The interaction of enflurane, halothane and the halothane metabolite trifluoroacetic acid with the binding of acidic drugs to human serum albumin. An in vitro study

The interaction of the volatile anaesthetics enflurane, halothane and the halothane metabolite trifluoroacetic acid with the binding of two highly bound acidic drugs (warfarin, phenytoin) to albumin has been studied in vitro by equilibrium dialysis. Trifluoroacetic acid (TFA) inhibited the binding of both drugs to human serum albumin (HSA). Halothane, on the other hand, increased the binding of warfarin to HSA, while enflurane inhibited only the binding of phenytoin. It seems that the binding of the acidic drugs warfarin and phenytoin to HSA is more sensitive to the structures of the gases than for the basic drug diazepam which was previously shown to be equally affected by both gases. Furthermore, it seems that drugs competing for the same binding site (warfarin, phenytoin) may respond differently to conformational changes of the site. It is suggested that drugs bound to the "diazepam site" are more easily affected by the volatile anaesthetics than drugs bound to the "warfarin site".     

Stereoselective binding of the glucuronide conjugates of carprofen enantiomers to human serum albumin

The stereoselective binding of carprofen enantiomers and carprofen glucuronide diastereomers to human serum albumin (HSA) was studied using an ultrafiltration method. Carprofen glucuronides exhibit a considerable and stereoselective affinity to HSA, although less than that seen for the parent enantiomers. The (S)-glucuronide showed a higher binding affinity to HSA than the (R)-glucuronide. The (S)-enantiomer of unmetabolized carprofen was bound to fatty acid free HSA to a much greater extent than the (R)-enantiomer. Warfarin reduced the binding of the glucuronides to a greater extent than did diazepam, but diazepam displaced the unconjugated enantiomers to a greater extent than did warfarin. These results suggest differences in binding region between the carprofen enantiomers and their glucuronides on the albumin molecule.     

Some novel observations of a drug (indoprofen)—albumin interaction

Indoprofen, a new-steroidal anti-inflammatory agent, binds to human serum albumin in an endothermic reaction at low degrees of saturation and with an exothermic reaction at higher drug to protein ratios. Although indoprofen appears to bind to the same primary site as diazepam, dialysis studies show an increased binding of both drugs in the presence of the other. This novel observation is probably due to the effect of the drugs on the N → B conformational change of human serum albumin.     

Use of 1-anilino-8-naphthalene sulfonate as a fluorescent probe in the investigation of drug interactions with human alpha-1-acid glycoprotein and serum albumin

We report a rapid method for the characterization of the human serum albumin (HSA) and alpha-1-acid glycoprotein (AAG) interactions with drugs. The binding of 1-anilino-8-naphthalene sulfonate (ANS) to AAG and HSA was measured by fluorescence spectroscopy. Fluorescence data indicated that ANS was bound tightly to at least one site on AAG, with an affinity constant of 1.35 x 10(6) M-1. The fluorescence of an ANS:AAG complex was quenched by the binding of various drugs. Fluorescence quenching of the HSA:ANS complex showed a single site with an affinity constant of 0.72 x 10(6) M-1. The interaction of AAG and HSA with ANS or other drugs was also studied by comparative equilibrium dialysis. [14C]Pipequaline was used as an AAG and HSA site marker. [14C]Pipequaline seems to share sites I (azapropazone) and II (diazepam and ibuprofen) of HSA. However, high concentrations of warfarin were unable to displace [14C]pipequaline. On the other hand, it was shown that palmitic acid decreased, whereas bilirubin increased the pipequaline binding.     

Binding of nonsteroidal anti-inflammatory agents and their effect on binding of racemic warfarin and its enantiomers to human serum albumin

The binding of racemic warfarin, its enantiomers, and several nonsteroidal anti-inflammatory agents to human serum albumin was investigated by equilibrium dialysis at 4 degrees C in pH 7.4 phosphate buffer. The primary binding constant for the S(-) enantiomer of warfarin was approximately two times greater than the corresponding binding of the R(+) enantiomer. The effect of azapropazone, phenylbutazone, naproxen, ibuprofen, mefenamic acid, and tolmetin on the binding of racemic warfarin and its enantiomers was studied. Warfarin was displaced by all of the nonsteroidal anti-inflammatory agents except tolmetin. Azapropazone caused the largest displacement of warfarin (39 to 46% free warfarin versus 2.5 to 6% free warfarin without competing drug), followed by phenylbutazone (23 to 43% free warfarin), naproxen (9 to 24% free warfarin), mefenamic acid (5 to 11.5% free warfarin), and ibuprofen (5 to 9% free warfarin). Azapropazone and phenylbutazone competed with warfarin for the same primary binding site on the albumin molecule. Naproxen appeared to affect warfarin binding at both primary and secondary sites. Ibuprofen and mefenamic acid interfered with the binding of warfarin at its secondary sites. In contrast to the other drugs studied, tolmetin caused an increase in the primary binding constant of warfarin. Structural analysis indicated that a common feature of those compounds which primarily bind at the warfarin site is a hydrophobic area bearing a widely delocalized negative charge.     

The role of buffer composition during equilibrium dialysis in determining concentrations of free diazepam in serum

Serum levels of free diazepam as determined with equilibrium dialysis depend on the composition of the dialyzing buffer with respect to Ca2+ and Cl-. Physiological concentrations of these ions double the obtained free serum levels, in line with data of diazepam containing albumin solutions. The diazepam binding capacity of serum was found to be higher than that of albumin alone, suggesting that albumin is probably not the only diazepam binding protein in serum. Evidence was also found that the diazepam binding to these other serum proteins is dependent on the presence of Ca2+ and Cl-.     

Displacement by anionic drugs of endogenous ligands bound to albumin in uremic serum

Impaired binding of anionic drugs to serum albumin in patients with uremia is thought to be due to the accumulation of endogenous substances that bind to albumin. In this study the displacement by the anionic drugs diazepam, warfarin, and salicylic acid, which are known to be representative drugs for the binding sites on the albumin molecule, of several endogenous ligands that bind to albumin in uremic serum was examined. The free fractions of the ligands bound to albumin were separated by ultrafiltration in the presence and the absence of test drugs and assayed by high-performance liquid chromatography. Diazepam displaced indoxyl sulfate (IS), hippuric acid (HA), and indole-3-acetic acid (IAA), and warfarin displaced IS, HA, ISAA, and 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid from serum albumin. However, salicylic acid did not displace the substance examined. The methods reported here are useful for determining the binding sites of the endogenous ligands on albumin and to clarify the drug-ligand interaction on albumin molecule in uremic serum.     

Effect of plasma protein binding on the uptake of methadone and diazepam in the isolated perfused rat lung

The pulmonary uptake of the basic (pKa 9.1) lipophilic amine (methadone) and a nonbasic (pKa 3.4) lipophilic amine (diazepam) was compared in a single pass isolated perfused rat lung (IPL) preparation. The radiolabeled drugs were infused into the IPL for 10 min followed by a 30-min drug-free perfusion. If the perfusate (Krebs-Ringer bicarbonate buffer) contained 4.5% bovine serum albumin, the rapid and extensive uptake observed for methadone (32.4% of infused amount) was similar to that reported for other basic lipophilic amines. Uptake of the nonbasic diazepam was slight (3.4% of infused amount). These results are consistent with the idea that basic amines accumulate in lung tissues to a great extent. However, if the bovine serum albumin was omitted from the perfusate, diazepam uptake in the IPL increased about 10-fold while methadone uptake increased only slightly. This observation, together with the extensive binding of diazepam to plasma albumin, suggested that plasma protein binding was a major factor in limiting the pulmonary accumulation of this nonbasic lipophilic amine. Since many nonbasic drugs are known to have a high affinity for plasma albumin, the observed dependence of pulmonary drug accumulation on basicity of the amine may be related to the plasma protein binding as well as the characteristics of the interaction of amines with pulmonary tissue.     

Stereospecific benzodiazepine receptor binding by the enantiomers of oxazepam sodium hemisuccinate

The ability of the enantiomers of oxazepam sodium hemisuccinate to displace ³H diazepam specifically bound to benzodiazepine receptors has been investigated in crude synaptosomal preparations from rat brain. The relative potencies of the enantiomers to displace ³n diazepam was consistent with their reported differential anticonvulsant activity in animals and anxiolytic activity in man.The benzodiazepines represent a class of drugs with important anxiolytic, hypnotic, muscle relaxant and anticonvulsant properties, (Randall, Schallek, Sternbach &; Ning, 1974). By using ³H diazepam as the ligand in binding studies with crude synaptosomal preparations it has recently been demonstrated that benzodiazepines bind to specific receptors in rat brain. Bound 5n diazepam is not displaced by putative neurotransmitters but the displacement potencies of other benzodiazepines is related to their anxiolytic activities (Squires &; Braestrup, 1977, Möhler &; Okada, 1977).If a drug exhibits stereoisomerism and the enantiomers have differing pharmacological potencies in vivo, then the pharmacological properties most relevant for these effects should also show stereospecificity (Enna, et al., 1976, Crow. &; Johnstone, 1977).The enantiomers of oxazepam sodium hemisuccinate (OXZ) have been reported to exhibit, differing anticonvulsant activity in animal studies (Mussini et al., 1972, de Angelis, Predominate &; Vertua, 1972) and (+) OXZ has recently been shown to be more effective than the racemic mixture in the treatment of anxious patients (Lescovelli, Castellani &; Perbellini, 1976) In view of these findings we have investigated the relative potencies of (+) and (-) OXZ in displacing specific-'H diazepam binding in preparations of rat brain.     

Microcalorimetric study for the binding of ionic drugs to human erythrocytes and the ghost membranes

The binding of phenothiazine derivatives (as cationic drugs) and non-steroidal anti-inflammatory drugs (as anionic drugs) to human erythrocytes and ghost membranes has been compared with respect to their thermodynamic characteristics, by flow microcalorimetry at pH 7.4 and 37 C. From enthalpyentropy correlation, it was shown that anionic and cationic drugs are bound to different binding sites on the membranes. Phenothiazines bind to a single common site of the erythrocyte membranes with relatively high binding affinities (K = 10(4)-10(5) M-1). The binding is entropy-driven and characterized by a small negative enthalpy (delta H) and a positive entropy change (delta S), reflecting hydrophobic interactions. However, the binding reaction for the intact erythrocytes shows large negative values for both delta H and delta S. The values of K for the membranes and delta H for the intact erythrocytes increased with the increase of the hydrophobic character of the substituent group at the C-2 position of the phenothiazine nucleus (H less than Cl less than CF3). The results indicate that phenothiazines bind and or penetrate to the inner membranes of the erythrocytes and react with intracellular components such as haemoglobin, while anti-inflammatory drugs bind to the surface protein on the membranes with a lower affinity (K = 10(3) M-1) than phenothiazines, reflecting the small negative delta H and positive delta S for the interaction with intact erythrocytes.     

Interaction of benzodiazepine derivatives with bovine serum albumin—II: Circular dichroism studies

The interaction of benzodiazepine derivatives with bovine serum albumin (BSA) was studied by circular dichroism (CD) measurements. Most of the investigated benzoidiazepines show biphasic extrinsic Cotton effects, which are largely influenced by raising pH from 6.60 to 8.20. Quantitative estimation of the CD data pointed out that there are several binding sites on the BSA molecule. The CD data do not differ very much from those found for the interaction of the drugs with human serum albumin (HSA). Therefore it is suggested that at least a part of the benzodiazepine binding sites on the BSA molecule has similar properties to the single binding site on the HSA molecule. The extrinsic Cotton effects of the benzodiazepines in the presence of BSA are influenced by fatty acids and sodium dodecyl sulfate in a similar way as by the pH-value of the solution. This is explained by a similar influence of the substances and of pH on the protein conformation.     

Human Serum Albumin Conformational Changes as Induced by Tenoxicam and Modified by Simultaneous Diazepam Binding

Abstract— The binding of tenoxicam to human serum albumin has been shown by affinity chromatography proton titration and equilibrium dialysis to be dependent on the neutral to basic conformational change of the protein. The influence of diazepam on the interaction was also investigated using the same techniques, suggesting that diazepam increases the association of tenoxicam to albumin. Affinity chromatography revealed that the reciprocal effect also occurs. Displacement studies indicated that diazepam causes a significant increase in the affinity of tenoxicam to its main binding site, albumin site I, which is different from the diazepam site (site II). Tenoxicam seemed to cause an allosteric change in the conformation of the protein during its own binding, as did warfarin. The mechanism of this effect was a pH-dependent conformational change of albumin induced by electrostatic forces within the protein. Diazepam induced a distant accommodation of the protein, an effect accompanied by an enhanced inhibition of the release of protons from albumin.     

Use of a human serum albumin-based high-performance liquid chromatography chiral stationary phase for the investigation of protein binding: detection of the allosteric interaction between warfarin and benzodiazepine binding sites

The interaction between the benzodiazepine and the warfarin binding sites in human serum albumin (HSA) has been investigated using an HSA-based HPLC chiral stationary phase (HSA-CSP). (R)-Warfarin and (S)-warfarin were added to the mobile phase and racemic mixtures of oxazepam, lorazepam, and their hemisuccinic derivatives were injected onto the HSA-CSP. The presence of (R)-warfarin in the mobile phase did not significantly affect the chromatographic retention (expressed as capacity factor, k') of the investigated benzodiazepine hemisuccinate derivatives. The presence of (S)-warfarin did not significantly affect the k' of oxazepam and oxazepam hemisuccinate, but resulted in a dramatic increase in the k' of (S)-lorazepam hemisuccinate and also improved the enantiomeric resolution of lorazepam. These results confirm the existence of an allosteric interaction between the benzodiazepine binding site and the warfarin binding site. Furthermore, the study indicates that chromatography on the silica-immobilized HSA can detect interactions between binding sites on the protein. This can be of great importance in the determination of drug-drug interactions.     

Stereoselective binding of tertatolol and of 4-hydroxytertatolol to human plasma proteins.

The binding of racemic tertatolol and 4-hydroxytertatolol and of their enantiomers was compared in alpha 1-acid glycoprotein and albumin solutions. The binding rate of S(-)tertatolol to alpha 1-acid glycoprotein was much greater than that of R(+)tertatolol, the binding of the racemate being intermediate. It was the reverse for the binding to albumin, although the differences were slight. The binding of 4-hydroxytertatolol racemate and enantiomers was very low as compared to the binding of tertatolol, and there were no statistically significant differences in the binding of the 4-hydroxytertatolol enantiomers to either alpha 1-acid glycoprotein or albumin.     

Evidence for the fatty acid-induced heterogeneity of the N and B conformations of human serum albumin

The influence of oleic acid on the interaction between albumin and warfarin, oxyphenbutazone or diazepam has been studied by circular dichroism and equilibrium dialysis. The pH dependences of the molar ellipticity of the drug-albumin complexes and of the free fraction of drug are completely changed by the presence of oleic acid. This phenomenon is attributed to an oleic acid-induced conformational change in both the neutral (N) and the basic (B) conformation of albumin, a change to which the warfarin-oxyphenbutazone binding area and the diazepam binding site is sensitive. The oleic acid-induced conformational states of albumin, the so-called N* and B* conformations, show binding properties that are different from the binding properties of the N and B conformations.     

Serum protein binding of model acidic drugs in smokers and non-smokers

The serum protein binding of sulfisoxazole (sulfafurazole, INN) and diazepam, drugs that bind to the two main binding sites for acidic drugs on albumin, has been studied in young smokers and non-smokers. No differences between the two groups could be detected with regard to the serum protein binding of either drug. Thiocyanate, present in relatively large amounts in the serum of smokers, does not seem to affect the drug-binding properties of serum proteins.     

Effects of phenylbutazone, tolbutamide, and clofibric acid on binding of racemic warfarin and its enantiomers to human serum albumin

The effect of phenylbutazone, tolbutamide, and clofibric acid on the binding of racemic warfarin and its enantiomers to human serum albumin was studied by equilibrium dialysis. Warfarin had one primary and two secondary binding sites on the albumin molecule. No difference in binding was detected at the primary binding site; the extent of R(+)-isomer binding at the secondary binding sites was 2.5 times greater than the corresponding S(-)-isomer binding. Phenylbutazone and warfarin appear to compete for the same primary binding site on the albumin molecule. Tolbutamide interferes with the binding of warfarin enantiomers at their secondary sites. Clofibric acid has a less pronounced effect on warfarin binding than does phenylbutazone or tolbutamide.     

Effect of beta-carboline derivatives on the binding of L-tryptophan and diazepam to bovine and human albumin.

The effects of 12 beta-carboline derivatives on the binding of L-tryptophan and diazepam to bovine and human albumin have been investigated to seek similarities between the indole binding site on albumin and the benzodiazepine receptor in the brain. The binding of L-tryptophan and diazepam was measured at 37 degrees and pH 7.4 by equilibrium dialysis. Norharmane was the most potent inhibitor of the binding of L-tryptophan and diazepam to both bovine and human albumin. The kinetics of the inhibitory effects of several of the beta-carbolines were studied. Norharmane decreased the value (n) for the number of binding sites for the binding of L-tryptophan to both bovine and human albumin. Norharmane and harmane decreased the apparent association (Ka) but increased n for the interaction of diazepam with bovine albumin. Norharmane also had a similar effect on the binding of diazepam to human albumin. The similarities between the inhibitory effects of the beta-carbolines on the binding of L-tryptophan and diazepam to albumin and the affinity of the beta-carbolines for the central benzodiazepine receptor point to some common structural requirements for binding to the receptor and to albumin.     

Species-dependent binding of disopyramide enantiomers

Serum protein binding of the basic enantiomers of disopyramide were studied in several animal species. (S)-(+)-Disopyramide was more highly bound than the (R)-(-)-enantiomer to serum protein in the man, gorilla, and pig. The reverse was true in cow serum, and in serum and albumin from sheep. Enantioselective differences in binding were due to differences in association constants. No enantioselective differences in binding were observed in serum protein from horse and goat, or in albumin from cow and pig. Disopyramide was highly bound to two sites on horse albumin. The association constant characterizing the binding of disopyramide to the first (major) site on horse albumin was 1.3 x 10(7) M-1. At predialysis concentrations of 10(-7) M, tris-(2-butoxyethyl)phosphate displaced disopyramide from sites on horse albumin and from sites on serum protein from the horse, man, gorilla, cow, and pig. At predialysis concentrations of 10(-5) M, warfarin and diazepam had no effect on disopyramide binding in these animal species. It is concluded that the enantioselective binding of disopyramide is species dependent, the site that is responsible for the moderate to high binding of disopyramide enantiomers is probably located on alpha 1-acid glycoprotein, and the sites that bind disopyramide in the horse are located on albumin and may be unique.     

Intravascular factors affecting diazepam binding to human serum albumin.

The binding of a benzodiazepine derivative (diazepam) to human serum albumin was studied by equilibrium dialysis. The influence on this interaction of various endogenous substances (fatty acids, uric acid and bilirubin) and the metabolites of diazepam (desmethyldiazepam and oxazepam) were investigated. Binding studies were analyzed by fitting a model which utilizes the two independently measured quantities, the free drug concentration and the total amount of drug, to the experimental data points with a least squares method. Oleic acid decreases the binding of diazepam at all molar ratios of fatty acid:albumin. In contrast, palmitic acid at ratios of < 1:1 enhanced diazepam binding. Both uric acid and bilirubin had negligible effects on diazepam binding. The metabolites were bound to a less extent than diazepam (diazepam > desmethyldiazepam > oxazepam), and both metabolites reduced diazepam binding to albumin. In vivo, the absolute ratio of specific fatty acids to each other is probably as important a source of variations as the quantitative changes in total fatty acids or metabolites.