Ilona Fitos

Separation of enantiomers of benzodiazepines on the Chiral-AGP column

The resolution of twenty-five 3-chiral and 5-chiral 1,4-benzodiazepines and related compounds was studied on a Chiral-AGP column. Relationship between the structure and enantioselective retention is discussed stressing the role of hydrophobic and hydrogen-bonding interactions as well as the importance of the conformation of the enantiomers. The majority of the benzodiazepines were separated with high separation factors and high resolution. The enantioselectivity was influenced by the nature and the concentration of the organic modifier in the mobile phase, as well as by the pH. Chiral chromatographic separation was compared with stereoselective binding on native AGP.

Probing Protein Binding Sites by Circular Dichroism Spectroscopy

Pharmacological and pharmacodynamic properties of biologically active natural and synthetic compounds are crucially determined via their binding to proteins of the human body. Several spectroscopic techniques are available to study these mainly non-covalent interactions. Circular dichroism (CD) spectroscopy, being sensitive to the chirality of ligand molecules induced by the asymmetric protein environment, has widely and successfully been applied for many decades. Chiral conformation of the ligand due to conformational adaptation to its binding site, or interaction between ligand molecules held in chiral arrangement relative to each other by the protein sites, results in one or more induced CD bands with different shape, sign and intensity. These extrinsic Cotton effects present in light absorbing region of the optically active or inactive ligand molecules give qualitative and quantitative information of the binding process. It can provide valuable data on the stereochemistry, number, location and nature of the binding sites. This paper is aimed to survey briefly the literature and the results of recent investigations undertaken in this field.

Stereoselective allosteric binding interaction on human serum albumin between ibuprofen and lorazepam acetate

The effect of ibuprofen enantiomers on the stereoselective binding of 3-acyloxy-1,4-benzodiazepines to human serum albumin (HSA) was studied using both native and Sepharose-immobilized protein. (S)-Lorazepam acetate exhibited considerably enhanced binding, especially in the presence of (+)-(S)-ibuprofen. The phenomenon is an indication of cooperative allosteric interaction between different binding sites during multiple cobinding of two ligands.

Chiral high-performance liquid chromatographic separations of vinca alkaloid analogues on α1-acid glycoprotein and human serum albumin columns

Separations of the stereoisomers of a series of tetracyclic and pentacyclic vinca alkaloid analogues having two or three chiral centres were performed on Chiral-AGP and Chiral-HSA high-performance liquid chromatographic columns. Phosphate buffers with pH 5-7 containing 5-35% acetonitrile or 2-propanol were used as mobile phases. The results were in accordance with previous binding data obtained with native AGP and on an HSA-Sepharose column. Whereas on Chiral-AGP the retention of the trans isomers having 1(R),12b(S)-indolo[2,3-a]quinolizidine or the corresponding 3(S),16(R)-eburnane absolute configurations was exceedingly high, on Chiral-HSA the trans isomers, independently of their absolute configurations, were more retained. Eburnane-type compounds could also be separated according to the configuration of the chiral centre at position 14. A comparison of the chromatographic properties of the vinca alkaloids on the Chiral-AGP and Chiral-HSA columns demonstrates that these compounds are bound with higher affinity to the AGP phase. The AGP column resolves a very broad range of vinca alkaloids compared with the HSA column. Higher stereoselectivity and a much better chromatographic performance were also obtained on the Chiral-AGP column.

Determination of Human Serum α1-Acid Glycoprotein and Albumin Binding of Various Marketed and Preclinical Kinase Inhibitors

There are about 380 protein kinase inhibitors in drug development as of today and 15 drugs have been marketed already for the treatment of cancer. This time 139 validated kinase targets are in the focus of drug research of pharmaceutical companies and big efforts are made for the development of new, druglike kinase inhibitors. Plasma protein binding is an important factor of the ADME profiling of a drug compound. Human serum albumin (HSA) and alpha(1)-acid glycoprotein (AAG) are the most relevant drug carriers in blood plasma. Since previous literature data indicated that AAG is the principal plasma binding component of some kinase inhibitors the present work focuses on the comprehensive evaluation of AAG binding of a series of marketed and experimental kinase inhibitors by using circular dichroism (CD) spectroscopy approach. HSA binding was also evaluated by affinity chromatography. Protein binding interactions of twenty-six kinase inhibitors are characterized. The contribution of AAG and HSA binding data to the pharmacokinetic profiles of the investigated therapeutic agents is discussed. Structural, biological and drug binding properties of AAG as well as the applicability of the CD method in studying drug-protein binding interactions are also briefly reviewed.

Species-dependency in chiral-drug recognition of serum albumin studied by chromatographic methods

Stereoselective binding of benzodiazepine and coumarin drugs to serum albumin from human and six mammalian species were studied by chiral chromatographic techniques. The applied methods were affinity chromatography on the albumins immobilized on Sepharose 4B, high-performance liquid chromatography (HPLC) separation on columns based on human serum albumin (HSA) and bovine serum albumin (BSA), and chiral HPLC analysis of ultrafiltrates of solutions containing the racemic drug and the native protein. Substantial differences in preferred configurations and conformations were detected among the species. The binding stereoselectivity of the 2,3-benzodiazepine drug, tofisopam, in human, is opposite to that in all other species. In the binding of 1,4-benzodiazepines, dog albumin is very similar to HSA. Highly preferred binding of (S)-phenprocoumon was found with dog albumin.

Stereoselective binding of a 2,3-benzodiazepine to human serum albumin. Effect of conformation on tofizopam binding

The binding of Tofizopam enantiomers to human serum albumin has been investigated by ultrafiltration and affinity chromatography. In solution, Tofizopam molecules exist in two conformations which slowly interconvert into each other. Both conformers of (R)-Tofizopam have the same binding constant of 4.8 X 10(3) M-1. The binding of the (S)-enantiomer, however, depends on the conformation. The minor and major (S)-conformers were characterized by association constants of 2.3 X 10(3) and 15.1 X 10(3) M-1, respectively. Thus, the stereoselectivity of binding differs for the two conformations of the enantiomers. Kinetic parameters for the interconversion of conformations have been determined. Tofizopam displaces both bound diazepam and warfarin.

Binding of vinca alkaloid analogues to human serum albumin and to α1-acid glycoprotein

Abstract The binding of a series of vinca alkaloid analogues having eburnane or indolo[2,3- a ]quinolizidine skeletons was studied with human serum albumin (HSA) by affinity chromatography and with α 1 -acid glycoprotein by means of competition experiments. On HSA the binding occurs at the benzodiazepine-indole binding site via hydrophobic interaction and shows slight stereoselectivity preferring the trans isomers. The binding to α 1 -AGP proved to be highly stereoselective in favour of the trans isomers having 3( S ),16( R )eburnane or 1( R ),12b( S )indolo[2,3- a ]quinolizidine absolute configurations.

Inverse stereoselectivity in the binding of acenocoumarol to human serum albumin and to α1-acid glycoprotein

Stereoselective binding of rac-acenocoumarol to human serum albumin (HSA) and alpha 1-acid glycoprotein (alpha 1-AGP) was investigated by affinity chromatography and by combined ultrafiltration (UF) and circular dichroism (CD) methods. For HSA, the ratio of the enantiomeric constants was KR/KS = 2, while for alpha 1-AGP, KS/KR = 3.

Selective effect of clonazepam and (S)-uxepam on the binding of warfarin enantiomers to human serum albumin

Both clonazepam and (S)-uxepam selectively increase the binding of (S)-warfarin to human serum albumin. By liquid affinity chromatography, improved resolution of rac-warfarin was achieved.