Stepwise frontal affinity chromatography model for drug and protein interaction.

Frontal affinity chromatography is an efficient technique that combines affinity interaction and high-performance liquid chromatography, and frontal analysis has been used in studying the interaction between drugs and proteins. Based on frontal analysis, stepwise frontal analysis has been established. The present study aimed to use the Lineweaver-Burk plot in stepwise frontal analysis by taking the weighted average of time data. Commercial human serum albumin (HSA) and alpha-1-acid glycoprotein (AGP) columns were used as an affinity column. Warfarin and digitoxin were chosen as model drugs for the HSA column, whereas verapamil and tamsulosin were selected as model drugs for the AGP column. The time data obtained by frontal analysis and stepwise frontal analysis were compared, and the results revealed good correlation (r2 = 0.9946-0.9998). Frontal analysis and stepwise frontal analysis were also used to analyze the equilibrium dissociation constants (Kd) of model drugs on the HSA and AGP columns. The Kd values were compared with literature values, which revealed the same order of magnitude. These results illustrate that conversion of the time data is reasonable and feasible. The Lineweaver-Burk plot can be used in the stepwise frontal analysis model to study the characteristics of the interaction between drugs and proteins. Graphical abstract ᅟ.

Drug discovery with an RBM20 dependent titin splice reporter identifies cardenolides as lead structures to improve cardiac filling.

Diastolic dysfunction is increasingly prevalent in our ageing society and an important contributor to heart failure. The giant protein titin could serve as a therapeutic target, as its elastic properties are a main determinant of cardiac filling in diastole. This study aimed to develop a high throughput pharmacological screen to identify small molecules that affect titin isoform expression through differential inclusion of exons encoding the elastic PEVK domains. We used a dual luciferase splice reporter assay that builds on the titin splice factor RBM20 to screen ~34,000 small molecules and identified several compounds that inhibit the exclusion of PEVK exons. These compounds belong to the class of cardenolides and affect RBM20 dependent titin exon exclusion but did not affect RBFOX1 mediated splicing of FMNL3. We provide evidence that cardenolides do not bind to the RNA interacting domain of RBM20, but reduce RBM20 protein levels and alter transcription of select splicing factors that interact with RBM20. Cardenolides affect titin isoform expression. Understanding their mode of action and harnessing the splice effects through chemical modifications that suppress the effects on ion homeostasis and more selectively affect cardiac splicing has the potential to improve cardiac filling and thus help patients with diastolic heart failure, for which currently no targeted therapy exists.

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%.

Analysis of drug interactions with modified proteins by high-performance affinity chromatography: binding of glibenclamide to normal and glycated human serum albumin.

High-performance affinity chromatography (HPAC) was used to examine the changes in binding that occur for the sulfonylurea drug glibenclamide with human serum albumin (HSA) at various stages of glycation for HSA. Frontal analysis on columns containing normal HSA or glycated HSA indicated glibenclamide was interacting through both high affinity sites (association equilibrium constant, K(a), 1.4-1.9 × 10(6)M(-1) at pH 7.4 and 37 °C) and lower affinity sites (K(a), 4.4-7.2 × 10(4)M(-1)). Competition studies were used to examine the effect of glycation at specific binding sites of HSA. An increase in affinity of 1.7- to 1.9-fold was seen at Sudlow site I with moderate to high levels of glycation. An even larger increase of 4.3- to 6.0-fold in affinity was noted at Sudlow site II for all of the tested samples of glycated HSA. A slight decrease in affinity may have occurred at the digitoxin site, but this change was not significant for any individual glycated HSA sample. These results illustrate how HPAC can be used as tool for examining the interactions of relatively non-polar drugs like glibenclamide with modified proteins and should lead to a more complete understanding of how glycation can alter the binding of drugs in blood.

Biotransformation of extracted digitoxin from Digitalis lanata by Streptomyces.

The biotransformation of digitoxin and some of its derivatives extracted from Digitalis lanata by Streptomyces isolated species was investigated. Cultures of a Streptomyces strain designated EUSA2003B, isolated from an Egyptian soil sample, efficiently induced selective 12beta-hydroxylation of the steroid aglycone of digitoxin (DT) and its alpha-acetyl and beta-methyl derivatives. The transformation reaction was performed within a 5-day fermentation process, products were isolated and their aglycone moiety was obtained by acid hydrolysis and their structures were elucidated by 13C and 1H NMR. The biotransformation resulted mainly digoxin (DG, approximately 87%), meanwhile, digoxigenone (DGON, approximately 7.0%) was also afforded as a side product. The present study revealed that: 1-Streptomyces isolate EUSA2003B harbors its specific 12beta-hydroxlase and has the capability to transform DT and it's alpha-acetyl and beta-methyl derivatives into their corresponding digoxins at reasonable yields. 2-The minor structural differences in the trisaccharide side chain seemed ineffective on the transformational capability of this organism. 3-The Streptomyces might also possess a specific glycosidase that splits the saccharidic side chain beside another dehydrogenase that oxidizes C3 at the steroid nucleus into its ketone form (DGON).

Infusion of albumin attenuates changes in serum protein binding of drugs in surgical patients compared with volume replacement with HAES.

BACKGROUND: In vitro studies have indicated that stabilizers present in pharmaceutical-grade albumin influence albumin-binding capacity for highly protein bound drugs. METHODS: A randomized study including 40 surgical patients, treated with either albumin or starch solutions, was performed. Volumes of colloids were given based on clinical indication. Blood samples were obtained. The serum samples were analyzed to determine the concentrations of albumin, tryptophan, N-acetyl-dl-tryptophan, caprylate and alpha-1-acid glycoprotein as well as in vitro drug binding of naproxen, warfarin and digitoxin. RESULTS: During surgery, the albumin concentration declined in the Starch group from 26.8 to 15.3 g/l. It remained unchanged in the Albumin group (29.2 g/l). The two groups were analyzed with the pre-operative sample acting as the control. In the starch group, the percent free concentration of the drugs increased significantly (P<0.01): for naproxen from 0.2% to 0.6%, for warfarin from 1.2% to 1.8% and for digitoxin from 6.8% to 11.1%. In the Albumin group, the % free fraction of naproxen doubled from 0.1% to 0.2% (P<0.05), whereas the % free fraction of warfarin decreased from 1.1% to 1.0% (P<0.05). The free fraction of digitoxin remained unchanged. CONCLUSIONS: Infusion of albumin during surgery resulted in maintained albumin values and almost maintained binding parameters for the study drugs, although some statistically significant changes were found. The use of starch solutions, however, led to in a reduction in albumin values and a significant reduction in binding parameters.

Nitric-oxide-dependent activation of pig oocytes: the role of the cGMP-signalling pathway.

Pig oocytes matured in vitro were parthenogenetically activated (78%) after treatment with 2 mM nitric oxide-donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP) for 24 h. Inhibition of soluble guanylyl cyclase with the specific inhibitors 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or 6-anilino-5,8-quinolinequinone (LY83583) suppressed the SNAP-induced activation in a dose-dependent manner (23% of activated oocytes after treatment with 400 microM ODQ; 12% of activated oocytes after treatment with 40 microM LY83583). 8-Bromo-cyclic guanosine monophosphate (8-Br-cGMP), a phosphodiesterase-resistant analogue of cGMP, enhances the effect of suboptimal doses (0.1 or 0.5 mM) of the NO donor SNAP. DT3, a specific inhibitor of cGMP-dependent protein kinase (PKG, PKG), is also able to inhibit the activation of pig oocytes after NO donor treatment. Involvement of the cGMP-dependent signalling pathway is specific for NO-induced oocyte activation, because both the guanylyl cyclase inhibitor ODQ and the PKG inhibitor DT3 are unable to inhibit activation in oocytes treated with the calcium ionophore A23187. These data indicate that the activation of pig oocytes with an NO donor is cGMP-dependent and that PKG plays an important role in this mode of oocyte activation.

Calcium-dependent regulation of NF-(kappa)B activation in cystic fibrosis airway epithelial cells.

Dysregulation of nuclear factor kappa B (NF-(kappa)B) and increased Ca(2+) signals have been reported in airway epithelial cells of patients with cystic fibrosis (CF). The hypothesis that Ca(2+) signaling may regulate NF-(kappa)B activation was tested in a CF bronchial epithelial cell line (IB3-1, CFTR genotype DeltaF508/W1282X) and compared to the CFTR-corrected epithelial cell line S9 using fluorescence microscopy to visualized in situ NF-(kappa)B activation at the single cell level. Upon stimulation with IL-1beta,we observed a slow but prolonged [Ca(2+)](i) increase (up to 10 min) in IB3-1 cells compared to S9 cells. The IL-1beta-induced [Ca(2+)](i) response was accompanied by an activation of NF-(kappa)B in IB3-1 but not in S9 cells. Pretreatment of IB3-1 cells with the ER Ca(2+) pump inhibitor thapsigargin inhibited the IL-1beta-induced [Ca(2+)](i) response. Treatment with either the calcium chelator BAPTA or an inhibitor of I(kappa)Balpha phosphorylation (digitoxin) led to a drastic [Ca(2+)](i) decrease accompanied by an inhibition of NF-(kappa)B activation of IL-1beta-stimulated IB3-1 cells in comparison to untreated cells. In IB3-1 cells cultured at low temperature (26 degrees C) for 16 h, the IL-1beta-induced [Ca(2+)](i) response was inhibited and no significant NF-(kappa)B activation was observed. To our knowledge, this is the first report of visualization of the Ca(2+)-mediated activation of NF-(kappa)B in individual living airway epithelial cells. Our results support the concept that [Ca(2+)](i) is a key regulator of NF-(kappa)B activation in CF airway epithelial cells.

Application of fluorescence correlation spectroscopy to hapten-antibody binding.

Two-photon fluorescence correlation spectroscopy 2P-FCS has received a large amount of attention over the past ten years as a technique that can monitor the concentration, the dynamics, and the interactions of molecules with single molecule sensitivity. In this chapter, we explain how 2P-FCS is carried out for a specific ligand-binding problem. We briefly outline considerations for proper instrument design and instrument calibration. General theory of autocorrelation analysis is explained and straightforward equations are given to analyze simple binding data. Specific concerns in the analytical methods related to IgG, such as the presence of two equivalent sites and fractional quenching of the bound hapten-fluorophore conjugate, are explored and equations are described to account for these issues. We apply these equations to data on two antibody-hapten pairs: antidigoxin IgG with fluorescein-digoxin and antidigitoxin IgG with Alexa488-digitoxin. Digoxin and digitoxin are important cardio glycoside drugs, toxic at higher levels, and their blood concentrations must be monitored carefully. Clearly, concentration assays based on IgG rely on accurate knowledge of the hapten-IgG binding strengths. The protocols for measuring and determining the dissociation constants for both IgG-hapten pairs are outlined and discussed.

Electrospray ionization mass spectrometry as a tool for determination of drug binding sites to human serum albumin by noncovalent interaction.

Most proteins in blood plasma bind ligands. Human serum albumin (HSA) is the main transport protein with a very high capacity for binding of endogenous and exogenous compounds in plasma. Many pharmacokinetic properties of a drug depend on the level of binding to plasma proteins. This work reports studies of noncovalent interactions by means of nanoelectrospray ionization mass spectrometry (nanoESI-MS) for determination of the specific binding of selected drug candidates to HSA. Warfarin, iopanoic acid and digitoxin were chosen as site-specific probes that bind to the main sites of HSA. Two drug candidates and two known binders to HSA were analyzed using a competitive approach. The drugs were incubated with the target protein followed by addition of site-specific probes, one at a time. The drug candidates showed predominant affinity to site I (warfarin site). Naproxen and glyburide showed affinity to both sites I and II. The advantages of nanoESI-MS for these studies are the sensitivity, the absence of labeled molecules and the short method development time.

Pharmaceutical-grade albumin: impaired drug-binding capacity in vitro.

BACKGROUND: Albumin is the most abundant protein in blood plasma, and due to its ligand binding properties, serves as a circulating depot for endogenous and exogenous (e.g. drugs) compounds. Hence, the unbound drug is the pharmacologically active drug. Commercial human albumin preparations are frequently used during surgery and in critically ill patients. Recent studies have indicated that the use of pharmaceutical-grade albumin is controversial in critically ill patients. In this in vitro study we investigated the drug binding properties of pharmaceutical-grade albumins (Baxter/Immuno, Octapharma, and Pharmacia & Upjohn), native human serum, and commercially available human serum albumin from Sigma Chemical Company. METHODS: The binding properties of the various albumin solutions were tested in vitro by means of ultrafiltration. Naproxen, warfarin, and digitoxin were used as ligands. HPLC was used to quantitate the total and free drug concentrations. The data were fitted to a model of two classes of binding sites for naproxen and warfarin and one class for digitoxin, using Microsoft Excel and Graphpad Prism. RESULTS: The drugs were highly bound to albumin (95-99.5%). The highest affinity (lowest K1) was found with naproxen. Pharmaceutical-grade albumin solutions displayed significantly lower drug-binding capacity compared to native human serum and Sigma albumin. Thus, the free fraction was considerably higher, approximately 40 times for naproxen and 5 and 2 times for warfarin and digitoxin, respectively. The stabilizers caprylic acid and N-acetyl-DL-tryptophan used in the manufacturing procedure seem to be of importance. Adding the stabilizers to human serum and Sigma albumin reduced the binding affinity whereas charcoal treatment of the pharmaceutical-grade albumin from Octapharma almost restored the specific binding capacity. CONCLUSION: This in vitro study demonstrates that the specific binding for warfarin and digitoxin is significantly reduced and for naproxen no longer detectable in pharmaceutical-grade albumin. It further shows that the addition of stabilizers may be of major importance for this effect.

Biosensor analysis of the interaction between immobilized human serum albumin and drug compounds for prediction of human serum albumin binding levels.

The interactions between a set of drugs, selected on the basis of reported human serum albumin (HSA) binding levels, and immobilized HSA were investigated using surface plasmon resonance technology. Major HSA binding sites were available after immobilization. The intensity of the signal obtained from the interaction of the drug with the HSA surface was correlated with the reported HSA binding level. Drugs were classified into groups corresponding to high, medium, or low HSA binding based on the injection of the drug at 80 microM concentration. A set of 10 drugs binding to alpha(1)-acid glycoprotein (AGP) was also investigated and correlated with reported AGP binding data. The throughput of the presented assay is 100 compounds/24 h, and the sample consumption is less than 100 microL (8 nmol).

Effect of nanoparticles on digitoxin uptake and pharmacologic activity in rat glomerular mesangial cell cultures.

Our experiments analyzed the uptake of free and nanoparticles (NP)-associated digitoxin (DGT) by rat glomerular mesangial cells. NP were prepared by the nanoprecipitation method using the biodegradable polyester, polycaprolactone (PCL). Prior to in vitro experiments, the systems were characterized by means of spectrofluorimetry, dynamic light scattering, and size exclusion chromatography (SEC). The loading efficiency was 80.30 +/- 1.03% of the initial DGT amount in the preparation, and the average particle size was 176 +/- 8 and 161 +/- 6 nm for DGT-NP and "empty" NP, respectively. SEC studies revealed noncovalent interactions among the different chemical compounds in the formulation. In vitro experiments were conducted at 37 degrees C and pH 7.5 by incubating "empty" NP, free DGT or DGT-NP (10 microg PCL/mL; 100 ng DGT/mL) with glomerular mesangial cells for 30 and 60 min. Uptake of DGT by the cells was favored by its incorporation into PCL-NP and showed time dependency. After 30 min of incubation, no significant differences of drug uptake were seen between free DGT (13.1 +/- 2.8%) or DGT-NP (17.4 +/- 4.9%); however, the uptake of DGT, when it was associated to the polymeric carrier, increased by approximately 2-fold (37.8 +/- 5.7%) at 60 min, whereas no significant changes were observed for free drug (20.0 +/- 6.8%). The pharmacologic activity of the drug was evaluated by measuring the planar cell surface area (PCSA). "Empty" NP, free drug, or DGT-NP did not produce significant variations on the PCSA as compared with control cells after a 30-min incubation. Nonetheless, DGT-NP reduced the PCSA to 82.51 +/- 8.42% of control values when the incubation lasted 60 min. The ability of cells to exclude the trypan blue dye and the leakage of lactate dehydrogenase into the medium revealed no signs of increased toxicity from incorporation of DGT into PCL-NP. Therefore, PCL-NP improved drug uptake by the cells without altering the pharmacologic activity and toxicity of the drug. Thus, they can be a useful approach to target drugs to the kidneys or the heart.

Interspecies differences in cancer susceptibility and toxicity.

One of the most complex challenges to the toxicologist represents extrapolation from laboratory animals to humans. In this article, we review interspecies differences in metabolism and toxicity of heterocyclic amines, aflatoxin B1, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and related compounds, endocrine disrupters, polycyclic aromatic hydrocarbons, tamoxifen, and digitoxin. As far as possible, extrapolations to human toxicity and carcinogenicity are performed. Humans may be more susceptible to the carcinogenic effect of heterocyclic amines than monkeys, rats, and mice. Especially, individuals with high CYP1A2 and 3A4 activities and the rapid acetylator phenotype may be expected to have an increased risk. Striking interspecies variation in susceptibility to aflatoxin B1 carcinogenesis is known, with rats representing the most sensitive and mice the most resistant species, refractory to dietary levels three orders of magnitude higher than rats. An efficient conjugation with glutathione, catalyzed by glutathione S-transferase mYc, confers aflatoxin B1 resistance to mice. Extremely large interspecies differences in TCDD-induced toxicity are known. The guinea pig is the most susceptible mammal known, with an LD50 in the range 1-2 micrograms TCDD/kg, whereas the hamster is the most resistant species with an LD50 greater than 3000 micrograms/kg. A number of experts have pointed out to the fact that humans appear to be less sensitive to TCDD than most laboratory animals. Human exposure to background levels of TCDD is not likely to cause an incremental cancer risk. A clear cause--effect relationship has been shown between environmental endocrine-disrupting contaminants and adverse health effects in wildlife, whereas the effects seem to be less critical for humans. Studies on DNA adduct formation and metabolism of the nonsteroidal antiestrogen tamoxifen indicate that rats and mice are orders of magnitude more susceptible than humans.

Non-absorption of ingested lipophilic and amphiphilic allelochemicals by generalist grasshoppers: the role of extractive ultrafiltration by the peritrophic envelope.

The role of the peritrophic envelope in the non-absorption of three allelochemicals ingested by generalist grasshoppers was examined. This study tested the hypothesis that the association of lipophilic and amphiphilic allelochemicals with lipid aggregates (mixed micelles) reduces their permeability through the peritrophic envelope, a process similar to extractive ultrafiltration. Each of three allelochemicals (digitoxin, ouabain, and xanthotoxin) were solubilized in a lysolecithin suspension and injected separately into the midgut lumens of adult Melanoplus sanguinipes (Orthoptera: Acrididae). The low permeability of digitoxin through the peritrophic envelope was consistent with the extractive ultrafiltration of this compound. By comparison, ouabain and xanthotoxin permeability coefficients were 7- and 12-fold higher, respectively, than those of digitoxin. The results of extractive ultrafiltration assays confirmed that digitoxin is effectively extracted in lysolecithin micelles, but that neither ouabain nor xanthotoxin aggregates efficiently with these micelles.

Characterisation of the binding of digitoxin and acetyldigitoxin to human serum albumin by high-performance affinity chromatography.

Zonal elution and high-performance affinity chromatography were used to examine interactions of the drugs digitoxin and acetyldigitoxin with the protein human serum albumin (HSA). This was done by injecting small amounts of digitoxin and acetyldigitoxin onto an immobilized HSA column in the presence of mobile phases that contained various concentrations of digitoxin, acetyldigitoxin or other solutes as competing agents. A fixed concentration of beta-cyclodextrin was also present in the mobile phase as a solubilising agent. It was found that digitoxin and acetyldigitoxin each had strong interactions at a single common binding site on HSA, but with slightly different equilibrium constants for this region. Neither compound showed any competition with warfarin or L-tryptophan, which were used as probes for binding at the warfarin-azapropazone and indole-benzodiazepine sites of HSA. These results confirmed the presence of a separate binding region on HSA for digitoxin-related compounds.

[Prolonged half-life of digitoxin in the elderly]. / Forlenget halveringstid for digitoksin hos de eldste.

Digitoxin is frequently used in Norway in the treatment of cardiac failure. Digitalis glycosides may give rise to a number of side effects difficult to separate from disease in the elderly. Six patients aged 77-93 years, treated with digitoxin 0.05 mg/day, were hospitalized due to digitalis intoxication. Mean digitoxin half-life was 25.2 days. This is significantly more than reported in other studies on younger patients. The symptoms of digitoxin intoxication disappeared on discontinuation of medication. The slow elimination of digitoxin may be related to reduced serum albumin concentration. When digitoxin is used in the treatment of heart failure in the very elderly patients, one should be aware of the possibility of digitoxin intoxication, even at a low dose.