Role Of The Monocambamate Metabolites On The Hydrolysis Of Bambuterol Enantiomers Catalyzed By Human Butyrylcholinesterase: A Systematic Study

Bambuterol(BMB) is a prodrug of terbutaline(TBT) belonging to the class of long-acting β2-adrenoceptor agonists, BMB is prescribed as an oral formulation in the therapy of asthma, bronchospasm, emphysema and chronic obstructive pulmonary disease. In vivo, BMB is metabolized in a two steps process into the active compound TBT by plasma butyrylcholinesterase(BCh E). In the first step BMB is hydrolyzed to bambuterol monocarbamate(MONO) which is metabolized in the second step by the same enzyme into the active compound TBT. As a result of this metabolism, the enzyme looses temporary its activity because the carbamate groups of BMB and MONO block the catalytic site of the enzyme and this carbamoyl-enzyme intermediate is only slowly reactivated by water. Therefore, the hydrolysis of BMB and MONO is accompanied by the inhibition of BCh E. In this work, BMB and MONO have been studied as substrates and inhibitors of BCh E and a complete picture of the hydrolytic process has been obtained. Particular focus was given to the role played by MONO in the inhibition of the enzyme, since it was previously poorly characterized. The results of this work can be summarized as follows:1) MONO enantiomers were synthesized by an optimized alkaline hydrolysis of BMB enantiomers and were purified by solid-phase extraction(SPE). The chemical and optical purity of the MONO exceed 99.3% and 99.4%, respectively.2) The study of the inhibition kinetics of BMB and MONO enantiomers revealed their timeand concentration-dependent mechanism of interaction with BCh E. The IC50 values of BMB and MONO enantiomers is within a nanomolar range, therefore all of them are powerful inhibitors. The comparison of the enzyme inhibition rates shows that(R)- and(S)-MONO are 15-fold slower than their respective BMB enantiomers, and for both BMB and MONO, the R-enantiomer is 4-fold faster than S-enantiomer, in other words, the inhibition of BCh E by both BMB and MONO is enantioselective, BMB and MONO have the same enantioselectivity.3) The study of the hydrolysis kinetics of BMB and MONO as substrates of BCh E performed at physiologically relevant concentrations showed the following ranking of hydrolysis rate:(R)-BMB > rac-BMB >(S)-BMB >(R)-MONO > rac-MONO >(S)-MONO. Because of the different hydrolysis rate between BMB and MONO, when BMB is the substrate, MONO accumulates until all BMB has been hydrolyzed and the inhibition of the enzyme by BMB reduces the hydrolysis rate of MONO.4) The mathematical model of the hydrolytic process generated to fit the experimental data indicate that: i) the(R)- and(S)- components in racemates compete kinetically for the hydrolysis/inhibition; ii) at physiologically relevant concentrations of enzyme and substrates, the hydrolysis of MONO enantiomers enhance the inhibitory power of BMB enantiomers about 27%(R) and 15%(S) and extend more than 1 hour the duration of inhibition.