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.
Competing Interests: Declaration of Competing Interest Research in our laboratory has received the following patent: U.S. Patent Application No: 15/495,410, allowed October 16th, 2018, Title: Compositions and methods for the treatment of Phenylketonuria (PKU); The University of the Sciences has licensed the technology to Abri LLC and the corresponding author owns minority stake in the company.
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