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.     

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)     

Binding of [3H]mianserin to bovine serum albumin,human serum albumin and α1-acid glycoprotein

Scatchard plots which were curvilinear with negative slopes were obtained when the binding of [³H]mianserin to bovine serum albumin (BSA), human serum albumin (HSA), defatted human serum albumin (D-HSA) and α₁-acid glycoprotein (α₁-AGP) was studied with equilibrium dialysis with constant protein concentrations and various ligand concentrations. Binding parameters were estimated graphically and with a non-linear least-squares computer program, assuming two classes of independent binding sites. α₁-AGP had the highest binding affinity (K) and binding capacity (nK). The binding parameters, n and K were not independent of protein concentration when the BSA concentration was varied. Linear atypical Scatchard plots with positive slopes were obtained when the protein concentration was varied for BSA, HSA and D-HSA, at a fixed ligand concentration.     

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.     

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.     

Binding of vinca alkaloid analogues to human serum albumin and to alpha 1-acid glycoprotein

The binding of a series of vinca alkaloid analogues having eburnane or indolo[2,3-a]quinolizidine skeletons was studied with human serum albumin (HSA) by affinity chromatography and with alpha 1-acid glycoprotein by means of competition experiments. On HSA the binding occurs at the benzodiazepine-indole binding site via hydrophobic interaction and shows slight stereoselectivity preferring the trans isomers. The binding to alpha 1-AGP proved to be highly stereoselective in favour of the trans isomers having 3(S),16(R)eburnane or 1(R),12b(S)indolo[2,3-a]quinolizidine absolute configurations.     

The binding characteristics of some adrenergic beta-receptor antagonists to human serum proteins

Binding of pindolol and 8 related compounds was studied in vitro by equilibrium dialysis. The overall binding in serum was compared with the binding to the main, isolated, serum proteins. Most substances show both saturable and non-saturable binding in serum. The saturable and main binding is to alpha 1-AGP, the low non-saturable binding corresponds to albumin and lipoprotein binding. The binding to alpha 1-AGP is characterized by approximately one binding site and association constants K ranging from 10(4) to 10(6) M-1. The binding of pindolol to alpha 1-AGP is strongly inhibited by propranolol, lidocaine, erythromycin, imipramine and TBEP. Significant correlations were found between log NK and log partition coefficient octanol-phosphate buffer suggesting that the protein binding of the 9 adrenergic beta-receptor antagonists to all serum proteins, including alpha 1-AGP, is predominantly hydrophobic in nature.     

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.     

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 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.     

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).     

Comparative study of interaction mode of diazepines with human serum albumin and alpha 1-acid glycoprotein.

The binding of nine diazepines to human serum albumin (HSA) and to alpha 1-acid glycoprotein (AGP) was investigated by means of fluorescence and circular dichroism (CD) spectroscopies. The binding parameters of diazepam obtained from fluorescence agreed with those obtained from CD measurements. Diazepines have one tight binding site on both HSA and AGP. The binding parameters (nK) of the diazepine: HSA systems are slightly higher than those of diazepine:AGP systems. The relationship between the binding parameters for these two serum proteins and the physicochemical parameters of diazepines was studied by multiple regression analysis to elucidate the binding mode. Moreover, the effects of long-chain fatty acids and cesium chloride on the binding of diazepines to HSA and AGP were also studied. The driving force for the binding of diazepines to both proteins appears to be hydrophobic interaction. In addition, steric effects and electrostatic interactions may also contribute to the binding of diazepines to HSA and AGP, respectively.     

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.     

Plasma protein binding of the reversible type A MAO inhibitor cimoxatone (MD 780515)

Binding of a new selective reversible type A MAO inhibitor cimoxatone (MD 780515) to plasma proteins was studied in vitro by equilibrium dialysis. Binding to 580 microM human serum albumin (HSA) and to total plasma proteins was 93-96% and independent of cimoxatone concentration (0.15-207 microM). The drug was mainly bound to HSA with two binding sites and a moderate association constant (K = 2.9 X 10(4) M-1). Free fatty acids did not modify cimoxatone binding to HSA. Cimoxatone was also moderately bound to isolated lipoprotein fractions; alpha 1-acid glycoprotein and gamma-globulins did not play an important role in the binding of cimoxatone. MD 770222, the O-demethyl metabolite, appeared to be bound to HSA at the same binding sites as cimoxatone. However, no interaction occurred between the two compounds for 580 microM HSA. L-Tryptophan, bilirubin, the benzodiazepines flunitrazepam and oxazepam, imipramine and aspirin, did not displace cimoxatone from its binding sites. On the other hand, warfarin and phenylbutazone decreased cimoxatone binding to 29 microM HSA but no interaction occurred with 580 microM HSA.     

Biosensor analysis of the interaction between immobilized human serum albumin and drug compounds for prediction of human serum albumin binding levels

The interactions between a set of drugs, selected on the basis of reported human serum albumin (HSA) binding levels, and immobilized HSA were investigated using surface plasmon resonance technology. Major HSA binding sites were available after immobilization. The intensity of the signal obtained from the interaction of the drug with the HSA surface was correlated with the reported HSA binding level. Drugs were classified into groups corresponding to high, medium, or low HSA binding based on the injection of the drug at 80 microM concentration. A set of 10 drugs binding to alpha(1)-acid glycoprotein (AGP) was also investigated and correlated with reported AGP binding data. The throughput of the presented assay is 100 compounds/24 h, and the sample consumption is less than 100 microL (8 nmol).     

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.     

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.     

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

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

In vitro behaviour of sesquiterpene lactones and sesquiterpene lactone-containing plant preparations in human blood, plasma and human serum albumin solutions

The interactions of the three sesquiterpene lactones (SLs) dihydrohelenalin acetate, dihydrohelenalin methacrylate and helenalin isobutyrate from Arnica montana and of parthenolide from Tanacetum parthenium as well as of three ethanolic Arnica preparations with human blood proteins using different matrices, human serum albumin (HSA), plasma and whole blood, were investigated. The extent of protein binding in human plasma or to human albumin differed significantly between individual SLs (dihydrohelenalin methacrylate < dihydrohelenalin acetate < helenalin isobutyrate < parthenolide), e. g., 30 % to 50 % of the SLs were bound to plasma. On the whole, SLs in the ethanolic preparations showed a lower degree of protein binding. Interestingly, although HSA has a reactive thiol group at its cysteine-34 position which is prone to react with the alpha,beta-unsaturated carbonyl of SLs, our studies showed that less than 6 % of SLs are bound to this position within 60 minutes. Thus, a reaction with other amino acids as well as non-covalent interactions with plasma proteins have to be considered. Considering the biological activity of SLs in whole blood, our studies demonstrate that knowledge of their plasma protein binding is important for interpreting the reported data of in vitro and ex vivo assays.