A site-oriented study of nine non-steroidal anti-inflammatory drugs (NSAIDs), suprofen, ibuprofen, diclofenac sodium, pirprofen, flurbiprofen, ketoprofen, phenylbutazone, oxyphenbutazone, and ketophenylbutazone, on human serum albumin (HSA) was carried out at pH 7.4 by various direct and indirect methods to gain insight into the high affinity binding sites of NSAIDs. The binding was determined by equilibrium dialysis, circular dichroism and fluorescence methods in order to strengthen the results. Irrespective of the method used, close agreement between the binding parameters obtained by the different methods was obtained. A site-oriented description, as revealed by the fluorescent probe displacement method, suggests that site II and site I probes bound to HSA were selectively displaced by NSAIDs with a carboxyl group and without a carboxyl group, respectively. The absence of a carboxyl group of the NSAIDs changed the binding site from site II to site I. Different binding models describing the competitive and independent binding of a NSAID and a site-specific probe bound simultaneously to HSA further describe the respective high affinity binding sites for NSAIDs, thus underscoring the necessity of a carboxyl group in order for a NSAID to bind to site II. The presence of tyrosine, lysine and histidine amino acid residues in the binding of carboxyl group-containing NSAIDs to HSA was evident, whereas tryptophan is believed to take part in the binding of non-carboxyl group-containing NSAIDs. The present findings support the proposal that two separate primary binding sites exist for different NSAIDs; hence, an attempt to correlate the present results with those in literature has been made.

A wide variety of drugs bind to human serum albumin (HSA) at its two principal sites, namely site I and site II. A number of reports indicate that drug binding to these two binding sites are not completely independent, and that interactions between ligands of these two discrete sites can play a role. In this study, the effect of the binding of long-chain fatty acids on the interactive binding between dansyl-L-asparagine (DNSA; site I ligand) and ibuprofen (site II ligand) at pH6.5 was examined. Binding experiments showed that the binding of sodium oleate (Ole) to HSA induces conformational changes in the molecule, which, in turn, changes the individual binding of DNSA and ibuprofen, as well as the mode of interaction between these two ligands from a ‘competitive-like’ allosteric interaction in the case of the defatted HSA conformer to a ‘nearly independent’ binding in the case of non-defatted HSA conformer. Circular dichroism measurements indicated that ibuprofen and Ole are likely to modify the spatial orientation of DNSA at its binding site. Docking simulations suggest that the long-distance electric repulsion between DNSA and ibuprofen on defatted HSA contributes to a ‘competitive-like’ allosteric interaction, whereas extending the distance between ligands and/or increasing the flexibility or size of the DNSA binding site in fatted HSA evokes a change in the interaction mode to ‘nearly independent’ binding. The present findings provide further insights into the structural dynamics of HSA upon the binding of fatty acids, and its effects on drug binding and drug-drug interactions that occur on HSA.

Hemoglobin (Hb) is an ideal material for use in the development of an oxygen carrier in view of its innate biological properties. However, the vascular retention of free Hb is too short to permit a full therapeutic effect because Hb is rapidly cleared from the kidney via glomerular filtration or from the liver via the haptogloblin-CD 163 pathway when free Hb is administered in the blood circulation. Attempts have been made to develop alternate acellular and cellular types of Hb based oxygen carriers (HBOCs), in which Hb is processed via various routes in order to regulate its pharmacokinetic properties. These HBOCs have been demonstrated to have superior pharmacokinetic properties including a longer half-life than the Hb molecule in preclinical and clinical trials. The present review summarizes and compares the pharmacokinetic properties of acellular and cellular type HBOCs that have been developed through different approaches, such as polymerization, PEGylation, cross-linking, and encapsulation.

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