DEVELOPMENT AND CHARACTERIZATION OF A PACKED AChE-micro-IMER FOR DRUG DISCOVERY IN ALZHEIMER’S DISEASE

In the search for new potential drugs for AD treatment, the immobilization of the target enzyme acetylcholinesterase (AChE) and the insertion of the enzyme reactor in a HPLC system have demonstrated to be an excellent tool to rapidly screen for inhibitors. With the purpose of reducing the amount of used enzyme, substrate and inhibitors and of avoiding aspecific interactions between analytes and matrix, in previous studies AChE was covalently immobilized on a silica (1) or monolithic (2) matrix by using an in situ technique. The aim of this work was to prepare a micro-reactor by using a batch-wise technique in the attempt to lower the costs and increase the versatility of the system. Immobilization by batch wise technique involves essentially two steps: immobilization of the target enzyme onto non-packed affinity support followed by packing the stationary phase into an empty column that can be used in a flow system as immobilized enzyme reactor (IMER). Advantages of this technique over the in situ immobilization are: the size of the obtained IMER can be easily varied by using a different amount of bulk material, the optimization of the immobilization procedure can be performed in a parallel on a limited amount of material in a non-flow system, reducing time wasting and costs. In the present study human recombinant AChE was covalently immobilized onto 30 mg of Bakerbond glutaraldehyde-P (Glut-P, J.T. Becker, USA), a wide pore silica based affinity support. Evaluated parameters for the immobilization were: pH, amount of glut-P and contact time. The packing of the AChE-Glut-P into an empty column (Amersham Biosciences, Sweden) gave a micro-IMER of 5 mm ID and 3 mm thickness. After insertion in an HPLC system, chromatographic parameters in terms of mobile phase composition, pH and flow rate were evaluated. Inhibition studies were performed by using well-known inhibitors whose potency was distributed over four orders of magnitude. A particular attention was paid at aspecific interaction between matrix and inhibitors and conditioning time. The results indicate that the small amount of AChE immobilized on a reduced amount of chromatographic support was appropriate for high testing speed.