Uricosuric Agents Affect Plasma and Kidney Concentration of Adefovir via Inhibition of Oat1 and Mrp2 in Rats

Abstract

Uricosuric agents lower serum uric acid levels by increasing urinary excretion via inhibition of urate transporter 1 (URAT1), urate reabsorption transporter in the renal proximal tubules. Probenecid and benzbromarone have been used as uricosurics, but these drugs inhibit organic anion transporters (OATs) in addition to URAT1. In this study, we investigated whether uricosuric agents interacted with adefovir, known as a substrate for OAT1, using Sprague–Dawley (SD) rats. Furthermore, involvement of other transporters, multi-drug resistance protein 2 (MRP2) in this interaction was examined using Mrp2-deficient rats. Probenecid and lesinurad increased plasma adefovir concentrations and decreased kidney-to-plasma partition coefficient (Kp) in these rats, presumably by inhibiting Oat1. Although benzbromarone had no effect on plasma adefovir concentration, it increased the Kp to 141% in SD rats. Since this effect was abolished in Mrp2-deficient rats, together with the MRP2 inhibition study, it is suggested that benzbromarone inhibits Mrp2-mediated adefovir excretion from the kidney. In contrast, dotinurad, a novel uricosuric agent that selectively inhibits URAT1, had no effect on the plasma and kidney concentrations of adefovir. Therefore, due to the lack of interaction with adefovir, dotinurad is expected to have low drug–drug interaction risk mediated by OAT1, and also by MRP2.

INTRODUCTION

The prevalence and incidence of gout are increasing in many countries.¹⁾ Approximately 20% men are diagnosed as hyperuricemia, a common pathogenic factor in the developing gout, with serum uric acid (UA) level greater than 7.0 mg/dL.^2–5) In the treatment of hyperuricemia, several guidelines recommend that serum UA levels should be below 6.0 mg/dL.^6–8) Serum UA levels are determined by both UA synthesis and excretion. UA is synthesized by xanthine oxidoreductase (XOR), so XOR inhibitors, including allopurinol, febuxostat and topiroxostat have been used for the treatment of hyperuricemia.^9,10) UA is excreted mainly in the kidney and intestine. In the kidney, UA is filtrated at the glomerulus followed by reabsorption (from the lumen) to renal epithelial cells via urate transporter 1 (URAT1, solute carrier family 22 member 12: SLC22A12).¹¹⁾ Therefore, URAT1 inhibitors are used as uricosuric agents for urate-lowering therapies by increasing the urinary excretion of UA. Uricosuric agents have been prescribed less than XOR inhibitors for several reasons. Probenecid is a well-known organic anion transporters (OATs) inhibitor that cause a drug–drug interactions (DDIs) by affecting the concentration of concomitant drugs such as indomethacin and benzylpenicillin in clinical settings.^12,13) Because benzbromarone causes rare but severe idiosyncratic hepatotoxicity, it has not been approved in many countries, and the measurement of liver function is required even in countries that have granted approval.¹⁴⁾ Recently, the novel URAT1 inhibitors, lesinurad and dotinurad have been launched and these drugs weakly inhibited the OAT1 and OAT3 in vitro.¹⁵⁾ Shen et al. showed that lesinurad has little effect on the renal clearance (CL_r) of furosemide which is a substrate of OAT1/3, but it decreased furosemide exposure.¹⁶⁾ The results did not strictly show whether lesinurad interacts with OAT1/3 substrates.

Adefovir is an anti-hepatitis B virus agent that causes severe renal impairment as an adverse effect.¹⁷⁾ Since adefovir is known to excrete mainly in urine via OAT1, dose-adjustment is needed in patients with impaired renal function to avoid the adverse effect.^13,18) Our previous study using Sprague–Dawley (SD) rats showed that probenecid and lesinurad increased the plasma concentration of adefovir.¹⁵⁾ These findings suggest that most of the uricosuric agents have a potential DDI risk via OATs.

Since probenecid-induced increase in plasma concentration of adefovir in Multi-drug resistance protein 2 (Mrp2)-deficient (TR-) rats was more remarkable than in Wistar rats,¹⁹⁾ we consider that involvement of Mrp2 should also be examined in the DDI study of adefovir. In the present study, we investigated the effects of uricosuric agents on Oat1 especially by focusing on the adefovir concentration in the kidney in addition to plasma. Furthermore, the involvement of MRP2 in the interaction of adefovir with uricosuric agents was also examined in Mrp2-deficient rats.

MATERIALS AND METHODS

Drugs and Materials

Adefovir was purchased from LKT Laboratories, Inc. (St. Paul, MN, U.S.A.). Probenecid was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Benzbromarone was purchased from Wako Pure Chemical Corporation (Osaka, Japan). Lesinurad was purchased from Selleck Chemicals, LLC. (Houston, TX, U.S.A.). Dotinurad (also called as FYU-981) and 6-hydroxybenzbromarone were synthesized by FUJI YAKUHIN CO., LTD. (Saitama, Japan). All other reagents used were high-quality, commercially available.

Animals

Six-week-old male SD rats and Eisai hyperbilirubinemic (EHBR, Mrp2-deficient) rats were obtained from Japan SLC, Inc. (Shizuoka, Japan). Rats were given 5–7 d to acclimate to the animal housing conditions before experimentation. The rats were housed in sterilized cages (3 or 4 per cage) under controlled environmental conditions (22 ± 4 °C, 60 ± 20% relative humidity, 12-/12-h light–dark cycle). The CE-2 pellet diet (CLEA Japan Inc., Tokyo, Japan) and tap water were available ad libitum. Applicable animal studies were performed according to the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and/or national guidelines. All studies were approved by the Animal Experiment Committee of Research Laboratories 2, FUJI YAKUHIN CO., LTD.

In Vivo Interaction Study of Adefovir and Uricosuric Agents in Rats

Seven-week-old rats that were fasted for 18 h before administration and randomly divided into each treatment group. Rats were orally received 100 mg/kg of probenecid, 67 mg/kg of lesinurad, 50 mg/kg of benzbromarone, 1.3 mg/kg of dotinurad, and 0.5% methylcellulose as control (n = 6). Drug dosages used in the present study were calculated based on their clinical maximal doses as described previously,¹⁵⁾ because DDI guideline or guidance from the U.S. Food and Drug Administration (FDA), European Medicines Agency (EMA), and Pharmaceuticals and Medical Device Agency (PMDA) shows the DDI risk must be calculated using an unbound C_max of clinical maximum dose^20–22) (Supplementary Table 1). Thirty minutes after drug gavage, 3 mg/kg of adefovir dissolved in saline was intravenously administered via the tail vein. Blood samples (approximately 200 µL) were collected from the jugular vein at 0.083, 0.25, 0.5, 1, 2, and 4 h after adefovir administration using a heparinized needle and kept on ice, and urine was collected for 0–4 h. Plasma was obtained from the blood by centrifugation at 1600 × g for 10 min at 4 °C. After a two-week interval, rats were orally administered uricosuric agents and adefovir at the same time course. At 0.5 h after adefovir administration, blood was collected from the abdominal aorta under anesthesia, and plasma was obtained in the same way. At the same time, the kidneys were removed and homogenized in a 4-fold volume of ice-cold saline.

Determination of Adefovir Concentration in Plasma, Urine and Kidney

Adefovir in these samples was detected as previously described.²³⁾ Briefly, adefovir was derivatized by incubation at 80 °C in 0.34% chloroacetaldehyde solution for 50 min and detected at excitation and emission wavelengths of 236 and 420 nm, respectively, using an InertSustain C18 column (GL Sciences Inc., Tokyo, Japan) and ACQUITY ultra performance liquid chromatography (UPLC) H-Class PLUS System (Waters Corporation. Milford, MA, U.S.A.) at 40 °C and the total flow rate was 0.4 mL/min. Isocratic mobile phases was used in the measurement of adefovir (50 mmol/L sodium acetate buffer, pH 5.5:acetonitrile = 95 : 5).

Determination of Uricosuric Agent Concentrations in Plasma

Plasma uricosuric agent concentrations were measured using the same samples as described in the previous section. Plasma samples were deproteinated with thrice volume of methanol and centrifuged at 18000 × g for 10 min at 4 °C. Concentrations of probenecid, benzbromarone, lesinurad, and dotinurad were detected at 250, 280, 291, and 322 nm UV spectra, respectively, using a Mightysil RP-18GP Aqua column (Kanto Chemical Co., Inc., Tokyo, Japan) and an Alliance 2695 HPLC system (Waters Corporation, Milford, MA, U.S.A.) at 40 °C and the total flow rate was 1.0 mL/min. Isocratic mobile phases were used in the measurement of probenecid and lesinurad (0.5% acetic acid:acetonitrile = 50 : 50), and dotinurad (5 mmol/L ammonium acetate:acetonitrile = 77 : 23). The gradient flow was used in the measurement of benzbromarone; 0.5% acetic acid and acetonitrile were used as mobile phases and the percentage of acetonitrile was 55% at 0–15 min, linear manner up to 65% at 15–25 min, and 55% at 25.1–30 min.

Pharmacokinetic Analysis

The pharmacokinetic parameters were calculated by the non-compartmental model using Phoenix WinNonlin Version 6.4 (Certara USA, Inc., Princeton, NJ, U.S.A.). The CL_r and the kidney-to-plasma partition coefficient (Kp) of adefovir were calculated using the following equations. CL_r = (urine adefovir concentration × urine volume)/area under the curve (AUC)_0–4h. Kp = kidney adefovir concentration at 4 h/plasma adefovir concentration at 4 h.

Inhibition Study of Uricosuric Agents Using MRP2-Overexpressing Membrane Vesicles

Membrane vesicles obtained from MRP2-overexpressing HEK293 cells (SOLVO Biotechnology, Hungary) were used in this study. 5(6)-Carboxy-2′,7′-dichlorofluorescein (CDCF) was used as substrate. Assay was performed in the presence of 5 µmol/L CDCF containing 4 mmol/L ATP or AMP to distinguish between transporter-mediated uptake and passive diffusion into the vesicles. In the ice-cold 96-well plate, 50 µL of membrane vesicle suspended in 7.5 µmol/L CDCF containing ice-cold assay buffer (250 mmol/L sucrose, 10 mmol/L MgCl₂, 10 mmol/L Tris–HCl, pH 7.0) were added. Test articles dissolved in dimethyl sulfoxide (DMSO), 0.75 µL, were added to the membrane mixture. Vehicle control contained DMSO instead of test article at a final concentration of 1%. Reactions were initiated at 37 °C by adding 25 µL of prewarmed 12 mmol/L MgATP (or 12 mmol/L AMP as a background control) in assay buffer. After 5 min, reactions were quenched by adding 200 µL of ice-cold washing buffer (250 mmol/L sucrose, 100 mmol/L NaCl, 10 mmol/L Tris–HCl, pH 7.0) and immediately filtered using the 96-well filter plate (FastRemover, GL Science Inc., Tokyo, Japan). The filters were washed five times with 200 µL of ice-cold washing buffer and air dried. Membrane vesicles on the filter were lysed by adding 100 µL of detector solution (reagent from PREDIVEZ vesicular transport assay kit, SOLVO Biotechnology). After 10 min, lysed solutions were filtered into the 96-well black plate (Nunclon, Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.). The concentration of substrate in the lysed solutions were determined by fluorescence (ex/em = 485/538 nm) using an FlexStation 3 (Molecular Devices, Inc., San Jose, CA, U.S.A.). Assay was performed in duplicate. Inhibition ratio of substrate transport activity was calculated using the following equation: inhibition ratio (% of control) = [1 − (A − B)/(C − D)] × 100, where A represents the amount of translocated substrate in the presence of test article and ATP, B represents the amount of translocated substrate in the presence of test article and AMP, C represents the amount of translocated substrate in the presence of 1% DMSO and ATP, and D represents the amount of translocated substrate in the presence of 1% DMSO and AMP. The concentration–response curves were analyzed using JMP (SAS Institute Inc., Cary, NC, U.S.A.) and a four-parameter logistic equation was applied to calculate the IC₅₀ with 95% confidence interval (CI).

Statistical Analysis

In the studies, the mean and standard deviation (S.D.) were calculated using Microsoft Excel (Microsoft Corporation, Redmond, DC, U.S.A.), and differences from the Control group were analyzed using the Dunnett’s multiple comparison test in JMP (SAS Institute Inc.) at significance levels of 5 and 1%.

RESULTS

Effect of Uricosuric Agents on the Plasma and Kidney Adefovir Concentration in SD Rats

The AUC_0–4h of adefovir was significantly increased by concomitant administration of probenecid and lesinurad. These treatments decreased CL_r to 44 and 64%, respectively (Table 1). In contrast, benzbromarone and dotinurad did not have any effect on the plasma adefovir concentration, but CL_r tended to decrease with benzbromarone to 70%.

Table 1. Pharmacokinetic Parameters of Adefovir in SD Rats with or without Each Uricosuric Agent

Group	C0 (µg/mL)	AUC0–4h (µg*h/mL)	CLr (mL/min/kg)
Control	8.70 ± 1.29	2.59 ± 0.26	5.32 ± 2.43
Probenecid	10.61 ± 1.95	5.83 ± 2.36**	2.32 ± 0.76*
Benzbromarone	10.30 ± 1.38	2.89 ± 0.37	3.72 ± 1.90
Lesinurad	10.56 ± 1.99	4.71 ± 1.62*	3.42 ± 2.23
Dotinurad	9.20 ± 1.13	2.62 ± 0.49	5.98 ± 3.06

C₀: plasma concentration at 0 h, AUC_0–4h: area under the curve from 0 to 4 h, CL_r: renal clearance. Data were analyzed using the Phoenix WinNonlin 6.4 software (Certara USA, Inc., Princeton, NJ, U.S.A.). The data represent mean ± S.D. (n = 6). * p < 0.05, ** p < 0.01 for significant differences from the Control group using the Dunnett’s multiple comparison test.

As shown in Fig. 1, the kidney-to-plasma partition coefficient (Kp) of adefovir in SD rats was significantly and slightly decreased by treatment with probenecid and lesinurad, respectively. Although benzbromarone did not affect plasma adefovir concentration, it increased the Kp. Dotinurad had little effect on the Kp.

Fig. 1. The Effects of Uricosuric Agents on Kp of Adefovir in SD Rats

Kp values in kidney are calculated from adefovir concentration in the kidney divided by that in plasma. Data are presented as mean + S.D. (n = 6). ** p < 0.01 for significant differences from the Control group using the Dunnett’s multiple comparison test.

Effect of Uricosuric Agents on the Plasma and Kidney Adefovir Concentration in Mrp2-Deficient Rats

The AUC_0–4h of adefovir was significantly increased by concomitant administration of probenecid, and CL_r were decreased to 53% (Table 2). Benzbromarone and lesinurad modestly increased plasma adefovir concentration. In contrast, dotinurad did not have any effect on the plasma adefovir concentration.

Table 2. Pharmacokinetic Parameters of Adefovir in Mrp2-Deficient Rats with or without Each Uricosuric Agent

Group	C0 (µg/mL)	AUC0–4h (µg*h/mL)	CLr (mL/min/kg)
Control	9.46 ± 1.68	3.12 ± 0.80	3.22 ± 1.50
Probenecid	11.73 ± 1.65	5.49 ± 1.39**	1.72 ± 0.50
Benzbromarone	10.36 ± 2.41	3.66 ± 0.75	2.68 ± 1.09
Lesinurad	9.64 ± 1.39	3.95 ± 0.45	2.47 ± 0.77
Dotinurad	9.45 ± 1.97	3.29 ± 0.27	2.64 ± 0.96

C₀: plasma concentration at 0 h, AUC_0–4h: area under the curve from 0 to 4 h, CL_r: renal clearance. Data were analyzed using the Phoenix WinNonlin 6.4 software (Certara USA, Inc., Princeton, NJ, U.S.A.). The data represent mean ± S.D. (n = 5 or 6). ** p < 0.01 for significant differences from the Control group using the Dunnett’s multiple comparison test.

In Mrp2-deficient rats without uricosuric agents, the Kp of adefovir was twofold higher than that in SD rats. Furthermore, the Kp in Mrp2-deficient rats was decreased by probenecid and lesinurad more potently than in SD rats (Fig. 2). In contrast, benzbromarone and dotinurad had no effect on Kp.

Fig. 2. The Effects of Uricosuric Agents on Kp of Adefovir in Mrp2-Deficient Rats

Kp values in kidney are calculated from adefovir concentration in the kidney divided by that in plasma. Data are presented as mean + S.D. (n = 6). ** p < 0.01 for significant differences from the Control group using the Dunnett’s multiple comparison test.

Pharmacokinetic Analysis of Uricosuric Agents in SD Rats and Mrp2-Deficient Rats

The plasma concentrations of uricosuric agents in the SD rats and Mrp2-deficient rats were measured (Fig. 3). The C_max of probenecid, benzbromarone, lesinurad and dotinurad in SD rats were 192.94 ± 39.41, 36.83 ± 6.50, 75.33 ± 18.76 and 3.80 ± 0.72 µg/mL, respectively. The C_max of these drugs in Mrp2-deficient rats were 162.57 ± 62.09, 57.67 ± 19.25, 105.98 ± 29.76 and 5.03 ± 0.77 µg/mL, respectively, showing the trend of higher value than SD rats except for probenecid.

Fig. 3. Plasma Concentration of Uricosuric Agents in SD Rats and Mrp2-Deficient Rats

The plasma concentrations of probenecid (A), benzbromarone (B), lesinurad (C) and dotinurad (D) in SD and Mrp2-deficient rats were shown as solid and dotted line, respectively. Each point represents the mean + S.D. (n = 6).

Inhibitory Effect of Uricosuric Agents on the Substrate Transport by MRP2-Overexpressing Membrane Vesicles

For each uricosuric agent, MRP2 inhibition was measured using MRP2-overexpressing membrane vesicles. Probenecid, benzbromarone (and 6-hydroxybenzbromarone), lesinurad and dotinurad at concentrations of 30–1000, 1–30, 10–300 and 10–300 µmol/L, respectively. Probenecid, benzbromarone, lesinurad and dotinurad concentration-dependently inhibited MRP2 and the IC₅₀ (95% CI) were 518 (217–1236), 3.84 (3.54–4.16), 32.4 (30.8–34.1) and 20.8 (10.9–39.7) µmol/l, respectively (Fig. 4). Furthermore, 6-hydroxybenzbromarone, an active metabolite of benzbromarone, concentration-dependently inhibited MRP2 and the IC₅₀ (95% CI) was 3.04 (2.12–4.37) µmol/L.

Fig. 4. Concentration–Response Curve of Probenecid (A), Benzbromarone (B), 6-Hydroxybenzbromarone (C), Lesinurad (D) and Dotinurad (E) on CDCF Transport by Membrane Vesicles from MRP2-Overexpressing HEK293 Cells

Each value is the mean of inhibition rate of CDCF transport relative to the vehicle control (n = 2).

Assessment of DDI Risk of Uricosuric Agents by OAT1 Inhibition

For each uricosuric agent, DDI risk by OAT1 inhibition was assessed using the in vitro IC₅₀ of OAT1 and plasma unbound concentration at clinical maximal dose (Table 3). According to the guidelines and guidance from the FDA, EMA, and PMDA,^20–22) a drug can cause clinically significant DDI risk when C_max,u/IC₅₀ values exceed 0.1, and only probenecid is applicable to this condition. Although lesinurad interacted with adefovir in rats, its DDI risk with OAT1 would be low in the clinical condition.

Table 3. DDI Risk Assessment of Uricosuric Agents by Transporter Inhibition Using Plasma Concentration and IC₅₀ Value of OAT1

Parameter	Probenecid	Benzbromarone	Lesinurad	Dotinurad
OAT1 IC50 (µmol/L)	10.9	3.14	6.99	4.08
Cmax (µmol/L)	78.8*	5.42**	20.2***	1.02****
Protein binding (%)	89 (83–95)	97.3 (96.3–98.3)	98.2 (97.9–98.5)	99.3 (99.2–99.4)
fu	0.11	0.027	0.018	0.007
Cmax,u/IC50	0.80	0.047	0.052	0.0018

IC₅₀: half maximal inhibitory concentration, C_max: maximum concentration at clinical dose, fu: unbound fraction rate, C_max,u: unbound maximum concentration (fu × C_max). If C_max,u/IC₅₀ exceeds 0.1, the drug has DDI risk at clinical dose and it is recommended to investigate further by human DDI study. OAT1 IC₅₀ was referred by Taniguchi et al. Clinical maximum doses (mg) were approved doses in Japan (probenecid, benzbromarone and dotinurad) or United States (lesinurad). C_max and protein binding were referenced information provided by the PMDA (probenecid, benzbromarone and dotinurad) or FDA (lesinurad). *: The data represents the mean concentration of q.i.d. administration, at a dose of 2 g, for 4 weeks (22.5 µg/mL). **: The data represent the C_max of single administration, at a dose of 100 mg (2.3 µg/mL). ***: The data represent the C_max of single administration, at a dose of 200 mg in Japanese subjects (8.17 µg/mL). ****: The data represent the C_max of single administration, at a dose of 4 mg (366.50 ng/mL).

DISCUSSION

UA is mainly excreted from the kidney. Approximately 90% of UA filtrated in glomeruli is reabsorbed via URAT1 in the proximal tubule.¹¹⁾ Uricosuric agents decrease serum UA via increasing urinary excretion of UA by inhibition of URAT1. In our previous in vitro study, uricosuric agents inhibited OAT1 and OAT3 in the proximal tubule, which are involved in UA secretion.¹⁵⁾ In fact, probenecid, a well-known uricosuric drug shows many DDIs via OATs, thereby increasing the plasma concentration of concomitant drugs such as indomethacin and benzylpenicillin.^12,13) Moreover, OAT1, OAT3, MRP2, and MRP4 are involved in tubular secretion of UA, so if a uricosuric agent inhibits these transporters, its hypouricemic effect will be attenuated. In addition to avoiding DDI risk, uricosuric agents that selectively inhibit URAT1 have advantages in terms of efficacy. In our previous study, probenecid and lesinurad increased plasma adefovir concentration, but the involvement of Mrp2 inhibition was not examined. In this study, we examined whether each uricosuric agent interacts with renal Oat1 and Mrp2 by measuring plasma and kidney concentrations of adefovir in SD and Mrp2-deficient rats.

Probenecid increased plasma adefovir levels and decreased Kp of adefovir in both SD and Mrp2-deficient rats (Figs. 1, 2). Moreover, the decrement of Kp was more potent in Mrp2-deficient rats. These results suggest that probenecid inhibits the renal uptake of adefovir via Oat1. Contrary to our results, Servais et al. previously reported that probenecid did not affect adefovir CL_r in normal Wistar rats, while probenecid decreased adefovir CL_r in Mrp2-deficient rats (TR- rats).¹⁹⁾ The differences between the outcomes of the two studies might arise from strain difference or the assessing time course, that is, the rat strains were SD and Wistar, and adefovir concentration was evaluated for 4 h and over 48 h by us and Servais et al., respectively. By inhibiting Oat1 that expresses almost whole kidney, probenecid may have decreased CL_r and subsequently increased AUC of adefovir (Tables 1, 2), considering the renal distribution of adefovir and its predominant renal excretion.¹⁸⁾

Lesinurad affected the plasma adefovir levels without effect on the Kp in SD rats (Fig. 1). On the contrary, lesinurad decreased the Kp in Mrp2-deficient rats (Fig. 2). Drug concentrations were not responsible for the different effects on the pharmacokinetics of adefovir, because the C_max of lesinurad was not significantly different between SD and Mrp2-deficient rats (Fig. 3). It seems that renal secretion of adefovir was attenuated by Mrp2-deficiency, leading to an increase in adefovir concentration in the kidney. In this model, moderate Oat1 inhibition can be observed as a decrease in Kp. Similar phenomenon was observed for probenecid as shown by more potent decrement of the Kp in Mrp2-deficient rats compared with SD rats.

In contrast to probenecid or lesinurad, benzbromarone had little effect on the plasma adefovir concentration (Tables 1, 2), while it increased the Kp in SD rats (Fig. 1). In the in vitro study, benzbromarone inhibited MRP2 at least 5-times more potently than other agents (Fig. 4). Further, 6-hydroxybenzbromarone, an active metabolite of benzbromarone, showed somewhat stronger inhibition and the C_max is not lower than benzbromarone in both SD rats and humans²⁴⁾ (Table 3, Supplementary Fig. 1). The results suggest that treated benzbromarone inhibits Mrp2 to cause renal accumulation of adefovir in SD rats. On the other hand, benzbromarone did not increase the Kp of adefovir in Mrp2-deficient rats (Fig. 2). In Mrp2-deficient rats, the Kp was twofold higher than that in SD rats despite the similar plasma levels of adefovir between the two strains, suggesting that Mrp2 made a significant contribution to the secretion of adefovir in rats. By using Mrp2-deficient rats, adefovir can be detected as a substrate of Mrp2, although it has presumable low affinity to the transporter. The characteristic of adefovir as a substrate of Mrp2 may contribute to its selective effect expression toward Kp but not on plasma concentration in SD rats.

Several factors should be considered to extrapolate the experimental results of rats to humans. The fractional excretion of adefovir in urine (fe) in human is close to that of rodents, approximately 90 and 80%, respectively.^18,25) In addition, as to the expression level of transporter, it is reported that expression levels of OAT1 and MRP2 in human is close to those of rats.^26,27) These reports suggest that rats are resemble to humans in view of the fate of adefovir. On the other hand, in contrast to rats, adefovir is shown to be a substrate of MRP4 but not of MRP2 in humans,²⁸⁾ suggesting that transporter selectivity of adefovir would be a factor of the species difference between rats and humans. A previous report showed that benzbromarone also inhibits urate transport via MRP4 with an IC₅₀ of 0.104 µmol/L,²⁹⁾ that is stronger than the results in our MRP2 inhibition study (Fig. 4). It is possible that inhibition of MRP2- and/or MRP4-mediated adefovir excretion by benzbromarone in renal epithelial cells caused adefovir accumulation. However, it seems difficult to estimate the DDI risk for MRP2 and/or MRP4 in humans precisely, because it is necessary to obtain the intrarenal parameters such as drug concentration and protein binding ratio to delineate renal handling of adefovir.

In regard to OAT1, the regulatory guidance/guideline shows that a drug can cause DDI in human when C_max,u/IC₅₀ values exceed 0.1.^20–22) The C_max,u/IC₅₀ value of each uricosuric agent was calculated using the OAT1 IC₅₀ value measured in our previous study (Table 3). As evidenced by the effect of probenecid on the pharmacokinetics of adefovir in rats, its C_max,u/IC₅₀ value for OAT1 exceeded the criteria (Table 3). In fact, probenecid, a well-known perpetrator of OAT1 caused DDI with adefovir in this study. Despite the effect of lesinurad on the pharmacokinetics of adefovir in rats, it does not have a DDI risk for OAT1 in humans (Table 3). DDI risk assessment of dotinurad suggests that it does not inhibit OAT1 in clinical conditions (Table 3). It was ascertained that it did not affect adefovir pharmacokinetics in either SD or Mrp2-deficient rats, even though plasma dotinurad concentrations in rats (3.80–5.03 µg/mL, Fig. 3) were 10-fold higher than that in humans (366.50 ng/mL, Table 3). These results indicate that the study in rats provided useful information for evaluating a DDI risk with adefovir.

Limitations

It is not verified whether adefovir is transported via Mrp2 or not. However, the fact that adefovir inhibits Mrp2 in killifish suggest that it can be a competitive substrate of Mrp2.³⁰⁾ In the present study, Kp of adefovir in Mrp2-deficient rats was twofold higher than that in SD rats. Together with the increased Kp by benzbromarone with Mrp2 inhibition, it can be implied that adefovir is a substrate of Mrp2.

Plasma concentration of adefovir and other compounds were measured until 4 h after administration to detect kidney concentrations of adefovir for calculating Kp. Additional blood and urine sampling at the later period and determining pharmacokinetic parameters using thereof may provide different results such as increased CL_r.

CONCLUSION

Our results show that adefovir is taken up and excreted from the rat kidney, and these processes are mediated by Oat1 and Mrp2 as evidenced by respective inhibitors, probenecid and benzbromarone (shown in graphical abstract). In contrast, novel uricosuric agents such as dotinurad and lesinurad do not inhibit these transporters, suggesting that in particular the former has lower DDI risk with adefovir.

Acknowledgments

We appreciate Mr. Kazuma Sekine for his assistance of pharmacokinetic evaluation.

Author Contributions

Participated in research design: Keisuke Motoki, Tetsuya Taniguchi, and Takashi Iwanaga.

Conducted experiments: Keisuke Motoki, Tetsuya Taniguchi, Miku Sakai, and Noriko Chikamatsu.

Performed data analysis: Keisuke Motoki, and Tetsuya Taniguchi.

Wrote or contributed to the writing of the manuscript: Keisuke Motoki, Tetsuya Taniguchi, Naoki Ashizawa, and Katsuhiro Yamano.

Conflict of Interest

The authors are employees of FUJI YAKUHIN CO., LTD.

Supplementary Materials

This article contains supplementary materials.

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