title: Formulation and PEGylation optimization of the therapeutic PEGylated phenylalanine ammonia lyase for the treatment of phenylketonuria.
abstract: Phenylketonuria (PKU) is a genetic metabolic disease in which the decrease or loss of phenylalanine hydroxylase (PAH) activity results in elevated, neurotoxic levels of phenylalanine (Phe). Due to many obstacles, PAH enzyme replacement therapy is not currently an option. Treatment of PKU with an alternative enzyme, phenylalanine ammonia lyase (PAL), was first proposed in the 1970s. However, issues regarding immunogenicity, enzyme production and mode of delivery needed to be overcome. Through the evaluation of PAL enzymes from multiple species, three potential PAL enzymes from yeast and cyanobacteria were chosen for evaluation of their therapeutic potential. The addition of polyethylene glycol (PEG, MW = 20,000), at a particular ratio to modify the protein surface, attenuated immunogenicity in an animal model of PKU. All three PEGylated PAL candidates showed efficacy in a mouse model of PKU (BTBR Pahenu2) upon subcutaneous injection. However, only PEGylated Anabaena variabilis (Av) PAL-treated mice demonstrated sustained low Phe levels with weekly injection and was the only PAL evaluated that maintained full enzymatic activity upon PEGylation. A PEGylated recombinant double mutant version of AvPAL (Cys503Ser/Cys565Ser), rAvPAL-PEG, was selected for drug development based on its positive pharmacodynamic profile and favorable expression titers. PEGylation was shown to be critical for rAvPAL-PEG efficacy as under PEGylated rAvPAL had a lower pharmacodynamic effect. rAvPAL and rAvPAL-PEG had poor stability at 4°C. L-Phe and trans-cinnamate were identified as activity stabilizing excipients. rAvPAL-PEG is currently in Phase 3 clinical trials to assess efficacy in PKU patients.
authors.affiliation: BioMarin Pharmaceutical, Novato, California, United States of America.
authors.forename: Sean M
authors.initials: SM
authors.lastname: Bell
authors.affiliation: BioMarin Pharmaceutical, Novato, California, United States of America.
authors.forename: Dan J
authors.initials: DJ
authors.lastname: Wendt
authors.affiliation: BioMarin Pharmaceutical, Novato, California, United States of America.
authors.forename: Yanhong
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: BioMarin Pharmaceutical, Novato, California, United States of America.
authors.forename: Timothy W
authors.initials: TW
authors.lastname: Taylor
authors.affiliation: BioMarin Pharmaceutical, Novato, California, United States of America.
authors.forename: Shinong
authors.initials: S
authors.lastname: Long
authors.affiliation: BioMarin Pharmaceutical, Novato, California, United States of America.
authors.forename: Laurie
authors.initials: L
authors.lastname: Tsuruda
authors.affiliation: BioMarin Pharmaceutical, Novato, California, United States of America.
authors.forename: Bin
authors.initials: B
authors.lastname: Zhao
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida, United States of America.
authors.forename: Phillip
authors.initials: P
authors.lastname: Laipis
authors.affiliation: BioMarin Pharmaceutical, Novato, California, United States of America.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Fitzpatrick
mesh_terms: D017033:Anabaena / Q000201:enzymology; D000818:Animals; D000906:Antibodies / Q000097:blood; D004195:Disease Models, Animal; D004339:Drug Compounding; D056947:Enzyme Replacement Therapy; D004797:Enzyme-Linked Immunosorbent Assay; D051379:Mice; D046937:Nostoc / Q000201:enzymology; D028528:Petroselinum / Q000201:enzymology; D010650:Phenylalanine Ammonia-Lyase / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000627:therapeutic use*; D010661:Phenylketonurias / Q000188:drug therapy* / Q000473:pathology; D011092:Polyethylene Glycols / Q000737:chemistry*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000627:therapeutic use
chemical_list: D000906:Antibodies; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D010650:Phenylalanine Ammonia-Lyase