Serum Protein Binding Of Drugs Research Articles

Luminescence studies of binding affinity of vildagliptin with bovine serum albumin

Vildagliptin (VDG)is a frontier drug for diabetes mellitus. It is prescribed both in the monotherapy as well as in an amalgamation with other antidiabetic drugs. Drug-serum protein binding is an essential parameter which influences ADME properties of the drug. In current study, binding of VDG with serum protein (bovine serum albumin: BSA) was investigated using multi-spectroscopic techniques. A computational approach was also employed to identify the binding affinity of VDG with BSA at both Sudlow I and II sites. An enzyme activity assay specific for esterase was also investigated to know the post-binding consequences of VDG with BSA. Fluorescence spectra of BSA samples treated with VDG shows static quenching with binding parameters for VDG-BSA complex show single class of equivalent binding stoichiometry(n = 1.331) and binding constant 1.1 x 104M−1 at 298.15 K. The binding constant indicates important role of non-polar interactions in the binding process. Fluorescence resonance energy transfer (FRET) analysis of VDG absorption spectra and emission spectrum of BSA confirmed no significant resonance in energy transfer. Synchronous fluorescence of BSA after binding with VDG show maximum changes in emission intensity at tryptophan (Trp) residues. Post binding with VDG, BSA conformation changes as suggested by circular dichorism (CD) spectra of BSA and this lead to enhanced protein stability as indicated by a thermal melting curve of BSA. Communicated by Ramaswamy H. Sarma

Binding of ethyl pyruvate to bovine serum albumin: Calorimetric, spectroscopic and molecular docking studies

Various in vitro and in vivo studies have shown the anti-inflammatory and anticancer potential role of ethyl pyruvate. Bio-distribution of drugs is significantly influenced by the drug-serum protein binding. Therefore, the binding mechanism of the ethyl pyruvate with bovine serum albumin was investigated using UV–vis absorption, fluorescence, circular dichroism, isothermal titration calorimetry and molecular docking techniques. Absorption and fluorescence quenching studies indicated the binding of ethyl pyruvate with protein. Circular dichroism spectra of bovine serum albumin confirmed significant change in the conformation of protein upon binding. Thermodynamic data confirmed that ethyl pyruvate binds to bovine serum albumin at the two different sites with high affinity. Binding of ethyl pyruvate to bovine serum albumin involves hydrogen bonding, van der Waal and hydrophobic interactions. Further, docking studies indicated that ethyl pyruvate could bind significantly at the three binding sites. The results will definitely contribute to the development of ethyl pyruvate as drug.

Mathematical investigation of two dimensional pattern formation

During the perinatal period the problems of drug disposition are pronounced. The adaptation from the maternal-fetal unit to extrauterine life leads to alterations in drug absorption, distribution, metabolism, and renal elimination. Since both morphology and function of the alimentary tract changes, drugs are absorbed more slowly in the neonate than in older infants. Moreover, the serum protein binding of drugs is reduced in neonates. Consequently a measured plasma concentration may reflect a higher plasma/tissue level in the newborn as compared with adults. If the distribution volume of a drug corresponds to the total body water or extracellular water space, the dosage may be calculated in relation to the individual variations in the body surface area. But this procedure is not applicable for the newborn infant due to impaired metabolic functions of the liver and renal elimination mechanisms. The capacity of drug oxidation and glucuronidation is not fully developed in the neonate. Also glomerular filtration and tubular secretion are reduced during the first weeks of life. Therefore the elimination half-lives of many drugs are considerably prolonged in the newborn compared with that in older infants. As a consequence, individual therapeutic drug monitoring based on pharmacokinetic concepts and assisted by computer programs is becoming increasingly important.

Quantification of interactions between drug leads and serum proteins by use of “binding efficiency”

To develop efficient and reliable methods for prediction of serum protein binding of drug leads, the kinetic characteristics for the interactions between selected compounds and human serum albumin and α 1-acid glycoprotein have been explored using a surface plasmon resonance biosensor. Conventional methods for quantification of interactions (i.e., using rate constants or affinities determined on the basis of a reasonable mechanistic model) were applicable for only a few of the compounds. The affinity of a primary interaction and the contribution of lower affinity secondary interactions could be estimated for some compounds, but the affinity of many compounds could not be quantified by either of these methods. To have a quantification method that could be used for all compounds, independent of affinity and complexity of interaction mechanisms, the concept of “binding efficiency,” analogous to “catalytic efficiency” used for enzymes, was developed. It allowed the quantification of the binding of compounds interacting with weak affinity and for which saturation is not reached within a concentration range where the compound is soluble or when the influence of interactions with secondary sites makes interpretations difficult. In addition, compounds with large fractional binding can be identified by this strategy and simply quantified relative to reference compounds. This approach will enable ranking and identification of structure–activity relationships of compounds with respect to their serum protein binding profile.

Study on Binding of Drug to Serum Protein

After being distributed in the circulating blood, drugs bind to serum proteins varying degrees. In general, such binding is reversible, and a dynamic equilibrium exists between the bound and unbound molecular species. It is believed that unless there is a specific transport system (e.g. receptor-mediated endocytosis, protein-mediated transport), only unbound drugs are able to penetrate through biomembranes, are distributed to tissues, and undergo metabolism and glomerular filtration. It is also believed that only unbound molecules present in target tissues can exert their pharmacological effects, and that the concentration of unbound molecules in tissues is in proportion to the drug serum concentration. Therefore, drug-serum protein binding is critically involved in the manifestation of the pharmacological effects of a drug as well as its pharmacokinetics. Among serum proteins, human serum albumin (HSA) and alpha(1)-acid glycoprotein (AGP) play important roles in protein binding for many drugs, which is of key importance to drug distribution in the body. In addition, they are widely used in clinical settings as blood preparations and drug delivery system carriers. It is thus of great importance from the viewpoint of pharmaceutical science to clarify the structure, function, and pharmaceutical properties of HSA and AGP. Accordingly, since starting my laboratory, the focus of my research has involved molecular pharmaceutical studies on the interactions of drugs and HSA and AGP for the purpose of applying these findings to clinical fields, such as drug treatment, diagnosis and drug discovery. In this review, the molecular properties of HSA and AGP will be briefly outlined. The static and dynamic topology of drug binding sites on these proteins, investigated by various spectroscopic techniques, X-ray crystallography, quantitative structure-activity relationships, molecular modeling, photo affinity labeling, site-directed mutagenesis etc., changes in the serum protein binding of drugs in pathological conditions, such as liver and kidney failure and various inflammation diseases and factors contributing to the changes will then be summarized. Finally, cases in which protein binding displacement can be applied to medical fields will also be introduced.

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

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

Interpretations of Laboratory Test Data on Serum Protein Binding

Serum proteins that are important in the serum protein binding of drugs are human serum albumin (HSA) and alpha1-acid glycoprotein (AGP). Several binding sites exist on HSA and AGP molecules. HSA, AGP, free fatty acid (FFA), blood urea nitrogen (BUN) and bilirubin, which can all be determined by laboratory test, affect the binding capacities of binding sites on these proteins. The increase and decrease of HSA and AGP influence the binding capacities of all binding sites. As an additional influence on the binding sites on protein molecules, the increment of FFA decrease the binding capacity of site II, while binding capacity of site I is enhanced by FFA. Increase in bilirubin remarkably decreases the binding capacity of site I. BUN data are associated with the amounts of several uremic toxins, 3-carboxy-4-methyl-5-propyl-2-furanpropanate (CMPF), indole-3-acetate (IA), indoxyl sulfate (IS) and hippurate (HA). With CMPF, the binding capacity of site I is decreased, while IA, IS, HA contribute to the binding inhibition of site II of HSA. If we can monitor binding capacities of binding sites of HSA and AGP, laboratory test data can be interpreted from a pharmaceutical perspective regarding protein binding, because changes in laboratory test data that are endogenous substance concentrations have an influence on the binding capacities of those binding sites.

A rapid spectrofluorimetric technique for determining drug-serum protein binding suitable for high-throughput screening.

To develop and validate a rapid method for determining the dissociation constants with which pharmaceutical candidates and drugs bind to serum albumin and to alpha1-acid glycoprotein with the goal of deducing the extent of binding. The quenching of the intrinsic tryptophan fluorescence of serum albumin and alpha1-acid glycoprotein was monitored by spectrofluorimetry and the data were used to calculate the apparent dissociation constant. Sodium warfarin was used to probe the warfarin-binding site of serum albumin and diazepam was used to probe the benzodiazepine binding site. Additionally, the binding of sodium salicylate, phenylbutazone, sulfinpyrazone, iophenoxic acid, theophylline, chloramphenicol, acetaminophen, lithium chloride and ampicillin were also investigated. Chlorpromazine hydrochloride and imipramine hydrochloride were used as probes for alpha1-acid glycoprotein. The assays were also extended to the multiwell format. The quenching curves were fitted to the quadratic binding equation to determine the dissociation constants. Intrinsic fluorescence measurements are an excellent predictor of the drug binding to human serum albumin and to alpha1-acid glycoprotein. These measurements detect binding to the warfarin and benzodiazepine binding sites of human serum albumin. The dissociation constants estimated using the method compare favorably to the dissociation constants previously reported by Epps et al. using extrinsic fluorescence methodology, and the results correlate well with equilibrium dialysis using drug displacement endpoints. These measurements can be carried out with small samples and do not require separation of the bound and unbound species. Additionally, the proposed methods eliminate membrane separations, are not compound specific and do not require analytical chromatography or mass spectrometry for quantitation. Spectrofluorimetry may prove to be a useful method for rapidly determining the protein binding of combinatorial libraries.

Alpha 1-acid glycoprotein and serum binding of drugs in healthy and diseased dogs.

Inter-individual variation in drug serum protein binding was studied in healthy dogs and in dogs with inflammatory diseases for lidocaine, oxprenolol and propranolol, which bind mainly to alpha 1-acid glycoprotein (alpha 1-AGP), and for diazepam, digitoxin and phenytoin, which bind mainly to albumin. For the drugs mostly bound to alpha 1-AGP, in both groups of dogs binding varied considerably, and it was markedly higher in dogs with inflammatory disease. For the other drugs, the variation in binding was smaller and did not differ between the two groups of dogs. In both groups of dogs, the alpha 1-AGP concentration varied widely; it was higher in the serum of the dogs with inflammation, while the concentration of albumin was lower in these animals. There was a significant negative correlation between percentage free lidocaine, oxprenolol or propranolol and alpha 1-AGP concentration, suggesting that the inter-individual variation in binding of these drugs is due to the variation in alpha 1-AGP concentration. There was a marked intra-individual variation in lidocaine binding and in serum alpha 1-AGP concentration, studied over a period of 3 weeks in healthy dogs; a significant negative correlation between percentage free lidocaine and alpha 1-AGP concentration was obtained.

Altered Drug–Serum Protein Binding in the Genetically Obese Zucker Rat

Drug–serum protein binding was evaluated in genetically obese Zucker rats, their lean littermates, and lean Sprague–Dawley rats. The free fraction (fp) of phenytoin was significantly higher in the obese rat (fp=0.177) compared to its lean littermate (fp=0.136), apparently due to displacement by free fatty acids. Conversely, diazepam and propranolol fp values were decreased in the obese Zucker rat (fp=0.107 and fp=0.122, respectively) compared to the lean Zucker rat (fp=0.140 and fp=0.174, respectively). Evidence strongly suggests that the increased binding of propranolol was not due to elevations in the serum concentrations of α1-acid glycoprotein (as is the case in the human obese population). Rather, the decreased fp for both diazepam and propranolol was a result of increased lipoprotein partitioning. Strain differences between the lean Zucker rat and lean Sprague–Dawley rat were also evident, with the serum binding of the Sprague–Dawley rat more closely resembling the obese Zucker rat.

Effect of Late Pregnancy on Salicylate, Diazepam, Warfarin, and Propranolol Binding

Serum protein binding was measured in six women 38 wk pregnant and in five control subjects. Three distinct binding sites for drugs on human serum albumin have been identified. To determine whether changes in binding during pregnancy occur for common drugs or only for drugs that bind to a specific binding site, serum protein binding of three drugs—diazepam (site I), warfarin (site III), and salicylate—and four fluorescent probes—dansylsarcosine (site I), l-anilino-8-naphthalenesulfonate (site I), 7-anilinocoumarin-4-acetic acid (site II), and 5-dimethylaminonaphthalene-1-sulfonamide (DNSA) (site III)—were determined in control and pregnant sera. Unbound fractions of diazepam and salicylate in pregnant women increased but the unbound fraction of warfarin did not change. Dissociation constants (Kd1) of all fluorescent probes but DNSA were almost the same in control and pregnant sera, while the Kd1 of DNSA in pregnant serum was approximately 50% of control. Binding capacities of all probes decreased, which was attributed to decreased serum albumin concentration. We concluded that serum protein binding of drugs that bind to site I or site II on albumin decreased largely because of the reduced serum albumin concentration during pregnancy and that the binding of drugs that bind to site III changed little because of compensating effects of the decrease in serum albumin concentration and the increase in binding affinity to serum albumin. Serum concentration of α1-acid glycoprotein and serum binding of propranolol did not change in pregnant women. Clinical Pharmacology and Therapeutics (1984) 36, 201–208; doi:10.1038/clpt.1984.163

Effect of late pregnancy on salicylate, diazepam, warfarin, and propranolol binding: use of fluorescent probes.

Serum protein binding was measured in six women 38 wk pregnant and in five control subjects. Three distinct binding sites for drugs on human serum albumin have been identified. To determine whether changes in binding during pregnancy occur for common drugs or only for drugs that bind to a specific binding site, serum protein binding of three drugs--diazepam (site I), warfarin (site III), and salicylate--and four fluorescent probes--dansylsarcosine (site I), 1-anilino-8-naphthalenesulfonate (site I), 7-anilinocoumarin-4-acetic acid (site II), and 5-dimethylaminonaphthalene-1-sulfonamide (DNSA) (site III)--were determined in control and pregnant sera. Unbound fractions of diazepam and salicylate in pregnant women increased but the unbound fraction of warfarin did not change. Dissociation constants (Kd1) of all fluorescent probes but DNSA were almost the same in control and pregnant sera, while the Kd1 of DNSA in pregnant serum was approximately 50% of control. Binding capacities of all probes decreased, which was attributed to decreased serum albumin concentration. We concluded that serum protein binding of drugs that bind to site I or site II on albumin decreased largely because of the reduced serum albumin concentration during pregnancy and that the binding of drugs that bind to site III changed little because of compensating effects of the decrease in serum albumin concentration and the increase in binding affinity to serum albumin. Serum concentration of alpha 1-acid glycoprotein and serum binding of propranolol did not change in pregnant women.

Effect of halothane, enflurane and their metabolites on the serum protein binding of drugs

Effect of halothane, enflurane and their metabolites on the serum protein binding of drugs

Serum protein binding of drugs during and after pregnancy in humans.

The serum protein binding of three weakly acidic drugs (salicylic acid, sulfisoxazole, and phenytoin), one week base (diazepam), and one steroid (dexamethasone) was determined in pregnant women at seven time periods during pregnancy and at two periods post partum, as well as in a group of nonpregnant women of childbearing age. The serum free fraction values (ratio of concentrations, free to total drug) of all drugs rose during pregnancy, primarily after 15 wk of gestation, and remained elevated for at least 1 to 5 days post partum. Pregnancy had the greatest effect on protein binding of sulfisoxazole, diazepam, and salicylic acid. The magnitude of this effect is such that quantitatively significant changes in the pharmacokinetic and pharmacodynamic characteristics of certain drugs may be expected to occur during pregnancy (in addition to possible changes caused by other pregnancy-related effects such as altered activity of drug-metabolizing enzyme systems). All drugs but dexamethasone exhibited significant negative correlations between free fraction values and serum albumin concentrations during pregnancy. The serum protein binding of salicylic acid, but not the other drugs tested, was more extensive in nonpregnant women who were not taking oral contraceptives than in those who were.

Serum protein binding of drugs and bilirubin in newborn infants and their mothers.

The serum protein binding of dexamethasone, diazepam, phenytoin, sulfisoxazole, and bilirubin was determined in full-term newborn infants and their mothers immediately after delivery. Diazepam and sulfisoxazole were bound more extensively in infant serum than in maternal serum, dexamethasone was bound more extensively in the maternal serum, and the serum protein binding of phenytoin and bilirubin was much the same in the infants and their mothers. These results are consistent with and explain clinical observations that diazepam concentrations are higher in cord than in maternal serum and that phenytoin levels in newborn and maternal sera are in the same range when the mothers had taken one of these drugs for some time before delivery. There were negative correlations between free fraction of drug and albumin concentration in serum only for phenytoin (infants and mothers), diazepam (infants only), and sulfisoxazole (mothers only). Except for dexamethasone (infants and mothers) and phenytoin (mothers), there were positive correlations between the free fraction values of the various compounds and those of salicylic acid. It was previously determined that salicylic acid is more extensively bound in newborn than in maternal serum. Induction or augmentation of labor with oxytocin was associated with decreased serum protein binding of diazepam and possibly of bilirubin in mothers. There was a significant correlation between serum free fraction values in newborns and their mothers only in the case of phenytoin. Clinical Pharmacology and Therapeutics (1980) 28, 58–63; doi:10.1038/clpt.1980.131

Serum protein binding of drugs.

Knowledge of the serum protein binding of drugs can be of clinical importance. After discussing the different methodological aspects of measuring protein binding, the effects of binding on differen...

Intraindividual Relationships between Serum Protein Binding of Drugs in Normal Human Subjects, Patients with Impaired Renal Function, and Rats

The serum protein binding of phenytoin, salicylic acid, sulfisoxazole, and warfarin was determined in normal human adults, in patients with impaired renal function (kidney donor and recipient), and in adult male Sprague-Dawley rats. The free fraction values for salicylate and sulfisoxazole were significantly correlated in all three groups. The other correlations were statistically significant in only one or two of these groups. There was a statistically significant negative correlation between albumin concentration and the free fraction values of salicylic acid and sulfisoxazole (but not of phenytoin and only under special circumstances with warfarin) in normal human subjects and of phenytoin, salicylic acid, and sulfisoxazole (but not warfarin) in rats. No such correlation was observed for any of the drugs in patients with impaired renal function. These observations show that no single weakly acidic drug can serve as an index for quantitatively determining the effect of disease or species differences on the serum protein binding of other weakly acidic drugs.