (Agilent, Wilmington, DE, USA).
UDCA, GUDCA and TUDCA were separated on a Synergi polar RP column (2.0 mm internal diameter × 150 mm length, 4 μm particle size) (Phenomenex, Torrance, CA, USA) with a mobile phase consisting methanol and water (65:35, v/v) containing 0.1% formic acid at a flow rate of 0.2 mL/min.
Retention time was 6.3 min for TUDCA, 7.1 min for GUDCA, 13.0 min for UDCA and 13.0 min for UDCA-d5 (IS). Mass peak was monitored using selected reaction monitoring (SRM) at m/z 498.2 → 80.2 for TUDCA, m/z 448.1 → 74 for GUDCA, m/z 391.3 → 391.3 for UDCA and m/z 396.3 → 396.3 for UDCA-d5 in a positive ion mode. Calibration was applied on a standard curve in the range of 0.2–40 μM for TUDCA, GUDCA and UDCA. Linearity, accuracy, intraday precision and interday precision were found to be within the acceptance criteria.
4.7. Pharmacokinetics of MTX
The femoral arteries, femoral veins and bile duct of rats were cannulated with PE50 or PE10 polyethylene tubing (Jungdo, Seoul, Korea) under anesthesia with zoletil and lompun (50 and 5 mg/kg, respectively, intramuscular injection) and heparinized saline (10 U/mL) was used to prevent blood clotting. Pharmacokinetic studies were started at 12 h after the last treatment.
Methotrexate solution (3 mg/kg in PBS) was administered intravenously to rats. Blood samples were collected from the femoral artery at 0, 2, 5, 10, 15, 30, 60, 90, 120, 240 and 360 min after intravenous bolus injection and centrifuged at 13,200 rpm for 10 min. Bile samples were also collected every 60 min for up to 360 min through the bile cannula. Urine samples were collected for 6 h through urinary bladder. Aliquots of 50 μL of plasma, bile and urine samples were added to 250 μL of acetonitrile containing 2 ng/mL of propranolol (IS), followed by 10 min vortex-mixing and centrifugation at 13,200 rpm, an aliquot (1 μL) of the supernatant was injected directly into LC-MS/MS system.
The area under the plasma concentration–time curve from zero to infinity (AUC
∞) was calculated by trapezoidal method and extrapolation method by dividing the terminal-phase rate constant with plasma concentration at the last time point [
44]. Standard methods were used to calculate total clearance (CL
total) and half-life by a non-compartmental analysis using WinNonlin (version 2.1, Pharsight, Mountain View, CA, USA). Urinary and biliary clearances of MTX (CL
urine and CL
bile, respectively) were estimated by dividing the total amount of MTX excreted into urine and bile for 0–6 h with AUC
6h. Glomerular filtration rate (GFR) was estimated from the creatinine clearance (CL
cr), which was calculated by dividing the total creatinine amount in urine for 6 h by the mean plasma concentration of creatinine in each rat [
45]. Creatinine concentration was assayed using kits from Young-Dong Diagnostics Co. (Seoul, Korea) from the service of Seoul Clinical Laboratories (Yongin, Korea).
4.8. Estimation of In Vivo Hepatic and Renal Uptake Clearances of MTX
To evaluate the in vivo hepatic (CLup,liver) and renal (CLup,kidney) uptake clearances of MTX, rats received a dose of 3 mg/kg of MTX via femoral vein catheter and blood samples (120 μL) were collected at 1, 2, 3, 4 and 5 min from the femoral artery. Animals were sacrificed at 5 min and the liver and kidney were dissected and weighed following an immediate and gentle wash with tissues soaked in ice-cold saline. The liver and kidney samples were homogenized with nine volumes of saline. The concentration of MTX in the plasma, liver and kidney was determined using LC-MS/MS system. Briefly, aliquots (50 μL) of plasma and 10% tissue homogenates were added to 250 μL of acetonitrile containing 2 ng/mL of propranolol (internal standard). The samples were vortexed for 10 min and centrifuged at 13,200 rpm for 10 min and an aliquot (1 μL) of the supernatant was injected directly into LC-MS/MS system. The values of CLup,liver and CLup,kidney were calculated by dividing the amount of MTX in the liver and kidney at 5 min with the plasma AUC from zero to 5 min. A period of 5 min was selected because the excretion of MTX from the liver and kidney was negligible for this period.
4.9. Estimation of In Vivo Biliary and Urinary Excretion Clearances of MTX
The femoral arteries, femoral veins and bile duct of rats were cannulated as described earlier. Rats received an intravenous bolus injection of MTX (1 mg/kg dissolved in 1 mL/kg PBS), followed by an intravenous infusion of MTX (0.6 mg/kg/h for 6 h, dissolved in 0.6 mg/0.5 mL PBS) to obtain steady-state concentrations of MTX in the liver. Bile was collected at an interval of 1 h for up to 6 h. Plasma and liver samples were immediately collected at 6 h. The concentrations of MTX in the plasma, liver, bile and urine samples were determined by LC-MS/MS method. In vivo biliary excretion clearance (CLexc,bile) was calculated by dividing the biliary excretion rate of MTX for 2–6 h (i.e., steady state of plasma MTX concentration) with the concentration of MTX in the liver at 6 h.
Urinary excretion clearance (CL
exc,urine) of MTX was estimated using the following equation: CL
exc,urine = CL
urine − CL
GF, where glomerular filtration CL of MTX (CL
GF) was calculated by multiplying f
u (the unbound fraction of MTX in plasma) by GFR [
46,
47]. CL
urine was estimated by dividing the total amount of MTX excreted into urine for 0–6 h period with AUC
6h.
4.10. LC-MS/MS Analysis of MTX
The concentration of MTX in each sample was analyzed using an Agilent 6430 Triple Quadrupole LC-MS/MS system equipped with an Agilent 1260 HPLC system (Agilent). The separation was carried out on a Synergi Polar RP column (2.0 mm internal diameter × 150 mm length, 4 μm particle size) (Phenomenex) using a mobile phase comprising water containing 0.1% (v/v) formic acid and acetonitrile containing 0.1% (v/v) formic acid at a ratio of 20:80. The flow rate was 0.25 mL/min. The operating parameters of the mass spectrometry were as follows: ion spray, 4000 V in negative mode; capillary temperature, 300 °C; vaporizer temperature, 300 °C; sheath gas pressure, 35 arbitrary units; auxiliary gas, 10 arbitrary units; and nitrogen gas flow rate, 10 L/min. The retention time was 1.8 min for MTX and 2.2 min for propranolol (IS). Quantitation was carried out by SRM at m/z 455.2 → 308.1 for MTX and m/z 260 → 116 for propranolol in the positive ionization mode and CE was 15 eV. Calibration was applied on a standard curve for MTX in the range of 0.02–50 μg/mL in plasma and kidney and liver homogenates, 0.5–1500 μg/mL in bile and 0.5–300 μg/mL in urine. Intra- and inter-day precision and accuracy had coefficients of variance of less than 15%.