Changes in the expression of drug-metabolising enzymes and drug transporters in mice with collagen antibody-induced arthritis

Abstract 1. We investigated the changes in the expression of drug-metabolising enzymes and drug transporters in the liver, small intestine and kidney of mice with collagen antibody-induced arthritis (CAIA) to determine whether changes in these expressions affect pharmacokinetics of drugs in patients with rheumatoid arthritis. 2. mRNA expression levels of cytochrome P450 (Cyp) 2b10, Cyp2c29 and Cyp3a11 were observed to be lower in the liver and small intestine of CAIA mice than in control mice. Compared with control mice, mRNA expression levels of multidrug resistance 1 b, peptide transporter 2 and organic anion transporter (Oat) 2 were high in the liver of CAIA mice. Changes in these expression levels were different among organs. However, elevated expression of Oat2 mRNA was not associated with an increase in protein expression and transport activity evaluated using [3H]cGMP as a substrate. 3. These results suggest that arthritis can change the expression of pharmacokinetics-related genes, but the changes may not necessarily be linked to the pharmacokinetics in patients with rheumatoid arthritis. On the other hand, we found Oat2 mRNA expression level was positively correlated with plasma interleukin-6 level, indicating that transcriptional activation of Oat2 may occur in inflammatory state.


Introduction
Inflammation affects the pharmacokinetics of various drugs, and previous studies have investigated the changes in the expression of drug-metabolising enzymes and drug transporters in the inflammatory state in vivo and in vitro (Coutant and Hall 2018;Wu and Lin 2019;Stanke-Labesque et al. 2020;Saib and Delavenne 2021). These include cytochrome P450 (CYP) 1A2, CYP2C19, CYP3A4, multidrug resistance (MDR) 1, multidrug resistance-associated protein (MRP) 2 and breast cancer resistance protein (BCRP), which are downregulated in the inflammatory state (Coutant and Hall 2018;Wu and Lin 2019;Stanke-Labesque et al. 2020;Saib and Delavenne 2021), mainly attributed to inflammatory cytokines such as interleukin (IL) and tumour necrosis factor (TNF). Treatment with IL-1b, IL-6, or TNF-a decreased the mRNA and protein expression levels of various drug-metabolising enzymes and drug transporters in human primary hepatocytes (Aitken and Morgan 2007;Le Vee et al. 2009), mouse hepatocytes (Dickmann et al. 2012) and rat hepatocytes (Sukhai et al. 2001;Lee et al. 2009).
Rheumatoid arthritis (RA) is an inflammatory disease that can affect the pharmacokinetics of drugs in patients. The area under the concentration-time curve (AUC) of simvastatin and verapamil, which are CYP3A substrates, in patients with RA was significantly higher than that in healthy subjects (Coutant and Hall 2018). On the other hand, several reports have suggested that pharmacokinetics of certain drugs that are substrates of drug-metabolising enzymes and drug transporters were not different in patients with RA (Coutant and Hall 2018;Ono et al. 2021). Additionally, the relationship between changes in the expression of these proteins and their function in patients with RA remains unclear. It is important to clarify the effects of changes in the expression of these proteins on the pharmacokinetics. A collagen antibody-induced arthritis (CAIA) mouse was developed as an animal model of RA. The CAIA model has several advantages compared to the classical model of collagen-induced arthritis (CIA) such as high incidence rate, rapid disease onset, and the ability to use a wide range of strains and genetically modified mice (Khachigian 2006). It has been reported that various CYP isoforms, such as Cyp1a2, Cyp2b10, Cyp2c29, Cyp3a11 were downregulated and the enzymatic activity of the corresponding CYP was decreased in the liver of mice with CAIA (Dickmann et al. 2012). However, changes in the expression levels of drug-metabolising enzymes and drug transporters in the liver and extrahepatic organs have not been comprehensively elucidated; it needs to be clarified because RA is characterised by systemic inflammation, and drug-metabolising enzymes and drug transporters that contribute to the pharmacokinetics of drugs are expressed in the small intestine and kidney as well as in the liver.
In this study, we examined the variations in gene expression of drug transporters and drug-metabolising enzymes in the liver, small intestine and kidney of mice with CAIA. We found that arthritis affects the expression of drug-metabolising enzymes and drug transporters, depending on the organ. These findings may be useful in considering the systemic pharmacokinetics of drugs in patients with RA.

Animals
Seven-week-old female BALB/c mice were obtained from Japan SLC, Inc. (Shizuoka, Japan). Mice were housed in Specific Pathogen Free condition under a 12-h light/dark cycle and with free access to tap diet and water. All mouse experiments were approved by the Animal Research Committee of Ritsumeikan University (approval date: 4 February 2020, document number: BKC2019-039).
Collagen antibody-induced arthritis (CAIA) model CAIA model was produced according to the manufacturer's instructions. Mice (7-8 weeks old) were randomly assigned to each group and intraperitoneally injected with 1.5 mg/ 0.15 mL of collagen antibody cocktail (CAIA group; Chondrex, Redmond, WA, USA) or 0.15 mL of phosphate-buffered saline (PBS) as a vehicle [lipopolysaccharide (LPS) group and control group] on day 0. On day 3, the mice were intraperitoneally injected with 25 lg/0.05 mL of LPS (CAIA and LPS groups) or 0.05 mL of PBS (control group). The LPS group was created as a comparison group according to the manufacturer's instructions. On day 7, the mice were anaesthetised under isoflurane, and the blood (with heparin), liver, small intestinal epithelium (jejunum) and kidney (without capsule) were collected. Body weight and arthritis score were measured once a day. The arthritis score was assigned based on visual evaluation according to the manufacturer's instructions (Chondrex) as follows: score 0, normal; score 1, any one joint among interphalangeal joint, metacarpophalangeal joint and carpal and tarsal joint has redness and swelling; score 2, two joint types have redness and swelling; score 3, all three joint types have redness and swelling; and score 4, the entire paw has severe redness and swelling. The scores for each paw were summed, and the maximum score was 16.

Enzyme-linked immunosorbent assay (ELISA)
The plasma cytokine levels were measured using ELISA MAX TM Deiuxe Set Mouse IL-1b, IL-6 and TNF-a according to the manufacturer's instructions (BioLegend, Inc., San Diego, CA, USA).

Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
The mRNA levels of each drug transporter and drug-metabolising enzyme were determined by RT-qPCR. RNAlater Solution (Thermo Fisher Scientific, Waltham, MA, USA) was used when the liver, small intestine and kidney tissues were collected, and TRIzol reagent (Thermo Fisher Scientific) was used to isolate total RNA. Reverse transcription was conducted using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) with 2 lg of total RNA. Quantitative PCR was carried out on StepOne Real-Time PCR System (Thermo Fisher Scientific) using a mixture of PowerUp SYBR Green Master Mix (Thermo Fisher Scientific), cDNA and each primer. The thermocycling conditions were as follows: 50 C for 2 min, 95 C for 2 min, 40 cycles of 95 C for 3 s and 60 C for 30 s, 95 C for 15 s, 60 C for 1 min, 60-95 C at a rate of 0.3 C/s for melting curve acquisition. The primers used are listed in Supplementary Table S1. The specificity of all primers was confirmed by in silico and in vitro agarose gel electrophoresis. No amplification with genomic DNA and 100 ± 20% amplification efficiency based on the slope of the standard curve were also confirmed. The DDC T method was employed to calculate the relative expression levels of target genes. As the reference gene, glyceraldehyde 3-phosphate dehydrogenase (Gapdh) was used.

Organic anion transporter (OAT) 2-mediated uptake assay
The transport activity of mouse Oat2 was evaluated using primary mouse hepatocytes, which were isolated using a two-step collagenase liver perfusion method (Klaunig et al. 1981). Hepatocytes were seeded at a density of 5 Â 10 5 cells/ mL/well in 12-well plates coated with collagen (Type I, Nitta Gelatine Inc., Osaka, Japan). Cells were cultured in DMEM/ F12 containing 10% foetal bovine serum, 10 mM nicotinamide, 2 mM L-glutamine, 100 nM dexamethasone, 50 lM 2mercaptoethanol, 1 lg/mL insulin, 520 lM L-ascorbic acid, 100 units/mL penicillin, 100 lg/mL streptomycin and 10 mM HEPES. Hepatocytes were used for the uptake assay after 4-6 h of seeding. The culture medium was removed and replaced with an incubation medium (3 mM KCl, 145 mM NaCl, 0.5 mM MgCl 2 Á6H 2 O, 1 mM CaCl 2 Á2H 2 O, 5 mM D-Glucose and 5 mM HEPES). After pre-incubation for 10 min at 37 C, the medium was replaced with an incubation medium containing [ 3 H]cGMP (PerkinElmer, Inc., Boston, MA, USA). The incubation condition of uptake assay was 100 nM [ 3 H]cGMP as substrate concentration in time dependence experiment and for 1 min as incubation time in concentration dependence experiment. After incubation, the cells were washed twice with ice-cold incubation medium and 0.5 M NaOH was added to lyse the cells. The lysates were neutralised, and radioactivity was detected using a liquid scintillation counter (Beckman Coulter, Inc., Brea, CA, USA). The protein content of the solubilised cells was determined using Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and the uptake (mol) was normalised to the protein content.

SDS-PAGE and Western blotting
The protein expression of Oat2 was examined by Western blotting. The liver was homogenised using 1.15% KCl for the whole liver homogenates or 250 mM sucrose in 50 mM Tris-HCl buffer (pH 7.4) for the crude membrane fraction. The homogenates for the crude membrane fraction were centrifuged for 10 min at 3,000 Â g, and the supernatants were centrifuged for 30 min at 15,000 Â g. The pellets were suspended in 50 mM mannitol in 50 mM Tris-HCl buffer (pH 7.4). The protein content was then determined using Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Inc.). The liver samples (60 lg of protein per lane) were electrophoretically separated on 10% polyacrylamide gel at 200 V. Then, the proteins were transferred to PVDF membranes (Merck KGaA, Darmstadt, Germany) for 45 min at 15 V and blocked with 5% skim milk in TBS-T [0.15 M NaCl, 0.1% Tween 20 and 10 mM Tris-HCl (pH 8.0)] for 1 h at room temperature. After blocking, the membranes were incubated with the following primary antibodies at 4 C: SLC22A7 rabbit polyclonal antibody (1/ 1,000, Proteintech Group, Inc., Rosemont, IL, US), ATP1A1 (Na þ /K þ -ATPase) rabbit polyclonal antibody (1/10,000, Proteintech Group, Inc.) and anti-GAPDH monoclonal antibody (1/2,000, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). The membranes were washed with TBS-T and incubated with goat anti-rabbit IgG (whole molecule)-peroxidase antibody (1/10,000, Sigma-Aldrich) or peroxidase-conjugated AffiniPure goat anti-mouse IgG (H þ L) (1/10,000, Proteintech Group, Inc.) for 1 h at room temperature. The bound antibody was detected and quantified using ImmunoStar V R LD (FUJIFILM Wako Pure Chemical Corporation) and Amersham Imager 680 (GE Healthcare Japan, Tokyo, Japan). Protein expression was normalised to that of GAPDH (whole liver) or ATP1A1 (Na þ /K þ -ATPase, crude membrane fraction).

Statistical analysis
All data were presented as the mean ± S.D. Statistical analysis was carried out using GraphPad Prism version 8.4.3 (GraphPad Software, La Jolla, CA, USA) using one-way ANOVA with Tukey's multiple comparisons test or Pearson correlation analysis. Statistical analysis was also carried out in Microsoft Excel using Student's t-test. Statistical significance was set at P < 0.05.

Results
Body weight, arthritis score and plasma cytokine level in mice with CAIA We used anti-collagen antibodies and LPS to mice to establish a mouse model of CAIA. We administered PBS twice or PBS and LPS to mice of the control group or comparison group (LPS group), respectively. The mean value of body weight in each group was maintained after the first administration but decreased after LPS administration (Figure 1(A)). Arthritis score is an index of severity of arthritis. In the CAIA group, arthritis was observed from day 3, and arthritis score gradually increased from day 3 to day 7. On day 7, arthritis score was 6.4 ± 2.1 compared to the maximum score of 16 (Figure 1(B)). Arthritis was not observed in the control and LPS groups during the experimental period (Figure 1(B)). Figure 1(C) shows the plasma levels of IL-1b, IL-6 and TNF-a in each group on day 7. Higher IL-1b level was observed in the CAIA group as compared with that in the LPS group, although the level was low in the two CAIA mice. IL-6 level was well identified in the CAIA group and was significantly higher than that in the control and LPS groups. TNF-a level did not differ among the three groups (Figure 1(C)). mRNA expression of CYP isoforms and nuclear receptors in the liver, small intestine and kidney of mice with CAIA Then, the effect of CAIA on the mRNA expression levels of CYP isoforms and nuclear receptors, which are transcription factors that regulate CYP isoforms and drug transporters, were investigated in the liver, small intestine and kidney. In the liver, the mRNA expression levels of Cyp1a2, Cyp2b10, Cyp2c29 and Cyp3a11 were significantly lower in the CAIA group than in the control group. Decreased expression of Cyp3a11 was observed in the CAIA group than in the LPS group. Cyp3a13 expression level was significantly higher in the CAIA group than in the control and LPS groups. Pregnane X receptor (Pxr) expression levels were higher in the CAIA group than in the LPS group (Figure 2(A)). In the small intestine, Cyp2b10, Cyp2c29 and Cyp3a11 mRNA expression tended to be lower than in the CAIA group than in the control group, similar to that in the liver (Figure 2(B)). In the kidney that do not express several CYP isoforms, Cyp2d22 and Cyp2e1 expression levels in the CAIA group did not differ from those in the control group, similar to those in the small intestine and liver. Moreover, constitutive androstane receptor (Car) expression was significantly lower in the CAIA group than in the control group (Figure 2(C)). mRNA expression of drug transporters in the liver, small intestine and kidney of mice with CAIA Figure 3 shows the mRNA expression levels of each drug transporter in the liver, small intestine and kidney of mice with CAIA. In the liver, higher mRNA expression of Mdr1b, peptide transporter (Pept) 2 and Oat2 was observed in the CAIA group than in the control group. The expression levels of Mrp2, Mdr1a, bile salt export pump (Bsep), Bcrp, organic anion transporting polypeptide (Oatp) 1b2, Oatp2b1, organic cation transporter (Oct) 1 and multidrug and toxin extrusion (Mate) 1 did not differ among the three groups (Figure 3(A)). In the small intestine, decreased mRNA expression of Bcrp was observed in the CAIA group as compared with in the control and LPS groups (Figure 3(B)). Mdr1b mRNA expression was significantly lower in the small intestine and kidney of CAIA mice than in the control group, contrary to that in the liver (Figure 3(B,C)).
The hepatic mRNA expression levels of Mdr1b, Pept2 and Oat2 were high in the CAIA group (Figure 3(A)). The intestinal and renal mRNA expression levels of Mdr1b and the intestinal expression levels of Bcrp were low in the CAIA group (Figure 3(B,C)). Then, the mRNA expression levels and plasma IL-6 levels in the individual mice of control, LPS and CAIA groups were plotted as correlation diagram to evaluate the relationship between the mRNA expression levels of these drug transporters and the degree of inflammation. The hepatic mRNA expression level of Oat2 was positively correlated with plasma IL-6 level ( Figure 4). On the other hand, the hepatic mRNA expression levels of Mdr1b and Pept2 were not correlated with plasma IL-6 level (Supplementary Figure S1). The intestinal mRNA expression level of Mdr1b and Bcrp was negatively correlated with plasma IL-6 level (Supplementary Figure S1). The renal mRNA expression level of Mdr1b was not correlated with plasma IL-6 level (Supplementary Figure S1).

OAT2-mediated transport of cGMP in hepatocytes of mice with CAIA
The mRNA expression level of Oat2, an uptake transporter, increased significantly in the liver of mice with CAIA ( Figure  3(A)) and the individual mRNA expression level was correlated with plasma IL-6 level ( Figure 4). Therefore, we evaluated the transport activity of OAT2 in mouse primary hepatocytes using [ 3 H]cGMP as a substrate. Figure 5(A) shows the time dependence of cGMP uptake in mouse primary hepatocytes. cGMP transport activity did not differ between the hepatocytes of CAIA mice and control mice. Next, we examined the concentration dependence of cGMP uptake ( Figure 5(B)). However, saturation was not observed in the concentration-dependent experiment.
Protein expression of OAT2 in the whole liver and crude membrane fraction of mice with CAIA Despite the high expression levels of Oat2 mRNA in the liver of CAIA mice, transport activity of OAT2 did not change (Figures 3(A) and 5). Therefore, the protein expression of OAT2 in the whole liver and crude membrane fraction of CAIA mice was examined. In the whole liver, the protein expression of OAT2 in the CAIA group was similar to that in the control and LPS groups. However, as compared to the control group, the protein expression of OAT2 tended to decrease in the CAIA and LPS groups in the crude membrane fraction of the liver (Figure 6).

Discussion
We investigated the changes in the expression of drugmetabolising enzymes and drug transporters in the liver, small intestine and kidney of CAIA mice to determine whether changes in these expressions affect pharmacokinetics of drugs in patients with rheumatoid arthritis. First, we confirmed the severity of arthritis and degree of inflammation in CAIA mice (Figure 1). Arthritis score and plasma cytokine level in CAIA mice were similar to those in a previous study (Dickmann et al. 2012). The IL-6 levels were not different from those in patients with RA (Kobayashi et al. 2010;Chung et al. 2011;Adlan et al. 2015). TNF-a level was low near the limit of detection. Under these conditions, the mRNA expression of Cyp1a2, Cyp2b10, Cyp2c29, Cyp3a11, Cyp3a13 and Pxr was changed in the liver of mice with CAIA, consistent with previous report (Figure 2; (Dickmann Figure 2. mRNA expression levels of CYP isoforms and nuclear receptors in the liver (A), small intestine (B) and kidney (C) of control, LPS and CAIA mice. The mRNA expression levels of CYP isoforms and nuclear receptors were evaluated by RT-qPCR. The relative expression levels were normalised to the expression level of Gapdh as the reference gene and calculated relative to those in the control group. Each column represents the mean ± S.D. (n ¼ 5); Ã p < 0.05, ÃÃ p < 0.01, ÃÃÃ p < 0.001 vs. control group; † p < 0.05, † † p < 0.01, † † † p < 0.001 vs. LPS group (Tukey's multiple comparison test). AhR: aryl hydrocarbon receptor; Car: constitutive androstane receptor; Cyp: cytochrome P450; Pxr: pregnane X receptor et al. 2012)). We also showed the altered mRNA expression of CYP isoforms in the small intestine and kidney of CAIA mice. In rats with CIA, a classical animal model of RA, mRNA expression of Cyp3a1 was decreased in the small intestine (Lin et al. 2017). Thus, arthritis affects the expression levels of CYP in the small intestine, kidney and liver. In addition, changes in the mRNA expression levels of several drug transporters were observed in mice with CAIA and were different among the organs examined (Figure 3). In rats with adjuvant-induced arthritis, another classical animal model for RA, the changes in the expression of several ABC transporters, Mdr1a, Mdr1b, Mrp2 and Bcrp were different among organs such as the liver, small intestine, kidney and brain (Kawase et al. 2014). Effects of arthritis on the expression of drug transporters are different among organs, although the mechanism remains unclear. It is possible that systemic Figure 3. mRNA expression levels of drug transporters in the liver (A), small intestine (B) and kidney (C) of control, LPS and CAIA mice. The mRNA expression levels of drug transporters were evaluated by RT-qPCR. The relative expression levels of drug transporters were normalised to the expression level of Gapdh as the reference gene and calculated relative to those in the control group. Each column represents the mean ± S.D. (n ¼ 5); Ã p < 0.05, ÃÃ p < 0.01, ÃÃÃ p < 0.001 vs. control group; † † p < 0.01 vs. LPS group (Tukey's multiple comparison test). BCRP: breast cancer resistance protein; BSEP: bile salt export pump; MATE: multidrug and toxin extrusion; MDR: multidrug resistance; MRP: multidrug resistance-associated protein; OAT: organic anion transporter; OATP: organic anion transporting polypeptide; OCT: organic cation transporter; PEPT, peptide transporter.
inflammation causes different effects on each organ, and the regulation of drug transporter expression differs in each organ.
The changes in the expression of drug-metabolising enzymes and drug transporters due to inflammation are mainly attributed to inflammatory cytokines such as IL-1b, IL-6 and TNF-a. In human and mouse primary hepatocytes, these cytokines showed potential to decrease the expression levels of various drug transporters and drug-metabolising enzymes (Aitken and Morgan 2007;Le Vee et al. 2009;Dickmann et al. 2012). However, unlike previous in vitro studies (Hisaeda et al. 2004;Le Vee et al. 2009;Diao et al. 2010), downregulation of Mrp2, Bsep, Oatp, Oat and Oct was not observed in our results. The inflammatory response or regulation of each gene expression may differ between in vitro and in vivo studies. Inflammation-induced downregulation of drug transporters and drug-metabolising enzymes has been reported to be caused by the activation of nuclear factor (NF)-jB, a transcription factor that responds to inflammatory cytokines (Wu and Lin 2019). The downregulation observed in our results might be caused by NF-jB activation. However, the mechanism underlying the upregulation of Mdr1b, Pept2 and Oat2 in the liver of CAIA mice is not clear. Further studies are needed to clarify the mechanism underlying the changes in the expression of drug transporters and drug-metabolising enzymes in the inflammatory state in vivo.
OAT2 is mainly expressed in the liver and kidney, and is a solute carrier transporter that contributes to the uptake of several drugs, including ibuprofen, diclofenac, warfarin and tolbutamide, in the human liver (Bi, Lin, et al. 2018;Kimoto et al. 2018). In our results, the mRNA expression level of Oat2 was significantly higher in the liver of CAIA mice; however, the protein expression and transport activity of OAT2 were not correlated ( Figures  3(A), 5 and 6). It has been reported that the protein expression and transport activity of several drug transporters do not necessarily correlate with the corresponding mRNA expression for several reasons, including post-transcriptional modification, intracellular trafficking and membrane localisation (Ohtsuki et al. 2012;Li et al. 2019). From our results in Figure 6, mouse OAT2 localisation is not clear at this stage. The mechanism is not clear, and further investigation is needed to clarify post-transcriptional modification, intracellular trafficking and membrane localisation of OAT2 in CAIA mice. We then examined the transport activity of OAT2 using cGMP as a probe substrate (Cropp et al. 2008;Marada et al. 2015). Saturation of transport activity was not observed in concentration dependence experiment of cGMP uptake ( Figure 5(B)), although the uptake of 100 nM [ 3 H]cGMP was decreased by 38.8 ± 13.2% by an OAT2 inhibitor, indomethacin, at 100 lM in our preliminary experiment using normal mice (data not shown). The basal transport activity of OAT2 may be low in mouse hepatocytes. It was reported that Oat2  mRNA expression was significantly increased in mice fed a high-fat diet (He et al. 2020) and in pigs with cadmiuminduced liver injury (Wang et al. 2021), although the protein expression and transport activity were not determined. In addition, decreased expression level of OAT2 indicated a high risk of hepatocellular carcinoma in patients with chronic hepatitis C (Yasui et al. 2014). It has been speculated that Oat2 is related to embryonic development of various tissues in mice (Pavlova et al. 2000). Therefore, OAT2 may play important pathological and physiological roles, regardless of its transport function. From our results of positive correlation between the individual mRNA expression level of Oat2 and plasma IL-6 level (Figure 4), Oat2 gene expression may be regulated by inflammation. Further investigation of the role of OAT2 under pathological and physiological conditions is needed.
Our results of the changes in the pharmacokinetics-related genes can associate with their function, and affect pharmacokinetics of certain drugs in patients with RA. In patients with RA, the pharmacokinetics of CYP3A substrates, such as simvastatin and verapamil, have been characterised. The AUC of simvastatin was approximately 3.5-fold higher than that in healthy subjects (Coutant and Hall 2018), whereas the AUC of simvastatin was decreased by infusion of tocilizumab, an anti-human IL-6 receptor antibody, suggesting that IL-6 was responsible to reduce CYP3A activity in patients with RA (Schmitt et al. 2011). The AUC of verapamil in patients with RA was 3-4-fold higher than that in healthy subjects (Mayo et al. 2000). Serum levels of IL-1ra, IL-6 and IL-8 in patients with RA are negatively correlated with the serum concentration of 4b-hydroxycholesterol, which is an endogenous metabolite formed by CYP3A4/5 (Wollmann et al. 2018). Our results on the mRNA expression of Cyp3a11 (comparable to human CYP3A4) in the liver of CAIA mice supported these findings. However, the clinical significance of the changes in the expression of drug transporters or drug-metabolising enzymes except CYP3A has not been fully clarified in patients with RA. Whether change in the expression of drug transporters and drug-metabolising enzymes directly leads to a change in their function should be examined. RA may affect the pharmacokinetics of multiple substrates of drug transporters and drug-metabolizing enzymes because their gene expression profiles were altered in our results of CAIA mice.
In conclusion, we demonstrated the changes in the gene expression of drug transporters and drug-metabolising enzymes in the liver, small intestine and kidney of CAIA mice. Gene expression levels differed significantly between CAIA mice and control mice. The changes in gene expression of CYP isoforms in the CAIA group were similar among organs, whereas the changes in gene expression of several drug transporters in the CAIA group were different among organs. The significant increase in gene expression of Oat2 in the liver of CAIA mice was not linked to protein expression and transport activity. The changes in the expression of several drug transporters and drug-metabolizing enzymes found in this study may variously affect the pharmacokinetics in patients with RA.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This work was supported in part by JSPS KAKENHI and Mochida Memorial Foundation for Medical and Pharmaceutical Research.

Toshiya Katsura
http://orcid.org/0000-0002-7789-8803 Figure 6. Protein expression of OAT2 in the whole liver (A) and liver crude membrane fraction (B) of control, LPS and CAIA mice. The protein expression of OAT2 was evaluated by Western blotting. The relative expression was normalised to the expression of GAPDH (whole liver) or Na þ /K þ -ATPase (liver crude membrane fraction) as the loading control. Each column represents mean ± S.D. (n ¼ 5). These data were not significant (Tukey's multiple comparison test).