Substrate Of Organic Anion Transporter Research Articles

Potential Involvement of Organic Anion Transporters in Drug Interactions with Shuganning Injection, a Traditional Chinese Patent Medicine.

Traditional Chinese medicine injections have been widely used in China for the treatment of various diseases. Transporter-mediated drug-drug interactions are a major contributor to adverse drug reactions. However, the research on transporter-mediated Traditional Chinese medicine injection-drug interactions is limited. Shuganning injection is a widely used Traditional Chinese medicine injection for treating various liver diseases. In this study, we investigated the inhibitory effect of Shuganning injection and its four main ingredients (baicalin, geniposide, chlorogenic acid, and oroxylin A) on 9 drug transporters. Shuganning injection strongly inhibited organic anion transporter 1 and organic anion transporter 3 with IC50 values < 0.1% (v/v), and moderately inhibited organic anion transporter 2, organic anion transporting-polypeptide 1B1, and organic anion transporting-polypeptide 1B3 with IC50 values < 1.0%. Baicalin, the most abundant bioactive ingredient in the Shuganning injection, was identified as both an inhibitor and substrate of organic anion transporter 1, organic anion transporter 3, and organic anion transporting-polypeptide 1B3. Oroxylin A had the potential to act as both an inhibitor and substrate of organic anion transporting-polypeptide 1B1 and organic anion transporting-polypeptide 1B3. In contrast, geniposide and chlorogenic acid had no significant inhibitory effect on drug transporters. Notably, Shuganning injection markedly altered the pharmacokinetics of furosemide and atorvastatin in rats. Using Shuganning injection as an example, our findings support the implementation of transporter-mediated Traditional Chinese medicine injection-drug interactions in the development of Traditional Chinese medicine injection standards.

Plasma Protein Binding Refinement of the Extended Clearance Classification System: Subclasses for Predicting Hepatic Uptake or Renal Clearance for Classes 1B and 3B.

The Extended Clearance Classification System (ECCS) was established to facilitate the timely anticipation of clearance rate determination according to the physicochemical characteristics of a given compound and invitro passive membrane permeability. Unfortunately, distinguishing between renal and hepatic uptake clearance mechanisms using ECCS class 3B is not possible. We determined the effects of plasma protein binding (PPB) on major hepatic organic anion transporting polypeptide (OATP) and renal organic aniontransporter (OAT) substrates. A modified ECCS could predict when renal or hepatic uptake mechanisms were the main clearance rate determinants (accounting for ≥ 70% of total clearance). A dataset of 66 human OATP and 41 OAT substrates was analyzed to determine the effect of PPB. A total of 63 acidic and zwitterionic, and high-molecular-weight (MW > 400Da) compounds, including 50 drugs in ECCS classes 1B and 3B, were reanalyzed considering their PPB. Statistical analyses revealed that hepatic uptake transporter (OATP1B1 and OATP1B3) substrates possess a high PPB rate of ≥ 90%, whereas OAT1 and/or OAT3 substrates possess low PPB rates of < 90%. By analyzing the 63 drugs on the basis of their PPB, the active hepatic uptakes of acids and zwitterions were determined to be the main clearance mechanisms, with PPB ≥ 90%, whereas renally eliminated drugs exhibited limited PPB (< 90%). Therefore, PPB is an effective parameter for defining clearance rate determination for acidic and zwitterionic drugs with high MWs. Using PPB as an additional parameter in ECCS, clearance mechanisms for class 1B and 3B compounds can be predicted, and OATP and OAT substrates may be readily distinguished.

Mechanistic Study on the Effect of Renal Impairment on the Pharmacokinetics of Vildagliptin and its Carboxylic Acid Metabolite.

To clarify the mechanism of renal impairment leading to different degrees of increased plasma exposure to dipeptidyl peptidase 4 inhibitor vildagliptin and its major metabolite, M20.7. The 5/6 nephrectomized (5/6 Nx) rat model, to simulate chronic renal failure (CRF) patients, combined with kidney slices and transporter studies in vitro were used to assess this pharmacokinetic differences. After intragastric administration to 5/6 Nx rats, vildagliptin showed increased plasma levels by 45.8%, and M20.7 by 7.51 times, which was similar to patients with severe renal impairment. The recovery rate of M20.7 in urine and feces increased by less than 20%, showing limited effect of renal impairment on vildagliptin metabolism. In vitro studies found M20.7 to be the substrate for organic anion transporter 3 (OAT3). However, the active uptake of M20.7 in renal slices showed no difference between the 5/6 Nx and normal rats. In OAT3 overexpressed cells, the protein-bound uremic toxins, 3-carboxy-4-methyl-5propyl-2-furanpropionate (CMPF), hippuric acid (HA) and indoxyl sulfate (IS), which accumulate in CRF patients, inhibited M20.7 uptake with IC50 values of 5.75, 29.0 and 69.5μM respectively, far lower than plasma concentrations in CRF patients, and showed a mixed inhibition type. The large increase in plasma exposure of M20.7 could be attributed to the accumulation of uremic toxins in CRF patients, which inhibited OAT3 activity and blocked renal excretion of M20.7, while vildagliptin, with high permeability, showed a slight increase in plasma exposure due to reduced glomerular filtration.

Assessing the Effect of Organic Anion Transporter Inhibition on Circulating Pyridoxic Acid, an Endogenous Transporter Biomarker, using Physiologically‐based Pharmacokinetic Modeling

Transporter‐mediated renal secretion plays a critical role in the systemic clearance of ~30% of the Food and Drug Administration (FDA)‐approved drugs and is attributed to clinically significant drug‐drug interactions (DDIs). For example, organic anion transporters (OATs) are inhibited by perpetrator drugs (e.g., probenecid), leading to higher circulating levels of OAT substrates (e.g., furosemide).1 Recently, OAT inhibition has been shown to be associated with increased plasma levels of endogenous substrates, e.g., pyridoxic acid (PDA) and homovanillic acid, which can serve as potential OAT biomarkers.2 The objective of our study was to develop and validate a physiologically‐based pharmacokinetic (PBPK) model of PDA to allow prospective evaluation of the magnitude of potential DDI of an OAT inhibitor‐substrate pair. Probenecid and furosemide were used as selective inhibitor and substrate of OATs, respectively. Physicochemical properties and in vitro data pertaining to the pharmacokinetics of probenecid and PDA were collated from literature. A PBPK model was first constructed to predict the plasma concentration‐time profiles of probenecid after oral administration using Simcyp (v20). Then, utilizing the fraction secreted versus filtered as well as the unbound fraction (fu = 0.092) data of PDA,2 the total elimination and synthesis rates of PDA at steady‐state were used to develop a baseline PBPK model. The effect of OAT inhibition by probenecid on the PDA levels was estimated and the predicted area under the plasma concentration‐time profile (AUC), change in clearance, and the highest plasma concentration (Cmax) values were compared with the observed data.2 The model was able to capture the changes within 2‐fold of the clinical data. Using the predicted AUC profile of PDA and assuming 100% inhibition at Cmax, the magnitude of inhibition was estimated across different time points. The biomarker‐informed magnitude of inhibition showed significant correlation (r2 = 0.9336) with the observed change in furosemide concentrations in the presence of probenecid (Fig. 1). PDA‐informed PBPK model developed here can be used to prospectively predict the magnitude of OAT inhibition, which is particularly applicable to assessing DDI potential of investigational drugs or prescribed drugs in special populations with sparse samples, without the requirement of an exogenous probe substrate.

Assessment of the Effects of Inhibition or Induction of CYP2C19 and CYP2C9 Enzymes, or Inhibition of OAT3, on the Pharmacokinetics of Abrocitinib and Its Metabolites in Healthy Individuals.

Background and ObjectiveAbrocitinib is a Janus kinase 1-selective inhibitor for the treatment of moderate-to-severe atopic dermatitis. Abrocitinib is eliminated primarily by metabolism involving cytochrome P450 (CYP) enzymes. Abrocitinib pharmacologic activity is attributable to the unbound concentrations of the parent molecule and 2 active metabolites, which are substrates of organic anion transporter 3 (OAT3). The sum of potency-adjusted unbound exposures of abrocitinib and its 2 active metabolites is termed the abrocitinib active moiety. We evaluated effects of CYP inhibition, CYP induction, and OAT3 inhibition on the pharmacokinetics of abrocitinib, its metabolites, and active moiety.MethodsThree fixed-sequence, open-label, phase I studies in healthy adult volunteers examined the drug–drug interactions (DDIs) of oral abrocitinib with fluvoxamine and fluconazole, rifampin, and probenecid.ResultsCo-administration of abrocitinib with fluvoxamine or fluconazole increased the area under the plasma concentration–time curve from time 0 to infinity (AUCinf) of the unbound active moiety of abrocitinib by 91% and 155%, respectively. Co-administration with rifampin decreased the unbound active moiety AUCinf by 56%. The OAT3 inhibitor probenecid increased the AUCinf of the unbound active moiety by 66%.ConclusionsIt is important to consider the effects of DDIs on the abrocitinib active moiety when making dosing recommendations. Co-administration of strong CYP2C19/2C9 inhibitors or CYP inducers impacted exposure to the abrocitinib active moiety. A dose reduction by half is recommended if abrocitinib is co-administered with strong CYP2C19 inhibitors, whereas co-administration with strong CYP2C19/2C9 inducers is not recommended. No dose adjustment is required when abrocitinib is administered with OAT3 inhibitors.Clinical Trials Registration IDsNCT03634345, NCT03637790, NCT03937258Supplementary InformationThe online version contains supplementary material available at 10.1007/s13318-021-00745-6.

077 Effect of Cytochrome P450 Inhibition and Induction and Organic Anion Transporter 3 Inhibition on the Pharmacokinetics of Abrocitinib Active Moiety in Healthy Individuals

Abrocitinib is a Janus kinase-1–selective inhibitor under development for treatment of moderate-to- severe atopic dermatitis. Abrocitinib is eliminated primarily by cytochrome P450 (CYP)2C19 (53%), CYP2C9 (30%), CYP3A4 (11%), and CYP2B6 (6%) metabolism, and its pharmacologic activity is attributable to the unbound concentrations of the parent molecule and its 2 active metabolites, which are substrates of organic anion transporter 3 (OAT3). The sum of potency-adjusted unbound exposures of abrocitinib and its 2 active metabolites is termed the abrocitinib active moiety.

Transport mechanism and affinity of [99mTc]Tc-mercaptoacetyltriglycine ([99mTc]MAG3) on the apical membrane of renal proximal tubule cells

Technetium-99m-labeled mercaptoacetyltriglycine ([99mTc]MAG3) is widely used for evaluation of transplanted kidneys, diagnosis of tubular necrosis, and scintigraphic studies of tubular function. [99mTc]MAG3 is a substrate for organic anion transporter (OAT)1 and OAT3 on the basolateral membrane side for renal secretion. We investigated the transport mechanism and affinity of [99mTc]MAG3 on the apical membrane of renal proximal tubule cells for renal secretion. Adenosine triphosphate-binding cassette (ABC) transporters for renal secretion of [99mTc]MAG3 were examined using ABC transporter vesicles expressing multiple drug resistance 1 (MDR1), breast cancer resistance protein (BCRP), multidrug resistance-associated protein (MRP)2, and MRP4. MK-571, a MRP inhibitor, was applied to measure the Km and Vmax of MRP2 and MRP4 in a vesicle transport assay. Single photon emission computed tomography (SPECT) was performed in normal rats and MRP2-deficient Eisai hyperbilirubinuria rats (EHBR) using [99mTc]MAG3 with and without MK-571. [99mTc]MAG3 uptake in adenosine triphosphate was significantly higher than that in adenosine monophosphate in vesicles that highly expressed MRP2 and MRP4. The affinity of [99mTc]MAG3 for MRP4 was higher than that for MRP2. Renal secretion via MRP2 and MRP4 was identified by comparing normal and EHBR rats with and without MK-571 on SPECT. [99mTc]MAG3 is transported via MRP2 and MRP4 on the apical membrane of renal proximal tubule cells. The affinity of MRP4 is higher than that of MRP2. Significance statement[99mTc]MAG3, widely used for evaluation of transplanted kidneys, diagnosis of tubular necrosis, and scintigraphic studies of tubular function, is transported via MRP2 and MRP4 on the apical membrane of renal proximal tubule cells. The affinity of MRP4 is higher than that of MRP2.

Leflunomide increased the renal exposure of acyclovir by inhibiting OAT1/3 and MRP2.

Rheumatoid arthritis patients can be prescribed a combination of immunosuppressive drug leflunomide (LEF) and the antiviral drug acyclovir to reduce the high risk of infection. Acyclovir is a substrate of organic anion transporter (OAT) 1/3 and multidrug resistance-associated protein (MRP) 2. Considering the extraordinarily long half-life of LEF’s active metabolite teriflunomide (TER) and the kidney injury risk of acyclovir, it is necessary to elucidate the potential impact of LEF on the disposition of acyclovir. Here we used a specific MRP inhibitor MK571 and probenecid (OAT1/3 and MRP2 inhibitor) to assess the effects of MRP2 and OAT1/3 on the pharmacokinetics and tissue distribution of acyclovir in rats. We showed that LEF and probenecid, but not MK571 significantly increased the plasma concentration of acyclovir. However, kidney and liver exposures of acyclovir were increased when coadministered with LEF, probenecid or MK571. The kidney/plasma ratio of acyclovir was increased to approximately 2-fold by LEF or probenecid, whereas it was increased to as much as 14.5-fold by MK571. Consistently, these drugs markedly decreased the urinary excretion of acyclovir. TER (0.5−100 μmol/L) dose-dependently increased the accumulation of acyclovir in MRP2-MDCK cells with an IC50 value of 4.91 μmol/L. TER (5 μmol/L) significantly inhibited the uptake of acyclovir in hOAT1/3-HEK293 cells. These results suggest that LEF/TER increased the kidney accumulation of acyclovir by inhibiting the efflux transporter MRP2, which increased its kidney/plasma ratio and renal injury risk. However, the inhibitory effects of LEF/TER on OAT1/3 reduced the tubular cells’ uptake of acyclovir and increased the plasma concentration.

18\u03b2-glycyrrhetyl-3-O-sulfate would be a causative agent of licorice-induced pseudoaldosteronism

Licorice-induced pseudoaldosteronism is a common adverse effect in traditional Japanese Kampo medicine, and 3-monoglucuronyl glycyrrhetinic acid (3MGA) was considered as a causative agent of it. Previously, we found 22α-hydroxy-18β-glycyrrhetyl-3-O-sulfate-30-glucuronide (1), one of the metabolites of glycyrrhizin (GL) in the urine of Eisai hyperbilirubinuria rats (EHBRs) treated with glycyrrhetinic acid (GA), and suggested that it is also a possible causative agent of pseudoaldosteronism. The discovery of 1 also suggested that there might be other metabolites of GA as causal candidates. In this study, we found 22α-hydroxy-18β-glycyrrhetyl-3-O-sulfate (2) and 18β-glycyrrhetyl-3-O-sulfate (3) in EHBRs’ urine. 2 and 3 more strongly inhibited rat type 2 11β-hydroxysteroid dehydrogenase than 1 did in vitro. When EHBRs were orally treated with GA, GA and 1–3 in plasma and 1–3 in urine were detected; the levels of 3MGA were quite low. 2 and 3 were shown to be the substrates of organic anion transporter (OAT) 1 and OAT3. In the plasma of a patient suffering from pseudoaldosteronism with rhabdomyolysis due to licorice, we found 8.6 µM of 3, 1.3 µM of GA, and 87 nM of 2, but 1, GL, and 3MGA were not detected. These findings suggest that 18β-glycyrrhetyl-3-O-sulfate (3) is an alternative causative agent of pseudoaldosteronism, rather than 3MGA and 1.

Role of Hepatic Organic Anion Transporter 2 in the Pharmacokinetics of R- and S-Warfarin: In Vitro Studies and Mechanistic Evaluation.

Interindividual variability in warfarin dose requirement demands personalized medicine approaches to balance its therapeutic benefits (anticoagulation) and bleeding risk. Cytochrome P450 2C9 ( CYP2C9) genotype-guided warfarin dosing is recommended in the clinic, given the more potent S-warfarin is primarily metabolized by CYP2C9. However, only about 20-30% of interpatient variability in S-warfarin clearance is associated with CYP2C9 genotype. We evaluated the role of hepatic uptake in the clearance of R- and S-warfarin. Using stably transfected HEK293 cells, both enantiomers were found to be substrates of organic anion transporter (OAT)2 with a Michaelis-Menten constant ( Km) of ∼7-12 μM but did not show substrate affinity for other major hepatic uptake transporters. Uptake of both enantiomers by primary human hepatocytes was saturable ( Km ≈ 7-10 μM) and inhibitable by OAT2 inhibitors (e.g., ketoprofen) but not by OATP1B1/1B3 inhibitors (e.g., cyclosporine). To further evaluate the potential role of hepatic uptake in R- and S-warfarin pharmacokinetics, mechanistic modeling and simulations were conducted. A "bottom-up" PBPK model, developed assuming that OAT2-CYPs interplay, well recovered clinical pharmacokinetics, drug-drug interactions, and CYP2C9 pharmacogenomics of R- and S-warfarin. Clinical data were not available to directly verify the impact of OAT2 modulation on warfarin pharmacokinetics; however, the bottom-up PBPK model simulations suggested a proportional change in clearance of both warfarin enantiomers with inhibition of OAT2 activity. These results suggest that variable hepatic OAT2 function, in conjunction with CYP2C, may contribute to the high population variability in warfarin pharmacokinetics and possibly anticoagulation end points and thus warrant further clinical investigation.

Transporter-mediated interaction of indican and methotrexate in rats

Indican (indoxyl-β-D-glucoside) is present in several Chinese herbs e.g. Isatis indigotica, Polygonum tinctorium and Polygonum perfoliatum. The major metabolite of indican was indoxyl sulfate (IS), an uremic toxin which was a known substrate/inhibitor of organic anion transporter (OAT) 1, OAT 3 and multidrug resistance-associated protein (MRP) 4. Methotrexate (MTX), an important immunosuppressant with narrow therapeutic window, is a substrate of OAT 1, 2, 3, 4 and MRP 1, 2, 3, 4. We hypothesized that IS, the major metabolite of oral indican, might inhibit the renal excretion of MTX mediated by OAT 1, OAT 3 and MRP 4. Therefore, this study investigated the effect of oral indican on the pharmacokinetics of MTX. Rats were orally given MTX with and without indican (20.0 and 40.0 mg/kg) in a parallel design. The serum MTX concentration was determined by a fluorescence polarization immunoassay. For mechanism clarification, phenolsulfonphthalein (PSP, 5.0 mg/kg), a probe substrate of OAT 1, OAT 3, MRP 2 and MRP 4, was intravenously given to rats with and without a intravenous bolus of IS (10.0 mg/kg) to measure the effect of IS on the elimination of PSP. The results indicated that 20.0 and 40.0 mg/kg of oral indican significantly increased the area under concentration–time curve0-t (AUC0-t) of MTX by 231% and 259%, prolonged the mean residence time (MRT) by 223% and 204%, respectively. Furthermore, intravenous IS significantly increased the AUC0-t of PSP by 204% and decreased the Cl by 68%. In conclusion, oral indican increased the systemic exposure and MRT of MTX through inhibition on multiple anion transporters including OAT 1, OAT 3 and MRP 4 by the major metabolite IS.

Inhibition of organic anion transporter (OAT) activity by cigarette smoke condensate

Cigarette smoke condensate (CSC) has previously been shown to impair activity and expression of hepatic drug transporters. In the present study, we provided evidence that CSC also hinders activity of organic anion transporters (OATs), notably expressed at the kidney level. CSC thus cis-inhibited OAT substrate uptake in OAT1- and OAT3-transfected HEK293 cells, in a concentration-dependent manner (IC50=72.1μg/mL for OAT1 inhibition and IC50=27.3μg/mL for OAT3 inhibition). By contrast, OAT4 as well as the renal organic cation transporter (OCT) 2 were less sensitive to the inhibitory effect of CSC (IC50=351.5μg/mL and IC50=226.2μg/mL, for inhibition of OAT4 and OCT2, respectively). OAT3 activity was further demonstrated to be blocked by some single chemicals present in cigarette smoke such as the heterocyclic amines AαC (IC50=11.3μM) and PhIP (IC50=1.9μM), whereas other major cigarette smoke components used at 100μM, like nicotine, the nitrosamine NNK and the polycyclic aromatic hydrocarbons benzo(a)pyrene and phenanthrene, were without effect. AαC and PhIP however failed to trans-stimulate activity of OAT3, suggesting that they were not substrates for this transporter. Taken together, these data establish OAT1 and OAT3 transporters as targets of cigarette smoke chemicals, which may contribute to smoking-associated pharmacokinetics alterations.

Uptake of perfluorooctanoic acid by Caco-2 cells: Involvement of organic anion transporting polypeptides

The mechanism underlying the intestinal absorption of perfluorooctanoic acid (PFOA) was investigated using Caco-2 cells. The uptake of PFOA from the apical membrane of Caco-2 cells was fast, and pH, temperature, and concentration dependent, but Na+ independent. Coincubation with sulfobromophthalein (BSP), glibenclamide, estron-3-sulfate, cyclosporine A or rifamycin SV, which are typical substrates or inhibitors of organic anion transporting polypeptides (OATPs), significantly decreased the uptake of PFOA. However, coincubation with probenecid or p-aminohippuric acid, typical substrates of organic anion transporters, did not decrease the uptake of PFOA. Furthermore, coincubation with l-lactic acid or benzoic acid, substrates of monocarboxylic acid transporters, did not decrease PFOA uptake. The relationship between the initial uptake of PFOA and its concentration was saturable, suggesting the involvement of a carrier-mediated process. The calculated Km and uptake clearance (Vmax/Km) values for PFOA were 8.3μM and 55.0μL/mg protein/min, respectively. This clearance value was about 3-fold greater than that of the non-saturable uptake clearance (Kd: 18.1μL/mg protein/min). Lineweaver–Burk plots revealed that BSP competitively inhibits the uptake of PFOA, with a Ki value of 23.1μM. These results suggest that the uptake of PFOA from the apical membranes of Caco-2 cells could be, at least in part, mediated by OATPs along with BSP.

Prediction of renal transporter-mediated drug-drug interactions for a drug which is an OAT substrate and inhibitor using PBPK modelling

A PBPK modelling approach was used to predict organic anion transporter (OAT) mediated drug-drug interactions involving S44121, a substrate and an inhibitor of OAT1 and OAT3. Model predictions were then compared to the results of a clinical DDI study which was carried out to investigate the interaction of S44121 with probenecid, tenofovir and ciprofloxacin. PBPK models were developed and qualified using existing clinical data, and inhibition constants were determined in vitro. The model predictions for S44121 as an OAT inhibitor were similar to the results obtained from the clinical DDI study, with no interaction observed for tenofovir or ciprofloxacin in the presence of S44121. An observed AUC ratio of 2.2 was obtained for S44121 in the presence of probenecid, which was slightly higher than the model predicted AUC ratio of 1.6. A DDI study in the monkey was also carried out for the interaction between S44121 and probenecid, since the monkey has previously been reported to be a good preclinical model for OAT-mediated DDI. However, this study highlighted a species difference in the major route of S44121 elimination between monkey (mainly hepatic metabolism) and human (mainly renal excretion of unchanged drug), rendering a comparison between the two DDI studies difficult. Overall, for S44121 the PBPK modelling approach gave a better prediction of the extent of DDI than the static predictions based on inhibitor Cmax and IC50, therefore this can be considered a potentially valuable tool within drug development.

Increased Plasma Exposures of Conjugated Metabolites of Morinidazole in Renal Failure Patients: A Critical Role of Uremic Toxins.

Morinidazole is a 5-nitroimidazole drug. Its sulfate conjugate M7 was a sensitive substrate of organic anion transporter 1 (OAT1) and OAT3, whereas N+-glucuronides M8-1 and M8-2 were only OAT3 substrates. In chronic renal failure (CRF) patients, plasma exposures of the three conjugates increased by 15-fold, which were also found in 5/6 nephrectomized (5/6 Nx) rats in this study. Although the transcriptions of Oat1 and Oat3 in 5/6 Nx rat kidneys decreased by 50%, no difference was observed on the three conjugate uptakes between control and 5/6 Nx rat kidney slices. Thus, the highly elevated endogenous uremic toxins in 5/6 Nx rats and humans, namely, 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), hippuric acid (HA), and indoxyl sulfate (IS), were considered as influential factors. In rat kidney slices, the uptake of M7, M8-1, and M8-2 was dose dependently reduced by HA and IS, whose plasma concentrations were elevated 5 times in 5/6 Nx rats. In OAT3-overexpressed cells, the three conjugate uptakes were inhibited by CMPF, HA, and IS with IC50 values of 19.2, 87.4, and 222 μM (M7); 8.53, 39.4, and 161 μM (M8-1); and 6.75, 24.1, and 78.3 μM (M8-2), respectively. In OAT1-overexpressed cells, CMPF, HA, and IS showed weak inhibition on M7 uptake with IC50 values of 187, 162, and 200 μM, correspondingly. Results suggest that the reduced mRNA expression of renal transporters in CRF patients may not influence the activities of these transporters. However, accumulated uremic toxins may inhibit the transporters, particularly OAT3, leading to plasma exposure changes of relevant substrates.

Quantitative Prediction of Human Renal Clearance and Drug-Drug Interactions of Organic Anion Transporter Substrates Using In Vitro Transport Data: A Relative Activity Factor Approach.

Organic anion transporters (OATs) are important in the renal secretion, and thus, the clearance, of many drugs; and their functional change can result in pharmacokinetic variability. In this study, we applied transport rates measured in vitro using OAT-transfected human embryonic kidney cells to predict human renal secretory and total renal clearance of 31 diverse drugs. Selective substrates to OAT1 (tenofovir), OAT2 (acyclovir and ganciclovir), and OAT3 (benzylpenicillin, oseltamivir acid) were used to obtain relative activity factors (RAFs) for these individual transporters by relating in vitro transport clearance (after physiologic scaling) to in vivo secretory clearance. Using the estimated RAFs (0.64, 7.3, and 4.1, respectively, for OAT1, OAT2, and OAT3, respectively) and the in vitro active clearances, renal secretory clearance and total renal clearance were predicted with average fold errors (AFEs) of 1.89 and 1.40, respectively. The results show that OAT3-mediated transport play a predominant role in renal secretion for 22 of the 31 drugs evaluated. This mechanistic static approach was further applied to quantitatively predict renal drug-drug interactions (AFE ∼1.6) of the substrate drugs with probenecid, a clinical probe OAT inhibitor. In conclusion, the proposed in vitro-in vivo extrapolation approach is the first comprehensive attempt toward mechanistic modeling of renal secretory clearance based on routinely employed in vitro cell models.

Endogenous metabolites that are substrates of organic anion transporter’s (OATs) predict methotrexate clearance

Variable pharmacokinetics of high-dose-methotrexate (MTX) is responsible for severe toxicities. Unpredictable overexposure still occurs during some courses despite having controlled the main factors known to play a role in its elimination. The aim of our study was to evaluate whether the urine metabolomic profile measured at the time of MTX administration is predictive of the drug’s clearance and/or of treatment-related toxicity. We analyzed the urine content of endogenous metabolites before MTX administration in a cohort of adult patients treated for lymphoid malignancies. Individual MTX clearance (MTXCL) was estimated from population pharmacokinetic analyses of therapeutic drug monitoring data. We determined the urine metabolite content by gas chromatography-mass spectrometry (GC–MS) and applied Partial Least Square (PLS) analysis to assess the relationship between the urine metabolome and MTXCL. External validation was applied to evaluate the performances of the PLS model. We used orthogonal partial least squares discriminant analysis (OPLS-DA) to distinguish patients with normal or delayed elimination, and patients with or without toxicity. Sixty-two patients were studied. We obtained a very good prediction of individual MTX clearance using a set of 28 metabolites present in patient urine at baseline. The mean prediction error and precision were −0.36% and 21.4%, respectively, for patients not included in the model. The model included a set of endogenous organic anions, of which the tubular secretion depends on organic anion transporter (OAT) function. Our analyses did not allow us to discriminate between patients with or without delayed elimination or those who did or did not experience toxicity. Urinary metabolomics can be informative about an individual’s ability to clear MTX. More broadly, it paves the way for the development of a biomarker of tubular secretion, easily measurable from endogenous substances.

Drug Transport by the Blood-Aqueous Humor Barrier of the Eye.

The ocular barriers (cornea, blood-retinal barrier, and blood-aqueous humor barrier) make treating eye diseases with therapeutic drugs challenging. The tight capillary endothelium of the iris and the ciliary body epithelium form the blood-aqueous humor barrier. The iris and ciliary body (iris-ciliary body) express a variety of drug transporters in the ATP-binding cassette and solute carrier (SLC) families. ATP-binding cassette family drug transporters that are present in the iris-ciliary body include P-glycoprotein, breast cancer resistance protein, and several multidrug resistance-associated proteins. SLC family drug transporters that are present in the iris-ciliary body include organic anion transporters, organic anion transporting polypeptides, bile acid transporters (apical sodium-dependent bile salt transporter and sodium taurocholate cotransporter), organic cation transporters (novel organic cation transporter and multidrug and toxin extrusion transporter) and peptide transporters. Freshly dissected iris-ciliary body preparations actively accumulate a variety of substrates of SLC drug transporters that are expressed in the tissue. The ciliary body in vitro supports active transport in the aqueous humor-to-blood direction of several substrates of organic anion transporters and multidrug resistance-associated proteins, consistent with the subcellular localization of these transporters in the ciliary body epithelium. In vivo data suggest that drug transporters in the iris-ciliary body reduce the permeation of drugs in the direction of blood-to-aqueous humor, thereby reducing ocular drug bioavailability, and are also involved in active drug elimination from the aqueous humor. An understanding of the influence on pharmacokinetics of drug transporters in the blood-aqueous humor barrier should help improve drug delivery and efficacy in the eye.

A Human Renal Proximal Tubule Cell Line with Stable Organic Anion Transporter 1 and 3 Expression Predictive for Antiviral-Induced Toxicity.

Drug-induced nephrotoxicity still hampers drug development, because current translation from in vitro or animal studies to human lacks high predictivity. Often, renal adverse effects are recognized only during clinical stages of drug development. The current study aimed to establish a robust and a more complete human cell model suitable for screening of drug-related interactions and nephrotoxicity. In addition to endogenously expressed renal organic cation transporters and efflux transporters, conditionally immortalized proximal tubule epithelial cells (ciPTEC) were completed by transduction of cells with the organic anion transporter (OAT) 1 or OAT3. Fluorescence-activated cell sorting upon exposure to the OAT substrate fluorescein successfully enriched transduced cells. A panel of organic anions was screened for drug-interactions in ciPTEC-OAT1 and ciPTEC-OAT3. The cytotoxic response to the drug-interactions with antivirals was further examined by cell viability assays. Upon subcloning, concentration-dependent fluorescein uptake was found with a higher affinity for ciPTEC-OAT1 (Km = 0.8 ± 0.1 μM) than ciPTEC-OAT3 (Km = 3.7 ± 0.5 μM). Co-exposure to known OAT1 and/or OAT3 substrates (viz. para-aminohippurate, estrone sulfate, probenecid, furosemide, diclofenac, and cimetidine) in cultures spanning 29 passage numbers revealed relevant inhibitory potencies, confirming the robustness of our model for drug-drug interactions studies. Functional OAT1 was directly responsible for cytotoxicity of adefovir, cidofovir, and tenofovir, while a drug interaction with zidovudine was not associated with decreased cell viability. Our data demonstrate that human-derived ciPTEC-OAT1 and ciPTEC-OAT3 are promising platforms for highly predictive drug screening during early phases of drug development.Electronic supplementary materialThe online version of this article (doi:10.1208/s12248-016-9871-8) contains supplementary material, which is available to authorized users.

Diclofenac and Its Acyl Glucuronide: Determination of In Vivo Exposure in Human Subjects and Characterization as Human Drug Transporter Substrates In Vitro.

Although the metabolism and disposition of diclofenac (DF) has been studied extensively, information regarding the plasma levels of its acyl-β-d-glucuronide (DF-AG), a major metabolite, in human subjects is limited. Therefore, DF-AG concentrations were determined in plasma (acidified blood derived) of six healthy volunteers following a single oral DF dose (50 mg). Levels of DF-AG in plasma were high, as reflected by a DF-AG/DF ratio of 0.62 ± 0.21 (Cmax mean ± S.D.) and 0.84 ± 0.21 (area under the concentration-time curve mean ± S.D.). Both DF and DF-AG were also studied as substrates of different human drug transporters in vitro. DF was identified as a substrate of organic anion transporter (OAT) 2 only (Km = 46.8 µM). In contrast, DF-AG was identified as a substrate of numerous OATs (Km = 8.6, 60.2, 103.9, and 112 µM for OAT2, OAT1, OAT4, and OAT3, respectively), two organic anion-transporting polypeptides (OATP1B1, Km = 34 µM; OATP2B1, Km = 105 µM), breast cancer resistance protein (Km = 152 µM), and two multidrug resistance proteins (MRP2, Km = 145 µM; MRP3, Km = 196 µM). It is concluded that the disposition of DF-AG, once formed, can be mediated by various candidate transporters known to be expressed in the kidney (basolateral, OAT1, OAT2, and OAT3; apical, MRP2, BCRP, and OAT4) and liver (canalicular, MRP2 and BCRP; basolateral, OATP1B1, OATP2B1, OAT2, and MRP3). DF-AG is unstable in plasma and undergoes conversion to parent DF. Therefore, caution is warranted when assessing renal and hepatic transporter-mediated drug-drug interactions with DF and DF-AG.