ARTICLE

Defining Pre-Clinical Drug Development Needs for Physiological Relevance

Introduction

Screening leads is one of the most important stages in the drug development pipeline, due to its role in foundational decision-making that guides downstream development activities.

Pharmaceutical companies invest hundreds of millions of dollars into the drug development process, and an ineffective lead can result in not only significant financial setbacks, but also lost confidence by investors and the opportunity cost for time that could have been used to research other leads.

Labs seek to utilize technologies that deliver high levels of accuracy for drug candidate screening and preclinical development. This translates to a combination of sensitivity and enhanced specificity to predict in vivo behavior of selected compounds.


An increasing drive to enhance predictability of the effects of the drug in the human body has led to paradigm shifts in preclinical drug development. In the context of this predictability need, there is a growing trend to use cell-based assays - as well as 3D cell and tissue model systems - to replace 2D models.

The transition is primarily motivated by the increased physiologic relevance of 3D models compared to 2D cell cultures, which offers a wide spectrum of benefits in the drug discovery process. As a result, the use of physiologically relevant model systems could potentially enhance attrition rates of drug candidates and allow them to "fail fast," preventing downstream failures and conserving both costs and effort.

Since 3D models are more complex, the transition from 2D necessitates certain prerequisites. For example, training and educating staff to handle and manage the complexity of these model systems, as well as availability and access to supporting technologies, techniques, and protocols. Although a number of technology providers are developing and commercializing 3D cell and tissue culture systems and supporting tools, PerkinElmer offers a leading high content imaging system, the Opera Phenix® Plus, along with the Columbus™ image data storage software and analysis system. Additional PerkinElmer detection solutions include assay technologies such as AlphaLISA®, DELFIA® TRF, HTRF® and LANCE® Ultra TR-FRET, and lites® luminescence, EnSight and EnVision® multimode plate readers, and Signals Screening software.

3D versus 2D screening

The 2D screening modalities are declining as a result of their inconsistent ability to select for clinically-relevant drug candidates. In vitro effects can vary significantly from in vivo, especially the interaction of particular proteins that may prefer certain physiologic conditions over others.

In order to seamlessly transition from in vitro or ex vivo to in vivo, and avoid late-stage failure in development, 3D cell culture models can enable failure earlier in the process, and subsequent selection of therapeutic candidates with greater efficacy. Although 2D drug models facilitate high throughput screening, a greater physiological relevance to the in vivo environment is accomplished with 3D models. Traditional 2D models involve use of a monolayer of cultured cells on a plastic surface that has been treated to adhere cell or tissue under investigation.

While 2D was the predominant cell culture format used for decades, it does not represent an in vivo type physiological environment like 3D models do. 3D models employ complex tissue type environments and can form multi-dimensional structures such as spheroids and organoids that closely mimic in vivo in a way that 2D cultures cannot. Enzymatic and protein-protein interactions can be very specific, so having the additional cell morphology characteristics can make a significant difference.

The complexity of 3D systems facilitates multiparametric study of biological data points, ranging from overall cell number to viability, proliferation characteristics, response to stimuli, and more. The ability to obtain data that mimics in vivo conditions has helped prevent late-stage drug failures.

3D screening adds variables

Adding an extra dimension to an assay configuration can lead to a surprising number of new variables. Firstly, the overall stability of the 3D assay is important, which can dictate testing time and environment considerations. Secondly, the selection of an appropriate medium or substrate becomes particularly key. Choices such as synthetic versus biologic matrix can impact results that eventually determine translation to clinical research.

Besides the biochemical complexity of a 3D model, other considerations need to be taken into account, for example whether to monitor protein expression, as well as factors such as assay parameters, signal-to-background optimization, data analysis, and data management. In addition, 3D cell culture scale-up is key to providing appreciable culture volume to support multiple assays, which currently is a laborious process.

Requirements for working with 3D models

In addition to the considerations above, new lab methods, instrumentation and software are critical to support these more complex assays.

For example, to enable reproducible and consistent growth of 3D cells, specialized microplate technologies and substrates have been developed by the technology providers, such as PerkinElmer’s CellCarrier™ ULA microplates which are designed to ensure minimal cell-plate adhesion, promoting uniform and single-spheroid formation. The growth and health of the resulting cultures can then be monitored using imaging systems like the EnSight multimode plate reader.

Furthermore, functionality, cell viability and proliferation of the spheroids or other 3D cell cultures can be evaluated by luminescence assays such as the ATPlite 3D and ATPlite 1step 3D reagent kits directly within the culture plate, avoiding sample transfer challenges and facilitating automation. An entire physiologically relevant workflow end-to-end can be supported via such solutions.

Based on the goal of the study, high content imaging can be leveraged for multiparametric studies to decipher sub-cellular content rich information from 3D cells. Advanced systems such as the Opera Phenix® Plus have been developed for 3D applications that can handle imaging thicker samples. Without photo damage to live cells, the system’s proprietary Synchrony Optics™ enables simultaneous multicolor confocal image acquisition at speed and high sensitivity.

Working with 3D models requires researchers to be able to account for the behavior and characteristics of multiple cell types and morphologies within a single assay. Although the interaction of the different cell types yields critical information, so does the behavior of each individual cell. In addition to reagent considerations, moving from 2D to 3D screening invariably requires more sophisticated instrumentation, including advanced singleplex and multiplex readers for analysis of biomarkers identified in 3D cell cultures.

PerkinElmer’s EnVision® Multimode Plate Reader is a strong solution, providing exceptional speed, ultra-high-throughput screening, and maximum sensitivity across all detection points. The EnVision system delivers high sensitivity for time-resolved fluorescence (TRF) applications, is compatible for use with Alpha, HTRF® and DELFIA® assay technologies, and its Enhanced Security software option includes tools to facilitate 21 CFR Part 11 compliance for integration into regulated GxP environments.

Furthermore, a lab’s informatics capabilities must be able to store vast volumes of data generated when working with 3D cells and analyze it effectively to aid decision making. With the Columbus image data storage and analysis system, large volumes of data can be safely stored and shared across teams to analyze data, leveraging cutting-edge machine-learning and AI capabilities, which can be further enhanced by the Signals Screening informatics platform leveraging the TIBCO Spotfire® advanced analytics tool. Capable of incorporating dimensionality reduction, advanced statistics, and on-the-fly imaging rendering for instant validation needs, Signals Screening also enables reduced processing time.

Conclusion

The transition from 2D to 3D cell-based assays has signaled a shift toward increased adoption of physiologically relevant models in drug screening and development. Although the more sophisticated 3D assays offer this dynamic relevance, they are also more complex to design and manage. Managing the complexity of 3D assays, which extends to the instruments themselves as well as the vast amounts of data collected, often requires labs to adopt a new suite of technologies specifically geared toward 3D cell-based assays.

PerkinElmer is prepared to support these needs through instrumentation which can capture the information-rich data these more complex and physiologically relevant models offer and software offerings, which allow for better multiparametric data visualization to mine the data and allow for better decision-making – to either fail faster or take leads forward with more confidence.

Resources

  1. https://journals.sagepub.com/doi/full/10.1177/2472555219830087
  2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4394490/
  3. https://www.frontiersin.org/articles/10.3389/fphar.2018.00006/full
  4. https://www.perkinelmer.com/product/envision-hts-plate-reader-2105-0010
  5. https://www.perkinelmer.com/product/high-content-profiler-hcprofiler
  6. https://www.perkinelmer.com/lab-solutions/resources/docs/BRO_Columbus-Brochure_008364_01.pdf
  7. https://www.perkinelmer.com/product/image-data-storage-and-analysis-system-columbus
  8. https://www.sciencedirect.com/topics/computer-science/dimensionality-reduction