Preformulation Studies with Phenylalanine Ammonia Lyase: Essential Prelude to a Microcapsule Formulation for the Management of Phenylketonuria

Phenylalanine ammonia lyase (PAL) metabolizes phenylalanine to transcinnamic acid (TCA). Our eventual goal is to develop a PAL microcapsule formulation to deplete phenylalanine in the gastrointestinal tract (g.i.t). The focus of this research is pre-formulation studies with PAL. PAL exhibited undesirable time dependent decrease in activity due to TCA mediated product inhibition. Addition of bovine serum albumin (BSA) completely relieved product inhibition. Ultrafiltration experiments revealed that BSA acted by binding and sequestering TCA. PAL exhibits maximum activity at a pH of 8.5 and will need to be buffered to retain activity in the g.i.t. Buffer studies showed that a pH 8.5, 0.4 M Bicine buffer containing BSA was able to maintain maximal PAL activity against simulated gastric and intestinal fluid additions. Buffered PAL with BSA was able to rapidly and completely deplete phenylalanine in simulated mouse g.i.t conditions. A small fraction of phenylalanine in the g.i.t is present as dipeptides. Our studies established for the first time that PAL cannot metabolize phenylalanine dipeptides. Our results explain why previous trials with PAL in the management of phenylketonuria produced low efficacy. They will guide design of a PAL microcapsule formulation that maintains maximal PAL activity during its transit through the g.i.t. (c) 2022 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.

Automated summary

The study aims to develop a PAL microcapsule formulation to deplete phenylalanine in the gastrointestinal tract. The results show that PAL activity decreases over time, but product inhibition can be relieved by adding bovine serum albumin. Additionally, PAL exhibits maximum activity at pH 8.5 and requires buffering to maintain activity.