Predicted values for human total clearance of a variety of typical compounds with differently humanized-liver mouse plasma data.

Prediction of human pharmacokinetics is important in the preclinical stage. Values for total clearance of compounds from plasma should be one of the most important pharmacokinetic parameters for predictions. Although several physiological and empirical methods including single-species allometry for prediction of values for human clearance of compounds using humanized-liver mice have been reported, further improvement of prediction accuracies would be still expected. To optimize these approaches, we proposed methods for unbound intrinsic clearance in virtually 100% humanized-liver mouse by incorporating unbound plasma fractions of compounds in differently humanized-liver mice. Comparisons of prediction accuracies of values for human clearance of 15 model compounds were performed among our current physiological and previously reported models and single-species allometry using humanized-liver mice. Incorporation of the actual unbound plasma fractions of compounds and correction of residual mice hepatocyte in humanized-liver mice showed comparable prediction accuracy to that by single-species allometry. After exclusion of 3 compounds with large species differences in values of clearance and unbound plasma fractions between mice and humans out of 15 compounds, prediction accuracies were improved in the methods investigated. The previously and present reported physiological methods could show the good prediction accuracy of values for clearance of drugs from plasma.

Overgangen fra digitoksin til digoksin i årene 2011­13.

BACKGROUND: The withdrawal of digitoxin and subsequent substitution with digoxin around 2012 may have led to an increased health risk for patients. The aim of this study was to follow individual patients during the switch. MATERIAL AND METHOD: Serum concentrations of digitoxin and digoxin, measured at the Department of Clinical Pharmacology at St Olavs University Hospital in the period 1 January 2011-31 December 2013 were reviewed. Patients who had switched from digitoxin to digoxin and whose serum concentrations of both drugs had been measured during this period were included. RESULTS: A total of 304 patients, 1686 samples and 1858 serum concentration analyses were included in the study. Therapeutic serum concentrations were measured in 171 patients (56.3 %) before the switch and 176 (57.9 %) after this had taken place. Altogether 108 patients (35.5 %) had therapeutic concentrations both before and after the change. For 58.9 % of the patients, the change resulted in a reduction in serum concentration of digitalis, calculated as digoxin equivalents. The proportion of patients with assumed supratherapeutic concentrations fell from 43.1 % to 33.9 %; however, the proportion of patients with toxic serum concentrations rose from 0.3 % to 3.0 %. INTERPRETATION: Although the switch led to a reduction in dose and serum concentration for many, a significant number of patients may have been put in harm's way.

[Drug safety associated with the change of digitalis drug in Norway]. / Legemiddelsikkerhet ved bytte av digitalispreparat i Norge.

BACKGROUND In 2011, following a period with delivery problems, the only registered digitoxin drug in Norway was replaced with digoxin. As a result, approximately 21 000 patients had to replace digitoxin with digoxin. There are important pharmacokinetic differences between digitoxin and digoxin (the general term for both drugs is digitalis), which must be taken into account when changing therapy. The aim of this study was to investigate compliance of drug security, during the transition from digitoxin to digoxin in Norway.MATERIAL AND METHOD Enquiries addressed to the Norwegian Poison Information Centre and reports of fatal adverse effects to the Regional Drug Information Centres (RELIS) regarding intake of digitalis were analysed. Serum concentrations of digitoxin and digoxin analyzed at Oslo University Hospital were reviewed. All data sources were reviewed for the years 2010-2014 and patients > 20 years were included.RESULTS The total number of enquiries addressed to the Norwegian Poison Information Centre, fatal adverse drug reactions reported to RELIS, and patient samples in the toxic range analyzed at Oslo University Hospital increased from 2012, timewise related to the transition to digoxin.INTERPRETATION Despite extensive information from the Norwegian Medicines Agency, a small, transient increase was observed in the number of overdoses and reported deaths from digitalis related to change in therapy. The cause of the overdose was in many cases unknown. This study revealed several cases of incorrect dosage, simultaneous use of digitoxin and digoxin, and washout time that was insufficient or lacking before initiation of digoxin.

Simultaneous quantification of digoxin, digitoxin, and their metabolites in serum using high performance liquid chromatography-tandem mass spectrometry.

The study describes an LC-MS/MS method for simultaneous quantification of the cardiac glycosidic drugs digoxin and digitoxin and several of their metabolites. The assay represents a useful reference method for immunoassay-based tests, which are easily biased by the presence of metabolites of the target analytes or structurally similar substances.

Rapid quantification of digitoxin and its metabolites using differential ion mobility spectrometry-tandem mass spectrometry.

This study focuses on the quantitative analysis of the cardiac glycoside drug digitoxin and its three main metabolites digitoxigenin-bisdigitoxose, digitoxigenin-monodigitoxose, and digitoxigenin using electrospray ionization-differential ion mobility spectrometry-tandem mass spectrometry (ESI-DMS-MS/MS). Despite large molecular weight differences, gas-phase separation of the four compounds in the DMS drift cell was not possible, even by utilizing additional volatile chemical modifiers. Baseline separation was achieved after adduct formation with alkali metal ions, however, and efficiency was shown to improve with increasing size of the alkali ion, reaching optimum conditions for the largest cesium ion. Subsequently, an assay was developed for quantification of digitoxin and its metabolites from human serum samples and its analytical performance assessed in a series of proof-of-concept experiments. The method was applied to spiked human serum pools with concentration levels between 2 and 80 ng/mL. After a short reversed-phase chromatographic step for desalting the sample, rapid DMS separation of the analytes was carried out, resulting in a total run time of less than 1.5 min. The instrumental method showed good repeatability; the calculated coefficients of variation ranged from 2% to 13%.

The role of digitalis pharmacokinetics in converting atrial fibrillation and flutter to regular sinus rhythm.

This report examined the role of digitalis pharmacokinetics in helping to guide therapy with digitalis glycosides with regard to converting atrial fibrillation (AF) or flutter to regular sinus rhythm (RSR). Pharmacokinetic models of digitoxin and digoxin, containing a peripheral non-serum effect compartment, were used to analyze outcomes in a non-systematic literature review of five clinical studies, using the computed concentrations of digitoxin and digoxin in the effect compartment of these models in an analysis of their outcomes. Four cases treated by the author were similarly examined. Three literature studies showed results no different from placebo. Dosage regimens achieved ≤11 ng/g in the model's peripheral compartment. However, two other studies achieved significant conversion to RSR. Their peripheral concentrations were 9-14 ng/g. In the four patients treated by the author, three converted using classical clinical titration with incremental doses, plus therapeutic drug monitoring and pharmacokinetic guidance from the models for maintenance dosage. They converted at peripheral concentrations of 9-18 ng/g, similar to the two studies above. No toxicity was seen. Successful maintenance was achieved, using the models and their pharmacokinetic guidance, by giving somewhat larger than average recommended dosage regimens in order to maintain peripheral concentrations present at conversion. The fourth patient did not convert, but only reached peripheral concentrations of 6-7 ng/g, similar to the studies in which conversion was no better than placebo. Pharmacokinetic analysis and guidance play a highly significant role in converting AF to RSR. To the author's knowledge, this has not been specifically described before. In my experience, conversion of AF or flutter to RSR does not occur until peripheral concentrations of 9-18 ng/g are reached. Results in the four cases correlated well with the literature findings. More work is needed to further evaluate these provocative findings.

Evaluation of a straightforward and rapid method for the therapeutic drug monitoring of digitoxin by LC-MS/MS.

OBJECTIVES: Therapeutic drug monitoring of digitoxin is strongly recommended but metabolites of digitoxin and digitoxin-like immunoreactive substances may interfere with widely used immunoassays. Recently evaluated assays on LC-MS/MS have the drawback of long turnaround time. We sought to evaluate a specific method on LC-MS/MS optimizing sample preparation thereby significantly reducing turnaround time. DESIGN AND METHODS: Linearity, functional sensitivity, and precision of the method were established. External quality control samples were used for the evaluation of accuracy of the LS-MS/MS method. In addition, digitoxin concentrations in 221 samples were measured by LC-MS/MS and immunoassay. RESULTS: Linearity was validated between 0.15 and 80 ng/mL. Limit of quantification was established at 0.14 ng/mL. Between-day imprecision lay between 1.4 and 4.9% and meets the conditions required for routine analysis. Comparison to results obtained by immunoassay revealed a mean difference of -1.2 ng/mL. CONCLUSIONS: By optimizing preparation steps turnaround time was shorter for LC-MS/MS than for immunoassay. This did not result in increased susceptibility to matrix effects. Analytical performance was sufficient for routine analysis. Therefore, the method is suitable for routine therapeutic drug monitoring of digitoxin.

[Dosing of digitoxin in clinical practice]. / Dosering av digitoksin i klinisk praksis.

BACKGROUND: In 2007, new Norwegian guidelines suggested that serum concentrations of digitoxin should be in the interval 8 - 15 nmol/l, which is about 50 % lower than previous recommendations. MATERIAL AND METHODS: We studied trends in dosing and serum concentrations of digitoxin in the period 2000 - 08, based on 13 054 serum samples sent to our laboratory for analysis in that period. RESULTS: The median serum concentration of digitoxin was stable until the end of 2006 (at about 25 nmol/l); then it gradually decreased from 2007 until the end of 2008 (to 19 nmol/l). The mean daily dose decreased from 66 microg to 56 microg in the study period. At a given dose, patients above 85 years of age had serum concentrations that were almost twice as high as those for patients younger than 55 years. This age effect was particularly pronounced from 65 years. INTERPRETATION: Serum concentrations of digitoxin have gradually decreased after 2007, but are often higher than the new reference range. To obtain serum concentrations within the new reference interval, doses at least as low as those currently recommended should be used, particularly in the oldest patients.

Cytotoxic effects of cardiac glycosides in colon cancer cells, alone and in combination with standard chemotherapeutic drugs.

Cardiac glycosides have been reported to exhibit cytotoxic activity against several different cancer types, but studies against colorectal cancer are lacking. In a screening procedure aimed at identifying natural products with activity against colon cancer, several cardiac glycosides were shown to be of interest, and five of these were further evaluated in different colorectal cancer cell lines and primary cells from patients. Convallatoxin (1), oleandrin (4), and proscillaridin A (5) were identified as the most potent compounds (submicromolar IC50 values), and digitoxin (2) and digoxin (3), which are used in cardiac disease, exhibited somewhat lower activity (IC50 values 0.27-4.1 microM). Selected cardiac glycosides were tested in combination with four clinically relevant cytotoxic drugs (5-fluorouracil, oxaliplatin, cisplatin, irinotecan). The combination of 2 and oxaliplatin exhibited synergism including the otherwise highly drug-resistant HT29 cell line. A ChemGPS-NP application comparing modes of action of anticancer drugs identified cardiac glycosides as a separate cluster. These findings demonstrate that such substances may exhibit significant activity against colorectal cancer cell lines, by mechanisms disparate from currently used anticancer drugs, but at concentrations generally considered not achievable in patient plasma.

Determination of digoxin and digitoxin in whole blood.

A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method has been developed and validated for the determination of digoxin and digitoxin in whole blood samples in autopsy cases. Samples were prepared by liquid-liquid extraction (LLE) with ethyl acetate/heptane/dichloromethane (3:1:1). LC separation was achieved using an Atlantis dC(18)-column (2.1 x 50 mm, 3 microm). The time between injections was 11 min. Mass detection was performed by positive ion mode electrospray LC-MS-MS on the ammonium adducts with two transitions for each analyte and one for the internal standard (digoxin-d(3)). Within-day precision was between 8.3 and 10.8%, between-day precision was between 8.7 and 14.2% and accuracy (bias) was between -17.3 and 11.5%. LOQ was 0.1 nmol/L (0.08 ng/mL), with an accuracy and precision of 19% and -17% (digoxin) and 18% and 3% (digitoxin). Matrix effects ranged from 104 to 117%. Good qualitative correlation with previous findings was achieved for 38 autopsy cases. The median (range, number of cases) B-digitoxin and B-digoxin found with the LC-MS-MS method in this very limited material were 9.3 (3.4-23.8, n = 24) and 5.6 (3.4-26.5, n = 4) nmol/L, respectively.

Sensitive and specific LC-MS assay for quantification of digoxin in human plasma and urine.

Digoxin, a commonly prescribed cardiac glycoside with a narrow therapeutic window, is routinely used in pharmacokinetic studies to assess the in vivo activity of the drug efflux pump P-glycoprotein. To minimize adverse events, a sub-therapeutic dose of digoxin is usually administered, producing low plasma concentrations requiring a sensitive detection technique. Commonly available immunoassay techniques do not provide the required sensitivity to measure these low plasma concentrations and are potentially non-specific in certain subject populations. Previously published mass spectrometric techniques require either large plasma volumes or a tandem mass spectrometer. To overcome these challenges we have developed a sensitive and specific LC-MS method for the quantification of digoxin in small volumes of human plasma and urine. Plasma (1 mL) was extracted with methyl t-butyl ether under basic conditions followed by LC-MS detection of the sodium adducts of digoxin (803.4 m/z) and digitoxin (787.4 m/z, internal standard). Linearity and accuracy were demonstrated across a wide range of digoxin plasma concentration (0.05-1.5 ng/mL). This specific, sensitive, validated digoxin LC-MS assay can be used to quantify sub-therapeutic digoxin plasma concentrations in men and women (pregnant and non-pregnant).

The association of ABCB1 polymorphisms and elevated serum digitoxin concentrations in geriatric patients.

OBJECTIVE: Digitoxin is a known substrate of the efflux pump P-glycoprotein (gene name: ABCB1). P-glycoprotein expression was shown to be modulated by single nucleotide polymorphisms in the ABCB1 gene, but it remains unclear whether these polymorphisms influence digitoxin blood levels. Our objective was to examine the association of ABCB1 C3435T genotype and elevated serum digitoxin concentrations (SDC) in a cohort of 77 geriatric patients consecutively admitted to a geriatric department over a 12-month period. METHODS: The impact of ABCB1 3435 CC, CT, and TT genotypes on SDC and SDC normalized for daily digitoxin dosage and body weight was assessed by multivariate regression analysis. RESULTS: Among participants, 18 (23%) had the CC, 36 (47%) the CT, and 23 (30%) the TT genotype. Adjusting for relevant covariates, no significant association of ABCB1 C3435T genotype and SDC or normalized SDC was detected. Mean SDC was 22.4 ng/ml (95% CI 18.9-25.9) for the TT, 21.8 ng/ml (95% CI 18.1-25.5) for the CT, and 25.7 ng/ml (95% CI 20.6-30.8) for the CC genotype. The means for normalized SDC were 5.2 kg.l(-1) (95% CI 4.3-6.1) for the TT, 6.1 kg.l(-1) (95% CI 4.7-7.5) for the CT, and 6.2 kg.l(-1) (95% CI 4.6-7.7) for the CC genotype. CONCLUSION: In this sample of frail geriatric patients, the impact of ABCB1 C3435T genotype on serum digitoxin concentration was not of major relevance. Regular monitoring of digitoxin blood levels and surveillance of appropriate drug use remain the best ways to prevent digitoxin intoxications in the elderly.

LC-MS assay for quantitative determination of cardio glycoside in human blood samples.

A method is described for liquid chromatography-mass spectrometry analysis of the cardio glycosides digoxin and digitoxin in biological samples. The method was optimized for use in the forensic field and, therefore, comprises the determination from whole blood and tissue samples. Sample cleanup by solid phase extraction (SPE) on a functionalized polymeric phase was sufficient to limit matrix suppression to <10% for all analytes. Chromatographic separation was achieved using an RP-8 column. Detection of the cardio glycosides was performed with electrospray ionization in the positive mode. The system was run in single ion monitoring mode, measuring the sodium adducts (M + Na)+ of the analyte and of the internal standard, respectively. The method was fully validated for the analysis of blood samples and was also successfully applied in forensic cases. The method was accurate and precise over a linear concentration range up to 50 ng/g blood. Lower limit of quantitation was 0.2 ng/g for digoxin and 2 ng/g for digitoxin, respectively. As deuterated analyte was used as internal standard, we also present a new microwave-enhanced method for the fast preparation of the labelled analyte within 20 min.

Role of p-glycoprotein inhibition for drug interactions: evidence from in vitro and pharmacoepidemiological studies.

OBJECTIVES: We determined in vitro the potency of macrolides as P-glycoprotein inhibitors and tested in hospitalised patients whether coadministration of P-glycoprotein inhibitors leads to increased serum concentrations of the P-glycoprotein substrates digoxin and digitoxin. METHODS: In vitro, the effect of macrolides on polarised P-glycoprotein-mediated digoxin transport was investigated in Caco-2 cells. In a pharmacoepidemiological study, we analysed the serum digoxin and digitoxin concentrations with and without coadministration of P-glycoprotein inhibitors in hospitalised patients. RESULTS: All macrolides inhibited P-glycoprotein-mediated digoxin transport, with concentrations producing 50% inhibition (IC(50)) values of 1.8, 4.1, 15.4, 21.8 and 22.7 micromol/L for telithromycin, clarithromycin, roxithromycin, azithromycin and erythromycin, respectively. Coadministration of P-glycoprotein inhibitors was associated with increased serum concentrations of digoxin (1.3 +/- 0.6 vs 0.9 +/- 0.5 ng/mL, p < 0.01). Moreover, patients receiving macrolides had higher serum concentrations of cardiac glycosides (p < 0.05). CONCLUSION: Macrolides are potent inhibitors of P-glycoprotein. Drug interactions between P-glycoprotein inhibitors and substrates are likely to occur during hospitalisation.