A high-throughput cell-free profiling platform integrating high metabolic diversity for chemical safety assessment

Abstract Roughlynew drugs and chemicals are commercialized each year successfully clearing research and developmentRandamp Dhurdles to meet the safety requirements of regulators such as the U SFood and Drug AdministrationThese successeshoweverare the exception rather than the rule making it advantageous for Randamp D programs to have methods in place that ensure unsafe compoundsfail fastwell before entering time and resource intensive animal or human testingIn vitro toxicity assays have assumed this early screening role but lack the ability to assess the contribution of potentially toxic metabolites that the body produces from otherwise safe parent chemicalsThe integration of metabolic profiling into current toxicity screening platforms is therefore crucial to chemical riskexposureand drug safety assessmentsThe cytochrome PCYPfamily is one of the most important families of enzymes involved in human metabolismmetabolizing aboutof all xenobiotics and drugs in the liverIntegrating the function of CYPs and other metabolic familiese gUGTsin drug metabolism and pharmacokineticsDMPKare key to increasing the safety of drugs as well as speeding their developmentCurrent approaches used by pharmaceutical companies and contract research organizations rely on cultured hepatocyteswhich co activate multiple enzymatic pathways in a single integrated platformAlthough this method mimics liver physiologythere are several drawbacks including limited incubation time and metabolite productionthe need to statistically evaluate significant background metabolomesand complex quality control challenges inherent in cell handling techniquesZymtronix has developed a novelcell free technology that offers increased metabolite productionenzyme specific metabolomes with little backgroundand improved quality control compared to cell based systemsallowing for reduced cost and ease of useThis Phase I project will generate a proof of concept product for a Toxcompatiblehigh throughputHTPmetabolic processing unit built on a proprietary enzyme anchoring technologyFirstwe will adapt our existing technology to a functionalizedwell microplate lidand then optimize and characterize the newly engineered scaffold using CYP ACYP Band UGT Amodel enzymesFinallywe will use liquid chromatography mass spectrometry analysis to demonstrate the conversion of six model compounds by the enzyme trioindividually and in combinationand compare the metabolome with that produced by pooled human liver microsomesThe project will prepare us for future Phase II researchin which we will explore a larger number of chemical substrates with an expanded set of CYPs UGTs to determine a critical set of metabolic enzymes that produce a diverse and physiologically relevant metabolomeThe product will be constructed inwell microplate format to facilitate HTP robotic handling and integration with downstream cell based assays Narrative Because metabolism significantly affects the toxicity of chemicals taken up by the bodydetermining the metabolic profile of xenobioticsincluding commercial chemicalsfood ingredients and pharmaceuticalsis a critical part of chemical riskexposureand drug safety assessmentsCurrently proposed cell based assay systems for evaluating the environmental health impact of metabolites are labor intensivecostly and pose challenges to quality control and analysisZymtronix is developing a tunable cell free toxicity screening product for the ADME market that is Toxcompatible with a proprietary enzyme immobilization technology that will offer improved quality controlreduced costand ease of use