Enantioselective Binding Of Chiral Drugs To Plasma Proteins

After systemic introduction, most drugs undergo some degree of reversible binding to plasma proteins. In general, only the free or unbound fraction of a drug is able to traverse cell membranes to exert a therapeutic effect, or to undergo enzymatic metabolism. Factors influencing the binding of a drug to plasma proteins may significantly affect its pharmacokinentics and pharmacodynamics. This is especially true when the drug is highly bound or has low intrinsic clearance (in which case, total hepatic clearance is proportional to the free fraction). A clear understanding of the plasma protein binding behavior of a therapeutic agent is therefore fundamental to its safe and rational use.In the case of chiral drugs, the possibility arises that the individual stereoisomers may bind to plasma proteins with different affinities since all proteins are inherently chiral, resulting in different free fraction. As over 50% of the drugs in current clinical use are chiral and the majority of synthetically derived chiral drugs are administered as mixtures of the constituent stereoisomers (most commonly the racemate), this potentiality is significant. Therefore, it is important to study of individual enantiomeric differences in the development of an objective picture of drug-protein interaction. In this paper, systematic studies were performed on the enantioselective binding of chiral drugs used widely to plasma proteins using stereoselective HPLC method. The studies are expected to provide a clear understanding of the differences in drug-protein interaction as well as in pharmacokinetic and pharmacodynamics properties between the two enantiomers of chiral drugs in the drug discovery and development process.1. Enantioselective binding of esmolol to plasma proteinsA stereoselective reversed-phase HPLC assay to determine 5- and i?-enantiomers of esmolol was developed. The conditions for the derivatization, using 2,3,4,6-tetra-O -acetyl- P -D-glucopyranosyl isothiocyanate(GITC) as a pre-column chiral derivatization reagent, were optimized. The assay was linear from 0.08-20fig/mL for each enantiomer. The analytical method afforded average recoveries of over 85% for each enantiomer. Intra-day and inter-day variation coefficient were less than 7%. The validated method was applied to quantify the enantiomers of esmolol for protein binding studies.In native human plasma, esmolol enantiomers were metabolized by hydrolysis of ester linkage to the acid metabolite enantiomers with different hydrolysis rates. Anticholinesterase drug pyridostigmine bromide was added in order to stabilize esmolol in human plasma. 20|ig/mL of pyridostigmine bromide was enough for efficiently inhibiting hydrolysis of esmolol and did not influence the protein binding of esmolol. Ultrafiltration techniques were applied to determined the protein binding of each enantiomer of esmolol in human plasma, human serum albumin (HSA) and ai-acid glycoprotein (AGP). The results clearly showed that stereoselective binding of two enantiomers of esmolol with higher protein binding of the 5-enantiomer than that of /?-enantiomer in human plasma. To asses the site of the stereoselective binding to plasma proteins, the binding of esmolol enantiomers to HSA and AGP was examined. The binding to AGP was stereoselective for 5-enantiomer with the bound fraction of 33.45-13.45% for 5-enantiomer and 25.78-11.55% for fl-enantiomer, whereas the binding to HSA was found not to be stereoselective (nK(s>: 241L/mol, nK(/{): 2371Vmol). Both esmolol enantiomer were bound to one class of binding site on AGP and binding association constants were 6.31xl04L/mol for 5-enantiomer, 4.33x104L/ mol for /?-enantiomer, respectively. Protein binding affects hydrolysis rate of esmolol enantiomers. The in vitro hydrolysis rate of /?-esmolol was slightlyfaster than that of 5-esmolol. The stereoselective binding might be one of reasons for this difference in the in vitro hydrolysis rate.2. Enantioselective binding of propafenone to plasma proteinsThe interaction of propafenone enantiomers with human plasma, HSA and AGP was investigated using stereoselective HPLC after pre-column derivatization with GITC and ultrafiltration techniques. Results showed that the binding of propafenone enantiomers was concentration dependent and stereoselective in human plasma. Nonspecific binding to HSA was found to be weak and not stereoselective (nK(s>: 2.03x103IVmol, nK^: 2.02x103IVmol). The considerably stronger binding of the 5-enantiomer found in human plasma was due to the AGP component. Each of propafenone enantiomer interacted with two classes of binding sites present on AGP: one with high-affinity and small binding capacity characterized by K...