Binding study of desethyloxybutynin using high-performance frontal analysis method

Plasma protein binding of N-desethyloxybytynin (DEOXY), a major active metabolite of oxybutynin (OXY), was investigated quantitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system which consists of HPFA column, extraction column and analytical column was developed to determine the unbound concentrations of DEOXY enantiomers in human plasma, in human serum albumin (HSA) solutions, and in human α1-acid glycoprotein (AGP) solutions. DEOXY is bound in human plasma strongly and enantioselectively. The unbound drug fraction in human plasma samples containing 5 μM (R)- or (S)-DEOXY was 1.19±0.001 and 2.33±0.044%, respectively. AGP plays the dominant role in this strong and enantioselective plasma protein binding of DEOXY. The total binding affinity (nK) of (R)-DEOXY and (S)-DEOXY to AGP was 2.97×107 and 1.31×107 M−1, respectively, while the nK values of (R)-DEOXY and (S)-DEOXY to HSA were 7.77×103 and 8.44×103 M−1, respectively. While the nK value of (S)-DEOXY is weaker than that of (S)-OXY (1.53×107 M−1), the nK value of (R)-DEOXY is 4.33 times stronger than that of (R)-OXY (6.86×106 M−1). This suggests that the elimination of an ethyl group weakens the binding affinity of the (S)-isomer because of the decrease in hydrophobicity, while the binding affinity of the (R)-isomer is enhanced by the decrease in steric hindrance. The total binding affinity of DEOXY to HSA is much lower than that of DEOXY-AGP binding as well as OXY-HSA binding (2.64×104 and 2.19×104 M−1 for (R)-OXY and (S)-OXY, respectively). The study on competitive binding between OXY and DEOXY indicated that DEOXY enantiomers and OXY enantiomers are all bound competitively at the same binding site of AGP molecule.