Knockout Transporter Cell Lines to Assess Substrate Potential Towards Efflux Transporters.

P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance transporter 2 (MRP2) are efflux transporters involved in the absorption, excretion, and distribution of drugs. Bidirectional cell assays are recognized models for evaluating the potential of new drugs as substrates or inhibitors of efflux transporters. However, the assays are complicated by a lack of selective substrates and/or inhibitors, as well simultaneous expression of several efflux transporters in cell lines used in efflux models. This project aims to evaluate an in vitro efflux cell assay employing model substrates and inhibitors of P-gp, BCRP and MRP2 with knockout (KO) cell lines. The efflux ratios (ER) of P-gp (digoxin, paclitaxel), BCRP (prazosin, rosuvastatin), MRP2 (etoposide, olmesartan) and mixed (methotrexate, mitoxantrone) substrates were determined in wild-type C2BBe1 and KO cells. For digoxin and paclitaxel, the ER decreased to less than 2 in the cell lines lacking P-gp expression. The ER decreased to less than 3 for prazosin and less than 2 for rosuvastatin in the cell lines lacking BCRP expression. For etoposide and olmesartan, the ER decreased to less than 2 in the cell lines lacking MRP2 expression. The ER of methotrexate and mitoxantrone decreased in single- and double-KO cells without BCRP and MRP2 expression. These results show that KO cell lines have the potential to better interpret complex drug-transporter interactions without depending upon multi-targeted inhibitors or overlapping substrates. For drugs that are substrates of multiple transporters, the single- and double-KO cells may be used to assess their affinities for the different transporters.

Characterization of P-glycoprotein orthologs from human, sheep, pig, dog, and cat.

The ATP-binding cassette transporter P-glycoprotein (P-gp) limits the oral bioavailability of many drugs. Although P-gp has been well studied in humans and mice, little is known about the substrate specificities of many of its species orthologs. To address this, we performed in vitro analysis of P-gp transporter function using HEK293 cells stably expressing human, ovine, porcine, canine, and feline P-gp. We also employed a human physiologically based pharmacokinetic (PBPK) model to assess variations in digoxin exposure resulting from altered P-gp function. Compared to human P-gp, sheep P-gp had significantly less digoxin efflux (2.3-fold ±0.04 vs. 1.8-fold ±0.03, p < .0001) and all species orthologs had significantly less quinidine efflux compared with human P-gp (p < .05). Human P-gp also had significantly greater efflux of talinolol compared to sheep and dog P-gp (1.9-fold ±0.04 vs. 1.6-fold ±0.06, p = .003 and 1.6-fold ±0.05, p = .0002, respectively). P-gp expression protected all lines against paclitaxel-induced toxicity, with sheep P-gp being significantly less protective. The inhibitor verapamil demonstrated dose-dependent inhibition of all P-gp orthologs. Finally, a PBPK model showed digoxin exposure was sensitive to altered P-gp activity. Overall, our study found that species differences in this major drug transporter exist and that the appropriate species ortholog of P-gp should be evaluated during veterinary drug development.

Quantitative Characterization of Clinically Relevant Drug-Metabolizing Enzymes and Transporters in Rat Liver and Intestinal Segments for Applications in PBPK Modeling.

Rats are extensively used as a preclinical model for assessing drug pharmacokinetics (PK) and tissue distribution; however, successful translation of the rat data requires information on the differences in drug metabolism and transport mechanisms between rats and humans. To partly fill this knowledge gap, we quantified clinically relevant drug-metabolizing enzymes and transporters (DMETs) in the liver and different intestinal segments of Sprague-Dawley rats. The levels of DMET proteins in rats were quantified using the global proteomics-based total protein approach (TPA) and targeted proteomics. The abundance of the major DMET proteins was largely comparable using quantitative global and targeted proteomics. However, global proteomics-based TPA was able to detect and quantify a comprehensive list of 66 DMET proteins in the liver and 37 DMET proteins in the intestinal segments of SD rats without the need for peptide standards. Cytochrome P450 (Cyp) and UDP-glycosyltransferase (Ugt) enzymes were mainly detected in the liver with the abundance ranging from 8 to 6502 and 74 to 2558 pmol/g tissue. P-gp abundance was higher in the intestine (124.1 pmol/g) as compared to that in the liver (26.6 pmol/g) using the targeted analysis. Breast cancer resistance protein (Bcrp) was most abundant in the intestinal segments, whereas organic anion transporting polypeptides (Oatp) 1a1, 1a4, 1b2, and 2a1 and multidrug resistance proteins (Mrp) 2 and 6 were predominantly detected in the liver. To demonstrate the utility of these data, we modeled digoxin PK by integrating protein abundance of P-gp and Cyp3a2 into a physiologically based PK (PBPK) model constructed using PK-Sim software. The model was able to reliably predict the systemic as well as tissue concentrations of digoxin in rats. These findings suggest that proteomics-informed PBPK models in preclinical species can allow mechanistic PK predictions in animal models including tissue drug concentrations.

Effect of Oregon grape root extracts on P-glycoprotein mediated transport in in vitro cell lines.

Purpose: This study aims to investigate the potential of Oregon grape root extracts to modulate the activity of P-glycoprotein. Methods: We performed 3H-CsA or 3H-digoxin transport experiments in the absence or presence of two sources of Oregon grape root extracts (E1 and E2), berberine or berbamine in Caco-2 and MDCKII-MDR1 cells. In addition, real time quantitative polymerase chain reaction (RT-PCR) was performed in Caco-2 and LS-180 cells to investigate the mechanism of modulating P-glycoprotein. Results: Our results showed that in Caco-2 cells, Oregon grape root extracts (E1 and E2) (0.1-1 mg/mL) inhibited the efflux of CsA and digoxin in a dose-dependent manner. However, 0.05 mg/mL E1 significantly increased the absorption of digoxin. Ten µM berberine and 30 µM berbamine significantly reduced the efflux of CsA, while no measurable effect of berberine was observed with digoxin. In the MDCKII-MDR1 cells, 10 µM berberine and 30 µM berbamine inhibited the efflux of CsA and digoxin. Lastly, in real time RT-PCR study, Oregon grape root extract (0.1 mg/mL) up-regulated mRNA levels of human MDR1 in Caco-2 and LS-180 cells at 24 h. Conclusion: Our study showed that Oregon grape root extracts modulated P-glycoprotein, thereby may affect the bioavailability of drugs that are substrates of P-glycoprotein.

[Digoxin alleviates pulmonary fibrosis by regulating phosphatidylinositol-3-kinase/Akt signaling through inhibiting the activation of fibroblast: an in vivo and in vitro experiment].

OBJECTIVE: To investigate the effect of digoxin on bleomycin-induced pulmonary fibrosis in mice, and investigate its possible mechanism through in vitro and in vivo experiments. METHODS: (1) In vivo experiment: 60 C57/BL6J mice were randomly divided into control group, pulmonary fibrosis model group (model group), pirfenidone (300 mg/kg) group, digoxin 1.0 mg/kg and 0.2 mg/kg groups, with 12 mice in each group. The pulmonary fibrosis model of mice was reproduced by single intratracheal infusion of bleomycin (5 mg/kg). The control group was given the same amount of sterile normal saline. From the next day after modeling, each group was received corresponding drugs by intragastric administration once a day for 28 days. Control group and model group were given the same amount of normal saline. The mice were sacrificed and the lung tissue was collected to detect the lung coefficient. After hematoxylin-eosin (HE) and Masson staining, the lung tissue morphology and collagen changes were observed under light microscope. Immunohistochemistry was used to detect the positive expressions of α-smooth muscle actin (α-SMA) and extracellular matrix (ECM) collagen (COL-I and COL-III) in lung tissue. The protein expressions of ECM fibronectin (FN), transforming growth factor-ß (TGF-ß) and phosphorylation of Smad3 (p-Smad3) in lung tissue were detected by Western blotting. (2) In vitro experiment: human embryonic lung fibroblast-1 (HFL-1) cells were cultured and divided into blank control group, fibroblast activation model group (model group), pirfenidone (2.5 mmol/L) group and digoxin 100 nmol/L and 50 nmol/L groups when cell density reached 70%-90%. After 3-hour treatment with corresponding drugs, except blank control group, the other groups were treated with TGF-ß for 48 hours to establish fibroblast activation model. The expressions of α-SMA, FN and p-Smad3 proteins and the phosphorylations of phosphatidylinositol-3-kinase (PI3K)/Akt pathway proteins PI3K and Akt (p-PI3K, p-Akt) were detected by Western blotting. RESULTS: (1) In vivo, compared with the control group, the alveolar structure of mice in the model group was significantly damaged, a large number of inflammatory cells infiltrated, collagen deposition in the lung interstitium was increased, the deposition of ECM in the lung tissue was also increased, and the expressions of α-SMA, FN, TGF-ß and p-Smad3 protein were increased, indicating that the model of bleomycin-induced pulmonary fibrosis in mice was successfully prepared. Compared with the model group, digoxin significantly inhibited airway inflammation and collagen fiber deposition, reduced ECM deposition, and decreased the protein expressions of α-SMA, FN, TGF-ß and p-Smad3, while the effect was better than that of the pirfenidone group, and the digoxin 1.0 mg/kg group had a better effect except FN [α-SMA (A value): 5.37±1.10 vs. 9.51±1.66, TGF-ß protein (TGF-ß/GAPDH): 0.09±0.04 vs. 0.33±0.23, p-Smad3 protein (p-Smad3/GAPDH): 0.05±0.01 vs. 0.20±0.07, all P < 0.01]. (2) In vitro, compared with the blank control group, the expressions of FN, α-SMA, p-Smad3 and PI3K/Akt signaling proteins in the model group were increased, indicating that the fibroblast activation model induced by TGF-ß was successfully reproduced. Compared with the model group, digoxin significantly inhibited fibroblast activation, and decreased the expressions of FN, α-SMA, p-Smad3, and PI3K/Akt pathway proteins, moreover, the effect was better than that of the pirfenidone group, and decreased FN, SMA and p-Akt protein expressions were more obvious in digoxin 100 nmol/L group [FN protein (FN/GAPDH): 0.21±0.15 vs. 0.88±0.22, α-SMA protein (α-SMA/GAPDH): 0.20±0.01 vs. 0.50±0.08, p-Akt protein (p-Akt/GAPDH): 0.30±0.01 vs. 0.65±0.10, all P < 0.01]. CONCLUSIONS: Digoxin could suppress the pulmonary fibrosis in mice induced by bleomycin, which might be associated with the regulation of fibroblast activation via suppressing PI3K/Akt signaling pathway in a dose-dependent manner.

Oral digoxin effects on exercise performance, K+ regulation and skeletal muscle Na+ ,K+ -ATPase in healthy humans.

We investigated whether digoxin lowered muscle Na+ ,K+ -ATPase (NKA), impaired muscle performance and exacerbated exercise K+ disturbances. Ten healthy adults ingested digoxin (0.25 mg; DIG) or placebo (CON) for 14 days and performed quadriceps strength and fatiguability, finger flexion (FF, 105%peak-workrate , 3 × 1 min, fourth bout to fatigue) and leg cycling (LC, 10 min at 33% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ and 67% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ , 90% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ to fatigue) trials using a double-blind, crossover, randomised, counter-balanced design. Arterial (a) and antecubital venous (v) blood was sampled (FF, LC) and muscle biopsied (LC, rest, 67% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ , fatigue, 3 h after exercise). In DIG, in resting muscle, [3 H]-ouabain binding site content (OB-Fab ) was unchanged; however, bound-digoxin removal with Digibind revealed total ouabain binding (OB+Fab ) increased (8.2%, P = 0.047), indicating 7.6% NKA-digoxin occupancy. Quadriceps muscle strength declined in DIG (-4.3%, P = 0.010) but fatiguability was unchanged. During LC, in DIG (main effects), time to fatigue and [K+ ]a were unchanged, whilst [K+ ]v was lower (P = 0.042) and [K+ ]a-v greater (P = 0.004) than in CON; with exercise (main effects), muscle OB-Fab was increased at 67% V O 2 peak ${\rm{V}}_{{{\rm{O}}}_{\rm{2}}{\rm{peak}}}$ (per wet-weight, P = 0.005; per protein P = 0.001) and at fatigue (per protein, P = 0.003), whilst [K+ ]a , [K+ ]v and [K+ ]a-v were each increased at fatigue (P = 0.001). During FF, in DIG (main effects), time to fatigue, [K+ ]a , [K+ ]v and [K+ ]a-v were unchanged; with exercise (main effects), plasma [K+ ]a , [K+ ]v , [K+ ]a-v and muscle K+ efflux were all increased at fatigue (P = 0.001). Thus, muscle strength declined, but functional muscle NKA content was preserved during DIG, despite elevated plasma digoxin and muscle NKA-digoxin occupancy, with K+ disturbances and fatiguability unchanged. KEY POINTS: The Na+ ,K+ -ATPase (NKA) is vital in regulating skeletal muscle extracellular potassium concentration ([K+ ]), excitability and plasma [K+ ] and thereby also in modulating fatigue during intense contractions. NKA is inhibited by digoxin, which in cardiac patients lowers muscle functional NKA content ([3 H]-ouabain binding) and exacerbates K+ disturbances during exercise. In healthy adults, we found that digoxin at clinical levels surprisingly did not reduce functional muscle NKA content, whilst digoxin removal by Digibind antibody revealed an ∼8% increased muscle total NKA content. Accordingly, digoxin did not exacerbate arterial plasma [K+ ] disturbances or worsen fatigue during intense exercise, although quadriceps muscle strength was reduced. Thus, digoxin treatment in healthy participants elevated serum digoxin, but muscle functional NKA content was preserved, whilst K+ disturbances and fatigue with intense exercise were unchanged. This resilience to digoxin NKA inhibition is consistent with the importance of NKA in preserving K+ regulation and muscle function.

P-glycoprotein-mediated transport in a mucus-supplemented Caco-2 cell model in the presence of different surfactants.

The aim of the present study was to investigate if mucus applied to Caco-2 cell monolayers protects cells from high concentrations of surfactants, while still allowing for an identification of the surfactant's inhibitory effects on P-glycoprotein (P-gp). Two types of porcine mucin and six surfactants (Polysorbate 20 (PS20) and 80 (PS80), Kolliphor EL (Kol. EL) and RH40 (Kol. RH40), Labrafil M 2125 CS (L.fil) and Labrasol (L.sol)) were applied to Caco-2 cells, and TEER, paracellular transport and P-gp mediated digoxin transport was measured. The results showed that 15% porcine mucin type II was incompatible with Caco-2 cell monolayer integrity, resulting in a dramatic drop in monolayer TEER and increased mannitol transport. In contrast, mucin type III was compatible with Caco-2 cell monolayers in the concentration range of 2.5-15% without substantially disturbing barrier properties. The highest concentration of mucin type III impaired the ability of all six surfactants to decrease P-gp mediated digoxin transport. Subsequently lowering the mucin concentration to 5% facilitated adequate protection of cells and enabled e.g., 5% PS20 to inhibit P-gp mediated digoxin transport. Overall, the present work is useful for early-stage permeability investigations on how mucus affects P-gp mediated transport in the presence of formulation excipients.

Selection of an Optimal In Vitro Model to Assess P-gp Inhibition: Comparison of Vesicular and Bidirectional Transcellular Transport Inhibition Assays.

The multidrug resistance protein 1 (MDR1) P-glycoprotein (P-gp) is a clinically important transporter. In vitro P-gp inhibition assays have been routinely conducted to predict the potential for clinical drug-drug interactions (DDIs) mediated by P-gp. However, high interlaboratory and intersystem variability of P-gp IC50 data limits accurate prediction of DDIs using static models and decision criteria recommended by regulatory agencies. In this study, we calibrated two in vitro P-gp inhibition models: vesicular uptake of N-methyl-quinidine (NMQ) in MDR1 vesicles and bidirectional transport (BDT) of digoxin in Lilly Laboratories Cell Porcine Kidney 1 cells overexpressing MDR1 (LLC-MDR1) using a total of 48 P-gp inhibitor and noninhibitor drugs and digoxin DDI data from 70 clinical studies. Refined thresholds were derived using receiver operating characteristic analysis, and their predictive performance was compared with the decision frameworks proposed by regulatory agencies and selected reference. Furthermore, the impact of various IC50 calculation methods and nonspecific binding of drugs on DDI prediction was evaluated. Our studies suggest that the concentration of inhibitor based on highest approved dose dissolved in 250 ml divided by IC50(I2/IC50) is sufficient to predict P-gp related intestinal DDIs. IC50 obtained from vesicular inhibition assay with a refined threshold of I2/IC50 ≥ 25.9 provides comparable predictive power over those measured by net secretory flux and efflux ratio in LLC-MDR1 cells. We therefore recommend vesicular P-gp inhibition as our preferred method given its simplicity, lower variability, higher assay throughput, and more direct estimation of in vitro kinetic parameters, rather than BDT assay. SIGNIFICANCE STATEMENT: This study has conducted comprehensive calibration of two in vitro P-gp inhibition models: uptake in MDR1 vesicles and bidirectional transport in LLC-MDR1 cell monolayers to predict DDIs. This study suggests that IC50s obtained from vesicular inhibition with a refined threshold of I2/IC50 ≥ 25.9 provide comparable predictive power over those in LLC-MDR1 cells. Therefore, vesicular P-gp inhibition is recommended as the preferred method given its simplicity, lower variability, higher assay throughput, and more direct estimation of in vitro kinetic parameters.

Human Retinal Organoids Provide a Suitable Tool for Toxicological Investigations: A Comprehensive Validation Using Drugs and Compounds Affecting the Retina.

Retinal drug toxicity screening is essential for the development of safe treatment strategies for a large number of diseases. To this end, retinal organoids derived from human pluripotent stem cells (hPSCs) provide a suitable screening platform due to their similarity to the human retina and the ease of generation in large-scale formats. In this study, two hPSC cell lines were differentiated to retinal organoids, which comprised all key retinal cell types in multiple nuclear and synaptic layers. Single-cell RNA-Seq of retinal organoids indicated the maintenance of retinal ganglion cells and development of bipolar cells: both cell types segregated into several subtypes. Ketorolac, digoxin, thioridazine, sildenafil, ethanol, and methanol were selected as key compounds to screen on retinal organoids because of their well-known retinal toxicity profile described in the literature. Exposure of the hPSC-derived retinal organoids to digoxin, thioridazine, and sildenafil resulted in photoreceptor cell death, while digoxin and thioridazine additionally affected all other cell types, including Müller glia cells. All drug treatments caused activation of astrocytes, indicated by dendrites sprouting into neuroepithelium. The ability to respond to light was preserved in organoids although the number of responsive retinal ganglion cells decreased after drug exposure. These data indicate similar drug effects in organoids to those reported in in vivo models and/or in humans, thus providing the first robust experimental evidence of their suitability for toxicological studies.

Digoxin Exhibits Neuroprotective Properties in a Rat Model of Dementia.

Alzheimer's disease (AD) is by far the most common cause of cognitive impairment in older adults. Current treatments are entirely focused on the symptoms of AD. A complex etiology for AD has been proposed recently, in which AD leads in elevated levels of inflammation. We previously studied digoxin's involvement in the sporadic-AD intracerebroventricular (ICV)-streptozotocin (STZ) animal model due to its anti-inflammatory and neuroprotective characteristics. 18 adult sprague-dawley rats were split into three groups: control (n = 6), STZ + Saline (n = 6), and STZ + Digoxin (n = 6). Twelve AD-induced rats were split into two groups using stereotaxy five days after STZ injection (3 mg/kg) into both lateral ventricles: one group got digoxin (0.1 mg/kg/day, i.p.) for three weeks, while the other group received saline. Following treatment, each subject was subjected to a passive avoidance learning (PAL) test, followed by brain tissue harvesting. The levels of tumor necrosis factor-alpha (TNF-α) and choline acetyl transferase (ChAT) were measured in the brain, and neurons were counted using Cresyl violet staining in cornu ammonis-1 (CA1) and cornu ammonis-3 (CA3) cornu ammonis (CA3). ICV-STZ significantly shortened PAL latency, increased brain TNF-α levels, decreased brain ChAT activity, and decreased hippocampus neuron number. On the other hand, digoxin significantly reduced all of these STZ-induced deleterious effects. Digoxin significantly rescued rats from memory loss caused by ICV-STZ by decreasing hippocampal cell death, neuroinflammation, and cholinergic deficiency. These findings suggest that digoxin may be beneficial in treating cognitive impairment and Alzheimer's disease.

Effects of membrane transport activity and cell metabolism on the unbound drug concentrations in the skeletal muscle and liver of drugs: A microdialysis study in rats.

The unbound concentrations of 14 commercial drugs, including five non-efflux/uptake transporter substrates-Class I, five efflux transporter substrates-class II and four influx transporter substrates-Class III, were simultaneously measured in rat liver, muscle, and blood via microanalysis. Kpuu,liver and Kpuu,muscle were calculated to evaluate the membrane transport activity and cell metabolism on the unbound drug concentrations in the skeletal muscle and liver. For Class I compounds, represented by antipyrine, unbound concentrations among liver, muscle and blood are symmetrically distributed when compound hepatic clearance is low. And when compound hepatic clearance is high, unbound concentrations among liver, muscle and blood are asymmetrically distributed, such as Propranolol. For Class II and III compounds, overall, the unbound concentrations among liver, muscle, and blood are asymmetrically distributed due to a combination of hepatic metabolism and efflux and/or influx transporter activity.

Intestinal Excretion, Intestinal Recirculation, and Renal Tubule Reabsorption Are Underappreciated Mechanisms That Drive the Distribution and Pharmacokinetic Behavior of Small Molecule Drugs.

Drug reabsorption following biliary excretion is well-known as enterohepatic recirculation (EHR). Renal tubular reabsorption (RTR) following renal excretion is also common but not easily assessed. Intestinal excretion (IE) and enteroenteric recirculation (EER) have not been recognized as common disposition mechanisms for metabolically stable and permeable drugs. IE and intestinal reabsorption (IR:EHR/EER), as well as RTR, are governed by dug concentration gradients, passive diffusion, active transport, and metabolism, and together they markedly impact disposition and pharmacokinetics (PK) of small molecule drugs. Disruption of IE, IR, or RTR through applications of active charcoal (AC), transporter knockout (KO), and transporter inhibitors can lead to changes in PK parameters. The impacts of intestinal and renal reabsorption on PK are under-appreciated. Although IE and EER/RTR can be an intrinsic drug property, there is no apparent strategy to optimize compounds based on this property. This review seeks to improve understanding and applications of IE, IR, and RTR mechanisms.

Effects of digoxin on full-type hyperactivation in bovine ejaculated spermatozoa with relatively lower survivability for incubation with stimulators of cAMP signaling cascades.

Previous researches of our laboratory reported that addition of cAMP analog cBiMPS and protein phosphatase inhibitor calyculin A (stimulators of cAMP signaling cascades) improved the capacity of incubation medium to induce full-type hyperactivation in bovine ejaculated spermatozoa. However, this modified medium was valid only for samples with relatively good survivability for incubation with stimulators of cAMP signaling cascades. Thus, it is necessary to make further modified medium for evaluation of potentials to exhibit full-type hyperactivation in bovine sperm samples with relatively lower survivability. Na+/K+-ATPase is an integral membrane protein and involved with the regulation of rodent sperm motility. To make further modification of the medium, we examined effects of Na+/K+-ATPase inhibition with digoxin on motility, full-type hyperactivation and protein tyrosine phosphorylation in bovine ejaculated spermatozoa with relatively lower survivability for incubation with stimulators of cAMP signaling cascades and also performed the immunodetection of bovine sperm Na+/K+-ATPase. The addition of Na+/K+-ATPase inhibitor digoxin to the incubation medium containing cBiMPS and calyculin A had the tendency to lessen the decreases in the percentages of motile spermatozoa in all of 12 samples after the incubation for 1-3 h and significantly increased the percentages of full-type hyperactivation in one group of 4 samples (Sample-A1) and another group of 4 samples (Sample-A2) after 1 and 2 h respectively, though it had no significant effects on full-type hyperactivation in the other group of 4 samples (Sample-B). In addition, incubation time-related changes in the sperm protein tyrosine phosphorylation (a good marker for sperm capacitation) were correlated with those in the percentages of full-type hyperactivation in Sample-A1 containing digoxin. Immunodetection showed that Na+/K+-ATPase is present in the middle and principal pieces of the flagella, indicating that Na+/K+-ATPase has possible relations with sperm motility. These results obtained with bull ejaculated spermatozoa with relatively lower survivability indicate that incubation method using digoxin is useful to evaluate potentials of sperm samples to exhibit full-type hyperactivation, that digoxin has effects on suppressing reduction of sperm motility, and that prolonged incubation with digoxin induces reduction of capacitation state which may suppress the maintenance of full-type hyperactivation.

Retinoschisin and Cardiac Glycoside Crosstalk at the Retinal Na/K-ATPase.

Purpose: Mutations in the RS1 gene, which encodes retinoschisin, cause X-linked juvenile retinoschisis, a retinal dystrophy in males. Retinoschisin specifically interacts with the retinal sodium-potassium adenosine triphosphatase (Na/K-ATPase), a transmembrane ion pump. Na/K-ATPases also bind cardiac glycosides, which control the activity of the pump and have been linked to disturbances in retinal homeostasis. In this study, we investigated the crosstalk between retinoschisin and cardiac glycosides at the retinal Na/K-ATPase and the consequences of this interplay on retinal integrity. Methods: The effect of cardiac glycosides (ouabain and digoxin) on the binding of retinoschisin to the retinal Na/K-ATPase was investigated via western blot and immunocytochemistry. Also, the influence of retinoschisin on the binding of cardiac glycosides was analyzed via enzymatic assays, which quantified cardiac glycoside-sensitive Na/K-ATPase pump activity. Moreover, retinoschisin-dependent binding of tritium-labeled ouabain to the Na/K-ATPase was determined. Finally, a reciprocal effect of retinoschisin and cardiac glycosides on Na/K-ATPase localization and photoreceptor degeneration was addressed using immunohistochemistry in retinoschisin-deficient murine retinal explants. Results: Cardiac glycosides displaced retinoschisin from the retinal Na/K-ATPase; however, retinoschisin did not affect cardiac glycoside binding. Notably, cardiac glycosides reduced the capacity of retinoschisin to regulate Na/K-ATPase localization and to protect against photoreceptor degeneration. Conclusions: Our findings reveal opposing effects of retinoschisin and cardiac glycosides on retinal Na/K-ATPase binding and on retinal integrity, suggesting that a fine-tuned interplay between both components is required to maintain retinal homeostasis. This observation provides new insight into the mechanisms underlying the pathological effects of cardiac glycoside treatment on retinal integrity.

Conformational states of the pig kidney Na+/K+-ATPase differently affect bufadienolides and cardenolides: A directed structure-activity and structure-kinetics study.

There is a renewed interest in the Na+/K+-ATPase (NKA, EC 3.6.3.9) either as a target for new therapeutic uses or for understanding the putative pathophysiological role of its mammalian endogenous ligands. Recent data indicate that bufalin binds to the pig kidney NKA in a way different from ouabain and digoxin, raising the question of a putative class difference between bufadienolides and cardenolides. The purpose of this work was to perform a study of the relationship between structure and both activity and kinetics, focusing mainly on the influence of the lactone ring in C17 (5 vs. 6 membered), the effect of C14-15 cyclization and the carbohydrate moiety in C3. We compared the potency of fourteen related cardiotonic steroids (CTS) for inhibition of the cycling pig kidney NKA in two different concentrations of K+, as well as the affinity for binding to the E2P conformation of the enzyme (Mg-Pi medium) and the potency for inhibiting the E2[2K] conformation of the NKA (K+-pNPPase activity). Cardenolides were clearly sensitive to the antagonistic effect of high K+ concentrations whereas bufadienolides were not or less sensitive. The C14-15 cyclization observed in some bufadienolides, such as resibufogenin and marinobufagin, caused a drastic fall in the affinity for binding to the NKA in the E2P conformation and increased the velocity of K+-pNPPase inhibition. The absence of a carbohydrate moiety in C3 increased the velocity of inhibition. Cardenolides were much more dependent on the E2P conformation for binding than bufadienolides since their ratios of E2[2K] IC50 to E2P Ki were higher than for bufadienolides. Therefore, the present data established the remarkable influence of C14-15 cyclization and of the carbohydrate moiety in C3 on both affinity and kinetics of CTS and indicate that, as a class, bufadienolides would harbor qualitative differences from cardenolides with respect to the NKA conformations to which they can bind.

A novel source of the cardiac glycoside digoxin from the endophytic fungus Epicoccum nigrum: isolation, characterization, production enhancement by gamma irradiation mutagenesis and anticancer activity evaluation.

AIMS: Different endophytic fungi were isolated and screened for their digoxin-producing ability. Strain improvement and different culture conditions were studied for more effective production of digoxin. METHODS AND RESULTS: Among the isolated fungi, an isolate produced digoxin in a concentration of 2·07 mg l-1 . The digoxin-producing fungal isolate was identified as Epicoccum nigrum Link according to the morphological features and phylogenetic analyses. The potentiality of the fungal strain for production enhancement of digoxin was performed by gamma radiation mutagenesis. Gamma irradiation dose of 1000 Gy intensified the digoxin yield by five-fold. Using this dose, a stable mutant strain with improved digoxin productivity was isolated and the stability for digoxin production was followed up across four successive generations. In the effort to increase digoxin magnitude, selection of the proper cultivation medium, addition of some elicitors to the most proper medium and several physical fermentation conditions were tested. Fermentation process carried out in malt extract autolysate medium (pH 6·5) supplemented by methyl jasmonate and inoculated with 2 ml of 6-day-old culture and incubated at 25°C for 10 days stimulated the highest production of digoxin to attain 50·14 mg l-1 . Moreover, cytotoxicity of digoxin separated from the fungal culture was tested against five different cancer cell lines. Based on the MTT assay, digoxin inhibited the proliferation of the five different cancer cell lines and the recorded 50% inhibitory concentration ranged from 10·76 to 35·14 µg ml-1 . CONCLUSIONS: This is the first report on the production and enhancement of digoxin using fungal fermentation as a new and alternate source with high productivity. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings offer new and alternate sources with excellent biotechnological potential for digoxin production by fungal fermentation. Moreover, digoxin proved to be a promising anticancer agent whose anticancer potential should be assessed in prospective cancer therapy.

Probe Cocktail to Assess Transporter Function in Sandwich-Cultured Human Hepatocytes.

PURPOSE: Probe substrates are used routinely to assess transporter function in vitro. Administration of multiple probe substrates together as a "cocktail" in sandwich-cultured human hepatocytes (SCHH) could increase the throughput of transporter function assessment in a physiologically-relevant in vitro system. This study was designed to compare transporter function between cocktail and single agent administration in SCHH. METHODS: Rosuvastatin, digoxin, and metformin were selected as probe substrates of hepatic transporters OATP1B1, OATP1B3, BCRP, P-gp, and OCT1. Total accumulation (Cells+Bile) and biliary excretion index (BEI) values derived from administration of the cocktail were compared to values obtained after administration of single agents in the absence and presence of a model inhibitor, erythromycin estolate. RESULTS: For rosuvastatin and metformin accumulation, the ratio of means [90% confidence interval (CI)] for cocktail to single agent administration was 100% [94%, 106%] and 90% [82%, 99%], respectively. Therefore, the cocktail and single-agent mode of administration were deemed equivalent per standard equivalence criterion of 80-120% for rosuvastatin and metformin accumulation, but not for digoxin accumulation (77% [62%, 92%]). The ratio of means [90% CI] for rosuvastatin BEI values between the two administration modes (105% [97%, 114%]) also was deemed equivalent. The ratio for digoxin BEI values between the two administration modes was 99% [78%, 120%]. In the presence of erythromycin estolate, the two administration modes were deemed equivalent for evaluation of rosuvastatin, digoxin, and metformin accumulation; the ratio of means [90% CI] was 104% [94%, 115%], 94% [82%, 105%], and 100% [88%, 111%], respectively. However, rosuvastatin and digoxin BEI values were low and quite variable in the presence of the inhibitor, so the BEI results were inconclusive. CONCLUSIONS: These data suggest that rosuvastatin and metformin can be administered as a cocktail to evaluate the function of OATP1B1, OATP1B3, BCRP, and OCT1 in SCHH, and that digoxin may not be an ideal component of such a cocktail.

Inhibitory Effects of Commonly Used Excipients on P-Glycoprotein in Vitro.

Pharmaceutical excipients are no longer considered inert and have been shown to influence the activity of metabolic enzymes and transporters, resulting in altered pharmacokinetics of substrate drugs. In this study, the effect of 25 excipients commonly used in drug formulations were investigated for their effect on P-glycoprotein (P-gp) activity. The effect of excipients on P-gp were assessed by measuring the change in the cellular accumulation of a P-gp substrate, digoxin, in MDCK-MDR1 (Madin Darby canine kidney transfected with multidrug resistance 1 gene) cells. The cells were exposed to low (10 µM) and high (200 µM) concentrations of excipient along with 10 µM digoxin. Excipient concentrations were chosen to span the range of concentrations previously used for investigating activities in vitro. At 10 µM of excipient, an increase in the intracellular digoxin concentration was seen with d-α-tocopherol poly-(ethylene glycol) succinate (Vit-E-PEG; p = 0.002), poly(ethylene oxide)20 sorbitan monooleate (Tween 80; p = 0.001), cetyltrimethylammonium bromide (CTAB; p = 0.021), poly(ethylene oxide)35 modified castor oil (Cremophor EL; p = 0.01), polyethylene glycol15-hydroxystearate (Solutol HS 15; p = 0.006), and poly(ethylene glycol) hexadecyl ether (Brij 58; p = 0.001). At 200 µM, Vit-E-PEG ( p < 0.0001), sodium 1,4-bis (2-ethylhexoxy)-1,4-dioxobutane-2-sulfonate (AOT; p < 0.0001), Tween 80 ( p < 0.0001), CTAB ( p = 0.004), poly(ethylene oxide)20 sorbitan monolaurate (Tween 20; p < 0.0001), Cremophor EL ( p < 0.0001), Solutol HS 15 ( p < 0.0001), Brij 58 ( p < 0.0001), and sodium carboxymethyl cellulose (NaCMC; p = 0.006) increased intracellular digoxin significantly. Concentration-dependent inhibition of P-gp was then investigated for selected excipients giving an IC50 for Vit-E-PEG (12.48 µM), AOT (192.5 µM), Tween 80 (45.29 µM), CTAB (96.67 µM), Tween 20 (74.15 µM), Cremophor EL (11.92 µM), Solutol HS 15 (179.8 µM), Brij 58 (25.22 µM), and NaCMC (46.69 µM). These data add to the growing body of evidence demonstrating that not all excipients are inert and will aid excipient choice for rational formulation development.

Discovery and characterization of a prevalent human gut bacterial enzyme sufficient for the inactivation of a family of plant toxins.

Although the human gut microbiome plays a prominent role in xenobiotic transformation, most of the genes and enzymes responsible for this metabolism are unknown. Recently, we linked the two-gene 'cardiac glycoside reductase' (cgr) operon encoded by the gut Actinobacterium Eggerthella lenta to inactivation of the cardiac medication and plant natural product digoxin. Here, we compared the genomes of 25 E. lenta strains and close relatives, revealing an expanded 8-gene cgr-associated gene cluster present in all digoxin metabolizers and absent in non-metabolizers. Using heterologous expression and in vitro biochemical characterization, we discovered that a single flavin- and [4Fe-4S] cluster-dependent reductase, Cgr2, is sufficient for digoxin inactivation. Unexpectedly, Cgr2 displayed strict specificity for digoxin and other cardenolides. Quantification of cgr2 in gut microbiomes revealed that this gene is widespread and conserved in the human population. Together, these results demonstrate that human-associated gut bacteria maintain specialized enzymes that protect against ingested plant toxins.