Enzymatic probe method for the determination of galantamine based on the interaction with acetylcholinesterase

M. VIANA, A. LAMOUNIER, R. ZIOLLI, G. ESCANDAR, R. AUCELIO

Buzios

Congreso; XXXVII Colloquium Spectroscopicum Internationale; 2011

Alzheimer's disease (AD) is associated with cholinergic insufficiency, therefore its treatment is based on the use of both cholinergic agonists and inhibitors of the acetylcholinesterase (AChE). Galantamine (Gal) is an alkaloid present in the Amaryllidaceae and has become part of a new generation of drugs to treat AD. Gal is a selective inhibitor of AChE, presenting an excellent therapeutic profile. The spectroscopic study of the AChE-Gal interaction in brain homogenates is useful to understand the mechanism of action of Gal in the nervous tissue. In addition, because of both the increasing of the population affected by AD and the potential human and environmental toxicity of Gal, the quantification of residues of the drug and its metabolites either in biological fluids or in water bodies has become important. In this work, a new approach, based on the use of AChE, is used to study the interaction of Gal in nervous tissue and to quantify Gal in urine and in stream water. A 23 experimental design allowed the definition of a theoretical model to describe the behavior of relevant factors in the formation of AChE-galanthamine complexes. The quenching behavior of the intrinsic AChE fluorescence by HBrGal (Gal hydrobromide present in the pharmaceutical Reminyl®) was described by a Stern-Volmer model. Gal specificity to the AChE sites was confirmed since no change in the fluorescence of AChE was observed in the presence of a relatively high concentration of the methamidophos (AChE inhibitor). Analytical tests were made with HBrGal fortified stream water and urine samples. The samples were incubated with 2.0 x 10-1 μKat of AChE present in a rat brain homogenate (in sodium phosphate buffer 0.01 mol L-1, pH 7.4) for 60 min at 37oC. Then, fluorescence intensity was measured at 280/340 nm. The quantification of HBrGal was made by the interpolation of the F0/F value, calculated for the samples, in the Stern-Volmer curve. In addition, an inhibition curve of the catalytic activity of AChE (Ellman method) was used for the quantification of HBrGal. Results were in agreement with the ones obtained using HPLC with fluorescence detection. The Stern-Volmer constant (KSV) was 5.0 x 107 L. mol-1 and the inhibition curve of AChE by Gal indicated a value for IC50 of 2.0 x 10-6 mol L-1. The feasibility of the method to quantify Gal and to study in vitro interactions between proteins present in biological materials and Gal is demonstrated.