Despite billions in annual investment, most drugs never reach the market because preclinical experiments fail to predict human effects. In this webinar, we will cover why in vivo models only deliver an approximation of what is likely to happen in the human body and why building more predictive, human-relevant models is key to reducing drug attrition. During the webinar we will introduce ADME scientists to the field of microphysiological systems (MPS), also known as organ-on-a-chip (OOC).

We will explore how MPS technology can be implemented to create human-specific tissue models such as the gut and liver and interconnect them into systems that generate clinically translatable data. We will demonstrate how a deeper and earlier knowledge of human drug ADME profiles enables issues to be addressed prior to costly preclinical studies.

The webinar will conclude by highlighting how the complementary use of MPS bridges the human-relevance gap, facilitating the confident selection of safer and more effective drug candidates, whilst also reducing costs.

This webinar is sponsored by CN Bio Innovations.

The African continent harbors unparalleled genetic diversity yet remains largely underrepresented in  pharmaceutical  research  and  development.  Pharmacogenomics  is  fundamental  to  precision medicine strategies, and although to date largely absent from implementation, representing the genetic diversity of the African population within the laboratory is critical to the democratization of stratified  and  effective  healthcare  in  Africa.  Afrocentric  preclinical  resources  could  support precision  medicine on  the  continent  and  reshape  the  global  underrepresentation  of  African genetics  in  science. Models  which  could  begin  to  address  this  include  primary  human hepatocytes,  immortalized  hepatic  cell  lines,  and human induced  pluripotent  stem  cell  (hiPSC)derived hepatocyte-like cells – derived from individuals of African genetic ancestry. The feasibility of an African hepatic modeling platform is slowly being realized due to the convergence of several technologies and methodologies which have traditionally been siloed on the continent.

Abstract coming soon.

Part 1: 2-Pyridine Carboxaldehyde for Semi-Automated Soft Spot Identification in Cyclic Peptides: Cyclic peptides are an attractive option as therapeutics due to their ability to disrupt crucial protein-protein interactions and their flexibility in display type screening strategies, but they come with their own bioanalytical challenges in metabolite identification. Initial amide hydrolysis of a cyclic peptide results in a ring opening event in which the sequence is linearized. Unfortunately, the mass of the singly hydrolyzed sequence is the same (M + 18.0106 Da) irrespective of the initial site of hydrolysis, or soft spot. Soft spot identification at this point typically requires time-consuming manual interpretation of the tandem mass spectra, resulting in a substantial bottleneck in the hit to lead process. To overcome this, derivatization using 2-pyridine carboxaldehyde, which shows high selectivity for the alpha amine on the N-terminus, was employed. This strategy results in moderate- to high-efficiency derivatization with a unique mass tag and diagnostic ions that serve to highlight the first amino acid in the newly linearized peptide. The derivatization method and analytical strategy are demonstrated on a whole cell lysate digest, and the soft spot identification strategy is shown with two commercially available cyclic peptides: JB1 and somatostatin. Effective utilization of the automated sample preparation and interpretation of the resulting spectra shown here will serve to reduce the hit-to-lead time for generating promising proteolytically stable peptide candidates.

Part 2: Rapid and Definitive Identification of Cyclic Peptide Soft Spots by Isotope-Labeled Reductive Dimethylation and Mass Spectrometry Fragmentation: Cyclic peptides have been an emerging therapeutic modality over the past few decades. To identify drug candidates with sufficient proteolytic stability for oral administration, it is critical to pinpoint the amide bond hydrolysis sites, or soft spots, to better understand their metabolism and provide guidance on further structure optimization. However, the unambiguous characterization of cyclic peptide soft spots remains a significant challenge during early-stage discovery studies as amide bond hydrolysis forms a linearized isobaric sequence with addition of a water molecule regardless of the amide hydrolysis location. In this study, an innovative strategy was developed to enable rapid and definitive identification of cyclic peptide soft spots by isotope-labeled reductive dimethylation and mass spectrometry fragmentation. The dimethylated immonium ion with enhanced MS signal at a distinctive m/z in MS/MS fragmentation spectra reveals the N-terminal amino acid on a linearized peptide sequence definitively, and thus significantly simplifies the soft spot identification workflow. This approach has been evaluated to demonstrate the potential of isotope-labeled dimethylation to be a powerful analytical tool in cyclic peptide drug discovery and development.

This webinar is organized by ISSX M&S and transporter FG jointly. It will cover from in vitro to the human PK prediction for transporter substrates with focus on the current challenge and possible solutions related to:

1) Critical In vitro methods /parameters needed for a reliable PBPK model predictions, data quality, gaps and how to overcome the limitations of in vitro system; 

2) Key model structure and population parameters (including special populations) for transporter substrates - gaps in addressing multiple transporter involvement and interplay with enzymes; 

3) Status of model validation for index substrates, applications in regulatory interaction and filing -challenges and future direction.

The webinar will include sufficient time for Q&A with invited field expert as panelists.

Background: siRNA is a promising therapeutic modality highlighted by several US FDA approvals since 2018, with many more oligonucleotide assets in clinical development. To support siRNA discovery and development, robust and sensitive quantitative platforms for bioanalysis must be established to assess pharmacokinetic/pharmacodynamic relationships and toxicology. Droplet digital PCR offers improved sensitivity and throughput, as well as reduced susceptibility to matrix effects, compared with other analytical platforms. Methodology: The authors developed a stem-loop reverse transcription droplet digital PCR method to measure siRNA in mouse plasma and liver extract using bioanalytical method qualification guidelines. Conclusion: This newly developed assay has been demonstrated to be a superior alternative to other platforms, with the added benefit of greater sensitivity, with dynamic range from 390 to 400,000 copies/reaction and readiness for FDA investigational new drug-enabling applications.

This ISSX webinar will feature three presentations! The webinar will also recognize graduate students and postdoctoral fellows from academia who carried out transporter-related research and presented their work in the form of a poster at the 2023 North American ISSX meeting. The graduate student presenters will be introduced by their mentors, who will also participate in the question and answer portion of the webinar.

PREDICTION AND VERIFICATION OF FETAL EXPOSURE TO DUAL BCRP/P-GP DRUG SUBSTRATES USING THE PROTEOMICS INFORMED EFFLUX RATIO-RELATIVE EXPRESSION FACTOR (ER-REF) APPROACH AND PBPK MODELING AND SIMULATION

To inform fetal drug safety, it is important to determine or predict fetal drug exposure throughout pregnancy. The former is not possible in the first or second trimester. In contrast, at the time of birth, fetal drug exposure, relative to maternal exposure, can be estimated as Kp,uu (unbound fetal umbilical venous (UV) plasma area under the curve (AUC)/unbound maternal plasma (MP) AUC), provided the observed UV/MP values, spanning the dosing interval, are available from multiple maternal-fetal dyads. However, this fetal Kp,uu cannot be extrapolated to other drugs. To overcome the above limitations, we have used an efflux ratio-relative expression factor (ER-REF) approach to successfully predict the fetal Kp,uu of P-gp substrates. Because many drugs taken by pregnant people are also BCRP substrates, here, we extend this approach to drugs that are effluxed by both placental BCRP and P-gp or P-gp alone. To verify our predictions, we chose drugs for which UV/MP data were available at term: glyburide and imatinib (both BCRP and P-gp substrates) and nelfinavir (only P-gp substrate). First, the ER of the drugs was determined using Transwells and MDCKII cells expressing either BCRP or P-gp. Then, the ER was scaled using the proteomics-informed REF value to predict the fetal Kp,uu of the drug at term. The ER-REF predicted fetal Kp,uu of glyburide (0.43), imatinib (0.42), and nelfinavir (0.40) fell within two-fold of the corresponding in vivo fetal Kp,uu (0.44, 0.37, and 0.46, respectively). These data confirm that the ER-REF approach can successfully predict fetal drug exposure to BCRP/ P-gp and P-gp substrates, at term.

METABOLOMICS-BASED BIOMARKER APPROACH FOR IDENTIFICATION AND VALIDATION OF CLINICAL ENDOGENOUS BIOMARKERS OF RENAL ORGANIC ANION TRANSPORTERS

Endogenous substrates of drug transporters can be used as phenotypic biomarkers to predict transporter-mediated drug-drug interactions (DDIs), drug toxicity, and inter-individual variability in transport function. Inhibition of renal organic anion transporter (OAT) 1 and 3-mediated uptake and multidrug resistance-associated protein (MRP) 2 and 4-mediated efflux can increase plasma concentration and decrease urine amount of endogenous substrates, respectively, which can be used as clinical biomarkers. We identified and validated clinically relevant biomarkers of OAT1/3 and MRP2/4 using a state-of-the-art metabolomics-based DMET biomarker discovery (MDBD) approach. Untargeted metabolomics of the plasma and urine samples collected from a clinical furosemide/probenecid pharmacokinetic DDI study (n=16) yielded >10,000 features (m/z values) that were either increased or decreased in plasma and urine samples in the presence of probenecid (dual OAT/MRP inhibitor). Features that were significantly increased in plasma (1.5 to 10-fold) and were either decreased or non-significantly increased in urine samples were characterized as putative OAT1/3 biomarkers. Similarly, identification of putative MRP2/4 biomarkers included features that were significantly decreased in urine (by >45%) with non-significant change in plasma. Putative OAT1/3 biomarkers (m/z range, 130-384) belonged to the following pathways: tryptophan metabolites, steroids, peptides, glucuronide conjugates, and fatty acids. MRP2/4 biomarkers (m/z range, 110-510) were majorly bile acids, fatty acids, steroids, glycosides, and peptides. In vitro validation of the identified OAT1/3 biomarkers confirmed 12 putative OAT1 substrates as OAT1 biomarkers (>1.4-fold uptake in transporter over-expressing cells, p-value <0.05), 17 as OAT3 biomarkers and 21 as dual OAT1/3 biomarkers. This high-throughput and robust MDBD approach to identify and validate biomarkers is applicable beyond transporters.          

HOW MUCH IS ENOUGH? BI-907828: A MDM2-P53 ANTAGONIST WITH LIMITED BBB PENETRATION BUT POTENT EFFICACY IN GLIOBLASTOMA MDM2–p53 inhibition may be effective in glioblastoma (GBM). This study evaluates the pharmacokinetics/pharmacodynamics of BI-907828, a potent antagonist of MDM2, in GBM, and demonstrates a translational paradigm with a focus on a unified “Delivery – Potency – Efficacy” relationship in drug development for central nervous system(CNS) tumors. BI-907828 was tested for cytotoxicity and MDM2–p53 pathway inhibition. Systemic pharmacokinetics and transport mechanisms controlling CNS distribution were evaluated in mice. BI-907828 free fractions in cell media, mouse and human specimens were measured to determine “active” unbound concentrations. Efficacy measures, including overall survival and target expression were assessed in mouse orthotopic GBM xenografts. BI-907828 exhibited potent inhibition of MDM2–p53 pathway and promoted cell death in GBM TP53 wild-type cells. MDM2-amplified cells are highly sensitive to BI-907828, with an effective unbound concentration of 0.1 nmol/L. The CNS distribution of BI-907828 is limited by blood–brain barrier (BBB) efflux mediated by P-gp, resulting in a Kp,uu_brain of 0.002. Despite this seemingly “poor” BBB penetration, weekly administration of 10 mg/kg BI-907828 extended median survival of orthotopic GBM108 xenografts from 28 to 218 days (P < 0.0001). This excellent efficacy can be attributed to high potency, resulting in a limited, yet effective, exposure in the CNS. These studies show that efficacy of BI-907828 in orthotopic models is related to high potency even though its CNS distribution is limited by BBB efflux. Therefore, a comprehensive understanding of all aspects of the “Delivery – Potency – Efficacy” relationship is warranted in drug discovery and development, especially for treatment of CNS tumors.

The ISSX New Investigator Group will present this Fireside Chat with Dr. Eric Chan, an influential scientist in the field. This chat session will be moderated by a member of the group and the discussion will include wide-ranging topics such as current scientific trends, leveraging your network, and thinking strategically about your career in industry, academia, and/or government.

About the Speaker:

Dr. Eric Chan is a professor at the Department of Pharmacy and Pharmaceutical Sciences, National University of Singapore. He received his Ph.D. from the same department and was a postdoctoral research scientist at S*BIO Pte Ltd, a joint venture between the Economic Development Board of Singapore and Chiron Corporation at Emeryville, California. As professor, his research interests are (1) metabolism-driven systems biology modelling of diseases, pharmacology, toxicology and mammalian host-bacteria interactions and (2) xenobiotic-derived reactive metabolite research with specific focus on interaction with biological proteins and physiological-based pharmacokinetics-pharmacodynamics (PBPK-PD) modelling of pharmacology and toxicology. He advocates #LearningWithoutBarrier and has been sharing his knowledge generously via his #WeeklyDoseOfPK in LinkedIn. 

Selective chemical inhibitors are critical for reaction phenotyping to identify drug-metabolizing enzymes that are involved in the elimination of drug candidates. Although relatively selective inhibitors are available for the major cytochrome P450 enzymes (CYP), they are quite limited for the less common CYPs and non-CYPs. To address this gap, we developed a multiplexed high throughput screening (HTS) assay using 20 substrate reactions of multiple enzymes to simultaneously monitor the inhibition of enzymes in a 384-well format. Four 384-well assay plates can be run at the same time to maximize throughput. This is the first multiplexed HTS assay for drug-metabolizing enzymes reported. The HTS assay is technologically enabled with state-of-the-art robotic systems and highly sensitive modern LC-MS/MS instrumentation. Virtual screening is utilized to identify inhibitors for HTS based on known inhibitors and enzyme structures. Screening of ~4000 compounds generated many hits for many drug-metabolizing enzymes. This webinar will highlight the characterization of selective inhibitors of CYP3A5 (loteprednol etabonate) and AO (erlotinib and dibenzothiophene). We encourage colleagues from other organizations to explore their proprietary libraries using a similar approach to identify better inhibitors that can be used across the industry.

This workshop is organized through the efforts of the ISSX Transporters Focus Group and is organized by Drs. Aleksandra Galetin, Xinning Yang, Bhagwat Prasad, Xiaoyan Chu, Hong Shen, Pei Feng Shawn Tan (student member), Mitesh Patel, Eva Gil Berglund, and Chitra Saran.

This virtual workshop will bring together scientists from academia, industry, and regulatory agencies to share their research findings, experience, and expert views on endogenous biomarkers for drug transporters, an active research area with rapid advances in recent years. 

The three-day workshop will cover several important topics in these areas: 

-application of transporter biomarker in drug development,
-understanding transporter function changes in specific populations facilitated by biomarker assessment, 
-methods to identify transporter biomarkers, and 
-emerging biomarkers of efflux transporters.  

Each session of the workshop consists of presentations from several expert speakers followed by roundtable discussions, with case study presentations incorporated into one session to showcase the experience and advances in using transporter endogenous biomarkers during drug development. In addition, there will be an interactive virtual poster session, which will be selected from the pool of abstracts from trainees and industry attendees.

Your workshop registration includes access to all presentation slides and recordings.