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.     

Protein binding of non-steroidal anti-inflammatory drugs in plasma and synovial fluid of arthritic patients

1 The protein binding of seven non-steroidal anti-inflammatory drugs (indomethacin, tolmetin, salicylic acid, ibuprofen, flurbiprofen, naproxen and GP53,633) and warfarin was investigated by equilibrium dialysis in simultaneous samples of synovial fluid and plasma from 12 arthritic patients. 2 The protein binding of all drugs studied except warfarin and flurbiprofen was significantly lower in synovial fluid than in plasma. 3 The decreased protein binding of these drugs is likely to explain the lower total drug concentrations found in synovial fluid in comparison to plasma. 4 The lower albumin concentration plays an important role in determination of reduced drug binding in synovial fluid compared to plasma and the fatty acid concentration in synovial fluid may also influence the protein binding of some of these drugs.     

Differentiated effects of liver cirrhosis on the albumin binding sites for diazepam,salicylic acid and warfarin

The protein binding of diazepam, salicylic acid and warfarin in serum of patients with liver cirrhosis has been studied by equilibrium dialysis and circular dichroism measurements and compared with that in normal serum. Diazepam and warfarin are mainly bound to two separate sites on the serum albumin molecule and salicylic acid to both sites. The binding of diazepam and salicylic acid was impaired in serum from the patients when compared with the binding in serum from healthy individuals, while the binding of warfarin was unchanged. The results were the same when the binding was studied with albumin isolated from patient serum and normal serum. After treatment of the albumin with charcoal at pH 3, however, the binding was improved.The apparent binding constants of the drugs in serum both from patients with liver cirrhosis and from healthy persons were increased by dilution of the sera in buffer. This effect is due to the presence of competitive inhibitors in the sera. The decreased binding of diazepam and salicylic acid in the serum from the patients with liver cirrhosis can in addition be related to the presence of allosteric inhibitors specifically affecting the diazepam binding site on albumin, while the warfarin site apparently is unaffected.     

Binding of non-steroid anti-inflammatory drugs and warfarin to liver tissue of rabbits in vitro

Liver slices (10mm 0.1mm thickness) were incubated at 37 degrees C in drug solutions up to 360 min. The drugs were dissolved in Ringer solution of pH 7.0. After 60, 180, 240 and 360 min each drug concentration was determined in the incubation fluid. For all drugs tested equilibrium was obtained after 240 min. The amount of drug taken up by the slices was calculated from the difference to initial concentration. In parallel, samples of the slices were homogenized together with their incubation fluids after the same incubation intervals. The free concentration was determined by ultrafiltration. For warfarin and several non-steroid anti-inflammatory drugs (NSAID), i.e. acetylsalicylic acid, ibuprofen, ketoprofen and oxyphenbutazone, there was no difference between the free drug concentrations in the homogenized and in non-homogenized samples. This suggests that the binding of these drugs to liver tissue was not altered by homogenization. Further was studied whether NSAIDS interfere with binding of warfarin to liver tissue. Acetylsalicylic acid, flurbiprofen, ibuprofen, ketoprofen, oxyphenbutazone and proquazon markedly increased the free concentration of warfarin both in liver slices and homogenates (p less than 0.01). The extent of displacement did not differ between slices and homogenates.     

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".     

Evidence for the existence of high affinity binding sites for indomethacin on human blood platelets

Using human blood-washed platelets and [3H]indomethacin, we demonstrated the presence of saturable, time- and temperature-dependent high affinity binding sites for non-steroidal anti-inflammatory drugs. The observed Kd value for indomethacin was 5 nM. Structural specificity of the non-steroidal anti-inflammatory drug site was studied with arylacetic acids, anthranilic acids, and compounds from other chemical families. Arylacetic acid drugs had affinities which were similar to the affinity of indomethacin. Affinity differences among the other drugs may be related to the presence or absence of the lipophilic substituent on the central ring. As expected, anti-inflammatory pyrrazole derivatives, aspirin, bucloxic acid, cortisol, nordihydroguaiaretic acid, and the chemotactic peptide formyl-Met-Leu-Phe were not recognized by the indomethacin binding site.     

The Effect of Co-administered Drugs on Oxaprozin Binding to Human Serum Albumin

The binding of the non-steroidal anti-inflammatory drug oxaprozin to human serum albumin was studied by bioaffinity high-performance liquid chromatography using a column based on immobilized human serum albumin. Displacement studies using marker compounds for the major drug binding sites showed that oxaprozin has a high affinity for the benzodiazepine/indole site and binds to the warfarin site but with a significantly lower affinity. Biochromatography and ultrafiltration techniques were used to screen for possible competition and allosteric interactions between oxaprozin and potential co-administered drugs including non-steroidal anti-inflammatory drugs, antipyretics, hypoglycaemics, inhibitors of angiotensin-converting enzyme, anaesthetics, metal ions and anticancer agents. Competition occurred mainly with drugs bound at the benzodiazepine site (benzodiazepines, various non-steroidal anti-inflammatories).     

In vitro binding study of gemfibrozil to human serum proteins and erythrocytes: interactions with other drugs

The binding of gemfibrozil to human serum, isolated proteins and erythrocytes was studied in vitro by equilibrium dialysis. Our results show that this drug is highly bound to serum (99%) at therapeutic levels. Human serum albumin was shown to be mainly responsible for this binding (98.6%) with a saturable process characterized by two binding sites with a moderate affinity. Like many acidic drugs with a carboxylic acidic group, gemfibrozil showed none or negligible binding to alpha 1 acid glycoprotein, lipoproteins and gamma-globulins. The drug binding to erythrocytes is very low (0.8%). The unbound fraction in blood remains constant (0.8%) within the range of therapeutic concentrations. Moreover, interactions were studied with bilirubin and palmitic acid at pathophysiological concentrations and acenocoumarol, salicylic acid, valproic acid, furosemide, phenylbutazone, tolbutamide, warfarin and sulfamethoxazol at therapeutic concentrations. Neither endogenous compounds nor the other drugs studied altered gemfibrozil binding in serum. Likewise, the binding of warfarin to serum and to human serum albumin (600 microM) is not influenced by gemfibrozil.     

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.     

Characterization of a binding site of UCN-01, a novel anticancer drug on alpha-acid glycoprotein

The binding site of 7-hydroxystaurosporine (UCN-01) on alpha-acid glycoprotein (AGP) was studied by fluorescence and ultracentrifugation experiments. Three ligands, propranolol, warfarin and progesterone were employed as marker ligands and quinaldine red was employed as a fluorescent probe. The presence of UCN-01, pro- pranolol, warfarin and progesterone resulted in a significant quenching of the fluorescence of quinaldine red, when bound to AGP, depending upon the potency of the binding to AGP. The construction of Klotz plots indicated that the displacement effects of propranolol, warfarin and progesterone on UCN-01-AGP binding were competitive in nature. These data suggest that the binding site of UCN-01 on the AGP partly overlaps the binding site for basic drugs, acidic drugs, as well as steroid hormones.     

Variation in the binding affinity of warfarin and phenprocoumon to human serum albumin in relation to surgery

Summary The binding equilibria of warfarin and phenprocoumon with defatted human serum albumin were studied by equilibrium dialysis in 33 mM sodium phosphate buffer, pH 7.4, 37°C. The binding isotherms for both ligands were consistent with binding to two similar and independent sites in the albumin molecule.The binding affinity of warfarin was markedly increased on adding palmitic acid up to palmitate 4 mol per mol albumin and then it decreased. The binding affinity of phenprocoumon varied similarly but to a lesser degree.Serum samples were obtained from 14 patients under-going knee joint surgery, six consecutive samples from each patient. The binding affinity of warfarin and phenprocoumon added in low concentrations to the serum samples was consistently less than to purified albumin. The binding affinity for warfarin increased slightly with increasing fatty acid concentrations during surgery, but the increase was much less than expected from the in vitro studies. The binding of phenprocoumon in the serum samples was not influenced by changing fatty acid concentration. The binding affinity for both drugs decreased markedly during the three days following surgery.     

Protein binding of GP53,633: A basic nonsteroidal anti-inflammatory drug

The protein binding of GP53,633 [2-tert.butyl-4(5)-phenyl-5(4)-(3-pyridyl)-imidazole], a basic non-steroidal anti-inflammatory drug (NSAID), has been investigated. Although GP53,633 is a base (pK_a 6.4) rather than an acid, the binding in plasma or serum was totally accounted for by binding to albumin. Scatchard analysis of the binding to albumin suggested the presence of one high affinity site and a number of low affinity sites. GP53,633 and its major metabolite, CGP8716, displaced site-I fluorescent probes (DNSA and warfarin) but not the site-II probe dansylsarcosine. Binding studies by equilibrium dialysis showed that GP53,633 and its metabolite displaced site-I drugs but not site-II drugs, and ¹⁴C-GP53,633 was itself displaced by site-I but not site-II drugs. As with other site-I drugs, the binding of GP53,633 was enhanced by addition of oleic acid at molar ratios of up to 2:1 with albumin. Albumin binding of GP53,633 was markedly increased by raising the pH from 6.0 to 8.5 suggesting that only the unionised drug can bind at site-I. The data are consistent with the major part of the binding energy at site-I being due to hydrophobic interactions and also suggest that there is a cationic centre on the protein at or near site-I which precludes the binding of positively charged drugs.     

Influence of long-chain free fatty acids on the binding of warfarin to bovine serum albumin

The effects of the concentration of long-chain free fatty acids and bovine serum albumin on the binding characteristics of warfarin with albumin have been studied by adsorption and fluorescence spectroscopy using the method of ultrafiltration. The binding affinity of warfarin is higher in a concentrated than in a dilute albumin solution. In a dilute solution of serum albumin (55 μM or less), the binding constant of the warfarin-albumin complex is increased in the presence of 100 μM (or less) of either lauric or oleic acid or a mixture of these two acids, but higher concentrations of the fatty acids decrease such binding. A mixture of saturated and unsaturated fatty acids shows the same effects as those by either fatty acid. Simultaneous measurements of the fluorescence peak intensity and polarization of fluorescence of warfarin bound to albumin in a dilute solution indicate that low and high concentrations of free fatty acids induce different conformational states of the same warfarin-binding sites on the albumin molecule. Such a dualistic behavior of free fatty acids could be best interpreted in terms of allosteric interactions involving heterotropic effects. On the other hand, in a concentrated solution of serum albumin, the binding constant of warfarin is decreased in the presence of both low and high concentrations of free fatty acids. Such a decrease in warfarin binding with concentrated albumin in the presence of free fatty acids is not due simply to a displacement of warfarin by free fatty acids from the warfarin-binding sites, but also could result simultaneously from a further conformational change of warfarin-binding sites caused by the interaction of warfarin and free fatty acids (a negative heterotropic effect).     

Protein binding of drugs in uremic and normal serum: The role of endogenous binding inhibitors

The protein binding of diazepam, indomethacin, salicylic acid, sulfadimetoxine and warfarin in serum of uremic patients has been studied by equilibrium dialysis and circular dichroism measurements and compared with that in normal serum. Comparisons have also been made with isolated human serum albumin (HSA) from uremic patients and healthy individuals. The binding of diazepam, salicylic acid, sulfadimetoxine and warfarin is impaired in the uremic sera, while the binding of indomethacin is apparently unchanged. The apparent binding constants of salicylic acid and warfarin in both uremic and normal sera are affected by dilution of the sera in buffer. The binding constants obtained with isolated albumins, however, are unaffected by dilution. The albumin isolated from uremic serum shows lower binding affinity for salicylic acid and warfarin than normal HSA, but the affinity was normalized by charcoal treatment at pH 3.0. It is shown that the binding both in normal and uremic sera is impaired compared with isolated defatted serum albumin due to the presence of competitive inhibitors. The inhibition is more pronounced in uremic serum. In addition, the binding to albumin in uremic sera is impaired by strongly bound allosteric inhibitors. It is also emphasized that determinations of association constants have to be related to the dilution of the serum, plasma or blood, respectively.     

Studies of the action of some anti-inflammatory drugs on complement mediated immune haemolysis.

1 The effects of various anti-inflammatory and non-anti-inflammatory drugs on complement mediated haemolysis have been studied. Drugs which were significantly protein bound were found to inhibit this form of immune lysis, but only at greater concentrations than achieved therapeutically. 2 Removal of the drugs by prolonged dialysis resulted in restoration of complement activity with the exceptions of phenylbutazone and warfarin sodium. 3 Reconstitution experiments indicated that C2 and some of the later components especially C7 were affected by the drugs. 4 Intra-articular injections of prednisolone (100 mg) in patients with rheumatoid arthritis, failed to produce significant changes in the synovial fluid complement system. 5 None of the drugs affected the binding of antibody to antigen, or the ability of sensitized sheep cells to fix complement.     

Effect of heparin and fatty acids on the binding of quinidine and warfarin in plasma

After an intravenous injection of heparin, the plasma protein binding of warfarin was greatly increased while the binding of quinidine seemed to be less affected. The increased binding of warfarin seemed partly due to the release of plasma non-esterified fatty acids, NEFA, but the level of NEFA alone could not explain the interindividual variations of plasma warfarin binding. Addition in vitro of palmitic acid to serum demonstrated an increased binding of warfarin and an unaltered binding of quinidine up to a serum NEFA level of 3.0 meq/l. Albumin isolated from post heparin plasma revealed an increased binding affinity for warfarin at the warfarin high affinity binding sites, and a slightly depressed affinity for quinidine. The low affinity binding sites on the albumin molecule for both drugs did not seem to be influenced by NEFA.     

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.     

Modifications in prolactin binding capacity in the rat liver induced by non-steroidal anti-inflammatory drugs

Modifications in prolactin specific binding in the rat liver induced by different non-steroidal anti-inflammatory drugs (indomethacin, piroxicam, ketoprofen, phenylbutazone, mefenamic acid and acetylsalicylic acid) have been studied. All caused a dose-dependent inhibition of prolactin binding capacity whereas no change was found in dissociation constant values. The inhibitory effect is reversible and highly specific, since insulin binding to the same membrane preparation is not affected. The degree of inhibitory activity on prolactin binding is related to the anti-inflammatory activity of each drug, which supports the hypothesis of an involvement of prostaglandin synthesis.     

Interactions of human serum albumin with meloxicam: Characterization of binding site

Human serum albumin (HSA) is the most prominent protein in plasma. The three-domain design of HSA provides a variety of binding sites for many ligands, including heme, bilirubin and drugs. Here, we report the effect of new generation, non-steroidal anti-inflammatory drug (NSAID) meloxicam on the albumin conformation and ligand binding. In the present work the interaction of meloxicam with HSA in aqueous solution at physiological pH has been investigated through circular dichroism and fluorescence spectroscopy. The strong quenching of the fluorescence clearly indicated that the binding of the drug to HSA changed the microenvironment of tryptophan residue and the tertiary structure of HSA. This was confirmed by the destabilization of the warfarin binding site. CD and fluorescence spectroscopic results showed marked reductions (about 40% decrease in the CD Cotton effect intensity, and ∼15% decrease of the fluorescence intensity) in the affinity of albumin for bilirubin upon meloxicam binding. The strong inhibition of warfarin and ANS bound to protein after meloxicam modification compared with aspirin confirms that the binding site of both drugs is not the same.