EP258: Pre-analytical considerations are important in the pharmacological assessment of enzyme therapeutics

Abstract

Enzyme therapeutics are distinct from other modalities as they have the potential to continue metabolizing their target analyte even after blood collection. This poorly characterized phenomenon is important as it may lead to an overestimation of the in vivo pharmacological effects of an enzyme therapeutic. With the recent development of highly effective metabolite lowering enzyme therapeutics, it is important to understand the impact of pre-analytical sample processing as a source of inaccurate metabolite measurements in pharmacological evaluation of both nonclinical and clinical studies. Pegzilarginase is a pegylated modified human arginase 1 enzyme in clinical development for Arginase 1 deficiency (ARG1-D). Given the potency of pegzilarginase, the potential for spurious measurements of plasma arginine levels was investigated. Continued metabolism of arginine was demonstrated ex vivo after blood draw leading to artefactually low arginine levels in normally processed plasma samples. These effects were ameliorated by the competitive arginase inhibitor nor-NOHA when added to the collection tube prior to blood collection. This approach has been adapted to ensure accurate measurement of plasma arginine in patients receiving pegzilarginase in clinical trials. Similarly, a recently developed engineered enzyme (AGLE-177), with novel specificity for homocysteine and homocystine has been shown to artefactually lower total homocysteine plasma levels ex vivo after blood collection. Mitigation of the effect, by adding an inhibitor to the blood collection tube, enabled accurate assessment of tHcy levels in non-clinical efficacy and toxicology studies. These studies highlight the need to consider continued enzymatic digestion of metabolites during the pre-analytical sample handling for in vivo pharmacological studies of enzyme therapeutics.