Inflammasome inhibition blocks cardiac glycoside cell toxicity.

Chronic heart failure and cardiac arrhythmias have high morbidity and mortality, and drugs for the prevention and management of these diseases are a large part of the pharmaceutical market. Among these drugs are plant-derived cardiac glycosides, which have been used by various cultures over millennia as both medicines and poisons. We report that digoxin and related compounds activate the NLRP3 inflammasome in macrophages and cardiomyocytes at concentrations achievable during clinical use. Inflammasome activation initiates the maturation and release of the inflammatory cytokine IL-1ß and the programmed cell death pathway pyroptosis in a caspase-1-dependent manner. Notably, the same fluxes of potassium and calcium cations that affect heart contraction also induce inflammasome activation in human but not murine cells. Pharmaceuticals that antagonize these fluxes, including glyburide and verapamil, also inhibit inflammasome activation by cardiac glycosides. Cardiac glycoside-induced cellular cytotoxicity and IL-1ß signaling are likewise antagonized by inhibitors of the NLRP3 inflammasome or the IL-1 receptor-targeting biological agent anakinra. Our results inform on the molecular mechanism by which the inflammasome integrates the diverse signals that activate it through secondary signals like cation flux. Furthermore, this mechanism suggests a contribution of the inflammasome to the toxicity and adverse events associated with cardiac glycosides use in humans and that targeted anti-inflammatories could provide an additional adjunct therapeutic countermeasure.

Glucitol-core containing gallotannins inhibit the formation of advanced glycation end-products mediated by their antioxidant potential.

Glucitol-core containing gallotannins (GCGs) are polyphenols containing galloyl groups attached to a 1,5-anhydro-d-glucitol core, which is uncommon among naturally occurring plant gallotannins. GCGs have only been isolated from maple (Acer) species, including the red maple (Acer rubrum), a medicinal plant which along with the sugar maple (Acer saccharum), are the major sources of the natural sweetener, maple syrup. GCGs are reported to show antioxidant, α-glucosidase inhibitory, and antidiabetic effects, but their antiglycating potential is unknown. Herein, the inhibitory effects of five GCGs (containing 1-4 galloyls) on the formation of advanced glycation end-products (AGEs) were evaluated by MALDI-TOF mass spectroscopy, and BSA-fructose, and G.K. peptide-ribose assays. The GCGs showed superior activities compared to the synthetic antiglycating agent, aminoguanidine (IC50 15.8-151.3 vs. >300 µM) at the early, middle, and late stages of glycation. Circular dichroism data revealed that the GCGs were able to protect the secondary structure of BSA protein from glycation. The GCGs did not inhibit AGE formation by the trapping of reactive carbonyl species, namely, methylglyoxal, but showed free radical scavenging activities in the DPPH assay. The free radical quenching properties of the GCGs were further confirmed by electron paramagnetic resonance spectroscopy using ginnalin A (contains 2 galloyls) as a representative GCG. In addition, this GCG chelated ferrous iron, an oxidative catalyst of AGE formation, supported a potential antioxidant mechanism of antiglycating activity for these polyphenols. Therefore, GCGs should be further investigated for their antidiabetic potential given their antioxidant, α-glucosidase inhibitory, and antiglycating properties.

Rapid detection of convallatoxin using five digoxin immunoassays.

CONTEXT: Cardiac glycosides of plant origin are implicated in toxic ingestions that may result in hospitalization and are potentially lethal. The utility of commonly available digoxin serum assays for detecting foxglove and oleander ingestion has been demonstrated, but no studies have evaluated the structurally similar convallatoxin found in Convallaria majalis (lily of the valley) for rapid laboratory screening, nor has digoxin immune Fab been tested as an antidote for this ingestion. OBJECTIVE: We aimed to (1) evaluate multiple digoxin assays for cross-reactivity to convallatoxin, (2) identify whether convallatoxin could be detected in vivo at clinically significant doses, and (3) determine whether digoxin immune Fab could be an effective antidote to convallatoxin. MATERIALS AND METHODS: Cross-reactivities of purified convallatoxin and oleandrin with five common digoxin immunoassays were determined. Serum from mice challenged with convallatoxin was tested for apparent digoxin levels. Binding of convallatoxin to digoxin immune Fab was determined in vitro. RESULTS: Both convallatoxin and oleandrin were detectable by a panel of commonly used digoxin immunoassays, but cross-reactivity was variable between individual assays. We observed measurable apparent digoxin levels in serum of convallatoxin intoxicated mice at sublethal doses. Convallatoxin demonstrated no binding by digoxin immune Fab. CONCLUSION: Multiple digoxin immunoassays detect botanical cardiac glycosides including convallatoxin and thus may be useful for rapid determination of severe exposures, but neutralization of convallatoxin by digoxin immune Fab is unlikely to provide therapeutic benefit.

Transport inhibition of digoxin using several common P-gp expressing cell lines is not necessarily reporting only on inhibitor binding to P-gp.

We have reported that the P-gp substrate digoxin required basolateral and apical uptake transport in excess of that allowed by digoxin passive permeability (as measured in the presence of GF120918) to achieve the observed efflux kinetics across MDCK-MDR1-NKI (The Netherlands Cancer Institute) confluent cell monolayers. That is, GF120918 inhibitable uptake transport was kinetically required. Therefore, IC50 measurements using digoxin as a probe substrate in this cell line could be due to inhibition of P-gp, of digoxin uptake transport, or both. This kinetic analysis is now extended to include three additional cell lines: MDCK-MDR1-NIH (National Institute of Health), Caco-2 and CPT-B2 (Caco-2 cells with BCRP knockdown). These cells similarly exhibit GF120918 inhibitable uptake transport of digoxin. We demonstrate that inhibition of digoxin transport across these cell lines by GF120918, cyclosporine, ketoconazole and verapamil is greater than can be explained by inhibition of P-gp alone. We examined three hypotheses for this non-P-gp inhibition. The inhibitors can: (1) bind to a basolateral digoxin uptake transporter, thereby inhibiting digoxin's cellular uptake; (2) partition into the basolateral membrane and directly reduce membrane permeability; (3) aggregate with digoxin in the donor chamber, thereby reducing the free concentration of digoxin, with concomitant reduction in digoxin uptake. Data and simulations show that hypothesis 1 was found to be uniformly acceptable. Hypothesis 2 was found to be uniformly unlikely. Hypothesis 3 was unlikely for GF120918 and cyclosporine, but further studies are needed to completely adjudicate whether hetero-dimerization contributes to the non-P-gp inhibition for ketoconazole and verapamil. We also find that P-gp substrates with relatively low passive permeability such as digoxin, loperamide and vinblastine kinetically require basolateral uptake transport over that allowed by +GF120918 passive permeability, while highly permeable P-gp substrates such as amprenavir, quinidine, ketoconazole and verapamil do not, regardless of whether they actually use the basolateral transporter.

Insulin interacts directly with Na⁺/K⁺ATPase and protects from digoxin toxicity.

Insulin has shown to have cardioprotective effect in diabetic patient after digoxin intoxication. The latter, prompted us to study whether insulin interacts directly with Na⁺/K⁺-ATPase. The interaction of insulin with Na⁺/K⁺-ATPase was explored using enzyme activity, Biacore and Western blot. We also used, flow cytometry, immunohistochemistry and chronotropy on both neonatal and adult rats cardiomyocytes. Insulin at concentration 1.7e⁻7 M blunted the effect of digoxin on Na⁺/K⁺-ATPase activity. In Western blot, the same insulin concentration decreased enzyme α subunit immunoreactivity. Insulin and digoxin decreased both enzyme α subunit immunoreactivity but insulin/digoxin co-treatment did not. Biacore confirmed a direct interaction between insulin and Na⁺/K⁺-ATPase. In neonatal rat cardiomyocytes, insulin plus digoxin induced cell apoptosis but not alone. In adult rat cardiomyocytes, insulin at optimal dose did not induce apoptosis but prevented the one induced by digoxin. In immunocytochemsitry both insulin and digoxin altered Na⁺/K⁺-ATPase α subunit immunoreactivity while their association did not. Finally, insulin increased the beating rate of neonatal rat cardiomyocytes (45±7 beats/min); so did digoxin (36±13 beats/min). The effect of insulin was prevented after pre-treated with digoxin. These results demonstrate that insulin interacts directly with Na⁺/K⁺-ATPase pump and alters the effect of digoxin. This would have important clinical relevance in cardiac complications related to type I and II diabetes.

Different transport activity of human triallelic MDR1 893Ala/Ser/Thr variant and its association with herb extracts.

Individual pharmacokinetic differences for herb-drug interaction have been associated with genetic variations of the multidrug resistance (MDR) gene. A high level expression of MDR protein increases cellular efflux and might decrease drug sensitivity. This study investigated the drug efflux activity difference of human MDR1 triallelic variant 2677G/T/A (rs2032582), as a nonsynonymous 893Ala/Ser/Thr, using Xenopus laevis oocytes and MDR1 overexpressing LLC-PK1 cells. Two MDR1 variants (2667T/893Ser and 2667A/893Thr) were generated using human MDR1 cDNA (2677G/893Ala). No significant difference in the expression of MDR1 893Ala/Ser/Thr was found in X. laevis oocytes. However, the MDR1 2667A/893Thr variant interestingly showed a significant decrease of efflux activity for both digoxin and daunorubicin compared with those of 893Ala and 893Ser variants. In further investigation assessing the inhibitory effects of three herbal extracts on MDR1, 893Ala and 893Ser showed significant decreases of efflux activities in treatments with P. cocos (p = 0.005 for 893Ser) and D. dasycarpus (p = 0.0009 for 893Ala; p = 0.002 for 893Ser) in X. laevis oocytes. The results in this study suggest that herbal medicines could interact with other drugs and change the therapeutic effects depending on the genetic polymorphisms of individuals.

Antidigoxin antiserum prevents endogenous digitalis-like compound-mediated reperfusion injury via modulating sodium pump isoform gene expression.

Endogenous digitalis-like compound (EDLC) is an endogenous ligand of the digitalis receptor and can remarkably inhibit Na+/K+-ATPase activity. Antidigoxin antiserum (ADA), a selective EDLC antagonist, may lessen myocardial reperfusion injury; however, the molecular mechanisms underlying the effect remain unclear. Therefore, this study investigated whether ADA may prevent myocardial reperfusion injury and modulate gene expression of sodium pump alpha isoforms. Cardiac function was examined in isolated rat hearts subjected to ischemia and reperfusion (I/R). The infarct size, EDLC level, Na+/K+-ATPase activity, and the levels of mRNA for sodium pump alpha isoforms were measured in vivo I/R rat hearts in the presence or absence of ADA. It was found that ADA significantly improved the recovery of cardiac function, decreased infarct size, decreased EDLC level, and recovered Na+/K+-ATPase activity in I/R hearts. Further studies showed that sodium pump alpha1, alpha2, and alpha3 isoform mRNA levels were significantly reduced in I/R hearts, and pretreatment with ADA induced a large increase in the mRNA levels. These results indicate that EDLC may participate in depressing Na+/K+-ATPase activity and sodium pump alpha isoform gene expression in I/R heart. It is suggested that treatment with ADA may prevent EDLC-mediated reperfusion injury via modulating sodium pump isoform gene expression.

Studies on cerebral protection of digoxin against ischemia/reperfusion injury in mice.

The present study was designed to investigate the possible neuroprotective effect of digoxin induced pharmacological preconditioning (PP) and its probable mechanism. Bilateral carotid artery occlusion (BCAO) of 17 min followed by reperfusion for 24 h was employed to produce ischemia and reperfusion (I/R) induced cerebral injury in male swiss albino mice. Cerebral infarct size was measured using triphenyltetrazolium chloride staining. Memory was assessed using elevated plus maze test. Degree of motor incoordination was evaluated using inclined beam walking test, rota rod test and lateral push test. Digoxin (0.08 mg/kg, i.p.) was administered 24 h before surgery in a separate group of animals to induce PP. BCAO followed by reperfusion, produced significant rise in cerebral infarct size along with impairment of memory and motor coordination. Digoxin treatment produced a significant decrease in cerebral infarct size and reversal of I/R induced impairment of memory and motor incoordination. Digoxin induced neuroprotective effect was abolished significantly by verapamil (15 mg/kg, i.p.), a L-type calcium channel blocker, ruthenium red (3 mg/kg, s.c.), an intracellular ryanodine receptor blocker and 3,4-dichlorobenzamil (Na(+)/Ca(2+) exchanger inhibitor). These findings indicate that digoxin preconditioning exerts a marked neuroprotective effect on the ischemic brain, which is possibly linked to digitalis induced increase in intracellular calcium levels eventually leading to the activation of calcium sensitive signal transduction cascades.

Effects of uremic serum and uremic toxins on hepatic uptake of digoxin.

Not only the renal clearance but also the hepatic clearance of drugs varies with the progression of renal failure. The aim of this study was to investigate the effects of human uremic serum and various uremic toxins on the hepatic uptake of digoxin (DX), a drug mainly excreted into bile in patients with severe renal failure, using isolated rat and human hepatocytes as model systems. Uremic serum inhibited the uptake of DX into rat hepatocytes in a concentration-dependent manner, whereas normal serum did not affect the uptake. In addition, 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), hippuric acid, indole-3-acetic acid, indoxyl sulfate, and p-cresol (PC) concentration dependently inhibited the uptake. CMPF and PC at the concentration of 400 microM, which is within the plasma concentration range attained in patients with renal failure, inhibited the uptake of DX into rat hepatocytes by 27% and 23%, respectively. In human hepatocytes, 10% uremic serum, 400 microM CMPF, and 400 microM PC inhibited the uptake of DX by 23.3%, 23.4%, and 28.2%, respectively. In conclusion, our results suggest that hepatic uptake of DX is likely to be inhibited by uremic toxins, such as CMPF and PC, present in the serum of patients with renal failure.

Establishment and characterization of the transformants stably-expressing MDR1 derived from various animal species in LLC-PK1.

PURPOSE: Stable transformants expressing human multidrug resistance 1 (MDR1), monkey MDR1, canine MDR1, rat MDR1a, rat MDR1b, mouse mdr1a, and mouse mdr1b in LLC-PK1 were established to investigate species differences in P-glycoprotein (P-gp, ABCB1) mediated efflux activity. METHODS: The seven cDNAs of MDR1 from five animals were cloned, and their transformants stably expressing the series of MDR1 in LLC-PK1 were established. Transport studies of clarithromycin, daunorubicin, digoxin, erythromycin, etoposide, paclitaxel, propranolol, quinidine, ritonavir, saquinavir, verapamil, and vinblastine were performed by using these cells, and efflux activity was compared among the species. RESULTS: Except for propranolol, all compounds showed efflux activity in all transformants, and were judged to be substrates of P-gp. There were slight interspecies and interisoforms differences in the substrate recognition. However, the efflux ratio among the series of the MDR1 stably expressing cells showed good correlation as represented between human and monkey MDR1, and poor correlation as represented between human MDR1 and mouse mdr1a, and human and canine MDR1. CONCLUSIONS: Results in the present study indicate that all MDR1 stably expressing cells have efflux activity for various P-gp substrates, and that interspecies differences and similarities of the P-gp substrate efflux activity may exist.

4-(3'alpha15'beta-dihydroxy-5'beta-estran-17'beta-yl)furan-2-methyl alcohol: an anti-digoxin agent with a novel mechanism of action.

The synthesis and some pharmacological properties of 4-(3'alpha-15'beta-dihydroxy-5beta-estran-17'beta-yl)furan-2-methyl alcohol (16) have been described. The compound was synthesized by reacting a synthetic 3alpha- benzyloxy-5beta-estr-15-en-17-one with the ethylene acetal of 4-bromo-2-furancarboxyaldehyde, followed by hydrolysis of the ethylene acetal and reduction of the aldehyde. Despite its resemblance to the structure of cardiac steroids (CS), 16 does not bind to the CS receptor on Na(+),K(+)-ATPase and does not increase the force of contraction of heart muscle. However, 16 inhibited the digoxin-induced increase in the force of contraction and arrhythmias in guinea pig papillary muscle and human atrial appendages. The steroid also inhibited digoxin-induced alteration in endocytosed membrane traffic, indicating a novel mechanism of action.

Effect of milk thistle (Silybum marianum) and black cohosh (Cimicifuga racemosa) supplementation on digoxin pharmacokinetics in humans.

Phytochemical-mediated modulation of P-glycoprotein (P-gp) and other drug transporters may underlie many herb-drug interactions. Serial serum concentration-time profiles of the P-gp substrate, digoxin, were used to determine whether supplementation with milk thistle or black cohosh modified P-gp activity in vivo. Sixteen healthy volunteers were randomly assigned to receive a standardized milk thistle (900 mg daily) or black cohosh (40 mg daily) supplement for 14 days, followed by a 30-day washout period. Subjects were also randomized to receive rifampin (600 mg daily, 7 days) and clarithromycin (1000 mg daily, 7 days) as positive controls for P-gp induction and inhibition, respectively. Digoxin (Lanoxicaps, 0.4 mg) was administered orally before and at the end of each supplementation and control period. Serial digoxin serum concentrations were obtained over 24 h and analyzed by chemiluminescent immunoassay. Comparisons of area under the serum concentration time curves from 0 to 3 h (AUC(0-3)), AUC(0-24), Cmax, apparent oral clearance of digoxin (CL/F), and elimination half-life were used to assess the effects of milk thistle, black cohosh, rifampin, and clarithromycin on digoxin pharmacokinetics. Rifampin produced significant reductions (p < 0.01) in AUC(0-3), AUC(0-24), and Cmax, whereas clarithromycin increased these parameters significantly (p < 0.01). Significant changes in digoxin half-life and CL/F were also observed with clarithromycin. No statistically significant effects on digoxin pharmacokinetics were observed following supplementation with either milk thistle or black cohosh, although digoxin AUC(0-3) and AUC(0-24) approached significance (p = 0.06) following milk thistle administration. When compared with rifampin and clarithromycin, supplementation with these specific formulations of milk thistle or black cohosh did not appear to affect digoxin pharmacokinetics, suggesting that these supplements are not potent modulators of P-gp in vivo.

Characterisation of (R/S)-propafenone and its metabolites as substrates and inhibitors of P-glycoprotein.

Digoxin is a drug with a narrow therapeutic index, which is a substrate of the ATP-dependent efflux pump P-glycoprotein. Increased or decreased digoxin plasma concentrations occur in humans due to the inhibition or induction of this drug transporter in organs with excretory function such as small intestine, liver and kidney. It is well known that serum concentrations of digoxin increase considerably in humans if propafenone is given simultaneously. However, it has not been investigated in detail whether propafenone and its metabolites are substrates and/or inhibitors of human P-glycoprotein. The aim of this study, therefore, was to investigate the P-glycoprotein-mediated transport and inhibition properties of propafenone and its major metabolites 5-hydroxypropafenone and N-desalkylpropafenone in Caco-2 cell monolayers. Inhibition of P-glycoprotein-mediated transport by propafenone and its metabolites was determined using digoxin as a P-glycoprotein substrate. No polarised transport was observed for propafenone and N-desalkylpropafenone in Caco-2 cell monolayers. However, 5-hydroxypropafenone translocation was significantly greater from basal-to-apical compared with apical-to-basal (P(app) basal-apical vs. P(app) apical-basal, 10.21+/-2.63 x 10(-6) vs. 4.34+/-1.84 x 10(-6) cm/s; P<0.01). Moreover, propafenone, 5-hydroxypropafenone and N-desalkylpropafenone inhibited P-glycoprotein-mediated digoxin transport with IC(50) values of 6.8, 19.9, and 21.3 microM, respectively. In summary, whereas propafenone and N-desalkylpropafenone are not substrates of P-glycoprotein, 5-hydroxypropafenone is translocated by human P-glycoprotein across cell monolayers. In addition, propafenone and its two major metabolites 5-hydroxypropafenone and N-desalkylpropafenone are inhibitors of human P-glycoprotein and therefore contribute to the digoxin-propafenone interaction observed in humans.

Inhibition of P-glycoprotein-mediated transport by extracts of and monoterpenoids contained in Zanthoxyli fructus.

Citrus (rutaceous) herbs are often used in traditional medicine and Japanese cuisine and can be taken concomitantly with conventional medicine. In this study, the effect of various citrus-herb extracts on P-glycoprotein (P-gp)-mediated transport was examined in vitro to investigate a possible interaction with P-gp substrates. Component monoterpenoids of the essential oil in Zanthoxyli fructus was screened to find novel P-gp inhibitors. LLC-GA5-COL150 cells transfected with human MDR1 cDNA encoding P-gp were used. Cellular accumulation of [3H]digoxin was measured in the presence or absence of P-gp inhibitors or test samples. Aurantii fructus, Evodiae fructus, Aurantii fructus immaturus, Aurantii nobilis pericarpium, Phellodendri cortex, and Zanthoxyli fructus were extracted with hot water (decocted) and then fractionated with ethyl acetate. The cell to medium ratio of [3H]digoxin accumulation increased significantly in the presence of the decoction of Evodiae fructus, Aurantii nobilis pericarpium, and Zanthoxyli fructus, and the ethyl acetate fraction of all citrus herbs used. The ethyl acetate fraction of Zanthoxyli fructus exhibited the strongest inhibition of P-gp among tested samples with an IC50 value of 166 microg/mL. Then its component monoterpenoids, geraniol, geranyl acetate, (R)-(+)-limonene, (R)-(+)-linalool, citronellal, (R)-(+)-citronellal, DL-citronellol, (S)-(-)-beta-citronellol, and cineole, were screened. (R)-(+)-citronellal and (S)-(-)-beta-citronellol inhibited P-gp with IC50 values of 167 microM and 504 microM, respectively. These findings suggest that Zanthoxyli fructus may interact with P-gp substrates and that some monoterpenoids with the relatively lower molecular weight of about 150 such as (R)-(+)-citronellal can be potent inhibitors of P-gp.

Effects of antidigoxin antiserum on endoxin levels, apoptosis and the expression of Bax and Bcl-2 protein in ischaemia-reperfusion myocardium.

The aim of the present study was to investigate the effects of antidigoxin antiserum (ADA), an endoxin special antagonist, on endoxin levels, apoptosis and the expression of the apoptosis-related protein bcl-2 and bax in myocardial ischaemia-reperfusion (MIR). The left anterior descending coronary artery was subjected to 30 min ischaemia followed by 45 min reperfusion in open-chest anaesthetized rats. The rats were divided randomly into seven groups: a sham-operated group, an MIR group, a vehicle control (normal saline) group, and groups receiving verapamil (5 mg/kg) or ADA (9, 18 and 36 mg/kg). The drugs were injected into rats via the femoral vein before reperfusion was commenced. Myocardial endoxin levels were measured by radioimmunoassay. Apoptotic cells was detected using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling method. The expression of the apoptosis-related proteins bcl-2 and bax was detected by immunohistochemistry and their semiquantification scores were recorded by a computer image analysis system. Myocardial endoxin levels, the number of apoptotic cells and bax protein expression were increased in the MIR group compared with the sham group. Although bcl-2 protein expression was elevated in the MIR group, there was no significant difference between the MIR and sham groups. However, the ratio of bcl-2/bax was significantly decreased in the MIR group. In the group receiving 36 mg/kg ADA, myocardial endoxin levels, the number of apoptotic cells and bax protein expression were significantly decreased; bcl-2 protein expression was enhanced. The bcl-2/bax ratio was increased. The results suggest that ADA inhibited myocardial apoptosis induced by MIR in rats. The mechanisms involved require further investigation, but the present study may suggest that ADA prevents bax upregulation and enhances bcl-2 upregulation by antagonizing the effects of endoxin.

Immediate control of life-threatening digoxin intoxication in a child by use of digoxin-specific antibody fragments (Fab).

Digoxin-immune antibody fragments (Fab) for treatment of digitalis intoxication was introduced in 1976. Many reports have been published concerning this therapy for children, but few have focused on its immediate reversal of cardiac as well as extracardiac life-threatening manifestations of digoxin toxicity. We present a case of life-threatening digitalis intoxication in a child with postoperative renal insufficiency, after a Sennings procedure for transposition of the great arteries. Digoxin administration according to the nationally recommended dosage and intervals unexpectedly resulted in serum levels in the toxic range. Severe cardiac arrhythmias, haemodynamic instability and a rapid-increasing serum potassium level resulted. This report demonstrates how administration of Fab according to the manufacturer's dosage recommendation reversed the tachyarrhythmia immediately and re-established a normal level of serum potassium within minutes.

From bench to bedside: utilization of an in vitro model to predict potential drug-drug interactions in the kidney: the digoxin-mifepristone example.

Drug interactions are a common source of drug-induced toxicity. For drugs with narrow therapeutic windows, such as digoxin, an understanding of the potential mechanisms by which drugs might interact is essential to clinical practice. This article describes the utility of a renal tubular cell culture model in the prediction of drug interactions involving P-glycoprotein. Digoxin is a cardiac glycoside that undergoes active secretion in the renal tubules by the MDR1 (P-glycoprotein) drug efflux pump. Mifepristone (RU486) is a recently introduced abortifacient that is largely unstudied in terms of drug-drug interactions. The authors used an in vitro model to study the effects of mifepristone on the renal tubular secretion and cellular uptake of digoxin by Madin-Darby canine kidney (MDCK) cells. Mifepristone significantly inhibited the renal tubular secretion of digoxin (p = 0.0005), without interfering with its ability to enter the renal tubular cell. Similar results were found with the P-glycoprotein substrate vinblastine. The findings suggest that drug interactions may result if mifepristone is administered with P-glycoprotein substrates, highlighting the usefulness of this model in the study of not only common but also rare combinations of drugs.

Digitalis-like factor response to hyperinsulinemia in human pregnancy, a model of insulin resistance.

Insulin resistance is strongly associated with hypertension and is postulated to participate in the elevation of blood pressure, although the mechanisms involved are not understood. Recently, we reported that acute increases in plasma insulin levels in normal subjects resulted in increased serum levels of a sodium pump inhibitor, termed the digitalis-like factor (DLF), which has been implicated in both experimental and essential human hypertension. This study looked at the DLF response to hyperinsulinemia, achieved by an oral glucose tolerance test (OGTT), in the setting of a naturally occurring and self-resolving state of human insulin resistance, during third-trimester pregnancy. This model allowed us the further opportunity to compare the DLF response to insulin in the same subjects postpartum, after resolution of their insulin resistance. Administration of an OGTT during pregnancy and postpartum in the same subjects elicited a comparable serum glucose response but a significantly greater insulin response during third-trimester pregnancy, consistent with diminished insulin sensitivity (integrated insulin response during pregnancy: 1611+/-236 vs postpartum: 685+/-101 pmol/l, P=0.004). The time courses of the glucose and insulin responses were identical whether women were pregnant or not. Plasma free fatty acids fell significantly and to a comparable degree during pregnancy and postpartum, but the response was slower during pregnancy. DLF levels increased in response to oral glucose in both pregnant and nonpregnant states. The response was more rapid during pregnancy than after. These findings showed that the increment of insulin induced by oral glucose during pregnancy caused a more rapid rise in circulating DLF levels than it did during the nonpregnant state. At the same time, the response of circulating fatty acids to glucose is retarded during pregnancy. This suggests that the insulin resistance of pregnancy impairs insulin's influence on intermediary metabolism but not its influence on DLF. As a vasoactive substance, DLF might contribute to the hypertension characteristic of insulin-resistant states.

Interaction of omeprazole, lansoprazole and pantoprazole with P-glycoprotein.

Proton pump inhibitors are a class of drugs which are widely prescribed for acid-related diseases. They are primarily metabolized by CYP2C19 and CYP3A4. It is unknown so far whether proton pump inhibitors are also substrates of the ATP-dependent efflux transporter P-glycoprotein. Moreover, it is not established whether proton pump inhibitors are also inhibitors of P-glycoprotein function. The aim of our study was therefore to characterize omeprazole, lansoprazole and pantoprazole as P-glycoprotein substrates and inhibitors. Polarized transport of these compounds was assessed in P-glycoprotein-expressing Caco-2 and L-MDR1 cells. Inhibition of P-glycoprotein-mediated transport was determined using the cyclosporine analogue PSC-833 (valspodar) as P-glycoprotein inhibitor. Inhibition of efflux transport by omeprazole, lansoprazole and pantoprazole was assessed using digoxin as P-glycoprotein substrate. At concentrations of 5 microM, basal-to-apical transport of omeprazole, lansoprazole and pantoprazole was greater than apical-to-basal transport in Caco-2 and L-MDRI cells. Addition of PSC-833 (1 microM) showed a clear effect only for lansoprazole, suggesting that other transporters contribute to omeprazole and pantoprazole cellular translocation. Furthermore, all of the tested compounds inhibited digoxin transport with IC50 values of 17.7, 17.9 and 62.8 microM for omeprazole, pantoprazole and lansoprazole, respectively. In summary, our data provide evidence that proton pump inhibitors are substrates and inhibitors of P-glycoprotein. These findings might explain some of the drug interactions with proton pump inhibitors observed in vivo.

Experimental urethane anaesthesia prevents digoxin intoxication: electrocardiographic and histological study in rabbit.

An electrocardiographic and histological study was performed in rabbit to detect the effects of urethane (ethyl carbamate) intraperitoneal (i.p.) anaesthesia in digoxin intoxication, since it has been previously shown that this anaesthetic and digitalis glycosides exert specific peripheral effects on the cardiovascular system involving central structures of the autonomic nervous system. We observed that i.p. urethane anaesthesia prevented the onset of the electrocardiographic signs of digitalis intoxication, as well as inhibiting the appearance of histological myocardial alterations after treatment with toxic digoxin doses. On the other hand, lethal arrhythmias and severe myocardial damage were observed in animals that had not undergone preliminary urethane anaesthesia. These results indicate that the effect exerted by urethane in preventing the toxic action of digoxin is probably due to a decrease of sympathetic activity in anaesthetized animals by centrally mediated sympathetic inhibition.