Small Molecule Metabolites in Drug Discovery and Development
An ISSX Virtual Workshop Event | March 1-3, 2023
Wednesday, March 1, 2023
10:00 am - 2:00 pm ET (US)
Session 1
Chairs: Aaron Teitelbaum, Bingming Chen, Rheem Totah, and Valerie Kramlinger
10:00 am - 10:05 am | Welcome and Introduction
10:05 am - 10:45 am | The Importance of Drug Metabolites: Examples of Active and Reactive Metabolites
Rheem Totah, University of Washington
Abstract: Most drugs are generally hydrophobic to achieve the delicate balance between solubility and partition. As a result, they tend to accumulate over time in the various compartments in the body. Several enzyme systems exist that are capable of chemically modifying hydrophobic drugs, and other xenobiotics, to more hydrophilic, and thus water-soluble, metabolites facilitating excretion from the body. These enzyme systems are loosely classified into phase I enzymes that generally perform oxidation, reduction, and hydrolysis reactions, and Phase II enzymes which usually conjugate the xenobiotics, or their metabolites, to hydrophilic endogenous compounds to further increase their water solubility. In some cases, the generated metabolites are active prolonging the pharmacological effect of the drug. In other cases, metabolites can be reactive and cause inhibition of the parent enzyme, or other enzymes, leading to complicated pharmacokinetics that can result in drug-drug interactions. Fully characterizing the metabolism of a drug will lead to a better understanding of pharmacokinetics, half-life, and pharmacological properties of these drugs. Examples illustrating various metabolism pathways resulting in further activity or reactivity will be presented.
About the Speaker: Dr. Rheem Totah is a Professor in the Department of Medicinal Chemistry, School of Pharmacy, at the University of Washington. She received her PhD in Medicinal Chemistry from the University of Kansas in Lawrence, Kansas, under the mentorship of Dr. Robert Hanzlik. She was a Plein Fellow for two years in the School of Pharmacy at the University of Washington before joining the faculty as an Assistant Professor. Dr. Totah leads an active research program funded by NIH and industry. Her research is focused on extrahepatic drug biotransformation, drug induced cardiotoxicity and the role of thiol methyl transferases in drug metabolism and cell function. In the past she served in several capacities and on ad hoc committees at NIH, NSF, and the NIH/FDA Tobacco Regulatory Affairs and is currently a standing member of the NIH Integrative Myocardial Physiology/Pathophysiology B study section. She currently serves on the steering committee for the ISSX Biotransformation, Mechanisms and Pathways focus group. She is also a member of the 2023, 25th North American meeting organizing committee, and the academic co-chair for the 37th National Medicinal Chemistry Symposium in 2024.
10:45 am - 11:30 am | Implementation of Innovative HRMS Instrumentation Strategies to Help Accelerate Metabolite Identification and Preclinical Drug Discovery
Mark Cancilla, Merck
Abstract: Merck Research Laboratories has been investigating the incorporation of advanced high-resolution mass spectrometry (HRMS) acquisition and ion dissociation techniques to more definitively identify unknown metabolites. A review of our established and future HRMS-based workflows are provided with real-world examples in aiding design and decision making in the drug discovery design-make-test cycles. First, the advantages and integration of variable window SWATH acquisition into routine metabolism workflows will be reviewed. The second discussion will center around the use of alternative fragmentation and information collection to attempt real time definitive drug metabolite structure elucidation. Our experiences with using ultraviolet photodissociation (UVPD) and electron activated dissociation (EAD) to provide increased informative fragmentation to identify sights-of-metabolism will be highlighted. Finally, the use of ion mobility spectrometry (IMS) to calculate collision cross sections (CCS) to identify structural isomers utilizing a deep neural network with a metabolite-specific compound library will be reviewed. We envision these HRMS platforms will allow us to provide more efficient support and valuable impact across our active drug discovery pipeline.
About the Speaker: Dr. Mark Cancilla is a Director at Merck & Co. He received his Ph.D. in analytical chemistry from the University of California at Davis and was a postdoctoral fellow at the University of California, Berkeley Chemistry Department. As Director of the Biotransformation and Distribution Group within the Preclinical Development Department at Merck, Mark’s team is responsible for supporting in vitro and in vivo metabolism and distribution investigations for the entire pre-clinical and clinical drug portfolio. His research focuses on implementing innovative analytical technologies and software driven solutions to better understand the biotransformation of complex therapeutics.
11:30 am - 11:45 am | Break
11:45 am - 12:30 pm | Modern Method of Metabolite ID: Part 2. 1D and 2D NMR
Gregory Walker, Pfizer
Abstract: In both drug discovery and development, the structural characterization of metabolites has had an increasing importance over the last fifteen years. Unquestionably, the definitive analytical techniques for the elucidation of metabolite structures are high resolution mass spectrometry and NMR spectroscopy. High resolution mass spectrometry provides an empirical formula with given ratios of atoms which can guide the interpretation of NMR data. NMR data sets deliver the order in which the atoms are arranged. These two techniques are most powerful when their data are used together for structural proofs. The relatively low abundance of metabolites in complex matrices like urine, feces and plasma necessitates the need for both good chromatographic separation techniques as well as maximal analytical sensitivity. While mass spectrometry has historically been the much more sensitive spectroscopic technique, large strides in sensitivity have been achieved in NMR since the advent of micro cryo-probes. These probes, in conjunction with more advanced two-dimensional NMR experiments and higher field instruments have greatly reduced the amount of material required for the full structural characterization of a metabolite. This workshop will cover a brief review of the fundamentals of qualitative 1D and 2D NMR spectroscopy as well as the quantitative nature of 1D NMR. Additionally, I will show examples of how these advances in instrumentation can be leveraged to help solve some of the challenges in drug metabolism studies.
About the Speaker: Gregory S. Walker is an Associate Research Fellow at Pfizer Inc. with 35 years of experience in both pharmaceutical science and drug metabolism. Currently, he manages the drug metabolism NMR facility at Pfizer's research headquarters in Groton, CT. For the past 30 years he has worked in NMR groups at The Upjohn Company, Pharmacia and Pfizer. Greg has specialized in the structural characterization of low level unknown organic molecules originating from complex matrices using a variety of chromatographic and spectroscopic techniques. His career highlights include establishing NMR as a global analytical resource for the Pfizer drug metabolism division, developing a generalized quantitative NMR assay for qualification of bio-generated analytical standards and 80+ external publications and invited presentations. Greg is a member of the APA organizing committee and was chair of the SMASH 2018 NMR meeting. Greg is a recognized expert in structural elucidation of metabolites and has taught numerous short courses for such organizations as the American Chemical Society, Applied Pharmaceutical Analysis (APA) and International Society for the Study of Xenobiotics (ISSX). Greg is a two-time recipient of the Pfizer Achievement Award, most recently for his work in quantitative NMR as it pertains to drug metabolism.
12:30 pm - 1:15 pm | Predicting Human Metabolite Levels From in vitro Data
Ernesto Callegari, Pfizer
Abstract: Since metabolites can play a role in efficacy, toxicity and drug-drug-interactions, assessment of metabolite exposures and their effects are required by regulatory agencies. The objective of this work is to develop mechanistic static and PBPK models to predict circulating metabolite levels in humans from in vitro data, which can be applied in drug discovery to prioritize metabolites for further assessment. A dataset of compounds from different chemical classes with measured metabolite and parent levels in clinical studies were used to test the modeling approaches. The concepts and equations of the mechanistic static model will be reviewed and results from both mechanistic static and PBPK models using a dataset of compounds will be presented.
About the Speaker: Ernesto Callegari is a Research Fellow in Pharmacokinetics Dynamics and Metabolism at Pfizer Groton CT Laboratories. He earned a B.S. in Pharmacy from the University of Connecticut and a Ph.D. in Bioorganic Chemistry from Brandeis University. Dr. Callegari then joined Pfizer Drug Metabolism. Over the past 25 years, Dr. Callegari has held several scientific and management leadership positions in drug metabolism at Pfizer. He applied his pharmaceutical industry experience to numerous projects from a variety of therapeutic areas across drug discovery, development and through post-marketing. Dr. Callegari is responsible for the DDI and drug metabolism strategy for multiple drug development candidates and has developed PBPK models that addressed regulatory queries and supported waivers for clinical DDI studies.
1:15 pm - 2:00 pm | Accessing Metabolites of Various Types – Biosynthesis, Isolation Methods
Elizabeth Gillam, The University of Queensland
Abstract : Metabolite identification (MetID) is an essential component of safe and efficient drug development pipelines. Typically, detailed structural identification requires access to up to ~mg quantities of authentic metabolites and considerably more may be needed for activity and toxicity testing of metabolites. Ideally, metabolites could be sourced from biotransformations using human or animal tissue fractions or biological samples such as urine. However, such sources rarely yield substantial quantities of metabolites, and the work-up of a given metabolite is complicated by both the biological milieu and the presence of other, confounding metabolites. Chemo-, regio- and stereo-selective modification of structurally complex drug molecules is challenging for chemocatalysis, but enzymes, as biological catalysts with malleable, three-dimensional binding sites, can easily discriminate between the sites and faces of a molecule. This presentation will explore biocatalysis using engineered recombinant enzyme systems as an alternative for producing metabolites in quantity and how this can be applied to relieve other medicinal chemistry bottlenecks by accelerating structure-activity relationship analysis and late-stage functionalization of drug candidates.
About the Speaker: Elizabeth Gillam is a Professor of Biochemistry at the University of Queensland (UQ, Brisbane, Australia) with over 30 years experience in working on cytochrome P450 enzymes. She earned her B.Sc. with Hons and a University Medal in Biochemistry at UQ and her D. Phil at Oxford University. Elizabeth then joined Fred Guengerich’s lab at Vanderbilt University where she was responsible establishing many of the first bacterial expression systems for major drug metabolizing enzymes, e.g. CYP3A4, CYP2D6 and several others. A Past Secretary and MAC chair of ISSX amongst other roles, she has used recombinant systems to study the molecular basis to human drug metabolism and adverse drug reactions throughout her career. For the last ~ 20 years, Elizabeth’s research has focused on engineering cytochrome P450 enzymes as biocatalysts for drug development, plant biotechnology and other chemical industries. Recently, her group has devised methods to efficiently navigate the substrate and reaction specificity landscape of P450s, and developed small, targeted libraries of stable, solvent-tolerant and highly active enzymes as off-the-shelf reagents for applications in the pharmaceutical industry. Elizabeth's current research involves applying synthetic biology approaches to P450 systems to improve the efficiency of metabolite synthesis for structural identification, and to expand the range of chemistries that can be achieved with these versatile biocatalysts.
2:00 pm - 2:30 pm | Break
2:30 pm - 3:30 pm | Roundtable Discussion with Speakers
