Drug recovery following buccal absorption of propranolol.

1. The effect of ampicillin, cloxacillin, flucloxacillin and sulphafurazole on bilirubin on bilirubin binding by pooled human umbilical cord serum and bovine serum albumin was studied in vitro using Sephadex gel filtration. 2. Sulphafurazole displaced bilirubin from binding; both cloxacillins displaced bilirubin from pooled cord serum but not bovine serum albumin. 4. No displacement of bilirubin by the cloxacillins from pooled cord serum could be detected at therapeutic plasma concentrations of these drugs. 5. Scatchard analysis of the interactions showed that displacement of bilirubin by these drugs occurred principally at the primary, high affinity, low capacity binding site.     

Ceftriaxone binding to human serum albumin: competition with bilirubin

Ceftriaxone, a cephalosporin, is bound reversibly to defatted human serum albumin from adults, with a first stoichiometric binding constant of 60,000 M-1, as found by equilibrium dialysis at pH 7.4, 37 degrees. A second molecule is weakly bound, with a binding constant of 500 M-1. Possible cobinding with bilirubin was studied by the peroxidase method and by equilibrium dialysis with and without added bilirubin. Results indicated competitive binding; formation of an albumin complex containing both bilirubin and ceftriaxone could not be demonstrated. Light absorption spectra of bilirubin-albumin showed little change on addition of ceftriaxone, in agreement with the competitive biding mechanism. Binding to serum albumin from newborn infants is weaker than to the protein from adults, with the first binding constant being about 36,000 M-1. Cobinding of ceftriaxone and bilirubin to albumin from newborn infants is likewise competitive. It is concluded that ceftriaxone is a strong bilirubin displacer with a potential of inducing bilirubin encephalopathy in predisposed newborns.     

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 effects of various drugs on the binding site of bilirubin in serum albumin

In the presence of various drugs, the concentration of free bilirubin which was released from the first binding site of bilirubin in serum albumin was evaluated, using a sensitive method based on static fluorescence quenching of dansyl serum albumin. Bilirubin bound to human serum albumin more strongly than to bovine serum albumin. Non-steroidal anti-inflammatory drugs, fenamates and allylphenyl propionic acids, affected the bilirubin-serum albumin interaction. Flufenamic acid and ketoprofen released bilirubin from human serum albumin. The bilirubin bound to the serum albumin was not influenced by the presence of indomethacin, but clidanac strongly dissociated bilirubin from the bilirubin-serum albumin complex. Sulfa-drugs, antibiotics, steroidal agents, warfarine, tolubutamide and phenytoin showed no significant effects on the bilirubin-serum albumin interaction. The fluorescence quenching method may be useful to evaluate the interaction of drug-bilirubin-serum albumin.     

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.     

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.     

Plasma protein binding of azapropazone in patients with kidney and liver disease.

1 The free fraction of azapropazone in the plasma of 37 healthy volunteers ranged from 0.0027 to 0.0070 (0.0044 +/- 0.0009, mean +/- s.d.). The principal binding protein was found to be albumin. 2 In 27 patients with various degrees of renal failure the free fraction values of azapropazone were markedly enhanced (0.0260 +/- 0.0239, mean +/- s.d.) and increased more than tenfold in some patients. There was a weak correlation (r = 0.46, P less than 0.05) between the free fraction and the clearance of endogenous creatinine. Such correlation was not found for serum creatinine, serum albumin, serum uric acid and serum urea nitrogen. 3 In 32 patients with chronic liver disease the free fraction values of azapropazone were also markedly higher (0.0210 +/- 0.0242, mean +/- s.d.) than in healthy subjects. There were statistical significant correlation between free fraction values and the prothrombin complex activity in the plasma (r = 0.40, P less than 0.05) and the total bilirubin concentration in the plasma (r = 0.90, P less than 0.001), respectively. Such correlation was not found for serum albumin, serum glutamic oxalacetic transaminase, serum gamma-glutamyl transpeptidase and serum alkaline phosphatase. 4 In patients with kidney and liver disease the free fraction values of azapropazone correlated well with those of the anticoagulant drug phenprocoumon (r = 0.93, P less than 0.001). However, the binding of the latter drug was less impaired. Bilirubin, when added in vitro, displaced both drugs from plasma proteins but this displacing effect was much smaller than the binding changes observed in patients with liver disease. 5 Kidney and liver disease caused a marked impairment of the plasma protein binding of azapropazone. In patients with kidney disease the degree of impairment of azapropazone binding cannot or only poorly (creatinine clearance) be predicted from the biochemical parameters of kidney function whereas in patients with chronic liver disease the total bilirubin concentration in the plasma may serve as an index of the binding defect.     

Interindividual Differences in the Protein Binding of Sulfonamides: The Effect of Disease and Drugs 1,2

Abstract: The variation in the plasma protein binding of sulfonamides in healthy adults was minimal. Additional evidence for consistency in binding came from a literature survey in which it was found that the plasma protein binding of several sulfonamides in man and laboratory animals was remarkably similar as reported by investigators from different countries. Some hospitalized patients, however, do show a binding deficiency which may be due to an interaction between drugs, disease and altered plasma proteins. Several drugs were found to compete with the sulfonamide for the same binding sites on the protein (albumin) molecule. The plasma protein binding of a sulfonamide in six anephric patients was studied before and after haemodialysis. As expected all showed a deficiency in binding. Unexpectedly, however, the binding was less in the post-dialysis plasma sample in three patients. In some patients there appeared to be a qualitative and a quantitative change in the binding sites on the protein. More definitive studies are needed with plasma protein fractions from healthy and ill subjects in the presence and absence of drugs. This may lead to a better understanding of the side effects to some drugs and could result in the development of useful displacing agents.     

Plasma protein binding of drugs in pregnancy and in neonates

Plasma protein binding of drugs has important implications for drug disposition and action since it is the first, and controlling, step in drug distribution. Physiological changes in pregnancy include significant changes in plasma composition which affect drug binding and subsequent drug response; the extent of these changes depends on the stage of gestation. Both albumin and alpha 1-acid glycoprotein fractions are reduced, and consequently the binding of both acidic and basic drugs may be affected. This may lead to difficulties in maintaining adequate plasma concentrations of highly protein-bound drugs, since the measurement of total drug concentration in plasma may no longer be a valid indicator for dose adjustment. The newborn infant displays a continually changing plasma profile. The presence of fetal proteins and endogenous substrates known to interfere with drug binding can lead to unexpected complications due to a higher than expected 'free' drug fraction. Furthermore, a decrease in the affinity of albumin for bilirubin during this period may lead to bilirubin displacement by drugs such as diazepam, sulphonamides and salicylate, resulting in clinical jaundice which would not occur beyond the neonatal period. Plasma composition and its effect on drug binding should be taken into account when prescribing highly protein bound drugs with narrow therapeutic: toxic ratios.     

Ceftriaxone binding to human serum albumin. Indirect displacement by probenecid and diazepam

In vitro protein binding studies were conducted to examine the interaction between ceftriaxone (CEF), probenecid (PROB) and diazepam (DIAZ). The presence of PROB and DIAZ at concentrations equal to molar albumin concentration caused a decrease in CEF affinity from 3.7 x 10(4) M-1 (control) to 1.1 x 10(4) (PROB) and 2.6 x 10(4) (DIAZ) M-1, but not in binding capacity in pooled human plasma. PROB and DIAZ at five times the molar albumin concentration also caused a decrease in CEF affinity from 4.5 x 10(4) M-1 (control) to 0.45 x 10(4) (PROB) and 3.0 x 10(4) (DIAZ) M-1 in isolated human serum albumin. DIAZ and PROB displaced one another, confirming their common binding site (Site II, the benzodiazepine site) on serum albumin. By contrast, CEF was unable to displace either PROB or DIAZ from defatted albumin. In the presence of elevated free fatty acid concentrations (four times the albumin concentration), CEF decreased the binding of both drugs. CEF free fraction (fp) in isolated human serum albumin (CEF fp = 7.7%) was increased by drugs which bind to Site I: sulfisoxazole (CEF fp = 68.1%), warfarin (CEF fp = 56.0%) and furosemide (CEF fp = 55.0%). At ten times the molar concentration of albumin, CEF displaced both warfarin (warfarin fp from 0.99 to 2.20%) and phenytoin (phenytoin fp from 17.7 to 23.4%) from defatted albumin. CEF appeared to bind to Site I (the warfarin site) on human serum albumin, and was displaced by PROB and DIAZ via a mechanism which did not involve direct competition at a common binding site.     

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.     

Interactions with the protein binding of 7-hydroxy-methotrexate in human serum in vitro

7-Hydroxy-methotrexate (7-OH-MTX), the major extracellular methotrexate (MTX) metabolite, is 90-95% bound in human serum, with albumin (HSA) as the major binding protein. Reports of an interaction with concomitantly administered non-steroidal antiinflammatory drugs (NSAIDs) during MTX therapy led us to investigate whether these compounds could reduce the binding of 7-OH-MTX in vitro. Equilibrium dialysis experiments demonstrated that naproxen and indomethacin concentration dependently reduced the binding of 1 microM 7-OH-MTX. After ingestion of 1000 mg naproxen, per cent unbound 7-OH-MTX in sera from volunteers increased 2-3-fold in vitro, positively correlated to naproxen concentrations (P less than 0.00015). In addition, etacrynic acid, bilirubin, sulphamethizole and acetylsalicylic acid displaced 7-OH-MTX from its binding protein(s) in a competitive manner. The data suggest that 7-OH-MTX interacts with several exogenous and endogenous substances associated with HSA in human serum. Displacement of 7-OH-MTX from HSA may contribute to the interaction between NSAIDs and MTX.     

Effect of glycyrrhizic acid on protein binding of diltiazem,verapamil, and nifedipine

The effects of glycyrrhizic acid (GLZ) on protein binding of diltiazem, verapamil, and nifedipine were investigated. Protein binding studies (human serum, human serum albumin (HSA) and alpha1-acid glycoprotein (AAG)) were conducted using the equilibrium dialysis method with and without addition of GLZ. The binding parameters, such as the number of moles of bound drug per mole of protein, the number of binding sites per protein molecule, and the association constant, were estimated using the Scatchard plot. The serum binding of nifedipine, verapamil, and diltiazem was displaced with addition of GLZ, and the decreases of Ks for serum were observed. GLZ decreased the association constants of three drugs for HSA and AAG, while the binding capacity remained similar with addition of GLZ. Although the characteristics of interaction were not clear, GLZ seemed to mainly affect HSA binding of nifedipine rather than AAG binding, while GLZ seemed to affect both AAG- and HSA-bindings of verapamil and diltiazem resulting in a serum binding displacement.     

Stereoselectivity and species difference in plasma protein binding of KE-298 and its metabolites.

In vitro protein binding of KE-298 and its plasma metabolites, deacetyl-KE-298 (M-1) and S-methyl-KE-298 (M-2), was high in rat (>97%), dog (>89%) and human plasma (>99%), respectively. Human serum albumin (>93%) was the main protein involved in the binding to plasma proteins, while the binding to human serum globulins was low (16-33%). The binding of KE-298 and its metabolites in all species of plasma was stereoselective. The (+)-(S)-enantiomers of these compounds bound rat, dog and human plasma proteins to a greater extent than did the (-)-(R)-enantiomers, except that the case of KE-298 was opposite in rat plasma. The stereoselective plasma levels of these compounds in rats, dogs, or humans would likely be due to stereoselective differences in binding to plasma albumin. The protein binding of M-1 in adjuvant-induced arthritis rat plasma was >97%, and the stereoselectivity was similar to the case of normal rat plasma. KE-298 and its metabolites remarkably displaced [14C]warfarin, which bound on albumin in a solution of diluted rat serum albumin. Similarly, there was a displacement of [14C]warfarin in solutions of dog and human serum albumin, and concomitantly the displacement of [14C]diazepam. [3H]Digitoxin was not displaced by any of the enantiomers in each albumin solution. No stereoselectivity was found in displacement by enantiomers of the three compounds. These results suggest that stereoselective protein binding can be attributed to quantitative differences in binding to albumin rather than to the different binding sites.     

Binding of dapsone and its analogues to human serum albumin

The binding of dapsone, 4,4'-sulfonylbis(aniline)(1), and its diacetylated derivative, 4,4"'-sulfonylbis(acetanilide)(2), to human serum albumin is reported. To assess the ability of these compounds to displace 4'-[(4-aminophenyl)sulfonyl]acetanilide (3) from albumin, a dialysis rate technique was used. Competition for the bilirubin binding site on albumin was measured with the peroxidase assay. Compounds 1 and 2 strongly displaced both 3 and bilirubin from human serum albumin. The association constants for 1 and 2 with respect to bilirubin binding were 1.29 X 10(3) and 1.15 X 10(4) M-1, respectively. These results suggest that the binding site for 3 and the bilirubin binding site are similar with respect to 1 and 2 and that the binding of dapsone and its derivatives probably does not involve the amino function.     

Endogenous metabolites as modulators of the transport of drugs by serum albumin

The ability of serum albumin to bind reversibly on its surface drugs and to transport them in the process of distribution and elimination has been well established. Some endogenous metabolites, particularly unesterified fatty acids, bile acids, L-tryptophan and bilirubin which are formed both under physiological conditions and in pathological states of the organism, e.g., in uremia can displace drugs from their areas of binding on albumin molecule. The detection of endogenous metabolites and the study of the mechanism through which they displace drugs from the specific areas on plasma proteins is the urgent task of pharmacology.     

Stereoselective degradation of the fenoprofen acyl glucuronide enantiomers and irreversible binding to plasma protein.

Stereoselective degradation of fenoprofen (FEN) glucuronides and irreversible binding of FEN enantiomers to human serum albumin via their glucuronides were studied. At different pH values, 37 degrees C, and in the absence of albumin, degradation half-lives were diastereomeric, resulting mainly from a combination of hydrolysis and acyl migration. Lower pH enhanced FEN glucuronide stability and reduced the extent of irreversible binding. The degradation rate of R-FEN glucuronide was greater than that of the S-glucuronide (S-FEN). When human serum albumin was added to the medium, stability was decreased as compared to protein-free buffer. FEN glucuronides were readily hydrolyzed to parent drug, indicating an esterase-like activity of the albumin molecule. In vitro irreversible binding was higher for R-FEN (1.22% +/- 0.36) than for S-FEN glucuronide (0.76% +/- 0.12), when a 0.1 mM concentration of each conjugate enantiomer was incubated under physiological conditions (pH 7.4, 37 degrees C). Incubation with unconjugated FEN did not lead to measurable irreversible binding. Analysis of plasma samples from a clinical study showed that enantioselective irreversible binding of FEN to plasma proteins also occurs in vivo. After administration of a single 600-mg dose of racemic FEN to six healthy volunteers, covalent binding of R- and S-FEN to plasma proteins was measured in all subjects. The percentage of S-FEN protein adduct was greater than that of its R-enantiomer adduct. Total amounts of FEN irreversibly bound to plasma protein in vivo were also very low (1.02 +/- 0.32 and 3.23 +/- 0.85 mol/mol protein x 10(-4) for R- and S-FEN, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of heparin injection on plasma protein binding of bilirubin and salicylate in rats

Intravenous heparin injection significantly increased the free bilirubin and salicylate fractions in the plasma of rats. This effect occurred within 2 min after injection of 500 U of heparin/kg and lasted for 15--45 min (bilirubin) or for greater than 45 min (salicylate). In vitro addition of heparin to plasma had no quantitatively significant effect on the protein binding of bilirubin and salicylate. The in vivo effect of heparin on protein binding was reversible by treating the plasma with activated charcoal, a procedure known to remove fatty acids from albumin. Since protein binding affects the pharmacokinetic characteristics and the pharmacological activity of drugs, the heparin--drug interaction may have significant clinical implications. Use of heparinized plasma for exchange transfusion in the treatment of neonatal jaundice may be hazardous.     

Differences in the binding of drugs to plasma proteins from newborn and adult man. II

Summary The binding of certain drugs to isolated fractions of plasma proteins obtained from newborn and adult man has been studied by equilibrium dialysis. For thiopental, desipramine, nitrofurantoin, sulfamethoxydiazine, meticillin and salicylic acid no difference was found between binding to the albumin fraction from newborns and adults. However, for thiopental, desipramine and promethazine binding to the globulin fraction was smaller in the newborns than in adults. Addition of bilirubin to the albumin fraction decreased the binding of nitrofurantoin, sulfamethoxydiazine and meticillin. No difference in the binding of meticillin to the albumin or globulin fractions from newborns and adults was found. The binding decreased, however, if both fractions were combined. Four mechanisms to explain the difference in binding between newborns and adults are discussed: (1) Displacement of drugs by bilirubin, (2) different binding properties of cord and adult albumin, (3) different properties of the globulins and (4) interaction of albumin with globulins in the newborn.This study was partially supported by grant Ku 156/4 of the Deutsche Forschungsgemeinschaft     

Cefmenoxime and bilirubin: competition for albumin binding

Certain drugs are known to compete with bilirubin for albumin binding; therefore, all drugs administered to neonates should be tested to determine the degree of competition. The effect of cefmenoxime on bilirubin-albumin binding was determined by comparing the oxidation rate of free bilirubin in the presence and absence of drug. The reserve albumin concentration (RAC) of pooled cord serum was also measured using the MADDS dialysis rate method. We show that cefmenoxime competes with bilirubin for albumin binding with a displacement constant, of 3.1 x 10(3) l/mol. The maximal displacement factor (MDF) is used to determine the clinical effect of the drug at usual serum concentrations. The MDF for cefmenoxime is 1.10, representing approximately a 10% increase in free bilirubin concentration. In comparison, the MDF for a known bilirubin displacing drug, sulfisoxazole, is 2.43. The MADDS method showed an estimated 28% decrease in the RAC at 150 mumol/l, the mean peak serum concentration (MPSC) of cefmenoxime. These results show while cefmenoxime affects bilirubin-albumin binding, the degree of the effect is relatively small. However, cefmenoxime may pose a hazard to very sick, premature infants, especially if the infant is jaundiced.