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.     

Binding of catecholamines to alpha-1 acid glycoprotein, albumin and lipoproteins in human serum

The binding of catecholamines in human serum was determined by equilibrium dialysis at 37 degrees. For serum concentrations of 10-15 nM the bound fractions were 28.8 +/- 2.2%, 25.7 +/- 1.7% and 22.2 +/- 2.2% for (+/-)-isoproterenol (IPR), (+/-)-norepinephrine (NE) and (+/-)-epinephrine (EPI), respectively. At higher serum concentrations saturation occurred. Alpha-1 acid glycoprotein (AAG) possessed one high affinity binding site and approximately 10 low affinity sites. The catecholamines were bound to AAG with the same order of potency for both classes of binding sites: IPR (Kd1: 100 microM Kd2: 2.2 mM) greater than NE (Kd1: 120 microM, Kd2: 6.5 mM) greater than EPI (Kd1: 140 microM, Kd2: 14 mM). Human serum albumin (HSA) and lipoproteins (SLP) interacted with the catecholamines in a non-saturable manner. IPR showed the strongest and EPI the weakest association to both of these serum protein fractions. (-)-Propranolol was able to inhibit the binding of IPR in serum and to isolated AAG, but not to HSA or to SLP. The present results show that AAG is an important catecholamine-binding protein in human serum. AAG, but not HSA or SLP, possesses binding sites shared by adrenergic receptor stimulators and blockers.     

The blood binding of cefotiam and cyclohexanol, metabolites of the prodrug cefotiam hexetil, in-vitro.

The binding of cefotiam and cyclohexanol to human serum, isolated proteins and erythrocytes has been studied in-vitro by equilibrium dialysis. The two molecules are 50% bound to serum proteins and the free fraction for both compounds remained constant within the therapeutic concentration range. Human serum albumin (HSA) was exclusively responsible for the cefotiam binding (48%) with a saturable process characterized by one binding site (n = 1.00 +/- 0.14) with a very weak affinity (Ka = 1457 +/- 352 M-1). Like other cephalosporins, cefotiam showed no binding to alpha 1-acid glycoprotein, lipoproteins or gamma-globulins. Cyclohexanol is mainly bound to HSA with a weak affinity (Ka approximately 1,800 M-1) but lipoproteins and alpha 1-acid glycoprotein bind about 30% of bound cyclohexanol in serum. Interactions with free fatty acids (FFA) or bilirubin were studied at physiopathological concentrations. HSA-bound cefotiam was displaced by FFA (1260 microM) and bilirubin (330 microM), whereas the cyclohexanol binding was inhibited only by FFA. The cefotiam binding site seems to be close to the warfarin site (site I) whereas cyclohexanol probably shares the diazepam site (site II) on HSA. There is no mutual inhibition of binding between cefotiam and cyclohexanol at therapeutic levels. The binding of both compounds to erythrocytes is low and restricted when measured in the presence of plasma.     

Tianeptine binding to human plasma proteins and plasma from patients with hepatic cirrhosis or renal failure.

1. The binding of tianeptine to human plasma, isolated plasma proteins, red blood cells and to plasma from patients with cirrhosis or renal failure was studied in vitro by equilibrium dialysis. 2. Tianeptine is highly bound to plasma (95%) at therapeutic concentrations (0.3-1 microM). No saturation of the binding sites was seen. 3. Human serum albumin (HSA) was shown to be mainly responsible for this binding (94%) with a saturable process characterized by one binding site with a moderate affinity (Ka = 4.2 x 10(4) M-1) and a non-saturable process with a low total affinity (nKa = 1.2 x 10(4) M-1). 4. Like many basic and amphoteric drugs, tianeptine showed a saturable binding to alpha 1-acid glycoprotein (AAG) with one site and a moderate affinity (Ka = 3.7 x 10(4) M-1). Its binding to lipoproteins and red blood cells (RBC) was weak and non-saturable. Over the range of therapeutic drug concentrations (0.3-1 microM), the unbound fraction in blood remains constant (4.5%). 5. Interactions were studied using non-esterified fatty acids (NEFA) at pathological concentrations; they altered tianeptine binding to plasma and to isolated HSA. Tianeptine seems to bind to a HSA site different from sites I (warfarin) and II (diazepam), but close to site II. It also shares the only basic-site on AAG. However, at therapeutic drug concentrations (0.3-1 microM), not all of these interactions occur. 6. The binding of tianeptine varied according to HSA, AAG and NEFA concentrations both in patients and healthy subjects. In patients with chronic renal failure having high NEFA concentrations the unbound fraction of tianeptine (fu) increased from 0.045 to 0.153 compared with normal (P less than 0.001). In cirrhotic patients, with relatively low HSA concentrations, the fu of tianeptine increased from 0.045 to 0.088 compared with normal (P less than 0.01). 7. Multiple regression analysis of all of the data indicated that the fu of tianeptine was related significantly to HSA, NEFA and AAG concentrations, with a particularly strong correlation with NEFA concentrations. Therefore, variation of HSA and NEFA concentrations in patients on maintenance therapy may cause an increase of tianeptine fu.     

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.     

Plasma protein binding of quinine: binding to human serum albumin, alpha 1-acid glycoprotein and plasma from patients with malaria.

The binding of quinine to human serum albumin (HSA), alpha 1-acid glycoprotein (AAG) and plasma obtained from healthy subjects (10 caucasians and 15 Thais) and from Thai patients with falciparum malaria (n = 20) has been investigated. In healthy volunteers, plasma protein binding expressed as the percentage of unbound quinine was 7.9-31.0% (69-92.1% bound). The mean percentage of unbound quinine found with essentially fatty acid-free HSA (40 g L-1) was 65.4 +/- 1.5% (mean +/- s.d.) and was comparable with the value (66.3 +/- 3.8%, mean +/- s.d.) for Fraction V HSA (40 g L-1). This suggests that fatty acids do not influence the plasma protein binding of quinine. Binding of quinine to 0.7 g L-1 AAG was high (mean unbound 61.0 +/- 5.0%), indicating that quinine is bound primarily to AAG and albumin, although other plasma proteins such as lipoproteins may be involved. The mean percentage of unbound quinine was slightly less in caucasians (14.8 +/- 6.7% unbound), compared with healthy Thai subjects (17.0 +/- 6.7% unbound). The higher binding of quinine in caucasian subjects was associated with a higher plasma AAG concentration observed in caucasians. Mean percentage of unbound quinine was significantly lower in Thai patients with malaria (10.9 +/- 4.0%) than in the healthy Thai subjects. The increase in the extent of quinine binding corresponded with the increase in the acute-phase reactant protein, AAG in the patients with malaria. Overall, when the data were combined there was a significant correlation (r = 0.846, P < 0.005) between the binding ratio (bound/unbound) of quinine and the plasma AAG concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early absorption and distribution analysis of antitumor and anti-AIDS drugs: lipid membrane and plasma protein interactions

The interactions of a set of compounds of potential importance for anticancer and AIDS chemotherapy with lipid membranes and plasma proteins were studied with a surface plasmon resonance (SPR) based optical biosensor, giving valuable information on the absorption and distribution of the compounds. The technique allowed both effective screening of compounds and more detailed kinetic and mechanistic analysis of specific interactions. The interaction with two different types of lipid membranes could be reliably measured at a drug concentration as low as 20 microM, allowing analysis of poorly soluble compounds. Distribution was evaluated by investigation of the interactions with two human plasma proteins, human serum albumin (HSA) and alpha(1)-acid glycoprotein (AGP). Two apparent binding sites were clearly defined for HSA: one with rapid and one with slow association and dissociation rates. The sites appear to differ in accessibility and recognition characteristics rather than in their capacities to form strong complexes with drugs.     

Acetaldehyde adducts with serum proteins: effect on diazepam and phenytoin binding

The in vitro effects of acetaldehyde treatment on the binding of phenytoin and diazepam to human serum albumin (HSA) and human serum proteins (HSP) have been investigated. The incorporation of acetaldehyde into proteins following incubation with different concentrations of [1,2-14 C]-acetaldehyde (0.5, 25, 100 mmol/l) was carried out. The proteins were then dialyzed so that only the stable adduct was retained. Binding of phenytoin and diazepam was then studied. Scatchard plot analysis showed a slight decrease (p less than 0.01 for HSP and 25 mmol/l acetaldehyde) in the number of binding sites for phenytoin when the acetaldehyde/protein ratio was increased. The affinity constant was also increased (p less than 0.01) with 100 mmol/l acetaldehyde. No change could be demonstrated in the number of diazepam binding sites on HSA; an increase in the binding capacity of HSP was shown following incubation with 25 mmol/l acetaldehyde. The fraction of drug bound at therapeutic levels has been also calculated for both drugs. An increase for diazepam but no change for phenytoin can be observed before or after treatment of proteins with acetaldehyde.     

Binding of pyridostigmine bromide,N,N-diethyl-m-toluamide and permethrin,alone and in combinations,to human serum albumin

In this study we examined the interaction of the anti-nerve agent drug pyridostigmine bromide (PB, 3,3-dimethylaminocarbonyloxy- N-methylpyridiniyum bromide), the insect repellent DEET ( N, N-diethyl- m-toluamide), and the insecticide permethrin [3-(2,2-dichloroethyl)-2,2-dimethylcyclopropanecarboxylic acid (3-phenoxyphenyl)methyl ester] in binding to human serum albumin (HSA). Concentrations between 500 ng/ml and 10 microg/ml PB, DEET and permethrin, alone or in combination, were incubated with HSA at 37 degrees C for 60 min. Concentrations of PB, DEET and permethrin were determined using high performance liquid chromatography (HPLC). The results showed that 81.2+/-4.2%, and 84.6+/-2.5% of the initial concentration of PB was bound to HSA when incubated alone or in combination with DEET or permethrin, respectively. DEET and permethrin did not significantly interact with HSA after 1 h of incubation. Incubation of combinations of two or three compounds did not significantly alter the binding pattern of any of the compounds with HSA. These results showed that PB is highly bound to albumin protein, while the competition between PB, DEET and permethrin on binding sites of HSA as a possible site of interaction following combined administration in vivo is not likely.     

Effect of serum, alpha-1 acid glycoprotein, lipoproteins and albumin on human mononuclear leucocyte beta-adrenoceptors

The effects of serum, alpha-1 acid glycoprotein (AAG), serum lipoproteins (SLP) and human serum albumin (HSA) on 3H-(-)-dihydroalprenolol (3H-(-)-DHA) binding and (-)-isoproterenol ((-)-IPR) induced cyclic AMP (cAMP) elevation in human peripheral blood mononuclear leucocytes (MNL) were investigated. The saturable binding of 3H-(-)-DHA was decomposed into two classes of binding sites with maximum binding capacity of approximately 1400 and 30000 sites/cell and with dissociation constants (Kd) of approximately 0.7 and 65 nM. Stimulation of the MNL beta-adrenoceptors by (-)-IPR caused a concentration dependent cAMP accumulation (EC50 approximately 0.2 microM) with maximum level approximately 250% above basal. For all single leucocyte preparations, 30-35 min. exposure to serum, AAG and SLP increased the number of beta-adrenoceptors with 100-200% and the maximal responsiveness to (-)-IPR with 30-90%. The presence of proteins did not change the Kd or the EC50. (-)-Alprenolol inhibited concentration dependently the serum induced increment in (-)-IPR-responsiveness. Serum, AAG and SLP did also increase the number of low affinity binding sites with 25-40% without effect on the Kd. HSA had no consistent effect on beta-adrenergic binding or stimulation. The present study shows that serum, AAG and SLP influence the number and function of MNL beta-adrenoceptors in vitro.     

Characterization of drug-protein binding process by employing equilibrium sampling through hollow-fiber supported liquid membrane and Bjerrum and Scatchard plots

The technique equilibrium sampling through membrane (ESTM) was extended to measuring the free drug concentration in solutions of drug and protein. Bjerrum and Scatchard plots were employed for characterizing individual drug binding to pure human blood proteins. Four drugs were investigated as a model system: fluvoxamine and ropivacaine which dominantly bind to alpha-acid glycoprotein (AGP), and R,S-ibuprofen and S-ketoprofen which highly bind to human serum albumin (HSA). The level of drug binding to AGP and HSA relied on drug and protein concentrations. Bjerrum and Scatchard plots revealed high affinity constants (K(a)) at low protein concentration. Both Bjerrum and Scatchard plots of fluvoxamine and ropivacaine binding to AGP showed one specific binding site (n(1)=1) with ropivacaine K(a) value close to 5 times higher than the K(a) of fluvoxamine at 22.9muM AGP concentration. Bjerrum plots of ketoprofen and ibuprofen gave total number of binding sites or bound molecules of 6-7, which did not depend on the drug or protein concentration. Scatchard plots of ketoprofen and ibuprofen exhibited two binding sites (n(1) and n(2)) at 0.15muM and 0.75muM HSA concentrations. On one hand, at 0.15muM HSA, ketoprofen and ibuprofen were bound to site I at n(1)=1.2 and n(1)=1.0, respectively. However, at 0.75muM HSA, ketoprofen and ibuprofen were bound to site I at n(1)=1.2 and n(1)=1.9, respectively. On the other hand, site II, at 0.15muM HSA, interacted with ketoprofen and ibuprofen at n(2)=5.6 and 6.7, respectively. However, at 0.75muM HSA, site II interacted with ketoprofen at n(2)=7.4 and ibuprofen at n(2)=6.2. It would be concluded that, upon mixing ketoprofen and ibuprofen in a HSA solution, a ketoprofen-ibuprofen interaction would most likely occur at site II in HSA.     

Effects of nonenzymatic glycosylation and fatty acids on tryptophan binding to human serum albumin.

The effects of bound fatty acids and nonenzymatic glycosylation (NEG) on tryptophan binding to human serum albumin (HSA) were examined utilizing a rate of dialysis technique. HSA with 0, 1, 2, 3, or 5 mol of palmitate bound per mol of HSA was glycosylated in vitro to a level exceeding that seen in diabetes. NEG was not inhibited by fatty acids, suggesting that Lys-525, the primary site for NEG, is not an essential component of the principal sites for long-chain fatty acid binding to HSA. Scatchard analysis of binding data showed an expected fatty acid dependent decrease in the number of available tryptophan binding sites, but showed that fatty acids did not affect tryptophan affinity. The binding data failed to show an effect of NEG on tryptophan binding. The lack of inhibition of tryptophan binding by NEG suggests that drug-binding Site II, the indole/benzodiazepine site, is resistant to both NEG and to any conformational changes in HSA which may occur with NEG. These data suggest that elevated plasma free tryptophan and the resulting altered serotonin metabolism seen in diabetes are independent of increased NEG and likely result from diabetic hyperlipidemia.     

Binding of perfluorooctanoic acid to rat and human plasma proteins

Perfluorooctanoic acid (PFOA) is a commercially important organic fluorochemical and is considered to have a long half-life in human blood. In this paper, PFOA binding to rat and human plasma proteins was investigated. On the basis of results from size-exclusion chromatography and ligand blotting, most PFOA was in protein-bound form in male and female rat plasma, and the primary PFOA binding protein in plasma was serum albumin. PFOA binding to rat serum albumin (RSA) in the gas phase was observed by electrospray ionization MS. (19)F NMR experiments revealed that binding to RSA caused peak broadening and chemical shift changes of PFOA resonances, and on the basis of this observation, the dissociation constant was determined to be approximately 0.3 mM. The dissociation constants for PFOA binding to RSA and human serum albumin (HSA) and the numbers of PFOA binding sites on RSA and HSA were also determined by a separation method using microdesalting columns. No significant difference was found between PFOA binding to RSA and PFOA binding to HSA. The dissociation constants for binding of PFOA to RSA or HSA and the numbers of PFOA binding sites were in the range of 0.3-0.4 mM and 6-9, respectively. On the basis of these binding parameters and the estimated plasma concentration of serum albumin, greater than 90% of PFOA would be bound to serum albumin in both rat and human blood.     

Affinity of various compounds for benzodiazepine binding sites in rat brain, heart and kidneys in vitro

Binding of several psychoactive, antiinflammatory, antihypertensive, and antiarrhythmic drugs to central and peripheral benzodiazepine (BZ) binding sites was studied in the brain, heart and kidneys of rats. Diazepam exhibited the highest affinity for all binding sites (Ki values at 0.01 microM level); another 1,4-BZ, oxazepam, had markedly lower affinity for peripheral binding sites (Ki 21-37 microM). Non-BZ compounds had low affinity for central BZ receptors; proquazone was the most potent (Ki 9.5 microM). The affinities of non-BZ compounds were higher for peripheral BZ binding sites. The Ki value for proquazone was approximately 0.1 microM; and many other antiinflammatory agents, and the vasodilators cyclandelate and nifedipine, produced Ki values in the micromolar level. beta-Blocking drugs, and several other antihypertensive and antiarrhythmic agents lacked affinity for both central and peripheral BZ binding sites. According to the results, the affinity for peripheral binding sites is independent of an affinity for central BZ receptors. Non-BZ compounds that bound to brain BZ receptors bound with equal affinity to both BZ1 and BZ2 subgroups of receptors. The compounds with affinity for peripheral BZ binding sites did not select between heart and kidneys, which suggests that these organs have similar binding sites. The role of the peripheral BZ binding sites has not yet been established. The findings of this study allow the selection of a more varied group of ligands to be used when investigating the physiological significance of these binding sites.     

Interactions of Aldosterone Antagonist Diuretics with Human Serum Proteins

Purpose. The purpose of this study was to investigate the binding mechanism of aldosterone antagonist diuretics with human serum proteins, human serum albumin (HSA) and ₁-acid glycoprotein (AGP), as well as to identify the binding sites of the drugs on these proteins. Methods. Binding activities of spironolactone (SP) and its pharmacologically active metabolite canrenone (CR) to serum and serum protein were examined by ultrafiltration and spectroscopic techniques. The data for the binding of these drugs to HSA were analyzed on the basis of a theoretical model of simultaneous binding of the ligands. Results. The binding percentages of antagonist diuretics SP and CR to human serum proteins were 88.0% and 99.2%, respectively, at therapeutic concentrations. SP bound strongly and almost equally to both HSA and AGP, but CR bound strongly only to HSA. In addition, the displacement results found using fluorescent probes and ultrafiltration methods demonstrated that SP bound to site I, particularly to the warfarin region on HSA, and to the basic binding site on AGP, while CR bound to the warfarin region on HSA. Conclusions. The limited results presented here stress the need for caution on coadministration of acidic drugs which bind to the warfarin region on HSA and basic drugs which bind to AGP with SP and its metabolite CR.     

Irreversible alkylation of human serum albumin by zileuton metabolite 2-acetylbenzothiophene-S-oxide: a potential model for hepatotoxicity

2-acetylbenzothiophene-S-oxide (2-ABT-S-oxide or M1) is a reactive metabolite of zileuton, a drug used in the treatment of asthma and is capable of conjugating with glutathione in vitro. Human serum albumin (HSA) is the most abundant protein in plasma and plays a critical role in detoxifying reactive oxygen species. The current research is focused on understanding the interaction between M1 and HSA. The stability studies revealed the half-life of M1 to be about 0.85 h in HSA, 1.82 h in human plasma, and 4.48 h in phosphate-buffered saline (PBS) as determined by first-order approximation. The alkylation rate constant k for HSA was 20 M(-1) min(-1). After quenching with acetonitrile, the half-life of M1 did not change significantly, indicating that M1 is covalently bound to HSA. LC-MS and LC-MS/MS analysis of human plasma revealed the M1 alkylated peptide P (m/z 870) formed by HSA conjugation and concomitant water elimination. The specific amino acid on HSA bound to M1 was identified as Cys-34. This alkylation is observed to be concentration- and incubation-time-dependent in human plasma. HSA oxidized by N, N'-diacetyl-L-cystine exhibits a compromised ability of HSA to react with M1. The alkylated HSA diminished the binding affinity for warfarin. Furthermore, the alkylation was found to be irreversible in the dialysis experiment. In addition, M1 decomposes to 2-ABT in the presence of HSA, presumably acting as an oxidant. The formation of 2-ABT in the incubation and the self-condensation of M1 in PBS indicate that the alkylation of Cys-34 is only one of a number of reactions that occur in the presence of HSA. Irreversible protein modification may potentially lead to a loss of its function. HSA irreversible alkylation represents a model for other proteins to be potentially toxic and thus may help explain zileuton hepatotoxicity.     

Comparative studies of the covalent binding of [14C]-2-chloro-4-acetotoluidide by liver and kidney slices of the starling

In tissue slices of female starlings, binding of [14C]-2-chloro-4-acetotoluidide (CAT) radioactivity to liver proteins was almost five times greater than binding to kidney proteins after 2 h of incubation. Binding to protein of liver slices increased in a log linear fashion with increasing CAT concentrations. Binding to protein of kidney slices also increased with increasing concentrations but not in a log linear fashion. Mixed-function oxidase inhibitors, SKF 525-A and alpha-naphthoflavone, decreased binding to liver slices but did not affect binding to kidney slices. Anaerobic incubation conditions inhibited binding to both tissues. P-Hydroxymercuribenzoate and sodium cyanide did not affect the binding of radioactivity associated with [14C]-CAT to proteins of either liver or kidney slices. Diethyl maleate increased binding of the radioactivity to proteins of the kidney slices but not to liver slices. Cysteine also increased binding in kidney slices. Binding in liver slices did not increase significantly with cysteine. The cysteine-induced increase in protein binding in kidney slices did not appear to depend on the formation of sulfate from the metabolism of cysteine. There was no sex-dependent difference in starlings as to the binding of radioactivity in either liver or kidney slices. Male chicken kidney slices bound a much higher amount of radioactivity associated with [14C]-CAT than male starling kidney slices, while the liver slices bound comparable amounts. Male hamster liver slices bound much more radioactivity than did male starling liver slices. However, hamster kidney slices bound much less than did starling kidney slices.(ABSTRACT TRUNCATED AT 250 WORDS)     

In-vitro study on the competitive binding of diflunisal and uraemic toxins to serum albumin and human plasma using a potentiometric ion-probe technique

The competitive binding of diflunisal and three well-known uraemic toxins (3-indoxyl sulfate, indole-3-acetic acid and hippuric acid) to bovine serum albumin (BSA), human serum albumin (HSA) and human plasma was studied by direct potentiometry. The method used the potentiometric drug ion-probe technique with a home-made ion sensor (electrode) selective to the drug anion. The site-oriented Scatchard model was used to describe the binding of diflunisal to BSA, HSA and human plasma, while the general competitive binding model was used to calculate the binding parameters of the three uraemic toxins to BSA. Diflunisal binding parameters, number of binding sites, n(i) and association constants for each class of binding site, K(i), were calculated in the absence and presence of uraemic toxins. Although diflunisal exhibits high binding affinity for site I of HSA and the three uraemic toxins bind primarily to site II, strong interaction was observed between the drug and the three toxins, which were found to affect the binding of diflunisal on its primary class of binding sites on both BSA and HSA molecules and on human plasma. These results are strong evidence that the decreased binding of diflunisal that occurs in uraemic plasma may not be solely attributed to the lower albumin concentration observed in many patients with renal failure. The uraemic toxins that accumulate in uraemic plasma may displace the drug from its specific binding sites on plasma proteins, resulting in increased free drug plasma concentration in uraemic patients.     

Protein binding of oxazepam and its glucuronide conjugates to human albumin

The binding of oxazepam and its glucuronide conjugates to human serum albumin (HSA), as well as the binding interactions of the drug and its metabolites, were examined by equilibrium dialysis and kinetic probe studies. Oxazepam and its S(+) glucuronide are bound to the HSA molecule with affinity constants of 3.5 X 10(5) M-1 and 5.5 X 10(4) M-1, respectively, which were independent of protein concentration over a range of 0.1 to 5.0 g/dl. The R(-) glucuronide bound weakly to albumin, with the binding parameter, N X K, increasing at lower albumin concentrations. Pre-acetylation of fatty acid free-HSA resulted in decreased binding of all three compounds, probably by altering the conformation of the binding sites. Kinetic probe studies with p-nitrophenyl acetate indicate that oxazepam and its S(+) glucuronide shared a common binding site on HSA, but that the R(-) glucuronide bound at another site. Oxazepam binding was unaffected by the presence of its glucuronide conjugates but was inhibited by fatty acids. The percentage of oxazepam bound to plasma proteins in patients with renal impairment (94%) was lower than in normal volunteers (97%). This lower binding can neither be attributed to lower albumin concentrations because of the large binding capacity of the protein and linearity of N X K nor to displacement by elevated concentrations of glucuronide conjugates, but it may be ascribed partly to increased plasma fatty acids.     

BINDING CHARACTERISTICS OF KNI-272 TO PLASMA PROTEINS,A NEW POTENT TRIPEPTIDE HIV PROTEASE INHIBITOR

The binding characteristics of KNI-272, a potent and selective human immuno-deficiency virus (HIV) protease inhibitor, were evaluated in rat and human plasma, and in solutions of human α₁-acid glycoprotein (AAG) and human serum albumin (HSA). The unbound fractions (F_u ) of KNI-272 were 12·13 and 2·24% in rat and human plasma, respectively, at the drug concentration of 1·0 μg mL⁻¹. Although KNI-272 binds to both AAG and HSA, the F_u of KNI-272 in AAG solution was 1·83%, and only one-quarter of that in HSA solution (F_u6·78%). Binding displacing agents, such as disopyramide, warfarin, diazepam, and digitoxin, were used to determine the binding site of KNI-272 on these plasma proteins. The F_u of KNI-272 in AAG solution increased 14-fold when disopyramide was added to the AAG solution. In addition, warfarin, diazepam, and digitoxin were added to HSA solution as representative drugs bound to distinct binding sites on HSA, namely sites I, II, and III, respectively. The F_u values of KNI-272 in HSA solution significantly increased when warfarin and diazepam were added. In particular, with the addition of warfarin to HSA solution, the F_u of KNI-272 increased to 16%. The modified Scatchard plots of KNI-272 binding to AAG and HSA both showed biphasic curves, and the KNI-272 binding sites at low concentration range on AAG and HSA disappeared with the addition of disopyramide and warfarin, respectively. Therefore, it is considered that KNI-272 binds to the identical site as disopyramide on AAG and site I on HSA in the low KNI-272 concentration range. By comparing the KNI-272 binding parameters obtained in human plasma and these protein solutions, we can assume that KNI-272 binding at low concentration in human plasma is mainly concerned with the binding on AAG. As KNI-272 concentration in plasma increases, HSA becomes concerned with KNI-272 binding.