Restoration of the ER stress response protein TDAG51 in hepatocytes mitigates NAFLD in mice.

Endoplasmic reticulum stress is associated with insulin resistance and the development of nonalcoholic fatty liver disease. Deficiency of the endoplasmic reticulum stress response T-cell death-associated gene 51 (TDAG51) (TDAG51-/-) in mice promotes the development of high-fat diet (HFD)-induced obesity, fatty liver, and hepatic insulin resistance. However, whether this effect is due specifically to hepatic TDAG51 deficiency is unknown. Here, we report that hepatic TDAG51 protein levels are consistently reduced in multiple mouse models of liver steatosis and injury as well as in liver biopsies from patients with liver disease compared to normal controls. Delivery of a liver-specific adeno-associated virus (AAV) increased hepatic expression of a TDAG51-GFP fusion protein in WT, TDAG51-/-, and leptin-deficient (ob/ob) mice. Restoration of hepatic TDAG51 protein was sufficient to increase insulin sensitivity while reducing body weight and fatty liver in HFD fed TDAG51-/- mice and in ob/ob mice. TDAG51-/- mice expressing ectopic TDAG51 display improved Akt (Ser473) phosphorylation, post-insulin stimulation. HFD-fed TDAG51-/- mice treated with AAV-TDAG51-GFP displayed reduced lipogenic gene expression, increased beta-oxidation and lowered hepatic and serum triglycerides, findings consistent with reduced liver weight. Further, AAV-TDAG51-GFP-treated TDAG51-/- mice exhibited reduced hepatic precursor and cleaved sterol regulatory-element binding proteins (SREBP-1 and SREBP-2). In vitro studies confirmed the lipid-lowering effect of TDAG51 overexpression in oleic acid-treated Huh7 cells. These studies suggest that maintaining hepatic TDAG51 protein levels represents a viable therapeutic approach for the treatment of obesity and insulin resistance associated with nonalcoholic fatty liver disease.

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.

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.

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.

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.

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.

Strategies to Diagnose Nonalcoholic Steatohepatitis: A Novel Approach to Take Advantage of Pharmacokinetic Alterations.

Nonalcoholic steatohepatitis (NASH) is the progressive form of nonalcoholic fatty liver disease (NAFLD) and is diagnosed by a liver biopsy. Because of the invasiveness of a biopsy, the majority of patients with NASH are undiagnosed. Additionally, the prevalence of NAFLD and NASH creates the need for a simple screening method to differentiate patients with NAFLD versus NASH. Noninvasive strategies for diagnosing NAFLD versus NASH have been developed, typically relying on imaging techniques and endogenous biomarker panels. However, each technique has limitations, and none can accurately predict the associated functional impairment of drug metabolism and disposition. The function of several drug-metabolizing enzymes and drug transporters has been described in NASH that impacts drug pharmacokinetics. The aim of this review is to give an overview of the existing noninvasive strategies to diagnose NASH and to propose a novel strategy based on altered pharmacokinetics using an exogenous biomarker whose disposition and elimination pathways are directly impacted by disease progression. Altered disposition of safe and relatively inert exogenous compounds may provide the sensitivity and specificity needed to differentiate patients with NAFLD and NASH to facilitate a direct indication of hepatic impairment on drug metabolism and prevent subsequent adverse drug reactions. SIGNIFICANCE STATEMENT: This review provides an overview of the main noninvasive techniques (imaging and panels of biomarkers) used to diagnose NAFLD and NASH along with a biopsy. Pharmacokinetic changes have been identified in NASH, and this review proposes a new approach to predict NASH and the related risk of adverse drug reactions based on the assessment of drug elimination disruption using exogenous biomarkers.

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.

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.

Predicting Drug Interactions with Human Equilibrative Nucleoside Transporters 1 and 2 Using Functional Knockout Cell Lines and Bayesian Modeling.

Equilibrative nucleoside transporters (ENTs) 1 and 2 facilitate nucleoside transport across the blood-testis barrier (BTB). Improving drug entry into the testes with drugs that use endogenous transport pathways may lead to more effective treatments for diseases within the reproductive tract. In this study, CRISPR/CRISPR-associated protein 9 was used to generate HeLa cell lines in which ENT expression was limited to ENT1 or ENT2. We characterized uridine transport in these cell lines and generated Bayesian models to predict interactions with the ENTs. Quantification of [3H]uridine uptake in the presence of the ENT-specific inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBMPR) demonstrated functional loss of each transporter. Nine nucleoside reverse-transcriptase inhibitors and 37 nucleoside/heterocycle analogs were evaluated to identify ENT interactions. Twenty-one compounds inhibited uridine uptake and abacavir, nevirapine, ticagrelor, and uridine triacetate had different IC50 values for ENT1 and ENT2. Total accumulation of four identified inhibitors was measured with and without NBMPR to determine whether there was ENT-mediated transport. Clofarabine and cladribine were ENT1 and ENT2 substrates, whereas nevirapine and lexibulin were ENT1 and ENT2 nontransported inhibitors. Bayesian models generated using Assay Central machine learning software yielded reasonably high internal validation performance (receiver operator characteristic > 0.7). ENT1 IC50-based models were generated from ChEMBL; subvalidations using this training data set correctly predicted 58% of inhibitors when analyzing activity by percent uptake and 63% when using estimated-IC50 values. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates and can thereby circumvent the BTB through this transepithelial transport pathway in Sertoli cells. SIGNIFICANCE STATEMENT: This study is the first to predict drug interactions with equilibrative nucleoside transporter (ENT) 1 and ENT2 using Bayesian modeling. Novel CRISPR/CRISPR-associated protein 9 functional knockouts of ENT1 and ENT2 in HeLa S3 cells were generated and characterized. Determining drug interactions with these transporters can be useful in identifying and predicting compounds that are ENT1 and ENT2 substrates and can circumvent the blood-testis barrier through this transepithelial transport pathway in Sertoli cells.

Multiple Computational Approaches for Predicting Drug Interactions with Human Equilibrative Nucleoside Transporter 1.

Equilibrativenucleoside transporters (ENTs) participate in the pharmacokinetics and disposition of nucleoside analog drugs. Understanding drug interactions with the ENTs may inform and facilitate the development of new drugs, including chemotherapeutics and antivirals that require access to sanctuary sites such as the male genital tract. This study created three-dimensional pharmacophores for ENT1 and ENT2 substrates and inhibitors using Kt and IC50 data curated from the literature. Substrate pharmacophores for ENT1 and ENT2 are distinct, with partial overlap of hydrogen bond donors, whereas the inhibitor pharmacophores predominantly feature hydrogen bond acceptors. Mizoribine and ribavirin mapped to the ENT1 substrate pharmacophore and proved to be substrates of the ENTs. The presence of the ENT-specific inhibitor 6-S-[(4-nitrophenyl)methyl]-6-thioinosine (NBMPR) decreased mizoribine accumulation in ENT1 and ENT2 cells (ENT1, ∼70% decrease, P = 0.0046; ENT2, ∼50% decrease, P = 0.0012). NBMPR also decreased ribavirin accumulation in ENT1 and ENT2 cells (ENT1: ∼50% decrease, P = 0.0498; ENT2: ∼30% decrease, P = 0.0125). Darunavir mapped to the ENT1 inhibitor pharmacophore and NBMPR did not significantly influence darunavir accumulation in either ENT1 or ENT2 cells (ENT1: P = 0.28; ENT2: P = 0.53), indicating that darunavir's interaction with the ENTs is limited to inhibition. These computational and in vitro models can inform compound selection in the drug discovery and development process, thereby reducing time and expense of identification and optimization of ENT-interacting compounds. SIGNIFICANCE STATEMENT: This study developed computational models of human equilibrative nucleoside transporters (ENTs) to predict drug interactions and validated these models with two compounds in vitro. Identification and prediction of ENT1 and ENT2 substrates allows for the determination of drugs that can penetrate tissues expressing these transporters.

A Two-Way Interaction between Methotrexate and the Gut Microbiota of Male Sprague-Dawley Rats.

Methotrexate (MTX) is a chemotherapeutic agent that can cause a range of toxic side effects including gastrointestinal damage, hepatotoxicity, myelosuppression, and nephrotoxicity and has potentially complex interactions with the gut microbiome. Following untargeted UPLC-qtof-MS analysis of urine and fecal samples from male Sprague-Dawley rats administered at either 0, 10, 40, or 100 mg/kg of MTX, dose-dependent changes in the endogenous metabolite profiles were detected. Semiquantitative targeted UPLC-MS detected MTX excreted in urine as well as MTX and two metabolites, 2,4-diamino-N-10-methylpteroic acid (DAMPA) and 7-hydroxy-MTX, in the feces. DAMPA is produced by the bacterial enzyme carboxypeptidase glutamate 2 (CPDG2) in the gut. Microbiota profiling (16S rRNA gene amplicon sequencing) of fecal samples showed an increase in the relative abundance of Firmicutes over the Bacteroidetes at low doses of MTX but the reverse at high doses. Firmicutes relative abundance was positively correlated with DAMPA excretion in feces at 48 h, which were both lower at 100 mg/kg compared to that seen at 40 mg/kg. Overall, chronic exposure to MTX appears to induce community and functionality changes in the intestinal microbiota, inducing downstream perturbations in CPDG2 activity, and thus may delay MTX detoxication to DAMPA. This reduction in metabolic clearance might be associated with increased gastrointestinal toxicity.

Nucleoside Reverse Transcriptase Inhibitor Interaction with Human Equilibrative Nucleoside Transporters 1 and 2.

Equilibrative nucleoside transporters (ENTs) transport nucleosides across the blood-testis barrier (BTB). ENTs are of interest to study the disposition of nucleoside reverse-transcriptase inhibitors (NRTIs) in the human male genital tract because of their similarity in structure to nucleosides. HeLa S3 cells express ENT1 and ENT2 and were used to compare relative interactions of these transporters with selected NRTIs. Inhibition of [3H]uridine uptake by NBMPR was biphasic, with IC50 values of 11.3 nM for ENT1 and 9.6 µM for ENT2. Uptake measured with 100 nM NBMPR represented ENT2-mediated transport; subtracting that from total uptake represented ENT1-mediated transport. The kinetics of ENT1- and ENT2-mediated [3H]uridine uptake revealed no difference in Jmax (16.53 and 30.40 pmol cm-2 min-1) and an eightfold difference in Kt (13.6 and 108.9 µM). The resulting fivefold difference in intrinsic clearance (Jmax/Kt) for ENT1- and ENT2 transport accounted for observed inhibition of [3H]uridine uptake by 100 nM NBMPR. Millimolar concentrations of the NRTIs emtricitabine, didanosine, lamivudine, stavudine, tenofovir disoproxil, and zalcitabine had no effect on ENT transport activity, whereas abacavir, entecavir, and zidovudine inhibited both transporters with IC50 values of ∼200 µM, 2.5 mM, and 2 mM, respectively. Using liquid chromatography-tandem mass spectrometry and [3H] compounds, the data suggest that entecavir is an ENT substrate, abacavir is an ENT inhibitor, and zidovudine uptake is carrier-mediated, although not an ENT substrate. These data show that HeLa S3 cells can be used to explore complex transporter selectivity and are an adequate model for studying ENTs present at the BTB. SIGNIFICANCE STATEMENT: This study characterizes an in vitro model using S-[(4-nitrophenyl)methyl]-6-thioinosine to differentiate between equilibrative nucleoside transporter (ENT) 1- and ENT2-mediated uridine transport in HeLa cells. This provides a method to assess the influence of nucleoside reverse-transcriptase inhibitors on natively expressed transporter function. Determining substrate selectivity of the ENTs in HeLa cells can be effectively translated into the activity of these transporters in Sertoli cells that comprise the blood-testis barrier, thereby assisting targeted drug development of compounds capable of circumventing the blood-testis barrier.

Folate receptor-beta expression as a diagnostic target in human & rodent nonalcoholic steatohepatitis.

INTRODUCTION: Nonalcoholic steatohepatitis (NASH) afflicts 20-36% of individuals with nonalcoholic fatty liver disease (NAFLD). A lipotoxic hepatic environment, altered innate immune signaling and inflammation are defining features of progression to NASH. Activated resident liver macrophages express folate receptor beta (FR-ß) which may be an indicator of progression from steatosis to NASH. The goals of this study were to characterize FR-ß protein expression in human NAFLD and rodent models of NASH, and demonstrate liver targeting of an FR-ß imaging agent to the liver of a rodent NASH model using FR-ß. METHODS: Rat liver lysates from methionine choline deficient (MCD) fed rats, high fat diet (HFD) and methionine choline sufficient (MC+) rat controls were analyzed for hepatic FR-ß protein. The FR-ß-targeted agent, Etarfolatide was injected into MCD and MC + -fed C57BL/6 mice for efficient FastSPECT hepatic imaging. Additionally, FR-ß expression across the stages of human NAFLD from normal to NASH was assessed. RESULTS: FastSPECT images show targeting of Etarfolatide to the liver of mice fed 8 weeks of MCD diet but not control-fed mice. The MCD rat model exhibited significantly increased protein expression of hepatic FR-ß in contrast to HFD or normal samples. Similarly human liver samples categorized as NASH Fatty or NASH Not Fatty showed elevated FR-ß protein when compared to normal liver. FR-ß transcript expression levels were elevated across both NASH Fatty and NASH Not Fatty samples. CONCLUSION: The findings in this study indicate that FR-ß expression in NASH may be harnessed to target agents directly to the liver.

Gene-by-Environment Interaction of Bcrp-/- and Methionine- and Choline-Deficient Diet-Induced Nonalcoholic Steatohepatitis Alters SN-38 Disposition.

Disease progression to nonalcoholic steatohepatitis (NASH) has profound effects on the expression and function of drug-metabolizing enzymes and transporters, which provide a mechanistic basis for variable drug response. Breast cancer resistance protein (BCRP), a biliary efflux transporter, exhibits increased liver mRNA expression in NASH patients and preclinical NASH models, but the impact on function is unknown. It was shown that the transport capacity of multidrug resistance protein 2 (MRP2) is decreased in NASH. SN-38, the active irinotecan metabolite, is reported to be a substrate for Bcrp, whereas SN-38 glucuronide (SN-38G) is a Mrp2 substrate. The purpose of this study was to determine the function of Bcrp in NASH through alterations in the disposition of SN-38 and SN-38G in a Bcrp knockout (Bcrp-/- KO) and methionine- and choline-deficient (MCD) model of NASH. Sprague Dawley [wild-type (WT)] rats and Bcrp-/- rats were fed either a methionine- and choline-sufficient (control) or MCD diet for 8 weeks to induce NASH. SN-38 (10 mg/kg) was administered i.v., and blood and bile were collected for quantification by liquid chromatography-tandem mass spectrometry. In Bcrp-/- rats on the MCD diet, biliary efflux of SN-38 decreased to 31.9%, and efflux of SN-38G decreased to 38.7% of control, but WT-MCD and KO-Control were unaffected. These data indicate that Bcrp is not solely responsible for SN-38 biliary efflux, but rather implicate a combined role for BCRP and MRP2. Furthermore, the disposition of SN-38 and SN-38G is altered by Bcrp-/- and NASH in a gene-by-environment interaction and may result in variable drug response to irinotecan therapy in polymorphic patients.

Transepithelial transport across the blood-testis barrier.

The blood-testis barrier protects developing germ cells by limiting the entry of xenobiotics into the adluminal compartment. There is strong evidence that the male genital tract can serve as a sanctuary site, an area of the body where tumors or viruses are able to survive treatments because most drugs are unable to reach therapeutic concentrations. Recent work has classified the expression and localization of endogenous transporters in the male genital tract as well as the discovery of a transepithelial transport pathway as the molecular mechanism by which nucleoside analogs may be able to circumvent the blood-testis barrier. Designing drug therapies that utilize transepithelial transport pathways may improve drug disposition to this sanctuary site. Strategies that improve disposition into the male genital tract could reduce the rate of testicular relapse, decrease viral load in semen, and improve therapeutic strategies for male fertility.

Misregulation of membrane trafficking processes in human nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) remodels the expression and function of genes and proteins that are critical for drug disposition. This study sought to determine whether disruption of membrane protein trafficking pathways in human NASH contributes to altered localization of multidrug resistance-associated protein 2 (MRP2). A comprehensive immunoblot analysis assessed the phosphorylation, membrane translocation, and expression of transporter membrane insertion regulators, including several protein kinases (PK), radixin, MARCKS, and Rab11. Radixin exhibited a decreased phosphorylation and total expression, whereas Rab11 had an increased membrane localization. PKCδ, PKCα, and PKA had increased membrane activation, whereas PKCε had a decreased phosphorylation and membrane expression. Radixin dephosphorylation may activate MRP2 membrane retrieval in NASH; however, the activation of Rab11/PKCδ and PKA/PKCα suggest an activation of membrane insertion pathways as well. Overall these data suggest an altered regulation of protein trafficking in human NASH, although other processes may be involved in the regulation of MRP2 localization.