Characterization of hOAT4 and mOat5 as functional orthologs for renal anion uptake and efflux transport.

Organic anions (OA) are compounds including drugs or toxicants that are negatively charged at physiological pH and are typically transported by Organic Anion Transporters (OATs). Human OAT4 (SLC22A11) is expressed in the apical membrane of renal proximal tubules. Although there is no rodent ortholog of hOAT4, rodents express Oat5 (Slc22a19), an anion exchanger that is also localized to the apical membrane of renal proximal tubule cells. The purpose of this study was to determine the functional similarity between mouse Oat5 and human OAT4. Chinese hamster ovary (CHO) cells expressing SLC22A11 or Slc22a19 were used to assess the transport characteristics of radiolabeled ochratoxin (OTA). We determined the kinetics of OTA transport; the resulting Kt and Jmax values were very similar for both hOAT4 and mOat5: Kt 3.9 and 7.2 µM, respectively, & Jmax 4.4 and 3.9 pmol/cm2, respectively. For the profile of OTA inhibition by OAs, IC50 values were determined for several clinically important drugs and toxicants. The resulting IC50 values ranged from 9 µM for indomethacin to ~600 µM for the diuretic hydrochlorothiazide. We measured the efflux of OTA from preloaded cells; both hOAT4 and mOat5 supported the efflux of OTA. These data support the hypothesis that OAT4 and Oat5 are functional orthologs and share selectivity for OTA both for reabsorption and secretion. Significance Statement This study compares the selectivity profile between human OAT4 and mouse Oat5. Our data revealed a similar selectivity profile for OTA reabsorption and secretion by these two transporters, thereby supporting the hypothesis that hOAT4 and mOat5, while not genetic orthologs, behave as functional orthologs for both uptake and efflux. These data will be instrumental in selecting an appropriate animal model when studying the renal disposition of anionic drugs and toxicants.

Addressing the Clinical Importance of Equilibrative Nucleoside Transporters in Drug Discovery and Development.

The US Food and Drug Administration (FDA), European Medicines Agency (EMA), and Pharmaceuticals and Medical Devices Agency (PMDA) guidances on small-molecule drug-drug interactions (DDIs), with input from the International Transporter Consortium (ITC), recommend the evaluation of nine drug transporters. Although other clinically relevant drug uptake and efflux transporters have been discussed in ITC white papers, they have been excluded from further recommendation by the ITC and are not included in current regulatory guidances. These include the ubiquitously expressed equilibrative nucleoside transporters (ENT) 1 and ENT2, which have been recognized by the ITC for their potential role in clinically relevant nucleoside analog drug interactions for patients with cancer. Although there is comparatively limited clinical evidence supporting their role in DDI risk or other adverse drug reactions (ADRs) compared with the nine highlighted transporters, several in vitro and in vivo studies have identified ENT interactions with non-nucleoside/non-nucleotide drugs, in addition to nucleoside/nucleotide analogs. Some noteworthy examples of compounds that interact with ENTs include cannabidiol and selected protein kinase inhibitors, as well as the nucleoside analogs remdesivir, EIDD-1931, gemcitabine, and fialuridine. Consequently, DDIs involving the ENTs may be responsible for therapeutic inefficacy or off-target toxicity. Evidence suggests that ENT1 and ENT2 should be considered as transporters potentially involved in clinically relevant DDIs and ADRs, thereby warranting further investigation and regulatory consideration.

In Vitro and In Vivo Models for Drug Transport Across the Blood-Testis Barrier.

The blood-testis barrier (BTB) is a selectively permeable membrane barrier formed by adjacent Sertoli cells (SCs) in the seminiferous tubules of the testes that develops intercellular junctional complexes to protect developing germ cells from external pressures. However, due to this inherent defense mechanism, the seminiferous tubule lumen can act as a pharmacological sanctuary site for latent viruses (e.g., Ebola, Zika) and cancers (e.g., leukemia). Therefore, it is critical to identify and evaluate BTB carrier-mediated drug delivery pathways to successfully treat these viruses and cancers. Many drugs are unable to effectively cross cell membranes without assistance from carrier proteins like transporters because they are large, polar, and often carry a charge at physiologic pH. SCs express transporters that selectively permit endogenous compounds, such as carnitine or nucleosides, across the BTB to support normal physiologic activity, although reproductive toxicants can also use these pathways, thereby circumventing the BTB. Certain xenobiotics, including select cancer therapeutics, antivirals, contraceptives, and environmental toxicants, are known to accumulate within the male genital tract and cause testicular toxicity; however, the transport pathways by which these compounds circumvent the BTB are largely unknown. Consequently, there is a need to identify the clinically relevant BTB transport pathways in in vitro and in vivo BTB models that recapitulate human pharmacokinetics and pharmacodynamics for these xenobiotics. This review summarizes the various in vitro and in vivo models of the BTB reported in the literature and highlights the strengths and weaknesses of certain models for drug disposition studies. SIGNIFICANCE STATEMENT: Drug disposition to the testes is influenced by the physical, physiological, and immunological components of the blood-testis barrier (BTB). But many compounds are known to cross the BTB by transporters, resulting in pharmacological and/or toxicological effects in the testes. Therefore, models that assess drug transport across the human BTB must adequately account for these confounding factors. This review identifies and discusses the benefits and limitations of various in vitro and in vivo BTB models for preclinical drug disposition studies.

Representative Rodent Models for Renal Transporter Alterations in Human Nonalcoholic Steatohepatitis.

Alterations in renal elimination processes of glomerular filtration and active tubular secretion by renal transporters can result in adverse drug reactions. Nonalcoholic steatohepatitis (NASH) alters hepatic transporter expression and xenobiotic elimination, but until recently, renal transporter alterations in NASH were unknown. This study investigates renal transporter changes in rodent models of NASH to identify a model that recapitulates human alterations. Quantitative protein expression by surrogate peptide liquid chromatography-coupled mass spectrometry (LC-MS/MS) on renal biopsies from NASH patients was used for concordance analysis with rodent models, including methionine/choline deficient (MCD), atherogenic (Athero), or control rats and Leprdb/db MCD (db/db), C57BL/6J fast-food thioacetamide (FFDTH), American lifestyle-induced obesity syndrome (ALIOS), or control mice. Demonstrating clinical similarity to NASH patients, db/db, FFDTH, and ALIOS showed decreases in glomerular filtration rate (GFR) by 76%, 28%, and 24%. Organic anion transporter 3 (OAT3) showed an upward trend in all models except the FFDTH (from 3.20 to 2.39 pmol/mg protein), making the latter the only model to represent human OAT3 changes. OAT5, a functional ortholog of human OAT4, significantly decreased in db/db, FFDTH, and ALIOS (from 4.59 to 0.45, 1.59, and 2.83 pmol/mg protein, respectively) but significantly increased for MCD (1.67 to 4.17 pmol/mg protein), suggesting that the mouse models are comparable to human for these specific transport processes. These data suggest that variations in rodent renal transporter expression are elicited by NASH, and the concordance analysis enables appropriate model selection for future pharmacokinetic studies based on transporter specificity. These models provide a valuable resource to extrapolate the consequences of human variability in renal drug elimination. SIGNIFICANCE STATEMENT: Rodent models of nonalcoholic steatohepatitis that recapitulate human renal transporter alterations are identified for future transporter-specific pharmacokinetic studies to facilitate the prevention of adverse drug reactions due to human variability.

Transporter Inhibition Profile for the Antivirals Tilorone, Quinacrine and Pyronaridine.

Pyronaridine, tilorone and quinacrine are cationic molecules that have in vitro activity against Ebola, SARS-CoV-2 and other viruses. All three molecules have also demonstrated in vivo activity against Ebola in mice, while pyronaridine showed in vivo efficacy against SARS-CoV-2 in mice. We have recently tested these molecules and other antivirals against human organic cation transporters (OCTs) and apical multidrug and toxin extruders (MATEs). Quinacrine was found to be an inhibitor of OCT2, while tilorone and pyronaridine were less potent, and these displayed variability depending on the substrate used. To assess whether any of these three molecules have other potential interactions with additional transporters, we have now screened them at 10 µM against various human efflux and uptake transporters including P-gp, OATP1B3, OAT1, OAT3, MRP1, MRP2, MRP3, BCRP, as well as confirmational testing against OCT1, OCT2, MATE1 and MATE2K. Interestingly, in this study tilorone appears to be a more potent inhibitor of OCT1 and OCT2 than pyronaridine or quinacrine. However, both pyronaridine and quinacrine appear to be more potent inhibitors of MATE1 and MATE2K. None of the three compounds inhibited MRP1, MRP2, MRP3, OAT1, OAT3, P-gp or OATP1B3. Similarly, we previously showed that tilorone and pyronaridine do not inhibit OATP1B1 and have confirmed that quinacrine behaves similarly. In total, these observations suggest that the three compounds only appear to interact with OCTs and MATEs to differing extents, suggesting they may be involved in fewer clinically relevant drug-transporter interactions involving pharmaceutical substrates of the other major transporters tested.

Drug Transporters at the Human Blood-Testis Barrier.

Transporters are involved in the movement of many physiologically important molecules across cell membranes and have a substantial impact on the pharmacological and toxicological effect of xenobiotics. Many transporters have been studied in the context of disposition to, or toxicity in, organs such as the kidney and liver; however, transporters in the testes are increasingly gaining recognition for their role in drug transport across the blood-testis barrier (BTB). The BTB is an epithelial membrane barrier formed by adjacent Sertoli cells (SCs) in the seminiferous tubules that form intercellular junctional complexes to protect developing germ cells from the external environment. Consequently, many charged or large polar molecules cannot cross this barrier without assistance from a transporter. SCs express a variety of drug uptake and efflux transporters to control the flux of endogenous and exogenous molecules across the BTB. Recent studies have identified several transport pathways in SCs that allow certain drugs to circumvent the human BTB. These pathways may exist in other species, such as rodents and nonhuman primates; however, there is (1) a lack of information on their expression and/or localization in these species, and (2) conflicting reports on localization of some transporters that have been evaluated in rodents compared with humans. This review outlines the current knowledge on the expression and localization of pharmacologically relevant drug transporters in human testes and calls attention to the insufficient and contradictory understanding of testicular transporters in other species that are commonly used in drug disposition and toxicity studies. SIGNIFICANCE STATEMENT: While the expression, localization, and function of many xenobiotic transporters have been studied in organs such as the kidney and liver, the characterization of transporters in the testes is scarce. This review summarizes the expression and localization of common pharmacologically-relevant transporters in human testes that have significant implications for the development of drugs that can cross the blood-testis barrier. Potential expression differences between humans and rodents highlighted here suggest rodents may be inappropriate for some testicular disposition and toxicity studies.

Predicting disruptions to drug pharmacokinetics and the risk of adverse drug reactions in non-alcoholic steatohepatitis patients.

The liver plays a central role in the pharmacokinetics of drugs through drug metabolizing enzymes and transporters. Non-alcoholic steatohepatitis (NASH) causes disease-specific alterations to the absorption, distribution, metabolism, and excretion (ADME) processes, including a decrease in protein expression of basolateral uptake transporters, an increase in efflux transporters, and modifications to enzyme activity. This can result in increased drug exposure and adverse drug reactions (ADRs). Our goal was to predict drugs that pose increased risks for ADRs in NASH patients. Bibliographic research identified 71 drugs with reported ADRs in patients with liver disease, mainly non-alcoholic fatty liver disease (NAFLD), 54 of which are known substrates of transporters and/or metabolizing enzymes. Since NASH is the progressive form of NAFLD but is most frequently undiagnosed, we identified other drugs at risk based on NASH-specific alterations to ADME processes. Here, we present another list of 71 drugs at risk of pharmacokinetic disruption in NASH, based on their transport and/or metabolism processes. It encompasses drugs from various pharmacological classes for which ADRs may occur when used in NASH patients, especially when eliminated through multiple pathways altered by the disease. Therefore, these results may inform clinicians regarding the selection of drugs for use in NASH patients.

Renal Transporter Alterations in Patients with Chronic Liver Diseases: Nonalcoholic Steatohepatitis, Alcohol-Associated, Viral Hepatitis, and Alcohol-Viral Combination.

Alterations in hepatic transporters have been identified in precirrhotic chronic liver diseases (CLDs) that result in pharmacokinetic variations causing adverse drug reactions (ADRs). However, the effect of CLD on the expression of renal transporters is unknown despite the overwhelming evidence of kidney injury in CLD patients. This study determines the transcriptomic and proteomic expression profiles of renal drug transporters in patients with defined CLD etiology. Renal biopsies were obtained from patients with a history of CLD etiologies, including nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), alcohol-associated liver disease (ALD), viral hepatitis C (HCV), and combination ALD/HCV. A significant decrease in organic anion transporter (OAT)-3 was identified in NASH, ALD, HCV, and ALD/HCV (1.56 ± 1.10; 1.01 ± 0.46; 1.03 ± 0.43; 0.86 ± 0.57 pmol/mg protein) relative to control (2.77 ± 1.39 pmol/mg protein). Additionally, a decrease was shown for OAT4 in NASH (24.9 ± 5.69 pmol/mg protein) relative to control (43.8 ± 19.9 pmol/mg protein) and in urate transporter 1 (URAT1) for ALD and HCV (1.56 ± 0.15 and 1.65 ± 0.69 pmol/mg protein) relative to control (4.69 ± 4.59 pmol/mg protein). These decreases in organic anion transporter expression could result in increased and prolonged systemic exposure to drugs and possible toxicity. Renal transporter changes, in addition to hepatic transporter alterations, should be considered in dose adjustments for CLD patients for a more accurate disposition profile. It is important to consider a multiorgan approach to altered pharmacokinetics of drugs prescribed to CLD patients to prevent ADRs and improve patient outcomes. SIGNIFICANCE STATEMENT: Chronic liver diseases are known to elicit alterations in hepatic transporters that result in a disrupted pharmacokinetic profile for various drugs. However, it is unknown if there are alterations in renal transporters during chronic liver disease, despite strong indications of renal dysfunction associated with chronic liver disease. Identifying renal transporter expression changes in patients with chronic liver disease facilitates essential investigations on the multifaceted relationship between liver dysfunction and kidney physiology to offer dose adjustments and prevent adverse drug reactions.

Machine Learning Models Identify New Inhibitors for Human OATP1B1.

The uptake transporter OATP1B1 (SLC01B1) is largely localized to the sinusoidal membrane of hepatocytes and is a known victim of unwanted drug-drug interactions. Computational models are useful for identifying potential substrates and/or inhibitors of clinically relevant transporters. Our goal was to generate OATP1B1 in vitro inhibition data for [3H] estrone-3-sulfate (E3S) transport in CHO cells and use it to build machine learning models to facilitate a comparison of seven different classification models (Deep learning, Adaboosted decision trees, Bernoulli naïve bayes, k-nearest neighbors (knn), random forest, support vector classifier (SVC), logistic regression (lreg), and XGBoost (xgb)] using ECFP6 fingerprints to perform 5-fold, nested cross validation. In addition, we compared models using 3D pharmacophores, simple chemical descriptors alone or plus ECFP6, as well as ECFP4 and ECFP8 fingerprints. Several machine learning algorithms (SVC, lreg, xgb, and knn) had excellent nested cross validation statistics, particularly for accuracy, AUC, and specificity. An external test set containing 207 unique compounds not in the training set demonstrated that at every threshold SVC outperformed the other algorithms based on a rank normalized score. A prospective validation test set was chosen using prediction scores from the SVC models with ECFP fingerprints and were tested in vitro with 15 of 19 compounds (84% accuracy) predicted as active (≥20% inhibition) showed inhibition. Of these compounds, six (abamectin, asiaticoside, berbamine, doramectin, mobocertinib, and umbralisib) appear to be novel inhibitors of OATP1B1 not previously reported. These validated machine learning models can now be used to make predictions for drug-drug interactions for human OATP1B1 alongside other machine learning models for important drug transporters in our MegaTrans software.

Genetic evidence that uptake of the fluorescent analog 2NBDG occurs independently of known glucose transporters.

The fluorescent derivative of glucose, 2-Deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)-amino]-D-glucose (2NBDG), is a widely used surrogate reagent to visualize glucose uptake in live cells at single cell resolution. Using CRISPR-Cas9 gene editing in 5TGM1 myeloma cells, we demonstrate that ablation of the glucose transporter gene Slc2a1 abrogates radioactive glucose uptake but has no effect on the magnitude or kinetics of 2NBDG import. Extracellular 2NBDG, but not NBD-fructose was transported by primary plasma cells into the cytoplasm suggesting a specific mechanism that is unlinked from glucose import and that of chemically similar compounds. Neither excess glucose nor pharmacological inhibition of GLUT1 impacted 2NBDG uptake in myeloma cells or primary splenocytes. Genetic ablation of other expressed hexose transporters individually or in combination with one another also had no impact on 2NBDG uptake. Ablation of the genes in the Slc29 and Slc35 families of nucleoside and nucleoside sugar transporters also failed to impact 2NBDG import. Thus, cellular uptake of 2NBDG is not necessarily a faithful indicator of glucose transport and is promoted by an unknown mechanism.

Physiological Characterization of the Transporter-Mediated Uptake of the Reversible Male Contraceptive H2-Gamendazole Across the Blood-Testis Barrier.

The blood-testis barrier (BTB) is formed by a tight network of Sertoli cells (SCs) to limit the movement of reproductive toxicants from the blood into the male genital tract. Transporters expressed at the basal membranes of SCs also influence the disposition of drugs across the BTB. The reversible, nonhormonal contraceptive, H2-gamendazole (H2-GMZ), is an indazole carboxylic acid analog that accumulates over 10 times more in the testes compared with other organs. However, the mechanism(s) by which H2-GMZ circumvents the BTB are unknown. This study describes the physiologic characteristics of the carrier-mediated process(es) that permit H2-GMZ and other analogs to penetrate SCs. Uptake studies were performed using an immortalized human SC line (hT-SerC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Uptake of H2-GMZ and four analogs followed Michaelis-Menten transport kinetics (one analog exhibited poor penetration). H2-GMZ uptake was strongly inhibited by indomethacin, diclofenac, MK-571, and several analogs. Moreover, H2-GMZ uptake was stimulated by an acidic extracellular pH, reduced at basic pHs, and independent of extracellular Na+, K+, or Cl- levels, which are intrinsic characteristics of OATP-mediated transport. Therefore, the characteristics of H2-GMZ transport suggest that one or more OATPs may be involved. However, endogenous transporter expression in wild-type Chinese hamster ovary (CHO), Madin-Darby canine kidney (MDCK), and human embryonic kidney-293 (HEK-293) cells limited the utility of heterologous transporter expression to identify a specific OATP transporter. Altogether, characterization of the transporters involved in the flux of H2-GMZ provides insight into the selectivity of drug disposition across the human BTB to understand and overcome the pharmacokinetic and pharmacodynamic difficulties presented by this barrier. SIGNIFICANCE STATEMENT: Despite major advancements in female contraceptives, male alternatives, including vasectomy, condom usage, and physical withdrawal, are antiquated and the widespread availability of nonhormonal, reversible chemical contraceptives is nonexistent. Indazole carboxylic acid analogs such as H2-GMZ are promising new reversible, antispermatogenic drugs that are highly effective in rodents. This study characterizes the carrier-mediated processes that permit H2-GMZ and other drugs to enter Sertoli cells and the observations made here will guide the development of drugs that effectively circumvent the BTB.

Novel method for kinetic analysis applied to transport by the uniporter OCT2.

The kinetics of solute transport shed light on the roles these processes play in cellular physiology, and the absolute values of the kinetic parameters that quantitatively describe transport are increasingly used to model its impact on drug clearance. However, accurate assessment of transport kinetics is challenging. Although most carrier-mediated transport is adequately described by the Michaelis-Menten equation, its use presupposes that the rates of uptake used in the analysis of maximal rates of transport (Jmax) and half-saturation constants (Kt) reflect true unidirectional rates of influx from known concentrations of substrate. Most experimental protocols estimate the initial rate of transport from net substrate accumulation determined at a single time point (typically between 0.5 and 5 min) and assume it reflects unidirectional influx. However, this approach generally results in systematic underestimates of Jmax and overestimates of Kt; the former primarily due to the unaccounted impact of efflux of accumulated substrate, and the latter due to the influence of unstirred water layers. Here, we describe the bases of these time-dependent effects and introduce a computational model that analyzes the time course of net substrate uptake at several concentrations to calculate Jmax and Kt for unidirectional influx, taking into account the influence of unstirred water layers and mediated efflux. This method was then applied to calculate the kinetics of transport of 1-methyl-4-phenylpryridinium and metformin by renal organic cation transporter 2 as expressed in cultured Chinese hamster ovary cells.NEW & NOTEWORTHY Here, we describe a mathematical model that uses the time course of net substrate uptake into cells from several increasing concentrations to calculate unique kinetic parameters [maximal rates of transport (Jmax) and half-saturation constants (Kt)] of the process. The method is the first to take into consideration the common complicating factors of unstirred layers and carrier-mediated efflux in the experimental determination of transport kinetics.

PF-07321332 (Nirmatrelvir) does not interact with human ENT1 or ENT2: Implications for COVID-19 patients.

The ongoing pandemic of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) and subsequently, coronavirus disease 2019 (COVID-19), has led to the deaths of over 6.1 million people and sparked a greater interest in virology to expedite the development process for antivirals. The US Food and Drug Administration (FDA) granted emergency use authorization for three antivirals: remdesivir, molnupiravir, and nirmatrelvir. Remdesivir and molnupiravir are nucleoside analogs that undergo biotransformation to form active metabolites that incorporate into new viral RNA to stall replication. Unlike remdesivir or molnupiravir, nirmatrelvir is a protease inhibitor that covalently binds to the SARS-CoV-2 3C-like protease to interrupt the viral replication cycle. A recent study identified that remdesivir and the active metabolite of molnupiravir, EIDD-1931, are substrates of equilibrative nucleoside transporters 1 and 2 (ENT1 and 2). Despite the ubiquitous expression of the ENTs, the preclinical efficacy of remdesivir and molnupiravir is not reflected in wide-scale SARS-CoV-2 clinical trials. Interestingly, downregulation of ENT1 and ENT2 expression has been shown in lung epithelial and endothelial cells in response to hypoxia and acute lung injury, although it has not been directly studied in patients with COVID-19. It is possible that the poor efficacy of remdesivir and molnupiravir in these patients may be partially attributed to the repression of ENTs in the lungs, but further studies are warranted. This study investigated the interaction between nirmatrelvir and the ENTs and found that it was a poor inhibitor of ENT-mediated [3 H]uridine uptake at 300 µM. Unlike for remdesivir or EIDD-1931, ENT activity is unlikely to be a factor for nirmatrelvir disposition in humans; however, whether this contributes to the similar in vitro and clinical efficacy will require further mechanistic studies.

Localization of Xenobiotic Transporters Expressed at the Human Blood-Testis Barrier.

The blood-testis barrier (BTB) is formed by basal tight junctions between adjacent Sertoli cells (SCs) of the seminiferous tubules and acts as a physical barrier to protect developing germ cells in the adluminal compartment from reproductive toxicants. Xenobiotics, including antivirals, male contraceptives, and cancer chemotherapeutics, are known to cross the BTB, although the mechanisms that permit barrier circumvention are generally unknown. This study used immunohistological staining of human testicular tissue to determine the site of expression for xenobiotic transporters that facilitate transport across the BTB. Organic anion transporter (OAT) 1, OAT2, and organic cation transporter, novel (OCTN) 1 primarily localized to the basal membrane of SCs, whereas OCTN2, multidrug resistance protein (MRP) 3, MRP6, and MRP7 localized to SC basal membranes and peritubular myoid cells (PMCs) surrounding the seminiferous tubules. Concentrative nucleoside transporter (CNT) 2 localized to Leydig cells (LCs), PMCs, and SC apicolateral membranes. Organic cation transporter (OCT) 1, OCT2, and OCT3 mostly localized to PMCs and LCs, although there was minor staining in developing germ cells for OCT3. Organic anion transporting polypeptide (OATP) 1A2, OATP1B1, OATP1B3, OATP2A1, OATP2B1, and OATP3A1-v2 localized to SC basal membranes with diffuse staining for some transporters. Notably, OATP1C1 and OATP4A1 primarily localized to LCs. Positive staining for multidrug and toxin extrusion protein (MATE) 1 was only observed throughout the adluminal compartment. Definitive staining for CNT1, OAT3, MATE2, and OATP6A1 was not observed. The location of these transporters is consistent with their involvement in the movement of xenobiotics across the BTB. Altogether, the localization of these transporters provides insight into the mechanisms of drug disposition across the BTB and will be useful in developing tools to overcome the pharmacokinetic and pharmacodynamic difficulties presented by the BTB. SIGNIFICANCE STATEMENT: Although the total mRNA and protein expression of drug transporters in the testes has been explored, the localization of many transporters at the blood-testis barrier (BTB) has not been determined. This study applied immunohistological staining in human testicular tissues to identify the cellular localization of drug transporters in the testes. The observations made in this study have implications for the development of drugs that can effectively use transporters expressed at the basal membranes of Sertoli cells to bypass the BTB.

Transport Turnover Rates for Human OCT2 and MATE1 Expressed in Chinese Hamster Ovary Cells.

MATE1 (multidrug and toxin extruder 1) and OCT2 (organic cation transporter 2) play critical roles in organic cation excretion by the human kidney. The transporter turnover rate (TOR) is relevant to understanding both their transport mechanisms and interpreting the in vitro-in vivo extrapolation (IVIVE) required for physiologically-based pharmacokinetic (PBPK) modeling. Here, we use a quantitative western blot method to determine TORs for MATE1 and OCT2 proteins expressed in CHO cells. MATE1 and OCT2, each with a C-terminal V-5 epitope tag, were cell surface biotinylated and the amount of cell surface MATE1 and OCT2 protein was quantified by western analysis, using standard curves for the V5 epitope. Cell surface MATE1 and OCT2 protein represented 25% and 24%, respectively, of the total expression of these proteins in CHO cells. The number of cell surface transporters was ~55 fmol cm-2 for MATE1 and ~510 fmol cm-2 for OCT2. Dividing these values into the different Jmax values for transport of MPP, metformin, and atenolol mediated by MATE1 and OCT2 resulted in calculated TOR values (±SE, n = 4) of 84.0 ± 22.0 s-1 and 2.9 ± 0.6 s-1; metformin, 461.0 ± 121.0 s-1 and 12.6 ± 2.4 s-1; atenolol, 118.0 ± 31.0 s-1, respectively. These values are consistent with the TOR values determined for a variety of exchangers (NHEs), cotransporters (SGLTs, Lac permease), and uniporters (GLUTs, ENTs).

Attenuated Ochratoxin A Transporter Expression in a Mouse Model of Nonalcoholic Steatohepatitis Protects against Proximal Convoluted Tubule Toxicity.

Ochratoxin A (OTA) is an abundant mycotoxin, yet the toxicological impact of its disposition is not well studied. OTA is an organic anion transporter (OAT) substrate primarily excreted in urine despite a long half-life and extensive protein binding. Altered renal transporter expression during disease, including nonalcoholic steatohepatitis (NASH), may influence response to OTA exposure, but the impact of NASH on OTA toxicokinetics, tissue distribution, and associated nephrotoxicity is unknown. By inducing NASH in fast food-dieted/thioacetamide-exposed mice, we evaluated the effect of NASH on a bolus OTA exposure (12.5 mg/kg by mouth) after 3 days. NASH mice presented with less gross toxicity (44% less body weight loss), and kidney and liver weights of NASH mice were 11% and 24% higher, respectively, than healthy mice. Organ and body weight changes coincided with reduced renal proximal tubule cells vacuolation, degeneration, and necrosis, though no OTA-induced hepatic lesions were found. OTA systemic exposure in NASH mice increased modestly from 5.65 ± 1.10 to 7.95 ± 0.61 mg*h/ml per kg BW, and renal excretion increased robustly from 5.55% ± 0.37% to 13.11% ± 3.10%, relative to healthy mice. Total urinary excretion of OTA increased from 24.41 ± 1.74 to 40.07 ± 9.19 µg in NASH mice, and kidney-bound OTA decreased by ∼30%. Renal OAT isoform expression (OAT1-5) in NASH mice decreased by ∼50% with reduced OTA uptake by proximal convoluted cells. These data suggest that NASH-induced OAT transporter reductions attenuate renal secretion and reabsorption of OTA, increasing OTA urinary excretion and reducing renal exposure, thereby reducing nephrotoxicity in NASH. SIGNIFICANCE STATEMENT: These data suggest a disease-mediated transporter mechanism of altered tissue-specific toxicity after mycotoxin exposure, despite minimal systemic changes to ochratoxin A (OTA) concentrations. Further studies are warranted to evaluate the clinical relevance of this functional model and the potential effect of human nonalcoholic steatohepatitis on OTA and other organic anion substrate toxicity.

Remdesivir and EIDD-1931 Interact with Human Equilibrative Nucleoside Transporters 1 and 2: Implications for Reaching SARS-CoV-2 Viral Sanctuary Sites.

Equilibrative nucleoside transporters (ENTs) are present at the blood-testis barrier (BTB), where they can facilitate antiviral drug disposition to eliminate a sanctuary site for viruses detectable in semen. The purpose of this study was to investigate ENT-drug interactions with three nucleoside analogs, remdesivir, molnupiravir, and molnupiravir's active metabolite, ß-d-N4-hydroxycytidine (EIDD-1931), and four non-nucleoside molecules repurposed as antivirals for coronavirus disease 2019 (COVID-19). The study used three-dimensional pharmacophores for ENT1 and ENT2 substrates and inhibitors and Bayesian machine learning models to identify potential interactions with these transporters. In vitro transport experiments demonstrated that remdesivir was the most potent inhibitor of ENT-mediated [3H]uridine uptake (ENT1 IC50: 39 µM; ENT2 IC50: 77 µM), followed by EIDD-1931 (ENT1 IC50: 259 µM; ENT2 IC50: 467 µM), whereas molnupiravir was a modest inhibitor (ENT1 IC50: 701 µM; ENT2 IC50: 851 µM). Other proposed antivirals failed to inhibit ENT-mediated [3H]uridine uptake below 1 mM. Remdesivir accumulation decreased in the presence of 6-S-[(4-nitrophenyl)methyl]-6-thioinosine (NBMPR) by 30% in ENT1 cells (P = 0.0248) and 27% in ENT2 cells (P = 0.0054). EIDD-1931 accumulation decreased in the presence of NBMPR by 77% in ENT1 cells (P = 0.0463) and by 64% in ENT2 cells (P = 0.0132), which supported computational predictions that both are ENT substrates that may be important for efficacy against COVID-19. NBMPR failed to decrease molnupiravir uptake, suggesting that ENT interaction is likely inhibitory. Our combined computational and in vitro data can be used to identify additional ENT-drug interactions to improve our understanding of drugs that can circumvent the BTB. SIGNIFICANCE STATEMENT: This study identified remdesivir and EIDD-1931 as substrates of equilibrative nucleoside transporters 1 and 2. This provides a potential mechanism for uptake of these drugs into cells and may be important for antiviral potential in the testes and other tissues expressing these transporters.

Cationic Compounds with SARS-CoV-2 Antiviral Activity and Their Interaction with Organic Cation Transporter/Multidrug and Toxin Extruder Secretory Transporters.

In the wake of the COVID-19 pandemic, drug repurposing has been highlighted for rapid introduction of therapeutics. Proposed drugs with activity against SARS-CoV-2 include compounds with positive charges at physiologic pH, making them potential targets for the organic cation secretory transporters of kidney and liver, i.e., the basolateral organic cation transporters, OCT1 and OCT2; and the apical multidrug and toxin extruders, MATE1 and MATE2-K. We selected several compounds proposed to have in vitro activity against SARS-CoV-2 (chloroquine, hydroxychloroquine, quinacrine, tilorone, pyronaridine, cetylpyridinium, and miramistin) to test their interaction with OCT and MATE transporters. We used Bayesian machine learning models to generate predictions for each molecule with each transporter and also experimentally determined IC50 values for each compound against labeled substrate transport into CHO cells that stably expressed OCT2, MATE1, or MATE2-K using three structurally distinct substrates (atenolol, metformin and 1-methyl-4-phenylpyridinium) to assess the impact of substrate structure on inhibitory efficacy. For the OCTs substrate identity influenced IC50 values, although the effect was larger and more systematic for OCT2. In contrast, inhibition of MATE1-mediated transport was largely insensitive to substrate identity. Unlike MATE1, inhibition of MATE2-K was influenced, albeit modestly, by substrate identity. Maximum unbound plasma concentration/IC50 ratios were used to identify potential clinical DDI recommendations; all the compounds interacted with the OCT/MATE secretory pathway, most with sufficient avidity to represent potential DDI issues for secretion of cationic drugs. This should be considered when proposing cationic agents as repurposed antivirals. SIGNIFICANCE STATEMENT: Drugs proposed as potential COVID-19 therapeutics based on in vitro activity data against SARS-CoV-2 include compounds with positive charges at physiological pH, making them potential interactors with the OCT/MATE renal secretory pathway. We tested seven such molecules as inhibitors of OCT1/2 and MATE1/2-K. All the compounds blocked transport activity regardless of substrate used to monitor activity. Suggesting that plasma concentrations achieved by normal clinical application of the test agents could be expected to influence the pharmacokinetics of selected cationic drugs.

Testicular disposition of clofarabine in rats is dependent on equilibrative nucleoside transporters.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children and adolescents. Although the 5-year survival rate is high, some patients respond poorly to chemotherapy or have recurrence in locations such as the testis. The blood-testis barrier (BTB) can prevent complete eradication by limiting chemotherapeutic access and lead to testicular relapse unless a chemotherapeutic is a substrate of drug transporters present at this barrier. Equilibrative nucleoside transporter (ENT) 1 and ENT2 facilitate the movement of substrates across the BTB. Clofarabine is a nucleoside analog used to treat relapsed or refractory ALL. This study investigated the role of ENTs in the testicular disposition of clofarabine. Pharmacological inhibition of the ENTs by 6-nitrobenzylthioinosine (NBMPR) was used to determine ENT contribution to clofarabine transport in primary rat Sertoli cells, in human Sertoli cells, and across the rat BTB. The presence of NBMPR decreased clofarabine uptake by 40% in primary rat Sertoli cells (p = .0329) and by 53% in a human Sertoli cell line (p = .0899). Rats treated with 10 mg/kg intraperitoneal (IP) injection of the NBMPR prodrug, 6-nitrobenzylthioinosine 5'-monophosphate (NBMPR-P), or vehicle, followed by an intravenous (IV) bolus 10 mg/kg dose of clofarabine, showed a trend toward a lower testis concentration of clofarabine than vehicle (1.81 ± 0.59 vs. 2.65 ± 0.92 ng/mg tissue; p = .1160). This suggests that ENTs could be important for clofarabine disposition. Clofarabine may be capable of crossing the human BTB, and its potential use as a first-line treatment to avoid testicular relapse should be considered.