Metabolic dysfunction-associated fatty liver disease and metabolic dysfunction-associated steatohepatitis (MASH) are the most prevalent liver diseases globally. As MASH progresses, changes in expression of xenobiotic transporters significantly alter drug disposition and subsequent increased risk of toxicity. Patients with MASH and co-occurring chronic hepatitis B virus are becoming increasingly prevalent, presenting a unique challenge in clinical practice. Adefovir (ADEF) is an antiviral drug used to treat hepatitis B virus infection and is a substrate of organic anion transporters (OATs). Using a fast-food diet and thioacetamide-induced (FFDTH) MASH model that reflects human OAT expression, we evaluated the effect of MASH on the disposition of ADEF (7 mg/kg retro-orbital). FFDTH mice exhibited characteristic hepatic macrosteatosis, hepatocyte ballooning, lobular inflammation, weight loss, and had elevated liver to body weight ratios, but there were no signs of kidney injury, such as inflammation or fibrosis. Renal OAT 3 expression in FFDTH mice was decreased, with reduced uptake of ADEF by proximal tubule cells and increased plasma retention. Elimination into the urine was decreased compared to healthy mice, due in part to decreased expression of both Mrp4 and Oat5. Glomerular filtration rate was also decreased in FFDTH mice, which could further explain the change in ADEF disposition. Despite these changes, no significant difference in hepatic or renal accumulation was observed. These data suggest that renal disposition of ADEF may be altered in humans with MASH due to the similarity of OAT transporter expression in the FFDTH model of MASH. SIGNIFICANCE STATEMENT: As metabolic liver disease and chronic hepatitis B increasingly coexist, understanding how metabolic dysfunction-associated steatohepatitis alters drug handling is critical for patient safety. The study findings demonstrate that metabolic dysfunction-associated steatohepatitis significantly impairs renal transporter-mediated elimination of adefovir, highlighting the need to consider altered kidney drug disposition and potential dose adjustments in this growing patient population.

Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease with an urgent need for reliable, noninvasive biomarkers that can serve as reasonably likely surrogate endpoints, as diagnosis remains limited by the invasiveness of liver biopsy and the inaccuracy of existing noninvasive tests. MASH disrupts hepatic and renal transporter expression and function, leading to increased systemic retention of drug substrates. Because ezetimibe-glucuronide (EZEG) is a substrate of transporters altered in MASH, we evaluated whether EZEG could serve as a noninvasive, mechanism-based exogenous diagnostic function test. Plasma and urine EZEG concentrations were quantified by liquid chromatography-tandem mass spectrometry in biopsy-confirmed patients with nonalcoholic fatty liver disease activity scores (NAS) of 0-5 after administration of a 1 mg subtherapeutic dose of ezetimibe. Patients with MASH (NAS = 5, n = 8) exhibited approximately 4.5-fold higher plasma EZEG concentrations than non-MASH individuals (NAS 0-4, n = 45), demonstrating excellent discrimination with an area under the receiver operating characteristic curve of 0.92 (95% CI, 0.79-1.00) using the 60-minute postdose concentration. Plasma EZEG was strongly associated with hepatocyte ballooning, the U.S. Food and Drug Administration-defining and most difficult-to-detect histologic feature of MASH, supporting mechanistic specificity. In contrast, urinary EZEG excretion showed only modest increases and poor diagnostic discrimination. These findings indicate that MASH-associated transporter dysfunction leads to systemic EZEG accumulation, supporting its use as a mechanism-based diagnostic function test and a promising noninvasive strategy for early detection and disease stratification. SIGNIFICANCE STATEMENT: Metabolic dysfunction-associated steatohepatitis (MASH) lacks reliable noninvasive tests for diagnosis and therapeutic development. This study demonstrates that plasma ezetimibe-glucuronide, measured after a subtherapeutic ezetimibe dose, sensitively detects MASH-associated transporter dysfunction and strongly correlates with hepatocyte ballooning, the defining histologic feature of MASH. This mechanism-based probe-drug approach provides a highly discriminative, noninvasive strategy to support early disease detection and patient stratification.

Regulatory guidances (eg, FDA and European Medicines Agency) require an understanding of the interactions of novel drugs, natural products, and environmental toxicants with key transporters to avoid compounds with undesirable side effects. Computational approaches to predict such interactions using machine learning models trained on in vitro data could prevent compounds that are transporter inhibitors with potential for drug-drug interactions from reaching the more costly development stages. We now describe the curation and machine learning model building for the transporters covered in the FDA guidance (organic anion transporter 1, organic anion transporter 3, organic cation transporter 2, organic anion transporting polypeptide 1B1, organic anion transporting polypeptide 1B3, P-glycoprotein, breast cancer resistance protein, multidrug and toxin extruder 1, and multidrug and toxin extruder protein 2K) that enabled the creation of MegaTrans, a web-based software product that enables users to input molecules and predict the inhibition of transporters of interest. Each dataset was used to generate machine learning models using extended-connectivity fingerprint 6 with 5-fold cross-validation. These models were also applied to a dataset (N = 39) derived from recent FDA-approved drugs and chemical property overlap with our models was determined using t-distributed stochastic neighbor embedding. In aggregate, the model predictions had an accuracy of 66%, and a second round of transporter models displayed an accuracy of 76%. Our application of literature in vitro data on transporters for building these machine learning models can be used for compound profiling, representing an approach which could minimize the need for in vitro screening experiments and help to prioritize resources. Such applications of machine learning models could also be integrated at the drug design stages in artificial intelligence approaches, including generative artificial intelligence, further broadening the impact. SIGNIFICANCE STATEMENT: MegaTrans is a computational tool to predict the inhibition of small molecules across a range of clinically relevant human drug transporters, allowing visualization of model metrics, highlighting atom-bond contributions, and visualizing chemical space overlaps between queries and model datasets.

Proteolysis-targeting chimeras (PROTACs) are gathering considerable interest due to their ability to address previously undruggable targets. We were keen to understand the potential for these very large molecules to interact with transporters that may influence absorption, distribution, metabolism, and excretion or toxicity properties and to what extent this may be predictable using machine learning models. Consequently, we tested a set of PROTACs against several human drug transporters, namely the equilibrative nucleoside (ENT) family transporters ENT1 and ENT2, which have been directly implicated in the uptake of anticancer or antiviral drugs into target cells. We describe the dramatic inhibition observed for ENT1 and ENT2 but not for the unrelated transporter organic anion transporter 4. In addition, we report dose-response relationships for ENT1 to show some PROTACs are nanomolar inhibitors. We also explored the chemistry space of small molecules tested against ENT1 and ENT2 and compared them with PROTACs to illustrate that they are found on the periphery and close to other larger small molecules. While PROTACs are thought of as a dissimilar class to small molecules, it may be possible to bring them closer to those Food and Drug Administration-approved orally available large molecules, and in turn, increase their oral bioavailability. The outcomes of these combined in vitro and computational assessments could influence PROTAC development, be useful for their repurposing as ENT1 inhibitors for several disease indications beyond their primary one, and be used for transporter machine learning model generation and evaluation. Significance Statement Proteolysis-targeting chimeras are an increasingly popular class of molecules for which we do not have a complete picture of their absorption, distribution, metabolism, and excretion or toxicity properties. For example, their interactions with uptake and efflux transporters are unknown. Here, we provide evidence that many proteolysis-targeting chimeras act as inhibitors of equilibrative nucleoside transporters 1 and 2. We hope to stimulate further study of their potential for inhibition of other transporters.

Equilibrative nucleoside transporters (ENTs) facilitate the equilibrative movement of nucleosides and nucleobases across cell membranes in a sodium-independent manner. ENT1 (SLC29A1) and ENT2 (SLC29A2) also transport nucleoside analogs and can affect the pharmacokinetics and pharmacodynamics of drugs used in cancer, viral infections, and inflammatory disorders. ENT1 and ENT2 may be differentiated functionally by their sensitivity to inhibition by nitrobenzylthioinosine (NBMPR), and we used this difference in NBMPR sensitivity to create a HeLa-based ENT2 inhibition assay. We then screened a library of 1600 diverse compounds composed of drugs and natural products for inhibition against ENT1 and ENT2, selecting a subset of compounds for side-by-side comparison of dose-response studies. We used these screening data to build machine learning models for ENT1 and ENT2 inhibition, employing dataset balancing and conformal prediction to adjust for the asymmetrical nature of the data. A random forest model predicted a prospective test set of 44 additional molecules (from the MedChem Express Drug Repurposing Library [2700 compounds]) as potential ENT1 inhibitors with 59% accuracy. This resulted in the identification of the Food and Drug Administration-approved drugs isradipine, avanafil, and istradefylline as inhibitors of ENT1. These new experimental and computational methods and models for these clinically relevant transporters can be used to evaluate drug-transporter interactions early in drug discovery, before testing in vivo. SIGNIFICANCE STATEMENT: Recent regulatory guidance have suggest the inclusion of the equilibrative nucleoside transporters (eg, ENT1 and ENT2) as transporters with emerging clinical relevance for in vitro and in vivo assessment. We have screened over 1600 diverse molecules, allowing us to build machine learning models that in turn were further used to make predictions to validate the models. Our combined experimental and machine learning approach resulted in the identification of multiple Food and Drug Administration-approved medications as inhibitors of ENT1 or ENT2.

Valsartan (VAL) is commonly prescribed for patients with cardiovascular disease (CVD) to lower blood pressure, reduce heart failure risk, and prevent heart attacks or strokes by blocking the effects of angiotensin II. Many patients with CVD also suffer from metabolic dysfunction-associated steatohepatitis (MASH), which disrupts several xenobiotic transporters, affecting the pharmacokinetics of numerous drugs. Medications used in patients to treat comorbidities associated with MASH may be subject to this altered disposition and potential toxicity. This study aimed to assess how MASH alters the pharmacokinetics of VAL using a rodent model that mimics human MASH. MASH was induced in rats via a methionine- and choline-deficient (MCD) diet. Rats received VAL-a substrate of organic anion-transporting polypeptide (OATP) 1B1/1B3 and reported for multidrug resistance-associated protein-2-(2 mg/kg) through intravenous injection to isolate hepatic transport processes, and bile, serum, and liver concentrations measured using liquid chromatography-tandem mass spectrometry. Consistent with MASH progression, MCD rats presented with more gross pathology, including increased liver-to-body weight ratios, along with macrosteatosis, hepatocyte ballooning, and lobular inflammation. In MCD rats, the expression of Oatp1b2 was significantly reduced, and Mrp2 was internalized, resulting in higher systemic exposure to VAL compared with controls. Additionally, cumulative biliary excretion of VAL was lower in MCD rats. To further assess VAL disposition in MASH, transport kinetics were evaluated in human embryonic kidney 293 cells overexpressing OATP1B1 or OATP1B3, revealing similar affinity for VAL between both transporters. These findings suggest that changes in OATP function in MASH may alter VAL pharmacokinetics, which may have implications for personalized treatments. SIGNIFICANCE STATEMENT: Although expression of drug transporters in metabolic dysfunction-associated steatohepatitis (MASH) has been explored, the combined effect between MASH and genetic loss of transporters on the disposition of sartan drugs has not been determined. This study applied liquid chromatography-tandem mass spectrometry analyses and immunohistological staining to assess drug disposition and identify alterations to drug transporters in rodents on a methionine- and choline-deficient diet. The observations made in this study have significant implications regarding its disposition in the context of hepatic dysfunction associated with MASH.

Human Organic Anion Transporter 4 (OAT4) is predominantly expressed in the kidneys, particularly in the apical membrane of the proximal tubule cells. This transporter is involved in the renal handling of endogenous and exogenous organic anions (OAs), making it an important transporter for drug-drug interactions (DDIs). To better understand OAT4-compound interactions, we generated single concentration (25 μM) in vitro inhibition data for over 1400 small molecules against the uptake of the fluorescent OA 6-carboxyfluorescein (6-CF) in Chinese hamster ovary (CHO) cells. Several drugs exhibiting higher than 50% inhibition in this initial screen were selected to determine IC50 values against three structurally distinct OAT4 substrates: estrone sulfate (ES), ochratoxin A (OTA), and 6-CF. These IC50 values were then compared to the drug plasma concentration as per the 2020 FDA drug-drug interaction (DDI) guidance. Several screened compounds, including some not previously reported, emerged as novel inhibitors of OAT4. These data were also used to build machine learning classification models to predict the activity of potential OAT4 inhibitors. We compared multiple machine learning algorithms and data cleaning techniques to model these screening data and investigated the utility of conformal predictors to predict OAT4 inhibition of a leave-out set. These experimental and computational approaches allowed us to model diverse and unbalanced data to enable predictions for DDIs mediated by this transporter.

Organic anions (OAs) are compounds including drugs or toxicants that are negatively charged at physiologic 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 Michaelis constant (Kt) and maximal rate of mediated substrate transport (Jmax) values were very similar for both hOAT4 and mOat5: Kt 3.9 and 7.2 μM, respectively, and 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 organic anion transporter (OAT4) and mouse Oat5. Our data revealed a similar selectivity profile for ochratoxin A (OTA) reabsorption and secretion by these two transporters, thereby supporting the hypothesis that hOAT4 and mOat5, although 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.

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.

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.

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.

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.

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/dbMCD (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.

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.

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.

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 timedependent 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.

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).

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.

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.