A better Eukaryotic system for:
• Rapid screening & identification of drug metabolites
• Rapid, inexpensive Production of drug metabolites for MIST studies
• P450 inhibition screening using various protocols
Identification and characterization of metabolites is a critical step in the long and expensive process of bringing a new chemical entity to market. For drug candidates, every significant metabolite must be identified, and sufficient quantities synthesized to meet MIST requirements.
Until now, the synthesis and purification of several milligrams of some metabolites represented a slow and expensive step in the development process, often involving incubation with human or rat liver microsomes (or expensive recombinant systems) – typically yielding complex HPLC profiles and requiring multiple isolation and purification procedures.
CypExpress™ provides for robust metabolite identification and production, while reducing the time and expense involved in this critical process.
• Coexpress a specific human Cyp AND human P450 oxidoreductase
• Easy to handle dry powder
• Stable at room temperature
• CypExpress™ retains catalytic activity for many hours, even overnight
• Indeed, CypExpress™ can be reused for multiple reaction cycles
• Addition of Glucose-6-Phosphate Dehydrogenase is not required
• Reactions can be performed in Tris or Phosphate buffers
• Added NADP+ & Glucose-6-Phosphate are not essential (but will boost activity)
• After the reaction, CypExpress™ can be pelleted at relatively low speeds
• Clean HPLC profiles for easy metabolite ID and purification
• Very high percent conversions can be obtained from high concentrations of substrate – making purification easier
• Highly controlled production process for lot-to-lot reproducibility
Using proprietary processes, CypExpress™ are expressed in Pichia yeast. Following optimal expression, the cells are inactivated, permeabalized and dried to a fine powder. CypExpress™ retains the cellular mechanisms to provide the P450 enzymes with the energy and cofactors to continue to function for long experiments, and can generate much larger amounts of metabolite than mammalian microsomes or other genetically engineered expression systems.
To use, just add CypExpress™ to your reaction, mix and agitate (e.g. overnight), and then spin down the CypExpress™ and isolate the product(s). The catalytic activity of traditional systems (microsomal or recombinants) is typically depleted within 20-30 minutes, making it difficult to obtain large quantities of metabolites.
Case Study Using 1A2
Case Study Using 3A4
Case Study Using 2C9
Case Study Using 2C19
Case Study Using 2D6
Case Study Using 1A1
Case Study Using 2E1
The CypExpress™ System
Facile Scale-up Biocatalytic Production of P450 Metabolites Using a Novel Recombinant Human Catalytic System. E. Martinez, et al., International Society for the Study of Xenobiotics. Orlando, FL, USA, 2015.
Evaluation of Dry Powder Preparations of Permeabilized Pichia pastoris Cells Expressing Human Cytochrome P450 2D6 Enzyme (CYP2D6) for Drug Metabolite Generation. S Li, et al. Gordon Research Conference on Drug Metabolism, Holderness, NH, USA, 2013.
Rapid Identification and Production of Metabolites Using Stabilized Dried Powder (SDP) of Human Cytochrome P450s (hCYPS) 2D6, 3A4, and 2C9 Engineered in Yeast. S Das and M Subramanian. Great Lakes Drug Metabolism and Disposition Group, Indianapolis, IN, USA, 2014.
Rapid Identification and Production of Metabolites Using CypExpressTM 2D6, 3A4 and 2C9. M Subramanian, et al. International Society for the Study of Xenobiotics, San Francisco, CA, USA, 2014.