Plasma protein binding assays

Y Ishihama, T Miwa, N Asakawa. Drug-plasma protein binding assay by electrokinetic chromatography-frontal analysis. Electrophoresis 23 951-955... 

Jordan KH, Bruner J, Doan M et al. (2000) Automated Plasma Protein Binding Assay Implementation on a Tecan Genesis 150 and and Zymark RapidPlate. Poster presented at the LRIG SouthEast October 25 2000 Judd RL and Pesce AJ (1982) Free drug concentrations are constant in serial fractions of plasma ultrafiltrates. Clin Chem 28 1726... 

Impact of pH and Temperature on the Plasma Protein Binding Assay... 

A similar strategy was employed to identify a DPP-IV inhibitor (6) with good in vivo potency in a mouse model of diabetes [44], Plasma protein binding, as assessed by shift assay (50% serum), was presented for all final compounds. The compound selected as having the best overall profile was active in vivo at 0.1 mg/kg. The activity at 1 h post-dose was consistent with the free drug principle - total plasma concentration 269 nM murine-free fraction 4% unbound plasma concentration 11 nM in vitro potency versus murine DPP-IV 6nM. 

Additional assays used in early pharmaceutical property profiles usually include plasma protein binding, individual cytochrome P450 assays, stability in the presence of serum, production of metabolites likely to be involved in covalent binding to biomolecules, and interaction with efflux pumps ... 

Plasma protein binding is also an important parameter in the pharmacokinetic field. Frontal analysis combined with capillary zone electrophoresis (CZE-FA) (67-69) is a powerful technique for high-throughput assay, because it is relatively rapid and easy to automate, in comparison with conventional methods such as dialysis, ultrafiltration, and ultracentrifugation. Recently, we introduced the EKC approach with ionic CDs to frontal analysis for anionic drugs that cannot be analyzed by conventional CZE-FA (70). In this approach, ionic CDs work as an EKC pseudostationary not for proteins but for small solutes. 

What makes prediction of drug elimination complex are the multiple possible pathways involved which explain why there is no simple in vitro clearance assay which predicts in vivo clearance. Because oxidative metabolism plays a major role in drug elimination, microsomal clearance assays are often used as a first line screen with the assumption that if clearance is high in this in vitro assay it is likely to be high in vivo. This assumption is often, but not always true because, for example, plasma protein binding can limit the rate of in vivo metabolism. However, compounds which have a low clearance in hepatic microsomes can be cleared in vivo via other mechanisms (phase II metabolism, plasmatic errzymes). Occasionally, elimination is limited by hepatic blood flow, and other processes like biliary excretion are then involved. The conclusion is that the value of in vitro assays needs to be established for each chemical series before it can be used for compound optimization. 

Data generated from metabolic clearance measurements using liver microsomes can lead to an overestimation of the tme in vivo clearance if the free versus bound fraction is not considered. A useful follow-up assay is therefore plasma protein binding measurement. The impact of cytochrome P-450 inhibition on metabolic clearance of the parent (and thus exposure) is more complicated and it remains rather difficult to make quantitative predictions from in vitro data alone. The reason is that there are generally multiple clearance pathways involved and genetic polymorphism needs to be considered as well. 

E. R. Garrett, K. Seyda, and P. Marroum, HPLC assays of the illicit design drug, Ecstasy, a modified amphetamine with applications to stability, partitioning and plasma protein binding, Acta Pharm. Nord., 3 9 (1991). 

Horst RL, Reinhardt TA, Beitz DC, Littledike ET. A sensitive competitive protein binding assay for vitamin D m plasma. Steroids 1981 37 581-91. 

Assays will usually be on plasma and urine. Plasma protein binding should be estimated simultaneously because renal disease can alter plasma protein binding of some drugs. 

The relationship between chemical structure, lipophilicity, and its disposition in vivo has been extensively studied. These include solubility, absorption potential, membrane permeability, plasma protein binding, volume of distribution, and renal and hepatic clearance. Activities used in quantitative structure-activity relationships (QSAR) include chemical measurements and biological assays. QSAR currently are applied in many disciplines, with many pertaining to drug design and environmental risk assessment. 

Consistent wdth a major role of the liver in maintaining normal somatomedin levels in the circulation, low levels of somatomedin-A activity have been found in unextracted plasma of patients with cirrhosis of the liver and chronic hepatitis (S14, T5). Significant correlations were seen between somatomedin-A and albumin, cholinesterase, and other indicators of liver function (T5). The decrease measured in this RRA appears to be due to low levels of both somatomedins and binding protein, since Zapf et al. (Z5) have shown an 89% decrease in immunoreactive IGF-I, a 74% decrease in total IGF by protein binding assay (which preferentially measures IGF-II), and a 57% decrease in specific binding of somatomedin tracer to stripped serum, in patients with cirrhosis. 

Many factors may affect the degree of plasma protein binding in vivo such as age, disease state, pregnancy, etc. [101], However, under well-controlled in vitro settings, only few factors might impact the outcome of the assay, such as pH and temperature. In order to fully understand the importance of these factors, we have conducted in-house studies that indicated that careful control of both factors is vital for accurate plasma protein binding results. 

The most widely used technique for the evaluation of hERG channel interaction is the voltage clamp. A detailed description of the experimental setup has been described elsewhere [119]. The hERG interaction is measured and reported as the % inhibition of the hERG current compared to the vehicle control at various concentrations of the NCE. The concentration that inhibits 50% (IC50) is calculated, whenever possible. The concentrations of NCE used in the assay are carefully selected based on the expected maximum plasma concentration (Cmax) at the pharmacologically active dose (usually based on studies in animal models) and the human plasma protein binding for the NCE. 

Gutenberger, S.K. Olson, D.P. Kagel, R.A. Comparison of reversed phase high-performance liquid chromatography and competitive protein binding assay in the quantification of cortisol in bovine plasma. J.Liq.Chromatogr., 1985, 8, 107-124 [plasma cow dexamethasone (IS)]... 

Hit to lead Physicochemical properties (solubility, Log P) PAMPA Plasma protein binding Efficacy species and human liver microsomal stability, CAR and PXR transactivation assay... 

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