Modification of the ultrafiltration technique to overcome solubility and non-specific binding challenges associated with the measurement of plasma protein binding of corticosteroids

Plasma protein binding (PPB) methodology suitable for application in the lead optimisation of a corticosteroid series known to demonstrate non-specific binding (NSB) and poor solubility has been established. The method involved a modification to standard ultrafiltration (UF) techniques. In parallel with each experimental plasma sample, a control plasma sample was also processed by ultrafiltration. The retentate from experimental and control plasma samples were mixed back into the filtrate of the partner sample. The resulting regenerated plasma samples, one representing the experimental filtrate and one representing the experimental retentate, were then analysed by LC/MS/MS. Varying degrees of NSB were demonstrated with a number of corticosteroids, and this effect was eliminated using the modified method. Validation using a panel of established corticosteroids showed good agreement with published PPB figures. The published PPB figure for fluticasone propionate (FP) was, however, found to be an underestimate, and this was subsequently confirmed, at clinically relevant plasma concentrations, to be 99.3%. The modified method was particularly suited to lead optimisation because it provided samples in a consistent matrix compatible with standard high throughput LC/MS/MS analysis.     

Identification of the substrates for plasma hyaluronan binding protein

Plasma hyaluronan biding protein (PHBP) is a novel serine protease, which has an amino acid sequence homology to that of hepatocyte growth factor activator (HGFA), and has a similar domain structure to that of urinary plasminogen activator (u-PA), found in human plasma. We searched the PHBP substrate in human plasma by measuring the digested protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that fibrinogen and fibronectin were the major substrates of PHBP. PHBP cleaved the alpha-chain at multiple sites and the beta-chain between lysine53 and lysine54 but not the gamma-chain of fibrinogen. Therefore, PHBP did not initiate the formation of the fibrin clot and did not cause the fibrinolysis directly. PHBP did not cleave (activate) prothrombin and plasminogen, but it converted the inactive single chain urinary plasminogen activator to the active two chain form.     

应用蛋白质芯片技术检测血清自身抗体的初步探讨

目的研究应用蛋白质与抗原分子微阵列技术检测血清自身抗体。方法运用胶体金免疫金银染色法在氧化型琼脂糖凝胶膜结合戊二醛(GA)分子衍生的活性表面,制作蛋白质与抗原分子微阵列,建立检测血清自身抗体的方法。结果在未经选择的自身免疫病患者中[包括系统性红斑狼疮(SLE)、类风湿关节炎(RA)和混合性结缔组织病(MCTD)],共检出有多种自身抗体阳性,其中抗ssDNA抗体、抗甲状腺球蛋白(TG)抗体、抗甲状腺过氧化切酶(TPO)抗体、子宫内膜抗体(EmAb)和类风湿因子(RF)等的检出频率较高;但心磷脂抗体(ACA)、抗肝细胞膜特异性脂蛋白(LSP)和抗精子抗体(AsAb)未检出1项阳性,经初步的方法学对比考证,与酶联免疫吸附试验(ELISA)测试结果有较好的重复性(结果一致性达90%)。结论本法敏感度高,结果直观且实验条件易于控制,为阵列芯片的可视化提供了一种很好的模式。     

Predicting plasma protein binding of drugs--revisited

Plasma protein binding of drugs has been studied for almost 100 years, but despite the accumulation of large amounts of data, the accurate prediction of this ADME parameter continues to be problematic. This review outlines recent efforts on the development of prediction tools for plasma protein binding of drugs, specifically human serum albumin, in the context of its relevance and its influencing factors. The issue of why it is difficult to achieve prediction of sufficient quality for a diverse dataset will also be considered.     

Importance of assay specificity for plasma protein binding determinations

Plasma or serum protein binding determinations of extensively bound drugs are subject to serious error if the analytical methods employed do not discriminate between such drugs and their degradation products, metabolites, and impurities. Experimental evidence is presented to demonstrate the magnitude of this problem with respect to warfarin.Supported in part by Grant GM20852 from the National Institutes of Health.This is paper XVI in the series Comparative pharmacokinetics of coumarin anticoagulants. Previous paper: A. Yacobi, C.-M. Lai, and G. Levy,J. Pharm. Sci. 64 (1975).     

Clopidogrel variability: role of plasma protein binding alterations

Aim

The large inter-individual variability in clopidogrel response is attributed to pharmacokinetics. Although, it has been used since the late 1990s the pharmacokinetic fate of clopidogrel and its metabolites are poorly explained. The variable response to clopidogrel is believed to be multi-factorial, caused both by genetic and non-genetic factors. In this study, we examined whether the inactive metabolite can alter the plasma protein binding of the active metabolite, thus explaining the large inter-individual variability associated with clopidogrel response.

Methods

Female subjects (n = 28) with stable coronary disease who were not taking clopidogrel were recruited. Serial blood samples were collected following 300 mg oral dose of clopidogrel, plasma was isolated and quantified for total and free concentrations of active and inactive metabolites. Inhibition of platelet aggregation was measured using the phosphorylated vasodilator stimulated phosphoprotein (VASP) assay.

Results

A significant correlation was observed between VASP and both free (r = 0.49, P < 0.05) and total (r = 0.49, P < 0.05) concentrations of the active metabolite. Surprisingly, we observed a significant correlation with both free (r = 0.42, P < 0.05) and total (r = 0.67, P < 0.001) concentrations of the inactive metabolite as well. Free fractions of the active metabolite rose with increasing protein binding of the inactive metabolite (P < 0.05).

Conclusions

The above in vivo data suggest that the inactive metabolite displaces the active metabolite from binding sites. Thus, the inactive metabolite might increase the free concentration of the active metabolite leading to enhanced inhibition of platelet aggregation. The plasma protein binding mechanism would offer an additional therapeutic strategy to optimize clopidogrel pharmacotherapy.     

Drug-protein interaction: plasma protein binding of furocoumarins.

The binding of six furocoumarins (angelicin, psoralen, 8-methoxypsoralen, 5-methoxypsoralen, 8-methylpsoralen, 4,5'8-trimethylpsoralen) to human serum and human serum albumin was studied by equilibrium dialysis using tritium labelled compounds. The results indicated that in serum all furocoumarins are bound mostly by albumin, the extent of binding being related to the structure of the furocoumarins; at any rate, high values of the bound drug, ranging from 84 to 97% were observed. The percentage of binding is strictly related to the water solubility of the compounds. A limited number of binding sites, n = 1-2.4, were detected in the albumin molecule, indicating a high specificity in the binding process. The association constants of the furocoumarins to albumin. Ka, ranged from 1.2 X 10(4) M-1 (8-methoxypsoralen) to 1.9 X 10(5) M-1 (4,5'8-trimethylpsoralen).     

Stereoselective plasma protein binding of ibuprofen enantiomers

Summary We have developed a novel and reproducible method for determining the plasma protein binding of the two ibuprofen enantiomers in the presence of each other. The method involves the use of radiolabelled racemic ibuprofen, equilibrium dialysis, derivatization of the enantiomers to diastereomeric amides, high-performance liquid chromatography, and radiochemical analysis.We have determined the plasma protein binding of R(–)- and S(+)-ibuprofen in 6 healthy male volunteers after the oral administration of 800 mg racemic ibuprofen.The mean time-averaged percentage unbound of the R(–)-enantiomer, 0.419 was significantly less than that of the S(+)-enantiomer, 0.643, consistent with stereoselective plasma protein binding.The percentage unbound of each ibuprofen enantiomer was concentration-dependent over the therapeutic concentration range and was influenced by the presence of its optical antipode.     

Prediction of plasma protein binding displacement and its implications for quantitative assessment of metabolic drug-drug interactions from in vitro data

Although displacement from plasma protein binding (dPB) is usually of little clinical significance, it should be taken into account when interpreting changes in total plasma concentrations of drugs subject to metabolically based drug-drug interactions (mDDI). The aim of this study was to develop an approach to predict changes in the free fractions (fu) of pairs of drugs that compete for plasma binding, knowing their binding affinity constants, and to consider the implications of associated concentration- and time-dependence of such changes with respect to drug exposure. Experimental fu values of valproic acid and phenytoin in the presence of ibuprofen, diflunisal, or naproxen were predicted successfully (within 0.99- to 1.36-fold) by the model. In addition, the simulation of time-dependent changes in fu of valproic acid following administration of ibuprofen indicated different extents of dPB during 'first-pass' through the liver after oral absorption and on systemic recirculation. To understand the impact of the time-dependent change in fu, a full physiologically based pharmacokinetic model, that accounts for concentration-time profile of displacee and displacer and their mutual effect on each other, is required. The approach developed in this study is a first step towards the development of such a model.     

Clinical significance of drug binding,protein binding,and binding displacement drug interactions

Drugs widely used in clinical psychopharmacology, with few exceptions, are hepatically eliminated and circulate in the blood bound to plasma proteins. Often, the degree of binding is high (>90%). This fact has led to a widespread belief that such drugs may frequently participate in drug-binding interactions. The logic is as follows: Coadministration of drugs can result in a highly bound drug displacing a less avidly bound drug from its binding sites. This leads to greater amounts of free, nonprotein-bound, drug available for distribution to the sites of action. As free drug is a major determinant of pharmacologic effects, these drug interactions could result in toxicity and/or enhanced efficacy. This reasoning simplifies a complex situation where compensatory changes occur in the body to buffer the impact of drug-binding interactions. There are few examples where the above events have been documented to occur with psychoactive drugs leading to substantial clinical consequences. Although protein-binding displacement interactions are generally of minimal clinical significance, this is an assumption not based on evidence, but rather the lack of it. The purpose of this review is to examine some relevant aspects of drug-protein binding and draw conclusions for clinical practice about the significance of drug binding and drug-binding displacement interactions. Psychopharmacology Bulletin.     

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

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

A kinetic method for the determination of plasma protein binding of compounds unstable in plasma: Specific application to enalapril

Traditional methods for the determination of plasma protein binding (PPB), such as equilibrium dialysis and ultrafiltration, normally operate on a timescale ranging from tens of minutes to several hours and are not suitable for measuring compounds that have significant chemical degradation on this timescale. One such compound is enalapril. Although stable in human plasma enalapril is subject to rapid esterase-catalyzed hydrolysis in rat plasma. A method has been developed which allows the extent of rat PPB of enalapril to be determined from initial rates kinetics of the adsorption of the unstable compound to dextran coated charcoal (DCC). The method has been applied to stable compounds, and the results are consistent with those from traditional equilibrium dialysis experiments. The experimental method is simple to run, requires no specialized equipment, and can potentially be applied to other compounds that show instability in plasma where traditional experimental techniques are unsuitable.