Human albumin binding of tamoxifen in the presence of a perfluorochemical erythrocyte substitute.

The binding of tamoxifen (an HSA site IV ligand) to human serum albumin (HSA) in the presence of a perfluorochemical (PFC) erythrocyte substitute has been examined. Standard centrifugation followed by supernatant ultrafiltration was used to study the binding of 0.1 and 0.5 microgram mL-1 tamoxifen at ambient conditions. Tamoxifen was extensively bound (greater than 99%) to the PFC emulsion through an association with the emulsifiers of the droplets. Tamoxifen was also extensively bound (greater than 99%) to HSA. The percent free tamoxifen increased upon HSA dilution. Tamoxifen was extensively bound by various mixtures of HSA and the PFC emulsion and the percent free drug was similar to those obtained with HSA alone. However, the position of drug binding (PFC emulsion vs HSA) varied significantly with changes in the ratio of PFC emulsion to HSA. This could be important in terms of the different distribution of HSA and PFC emulsion in the body. Studies with PFC emulsion components indicated that any displacement of HSA-bound tamoxifen by the PFC emulsion was due to the oleic acid and, to a much smaller degree, Pluronic F-68 components. Other HSA site IV ligands are expected to be similarly displaced.     

Dosage plan of a flurbiprofen injection product using inhibition of protein binding by lipid emulsion in rats

Flurbiprofen-axetil (FP-ax), a bolus injection product of a non-steroidal anti-inflammatory drug (NSAID), is a prodrug of flurbiprofen, an NSAID. As flurbiprofen strongly binds to site II of human serum albumin (HSA), the free (unbound) concentration of flurbiprofen after injection of FP-ax is low. We have examined the inhibitory effect of free fatty acid (FFA), a binding inhibitor for site II of HSA, on the binding of flurbiprofen in-vitro and in-vivo by ultrafiltration, to establish an effective dosage of FP-ax. In-vitro, fatty acid mixtures (FAs) inhibited the binding of flurbiprofen to rat serum albumin. The free fraction of flurbiprofen was remarkably increased by FAs in rat serum. In-vivo, FP-ax was injected into a control group (low FFA concentration in serum) and a lipid emulsion group (high FFA concentration in serum). The area under the curve of the free concentration of flurbiprofen during the alpha phase and the distribution volume of the central compartment of flurbiprofen were significantly higher in the lipid emulsion group than the control group (5.0- and 1.2-times, respectively). When FP-ax was administered at high FFA concentration, the free concentration of flurbiprofen and distribution of flurbiprofen to tissues increased transiently. This administration method may be useful for patients with cancer pain, having a potent analgesic effect.     

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.     

Inhibitory effects of amino-acid fluids on drug binding to site II of human serum albumin in vitro

The effects of amino-acid fluids on ligand binding to human serum albumin (HSA) were investigated by fluorescence and ultrafiltration techniques. Warfarin and dansylsarcosine were used as the site marker fluorescence probes for site I and site II of HSA, respectively. Amino-acid fluids specifically decreased the fluorescence intensity induced by dansylsarcosine-HSA binding without any effects on that induced by warfarin-HSA binding. The ultrafiltration technique clarified that the free fraction of the site II drug, diazepam, in human serum was increased in the presence of amino-acid fluids, while no effect was observed in the free fraction of the site I drug, warfarin. The potencies of the effect on binding to site II, observed by fluorescence and ultrafiltration techniques, correlated well with the L-tryptophan contents in amino-acid fluids or with those in L-tryptophan solutions. Based on the comparison between the effects of amino-acid fluids and L-tryptophan solutions, we confirmed that L-tryptophan in amino-acid fluids specifically inhibits drug binding to site II of HSA.     

Determination of Protein Binding of a Highly Lipophilic Drug,Isocarbacyclin Methyl Ester (TEI-9090), Using a Polydimethylsiloxane-coated Glass Beads Assay

Abstract— An adsorption technique with polydimethylsiloxane-coated glass beads (PDMS-GB) was developed to determine the protein binding of a highly lipophilic and hydrophobic drug. The present assay method is based on the quantitative adsorption of unbound drug to the PDMS-GB. This method of batch separation in a glass assay tube has an advantage of simplicity and rapidity. To evaluate the reliability of PDMS-GB assay, we compared the protein binding of diazepam in serum in-vitro measured by ultrafiltration and PDMS-GB assay. There was no significant difference between the extent of binding measured by each method. Using PDMS-GB assay, we determined the protein binding of the prostaglandin I₂ (PGI₂) analogue isocarbacyclin methyl ester (TEI-9090), whose binding cannot be measured by commonly employed techniques (equilibrium dialysis, ultrafiltration, gel filtration or ultracentrifugation) because of a high degree of adsorption to membranes, resins or tubes. The percentage of TEI-9090 bound in human serum, 4% human serum albumin (HSA, fatty acid-free) and dog serum were ～98, ～87 and ～95%, respectively, and these values were independent of TEI-9090 concentration up to 10 ng mL^?1. The binding of isocarbacyclin (TEI-7165) to serum protein in man, dogs, rabbits and rats, determined by ultrafiltration, was also high (>90%). While the displacement of TEI-9090 and TEI-7165 binding to HSA by aspirin, salicylic acid and indomethacin was not observed, clofibric acid and free fatty acids significantly inhibited the protein binding of both compounds. These results indicate that the binding site of TEI-9090 and TEI-7165 on HSA could be identical with the possible binding site of PGI₂.     

Protein binding of diazepam and digitoxin in uremic and normal serum.

The protein binding of diazepam and digitoxin in serum from uremie patients has been studied by equilibrium dialysis and compared to that in normal serum. Comparisons have also been made with isolated human serum albumin (HSA) from uremie and normal individuals. Diazepam and digitoxin are bound to different sites on HSA. Their binding was impaired in the serum from the patients when compared to that in the normal serum owing to decreased affinity constants for the binding to the primary sites on albumin. In the uremic serum the number of binding sites for diazepam is increased compared with the number in normal serum and HSA. For digitoxin the number of binding sites is larger both in the normal and the patient serum than that obtained with HSA. The fact that apparently an increased number of binding sites is made use of, is probably due to the presence of substances which inhibit the binding to the primary sites. The binding of the drugs was improved after charcoal-treatment of the uremie albumin at pH 3.0.     

Bestimmung der Bindung von Chlordiazepoxid,Diazepam, Nitrazepam und Phenprocoumon an Human- und Rinderserumalbumin

Determination of the Binding of Chlorodiazepoxide, Diazepam, Nitrazepam and Phenprocoumon to Human and Bovine Serum Albumin Applying a modified ultrafiltration method, the binding of Chlorodiazepoxide, Diazepam, Nitrazepam and Phenprocoumon to human serum albumin (HSA) and bovine serum albumin (BSA) was determined (4 % buffered solutions, pH 7.4). Different degrees of binding to HSA and BSA were observed.     

The effects of age and smoking on the plasma protein binding of lignocaine and diazepam.

In 63 healthy ambulant subjects 18 to 88 years of age, the plasma protein binding of diazepam (principally bound to albumin) decreased with age. Diazepam binding in plasma correlated positively with plasma albumin concentration which also decreased with age. In contrast, the plasma protein binding of the basic drug, lignocaine (predominantly bound to alpha 1-acid glycoprotein [AAG]), tended to increase slightly with age. Lignocaine binding in plasma correlated positively with plasma AAG concentration which also increased slightly with age. Smoking did not affect the plasma protein binding of diazepam or lignocaine or the plasma concentrations of albumin, AAG or nonesterified fatty acids. These results suggest that age-related changes in plasma protein binding of lignocaine and diazepam are determined in part by age-related changes in the concentrations of the binding proteins in plasma. The ageing process alone causes only small changes in the plasma protein binding of these drugs compared with the effect of disease states, however.     

Protein binding of methotrexate to human albumin and serum. A first derivative spectroscopic analysis.

Usual methods allowing the measurement of the free concentration of a drug in serum, i.e. equilibrium dialysis, ultrafiltration and ultracentrifugation, are generally based on a physical separation of the bound and free fractions. During this, variations or errors may occur which are probably at the origin of the variability of the previously published results for methotrexate (CAS 59-05-2). In order to verify these results as well as to experience a technique recently applied to rifampicine the first derivative spectroscopic analysis was used to estimate the bound and free fractions of methotrexate in human serum and serum albumin (HSA). Free drug concentrations were measured at 377 nm and the bound form at 372.5 nm. In human serum, bound methotrexate was 54.1% on average for total concentrations ranging from 10(-5) mol/l to 10(-3) mol/l, without any saturation. With HSA, a saturation occurred. Scatchard analysis showed one family of binding sites characterized by 2 binding sites and an affinity constant of 3200 mol/l, in mean, values close to that previously calculated using equilibrium dialysis.     

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.     

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".     

Nonenzymatic glucosylation of human serum albumin and its influence on binding capacity of sulfonylureas

To estimate the functional change occurring in human serum albumin by nonenzymatic glucosylation, glucosylated human serum albumin was prepared by in vitro incubation with glucose. The rate of glucosylation proceeded as a first-order reaction. The binding of sulfonylureas to serum albumin was determined by equilibrium gel filtration. Through this method, it was possible to estimate the binding capacity of a low water solubility drug with a high affinity to protein. The amounts of the sulfonylureas bound to glucosylated HSA decreased by 44% with tolazamide and acetohexamide, 50% with glibenclamide, and 52% with tolbutamide, compared to human serum albumin (HSA). This suggests that a high concentration of glucosylated HSA in diabetic patients may possibly cause an increase in free drug concentration exceeding normal levels. This study shows that the decrease in the binding capacity of sulfonylureas with protein is due to the modification of albumin molecules by the covalent binding of glucose.     

Binding of prazosin to alpha 1-acid glycoprotein and albumin: effect of protein purity and concentrations

Prazosin is extensively bound in human serum/plasma. In the present study a bound fraction of 93-95% was observed at 37 degrees for therapeutic drug concentrations. Both alpha 1-acid glycoprotein (AAG) and albumin (HSA) are established as transport proteins for prazosin, but their individual contribution to the extent and variability of protein binding in serum/plasma is unclear. The present study showed that AAG possesses one binding site per molecule with high affinity (Kd approximately 0.8 microM) for prazosin. HSA, essentially globulin-free, bound prazosin with lower affinity (Kd approximately 30 microM) with an average of 0.3 binding sites per molecule. However, less purified HSA, containing globulins, exhibited apparently higher affinity (Kd approximately 8 microM), but lower binding capacity (0.07 sites per molecule) for prazosin. In mixtures of highly purified proteins, the concentrations of AAG, and not HSA, determined the extent and variability of prazosin binding.     

Pipequaline transport from blood to brain and liver: role of plasma protein-bound drug

Brain uptake of pipequaline (45319 RP) has been studied in-vivo after a single capillary transit by intracarotid injection to rats. Pipequaline is extensively bound to plasma proteins: i.e. human serum albumin (HSA), alpha-1-acid glycoprotein (AAG), lipoproteins and blood cells, mainly erythrocytes. The dialysable drug fraction as measured in-vitro by equilibrium dialysis at 37 degrees C, was inversely related to the concentration of binding component. Similarly, the brain uptake of pipequaline was inversely related to the protein concentration of the injected solution. However, the measured brain uptake of pipequaline was higher than those predicted by in-vitro measurements of dialysable drug for all proteins and erythrocytes, except HSA. These results show that a fraction of bound pipequaline as measured in-vitro is available for transport through the blood brain barrier. HSA-bound pipequaline is an exception as it is restricted to the vascular space. Pipequaline was totally cleared by the liver through a single passage.     

Binding of cosalane--a novel highly lipophilic anti-HIV agent--to albumin and glycoprotein

Cosalane is a potent inhibitor of HIV replication with multiple sites of action. The purposes of this study were to (a) determine the extent and nature of cosalane binding to mucin, alpha(1)-acid glycoprotein (AAG), plasma, and human (HSA) and bovine serum (BSA) albumin, and (b) determine the primary site(s) of cosalane binding to HSA. Plasma protein binding of cosalane was studied by a gel filtration technique. Cosalane binding to HSA was also determined in the presence of salicylic acid. Competitive inhibition studies were conducted using warfarin, digitoxin, and diazepam to determine the primary HSA binding site(s) of cosalane. The drug was bound extensively to HSA and BSA and required 500-550 moles to saturate 1 mole of protein. Stoichiometries of cosalane binding to alpha(1)-acid glycoprotein (AAG) and mucin were between 30 and 50 mol/mol of either glycoprotein. The binding isotherm deviated from a rectangular hyperbola, suggesting self-association of the ligand. Salicylic acid decreased cosalane binding to HSA by one order of magnitude. Inhibition studies of cosalane to HSA revealed that the compound binds primarily to warfarin site with a K(i) of 1.24 +/- 0.24 nM. In summary, cosalane binds extensively to serum albumins and to a lesser extent to both AAG and mucin.     

Circular dichroism studies on the inhibiting effect of oleic acid on the binding of diazepam to human serum albumin

The effect of a free fatty acid (oleic acid) on the binding of a benzodiazepine derivative (diazepam) to human serum albumin (HSA)¹ has been studied using the technique of circular dichroism. Both qualitative and quantitative results suggest that oleic acid significantly affects the binding of diazepam, even at low molar ratios to albumin (below 1:1). It is suggested that the displacement of bound diazepam occurs primarily through an allosteric mechanism.     

Binding of Prazosin to α1-Acid Glycoprotein and Albumin: Effect of Protein Purity and Concentrations

Abstract: Prazosin is extensively bound in human serum/plasma. In the present study a bound fraction of 93–95% was observed at 37° for therapeutic drug concentrations. Both α₁-acid glycoprotein (AAG) and albumin (HSA) are established as transport proteins for prazosin, but their individual contribution to the extent and variability of protein binding in serum/plasma is unclear. The present study showed that AAG possesses one binding site per molecule with high affinity (Kd≈0.8 μM) for prazosin. HSA, essentially globulin-free, bound prazosin with lower affinity (Kd≈30 μM) with an average of 0.3 binding sites per molecule. However, less purified HSA, containing globulins, exhibited apparently higher affinity (Kd≈8 μM), but lower binding capacity (0.07 sites per molecule) for prazosin. In mixtures of highly purified proteins, the concentrations of AAG, and not HSA, determined the extent and variability of prazosin binding.     

Cefazolin serum protein binding and its inhibition by bilirubin, fatty acids and other drugs

This paper describes the protein binding of cefazolin to human serum and to human serum albumin (HSA) using equilibrium dialysis. The drug is exclusively bound to HSA with a moderate affinity, Ka = 16,600 +/- 1600 M-1, and one saturable binding site, n = 0.73 +/- 0.02. Moreover cefazolin shows a dose-dependent binding leading a possible increase of the free fraction (when its total concentration increases). This antibiotic is displaced by free fatty acids (FFA) and bilirubin. Cefazolin binding to human serum and human serum albumin (HSA) was studied in presence of acidic drugs. At low concentrations clofibric acid and phenylbutazone both exhibiting high affinity for HSA displace strongly cefazolin. Valproic and salicylic acids, sulfamethoxazole, cefoperazone which have approximately the same affinity as cefazolin, must be used at higher concentrations to displace this antibiotic. A particular phenomenon was observed with cefazolin on HSA when associated with furosemide. A low concentration (5-25 microM) of this drug induces a positive cooperativity of binding between cefazolin and HSA. But at a molar ratio of furosemide to albumin greater than one, such cooperative interaction disappears and a competitive inhibition of cefazolin binding occurs. For all drugs studied, a competitive inhibition was found except for tryptophan. Finally, it is concluded that cefazolin shares the warfarin binding site on HSA.     

Determination of the binding of perazine and perazine sulfoxide to human serum albumin

Determination of the Binding of Perazine and Perazine Sulfoxide to Human Serum Albumin The binding of Perazine and its sulfoxide to 4% human serum albumin (HSA) in buffered phosphate solution (pH 7.4) was investigated by means of ultrafiltration (flat chamber) and modified gel filtration. It was found that within the physiological concentration range the sulfoxide is riot bound by albumin, whereas perazine even in strongly diluted solutions (ng range) showed a high affinity to albumin. The results were compared with literature data concerning the binding of perazine dimalonate (PDM) and desmethylperazine to human serum albumin (HSA).     

Elucidation of the human serum albumin (HSA) binding site for the Cu-PTSM and Cu-ATSM radiopharmaceuticals

The Cu-PTSM (pyruvaldehyde bis(N(4)-methylthiosemicarbazonato)copper(II)) and Cu-ATSM (diacetyl bis(N(4)-methylthiosemicarbazonato)copper(II)) radiopharmaceuticals exhibit strong, species-dependent binding to human serum albumin (HSA), while Cu-ETS (ethylglyoxal bis(thiosemicarbazonato)copper(II)) appears to only exhibit nonspecific binding to human and animal serum albumins. This study examines the structural basis for HSA binding of Cu-PTSM and Cu-ATSM via competition with drugs having known albumin binding sites. Warfarin, furosemide, ibuprofen, phenylbutazone, benzylpenicillin, and cephmandole were added to HSA solutions at drug:HSA mole ratios from 0 to 8:1, followed by quantification of radiopharmaceutical binding to HSA by ultrafiltration. Warfarin, a site IIA drug, progressively displaced both [(64)Cu]Cu-PTSM and [(64)Cu]Cu-ATSM from HSA. At 8:1 warfarin:HSA mole ratios, free [(64)Cu]Cu-PTSM and [(64)Cu]Cu-ATSM levels increased 300-500%. This was in contrast to solutions containing ibuprofen, a site IIIA drug; no increase in free [(64)Cu]Cu-PTSM or [(64)Cu]Cu-ATSM was observed except at high ibuprofen:HSA ratios, where secondary ibuprofen binding to the IIA site may cause modest radiopharmaceutical displacement. By contrast, and consistent with earlier findings suggesting Cu-ETS exhibits only nonspecific associations, [(64)Cu]Cu-ETS binding to HSA was unaffected by the addition of drugs that bind in either site. We conclude that the species-dependence of Cu-PTSM and Cu-ATSM albumin binding arises from interaction(s) with the IIA site of HSA.