Role of OATP4C1 in Renal Handling of Remdesivir and its Nucleoside Analog GS-441524: The First Approved Drug for Patients with COVID-19

– Purpose. Remdesivir and its active metabolite are predominantly eliminated via renal route; however, information regarding renal uptake transporters is limited. In the present study, the interaction of remdesivir and its nucleoside analog GS-441524 with OATP4C1 was evaluated to provide the detailed information about its renal handling. Methods. We used HK-2 cells, a proximal tubular cell line derived from normal kidney, to confirm the transport of remdesivir and GS-441524. To assess the involvement of OATP4C1 in handling remdesivir and GS-441524, the uptake study of remdesivir and GS-441524 was performed by using OATP4C1-overexpressing Madin-Darby canine kidney II (MDCKII) cells. Moreover, we also evaluated the IC 50 and K i value of remdesivir. Results. The time-dependent remdesivir uptake in HK-2 cells was observed. The results of inhibition study using OATs and OCT2 inhibitors and OATP4C1 knockdown suggested the involvement of renal drug transporter OATP4C1. Remdesivir was taken up by OATP4C1/MDCKII cells. OATP4C1-mediated uptake of remdesivir increased linearly up to 10 min and reached a steady state at 30 min. Remdesivir inhibited OATP4C1-mediated transport in a concentration-dependent manner with the IC 50 and apparent K i values of 42 ± 7.8 μM and 37 ± 6.9 μM, respectively. Conclusions. We have provided novel information about renal handling of remdesivir. Furthermore, we evaluated the potential drug interaction via OATP4C1 by calculating the K i value of remdesivir. OATP4C1 may play a pivotal role in remdesivir therapy for COVID-19, particularly in patients with kidney injury.


INTRODUCTION
Remdesivir, a nucleotide analog that inhibits viral RNA-dependent RNA polymerase (RDRP), for treating adults and children hospitalized with severe coronavirus disease 2019 (COVID-19), was approved in the United States on May 1 and in Japan on May 7, 2020 (Fig. 1A).
Several randomized controlled trials (RCTs) were conducted to investigate the effect of remdesivir on patients with COVID-19 (1-4). Yokoyama et al. reported the efficacy of remdesivir for patients with COVID-19 by conducting a network meta-analysis (5). Since the risk of severe symptoms is markedly high in elderly patients with COVID-19, the safety information of therapeutic drugs is essential in addition to the management of the patients' symptoms. Serious adverse events were reported to occur among 23% of the patients who were administered remdesivir (6). One of the most common serious adverse events, whose mechanism remains unknown, is acute kidney injury (6). Thus, appropriate administration method, including drug dose and duration, as well as drug safety information, such as side effects, need to be further assessed.
To assess the safety of drugs, a detailed understanding of drug profiles, particularly pharmacokinetics, is necessary. The summary on compassionate use provided by European Medicines Agency (EMA) states that remdesivir is a substrate of organic anion transporting polypeptide (OATP) 1B1 and P-glycoprotein (P-gp), which regulate both beneficial and adverse effects of various drugs (7). Moreover, remdesivir interacts with various drug transporters and cytochrome P450s (CYPs) such as CYP3A4, OATP1B3, bile salt export pump (BSEP), multidrug resistance-associated protein (MRP) 4, and Na + -taurocholate cotransporting polypeptide (NTCP) (7).
In contrast, pharmacokinetic data among individuals with normal kidney function demonstrated that remdesivir and its active metabolite are predominantly (74%) eliminated via the renal route; however, information regarding renal uptake transporters is limited. In the overexpressing cell system, human organic anion transporters (OAT) 1 and 3 did not transport remdesivir and its major systemic metabolites, GS-704277 and the nucleoside analog GS-441524 ( Fig. 1B)   OATP4C1 is the only member of OATP family found to be predominantly expressed in the kidney. OATP4C1, which is localized in the basolateral membrane of the proximal tubule, plays a pivotal role in urinary secretion of cardiac glycosides (digoxin and ouabain), thyroid hormones (triiodothyronine [T 3 ] and thyroxine), cAMP, methotrexate, sitagliptin, estrone 3-sulfate, chenodeoxycholic acid, and glycocholic acid (8)(9)(10).
In the present study, we evaluated the interaction of remdesivir and its nucleoside analog GS-441524 with OATP4C1 to provide the detailed information on renal handling of remdesivir. We assume that the data we provided will contribute to the safety use of remdesivir for patients with COVID-19.

Materials
Remdesivir (Fig. 1A) and GS-441524 (Fig. 1B) were purchased from Selleck Chemicals (Houston, TX). All other chemicals were commercially available and were of the highest possible purity.

Cell culture
Human kidney proximal tubule cell line HK-2 (CRL2190) and Canine kidney cell line MDCKII (CRL2936) were purchased from ATCC. OATP4C1/MDCKII and mock cells, which were established in our laboratory, were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and G418 (0.5 mg/mL) according to our previous report (11). HK-2 cells were cultured in DMEM/Ham's F12 supplemented with 10% FBS and penicillin-streptomycin (100 U/mL). All cells were cultured under an atmosphere of 5% CO 2 and 95% air at 37°C.

Transport studies
The cellular uptake in monolayer cultures grown on 24-well plates was measured. After the first wash, cells were pre-incubated in Krebs-Henseleit (KH) buffer (118 mM NaCl, 23.8 mM NaHCO 3 , 4.83 mM KCl, 0.96 mM KH 2 PO 4 , 1.20 mM MgSO 4 , 12.5 mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulpho nic acid, 5.0 mM D-glucose, and 1.53 mM CaCl 2 , pH 7.4). Moreover, the cellular uptake was initiated by adding T 3 (with or without any of trial drugs) or remdesivir or GS-441524. At the indicated times, the uptake was terminated by replacing the uptake buffer with ice-cold KH buffer and then washing twice with ice-cold KH buffer. Concentration of T 3 or remdesivir or GS-441524 was measured via liquid chromatography/tandem mass spectrometry (LC/MS/MS). Furthermore, the cellular uptake was calculated by dividing the uptake amount by the protein count of the cells.
siRNA Transfection HK-2 cells were seeded in 24-well cell culture plates at a density of 5.0 × 10 4 cells/well with antibiotics-free DMEM/Ham's F12. Transfection of siRNAs was performed using Lipofectamine ® RNAiMAX Reagent (Thermo Fisher Scientific) 24 h after seeding. SLCO4C1 siRNA (SASI_Hs01_00203727) and negative control (MISSION ® siRNA Universal Negative Control #SIC001) were obtained from Sigma-Aldrich (St. Louis, MO). Each siRNA diluted in Opti-MEM I Reduced Serum medium and Lipofectamine ® RNAiMAX Reagent were mixed gently and incubated at room temperature for 5 min. These mixtures were added to each well. The cells were incubated for 48 h at 37°C under 5% CO 2 and then used for experiments.

RT-PCR
Total RNA was isolated from the cultured cells using TriPure™ Isolation Reagent (Sigma-Aldrich), and cDNA was prepared from total RNA using ReverTra Ace ® qPCR RT Master Mix (TOYOBO, Osaka, Japan) according to the manufacturer's protocol. Quantitative-PCR was performed with KAPA SYBR Fast qPCR Kit (NIPPON Genetics Co., Ltd., Tokyo, Japan) using 20 ng of cDNA. Primer sequences are shown in the supporting information (Supplementary Table  1).
Amplification and detection for Quantitative-PCR were carried out on a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA). The relative mRNA expression was determined by the 2-∆∆ Ct method. HPRT was used as a housekeeping gene to normalize the relative expression level in samples.

Inhibitory effects of remdesivir
We calculated IC 50 values of remdesivir using a nonlinear least-squares regression analysis of the competition curves using the software KaleidaGraph (Synergy Software Inc., Reading, PA). (Eq. 1). Then, IC 50 values were converted to absolute inhibition constant (K i ) according to previous report (12) (Eq. 2). The following equation was used: where V is the transport amount (% of control), [I] is the inhibitor concentration, [S] is the substrate concentration, K m is the Michaelis-Menten constant, and n is the Hill coefficient.

Statistical analysis
Data are expressed as mean ± standard error (S.E.). Multiple statistical comparisons were made by the one-way analysis of variance (ANOVA) followed by Tukey's test. When appropriate, the differences between groups were tested for significance using the unpaired Student's t-test. Statistical significance was indicated by p values less than 0.05.

Remdesivir and GS-441524 uptake by HK-2 cells
HK-2 cells represent the proximal tubular cell line derived from normal kidney. We used these cells to confirm the accumulation of remdesivir and GS-441524 in renal proximal tubular cells. The results indicated that both remdesivir and GS-441524 uptake reached the steady state at 20 min ( Fig. 2A and 2B). At this incubation time point, the uptake of remdesivir and GS-441524 at 37°C was 20 and 2.2 pmol/mg protein, respectively. Corresponding uptake values at 4°C were 4.8 and 1.9 pmol/mg protein for remdesivir and GS-441524, respectively. Remdesivir uptake at 37°C was markedly increased comparing to the uptake at 4°C. Remdesivir uptake by HK-2 cells increased linearly up to 3 min; however, GS-441524 uptake by HK-2 cells did not increase in time-dependent manner.

Search of the responsible transporter for remdesivir uptake
Based on the results presented in Figure 1, we evaluated the remdesivir uptake by HK-2 using transporter inhibitors for OATs (p-aminohippuric acid (PAH) and probenecid) and OCT2 (ranitidine). The result revealed that the remdesivir uptake by HK-2 cells was not significantly inhibited by OAT and OCT inhibitors (Fig. 3A). In addition, we evaluated the influence of siRNA-mediated silencing of OATP4C1 on remdesivir uptake. The results showed that the uptake of remdesivir decreased by 50% in HK-2 cells treated with OATP4C1-siRNA (Fig. 3B). OATP4C1 mRNA level significantly decreased to 0.14-fold under the same condition (Fig. 3C). Thereafter, we also assessed the mRNA expression of organic anion transporters including OATP4C1 in HK-2 cells. The results indicated that only OATP4C1 expression was observed in HK-2 cells in our laboratory (Suppl. Fig. 1). Our HK-2 cells exhibited a time-dependent uptake of T 3 (a typical substrate of OATP4C1) (Suppl. Fig. 2). Thus, we decided to perform the remdesivir uptake study using OATP4C1 expressing MDCKII cells.

Time-dependent uptake of remdesivir and GS-441524 by OATP4C1
We examined the uptake of remdesivir and GS-441524 by OATP4C1 in OATP4C1overexpressing MDCKII cells. Remdesivir was taken up by OATP4C1/MDCKII cells more rapidly than the mock cells (Fig. 4A). OATP4C1-mediated uptake of remdesivir reached a steady state at 30 min (Fig. 4A). At this incubation time point, the uptake values of remdesivir by mock (vector transfected) cells and OATP4C1/MDCKII cells were 4.3 and 5.1 pmol/mg protein, respectively (Fig. 4A), and the OATP4C1-mediated uptake of remdesivir increased linearly up to 10 min (Fig. 4A); however, OATP4C1-mediated uptake of GS-441524 was not observed (Fig. 4B).

Inhibitory effects of remdesivir and GS-441524 on OATP4C1-mediated transport
To elucidate the potential drug interactions via OATP4C1, we performed the inhibition study of remdesivir and GS-441524. Herein, the inhibitor concentrations were set much higher than those used in clinics to avoid our overlooking the interaction (evaluated concentrations are 1~200 μM, 200 × Cmax (unbound) of remdesivir is 218.4 μM based on the Cmax of 5440 ng/ml (loading dose) and 12 % fraction unbound).
Moreover, the OATP4C1-mediated transport was inhibited by remdesivir, but not by GS-441524 ( Fig. 5A and 5B). Thereafter, we evaluated the IC 50 and apparent K i values of remdesivir (Eq. 1 and Eq. 2, see Materials and Methods), and observed that remdesivir inhibited OATP4C1-mediated transport in a concentration-dependent manner (Fig. 5A). The IC 50 and apparent K i values of remdesivir were 42 ± 7.8 μM and 37 ± 6.9 μM, respectively.

DISCUSSION
Remdesivir is contraindicated in patients with severe renal impairment; however, presently, no dose modification is recommended in patients with mild and moderate renal impairment. The pharmacokinetics data regarding the elimination and excretion of remdesivir is as follows (the results from Study GS-US-399-4231). Majority of the radioactive dose was recovered from urine (approximately 74%); the predominant species detected in urine were GS-441524 (49%), followed by remdesivir (10%) and other metabolites, accounting for 6% of total radioactive dose (each less than 2%). Furthermore, M14 accounted for 12% of the radioactive dose in feces, and all other metabolites were in trace amounts, accounting for 1% of total radioactivity (each less than 0.5%) (7). Thus, remdesivir and its major metabolites were excreted through urine, and based on the preclinical studies using [ 14 C]remdesivir (7), the detailed understanding of its renal transport was required. Remdesivir interacts with various drug transporters and cytochrome P450s (CYPs) (7). It is a substrate for OATP1B1 and P-gp (Study AD-399-2007) (7). Moreover, it also inhibits OATP1B1 and OATP1B3 with IC 50 values of 2.8 and 2.1 μM, respectively (Study AD-399-2005) (7) as well as BSEP-, MRP4-, and NTCP-mediated transport, with IC 50 values of 22, 5.1, and 72 μM, respectively (7); however, only OAT1 and OAT3 were assessed in the overexpressing cell system among renal uptake transporters, and these transporters did not transport remdesivir and its major systemic metabolites, GS-704277, and the nucleoside analog GS-441524 (Study PC-399-2020; IB Edition 5 21 February 2020 Section 3.3.6.2) (7).
In our present study, we evaluated the interactions of remdesivir and GS-441524 with renal transporters by using HK-2 and OATP4C1 overexpressing cell lines. From the results, we observed the time-and temperature-dependent remdesivir uptake in HK-2 cells ( Fig. 2A); however, time-and temperature-dependent increase in GS-441524 uptake was not observed (Fig. 2B). These results suggested the involvement of transporter molecules in the uptake of remdesivir. Based on the results of inhibition study using OATs and OCTs inhibitors, we speculated the involvement of renal drug transporter OATP4C1 (Fig. 3A). The effect of SLCO4C1 knockdown on remdesivir uptake in HK-2 cells and the expression profile of renal organic anion transporters strongly supported this hypothesis (Fig. 3B, 3C, and Suppl. Fig. 1).
Furthermore, we evaluated the remdesivir and GS-441524 uptake by OATP4C1 using overexpressing system. Our results indicated that remdesivir is the preferred substrate of OATP4C1 compared to GS-441524 ( Fig. 4A and 4B). Since remdesivir is a relatively hydrophilic compound, which has both carboxy and hydroxy groups, it theoretically requires a membrane transporter for its renal excretion (12,13); however, the half-life of remdesivir was short in blood and it was quickly converted into the active metabolite in peripheral blood mononuclear cells within 2 hours after administration (13). As aforementioned, the predominant metabolite detected in urine was GS-441524 (49%), followed by remdesivir (10%) (7). Further studies are required to elucidate the impact of OATP4C1-medated transport for remdesivir in clinical settings. In contrast, no information is available about remdesivir use among individuals with eGFR less than 30 ml/min/1.73 m 2 (14). Based on the toxicology studies in rhesus monkeys, remdesivir revealed kidney injury with 5~20 mg/kg administration, which is higher than the clinical dose (7). Based on the available data from several clinical trials, no significant renal adverse events were reported (1,15); however, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to acute kidney injury in up to 20%-40% of critically ill patients; herein, the elevation of both remdesivir and its metabolite concentration in plasma should be considered. Previously, our group reported that the expression of renal drug transporters including OATP4C1 were reduced in the model animal with renal failure (16). Since we revealed that remdesivir was a substrate of OATP4C1, the elevation of remdesivir concentration in plasma may be a result of OATP4C1 dysfunction in the patients with renal failure.
Furthermore, we performed an inhibition study to provide information on potential drug interaction via OATP4C1 by remdesivir and GS-441524. Our data revealed that remdesivir inhibited OATP4C1-mediated transport in a concentration-dependent manner (Fig. 5A). The apparent K i value of remdesivir was 37 ± 6.9 μM, which is relatively higher than the clinical concentration (4~9 μM as total) (Fig. 5A). Thus, based on these results, remdesivir may not affect the OATP4C1-mediated drug transport in patients with normal kidney function; however, drug interaction via OATP4C1 caused by remdesivir may be observed in patients with renal impairment due to elevated remdesivir accumulation in the body.

CONCLUSIONS
We have provided the novel information about renal handling of remdesivir. Additionally, we evaluated the potential drug interaction via OATP4C1 by calculating the IC 50 value of remdesivir. OATP4C1 may play a pivotal role in remdesivir therapy for COVID-19, particularly in patients with kidney injury.