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The Delaware Valley Drug Metabolism: Vendor Show on Wednesday, March 27.

Non-presenting Vendors

  • QPS LLC
  • QRD Pharma
  • BioRepository Resources
  • LabLogic Systems, Inc
  • Discovery Life Sciences / Gentest
  • Eurofins Discovery Services

Schedule

7:30 – 8:30 AM

Registration, networking, visit vendor exhibits, continental breakfast starting at 8:00

8:30 – 8:40 AM

Introduction

8:40 – 9:05 AM

Enhancing Metabolite Identification: Leveraging the Power of Electron-Activated Dissociation (EAD) for Confident Structural Elucidation
David Colquhoun, Ph.D., Market Development Manager, Biomarker Research, SCIEX
Abstract: This work presents a streamlined workflow using the ZenoTOF 7600 system and Molecule Profiler software for metabolite identification. Confident metabolite structure assignments were achieved by utilizing both CID and EAD data. EAD provided more informative MS/MS spectra, enhancing the software-based identification of drug metabolites.

Accurate mass spectrometry, particularly automated LC-MS/MS workflows using CID, has been crucial for investigating candidate modality metabolism in pharmaceutical drug development. Recent advancements, including improvements in duty cycle, enabled the integration of EAD into LC-MS/MS workflows, resulting in more confident characterizations of compounds of interest.

The study utilized the SCIEX OS software platform for data generation and analysis, employing the ZenoTOF 7600 system and Molecule Profiler software. Molecule Profiler allows the consolidation and ranking of structures based on EAD and CID data, making it a valuable tool for comparing MS/MS spectra and identifying unique fragments in a single results file.

9:05 – 9:30 am

Göttingen Minipigs as an Alternative Animal Model to Non-human Primates in Biomedical Research
John Cameron, M.S., Ellegaard BioResearch
Abstract: Over the years, the accepted standard animal models for drug development have been rodent (rat or mouse) and a non-rodent species (dog or NHP). Fairly recently, the supply of NHP have dwindled, causing the cost to skyrocket. With respect to the dog, the industry has become more aware of the ethical considerations, due to their companion status. Because of this, the industry has been looking for a viable alternative non-rodent species to the NHP and dog. Due to its anatomical and physiological similarities to humans, the minipig has emerged as a very viable alternative. The minipig for years has been used for dermal studies, but in recent years it has come to light that many of the organ systems of the minipig are similar to human. In my presentation, I plan to highlight why the minipig should be used as the non-rodent specie instead of either the NHP or dog.

In addition, I plan to speak briefly on two new transgenic strains recently developed. First, the Humanized minipig, which is used for therapeutic Ab studies. This model, if it cross-reacts, will be a better model than the NHP, as it does not produce ADAs to low and moderately immunogenic human antibodies. And finally, one of the biggest obstacles to using the minipig as a non-rodent specie is its size. I plan to briefly mention the newest transgenic strain, the micropig. This is genetically altered by introducing a mutation in the growth hormone receptor, resulting in a minipig that is less than half the size/weight of the standard minipig. The result will be a minipig that is anatomically and physiologically similar to humans, but the size of a beagle dog. This will revolutionize the industry. In fact, in Europe the default non-rodent specie is the minipig.

9:30 – 9:55 am

Pathways Less Traveled – In Vitro Assays to Investigate Non-CYP Biotransformation Pathways
Tom Fleischmann, Q2 Solutions
Abstract: In vitro assays with microsomes and hepatocytes are used to evaluate biotransformation pathways of drugs. The major pathway of xenobiotic metabolism are catalyzed by phase I enzymes, which are mainly cytochrome P450 (CYPs) through chemical reactions such as oxidation, reduction, or hydrolysis. However, chemical modifications to diminish the CYP-mediated drug interaction have resulted in the synthesis of compounds with structural features that favor metabolism by non-CYP pathways. Non-CYP enzymes include UGT, flavin monooxygenase (FMO), monoamine oxidase (MAO), aldehyde oxidase (AOX1), xanthine oxidase (XO), and microflora found in the GI tract. Examples of in vitro assays used to characterize non-CYP pathways will be presented.

9:55 – 10:25 am

Coffee break, networking, visit vendor exhibits

10:25 – 10:50 am

DDI Potential of Major Pharmaceutical Excipients on Transporters and Enzymes
Mark S. Warren, Ph.D., Sr. Study Director & Sr. Principal Investigator, BioIVT
Abstract: Predicting and minimizing the negative impacts of drug-drug interactions has been a goal of regulatory agencies and the pharmaceutical industry for the last couple of decades. Investigating the substrate and inhibition potential of new medications against transporters and metabolic enzymes is routinely requested by regulatory agencies as those drugs progress into the clinic. Less attention is paid to other compounds that are co-administered with those medications, such as pharmaceutical excipients. These excipients are added for a variety of reasons, such as to enhance drug stability or solubility, act as fillers, or aid in the manufacturing process by increasing powder flowability. Pharmaceutical excipients are believed to be inactive ingredients which are added to therapeutic drug products but are not intended to exert therapeutic effects by themselves. However, these excipients may inhibit intestinal transporters that can be responsible for enhancing or reducing the absorption of not only the drug that is administered along with the excipient, but also the absorption of co-medications that have never been tested with that excipient – and thus may be an unexpected source of drug-drug interactions. In addition, excipients may interact with intestinal CYP450 enzymes, altering metabolic profiles. Generic products may contain different excipients than the original branded medication, which may also contribute to unexpected drug-drug interactions. Numerous excipients were tested and shown to inhibit CYP3A4 at relevant concentrations. In addition, these excipients also were shown to inhibit the intestinal drug uptake transporter OATP2B1, as well as intestinal drug efflux transporters BCRP and P gp.

10:50 – 11:15 am

A Workflow for Oligonucleotide Metabolite Identification Studies
Ruisong Pei, Ph.D., Labcorp
Abstract: In recent years, oligonucleotides have emerged as a promising therapeutic area; in particular, for diseases where conventional drugs have not found success [1, 2]. In many cases oligonucleotides are modified from base nucleoside structures to improve delivery, increase stability, and impact their pharmacokinetic profiles [1, 2, 3]. Metabolite identification of the oligonucleotides is an essential step in understanding their stability, metabolism, safety, and elimination within biological systems. Although oligonucleotides are becoming established as a crucial pillar of drug development, best practices for metabolism, and specifically metabolite identification, studies are still being assessed. This abstract presents a metabolite identification workflow for oligonucleotides that includes the key steps and analytical methods involved. The first step in an oligonucleotide metabolite identification study is administration of either cold or radiolabeled oligonucleotide to a system of interest in which the oligonucleotide undergoes biotransformation to various metabolites. Subsequently, relevant matrices are collected for analysis, such as plasma, urine, or tissues. Following collection, fairly extensive sample preparation steps are utilized. In the majority of cases, solid-phase extraction (SPE) is the preferred method of sample clean-up. Sometimes a liquid-liquid extraction step is performed prior to the SPE step or occasionally in isolation; however, liquid-liquid extraction alone tends to produce more difficult to analyze samples. Optimization of SPE steps and conditions can impact oligonucleotide recovery and effectiveness of sample clean-up, but can be dependent on matrix and structure and so must be investigated on a case by case basis [4]. Prepared samples are then analyzed using high-performance liquid chromatography (HPLC) coupled with high-resolution accurate mass (HRAM) mass spectrometry (MS). LC-MS/MS analysis allows for separation and structural characterization of oligonucleotide metabolites with HRAM being required for confident identification of metabolites. In addition to the traditional manual data interrogation for metabolite identification, software such as BioPharma Finder™ software (Thermo Scientific™) and Mass-MetaSite can provide data mining tools and fragment matching based on a known oligonucleotide structure. The ongoing development and improvement of software tools will be a critical element in the success of oligonucleotide metabolite identification studies moving forward. In conclusion, this abstract details a metabolite identification workflow for oligonucleotides. By combining careful sample preparation steps, the advantages of high-resolution mass spectrometry, and powerful software solutions, this workflow enables the identification and characterization of oligonucleotide metabolites in relevant biological matrices for drug development.
References:
1. Takakusa, H., et al. Drug Metabolism and Pharmacokinetics of Antisense Oligonucleotide Therapeutics: Typical Profiles, Evaluation Approaches, and Points to Consider Compared with Small Molecule Drugs, Nucleic Acid Therapeutics, 2023; 33(3):83-94
2. Shadid, M., et al. Antisense oligonucleotides: absorption, distribution, metabolism, and excretion, Expert Opinion on Drug Metabolism & Toxicology, 2021; 17(11):1281-1292
3. Kilanowska, A., et al. Studying in vitro metabolism of the first and second generation of antisense oligonucleotides with the use of ultra-high-performance liquid chromatograph coupled with quadrupole time-of-flight mass spectrometry, Analytical and Bioanalytical Chemistry, 2020; 412:7453-7467
4. Sips, L. et al. LC-MS quantification of oligonucleotides in biological matrices with SPE or hybridization extraction, Bioanalysis, 2019; 11(21):1941-1954

11:15 – 11:40 am

Validated Biomarker Assay for the Analysis of Coproporphyrin I and Coproporphyrin III in Human Plasma
Cindy Rewerts, M.S., Sr. Advisor, Aliri
Abstract: Coproporphyrin I (CP‐I) and Coproporphyrin III (CP‐III) are potential endogenous biomarkers for hepatic organic anion transporting polypeptide (OATP)1B1/1B3 function. We developed and validated a bioanalytical assay for monitoring these biomarkers to assess OATP1B1/1B3 inhibition in place of a standalone prospective clinical drug-drug interaction (DDI) study. Currently, investigational drugs that alter the pharmacokinetics of other medications are subject to additional testing to understand how to manage a DDI risk safely. By monitoring the effect of an investigation drug on the levels of these endogenous substrates of OATP1B1/1B3 in early clinical development, the potential need for a dedicated clinical DDI study could be avoided.

Coproporphyrin I and Coproporphyrin III are extracted from human plasma using a supported liquid extraction methodology. After recovering the analytes with ethyl acetate, the samples were evaporation and reconstitution with formic acid and EDTA solution. The resulting extracts were analyzed by LC-MS/MS detection. The analytes were separated from potential interferences using a reverse phase column and gradient conditions. A Sciex 6500+ mass spectrometer operating in positive electrospray mode was used to detect CP-I, CP-III, and the internal standards. Because CP-I and CP-III are endogenous compounds, calibrators and quality control samples were prepared in charcoal-stripped human plasma. The analytical range of the assay was 50.0 to 5000 pg/mL for both biomarkers.

Understanding the dose-dependent changes in these endogenous substrates of OATP1B1/1B3 makes it possible to identify investigation drugs with a potential DDI risk before conducting clinical DDI studies in patients. To aid in this decision-making process, a method of analysis for coproporphyrin I and coproporphyrin III in human plasma was developed and validated to support regulated clinical studies. This method was validated following the principles outlined in the FDA M10 bioanalytical method validation guidance and from the AAPS “Biomarker Assay Validation by Mass Spectrometry” white paper. Included in the validation were experiments to measure accuracy and precision in an authentic and surrogate matrix, parallelism, and stability experiments.

11:40 – 1:00 pm

Lunch, networking, visit vendor exhibits

1:00 – 1:25 pm

Democratizing the Use of Modeling and Simulation with InSilicoTrials’ Biosimulation Platform
Michael Eckstut, Sr. Advisor, InSilicoTrials
Abstract: The use of in silico trials (biosimulation) is expected to continue to play an increasingly important role in the development and regulatory evaluation of new therapeutic compounds. Among the advantages that in silico approaches offer, is that they permit testing of drug candidates using virtual patients or computational emulations of preclinical experiments, allowing researchers (and regulators) to refine, reduce or even replace time-consuming and costly benchtop/in vitro/ex vivo experiments as well as the involvement of animals and humans in in vivo studies.

InSilicoTrials Technologies has developed a cloud-based bio-simulation platform aimed at democratizing the use of modeling and simulation. This platform supports the creation of dashboard applications for decision-makers, streamlining the transition from traditional methods to advanced in silico trials. The platform hosts healthcare simulation tools – coming from our network of 70+ (and growing!) world-renowned scientific partners – for different bench, preclinical and clinical evaluations, and for diverse disease areas.

This presentation will also discuss two use cases of in silico trials conducted on the InSilicoTrials.com platform. The first illustrates the potential of bio simulation to improve the early preclinical assessment of drug-induced cardiotoxicity risks. The second use case demonstrates the application of virtual patient generation to evaluate treatment effects in multiple sclerosis, showcasing the platform’s capability to enhance and expedite clinical decision-making.

1:25 – 1:50 pm

Total Bioanalytical Solutions for Antibody-Drug Conjugates
David A. Johnson, Ph.D., Sr. Director, DMPK, BioAgilytix
Abstract: Antibody drug conjugates (ADCs) are a somewhat new therapeutic modality compared with small molecule medicines. The concept is that linking a targeting protein to a toxic payload can increase the therapeutic index by lowering systemic exposure to the toxic payload. While this concept has been around for several decades, the practicality of designing and creating these complex molecules has gained traction only in the last 10-15 years. With the increased momentum behind the development of ADCs, there has been an equal increase in the bioanalytical needs for this therapeutic class.

Bioanalytical assay creation involves a sensitive LC-MS/MS method to detect low levels of toxic payload in plasma. This method typically needs to be adapted for analysis of the payload in the targeted tumor tissue as well, and that analysis is complicated by the need to collect a small (1-5 mg) tumor biopsy. Additionally, ligand binding assays for the intact ADC need to be developed, and these assays are impacted by the heterogenous distribution of different numbers of payload molecules attached to the targeting protein. Finally, the stimulation of the immune system by these molecules needs to be assessed, with general anti-drug antibodies being differentiated from neutralizing antibodies.

This presentation will describe development of each of the bioanalytical assays needed to support the advancement of ADCs from the candidate selection phase through the regulatory submission process.

1:50 – 2:20 pm

Coffee break, networking, visit vendor exhibits

2:20 – 2:50 pm

Advances in High End Mass Spectrometry Solutions to Study Metabolism and Distribution Studies for Drug Discovery and Development
Kieron Faherty Ph.D., Americas Field Marketing Manager, Separations and Mass Spectrometry, Waters Corporation
Abstract: Studies used to determine the metabolic fate of a drug molecule that employ HPLC separations of 15-45 minutes are challenged to remain compatible with modern high-throughput drug discovery. However, technologies such as UPLC, ion mobility (IM) spectrometry and high-resolution MS (HRMS), when combined, can achieve high throughput without compromising quality. Ultra-high Performance Liquid Chromatography (UPLC) provides excellent separation efficiency allowing rapid analysis. IM offers an additional separation, resolving co-eluting interferences, and structurally relevant CCS values. Meanwhile HRMS provides excellent data for metabolite identification. Increasing adoption of new techniques like mass spectrometry imaging, further provided detailed information on the spatial distribution of parent drug and its metabolites in ex vivo tissue without having to use radiolabelling. Here we present recent advances in UPLC-IM-HRMS-based metabolite identification and distribution workflow using the example of gefitinib metabolism in mouse.

2:50 – 3:20 pm

Modality specific approaches in in vitro ADME studies
Krisztina Heredi-Szabo, Ph.D., Principal Scientist & Sr. Study Director, Transporter Services, Charles River Labs, Hungary
Abstract: While still small molecules dominate the pipeline of many drug discovery companies, new chemical modalities are emerging to provide better and more specific treatments for several diseases. These new groups of molecules are advancing fast in the development, therefore reliable and solid in vitro ADME results are needed for characterization. New regulatory guidance documents are released around these modalities, but in many cases still the same rules apply for them as for small molecules.

In the current presentation, besides a short introduction to the emerging molecule groups the most frequently raised questions and possible answers will be presented based on the regulatory guidance documents and on in-house experience. Through case studies, we demonstrate different approaches tailored based on the chemical structure and physical-chemical properties of the various molecule groups for in vitro ADME studies.

3:20 – 3:50 pm

Quantitative Whole‑Body Autoradiography (QWBA) in Support of Pre-clinical ADME and Radiation Dosimetry for Clinical hAME Studies
Michael J. Potchoiba, QWBA COE Functional Leader & Study Director, Frontage Laboratories, Inc.
Abstract: Evaluating the distribution of radiolabeled drug candidates by QWBA provides pharmacokinetic data required for predicting the potential tissue deposition of an absorbed dose of radioactivity in human subjects. QWBA is unique in allowing visual and quantitative evaluation of radioactivity in small anatomical structures which otherwise could not be detected or measured by conventional radiometric techniques. QWBA studies can be designed to investigate if a drug candidate crosses the blood-brain barrier, its accumulation or retention in tissues, distribution into tumors, detection of tumor growth inhibition, distribution to a target tissue of interest, placental transfer, and lacteal excretion.

Regulatory agencies mandate pre clinical tissue distribution studies before permitting the study of radiolabeled drug candidates in human subjects. QWBA tissue concentration data supports radiation dosimetry required by regulatory agencies. Radiation absorbed doses (RADhuman) for a single administration of a carbon 14 drug candidate to human subjects must not exceed 3000 mrem for active blood forming organs, gonads, eye lens, and whole body. RADhuman for all other tissues must not exceed 5000 mrem after a single dose of radioactivity. The ICRP established a 1 mSv limit for human whole-body exposure. Conducting a QWBA study is essential to ensure the safety of human subjects administered a dose of carbon 14 radioactivity. Radiation dosimetry is typically based on a 100 µCi single dose of radioactivity administered to human adult subjects.

In summary, quantifying the distribution of radiolabeled drug candidates in tissues, determining elimination rates, and calculating radiation dosimetry prior to conducting human radiolabeled clinical trials are important for drug development.

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