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)     

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.     

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.     

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.     

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.     

The stereoselectivity of the 'single drug binding site' of human alpha 1-acid glycoprotein (orosomucoid)

The stereoselective binding of six pairs of basic, one pair of acidic drug enantiomers, and one pair of diastereomers for human alpha 1-acid glycoprotein was investigated by means of competition experiments against [3H]propranolol- or [14C]nicardipine-labelled binding sites using equilibrium dialysis to separate free from bound marker ligand. The affinity constants (Ka) for association of [3H]propranolol and [14C]nicardipine with alpha 1-AGP were 1.2 +/- 0.6 X 10(5) M-1 and 3.4 +/- 1.4 X 10(5) M-1, respectively, and control binding amounted to 57 +/- 7 and 91 +/- 2%, respectively. The following selectivity factors, calculated as the ratio of the higher over the lower enantiomer concentrations displacing 15% of control radiomarker binding (IC15-value), were obtained against propranolol and nicardipine: (-)/(+) propranolol: 1.9 and 1.7.; (+)-/(-)-disopyramide: 2.8 and 1.4; (+)-/(-)-verapamil: 1.6 and 1.9; (+)-(S)-/(-)-(R)-202-791, a dihydropyridine derivative: 2.6 and 2.0; (-)-/(+)-asocainol: 1.7 and 3.0; (+)-/(-)-tilidine: 1.1 and approximately equal to 2; (-)-(S)-/(+)-(R)- warfarin: 1.6 and 2.4; (+/-)-cis/(+/-)-trans-trans-tilidine: 1.7 and 1.8. When the calculation of radioligand-free fractions is also taken into account, it is apparent that only the tilidine isomers show no selectivity at propranolol-marked, and the disopyramide isomers at nicardipine-marked alpha 1-AGP-binding sites, in all other cases, a weak selectivity is detectable, which is, however, far below the values obtained for most neurotransmitter receptors. It is concluded that the single drug binding site of alpha 1-AGP is only slightly stereoselective and that the stereoselective binding of the drugs investigated is probably of no clinical consequence.     

Characterization of a binding site of UCN-01, a novel anticancer drug on alpha-acid glycoprotein

The binding site of 7-hydroxystaurosporine (UCN-01) on alpha-acid glycoprotein (AGP) was studied by fluorescence and ultracentrifugation experiments. Three ligands, propranolol, warfarin and progesterone were employed as marker ligands and quinaldine red was employed as a fluorescent probe. The presence of UCN-01, pro- pranolol, warfarin and progesterone resulted in a significant quenching of the fluorescence of quinaldine red, when bound to AGP, depending upon the potency of the binding to AGP. The construction of Klotz plots indicated that the displacement effects of propranolol, warfarin and progesterone on UCN-01-AGP binding were competitive in nature. These data suggest that the binding site of UCN-01 on the AGP partly overlaps the binding site for basic drugs, acidic drugs, as well as steroid hormones.     

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).     

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.     

Investigation of the binding of various tricyclic neuroleptics and antidepressants to alpha 1-acid glycoprotein

Recent studies have shown that the interaction of various tricyclic neuroleptics and antidepressants with isolated alpha 1-acid glycoprotein occurs at one common binding site and with relatively high association constants. The aim of the present study was to find differences in the binding of some phenothiazines, thioxanthenes, and several other drugs reported to bind alpha 1-AGP. The findings suggest that the affinity of the phenothiazines and thioxanthenes depends primarily on the existence of the tricyclic skeleton and is generally increased by the basic side chain. Substituents at position 3 of the phenothiazine nucleus influence the affinity in a variable way. The losses of radioactivity by non-specific absorptions to the dialysing chambers were considered for the calculation of the association constants. No correlation between association constants and the antipsychotic potency of neuroleptic drugs could be detected.     

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.     

Auramine O as a fluorescent probe for the binding of basic drugs to human alpha 1-acid glycoprotein (alpha 1-AG). The development of a simple fluorometric method for the determination of alpha 1-AG in human serum

A cationic fluorescent dye, auramine O (AO), exhibited an intense increase in fluorescence after binding to human alpha 1-acid glycoprotein (alpha 1-AG). The interaction between AO and the protein was studied by fluorescence spectroscopy and by equilibrium dialysis. AO binds to the protein via a single site with a dissociation constant of 24 microM. Various basic drugs such as chlorpromazine, imipramine, desipramine, quinidine, propranolol and lidocaine, which are known to bind to the protein, competitively inhibited the AO binding to the protein. The dissociation constants of these basic drugs obtained from such inhibitory experiments were comparable to those obtained with other methods (equilibrium dialysis, quenching of protein intrinsic fluorescence, and the difference spectrophotometric method) and from the literature. It is concluded that AO may be a useful fluorescent probe that binds to a single basic drug binding site on alpha 1-AG. In addition, a simple fluorometric method for the determination of alpha 1-AG in serum was developed using AO, and the validity of this method was confirmed by comparing it with the conventional radial immunodiffusion method.     

Binding of oxprenolol and propranolol to serum,albumin and α1-acid glycoprotein in man and other species

Species differences in binding of basic drugs have only occasionally been studied and we have therefore measured the binding of the β-adrenergic blockers oxprenolol and propranolol in (1) serum of healthy humans, dogs, rats and rabbits and of rabbits with experimental arthritis, (2) a solution of albumin of these species and (3) a solution of human α₁-AGP. In humans, dogs, rats and arthritic rabbits, binding of oxprenolol and propranolol was much higher in serum than in albumin solution; in healthy rabbits serum binding was very low and not different from albumin binding. For both drugs, concentration-dependency was seen in serum of dogs, humans and rats and of arthritic rabbits; a similar concentration-dependency was found for human α₁-AGP solution, but not for human albumin and for serum of healthy rabbits.Tris (2-butoxyethyl)-phosphate (TBEP), a known displacer of drugs from α₁-AGP in humans, decreased binding in serum of all species except the rabbit. For both β-blockers, species differences in capacity constants were found; species differences in affinity constants were present only for propranolol. These results suggest that in humans, dog and rat, but much less in rabbits, oxprenolol and propranolol bind mainly to α₁-AGP and that binding to α₁-AGP is more important for oxprenolol than for propranolol.     

Spectroscopic study of the interaction of coumarin anticoagulant drugs with polyvinylpyrrolidone

The interaction of coumarin anticoagulants with polyvinylpyrrolidone (PVP) was investigated using a fluorescence technique. The fluorescence intensities of warfarin and phenprocoumon were greatly enhanced following binding to PVP, while the fluorescence of 4-hydroxycoumarin was little enhanced in the presence of PVP. The enhanced fluorescence of warfarin and phenprocoumon bound to PVP can be explained by their incorporation into the hydrophobic environment in the PVP and by a decrease in the internal rotation of the alpha-substituted benzyl group in the drugs. The binding parameters of warfarin and phenprocoumon were estimated by the Klotz method; the binding constants for phenprocoumon and warfarin were found to be 2.6 X 10(4) and 2.2 X 10(4) M-1, respectively. The 13C-NMR measurements suggest the lactone moiety in the 4-hydroxycoumarin and the substituted benzene ring play an important role in the binding to PVP.     

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.     

In vitro binding study of gemfibrozil to human serum proteins and erythrocytes: interactions with other drugs

The binding of gemfibrozil to human serum, isolated proteins and erythrocytes was studied in vitro by equilibrium dialysis. Our results show that this drug is highly bound to serum (99%) at therapeutic levels. Human serum albumin was shown to be mainly responsible for this binding (98.6%) with a saturable process characterized by two binding sites with a moderate affinity. Like many acidic drugs with a carboxylic acidic group, gemfibrozil showed none or negligible binding to alpha 1 acid glycoprotein, lipoproteins and gamma-globulins. The drug binding to erythrocytes is very low (0.8%). The unbound fraction in blood remains constant (0.8%) within the range of therapeutic concentrations. Moreover, interactions were studied with bilirubin and palmitic acid at pathophysiological concentrations and acenocoumarol, salicylic acid, valproic acid, furosemide, phenylbutazone, tolbutamide, warfarin and sulfamethoxazol at therapeutic concentrations. Neither endogenous compounds nor the other drugs studied altered gemfibrozil binding in serum. Likewise, the binding of warfarin to serum and to human serum albumin (600 microM) is not influenced by gemfibrozil.     

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.     

Spectroscopic techniques in the study of protein binding. A fluorescence technique for the evaluation of the albumin binding and displacement of warfarin and warfarin-alcohol

1. The binding of racemic mixtures of warfarin and warfarin-alcohol to human serum albumin (HSA) is accompanied by an increase in the fluorescence quantum yield of these compounds. This property has been used to measure the characteristics of the binding of warfarin and warfarin-alcohol to HSA at 22 degrees C and 37 degrees C. Within the limits of the technique, no significant differences between the number of binding sites and strength of binding at the tight site at either temperature were observed. 2. The fluorescence of warfarin and warfarin-alcohol was used to label their binding site on HSA and to study the effects of other drugs on their binding. The results indicate that these two molecules are bound to the same site on HSA. 3. The validity of using changes in the fluorescence of warfarin as a measure of its displacement from HSA was investigated. Good correlations were observed between drug-induced decreases in the fluorescence of bound warfarin and displacement as measured by equilibrium dialysis. The displacement of warfarfin, as detected by fluorescence, correlates well with the increase in free warfarin resulting from addition of therapeutic drug concentrations to undiluted human serum. 4. The most potent displacing agents, by all the methods used, were iophenoxic acid, phenylbutazone and oxyphenylbutazone. The first of these is no longer used clinically, but the latter two are and have been reported to cause hypoprothrominaemia by displacing warfarin from HSA. The present study indicates that changes in the fluorescence of warfarin bound to HSA can be used to measure displacement of bound warfarin and to screen drugs that may cause clinically significant interactions by this mechanism.     

The interaction of enflurane, halothane and the halothane metabolite trifluoroacetic acid with the binding of acidic drugs to human serum albumin. An in vitro study

The interaction of the volatile anaesthetics enflurane, halothane and the halothane metabolite trifluoroacetic acid with the binding of two highly bound acidic drugs (warfarin, phenytoin) to albumin has been studied in vitro by equilibrium dialysis. Trifluoroacetic acid (TFA) inhibited the binding of both drugs to human serum albumin (HSA). Halothane, on the other hand, increased the binding of warfarin to HSA, while enflurane inhibited only the binding of phenytoin. It seems that the binding of the acidic drugs warfarin and phenytoin to HSA is more sensitive to the structures of the gases than for the basic drug diazepam which was previously shown to be equally affected by both gases. Furthermore, it seems that drugs competing for the same binding site (warfarin, phenytoin) may respond differently to conformational changes of the site. It is suggested that drugs bound to the "diazepam site" are more easily affected by the volatile anaesthetics than drugs bound to the "warfarin site".     

Selective binding of tamsulosin to genetic variants of human alpha1-acid glycoprotein

We investigated the characteristics of binding of tamsulosin to alpha(1)-acid glycoprotein (AGP) genetic variants. The binding of tamsulosin to each of the human AGP variants was determined by ultrafiltration, and the binding characteristics for each variant were compared using binding parameters and inhibition of the binding by disopyramide and warfarin. The affinities of tamsulosin binding to a F1/S variant mixture and total AGP variants were relatively high (dissociation constants 1.6 microM). On the other hand, the dissociation constant for variant A was 14.9+/-2.53 microM. The binding of tamsulosin was competitively inhibited by warfarin but not by disopyramide. Tamsulosin appears to be a suitable compound for studying the characteristics of drug binding to human AGP F1/S variants under clinical conditions.