Multiple human serum binding of two thienopyridinic derivatives,ticlopidine and PCR 2362, and their distribution between HSA, α1-acid glycoprotein and lipoproteins

The binding of two drugs, ticlopidine and PCR 2362, chemically related to thienopyridin, potent antiaggregant agents, was studied in vitro to serum and to the corresponding isolated proteins, HSA, α₁-AGP, VLDL, LDL and HDL, using equilibrium dialysis at pH 7.4 and 37°. The binding of these drugs to HSA and lipoproteins was non-saturable. The binding capacity of the lipoproteins was much greater than that of HSA and appeared to be dependent on lipid content. The binding capacities of the apoproteins were less than 10% of that observed for the native lipoproteins suggesting that drug-lipoprotein binding involves drug solubilisation in the lipid phase of lipoproteins rather than a classical binding to definite sites. However drug binding to α₁-AGP was saturable with n = 3 for both and K = 89,000 and 33,000 for ticlopidine and PCR 2362, respectively. At physiological concentration, α₁-AGP binding capacity represented 15% of total serum binding capacity which could double in pathological states, in which the level of this protein is increased.     

Differences in the serum protein binding of prazosin in man and rat

The serum protein binding of prazosin in man and rat has been studied in vitro by equilibrium dialysis. Prazosin was more extensively bound in human serum than in rat serum with binding ratios (B/F) of 14.3 +/- 3.4 and 4.4 +/- 0.2 (corresponding to 93.4 and 81.4% bound), respectively. This difference in binding between the species was partly due to qualitative differences between human and rat serum albumin, but also to the lower concentration of albumin in rat serum. Rat serum albumin (RSA) apparently showed two different classes of binding sites for prazosin, one with high (KD = 5.78 X 10(-6) M) and one with low (KD = 1.1 X 10(-4) M) affinity; the former is suggested as representing alpha 1-acid glycoprotein (alpha 1-AGP) with one binding site for prazosin per molecule, the latter as representing RSA with 0.28 binding sites per molecule. Human serum albumin (HSA) and human alpha 1-AGP both showed one class of binding sites with KD values of 2.7 X 10(-5) and 1.95 X 10(-6) M, respectively. HSA possessed 0.5 and human alpha 1-AGP 1 binding site for prazosin per molecule. The binding parameters obtained for the isolated serum proteins overestimated to some degree the total serum protein binding of prazosin in man. This was explained by a specific deviation from the law of mass action. HSA was the major binding protein in human serum at therapeutic concentrations, with ca. 60% of the total binding, the remaining 40% being bound to alpha 1-AGP. Anticipating that the high affinity binding site on the RSA preparation represents the binding of prazosin to alpha 1-AGP, then this protein accounts for 70% of the binding in rat serum, while rat serum albumin accounts for approximately 23%. The binding of prazosin to lipoproteins was insignificant in both species. The observed differences between man and rat in the serum protein binding of prazosin implicate differences in the two species with respect to prazosin pharmacokinetics and the pharmacological effect.     

Prazosin binding to human alpha 1-acid glycoprotein (orosomucoid), human serum albumin, and human serum. Further characterization of the 'single drug binding site' of orosomucoid

The plasma protein binding of the alpha 1-adrenergic blocking agent prazosin was investigated by means of circular dichroism (CD) and equilibrium dialysis (ED) measurements. The interaction of prazosin with human alpha 1-acid glycoprotein (alpha 1-AGP) results in pronounced negative extrinsic Cotton effects at 255 nm and a smaller negative band at 285 nm which are associated with the binding of prazosin to only one site of the protein. Various basic drugs, and warfarin also, at 50 microM displace prazosin 10 microM from its binding site on alpha 1-AGP and reduce the CD-spectra at 255 nm by 26% (disopyramide), 52% (mepivacaine), about 70% (verapamil, biperiden), and 90-100% (trihexyphenidyl, warfarin). (+/-)-Propranolol reduces the CD-spectra by 76%, its (-)-isomer by 89%, and the (+)-isomer by 65%. ED experiments indicated that the binding of prazosin to alpha 1-AGP is saturable with an association constant of 48 000 M-1 and 0.85 binding sites per protein molecule. Displacement of prazosin from alpha 1-AGP by the same drug as used for the CD experiments at displacer/prazosin ratios of 5 resulted in comparable reductions of the fraction bound as obtained by the CD experiments. Prazosin was also highly bound to human serum albumin (600 microM) with about 80-85% bound at prazosin concentrations from 1-100 microM. Since prazosin binding to human serum is only slightly higher (80-90%) it is concluded that prazosin binding in serum is largely mediated by the albumin fraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparative study of the interaction of warfarin with human alpha 1-acid glycoprotein and human albumin

The interaction of warfarin with human alpha 1-acid glycoprotein (alpha 1-AGP) and human albumin (HA) has been investigated using fluorescence and circular dichroism techniques. The fluorescence of warfarin is greatly enhanced following binding to alpha 1-AGP or HA, the binding constant for a single site being estimated by the Scatchard method. The binding constants for the two serum proteins are similar, but the thermodynamic parameters differ. The binding constants increase as the pH is raised to 9.0. Various basic drugs, such as chlorpromazine, propranolol and imipramine, markedly inhibited the binding of warfarin to alpha 1-AGP. But, some acidic drugs, including phenylbutazone, effectively displaced warfarin bound to HA. The difference in CD spectra observed for alpha 1-AGP and HA indicated that the drug-binding sites of the two proteins might have different asymmetries. It thus appears that the mode of interaction of warfarin with the two proteins differs.     

The interaction of human and bovine serum proteins with CYP3A in human liver microsomes: inhibition of testosterone 6beta-hydroxylation by albumin,alpha-globulins,alpha(1)-acid glycoprotein and gamma-globulins

The effects of human and bovine serum proteins on CYP3A activity, using testosterone as the probe substrate, were investigated in human liver microsomes. Serum albumin, alpha-globulins, and alpha(1)-acid glycoprotein (alpha(1)-AGP) of both species significantly inhibited testosterone 6beta-hydroxylation. When the inhibitory effects of serum proteins were compared with serum protein binding data, human alpha-globulins, with a ratio (relative metabolic activity/unbound fraction) of 0.3, showed higher, and bovine alpha(1)-AGP, with the ratio of 1.4, showed lower inhibitory effects than those expected from protein binding of testosterone. The effects of the other serum proteins were close to those expected from protein binding, according to the free drug hypothesis. The K(i) values obtained from the Dixon plots were 0.32% (w/v, 48 microM) for human serum albumin (HSA), 0.48% for human alpha-globulins, and 0.23% (52 microM) for human alpha(1)-AGP. K(i) values of bovine serum albumin, bovine alpha-globulins and bovine alpha(1)-AGP were 3-5 times higher than those of the respective human proteins. The results suggest a direct interaction of some of these serum proteins with the active site of the CYP3A isoform. Since the bovine serum proteins showed weaker inhibitory effects than human serum proteins, the wide use of BSA, which is viewed as interchangeable with HSA, needs to be cautioned.     

Assessment of the contribution of alpha 1-acid glycoprotein to the serum binding of basic drugs using serum treated with sulphosalicylic acid and DEAE-cellulose

Treatment of human serum with DEAE-cellulose in acid conditions almost completely removed alpha 1-acid glycoprotein (alpha 1-AG) with little change in the concentration of albumin and beta-lipoprotein, while treatment with sulphosalicylic acid removed almost all the proteins except alpha 1-AG. The binding of various drugs to serum treated as above was measured by equilibrium dialysis and the contribution of alpha 1-AG to drug binding by human serum was assessed. Sulphosalicylic acid-treated serum exhibited a saturable binding for propranolol, which was considered to be due to the binding to alpha 1-AG while DEAE-cellulose-treated serum mostly exhibited non-saturable binding, for which albumin and beta-lipoprotein may be responsible. With this treated serum, alpha 1-AG was estimated to contribute approximately 40% to the binding of therapeutic concentrations of propranolol, 15% to that of imipramine and 15-20% to that of desipramine, respectively, in serum samples pooled from healthy adults. However, no contribution of alpha 1-AG was observed in the binding of salicylic acid to the serum. Dissociation constants of the propranolol binding to the high affinity site in control serum, sulphosalicylic acid-treated serum and purified alpha 1-AG showed similar values (3.7-6.7 microM). These results suggest that treatment of serum with sulphosalicylic acid and DEAE cellulose is useful in assessing the contribution of alpha 1-AG to the serum binding of basic drugs.     

The binding protein of erythromycin in human serum

Erythromycin binding to human serum albumin and to alpha 1-acid glycoprotein was measured under conditions of binding equilibrium. At therapeutical concentrations of erythromycin the binding to albumin is not saturable. The fraction of total erythromycin bound to alpha 1-acid glycoprotein is proportionally related to the protein concentration and is bound to a single class of binding sites with an apparent association constant Ka = 0.16 X 10(6) M-1 (38 degrees). About one mole of erythromycin is bound per mole of alpha 1-acid glycoprotein. The binding affinity can be enhanced and vice versa lowered by increasing the concentrations of NaCl and urea, respectively. The semilogarithmic plot of bound/free ratios vs log concentration of NaCl or urea exhibits linear relationships. Erythromycin binding can be competitively inhibited by mersalyl (Ki = 11-16 microM) but not by other SH-reagents or by neuraminidase treatment. A marked reduction of erythromycin binding to alpha 1-acid glycoprotein is seen with dithiothreitol. alpha 1-acid glycoprotein is the main erythromycin binding protein in human serum.     

Inverse stereoselectivity in the binding of acenocoumarol to human serum albumin and to alpha 1-acid glycoprotein

Stereoselective binding of rac-acenocoumarol to human serum albumin (HSA) and alpha 1-acid glycoprotein (alpha 1-AGP) was investigated by affinity chromatography and by combined ultrafiltration (UF) and circular dichroism (CD) methods. For HSA, the ratio of the enantiomeric constants was KR/KS = 2, while for alpha 1-AGP, KS/KR = 3.     

Fluorescent investigations of binding of phenprocoumon to alpha 1-acid glycoprotein

The fluorescence of phenprocoumon is greatly enhanced on binding to a single site on alpha 1-acid glycoprotein (alpha 1-AGP). Advantage is taken of this phenomenon to estimate a binding constant for the binding of phenprocoumon to alpha 1-AGP. The fluorescence intensity and binding constant of the phenprocoumon: alpha 1-AGP complex decreased with pH from 6.5 to 8.5, suggesting that phenprocoumon binding to alpha 1-AGP is significantly affected by microenvironmental change in alpha 1-AGP. A variety of drugs, including chlorpromazine and dicumarol, significantly inhibited phenprocoumon binding to alpha 1-AGP. Fatty acids seem to displace phenprocoumon from its binding site on alpha 1-AGP, whereas the addition of neutral salts and sialic acid did not cause the displacement of phenprocoumon. It is concluded that the phenprocoumon binding site is located in the hydrophobic protein structure of alpha 1-AGP.     

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.     

Interaction of plasma proteins with cytochromes P450 mediated metabolic reactions: inhibition by human serum albumin and alpha-globulins of the debrisoquine 4-hydroxylation (CYP2D) in liver microsomes of human, hamster and rat

The effect of human serum albumin (HSA), alpha1-acid glycoprotein (alpha1-AGP), and alpha- and gamma-globulins on the in vitro metabolism of debrisoquine in human, hamster and rat liver microsomes was studied. Interaction of albumin with cytochrome P450 mediated phenytoin metabolism has been reported. Since plasma protein binding of phenytoin is high, in the present study a weakly protein bound drug, debrisoquine, was studied. Debrisoquine is a substrate of CYP2D6. The debrisoquine 4-hydroxylation was measured using a radio-TLC method. Among the four plasma proteins, alpha-globulins had the strongest inhibitory effect on the debrisoquine 4-hydroxylase activity. The inhibition with 2% alpha-globulins was 42+/-18% for human and higher for hamster and rat liver microsomes (65-71%). HSA had less effect than alpha-globulins. In the presence of HSA, the decrease in activity was between 18 and 35% for all liver microsomes studied. The debrisoquine 4-hydroxylase activity was not significantly changed by alpha1-AGP or gamma-globulins. Using an ultra-filtration method, the protein binding of debrisoquine to 4% HSA, 0.5% alpha1-AGP, 2% alpha-globulins and 2% gamma-globulins was found to be 22, 20, 22 and 5%, respectively. Since the observed inhibition is inconsistent with level of protein binding, it appears, particularly in the case of alpha-globulins, that the plasma proteins interact with CYP2D directly.     

The binding of amsacrine to human plasma proteins

Determination of amsacrine plasma protein binding by both equilibrium dialysis and ultracentrifugation gave similar results and indicated that amsacrine is highly bound (approximately 97%) in human plasma. This binding is independent of amsacrine concentration over the range 1-100 mumol litre-1, but is very sensitive to plasma pH and, to a lesser extent, to temperature. Approximately 20% of the drug appeared to be covalently bound to plasma proteins. Amsacrine was bound by all plasma proteins investigated including albumin, alpha 1-acid glycoprotein and various gamma-globulins. The binding to albumin appeared to occur by two processes, a saturable process at a single site with a KD of 13.9 mumol litre-1 and a non-saturable process. Despite differences in individual protein concentrations, no significant difference was observed in the unbound amsacrine fraction in plasma from patients receiving this drug for treatment of acute myelogenous leukaemia and plasma from healthy individuals.     

Stereoselective binding of propranolol enantiomers to human alpha 1-acid glycoprotein and human plasma.

The binding of propranolol enantiomers to human albumin (ALB), alpha 1-acid glycoprotein (alpha 1-AGP) and plasma was studied. (-) propranolol is more bound than (+)propranolol to alpha 1-AGP (P less than 0.001) and to plasma (P less than 0.05). In solutions containing ALB at a constant concentration (580 mumol/l) and alpha 1-AGP at increasing concentrations, the binding of both isomers increases but the stereo selectivity is evident throughout the alpha 1-AGP concentration range examined (25-100 mumol/l).     

Decrease in penbutolol central response as a cause of changes in its serum protein binding

Penbutolol is a beta-adrenoceptor antagonist that is extensively bound to alpha 1-acid glycoprotein (alpha 1-AGP), a protein that increases in inflammatory diseases thereby binding more drug in such conditions. Changes in serum binding can lead to modifications in the pharmacokinetics and pharmacodynamics of a drug, therefore, the central effect (as the anticonvulsant response) and brain uptake of penbutolol given intravenously to mice with experimental inflammation have been measured. A significant decrease of the central effect of penbutolol and its brain uptake was seen in diseased when compared with control animals (P less than 0.01). A parallel decrease in free fraction of penbutolol in diseased vs normal animals was detected. These results suggest that there is an increase in serum binding of basic drugs related to increments in alpha 1-AGP concentration, which reduces their central pharmacological effect.     

Distribution of moricizine in human blood: binding to plasma proteins and erythrocytes.

Using equilibrium dialysis and incubation experiments, we determined the binding of moricizine to human plasma, isolated plasma proteins, and erythrocytes. The mean (% +/- SD) plasma protein binding at various moricizine concentrations ranged from 81.2 +/- 2.1 to 89.9 +/- 2.1%. There was no apparent relationship between drug concentration and extent of binding in pooled plasma over the concentration range tested. However, protein concentration-dependent binding was observed with albumin and alpha 1-acid glycoprotein (alpha 1-AGP). The unbound fraction of moricizine fell from 61 to 19% and from 70 to 17% with increasing albumin (5 and 50 g/L, respectively) and alpha 1-AGP (0.2 and 1.2 g/L) concentrations. The binding of moricizine to beta-lipoprotein (5 g/L) was 70.6 +/- 3.1% and to gamma-globulin (12 g/L) was 13.6 +/- 3.3%. Moricizine partitioned into erythrocytes, showing an erythrocyte/plasma drug concentration ratio of 1.325 +/- 0.070 and erythrocyte/buffer ratio of 8.561 +/- 0.620. An estimation could be made that 57% of total drug in whole blood was associated with erythrocytes, 39% bound to plasma proteins, and 4% was free. The results of this study demonstrated that erythrocytes, albumin, and alpha 1-AGP were the major binding components in blood.     

The uptake of cardiac glycosides in relation to their actions in isolated cardiac muscle

1. The uptake of (3)H-digitoxin, (3)H-ouabain and (3)H-dihydro-ouabain by isolated guinea-pig atria has been studied and compared with the inhibition of the sodium pump and with the inotropic effect.2. Analysis of the curve relating the uptake of digitoxin and ouabain at equilibrium to the bath concentration enabled a non-saturable and a saturable binding site to be distinguished.3. The uptake of inactive doses of dihydro-ouabain was only by a non-saturable mechanism.4. The uptake of labelled digitoxin and ouabain was reduced in the presence of another glycoside. The amount of bound glycoside was nearly equivalent to the estimated non-saturable uptake.5. The uptake was reduced at 4 degrees C to the clearance of the non-saturable site.6. ED50 of digitoxin and of ouabain for inhibition of the sodium pump were measured and compared to the ED50 for inotropic effect and to the concentrations producing a half-saturation of the saturable binding site.7. It is concluded that binding to the saturable site may be responsible for the cardiac actions of the glycosides.     

Protein binding of chloroquine enantiomers and desethylchloroquine.

The protein binding of racemic chloroquine, its enantiomers and desethylchloroquine to plasma, purified human albumin, and alpha 1-acid glycoprotein (alpha 1-AGP) was determined by equilibrium dialysis. The binding was not concentration dependent. (+)-Chloroquine bound more to plasma (66.6 +/- 1.9%) and albumin (45.9 +/- 0.8%) than (-)-chloroquine (48.5 +/- 2.4% and 35.3 +/- 0.6%, respectively). These differences were statistically significant. (-)-Chloroquine bound more to alpha 1-AGP (47.5 +/- 0.7%) than (+)-chloroquine (34.5 +/- 0.5%). The binding of desethylchloroquine to alpha 1-AGP is higher than to albumin (38.9 +/- 0.9% and 21.1 +/- 0.4%, respectively.     

Binding of indapamide to serum proteins and erythrocytes

The binding of indapamide to isolated serum proteins and erythrocytes was studied in order to understand its blood distribution. In serum, indapamide was mainly bound to alpha 1-acid glycoprotein with a high affinity (K = 73.4/mM), and to albumin and lipoproteins. Indapamide was bound to erythrocytes via a saturable process with a high affinity (K = 385/mM and N = 57 microM for an hematocrit value of 0.48), and erythrocytes were the main binding component in blood (more than 80% of indapamide was associated to erythrocytes in blood). The binding to serum proteins affected indapamide distribution in blood, and alpha 1-acid glycoprotein was shown to be the more effective protein in decreasing the amount of indapamide associated to erythrocytes.     

Multiple binding of bepridil in human blood

Using radiolabelled bepridil, equilibrium dialysis experiments and direct ligand-binding studies were conducted to characterize the binding of bepridil to some human serum proteins, and to blood cells and platelets, respectively. Bepridil was bound to serum albumin with K = 26 +/- 1.6 X 10(3) mol-1 X 1, n = 1.07 +/- 0.04; to alpha 1-acid glycoprotein with K = 442 +/- 68 X 10(3) mol-1 X 1, n = 0.90 +/- 0.04; and with a lesser extent to lipoproteins and gamma-globulins. Bepridil, propranolol, quinidine and erythromycin were found to share the same site on alpha 1-acid glycoprotein. The binding of bepridil to both RBC and WBC was nonspecific with the following parameters: NK = 10.55 +/- 0.10 per 5 X 10(6) RBC/mm3 and NK = 0.34 +/- 0.01 per 10(3) WBC/mm3, whereas the binding to platelets was saturable with N = 10.97 +/- 1.06 mumol/l per 108 +/- 10(3) platelets/mm3 and K = 40 +/- 8 X 10(3) mol-1 X 1. The binding parameters were used to simulate the distribution of bepridil between serum proteins, blood cells and platelets over the therapeutic range and showed that serum albumin, alpha 1-acid glycoprotein and RBC were the major binding components.     

Binding of minaprine to human serum proteins and erythrocytes

The binding of minaprine to human serum, isolated proteins and erythrocytes was studied in vitro. This drug shows both a saturable and a nonsaturable binding process in serum. The drug is bound to alpha 1-acid glycoprotein in a saturable fashion with a moderate affinity whereas albumin is involved in the nonsaturable and major binding. The combination of these two processes results in a slight decrease (7%) in the drug binding to serum within the range of therapeutic concentrations. Competitive binding with metabolites of minaprine or with other basic drugs seems unlikely when these compounds are present in serum at therapeutic levels. The binding of minaprine to erythrocytes remains constant within the range of therapeutic concentrations.