Plasma Digoxin Concentrations Research Articles

Machine Learning-Based Prediction of Digoxin Toxicity in Heart Failure: A Multicenter Retrospective Study.

Digoxin toxicity (plasma digoxin concentration ≥0.9 ng/mL) is associated with worsening heart failure (HF). Decision tree (DT) analysis, a machine learning method, has a flowchart-like model where users can easily predict the risk of adverse drug reactions. The present study aimed to construct a flowchart using DT analysis that can be used by medical staff to predict digoxin toxicity. We conducted a multicenter retrospective study involving 333 adult patients with HF who received oral digoxin treatment. In this study, we employed a chi-squared automatic interaction detection algorithm to construct DT models. The dependent variable was set as the plasma digoxin concentration (≥ 0.9 ng/mL) in the trough during the steady state, and factors with p < 0.2 in the univariate analysis were set as the explanatory variables. Multivariate logistic regression analysis was conducted to validate the DT model. The accuracy and misclassification rates of the model were evaluated. In the DT analysis, patients with creatinine clearance <32 mL/min, daily digoxin dose ≥1.6 µg/kg, and left ventricular ejection fraction ≥50% showed a high incidence of digoxin toxicity (91.8%; 45/49). Multivariate logistic regression analysis revealed that creatinine clearance <32 mL/min and daily digoxin dose ≥1.6 µg/kg were independent risk factors. The accuracy and misclassification rates of the DT model were 88.2 and 46.2 ± 2.7%, respectively. Although the flowchart created in this study needs further validation, it is straightforward and potentially useful for medical staff in determining the initial dose of digoxin in patients with HF.

A simple and sensitive LC-MS/MS method for therapeutic drug monitoring of digoxin in children

This short communication introduced a simple and sensitive LC-MS/MS method for therapeutic drug monitoring of digoxin in children with the lower limit of quantitation of 0.2 ng/mL based on 30 μL of plasma. The plasma sample was pretreated by one-step protein precipitation. Then the chromatographic separation was performed on a short C-18 column with a total run time of 2.4 min. The detection was achieved through multiple reaction monitoring using positive ionization mode on a triple quadrupole mass spectrometer. The linear range of digoxin in human plasma was among 0.2–6.4 ng/mL. The intra-day and inter-day accuracies of digoxin ranged from –6.0 % to 10.1 % and imprecisions were less than 8.8 %. The extraction recovery rate of digoxin in plasma samples was above 90 %. Matrix factor normalized by internal standard was within acceptance criteria. This method was fully verified and applied to determine the plasma digoxin concentrations of 43 pediatric patients. It is approved appropriate and practical for the therapeutic drug monitoring of digoxin in routine clinical laboratory practice, especially for children.

Interaction between Chinese medicine and digoxin: Clinical and research update.

Background: Digoxin is one of the most widely and commonly used cardiac drug, which plays an irreplaceable role in treating heart failure and arrhythmia. The 2010 Edition of Pharmacopoeia of the People's Republic of China stipulates that the effective range of digoxin plasma concentration is 0.5-2.0ng/mL and it is toxic at plasma concentration >2ng/mL. Its effective plasma drug concentration is close to the toxic concentration, and large individual differences in the effects of the drug have been observed. It is often used in combination with other drugs, but drug interactions have a great impact on the plasma concentration of digoxin and lead to adverse reactions (ADRs), such as poisoning. Most of the reported drug interactions are with Western drugs. However, there are many combinations of traditional Chinese medicine (TCM) and Western drugs, TCM interacting with digoxin comprises monomer components, single medicines, and Chinese patent medicines. Aim of the study: We aimed i) to provide an overview of the TCM formulations affecting the pharmacology of digoxin and their mechanisms of action and ii) to provide a theoretical reference for the safe and rational use of digoxin in combination with TCM in clinical practice and to avoid ADRs. Methods: A literature search of electronic databases, including PubMed, MEDLINE, Cochrane Library, Web of Science, China National Knowledge Infrastructure, and WANFANG Data, was performed to search for articles published between 1 January 1960, and 1 August 2022. Search terms used included "digoxin," "traditional Chinese medicine," "Chinese patent medicine," and "adverse reactions" and their combinations. Results: A total of 49 articles were obtained, including clinical reports, pharmacological experiments and in vitro experiments. The mechanisms of action affecting the pharmacology of digoxin are complex. TCM formulations may affect the pharmacology of digoxin in vivo by influencing gastrointestinal motility or gastric juice pH, regulating P-glycoprotein levels, exerting cumulative pharmacological effects, and enhancing the sensitivity of the heart to digoxin. Although studies have shown that some TCM formulations interact with digoxin, they may be influenced by the complexity of the composition and the pharmacological effects of the TCM, the sensitivity of digoxin concentration determination methods, etc. The results of existing studies are controversial and further in-depth studies are required. Conclusion: Combinations of digoxin and TCM formulations are commonly used. This article serves as a reference to understand the interactions between TCM formulations and digoxin to avoid the occurrence of ADRs and improve the efficacy and safety of digoxin.

Clinical assessment of gepotidacin (GSK2140944) as a victim and perpetrator of drug-drug interactions via CYP3A metabolism and transporters.

Gepotidacin is a novel triazaacenaphthylene antibiotic in phase III development. Based on nonclinical in vitro characterization of gepotidacin metabolism, two phase I studies were conducted in healthy participants to investigate clinical drug-drug interactions (DDIs). We assessed gepotidacin as a DDI victim with a potent cytochrome P450 (CYP) 3A4/P-glycoprotein (P-gp) inhibitor (itraconazole), potent CYP3A4 inducer (rifampicin), and nonspecific organic cation transporter (OCT)/multidrug and toxic extrusion transporter (MATE) renal transport inhibitor (cimetidine) via single doses of gepotidacin before and after co-administration with multiple doses of the modulator drugs. Gepotidacin DDI perpetrator potential for P-gp inhibition (digoxin) and CYP3A4 inhibition (midazolam) was evaluated via single doses of the two-drug cocktail without and with gepotidacin. The DDI magnitudes were interpreted based on area under the concentration-time curve (AUC). A weak DDI (AUC increase 48%-50%) was observed for gepotidacin co-administered with itraconazole. A clinically significant decrease in gepotidacin plasma AUC (52%) was observed with rifampicin coadministration, indicating a moderate DDI. There was no DDI for gepotidacin with cimetidine; a unique biomarker approach showed increased serum creatinine (24%), decreased renal clearance of creatinine (21%), and N1-methylnicotinamide (39%), which confirmed extensive MATE inhibition and partial OCT2 inhibition. Gepotidacin was not a P-gp DDI perpetrator, although the maximum plasma concentration of digoxin increased (53%) and is potentially clinically relevant given its narrow therapeutic index. Gepotidacin demonstrated weak CYP3A4 inhibition with midazolam (<2-fold AUC increase). There were no new safety-risk profile findings. These results will inform the safe and efficacious clinical use of gepotidacin when co-administered with other drugs.

Therapeutic Monitoring of Plasma Digoxin for COVID-19 Patients Using a Simple UPLC-MS/MS Method

Background: Cardiovascular diseases (CVD) have been reported in 8%-16% of patients with 2019 coronavirus disease (COVID-19). Digoxin is one of the main drugs to treat CVD. Objective: The clinician conducted therapeutic drug monitoring (TDM) of digoxin according to the drug usage on patients to monitor the concentration of digoxin, so as to avoid its toxic and side effects, and provide a theoretical reference for clinical usage of digoxin in patients with COVID-19. Methods: A method for quantifying digoxin concentration in plasma with ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) was developed. After simple protein precipitation of plasma with methanol, digoxin and its internal standard (digoxin-d3) were detected in the positive ion mode using multiple reaction monitoring. Results: Plasma digoxin in the range of 0.2-10 ng/mL had good linearity. The UPLC-MS/MS method was validated with inter-run accuracies ranging from 91.3% to 107.4% and precision less than 13%. Nine plasma samples (5 at valley concentration and 4 at follow-up after stopping dosing) from three patients with COVID-19 were tested. The mean plasma digoxin concentration was 0.73 ng/mL (ranged from 0 to 1.31 ng/mL). Digoxin was detected at the concentration of 0.93 ng/mL after stopping drug administration for 14 days. Conclusion: In this study, we established a simple UPLC-MS/MS method using protein-precipitation to perform TDM of digoxin in patients with COVID-19, and found that about 56% of digoxin plasma concentration was within the treatment window (0.8-2.0 ng/mL). Digoxin can be remained in the body for nearly 14 days in severe patients with COVID-19 after stopping dosing.

Digoxin absorption decreased independently of P-gp activity in rats with irinotecan-induced gastrointestinal damage

BackgroundIrinotecan (CPT-11) is clinically known to cause severe diarrhea and gastrointestinal damage. Recently, we have reported that CPT-11-induced gastrointestinal damage is associated with the upregulation of intestinal P-glycoprotein (P-gp) expression and decreased absorption of its substrate, dabigatran etexilate (DABE), using a rat model. However, the P-gp activity or its contribution to the decreased absorption remains unclear. The aim of this study was to quantitatively evaluate how P-gp activity changes in rats with CPT-11-induced gastrointestinal damage, as assessed by the absorption of digoxin (DGX), a typical P-gp substrate.MethodsMale Sprague-Dawley rats were intravenously administered CPT-11 at a dose of 60 mg/kg/day for 4 days to induce gastrointestinal damage. Then, the rats were administered DGX orally (40 μg/kg), after some of them were orally administered clarithromycin (CAM; 10 mg/kg), a P-gp inhibitor. DGX (30 μg/kg) was administered intravenously to determine the bioavailability (BA). The rats’ DGX plasma concentration profiles were determined using LC-MS/MS.ResultsCPT-11 treatment decreased the maximum concentration (Cmax) and area under the plasma concentration-time curve (AUCpo) of DGX, which does not contradict to the DABE study. Although in the CPT-11-treated group the BA of DGX was significantly decreased to 40% of the control value, CAM did not affect the BA of DGX in the CPT-11-treated group.ConclusionsIncreased P-gp expression in rats with CPT-11-induced gastrointestinal damage is not necessarily associated with increased P-gp activity or contribution to the drug absorption in vivo. The decreased DGX absorption observed in this study might be attributable to other factors, such as a reduction in the absorptive surface area of the gastrointestinal tract.

Evaluating plasma Digoxin concentration after an intravenous loading dose in patients with renal failure

Background: Digoxin is a medication of Glycoside family which is commonly prescribed for patients with atrial fibrillation, atrial flutter, and heart failure. With a narrow therapeuticlevel(0.5–2 ng/mL), careful monitoring of digoxin blood level is necessary. Symptoms of digoxin toxicity include nausea, vomiting, visual changes, altered mental status, hyperkalemia, and cardiovascular collapse. As renal failure decreases the clearance of digoxin, and impaired renal function is common in heart failure, this population is at higher risk of toxicity. Methods & materials: In this prospective study, patients with chronic kidney disease- nondialysis (CKD-ND) who were admitted with heart failure or atrial fibrillation at university hospital, were enrolled. Digoxin-naive patients were treated with a 10 mcg/kg intravenous digoxin loading dose. Serum digoxin level was measured 6 to 12 hours after the last loading dose. Patients were followed for 48 hours for signs of toxicity. Correlation between therapeutic digoxin level and degree of renal failure was evaluated. The effect of serum electrolyte (magnesium, calcium, and potassium) concentration on digoxin level was determined. Pregnant women were excluded from study. Results: From 2018 to 2020, 87 CKD patients, (60 (69%) men and 27 (31%) women) aged from 31 to 92 years old, with a mean age of 70.51 ± 14.06 years were admitted to the cardiac unit. Near 80% of the cohort were CKD stage 3 and 4 patients. About half of patients had digoxin levels in therapeutic range (45 cases = 51.7%), followed by 24.1% with supra therapeutic and the other 24.1% with toxic levels. There was significant relationship between GFR and serum digoxin concentration (p-value = 0.038); the lower the GFR was, the higher was the digoxin level and also between serum creatinine and digoxin level (p value: .04). Serum digoxin concentration had significant correlation with serum magnesium level (p-value = 0.006). Conclusion: this study demonstrated that monitoring plasma Digoxin concentration after an intravenous loading dose in patients with renal failure can be very helpful to prevent digoxin toxicity. The lower the GFR was, the higher the digoxin level and the risk for toxicity if the bolus and even maintenance do not monitor meticulously.

A case of intoxication with tea made from Digitalis purpurea.

We present the case of a 34-year-old woman with recurrent depressive disorder who ingested purple foxglove with suicidal intent. She bought a foxglove plant (Digitalis purpurea) over the internet and used all of its leaves to make a tea that she then drank over a period of a few hours. Seventeen hours later, she developed abdominal pain, emesis and bradycardia and was admitted via the emergency department to the intensive care unit for further treatment and monitoring. The plasma digoxin concentration measured 3.53 nmol/l (therapeutic reference range 0.77–1.50 nmol/l) 21 hours after ingestion of the tea. She remained heamodynamically and neurologically stable, was treated with antiemetics and simple analgesia and did not require digoxin-specific antibodies. Despite normal renal function, her plasma digoxin half-life was prolonged (estimated 76 h), reflecting the long half-life of the parent compound digitoxin which is the main cardiac glycoside in Digitalis purpurea. She was transferred to psychiatric care 48 h after admission. In this report, we compare this case to other similar cases, which to date have only been rarely reported in the literature.

Ultrasensitive electrochemical detection of digoxin using graphite oxide and Au-NPs composites

Digoxin is a cardiac glycoside which is widely used as medication in treatment of congestive heart failure and arrhythmias. Unfortunately, digoxin has small therapeutic range (2 ng/mL), excess digoxin concentration in blood plasma can cause intoxication. Therefore, simple and sensitive procedure is required for strict monitoring of digoxin in blood level to minimize the risk of toxicity. In this paper, we report detection of digoxin using Au-NPs and GO composites synthesized with wet chemistry. X-ray diffraction (XRD), scanning electron microscopy (SEM) and Raman spectroscopy were employed for the confirmation of Au-NPs attachment on the surface of GO. The synthesized Au-NPs-GO composites were deposited on the surface of glassy carbon electrode (GCE) for electrochemical characterization. The electrodes were further incubated with mercaptoacetic acid (MAA) and digoxin antibody (Ab) for the formation of Ab-MAA-Au-NPs-GO modified electrode. MAA conjugation resulted in a 35 % increase in Ipc and 63 % decrease in charge transfer resistance (Rct). Antibody immobilization on MAA modified electrode resulted a sudden drop in both the cathodic peak (anodic peak) currents Ipc(Ipa). Increase in Rct (940 Ω) after Ab functionalization was due to its proteinic nature, which confirmed the formation of Ab-MAA-Au-NPs-GO modified GCE sensors. Work was further extended to probe the charge transfer characteristics in different dilution of digoxin. 17 % (10 %) decrease in Ipc(Ipa) as the digoxin drug was increased from 0.1 ng/mL to 1000 ng/mL. Linear sweep voltammetry (LSV) was also performed at the end for analytic performance of Ab modified sensor for various digoxin drug dilutions. Two linear regressions for current response against digoxin drug dilution were recorded, which confirmed sensitivity of the sensor was governed by interaction between Ab and digoxin drug coordinated by potassium ions at the electroactive sites. The sensitivity was 154.81 μAcm−2 ng-1 for 2-0.1 ng range and 10.71 μAcm−2 ng-1 in the range of 1000−2 ng. The limit of detection (LoD) was 0.19 ng/mL, which suggested that proposed Ab-MAA-Au-NPs-GO modified GCE electrochemical sensor can be successfully applied for future digoxin drug detection applications.

The Development and Validation of a Novel "Dual Cocktail" Probe for Cytochrome P450s and Transporter Functions to Evaluate Pharmacokinetic Drug-Drug and Herb-Drug Interactions.

This study was designed to develop and validate a 10 probe drug cocktail named “Dual Cocktail”, composed of caffeine (Cyp1a2 in rat and CYP1A2 in human, 1 mg/kg), diclofenac (Cyp2c11 in rat and CYP2C9 in human, 2 mg/kg), omeprazole (Cyp2c11 in rat and CYP2C19 in human, 2 mg/kg), dextromethorphan (Cyp2d2 in rat and CYP2D6 in human, 10 mg/kg), nifedipine (Cyp3a1 in rat and CYP3A4 in human, 0.5 mg/kg), metformin (Oct1/2 in rat and OCT1/2 in human, 0.5 mg/kg), furosemide (Oat1/3 in rat and OAT1/3 in human, 0.1 mg/kg), valsartan (Oatp2 in rat and OATP1B1/1B3 in human, 0.2 mg/kg), digoxin (P-gp in rat and human, 2 mg/kg), and methotrexate (Mrp2 in rat and MRP2 in human, 0.5 mg/kg), for the evaluation of pharmacokinetic drug–drug and herb-drug interactions through the modulation of a representative panel of CYP enzymes or transporters in rats. To ensure no interaction among the ten probe substrates, we developed a 2-step evaluation protocol. In the first step, the pharmacokinetic properties of five individual CYP probe substrates and five individual transporter substrates were compared with the pharmacokinetics of five CYP cocktail or five transporters cocktails in two groups of randomly assigned rats. Next, a pharmacokinetic comparison was conducted between the CYP or transporter cocktail group and the dual cocktail group, respectively. None of the ten comparison groups was found to be statistically significant, indicating the CYP and transporter substrate sets or dual cocktail set could be concomitantly administered in rats. The “Dual Cocktail” was further validated by assessing the metabolism of nifedipine and omeprazole, which was significantly reduced by a single oral dose of ketoconazole (10 mg/kg); however, no changes were observed in the pharmacokinetic parameters of other probe substrates. Additionally, multiple oral doses of rifampin (20 mg/kg) reduced the plasma concentrations of nifedipine and digoxin, although not any of the other substrates. In conclusion, the dual cocktail can be used to characterize potential pharmacokinetic drug–drug interactions by simultaneously monitoring the activity of multiple CYP isoforms and transporters.

Effect of purple grape juice on the pharmacokinetics of digoxin: Results of a food-drug interaction study

Digoxin is a glycosidic, cardiotonic plant extract with a narrow therapeutic window. The purpose of the study was to investigate the effects of purple grape juice on the pharmacokinetics of digoxin in rats. A randomized, controlled, single- and multiple-dose study was conducted to evaluate the pharmacokinetic profiles of orally and intravenously administered digoxin. The plasma concentration of digoxin was determined by radioimmunoassay using a gamma counter, and a Caco-2 cell transport model was used to investigate the potential mechanism by which purple grape juice affects the pharmacokinetics of digoxin. The results show that multiple-dose purple grape juice caused a remarkable increase in the AUC, Cmax, and Ka of orally administered digoxin (p < 0.05); only a single dose increased the digoxin AUC0-∞ by 72.80% (p < 0.05). Other parameters were not significantly affected. The study of intravenously administered digoxin found no significant difference in the pharmacokinetic characteristics of the two dosing groups (p > 0.05). The Caco-2 transwell experiments indicated that both the pure purple grape juice and its ethyl acetate extract significantly inhibited digoxin basolateral-to-apical (B→A) transport at concentrations of 10%, 30%, and 50% with dose dependency. These results confirmed that the improvement in bioavailability of digoxin resulted from the inhibition of P-gp by purple grape juice at the level of the gastrointestinal wall. Purple grape juice can increase the bioavailability of orally administered digoxin, especially with multiple doses. This information suggests that co-administration of oral digoxin and purple grape juice may require a dose adjustment. .

No Relevant Pharmacokinetic (PK) Interaction between Rezafungin and Nine Probe Drugs: Results from a Drug-Drug Interaction (DDI) Study

Introduction Rezafungin (RZF) is a new echinocandin being developed to treat patients with systemic fungal infections and to prevent invasive fungal infections in allogeneic hematopoietic stem cell transplant (HSCT) recipients and other patients at high risk of infection. RZF is differentiated by stable and efficacy-driving pharmacokinetics (PK) that allow for once-weekly dosing. Several in vitro studies have been conducted to assess the potential for rezafungin to alter the PK of other drugs. Based on the results, possible interactions were identified in which the risk of drug-drug interactions (DDIs) could not be ruled out relative to the assumed Ph3 dose of 400 mg once weekly and known PK exposure in healthy subjects. Clinical in vivo evaluations of drug interaction potential were performed proactively. Methods This was an open-label, inpatient study of 26 healthy subjects to assess DDIs between RZF and probe drugs selected for known interactions with CYP enzymes and transporters (and consequently commonly used in evaluations of PK interaction), as well as drugs likely to be coadministered with RZF. A loading dose of RZF 600 mg was administered on the first dosing day, to approximate a steady state plasma concentration of multiple once-weekly 400-mg doses, followed by 400-mg doses on days 10 and 15. Probe drugs were administered as part of a cocktail containing ≥2 drugs, once prior to and once after receiving RZF on a schedule designed to allow for washout between doses and to limit interactions with other drugs (Figure 1). Samples were analyzed to determine RZF, tacrolimus, repaglinide, metformin, rosuvastatin, pitavastatin, caffeine, efavirenz, midazolam and digoxin concentrations in plasma (except for tacrolimus which was in whole blood) to characterize the PK profile of each analyte. PK exposure parameters (area under curve [AUC] and maximum concentration [Cmax]) were calculated from the plasma concentration-time profiles by noncompartmental analysis. Results When rezafungin was dosed concomitantly with metformin, pitavastatin, caffeine, efavirenz, midazolam, digoxin, tacrolimus, repaglinide, and rosuvastatin, no relevant change in systemic concentrations of these probe drugs was observed (Figure 2). PK exposure of all drugs were similar with or without RZF. Maximum changes in mean Cmax or AUC were Conclusion No meaningful PK interactions occurred between RZF and the 9 probe drug substrates tested, providing evidence that no RZF dose adjustment is expected when co-administered with these commonly used drugs.

Quantitative Detection of Digoxin in Plasma Using Small-Molecule Immunoassay in a Recyclable Gravity-Driven Microfluidic Chip.

Immunoassays are critical for clinical diagnostics and biomedical research. However, two major challenges remaining in conventional immunoassays are precise quantification and development of immunoassays for small‐molecule detection. Here, a two signal‐mode small‐molecule immunoassay containing an internal reference that provides high stability and reproducibility compared to conventional small‐molecule immunoassays is presented. A system is developed for quantitative monitoring of the digoxin concentration in plasma in the clinically relevant range (0.6–2.6 nm). Furthermore, the model system is integrated into a simple gravity‐driven microfluidic chip (G‐Chip) requiring only 10 µL plasma. The G‐Chip allows fast detection without any complex operation and can be recycled for at least 50 times. The assay, and the G‐Chip in particular, has the potential for further development of point‐of‐care (POC) diagnostics.

Physiologically based pharmacokinetic modelling of acute digoxin toxicity and the effect of digoxin-specific antibody fragments

Context: Recommended doses of digoxin-specific antibody fragments (digoxin-Fab) for treatment of acute digoxin poisoning are pharmacokinetically unsubstantiated and theoretically excessive. Physiologically based pharmacokinetic (PBPK) modelling creates clinical simulations which are closely related to physiological and pharmacokinetic behaviour. This paper details the formulation of a PBPK model of digoxin and explores its use as a simulation tool for acute digoxin toxicity and its management.Materials and methods: A PBPK model of digoxin was constructed and validated for acute digoxin poisoning management by comparing simulations with observed individual acute overdose patients. These simulations were compared with standard two-compartment PK model simulations.Results: PBPK model simulations showed good agreement with post-absorption plasma concentrations of digoxin measured in 6 acute overdose patients. PBPK predictions were accurate to 1.5-fold or less of observed clinical values, proving to be more accurate than two-compartment simulations of the same patients which produced up to a 4.9-fold change.Conclusions: Compared to conventional two-compartment modelling, PBPK modelling is superior in generating realistic simulations of acute digoxin toxicity and the response to digoxin-Fab. Simulation capacity provides realistic, continuous data which has the potential to substantiate alternative, less expensive, and safer digoxin-Fab dosing strategies for the treatment of acute digoxin toxicity.

Enzalutamide and analytical interferences in digoxin assays

Objective: We report two cases of elevated digoxin plasma levels in patients receiving enzalutamide.Cases reported: The first patient, an 84-year-old male treated with enzalutamide, was hospitalized due to deterioration in his general state. Atrial fibrillation was discovered and treatment with digoxin was initiated. Supratherapeutic digoxin concentrations (4 µg/L and 3.5 µg/L 3 days later) led to treatment being stopped despite the lack of clinical or biological signs of overdose. The second patient, an 84-year-old male treated with digoxin and enzalutamide, was hospitalized for the same reasons. Digoxin concentration upon admission was 2.8 μg/L. Despite stopping treatment, digoxin blood levels were observed to have increased on D3 and D7 following admission (3 and 3.6 μg/L, respectively). However, no clinical or biological findings indicated an overdose. Blood samples were sent to the Pharmacology and Toxicology Laboratory for analysis.Methods: The second patient’s digoxin plasma level was determined using the chemiluminescent microparticle immunoassay (CMIA®, Abbott, Illinois) method. Enzalutamide levels were determined using HPLC-UV/DAD method. An interference study was performed using different assay methods by adding enzalutamide to control plasma at various concentrations from a Xtandi® (40mg) capsule.Results: Plasma concentration of digoxin at D7 for patient 2 was identical in both laboratories (3.5 vs. 3.6 µg/L). Enzalutamide was found in the patient’s plasma (12,5 mg/L). Adding 4, 10, 20, and 40 mg/L of enzalutamide to the untreated plasma showed that the plasma concentration of digoxin was positive (from 0.35 to 3.69 µg/L) using the CMIA method.Conclusions: Our results highlight the analytical interferences of enzalutamide with digoxin assays using the CMIA method.

HDAC2 was involved in placental P-glycoprotein regulation both in vitro and vivo

IntroductionPlacental P-glycoprotein (P-gp) plays a significant role in regulating drugs' transplacental transfer rates. Investigations on placental P-gp regulation could provide more therapeutic targets for individualized and safe pharmacotherapy during pregnancy. Currently, the epigenetic regulation of placental P-gp is rare. Our previous study has demonstrated that HDACs inhibition could up-regulate placental P-gp and HDAC1/2/3 might be involved in this process. The present study was carried out to further explore whether HDAC1/2/3 were indeed involved in the regulation of placental P-gp or not and screen out the subtype engaged in this process. MethodsBeWo and JAR cells were transfected with HDAC1/2/3 specific siRNA. After 48 h of transfection, cells were harvested for real-time quantitative PCR (qRT-PCR), Western blot, immunofluorescence and fluorescent dye efflux assay to evaluate P-gp expression, localization, and efflux activity, respectively. Hdac2 siRNA was intraperitoneally injected to pregnant mice every 48 h from E7.5 to E15.5 and digoxin was administered by gavages 1 h prior to euthanasia at E16.5. Placental Hdac1/2/3 and P-gp expression were determined by qRT-PCR and Western blot. Maternal plasma and fetal-unit digoxin concentrations were detected by enzyme-multiplied immunoassay. ResultsIn vitro, HDAC2 inhibition could significantly elevate P-gp expression and reduce intracellular accumulation of P-gp substrates (DiOC2 (3) and Rh 123) both in BeWo and JAR, while knockdown of HDAC1/3 had no influence on P-gp expression and its efflux activity. Additionally, in vivo, Hdac2 silencing in pregnant mice also elevated placental P-gp expression and decreased digoxin transplacental transfer rate. ConclusionHDAC2 inhibition could result in induction of placental P-gp expression and functionality both in vitro and in vivo.

The effect of 17α-ethynylestradiol induced intrahepatic cholestasis of pregnancy on placental P-glycoprotein in mice: Implications in the individualized transplacental digoxin treatment for fetal heart failure

IntroductionPlacental P-glycoprotein (P-gp) plays a significant role in controlling transplacental digoxin transfer rate. Investigations on P-gp regulation in placenta of women with different pregnant pathological states are of great significance to individualized transplacental digoxin treatment for fetal heart failure (FHF). This study aimed to explore the effect of 17α-ethynylestradiol induced intrahepatic cholestasis of pregnancy (ICP) on placental P-gp in mice. MethodsICP model in mice was induced by subcutaneous injection of 17α-ethynylestradiol dissolved in propylene glycol once daily from E12.5 to E16.5. Maternal plasma ALT, AST, TB, DBIL, γ-GT, LDH, ALP and TBA concentrations were measured. HE staining was applied for observation of maternal liver cells degeneration, necrosis and intrahepatic cholestasis. Placental Abcb1a/Abcb1b/HIF-1α mRNA and P-gp/HIF-1α protein expression were determined by real-time quantitative PCR and western-blot. Maternal plasma and fetal-unit digoxin concentrations were detected by a commercial kit assay. ResultsThe ICP group showed higher levels of maternal plasma ALT, AST, TB, DBIL, γ-GT, LDH, ALP and TBA concentrations, reduction in fetal survival rates, lower placental and fetal weights, and typical liver cells degeneration, necrosis and intrahepatic cholestasis. The placental Abcb1a mRNA and P-gp expression of ICP group were significantly elevated, while transplacental digoxin transfer rates were significantly decreased. Both placental HIF-1α mRNA and protein expression was significantly elevated in the ICP group, and there was a positive correlation between Abcb1a mRNA and HIF-1α mRNA. Conclusions17α-ethynylestradiol induced ICP could up-regulate placental P-gp expression and reduce transplacental digoxin transfer rate in mice, which might be partly associated with higher expression of HIF-1α.

Unravelling the complex drug-drug interactions of the cardiovascular drugs, verapamil and digoxin, with P-glycoprotein.

Drug-drug interactions (DDIs) and associated toxicity from cardiovascular drugs represents a major problem for effective co-administration of cardiovascular therapeutics. A significant amount of drug toxicity from DDIs occurs because of drug interactions and multiple cardiovascular drug binding to the efflux transporter P-glycoprotein (Pgp), which is particularly problematic for cardiovascular drugs because of their relatively low therapeutic indexes. The calcium channel antagonist, verapamil and the cardiac glycoside, digoxin, exhibit DDIs with Pgp through non-competitive inhibition of digoxin transport, which leads to elevated digoxin plasma concentrations and digoxin toxicity. In the present study, verapamil-induced ATPase activation kinetics were biphasic implying at least two verapamil-binding sites on Pgp, whereas monophasic digoxin activation of Pgp-coupled ATPase kinetics suggested a single digoxin-binding site. Using intrinsic protein fluorescence and the saturation transfer double difference (STDD) NMR techniques to probe drug-Pgp interactions, verapamil was found to have little effect on digoxin-Pgp interactions at low concentrations of verapamil, which is consistent with simultaneous binding of the drugs and non-competitive inhibition. Higher concentrations of verapamil caused significant disruption of digoxin-Pgp interactions that suggested overlapping and competing drug-binding sites. These interactions correlated to drug-induced conformational changes deduced from acrylamide quenching of Pgp tryptophan fluorescence. Also, Pgp-coupled ATPase activity kinetics measured with a range of verapamil and digoxin concentrations fit well to a DDI model encompassing non-competitive and competitive inhibition of digoxin by verapamil. The results and previous transport studies were combined into a comprehensive model of verapamil-digoxin DDIs encompassing drug binding, ATP hydrolysis, transport and conformational changes.

Calorie Restriction Increases P-Glycoprotein and Decreases Intestinal Absorption of Digoxin in Mice.

There is wide variation in how patients respond to therapeutics. Factors that contribute to pharmacokinetic variations include disease, genetics, drugs, age, and diet. The purpose of this study was to determine the effect of calorie restriction on the expression of Abcb1a in the intestine and whether calorie restriction can alter the absorption of an Abcb1a substrate (i.e., digoxin) in mice. Ten-week-old C57BL/6 mice were given either an ad libitum diet or a 25% calorie-restricted diet for 3 weeks. To determine digoxin absorption, mice were administered [(3)H]-labeled digoxin by oral gavage. Blood and intestine with contents were collected at 1, 2, 4, and 12 hours after digoxin administration. Concentrations of [(3)H]-digoxin in plasma and tissues were determined by liquid scintillation. Calorie restriction decreased plasma digoxin concentrations (about 60%) at 1, 2, and 4 hours after administration. Additionally, digoxin concentrations in the small intestine of calorie-restricted mice were elevated at 4 and 12 hours after administration. Furthermore, calorie restriction increased Abcb1a transcripts in the duodenum (4.5-fold) and jejunum (12.5-fold). To confirm a role of Abcb1a in the altered digoxin pharmacokinetics induced by calorie restriction, the experiment was repeated in Abcb1a/b-null mice 4 hours after drug administration. No difference in intestine or plasma digoxin concentrations were observed between ad libitum-fed and calorie-restricted Abcb1a/b-null mice. Thus, these findings support the hypothesis that calorie restriction increases intestinal Abcb1a expression, leading to decreased absorption of digoxin in mice. Because Abcb1a transports a wide variety of therapeutics, these results may be of important clinical significance.

The effect of maternal obesity on the expression and functionality of placental P-glycoprotein: Implications in the individualized transplacental digoxin treatment for fetal heart failure

IntroductionsPlacental P-glycoprotein (P-gp) plays a significant role in controlling digoxin transplacental rate. Investigations on P-gp regulation in placenta of women with different pregnant pathology are of great significance to the individualized transplacental digoxin treatment for fetal heart failure (FHF). This study aimed to explore the effect of maternal obesity on the expression and functionality of placental P-gp both in human and in mice. MethodsPlacenta tissues from obese and lean women were collected. Female C57BL mice were fed with either a normal chow diet or a high-fat diet for 12 weeks before mating and throughout pregnancy. Maternal plasma glucose, HDL-C, LDL-C, TC, TGs, insulin, IL-1β, IL-6 and TNF-α concentrations was detected. Placental ABCB1/Abcb1a/Abcb1b/IL-1β/IL-6/TNF-α mRNA and P-gp/IL-1β/IL-6/TNF-α protein expression were determined by real-time quantitative PCR and western-blot, respectively. Maternal plasma and fetal-unit digoxin concentrations were detected by a commercial kit assay. ResultsBoth ABCB1 gene mRNA and protein expression of obesity group was significantly lower than that of control group in human. The high-fat dietary intervention resulted in an overweight phenotype, a significant increased Lee's index, higher levels of plasma glucose, HDL-C, LDL-C, insulin and TGs, increased peri-renal and peri-reproductive gland adipose tissue weight, and larger size of adipose cell. Compared with control group at the same gestational day (E12.5, E15.5, E17.5), placental Abcb1a mRNA and P-gp expression of obese group were significantly decreased in mice, while digoxin transplacental rates were significantly increased. Higher maternal plasma IL-1β/TNF-α concentrations and placental IL-1β/TNF-α expression were observed in obesity groups in comparison with control group at the same gestational age. ConclusionsMaternal obesity could inhibit placental P-gp expression and its functionality both in human and in mice, which might be resulted from a heightened inflammatory response.