Visual monitoring and optical recognition of digoxin by functionalized AuNPs and triangular AgNPs as efficient optical nano-probes.

In this study, we presented elective, sensitive, and rapid UV-Vis spectrophotometry and calorimetric assay for the recognition of digoxin. Therefore, cysteamine-gold nanoparticles (Cys A-AuNPs) in the presence of cysteine acid amine and Silver nanoparticles in the presence of tetramethyl benzidine and hydrogen peroxide (AgNPs-TMB [3,3',5,5'-tetramethylbenzidine]-H2 O2 ) were synthesized and utilized as the desired probe. Finally, color variation of probes was observed in the absence and presence of digoxin. Obtained results indicate that the color of Cys A-AuNPs changed from dark pink to light in the absence and the presence of digoxin, respectively. Also, the color of AgNPs-TMB-H2 O2 changed from dark blue to light blue, in the absence and the presence of digoxin, respectively. Moreover, UV-Vis spectroscopies results indicate digoxin with a low limit of quantification of 0.125 ppm in human plasma samples which linear range was 0.125 to 11 ppm.

Estradiol regulation of P-glycoprotein expression in mouse kidney and human tubular epithelial cells, implication for renal clearance of drugs.

P-glycoprotein (P-gp/ABCB1) is an ATP-binding cassette drug efflux transporter expressed in a variety of tissues that affects the pharmacokinetic disposition of many drugs. Although several studies have reported gender-dependent differences in the expression of P-gp, the role of sex hormones in regulating the expression of P-gp and its transport activity has not been well understood. In this study, we demonstrated that 17ß-estradiol has the ability to induce the expression of P-pg in mouse kidneys and cultured human renal proximal tubular epithelial cells. After intravenous injection of a typical P-gp substrate, digoxin, renal clearance in female mice was approximately 2-fold higher than that in male mice. The expression of murine P-gp and its mRNA (Abcb1a and Abcb1b) were also higher in female mice than in male mice. The expression of P-gp in cultured renal tissues prepared from female and male mice was significantly increased by 17ß-estradiol, but not testosterone. Similar 17ß-estradiol-induced expression of P-gp was also detected in cultured human tubular epithelial cells, accompanied by the enhancement of its transport activity of digoxin. The present findings suggest the contribution of estradiol to female-predominant expression of P-gp in renal cells, which is associated with sex-related disparities in the renal elimination of digoxin.

Antibody-Bridged Beacon for Homogeneous Detection of Small Molecules.

In conventional competitive immunoassays for small molecules (SM), antibodies are either immobilized to solid phases or labeled with magnetic particles or probes. The former involves laborious blocking and washing steps, whereas the latter requires complicated labeling and purification steps. To circumvent these limitations, we describe here a new type of molecular beacon, termed antibody-bridged beacon (AbB), enabling homogeneous detection of SM without any immobilization or labeling of the antibody. The AbB is formed by the binding of an antibody to a pair of SM-labeled oligonucleotide probes that each comprise a stem sequence conjugated by either a fluorophore or a quencher. Competitive binding of the SM target to the antibody destructs the stem-loop structure of AbB, restoring the quenched fluorescence. A minimum binding energy of stem sequences is required for efficient formation of the desired stem-loop structure of AbB. A systematic study of the impact of stem sequences on the fluorescence background and quenching efficiency provided useful benchmarks, e.g., binding energy of -11 kcal/mol, for the construction of AbB. The optimized AbB showed fast signal responses, as demonstrated in the analyses of two small molecule targets, biotin and digoxin. Low nanomolar limits of detection were achieved. The novel AbB strategy, along with the guidelines established for the construction and application of AbB, offers a promising approach for homogeneous detection of small molecules, obviating immobilization or labeling of antibodies as required by other competitive immunoassays.

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

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

Clinical judgement perplexed by initially undisclosed use of herbal medicine and unexpected cross-reactivity of immunoassay.

We report a case of symptomatic bradycardia caused by consumption of a Chinese herbal medicine which was initially undisclosed to the attending emergency physician. The scientific name of the herb is Panax japonicus. Electrocardiogram revealed sinus bradycardia. Laboratory tests were normal except for the detection of a high serum digoxin level. Further interrogation of the patient eventually disclosed ingestion of the herb which, however, did not contain any digoxin. Other active ingredients in the herb include various types of ginsenoside. These are digoxin-like substances that had caused the observed false-positive detection of digoxin by fluorescence polarization immunoassay due to cross-reactivity. Our case-report provides an important insight about a blind-spot in the field of laboratory medicine (clinical pathology), namely, the false positive detection of digoxin due to crossreactivity in the immunoassay when we come across digoxin-like substances in clinical scenarios, which has barely received attention in the medical literature. It also conveys a clear educational message that with full understanding of the laboratory methodology and its mechanistic rationale there are actually some tricks-of-the-trade that allow us to optimize the specificity of the biochemical tests and the treatment of digoxin-like substances overdose.

Clinical consequences of a miscalibrated digoxin immunoassay.

BACKGROUND: A routine audit revealed that the analytical method used to measure digoxin concentrations by our statewide pathology provider in 2009 was underestimating digoxin concentrations by 10%. The assay was recalibrated by the manufacturer in 2010, but clinical outcomes of the underestimation were never measured. This is a pilot study to describe the prescribing behavior around out-of-range digoxin concentrations and to assess whether miscalibrated digoxin immunoassays contribute to clinically relevant effects, as measured by inappropriate alterations in digoxin doses. METHODS: About 30,000 digoxin concentrations across the State Hospital system were obtained in 2 periods before and after recalibration of the digoxin assay. Digoxin concentration means were calculated and compared and were statistically significantly different. Subsequently, a single-centered retrospective review of 50 randomly chosen charts was undertaken to study the clinical implications of the underestimated concentrations. RESULTS: Mean digoxin concentrations for 2009 and 2011 were significantly different by 8.8% (confidence interval, 7.0%-10.6%). After recalculating the 2009 concentrations to their "corrected" values, there was a 16% increase in the number of concentrations within the range when compared with the 2011 concentrations (41.48% versus 48.04%). However, overall, this did not cause unnecessary dose changes in patients who were "borderline" or outside the therapeutic range when compared with controls (P = 0.10). The majority of decisions were based on the clinical impression rather than concentration alone (85.1% versus 14.9%), even when the concentration was outside the "therapeutic range." CONCLUSIONS: Although recalculating digoxin concentrations measured during 2009 to their corrected values produced a significant change in concentration and values inside and outside the range, this does not seem to have had an influence on patient treatment. Rather, clinicians tended to use the clinical impression to dose digoxin.

Strip biosensor for amplified detection of nerve growth factor-beta based on a molecular translator and catalytic DNA circuit.

We have demonstrated a new visual detection approach based on a molecular translator and a catalytic DNA circuit for the detection of nerve growth factor-beta (NGF-ß). In this assay, a molecular translator based on the binding-induced DNA strand-displacement reaction was employed to convert the input protein to an output DNA signal. The molecular translator is composed of a target recognition element and a signal output element. Target recognition is achieved by the binding of the anti-NGF-ß antibody to the target protein. Polyclonal anti-NGF-ß antibody is conjugated to DNA1 and DNA2. The antibody conjugated DNA1 is initially hybridized to DNA3 to form a stable DNA1/DNA3 duplex. In the presence of NGF-ß, the binding of the same target protein brings DNA1 and DNA2 into close proximity, resulting in an increase in their local effective concentration. This process triggers the strand-displacement reaction between DNA2 and DNA3 and releases the output DNA3. The released DNA3 is further amplified by a catalytic DNA circuit. The product of the catalytic DNA circuit is detected by a strip biosensor. This proposed assay has high sensitivity and selectivity with a dynamic response ranging from 10 fM to 10 pM, and its detection limit is 10 fM of NGF-ß. This work provides a sensitive, enzyme-free, and universal strategy for the detection of other proteins.

Evaluation of digoxin-boronate ester formation through in-capillary derivatisation-large volume sample stacking-capillary zone electrophoresis.

Determination of digoxin through in-capillary derivatisation based on the formation of o-tolyl- and 2-naphthyl-anionic boronate esters in combination with large volume sample stacking-capillary electrophoresis is proposed. The derivatisation reaction was performed at basic pH values to obtain compounds with a charge and chromophore group during the stacking process. After stacking, the species were separated and detected at 225 nm using p-nitrophenol as an internal standard. Stacking and derivatisation parameters such as pre-concentration time, preconcentration voltage and injection time (relation between the analyte and the derivatisation agent) were evaluated using a Box-Behnken design. Under optimal conditions, the proposed method exhibits a linear range of 1.08-50.00 µM with a limit of detection of 0.36 µM; additionally, adequate repeatability and reproducibility was obtained (%RSD ≤ 5.0%). The methodology was validated by comparing it to an HPLC-UV established methodology and was successfully applied for the determination of digoxin in pharmaceutical tablets and blood serum samples, showing a positive performance for these matrices.

Impact of interferences including metabolite crossreactivity on therapeutic drug monitoring results.

Therapeutic drug monitoring is an integral part of services offered by toxicology laboratories because certain drugs require routine monitoring for dosage adjustment to achieve optimal therapeutic response and avoid adverse drug reactions. Immunoassays are widely used for therapeutic drug monitoring. However, immunoassays suffer from interferences from both exogenous and endogenous compounds including metabolites of the parent drug. Digoxin immunoassays are affected more commonly than any other immunoassays used for therapeutic drug monitoring. Digoxin immunoassays are affected by endogenous digoxin-like immunoreactive substances and exogenous compounds such as various drugs, certain herbal supplements, and Digibind. Carbamazepine is metabolized to carbamazepine 10, 11-epoxide, and the crossreactivity of this metabolite with carbamazepine immunoassay may vary from 0% to 94%. Immunoassays used for measuring concentrations of tricyclic antidepressants are affected by tricyclic antidepressant metabolites and by a number of other drugs. Immunoassays for immunosuppressants are also subjected to significant interferences from metabolites, and liquid chromatography combined with mass spectrometry or tandem mass spectrometry is recommended for therapeutic drug monitoring of immunosuppressants. However, liquid chromatography combined with mass spectrometry may also suffer from interferences, for example, due to ion suppression or from isobaric ions.

Rapid, presumptive identification of seed-based toxins using direct analysis in real time mass spectrometry (DART-MS) and its variants.

Detection of seed-based toxins is a need for forensic chemists when suspected poisonings occur. The evidence that is found is often physically unidentifiable, as the seeds are mashed to extract the toxin. This work investigates potential strategies for rapid detection of seed-based toxins and seed mashes containing these toxins using chemical signatures obtained by direct analysis in real time mass spectrometry (DART-MS). Seven toxins (digoxin, digitoxin, hypaconitine, hyoscyamine, lanatoside, oleandrin, and scopolamine) and six seeds containing these toxins were studied. While detection of four of the toxins was readily attainable, detection of digoxin, digitoxin, and lanatoside was hindered by the inability to thermally desorb these larger compounds under normal operating conditions. The use of DART-MS variants capable of higher desorption temperatures (thermal desorption (TD)-DART-MS and infrared thermal desorption (IRTD)-DART-MS) enabled detection of these compounds. Detection of toxins from direct analysis of seed mashes and methanolic seed mash extracts was found to be compound and technique dependent. Principal component analysis (PCA) of generated mass spectra enabled differentiation of seed species, even in cases where the toxins were undetectable.

Ultrafast mass spectrometry based bioanalytical method for digoxin supporting an in vitro P-glycoprotein (P-gp) inhibition screen.

The evaluation of interactions between drug candidates and transporters such as P-glycoprotein (P-gp) has gained considerable interest in drug discovery and development. Inhibition of P-gp can be assessed by performing bi-directional permeability studies with in vitro P-gp-expressing cellular model systems such as Caco-2 (human colon carcinoma) cells, using digoxin as a substrate probe. Existing methodologies include either assaying (3)H-digoxin with liquid scintillation counting (LSC) detection or assaying non-labeled digoxin with liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis at a speed of several minutes per sample. However, it is not feasible to achieve a throughput high enough using these approaches to sustain an early liability screen that generates more than a thousand samples on a daily basis. To address this challenge, we developed an ultrafast (9 s per sample) bioanalytical method for digoxin analysis using RapidFire™, an on-line solid-phase extraction (SPE) system, with MS/MS detection. A stable isotope labeled analog, d3-digoxin, was used as internal standard to minimize potential ionization matrix effect during the RF-MS/MS analysis. The RF-MS/MS method was more than 16 times faster than the LC-MS/MS method but demonstrated similar sensitivity, selectivity, reproducibility, linearity and robustness. P-gp inhibition results of multiple validation compounds obtained with this RF-MS/MS method were in agreement with those generated by both the LC-MS/MS method and the (3)H-radiolabel assay. This method has been successfully deployed to assess P-gp inhibition potential as an important early liability screen for drug-transporter interaction.

Rapid detection of oleander poisoning by Dimension Vista digoxin assay (Flex Reagent Cartridge).

Oleander poisoning can be detected by digoxin immunoassays and for last two decades the fluorescence polarization immunoassay (FPIA) has been used for rapid detection of oleander poisoning in clinical laboratories. Recently, Abbott Laboratories (Abbott Park, IL) discontinued this assay. Therefore, we explored the possibility of using another digoxin assay (Dimension Vista Flex Reagent Cartridge, Tina Quant, EMIT 2000 and old FPIA assay for comparison) for rapid detection of oleander poisoning. When aliquots of drug-free serum pools were supplemented with pure oleandrin or oleander extract, we observed the highest apparent digoxin values using Dimension Vista digoxin assay (Flex Reagent Cartridge). We also observed significant apparent digoxin values in vivo in sera of mice both 1 and 2 hr after feeding with oleander extract. When a serum pool prepared from patients taking digoxin was further supplemented with various amounts of oleander extract, the highest falsely elevated digoxin values were observed with Dimension Vista digoxin assay. Monitoring free digoxin using Dimension Vista digoxin assay (Flex Reagent Cartridge) did not eliminate this interference. Digibind neutralized digoxin-like factors of oleander extract and such effect can be monitored by observing significant reduction in apparent free digoxin levels in the presence of Digibind as measured in the protein-free ultrafiltrate using Dimension Vista digoxin assay (Flex Reagent Cartridge).

Variability of yellow tulp (Moraea pallida Bak.) toxicity.

Yellow tulp (Moraea pallida Bak.), collected predominantly during the flowering stage from a number of sites in South Africa, showed large variation in digoxin equivalent values, indicating variability in yellow tulp toxicity. Very low values were recorded for tulp collected from certain sites in the Northern Cape.

Chemoinformatic Screening for the Selection of Potential Senolytic Compounds from Natural Products.

Cellular senescence is a cellular condition that involves significant changes in gene expression and the arrest of cell proliferation. Recently, it has been suggested in experimental models that the elimination of senescent cells with pharmacological methods delays, prevents, and improves multiple adverse outcomes related to age. In this sense, the so-called senoylitic compounds are a class of drugs that selectively eliminates senescent cells (SCs) and that could be used in order to delay such adverse outcomes. Interestingly, the first senolytic drug (navitoclax) was discovered by using chemoinformatic and network analyses. Thus, in the present study, we searched for novel senolytic compounds through the use of chemoinformatic tools (fingerprinting and network pharmacology) over different chemical databases (InflamNat and BIOFACQUIM) coming from natural products (NPs) that have proven to be quite remarkable for drug development. As a result of screening, we obtained three molecules (hinokitiol, preussomerin C, and tanshinone I) that could be considered senolytic compound candidates since they share similarities in structure with senolytic leads (tunicamycin, ginsenoside Rb1, ABT 737, rapamycin, navitoclax, timosaponin A-III, digoxin, roxithromycin, and azithromycin) and targets involved in senescence pathways with potential use in the treatment of age-related diseases.

Persistence of hapten-antibody complexes in the circulation of immunized animals after a single intravenous injection of hapten.

To study the fate of a low molecular weight antigen (hapten) in the circulation of animals whose sera contain antibodies specific for that low molecular weight antigen, a single injection of digoxin-(3)H (0.4 mg/kg) was administered intravenously to 18 rabbits. Thirteen animals (nine nonimmunized and four immunized with bovine serum albumin) served as control animals. In five rabbits which had been immunized with a digoxin-bovine serum albumin conjugate and whose sera contained digoxin-specific antibodies, the mean 12-h serum digoxin concentration was 8,300 ng/ml (control: 92 ng/ml) and the mean serum concentration 12 mo after the single injection of digoxin-(3)H was 85 ng/ml. In digoxin-immunized rabbits, less than 10% of the digoxin-(3)H was excreted in the first 10 days (control: 77% recovered in urine and feces) and the mean biological half-life of digoxin, as calculated from serum digoxin-(3)H disappearance curves, was 72 days (control: 3.4 days). In sera of digoxin-immunized rabbits, more than 90% of the circulating digoxin-(3)H was immunoglobulin bound, as determined by the double-antibody and dextran-coated charcoal methods. The serum disappearance rate of (125)I-antidigoxin antibodies was similar in nonimmunized and in immunized animals and in the presence or absence of digoxin. It is concluded that the biological half-life of a hapten may be markedly prolonged when the hapten is bound to specific antibody. The persistence of antibody-hapten complexes in the circulation suggests that these complexes may not be deposited in tissues and raises the possibility that low molecular weight determinants may be capable of preventing or reversing the deposition of immune complexes, containing macromolecular antigens, in the tissues of experimental animals and man.

Determination and pharmacokinetics of periplocin in rat plasma by LC-MS.

A simple and sensitive LC-MS method for the determination of periplocin in rat plasma was developed and validated. The chromatographic separation was carried out using a reverse-phase Kromasil C(18) column(150 × 4.6 mm, i.d., 5 µm) with a mobile phase composed of methanol-water (76:24, v/v). The flow rate of mobile phase was 0.8 mL/min. The calibration curve was linear within the concentration range 1-1000 ng/mL. The intra- and inter-day precisions across three validation days over the entire concentration range was lower than 9.2% in terms of relative standard deviation. Accuracy determined at three quality control concentrations ranged from -2.0 to 6.0% in terms of relative error. The validated method was applied to the pharmacokinetic study of periplocin in rat plasma after intravenous and intramuscular administration.

Designing and construction of genetically encoded FRET-based nanosensor for qualitative analysis of digoxin.

Nanobiotechnological improvements defined on the utilization of biological materials and principles have enormously partaken to revolutionize physical, chemical, and biological sciences. However, the exploration of plant nanobiotechnology is still in its outset. The search for novel tools to monitor plant biomolecules is an emerging issue for the nanobiotechnologists. Given this, a genetically encoded FRET-based nanobiosensor has been developed to monitor the popular plant cardiac glycoside - digoxin, which is used as the most common prescription drug for heart-related illnesses across the world. Digoxin is sourced from the leaves of the foxglove plant (Digitalis purpurea L.) and has a significant demand in the medical sector. Moreover, with the rising popularity of the herbal formulations in the global market, attention towards the authentication and quality control of the herbal drugs is important. Furthermore, digoxin has a very narrow therapeutic range, i.e., 0.6 nM - 2.6 nM. Therefore, strict monitoring of blood digoxin levels is necessary. Besides, previously used techniques for drug authentication and quantification of small-molecule drugs in blood samples are not the best choice available. The nanobiosensor is based on the FRET (Fluorescence Resonance Energy Transfer) mechanism, and it is constructed in such a way that it gives a changed FRET output in the presence of digoxin. Two fluorophores, enhanced cyan fluorescent protein (ECFP) and Venus, were attached on either end of the sensory domain - human nuclear receptor ROR-gamma (RORγt). The developed nanobiosensor was named as fluorescent indicator protein for digoxin, (FLIP-digoxin). The ligand binding affinity of FLIP-digoxin was calculated as 425 µM. Affinity mutants of the FLIP-digoxin were also generated to measure digoxin in wide concentration ranges. This sensor offers high-throughput qualitative analysis of digoxin in Digitalis preparations procured from local drug stores. It confirms the authenticity of the preparations through the detection of digoxin. The FLIP-1n was also able to monitor digoxin concentration in serum samples in lesser than 5 min. The nanobiosensor was found pH stable, digoxin-specific, non- interfered by the biological serum species and can perform high throughput screening of the Digitalis powder, infusion and tincture preparations.

A novel self-powered aptasensor for digoxin monitoring based on the dual-photoelectrode membrane/mediator-free photofuel cell.

Self-powered sensor is considered as a promising, rapid, portable and miniaturized detection device that can work without external power input. In this work, a novel dual-photoelectrode self-powered aptasensor for digoxin detection was designed on the basis of a photofuel cell (PFC) composed of a black TiO2 (B-TiO2) photoanode and a CuBr photocathode in a single-chamber cell. The sensing platform avoided the use of membrane, free mediator, bioactive components and costly metal Pt electrodes. The large inherent bias between the Fermi energy level of B-TiO2 and that of CuBr improved the electricity output of PFC that the open circuit potential (OCP) and the maximum power density (Pmax) reached 0.58 V and 6.78 µW cm-2 respectively. Based on the excellent output of PFC, digoxin aptamer was immobilized on photoanode as the recognition element to capture digoxin molecules, which realized the high sensitive and selective detection of digoxin. The self-powered aptasensor displayed a broad linear in the range from 10-12 M to 10-5 M with a detection limit (3 S/N) of 0.33 pM. This work paved a luciferous way for further rapid, portable, miniaturized and on-site self-powered sensors.