Aim: The aim was to evaluate drug-plasma binding (DPB).by employing Hollow Fiber-in-Syringe Equilibrium Sampling Through Supported Liquid Membrane (HFiS ESTSLM) and RP-HPLC analysis. Materials & methods: HFiS ESTSLM and RP-HPLC were used to evaluate DPB of three weak basic drugs (Metoprolol, Diphenhydramine, and Sildenafil) with differing hydrophilicity and binding ability to blood plasma. Results: The results exhibited an increasing drug-dependent magnitude of DPB for the three model drugs. This trend of DPB confirmed that HFiS ESTSLM has the required sensitivity for determining DPB of the drugs. The DPB was drug concentration-dependent within the tested drug concentration range, especially at high concentration. Conclusion: HFiS ESTSLM and RP-HPLC offered a simple, easy and cost-effective procedure to evaluate DPB of these basic drugs.
[Box: see text].

In this work, the effect of γ-irradiated surfactants on drug-protein binding has been assessed. Irradiated aqueous solutions of Pluronic F-127, Pluronic L-35, Tween 20, and Tween 80 surfactants were used. Gamma irradiation was carried out for three different doses, to these four surfactants viz., 6, 30, and 36 kGy. Two drugs, Ornidazole (ONZ) and Telmisartan (TMS) were used for the binding study. The effect of four irradiated surfactants in the presence of drug - Bovine serum albumin (BSA) protein was analyzed. The drug solutions in methanol-aqueous media were combined with BSA in the initial step. In the next two succeeding steps, drug-BSA interaction in the presence of unirradiated and irradiated surfactants were carried out. The results of drug-BSA due to addition of irradiated and unirradiated surfactants were compared. The interaction processes were assessed through UV Spectroscopy, DLS, zeta potential, turbidity, and docking studies. Improved binding was observed for both the drugs and four surfactants for irradiated surfactants as determined from the binding constant values using UV spectroscopic studies. The DLS measurements demonstrated no general trend of increase or decrease in micellar size with absorbed dose. The binding and change in the size of micelles were observed to be highly drug and surfactant-specific. Among the four surfactants, irradiated Pluronic F-127 showed higher binding affinity. To the best of our knowledge, this is the first report on the effect of γ-irradiated surfactant on drug-BSA interaction. This can be applied to other drug-protein systems to tune their interaction to the required level.Communicated by Ramaswamy H. Sarma.

Odanacatib (ODN) is a selective cathepsin K inhibitor that acts as an anti-resorptive agent to treat osteoporosis. ODN is also found effective in reducing the effect of severe periodontitis. The interaction between ODN and human serum albumin (HSA) was investigated using spectroscopic, microscopic, and in silico approaches to characterize their binding. The fluorescence intensity of HSA increased upon the addition of increasing concentrations of ODN accompanied by blueshift in the fluorescence spectrum, which suggested hydrophobic formation around the microenvironment of the fluorophores upon ODN binding. A moderate binding affinity was obtained for ODN-HSA binding, with binding constant (Ka) values of ∼104 M-1. Circular dichroism results suggested that the overall secondary and tertiary structures of HSA were both only slightly altered upon ODN binding. The surface morphology of HSA was also affected upon ODN binding, showing aggregate formation. Drug displacement and molecular docking results revealed that ODN preferably binds to site III in subdomain IB of HSA, while molecular dynamics simulations indicated formation of a stable protein complex when site III was occupied by ODN. The ODN-HSA complex was mainly stabilized by a combination of hydrogen bonding, hydrophobic interactions, and van der Waals forces. These findings provide additional information to understand the interaction mechanism of ODN in blood circulation and may help in future improvements on the adverse effects of ODN.

Ultrafast affinity extraction (UAE) is a form of microscale affinity HPLC that can be employed to quickly measure equilibrium constants for solute-binding agent interactions in solution. This study used chromatographic and equilibrium theory with universal plots to examine the general conditions that are needed in UAE to obtain accurate, precise, and robust measurements of equilibrium constants for such interactions. The predicted results were compared to those obtained by UAE in studies that examined the binding of various drugs with two transport proteins: human serum albumin and α1-acid glycoprotein. The most precise and robust conditions for these binding studies occurred for systems with intermediate values for their equilibrium free fraction for the solute (F0 ≈ 0.20-0.80). These trends showed good agreement with those seen in prior studies using UAE. It was further determined how the apparent free fraction of a solute was related to the dissociation rate of this solute, the time allowed for solute dissociation during UAE, and the equilibrium free fraction for the solute. These results also agreed with experimental results, as obtained for the binding of warfarin and gliclazide with human serum albumin. The final section examined how a change in the apparent free fraction, as caused by solute dissociation, affected the accuracy of an equilibrium constant that was measured by UAE. In addition, theoretical plots were generated to allow the selection of conditions for UAE that provided a given level of accuracy during the measurement of an equilibrium constant. The equations created and trends identified for UAE were general ones that can be extended in future work to other solutes and binding agents.

Modification of proteins can occur during diabetes due to the formation of advanced glycation end-products (AGEs) with reactive dicarbonyls such as glyoxal (Go) and methylglyoxal (MGo). Human serum albumin (HSA) is a serum protein that binds to many drugs in blood and that is known to be modified by Go and MGo. This study examined the binding of various sulfonylurea drugs with these modified forms of HSA by using high-performance affinity microcolumns prepared by non-covalent protein entrapment. Zonal elution experiments were employed to compare the retention and overall binding constants for the drugs with Go- or MGo-modified HSA vs normal HSA. The results were compared to values from the literature, such as measured or estimated using affinity columns containing covalently immobilized HSA or biospecifically-adsorbed HSA. The entrapment-based approach provided estimates of global affinity constants within 3-5 min for most of the tested drugs and with typical precisions of ±10-23%. Each entrapped protein microcolumn was stable for over at least 60-70 injections and one month of use. The results obtained with normal HSA agreed at the 95% confidence level with global affinity constants that have been reported for the given drugs in the literature. It was found for HSA that had been modified with clinically-relevant levels of either Go or MGo that an increase in the global affinity constant of up to 2.1-fold occurred for some of the tested drugs. The information acquired in this study can be used in the future to adapt this entrapment-based approach to study and evaluate interactions between other types of drugs and normal or modified binding agents for clinical testing and biomedical research.

Ultrafast affinity extraction (UAE) and affinity microcolumns containing immobilized human serum albumin (HSA) were employed to evaluate the effect of advanced stage glycation on HSA and its binding to warfarin, a common site-specific probe for Sudlow site I of this protein. The modification of HSA by glyoxal (GO) and methylglyoxal (MGO) was considered, where GO and MGO are known to be important in the formation of many types of advanced glycation end products. Free drug fractions were measured by UAE for warfarin in solutions containing normal HSA or HSA that had been modified by GO or MGO at levels seen in serum during diabetes. The free fractions measured with the GO-modified HSA gave association equilibrium constants that ranged from 2.42-2.63 × 105 M-1 at pH 7.4 and 37 °C. These values were not significantly different from a value of 2.33 (±0.15) × 105 M-1 that was determined by the same method for warfarin with normal HSA. Similar studies using MGO-modified HSA gave association equilibrium constants for warfarin in the range of 3.07-3.31 × 105 M-1, which were 1.32- to 1.42-fold higher than the value seen for normal HSA (differences that were significant at the 95% confidence level). These results will be valuable in future binding studies based on affinity chromatography or other methods that employ warfarin as a probe to examine drug interactions at Sudlow site I of HSA and modified forms of this protein. This work also illustrates how UAE can be used, with analysis times of only minutes, to detect and measure small changes in the binding by drugs with unmodified or modified forms of a soluble binding agent or protein.

Biomolecules such as serum proteins can interact with drugs in the body and influence their pharmaceutical effects. Specific and precise methods that analyze these interactions are critical for drug development or monitoring and for diagnostic purposes. Affinity capillary electrophoresis (ACE) is one technique that can be used to examine the binding between drugs and serum proteins, or other agents found in serum or blood. This article will review the basic principles of ACE, along with related affinity-based capillary electrophoresis (CE) methods, and examine recent developments that have occurred in this field as related to the characterization of drug-protein interactions. An overview will be given of the various formats that can be used in ACE and CE for such work, including the relative advantages or weaknesses of each approach. Various applications of ACE and affinity-based CE methods for the analysis of drug interactions with serum proteins and other binding agents will also be presented. Applications of ACE and related techniques that will be discussed include drug interaction studies with serum agents, chiral drug separations employing serum proteins, and the use of CE in hybrid methods to characterize drug binding with serum proteins.

High-performance affinity microcolumns were used to characterize binding by the anti-diabetic drugs repaglinide and nateglinide with normal and glycated forms of human serum albumin. The microcolumns contained only nmol amounts of protein and provided a detailed analysis of these drug interactions with good precision and in a matter of minutes per experiment. The overall binding by repaglinide to normal and glycated albumin fits a model with two types of binding sites: a set of one or two moderate-to-high affinity regions and a larger set of weaker regions with association equilibrium constants of ∼105 and 103  M-1 , respectively, at pH 7.4 and 37°C. Competition studies gave site-specific association constants for repaglinide and nateglinide at Sudlow site I of 4.2 × 104 and 5.0 × 104  M-1 for normal albumin, with a decrease of 26%-30% being seen for nateglinide with glycated albumin and no significant change being noted for repaglinide. At Sudlow site II, repaglinide and nateglinide had association constants for normal albumin of 6.1 × 104 and 7.1 × 105  M-1 , with glycated albumin giving an increase in the association constant at this site for repaglinide of 1.6- to 1.8-fold and a decrease for nateglinide of 51%-58%.

Ultrafast affinity extraction (UAE) has recently been developed and employed for measuring non-bound (or free) fractions and binding or rate constants for drugs and other targets with soluble binding agents such as serum proteins. This study examined the long-term stability of 10 mm × 2.1 mm i.d. affinity microcolumns when used in UAE at both low and high flow rates (e.g., 0.5 and 3.5 mL/min) over an extended series of injections. This stability was investigated by using immobilized human serum albumin (HSA) and samples containing the drug warfarin with or without soluble HSA as a model system. The free warfarin fractions measured at 0.5 mL/min in the presence of soluble HSA were stable up to 150 injections and changed by <10% at 3.5 mL/min. The association equilibrium constant for warfarin with HSA that was estimated by UAE at 3.5 mL/min had no significant change over 50 injections and a change of only ∼18-22% over 100-150 injections. The dissociation rate constant for warfarin from HSA was found by combining UAE results at 0.5 and 3.5 mL/min and employing a new two-point approach, with no significant changes in this value being seen even after 200 injections. The effects of extended microcolumn use on the retention time, peak width, and peak asymmetry for warfarin, and on the backpressure of the microcolumn, were also considered. These results indicated that UAE and HSA microcolumns could be used to provide consistent values for free solute fractions, binding constants, and rate constants over a large series of injections. These results should be useful in future work by providing guidelines for the assessment, further development, and use of UAE in characterizing interactions involving other drugs and binding agents in solution-based samples.

The pseudoguaianolide-type sesquiterpene lactone (SL) britannin (BRT), found in different Inula species, has been characterized as a potent anticancer agent acting via modulation of the transcription factor NFkB and the Nrf2-Keap1 signaling pathway. In addition, a BRT-induced down-regulation of the immune checkpoint PD-L1 (programmed cell death ligand 1) expressed on cancer cells has been evidenced. Here we have performed a docking analysis of the direct binding of BRT to the PD-L1 protein, both in its monomeric and dimeric state. BRT appears to form stable complexes with PD-L1, with a preference for the dimeric form, binding at the interface of the two monomers. The calculated empirical energy of interaction (ΔE) value reaches -63.1 kcal/mol for the BRT-PD-L1 dimer complex, not far from the value calculated with the reference PD-L1 ligand BMS-202 (ΔE = -73.4 kcal/mol) under identical conditions. We also studied the potential PD-L1 dimer binding of 15 pseudoguaianolide sesquiterpene lactones analogues to BRT, including helenalin, gaillardin, bigelovin, coronopilin, and others. The docking analysis predicted that the SL chamissonolide (CHM) can also form equally stable complexes with PD-L1 dimer (ΔE = -64.8 kcal/mol). Preliminary compound structure-PD-L1 binding relationships have been delineated. This computational study supports the proposed interaction of BRT with PD-L1 and provides a guidance to the design of novel PD-L1 binders incorporating a SL-like tricyclic core unit.