Factors influencing the protein binding of azosemide using an equilibrium dialysis technique

Various factors most likely to influence the plasma protein binding of azosemide to 4% human serum albumin (HSA) were evaluated using equilibrium dialysis at the initial azosemide concentration of 10 μg mL^?1. It took approximately 8h of incubation to reach an equilibrium between 4% HSA and isotonic phosphate buffer of pH 7.4 containing 3% dextran (the ‘buffer’) using a Spectra/Por 2 membrane (molecular weight cut-off 12000–14000) in a water bath shaker kept at 37°C and a rate of 50 oscillations min^?1. Azosemide was fairly stable both in 4% HSA and in the ‘buffer’ for up to 24h. The binding of azosemide to 4% HSA was constant (95.5 ± 0.142%) at azosemide concentrations ranging from 5 to 100 μg mL^?1. However, the extent of binding was dependent on HSA concentration: the values were 88.4, 91.0, 92.2, 94.2, 94.9, 94.9, and 94.9% at albumin concentrations of 0.5, 1, 2, 3, 4, 5, and 6% respectively. The binding was also dependent on incubation temperature; the binding values were 97.0, 94.9, and 94.9% when incubated at 6, 28, and 37°C, respectively. The binding of azosemide was also influenced by buffers containing various chloride ion concentrations and buffer pHs. The binding values were 95.3, 94.9, and 93.6% for the chloride ion concentrations of 0, 0.249, and 0.546%, respectively, and the unbound values were 6.8, 5.1, 3.8, 3.4, and 3.3% for buffer pHs of 5.8, 6.4, 7.0, 7.4, and 8.0, respectively. The binding of azosemide was independent of the quantity of heparin (up to 40 UmL^?1), AAG (up to 0.16%), sodium azide (NaN₃, up to 5%), its metabolite, Ml (up to 10 μg mL^?1), and anticoagulants (EDTA and citrate).     

Factors influencing the protein binding of a new phosphodiesterase V inhibitor, DA-8159, using an equilibrium dialysis technique

Various factors influencing the protein binding of DA-8159 to 4% human serum albumin (HSA) were evaluated using an equilibrium dialysis technique at an initial DA-8159 concentration of 5 microg/mL. It took approximately 8 h incubation to reach an equilibrium between 4% HSA and an isotonic phosphate buffer of pH 7.4 containing 3% of dextran ('the buffer') using a Spectra/Por 2 membrane (mol. wt. cut-off: 12,000--14,000) in a water bath shaker kept at 37 degrees C and at a rate of 50 oscillations per min. The extent of binding was dependent on DA-8159 concentrations, HSA concentrations, incubation temperature, buffer pH, and alpha-1-acid glycoprotein (AAG) concentrations. The binding of DA-8159 in heparinized human plasma (93.9%) was significantly higher than in rats (81.4%), rabbits (80.4%), and dogs (82.2%), and this could be due to differences in AAG concentrations in plasma.     

Factors influencing the protein binding of vancomycin.

Various factors influencing the protein binding of vancomycin were examined using equilibrium dialysis method. Four per cent human serum albumin (HSA) and/or 0.08 per cent alpha-1-acid glycoprotein (AAG), dissolved in isotonic phosphate buffer, were dialyzed against isotonic phosphate buffer of pH 7.4 using Spectrapor 2 membrane. The protein binding of vancomycin to 0.08 per cent AAG was dependent on vancomycin concentrations; the values ranged from 21.1 per cent at the vancomycin concentration of 20 micrograms ml-1 to 5.30 per cent at 2400 micrograms ml-1. However, binding to 4 per cent HSA was relatively constant, 8.79 +/- 2.43 per cent over a vancomycin concentration range of 20-2400 micrograms ml-1. The values to 4 per cent HSA alone and 0.08 per cent AAG alone did not predict the greater binding of vancomycin in the presence of both proteins, especially at higher concentrations of vancomycin; the values to 4 per cent HSA with 0.08 per cent AAG were constant, 26.3 +/- 3.74 per cent, at the vancomycin concentration range of 20-2400 micrograms ml-1. This suggested an interaction between the proteins, which resulted in enhanced binding of vancomycin. The protein binding of vancomycin to 4 per cent HSA with 0.08 per cent AAG was not influenced by the different incubation temperatures (4 degrees, 22 degrees, and 37 degrees), quantities of heparin (up to 40 units ml-1) or AAG (up to 0.16 per cent), or buffers (isotonic phosphate buffer of pH 7.4, phosphate buffer of pH 7.4 and 0.9 per cent NaCl solution) at the vancomycin concentration of 80 micrograms ml-1. Vancomycin was found to be stable in human serum albumin or in isotonic phosphate buffer of pH 7.4.     

Binding of Sulfinpyrazone and its Metabolites in Human Serum and in Solutions of Human Serum Albumin

Abstract: The binding of sulfinpyrazone, its sulfone metabolite and its sulfide metabolite to serum protein was studied by equilibrium dialysis. At 20 μg/ml 99.1% of the parent compound was bound in serum, whereas 99.8% of the sulfide and 98.3% of the sulfone were bound at this concentration. The binding of the three compounds were studied in diluted serum and in solutions of human serum albumin (HSA). There was no evidence of binding to proteins other than albumin. The association constants to primary and secondary binding sites and the number of binding sites were calculated. For the sulfide a lower K₁-value in serum (0.76·10⁶ M^?1) than in the HSA solution (1.8·10⁶ M^?1) indicated the possible presence of a competitively bound substance in serum. In undiluted serum no displacing effect of the sulfide on sulfinpyrazone binding was found when both compounds were present in a concentration of 20 μg/ml, but in a HSA solution a pronounced sulfide induced displacement of the sulfinpyrazone from its primary binding site was shown. Acetylation of HSA depressed the binding of sulfinpyrazone but in undiluted serum there was no other effect on sulfinpyrazone binding by the addition of acetylsalicylic acid than could be explained by the displacing effect of salicylic acid. At concentrations at 20 μg/ml of sulfinpyrazone and above 50 μg/ml of the displacing agent significant displacement was demonstrated with phenylbutazone, tolbutamide and salicylic acid.     

Protein binding of sotalol enantiomers in young and elderly human and rat serum using ultrafiltration

The protein binding of sotalol (STL) enantiomers was evaluated using an ultrafiltration technique with serum from young (32±2 years, n=5) and elderly (73±6 years, n=5) male and female humans, and young (8 weeks, n=4) and elderly (60 weeks, n=3) male Sprague—Dawley rats. Serum samples were collected and immediately frozen at ?20°C. Within 1 week, the serum samples were thawed at room temperature, and adjusted to pH 7.4 using 0.05 M phosphate buffer, pH 5.0. Aliquots were spiked with 250 ng mL^?1 and 500 ng mL^?1 of each STL enantiomer, placed in ultrafiltration sets (Microsep, 30K molecular weight cut-off), capped, equilibrated to 37°C, and centrifuged at 1850g for 1.5h at 37°C. Aliquots of ultrafiltrate and unspun serum were analysed for STL enantiomer concentration using a stereospecific HPLC assay. In all groups, bound fraction was less than 7% for both STL enantiomers. There were no significant differences in bound fraction between groups, or between enantiomers. Adsorption of STL enantiomers to the ultrafiltration device and membrane, evaporative loss of serum samples during centrifugation, and protein concentration in each ultrafiltrate sample were all negligible. It is concluded that the binding of STL in human and rat serum at therapeutic concentrations and physiological temperature and pH is negligible and non-stereoselective.     

Pharmacokinetics of reboxetine in healthy volunteers. Single oral doses,linearity and plasma protein binding

The pharmacokinetics of reboxetine, a new antidepressant agent, were found to be close to linear in a crossover study comparing administration of single 2, 3, 4 and 5 mg capsule doses in 15 healthy male volunteers, and in the same study the capsules were bioequivalent to the proposed therapeutic tablet formulation (4mg). Kinetic analysis was based on HPLC assay of reboxetine in plasma and urine collected up to 72 h after each administration. Plasma levels indicated a rapid absorption (t_max?2h) and an elimination half-life of about 13 h. Clearance and volume of distribution were modest (ratios to bioavailability: CL/F?29 mL min^?1; V_z/F?32L); urinary excretion was ～9% of dose, corresponding to a renal clearance of only 3 mL min^?1 (a value consistent with the rate of glomerular filtration of unbound drug). In vitro, binding to plasma proteins, estimated from radioactivity levels following dialysis of ¹⁴C-labelled reboxetine, appeared to be dominated by α₁-acid glycoprotein without marked saturation up to plasma concentrations of over 500 ng mL^?1 (2.8–3.1% unbound with human plasma from three additional volunteers; 1.8–2.0% for 2gL^?1 orosomucoid α₁-acid glycoprotein, and 46.4–47.4% for 40 gL^?1 albumin), whilst the mean C_max in the current study was much lower (164 ng mL^?1 after a 5 mg dose).     

Stereoselective binding properties of naproxen glucuronide diastereomers to proteins

The stability of naproxen glucuronide (NAP-G) diastereomers was investigated in buffer, 0.3% and 3% human serum albumin (HSA) solutions, and human plasma.R-NAP-G was found to be less stable in phosphate buffer than itsS-diastereomer, whereas incubation media containing protein in general increased the degradation rate of NAP-G but also caused a change of the stereoselective stability where theR-NAP-G was more stable thanS-NAP-G. Reversible binding of NAP-Gs to HSA (0.3%) was investigated and compared with the corresponding properties of naproxen (NAP) enantiomers. NAP-G diastereomers exibited a considerable and stereoselective affinity to HSA, although less than that observed for the NAP enantiomers.In vitro irreversible binding of NAP-Gs to HSA, human and rat plasma proteins was also investigated. Irreversible binding was higher forR-NAP-G (50 μM) than forS-NAP-G (50 μM) in all incubation media. This stereoselective difference was observed with HSA containing medium as well as in rat and human plasma. Incubation with unconjugated NAP did not lead to irreversible binding. Preincubation of HSA with acetylsalicylic acid (≈ 11 mM) and glucuronic acid (50 mM) decreased the extent of irreversible binding suggesting involvement of lysine residues for covalent binding. Preincubation withS-NAP also decreased the irreversible binding yield. This paper is dedicated to Professor Richard Neidlein, Pharmaceutical Chemistry Institute, Heidelberg, in commemoration of his 65th birthday. Supported in part by National Institutes of Health Grants GM 36633 and DK 26307.     

Protein Binding of Phenprocoumon in the Absence and Presence of Furosemide

Abstract A modification of a fluorometric assay for phenprocoumon is described. The sensitivity of the method has been increased so that the lower limit is 5 ng/ml. By an ultrafiltration technique, phenprocoumon (PPC), in therapeutic concentrations, was bound to the extent of 99.9%, both in undiluted plasma and in Krebs–Henseleit (K–H) solutions containing 4 g human serum albumin (HSA)/100 ml. An increase in the pH from 6.05 to 8.02 in phosphate buffers containing 0.2 g HSA/100 ml yielded an increase in the apparent association constant for PPC binding to HSA from 0.562–10⁶ 1/mol to 1.195–10⁶1/mol, while the number of PPC mol bound per mol albumin remain unchainged (1.05 and 1.03 respectively). A reduced binding of PPC in undiluted plasma containing increasing concentrations of furosemide have been observed. Scatchard plots based on binding studies in K–H solutions containing 0.2 g HSA/100 ml indicate a competitive nature of the albumin binding of PPC and furosemide.     

Stability,tissue metabolism,tissue distribution and blood partition of azosemide

Stability of azosemide after incubation in various pH solutions, human plasma, human gastric juice, and rat liver homogenates, metabolism of azosemide after incubation in 9000g supernatant fraction of various rat tissue homogenates in the presence of NADPH, tissue distribution of azosemide and M1 after intravenous (IV) administration of azosemide, 20 mg kg^?1, to rats, and blood partition of azosemide between plasma and blood cells from rabbit blood were studied. Azosemide seemed to be stable for up to 48 h incubation in various pH solutions ranging from two to 13 at an azosemide concentration of 10 μg mL^?1; more than 93.4% of azosemide was recovered and a metabolite of azosemide, M1, was not detected. However, the drug was unstable in pH 1 solution: 75.8% of azosemide was recovered and 2.16 μg mL^?1 of M1 (expressed in terms of azosemide) was formed after 48 h incubation in pH 1 solution at an azosemide concentration of 10 μg mL^?1. Azosemide was stable in both human plasma and rat liver homogenates for up to 24 h incubation at an azosemide concentration of 1 μg mL^?1, and in human gastric juice for up to 4 h incubation at an azosemide concentration of 10 μg mL^?1. However,-all rat tissues stdied had metabolic activity for azosemide in the presence of NADPH, with heart having a considerable metabolic acitivity: approximately 22% of azosemide disappeared and 9.32 μg of M1 was formed per gram of heart (expressed in terms of azosemide) after 30 min incubation of 50 μg of azosemide in 9000g supernatant fraction of heart homogenates. The tissue to plasma ratios of azosemide (T/P) were greater than unity only in the liver (1.26) and kidney (1.74); however, M1 showed high affinity for all tissues studied except the brain and spleen when each tissue was collected at 30 min after IV administration of azosemide to rats. The equilibrium plasma to blood cell concentration ratios of azosemide were independent of azosemide blood concentrations: the values were 2.78–4.25 at azosemide blood concentrations of 1, 10, and 20 μg mL^?1 three rabbits. There was negligible ‘blood storage effect’ of azosemide, especially at low blood concentrations of azosemide, such as 1 and 10 μg mL^?1.     

Characterization of the comparative drug binding to intra- (liver fatty acid binding protein) and extra- (human serum albumin) cellular proteins

Abstract

1.?This study compared the extent, affinity, and kinetics of drug binding to human serum albumin (HSA) and liver fatty acid binding protein (LFABP) using ultrafiltration and surface plasmon resonance (SPR).

2.?Binding of basic and neutral drugs to both HSA and LFABP was typically negligible. Binding of acidic drugs ranged from minor (f_u?>?0.8) to extensive (f_u?<?0.1). Of the compounds screened, the highest binding to both HSA and LFABP was observed for the acidic drugs torsemide and sulfinpyrazone, and for β-estradiol (a polar, neutral compound).

3.?The extent of binding of acidic drugs to HSA was up to 40% greater than binding to LFABP. SPR experiments demonstrated comparable kinetics and affinity for the binding of representative acidic drugs (naproxen, sulfinpyrazone, and torsemide) to HSA and LFABP.

4.?Simulations based on in vitro kinetic constants derived from SPR experiments and a rapid equilibrium model were undertaken to examine the impact of binding characteristics on compartmental drug distribution. Simulations provided mechanistic confirmation that equilibration of intracellular unbound drug with the extracellular unbound drug is attained rapidly in the absence of active transport mechanisms for drugs bound moderately or extensively to HSA and LFABP.     

丹酚酸B与大鼠血浆蛋白结合率的测定

建立测定丹酚酸B与大鼠血浆蛋白结合率的方法; 体外采用平衡透析法, 模拟丹酚酸B与血浆蛋白的结合过程, 体内采用超滤法, 并以高效液相色谱法进行测定。透析内液用甲醇沉淀蛋白, 透析外液过滤后直接测定。结果透析外液线性范围为0.5～20 μg·mL⁻¹, 透析内液的线性范围为2～200 μg·mL⁻¹, 提取回收率68.6%～81.9%, 日内日间精密度均小于8.5%, 丹酚酸B的体外血浆蛋白结合率为75.2%, 体内血浆蛋白结合率为92.1%, 丹酚酸B与大鼠血浆蛋白结合率较高。建立的方法灵敏度高, 重现性好, 操作简单, 能够满足分析要求。     

Serum protein binding of noscapine: Influence of a reversible hydrolysis

Abstract— The binding of the antitussive drug noscapine to human serum, pure albumin and α₁-acid glycoprotein has been investigated by ultrafiltration and equilibrium dialysis, using radiolabelled noscapine. The binding in serum pooled from volunteers was 93 ± 0.2% (at 100 ng mL^?1). After incubation for 24 h the binding decreased to about 85% (ultrafiltration 87.0 ± 1.0%; equilibrium dialysis 84.3 ± 1.2%), because of the conversion of noscapine to noscapinic acid. Only unbound drug underwent this hydrolysis, and as noscapine is extensively bound in healthy volunteers, this elimination process is probably unimportant. The major binding protein of noscapine was albumin (K = 3060 M^?1, n = 5.62), but the binding to ai-acid glycoprotein was also substantial (K = 31500 M^?1, n = 1.73). The interindividual variation in binding was low and binding was linear at the concentrations observed after therapeutic doses (0–500 ng mL^?1).     

Unexpected difference between human serum albumin and human serum toward l-tryptophan binding

The binding of l-tryptophan to human serum albumin (HSA) Fr. V in Krebs-Ringer phosphate buffer and to HSA in serum was studied by equilibrium dialysis. At constant albumin concentration (in the in vivo range) and various l-tryptophan concentrations, binding was greater in serum, with an apparent association constant (k) equal to 4.88 × 10⁴ I/mole and the number of binding sites (n) equal to 0.62. The corresponding kinetic parameters obtained at constant HSA Fr. V concentration and various l-tryptophan concentrations in Krebs-Ringer phosphate bufferwere 7.61 × 10³ I/mole and 0.28, respectively. The Scatchard plot obtained from experiments with various albumin concentrations and a constant l-tryptophan concentration had a negative slope in serum and a positive slope in Krebs-Ringer phosphate buffer.     

Application of headspace solid phase microextraction for study of noncovalent interaction of borneol with human serum albumin

Aim:

To investigate noncovalent interactions between borneol and human serum albumin (HSA) under near-physiological conditions.

Methods:

A 65-μm polydimethylsiloxane (PDMS) fiber was selected for sampling. The extraction temperature was kept at 37 °C, and the extraction time was optimized at 10 min. Borneol solutions of different concentrations were equilibrated in 600 μmol/L HSA and 67 mmol/L phosphate buffer solution (pH 7.4, 37 °C) for 24 h prior to solid phase microextraction (SPME) using headspace mode. The binding properties were obtained based on the calculation of extracted borneol amount using gas chromatography (GC) determination.

Results:

The headspace SPME extraction method avoided disturbance from the HSA binding matrix. The recovery showed good linearity for the borneol concentrations over the range of 0.4–16.3 μmol/L with a regression coefficient (R²) of 0.9998. The limit of detection and lower limit of quantitation were determined to be 0.01 μmol/L and 0.4 μmol/L, respectively. The binding constant and the percentage binding rate were estimated to be 2.4×10³(mol/L)^-1 and 59.5%, respectively.

Conclusion:

Headspace SPME coupled to GC is a simple, sensitive and rapid method for the study of borneol binding to HSA. The method may be applied in the determination of other protein binding properties in human plasma.     

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.     

Rat Protein Binding and Cerebral Phospholipid Affinity of the H3-receptor Antagonist Thioperamide

The binding of thioperamide, a known H₃-receptor antagonist, to rat plasma proteins and its affinity for rat cerebral phospholipids are investigated. Thioperamide is strongly bound to plasma proteins (95?80% at plasma concentrations of 3.5?400 μg mL^?1), and its binding can be resolved into two components: a high-affinity, saturable component and a non-specific component. The drug has a high affinity for cerebral phospholipids, with a partition coefficient of approximately 100 (log K = 2.06 ± 0.14), which should promote brain penetration and accumulation. Protein binding and cerebral phospholipid affinity can suggest the explanation of some differences reported in the literature on thioperamide distribution data: at low plasma concentrations of the drug, its protein binding (95% at 3.5 μg mL^?1) can prevent brain accumulation, while at higher concentrations the free plasma fraction suddenly increases (> 10% at 18 μg mL^?1) and it allows passive distribution to lipophilic tissues such as brain tissue.     

Modification of human serum albumin binding of methotrexate by folinic acid and certain drugs used in cancer chemotherapy

Summary The binding of methotrexate (MTX) and citrovorum factor (CF) to human serum albumin (HSA) was investigated. The affinity constant for MTX was 820 M^–1, with 2 binding sites, and for CF 2340 M^–1, with 1.5 binding sites. MTX and CF, which are used together in high dose therapy, compete for HSA binding. Competition for HSA binding between MTX and adriamycin, bleomycin and cyclophosphamide, drugs often used in association with MTX in cancer chemotherapy, was also demonstrated. The clinical importance of such competition depends on the drug/protein concentration ratio which is extremely variable.     

Binding of gefitinib, an inhibitor of epidermal growth factor receptor-tyrosine kinase, to plasma proteins and blood cells: in vitro and in cancer patients

Summary Gefitinib exhibits wide inter-subject pharmacokinetic variability which may contribute to differences in treatment outcome. Unbound drug concentrations are believed to be more relevant to pharmacological and toxicological responses than total drug. Thus it is desirable to determine gefitinib binding in plasma and factors affecting this process. An equilibrium dialysis method using 96-well microdialysis plates was optimized and validated for determining the fraction unbound (fu) gefitinib in human plasma. Gefitinib binding in plasma from four different species and isolated protein solutions as well as drug partitioning in human blood cells were investigated. Unbound gefitinib plasma concentrations were measured in 21 cancer patients receiving daily oral gefitinib 250 mg or 500 mg. It was found that gefitinib was extensively bound in human rat mouse and dog plasma with mean fu values of 3.4%, 3.8%, 5.1% and 6.0% respectively. In isolated protein solutions approximately 90% and 78% of gefitinib was bound to human serum albumin (HSA) (40 mg/dL) and alpha1-acid glycoprotein (AAG) (1.4 mg/dL) with binding constants of 1.85 × 10⁴ M⁻¹ and 1.13 × 10⁵ M⁻¹ respectively. In whole blood 2.8% of gefitinib existed as the free drug while 79.4% and 17.8% was bound to plasma proteins and blood cells respectively. In plasma from cancer patients fu at pre-treatment varied 2.4-fold (mean 3.4 ± 0.6%; range 2.2–5.4%) and fu was constant over the 28-days of treatment (P > 0.05). Pre-treatment AAG concentration was negatively correlated with pre-treatment fu (R² = 0.28, P = 0.01). In conclusion gefitinib is highly protein bound (∼ 97%) in human plasma. Variable AAG concentrations observed in cancer patients may affect gefitinib fu with implications for inter-subject variation in drug toxicity and response.     

Pharmacokinetics, blood partition and protein binding of DA-7867, a new oxazolidinone

The pharmacokinetics after single intravenous and single and consecutive 2 week oral administration, tissue distribution, in vitro tissue metabolism, stability, blood partition and protein binding of DA‐7867, a new oxazolidinone, were evaluated. After intravenous administration at a dose of 10mg/kg to rats, DA‐7867 was eliminated slowly with time‐averaged total body clearance of 0.915ml/min/kg. After consecutive 2 week oral administration at a dose of 2mg/kg/day to rats, DA‐7867 was accumulated in rats; the AUC was significantly greater (1430 versus 1880µg min/ml) than that after single oral administration at a dose of 2mg/kg. The rat tissues studied had low affinity to DA‐7867; the tissue‐to‐plasma ratios were smaller than unity after both intravenous and oral administration at a dose of 20mg/kg. The rat tissues studied had almost negligible metabolic activity for DA‐7867 based on 30min incubation of DA‐7867 with 9000 g supernatant fraction of rat tissues. DA‐7867 was stable for up to 24h incubation in various buffer solutions having pHs from 1 to 11, Sørensen phosphate buffer of pH 7.4, and rat plasma, urine and liver homogenate and 3h incubation in five human gastric juices. The binding of DA‐7867 to 4% human serum albumin was 50.6% at DA‐7867 concentrations ranging from 0.5 to 20µg/ml. The equilibrium of DA‐7867 between plasma and blood cells of rabbit blood reached fast (within 30s manual mixing), and the plasma‐to‐blood cell concentration ratios were independent of initial blood concentrations of DA‐7867, 1–20µg/ml; the values ranged from 1.39 to 1.63. Protein binding of DA‐7867 in five fresh rats plasma was 72.3%. Copyright © 2004 John Wiley & Sons, Ltd.     

Binding Study of the Fluorescence Probe l-Anilino-8-naphthalenesulfonate to Human Plasma and Human and Bovine Serum Albumin Using Potentiometric Titration

The binding of l-anilino-8-naphthalenesulfonate (ANS) to bovine serum albumin (BSA), human serum albumin (HSA), and human plasma has been studied by potentiometric titration utilizing a laboratory constructed ion selective electrode (ISE) of ANS. Three classes of ANS binding sites were found on BSA, HSA, and plasma at 25 and 37°C. Computer analysis of the data resulted in estimates for the association constants, number of binding sites (HSA, BSA), and binding capacity of each class. The association constants for the first class of binding sites at 25°C were found to be 7.53 (±0.59) × 10⁵, 2.70 (±0.20) × 10⁵, and 2.64 (±0.26) × 10⁵ M ^–l for BSA, HSA, and plasma, respectively. Lower values for the association constants of all binding classes were estimated at the higher temperature (37°C). The binding capacity for ANS decreased in the order BSA, plasma, HSA.