Recent Advances in Synthetic Drugs and Natural Actives Interacting with OAT3

Organic anion transporter 3 (OAT3) is predominantly expressed in the kidney and plays a vital role in drug clearance. Consequently, co-ingestion of two OAT3 substrates may alter the pharmacokinetics of the substrate. This review summarizes drug–drug interactions (DDIs) and herbal–drug interactions (HDIs) mediated by OAT3, and inhibitors of OAT3 in natural active compounds in the past decade. This provides a valuable reference for the combined use of substrate drugs/herbs for OAT3 in clinical practice in the future and for the screening of OAT3 inhibitors to avoid harmful interactions.


Introduction
The two primary superfamilies, ATP binding cassette (ABC) and solute carrier (SLC), play a crucial role in the absorption, distribution, and elimination of various endogenous substances such as hormones and signal molecules, as well as exogenous substances and drugs [1,2]. Generally, SLC transporters mediate the influx of substances from the blood into the epithelium, while ABC transporters mediate the efflux of substances [3][4][5]. The SLC22 family comprises six primary subfamilies: organic anion transporter (OAT), OATlike, OAT-related, organic cation transporter (OCT), organic cation and carnitine transporter (OCTN), and OCT/OCTN-related [6,7].
OAT3 is considered the primary transporter in OAT due to its broad substrate specificity. OAT3 (encoded by SLC22A8) is widely distributed in the kidney, liver, choroid plexus [8], olfactory mucosa, brain [9], retina, and placenta [10], but it is pre-dominantly expressed on the basolateral membrane of renal tubular cells [11,12]. OAT3 utilizes dicarboxylate (e.g., α-Ketoglutarate) to exchange organic anions, with the concentration gradient as the driving force [13]. It first transports organic anions (OAs) from the blood to the proximal tubules through the basolateral membrane and then discharges OAs into the urine through the apical membrane of tubular epithelial cells, reabsorbing specific compounds in the glomerular filtrate back into the internal circulation [14,15].
OAT3 plays a crucial role in the uptake, distribution, and excretion of various endogenous/exogenous substances, but current research has primarily focused on the interactions between OAT3 and clinical drugs [16][17][18]. When two or more drugs are taken simultaneously, OAT3 becomes a target of drug competition [19,20], potentially altering the toxicity [21], pharmacokinetics [22], and function of these drugs and resulting in potential drug-drug interactions (DDIs) [23,24]. However, when herbal medicines and OAT3 substrate drugs are taken together, they may lead to severe herbal drug interactions (HDIs), such as liver injury [25], increased clotting time, headache, insomnia, and even coma and death [26,27]. Research has shown that not all of the interactions mentioned above are negative. For example, enalaprilat and quinaprilat are two types of antihypertensive drugs. When used in combination with some synthetic drugs or flavonoids, their transport through OAT3 is inhibited, leading to synergistic blood pressure reduction [28,29]. OAT3 has a significant role in DDIs and HDIs [13], with effects that can be either beneficial or adverse. In this review, we summarize the DDIs and HDIs mediated by OAT3, as well as OAT3 inhibitors in natural products over the past decade. Through this review, we can predict and avoid DDIs/HDIs mediated by OAT3.
Imipenem is an antibiotic with antibacterial effects, but when administered alone, it is rapidly degraded by dehydropeptidase-1 or renal dipeptidase (also termed DPEP1) [44]. Cilastatin and imipenem are both substrates of OAT3. When administered together intravenously, the plasma concentration-time curve (AUC) for imipenem is significantly increased by 1.65-fold, while the plasma/serum concentration half-time (t 1/2β ) of imipenem increased by 2.35-fold. Cilastatin also inhibits the uptake of imipenem mediated by OAT3 in the kidney, thereby reducing the hydrolysis of and acting as a complement to imipenem [35,45,46].
In addition to its role in DDIs, OAT3 inhibition has been shown to have a renoprotective effect against nephrotoxic drugs [47]. For example, diclofenac [48], a commonly used nonsteroidal anti-inflammatory drug (NSAID), can cause kidney damage [49]. However, when diclofenac is co-administered with cilastatin, an OAT3 inhibitor, the AUC 0-12h of diclofenac increases by 35.4% and its plasma clearance (CL p ) significantly decreases. Coadministration of cilastatin with diclofenac acyl glucuronide, a metabolite of diclofenac, leads to an increase in the concentration of diclofenac acyl glucuronide in the plasma by 46.7%, but a slight decrease in AUC 0-12h of diclofenac acyl glucuronide in the kidney by 18.0%. Moreover, diclofenac acyl glucuronide but not diclofenac exhibited OAT-dependent cytotoxicity and was identified as an OAT1/3 substrate. The accumulation of diclofenac acyl glucuronide in primary proximal tubule cells (RPTCs) is reduced, and the cytotoxicity caused by diclofenac acyl glucuronide is also reduced [36].
Methotrexate (MTX) is a drug used for the high-dose treatment of various malignant tumors or the low-dose treatment of rheumatoid arthritis and psoriasis [50,51]. Delayed elimination of MTX can lead to severe toxicity, such as bone marrow suppression, mucositis, and kidney damage [37]. Kidney clearance is the main pathway for MTX clearance (65-80%). Proton pump inhibitors (PPIs) inhibit the hOAT3-mediated uptake of MTX with IC 50 values of 0.40-5.5 µM [37]. Rhein, the main metabolite of diacerein, markedly inhibited MTX accumulation in rat kidney slices and hOAT3-HEK293 cells, indicating that OAT3 is involved in DDIs in the kidney. When the two drugs were co-administered orally, the maximal plasma/serum concentration (C max ) and AUC of MTX were increased by 2.5-and 4.4-fold, respectively, and the CL of MTX was decreased by 66.7%. When the two drugs were co-administered intravenously, the t 1/2β of MTX was prolonged by 86.7%, the CLP was decreased by 57.6%, and the AUC was increased by 183.4%. Rhein alleviated MTXinduced renal toxicity in vivo mainly by inhibiting OAT3 to reduce the renal clearance of MTX [38]. Masahiro Iwaki et al. found that seven NSAIDs-Glu exhibit a concentrationdependent inhibitory effect on MTX uptake through OAT3, with diclofenac-Glu having the most effective inhibitory ability (IC 50 = 3.17 µM) [39]. In addition to the above-mentioned drugs, tranilast, an anti-allergic drug, can inhibit MTX transport.
OAT3 renal sections and HEK293T-OAT3 cell uptake experiments have shown that tranilast can inhibit MTX uptake by OAT3. Pharmacokinetic studies in rats have shown that when MTX (5 mg/kg) is administered orally alongside tranilast (10 mg/kg), the C max and AUC 0-24h of MTX increased to 2.14-and 1.46-fold, respectively, while CL z/f and V z/f decreased by 24.90% and 39.23%, respectively [40].
Quinapril is an angiotensin-converting enzyme (ACE) inhibitor used to treat hypertension and congestive heart failure. In a pharmacokinetic study on rats, co-administration of quinapril (3 mg/kg) with gemcabene (30 mg/kg) resulted in a 40% decrease in the urinary excretion of quinaprilat (quinaprilat, the metabolite of the quinapril, is also the substrate of OAT3) and a 53% increase in the AUC 0-24h of quinaprilat, leading to a reduction in blood pressure. Subsequent studies discovered that gemcabene inhibited quinaprilat uptake by hOAT3 and rOat3 at IC 50 values of 35 and 48 µM. Moreover, gemcabene acylglucuronide, the major metabolite of gemcabene glucuronidation, also inhibited the hOAT3and rOat3-mediated uptake of quinaprilat at IC 50 values of 197 and 133 µM, respectively. This indicated the mechanism by which concomitant intake of gemcabene with quinapril can reduce blood pressure [28]. In another study, Ni et al. investigated the effects of several common drugs on the OAT3-mediated uptake of enalaprilat (another oral ACE). Benzbromarone was found to be the most effective inhibitor of OAT3 (IC 50 = 0.14 µM), while diclofenac sodium was the weakest inhibitor (IC 50 = 6.13 µM) [29].
One study showed that concurrent treatment with mizoribine and bezafibrate can result in rhabdomyolysis. It was suggested that both bezafibrate and mizoribine are substrates of OAT3, and when bezafibrate was co-administered orally with mizoribine, the t 1/2β of bezafibrate increased to 1.39-fold, and the renal clearance (CL r ) decreased to 0.81-fold. However, when bezafibrate was co-administrated with mizoribine intravenously, the AUC and t 1/2β of bezafibrate increased to 1.29-and 1.25-fold, respectively. This study also suggested that mizoribine can competitively inhibit the uptake of bezafibrate by OAT3 [41].
Similarly, in a study in which benzylpenicillin and acyclovir were co-administered intravenously in rats, the cumulative urinary excretion of acyclovir decreased by 45% and CL R decreased by 44%, while the t 1/2β of acyclovir increased by 1.9-fold. This indicates that benzylpenicillin can inhibit the renal excretion of acyclovir by inhibiting OAT3 [42].
Wen et al. found that when piperacillin and tazobactam were administered simultaneously, both CL p and CL R of tazobactam were decreased, and the AUC, t 1/2β , and k m of tazobactam were increased to 2.15-, 1.24-, and 1.56-fold, respectively. Moreover, piperacillin can competitively inhibit the uptake of tazobactam mediated by OAT3 [43].

OAT3 and Herb-Drug Interactions (HDIs)
Natural medicines containing flavonoids and other functional components, particularly polyphenols, are increasingly used in clinical therapy [52]. In the elderly population with chronic diseases such as hypertension, hyperlipidemia [53], and hyperuricemia, it is common to use prescription drugs, over-the-counter drugs, and natural products simultaneously [47,54]. A survey showed that 78% of elderly respondents take both prescription drugs and dietary supplements, and 32.6% of them experience potential adverse drug reactions when they were taking a combination of herbs and other drugs [55]. Some herbs may directly cause organ toxicity, alter the pharmacokinetics or efficacy of prescription drugs mediated by OAT3, or inhibit enzymes involved in drug metabolism [26].
Purarin (PUR) is an isoflavone component extracted from Pueraria lobata [56]. When PUR and MTX are orally administered in combination, the C max , AUC, and t 1/2β of MTX are increased by 79%, 74%, and 70%, respectively. When PUR and MTX are co-administered intravenously, the AUC and t 1/2β of MTX increase by 59% and 37%, respectively, and a 37% decrease in CL p was observed. PUR also simultaneously inhibits MTX uptake in rat kidney slices and HEK293-OAT3 cells. Therefore, the combined administration of PUR can inhibit OAT3 and enhance MTX exposure (Table 2) [57].  Oral administration of rhubarb extract 5 g crude drug/kg/day and furosemide 10 mg/kg for 7 days in rats -AUC 0-t ↑ [62] Total body clearance (CL/F); area under the concentration-time curve (AUC); maximum serum concentration (C max ); plasma clearance (CL p ); plasma/serum concentration half-time (t 1/2β ); renal clearance (CL R ); ↓, reduced level; ↑, increased level; -, data were not reported.
Steviol glucuronide is the major metabolite of steviol, and its uptake is primarily mediated by OAT3 (Wang et al.). Inhibition studies have shown that three drugs (with IC 50 values between 2.92 and 9.97 µM) can inhibit steviol glucuronide uptake mediated by OAT3 in a concentration-dependent manner, and may also alter its renal clearance [58]. Steviol acyl glucuronide, the main circulating metabolite of steviol glycosides after oral administration, is also a substrate of OAT3 [63]. Therefore, if the activity of OAT3 is inhibited, it may alter the pharmacokinetic characteristics of steviol acyl glucuronide. Zhou et al. showed that probenecid and glimepiride inhibit hOAT3/rOat3-mediated steviol acyl glucuronide transport in a concentration-dependent manner (IC 50 < 5 µM) ( Table 2). When administered in combination with probenecid or glimepiride, the C max of steviol acyl glucuronide was increased to 10.8-or 9.7-fold, respectively. The AUC 6-8h of steviol acyl glucuronide was increased to 2.9-or 2.5-fold, respectively. Therefore, people with impaired renal function should be cautious when taking steviol glycoside products [59].
The increased renal transport of imipenem mediated by OAT3 can lead to certain renal toxicity. Apigenin, a flavonoid compound widely distributed in traditional Chinese medicine, is a potent OAT3 inhibitor [64]. In cases with the combined administration of apigenin and imipenem, the survival rate of hOAT3-HEK93 cells significantly increased, and the IC 50 value of imipenem within the cells increased to 8.1-fold. At the same time, the AUC 0-6h of imipenem increased by 46%, while the CL p and CL r of imipenem decreased by 29% and 52%, respectively. The intracellular accumulation of imipenem decreased, thereby partially reducing the cytotoxicity of imipenem. These results indicate that the inhibition of apigenin on hOAT3 can protect the cytotoxicity induced by imipenem in vitro. Therefore, apigenin can be used as a potential clinical drug to alleviate adverse renal reactions caused by imipenem [60].
Natural drugs containing flavonoids are often taken concomitantly with prescribed or over-the-counter drugs, which may lead to potential interactions [65]. For instance, Ni et al. reported the inhibitory effects of various flavonoids with distinct structures on the activity of OAT3 transport drugs [66]. Among these flavonoids, galangin exhibited the strongest inhibitory effect on the activity of OAT3 with an IC 50 value of 0.03 µM, while myricetin exhibited the weakest inhibitory effect, with an IC 50 value of 22.58 µM (Table 2) [29].
Lu et al. [61] reported that dichloromethane extract from Juncus effusus can alter the pharmacokinetics of furosemide (FS), an OAT3 substrate drug, in rats. The AUC 0-t of FS was increased by 80% and 55% after oral or intravenous administration of Juncus effusus (D), respectively. Therefore, caution is needed to avoid potential side effects resulting from the interaction between OAT3 substrate drugs and Juncus effusus (D). Ma et al. observed a 32% and 52% increase in the AUC 0-t of FS following single or multiple dose co-administration of FS and rhubarb extract, respectively [62].

Natural Bioactive Inhibitors of OAT3
Many natural bioactive compounds, including flavonoids and their metabolites such as sulfates and glucuronides, have been identified as potent inhibitors and/or substrates of OAT3 [67,68]. The study of OAT3 inhibitors can help us predict and avoid potential OAT3mediated DDIs/HDIs [69], and these inhibitors may also have potentially beneficial effects on renal diseases, such as AAI-induced kidney disease [70]. Table 3 summarizes the natural bioactive compounds identified as OAT3 inhibitors in the last decade. These compounds are classified into nine groups: phenolic acids, flavonoids, alkaloids, anthraquinones, phenols, phenylpropanoids, terpenoids, phenanthrenoids, and others.  Total body clearance (CL/F), area under the concentration-time curve (AUC), maximum serum concentration (C max ), plasma clearance (CL p ), plasma/serum concentration half-time (t 1/2β ), renal clearance (CL R ), ↓ reduced level, ↑ increased level. H represents hexane extract, D for dichloromethane extract, B for n-Butanol extract, and A for aqueous phase extract.

Phenolic Acids
Salvia miltiorrhiza has been used to treat cardiovascular diseases, but its biochemical mechanisms are still unclear. However, studies by Wang et al. have shown that six hydrophilic components from Salvia miltiorrhiza, tanshinol, rosmarinic, lithospermic acid, salvianolic acid A, salvianolic acid B, and protocatechuic acid (Figure 1), have significant inhibitory effects on substrate uptake mediated by mOAT3. Among these, lithospermic acid (K i = 31.3 µM), rosmarinic (K i = 4.3 µM), and salvianolic acid A (K i = 21.3 µM) produce virtually complete inhibition [79]. Wang et al. investigated the effects of nine compounds extracted from natural diets and herbs on hOAT3-mediated uptake. The results indicated that four of them significantly inhibited the transport of hOAT3, with 1,3-dicaffeoylquinic acid and ginkgolic acid (17:1) exhibiting 41% inhibition, while a 30-35% reduction in hOAT3 mediated uptake was observed in response to 1,5-dicaffeoylquinic acid and ginkgolic acid (15:1) [72].
Wang et al. investigated the effects of nine compounds extracted from natural diets and herbs on hOAT3-mediated uptake. The results indicated that four of them significantly inhibited the transport of hOAT3, with 1,3-dicaffeoylquinic acid and ginkgolic acid (17:1) exhibiting 41% inhibition, while a 30-35% reduction in hOAT3 mediated uptake was observed in response to 1,5-dicaffeoylquinic acid and ginkgolic acid (15:1) [72].
Wang et al. evaluated 22 substances isolated from Semen cassiae, and 6 anthraquinones significantly (IC50 < 10 µM) inhibited OAT3-mediated transport. In vivo experimental results demonstrated that Semen cassiae extract can nearly eliminate the alterations in renal histology induced by mercury chloride in rats. With docking and validation of OAT3 inhibitors, it may become a drug for treating Hg-induced renal injury [75].
Wang et al. evaluated 22 substances isolated from Semen cassiae, and 6 anthraquinones significantly (IC50 < 10 µM) inhibited OAT3-mediated transport. In vivo experimental results demonstrated that Semen cassiae extract can nearly eliminate the alterations in renal histology induced by mercury chloride in rats. With docking and validation of OAT3 inhibitors, it may become a drug for treating Hg-induced renal injury [75].  Wang et al. evaluated 22 substances isolated from Semen cassiae, and 6 anthraquinones significantly (IC 50 < 10 µM) inhibited OAT3-mediated transport. In vivo experimental results demonstrated that Semen cassiae extract can nearly eliminate the alterations in renal histology induced by mercury chloride in rats. With docking and validation of OAT3 inhibitors, it may become a drug for treating Hg-induced renal injury [75].

Phenols
As shown in Figure 5, two phenolic compounds, 9-Dehydroxyeurotinone and 2-O-Methyl-9-dehydroxyeurotinone from extracts of Semen cassiae are potent inhibitors of OAT3 (IC50 < 10 µM). In addition, 9-dihydroxyurotinone is a noncompetitive inhibitor of OAT3 [75]. Tatsuya et al. simultaneously injected 6-CF (1 mg/kg) and EGCG (60 mg/kg) intravenously in rats and detected the concentration of 6-CF in the blood and urine. Their results demonstrated that simultaneous injection elevated the plasma concentration of 6-CF by 8fold after 1 h, while the AUC0-1h was significantly increased to 2.2-fold. In contrast, EGCG significantly reduced the CLr of 6-CF, indicating that EGCG can inhibit OAT3 [76].

Terpenes
There is currently only one terpene compound, 3-Oxo-16α-hydroxy-olean-12-en-28β-oic acid, which was isolated from Eclipta prostrata L. It was identified as an OAT3 Tatsuya et al. simultaneously injected 6-CF (1 mg/kg) and EGCG (60 mg/kg) intravenously in rats and detected the concentration of 6-CF in the blood and urine. Their results demonstrated that simultaneous injection elevated the plasma concentration of 6-CF by 8-fold after 1 h, while the AUC 0-1h was significantly increased to 2.2-fold. In contrast, EGCG significantly reduced the CLr of 6-CF, indicating that EGCG can inhibit OAT3 [76].

Phenols
As shown in Figure 5, two phenolic compounds, 9-Dehydroxyeurotinone and 2-O-Methyl-9-dehydroxyeurotinone from extracts of Semen cassiae are potent inhibitors of OAT3 (IC50 < 10 µM). In addition, 9-dihydroxyurotinone is a noncompetitive inhibitor of OAT3 [75]. Tatsuya et al. simultaneously injected 6-CF (1 mg/kg) and EGCG (60 mg/kg) intravenously in rats and detected the concentration of 6-CF in the blood and urine. Their results demonstrated that simultaneous injection elevated the plasma concentration of 6-CF by 8fold after 1 h, while the AUC0-1h was significantly increased to 2.2-fold. In contrast, EGCG significantly reduced the CLr of 6-CF, indicating that EGCG can inhibit OAT3 [76].

Others
Lu et al. [61] evaluated the effects on OAT3 of 172 extracts and showed that 14 were strong inhibitors of OAT3 (IC50 between 0.3 and 4.8 µg/mL). Generally speaking, the n-Butanol extract from plants is more effective. For example, the n-Butanol extract IC50 value for Anchusa azurea, Symphytum asperum, and Echium russicum are 0.343, 0.406, 0.460, respectively.
With regards to the effects of natural bioactive compounds, most studies used the IC50 value as an indicator for OAT3 inhibition. However, depending on the potential differences in protein binding and tissue-specific concentrations, IC50 alone may not be sufficient to indicate that exposure to the active compound can cause effective effects. Therefore, further in vivo studies are needed to verify the efficacy of these natural bioactive compounds.
In conclusion, when these substances are administered alongside other drugs or herbs, they may change the pharmacokinetics of the drug or herbal elimination and have

Others
Lu et al. [61] evaluated the effects on OAT3 of 172 extracts and showed that 14 were strong inhibitors of OAT3 (IC50 between 0.3 and 4.8 µg/mL). Generally speaking, the n-Butanol extract from plants is more effective. For example, the n-Butanol extract IC50 value for Anchusa azurea, Symphytum asperum, and Echium russicum are 0.343, 0.406, 0.460, respectively.
With regards to the effects of natural bioactive compounds, most studies used the IC50 value as an indicator for OAT3 inhibition. However, depending on the potential differences in protein binding and tissue-specific concentrations, IC50 alone may not be sufficient to indicate that exposure to the active compound can cause effective effects. Therefore, further in vivo studies are needed to verify the efficacy of these natural bioactive compounds.
In conclusion, when these substances are administered alongside other drugs or herbs, they may change the pharmacokinetics of the drug or herbal elimination and have

Others
Lu et al. [61] evaluated the effects on OAT3 of 172 extracts and showed that 14 were strong inhibitors of OAT3 (IC 50 between 0.3 and 4.8 µg/mL). Generally speaking, the n-Butanol extract from plants is more effective. For example, the n-Butanol extract IC 50 value for Anchusa azurea, Symphytum asperum, and Echium russicum are 0.343, 0.406, 0.460, respectively.
With regards to the effects of natural bioactive compounds, most studies used the IC 50 value as an indicator for OAT3 inhibition. However, depending on the potential differences in protein binding and tissue-specific concentrations, IC 50 alone may not be sufficient to indicate that exposure to the active compound can cause effective effects. Therefore, further in vivo studies are needed to verify the efficacy of these natural bioactive compounds.
In conclusion, when these substances are administered alongside other drugs or herbs, they may change the pharmacokinetics of the drug or herbal elimination and have certain clinical consequences, including reduced therapeutic efficacy or specific side effects caused by interactions. Therefore, caution should be exercised when administering multiple drugs involving the above substances.

Conclusions
OAT3-mediated DDIs/HDIs have both potential benefits and risks. On the one hand, they can enhance drug efficacy by prolonging its half-life, but on the other hand, they may lead to nephrotoxicity by increasing drug accumulation in the kidneys. Therefore, it is important to pay attention to the potential DDIs/HDIs mediated by OAT3 during drug/herb development and in clinical practice. In this regard, the identification of natural active OAT3 inhibitors, which are summarized into nine categories, can help predict and avoid potential interactions. A better understanding of the regulatory pathways involved can also lead to the development of new methods to alleviate or avoid DDIs/HDIs mediated by OAT3. While taking advantage of OAT3-mediated interactions can be beneficial in clinical treatment, it is also important to remain vigilant with respect to potential interactions with unreported drugs. The key role played by OAT3 in DDIs/HDIs has been confirmed, and this related review may also provide a reference for future clinical application of drugs as OAT3 substrates to avoid adverse effects caused by DDIs/HDIs. In addition, there is limited research on natural OAT3 inhibitors, with most studies limited to the in vitro level. The effects of and mechanisms of action for specific natural compounds on OTA3 inhibition in vivo require further research.
Author Contributions: Writing-original draft preparation, Y.C. and H.L.; writing-review and editing, Y.C., K.W. and Y.W.; funding acquisition, Y.W. and Y.C. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.