OPTI-EXPRESSION Technology is a proprietary, transient transfection technology that allows simultaneous transfection of multiple transporter genes into polarized mammalian cell monolayers. This patented technology provides the following benefits when investigating the role of transporters in pharmacokinetic research:
- More efficient and consistent gene expression than conventional stable transfection methods
- Rapid establishment of custom transporter assays and multi-transporter models
- Generates exactly matched controls for more accurate assessment of transporter activity
- Allows quantitative expression of transporters, in order to mimic in vivo expression levels
- Ideal for conducting DDI risk assessment studies, transporter screening and mechanistic studies
Research Services Using OPTI-EXPRESSION Technology
BioIVT provides collaborative research services to assess transporter-mediated disposition in drug discovery and development. From lead candidate selection, to IND-enabling studies, through clinical development, we provide customized research programs to investigate transporters of interest in critical organs including liver, kidney, intestine, and the blood-brain barrier. We help design research studies based on our clients’ needs and objectives, and select from over 100 well-characterized transporter assays and panels, including the following programs:
- Single Transporter Assays: Studies with 1 transporter overexpressed
- Multi-Transporter Models: Studies with up to 8 transporters overexpressed simultaneously
- OPTI-REGULATORY Panel: Includes the 11 transporters recommended for evaluation by key regulatory agencies
- OPTI-DDI Panel: Includes transporters typically used in DDI risk assessment
- OPTI-TARGET Panel: Targets/identifies transporters involved in drug therapy, toxicity, and delivery
- OPTI-ADME Panel: Identifies transporters that affect the ADME properties of potential drug candidates
- OPTI-SAFETY Panel: Identifies potential safety problems early in the drug selection process
- OPTI-CNS Panel: Identifies and characterizes innovative targets for the treatment of neurological diseases
Transporter Kits Using OPTI-EXPRESSION Technology
We use OPTI-EXPRESSION Technology to produce TRANSFLEX™ Plates giving researchers a convenient way to conduct transporter assays in their own laboratories. Our assay-ready kits include transporter plates with MDCK-II cells that overexpress transporter proteins of interest. They require minimal cell culture and maintenance, and are available in the following customizable 96-well formats:
- Single-Transporter Assays: Cells expressing one transporter
- Multi-Transporter Models: Cells expressing more than one transporter
Our Process and the Importance of Exactly Matched Controls
The control cells used in our assays come from the same cell culture as the experimental cells, and are treated the same as the experimental cells throughout the transfection process. In our process, MDCK-II cells from a single batch are grown on 96-well membrane-bottom plates until they form monolayers. Cells destined to be experimental cells are transfected with a plasmid encoding the transporter of interest, and cells destined to be control cells are transfected with a plasmid encoding green fluorescent protein (GFP). Following transfection, the cells are cultured for approximately 48 hours before the transporter assays are performed.
During the transporter assays, the experimental cells and the control cells are subjected to identical assay conditions, with each assay condition run in triplicate. This approach prevents unwanted differences between experimental and control cells, such as variations caused by genetic drift, passive permeability or flux from interfering endogenous transporters, and avoids the decreased expression (i.e., silencing) of exogenous protein that often plagues ‘stable’ cell lines with increasing passage number.
Our approach provides excellent assay reproducibility. Compared to stably transfected cell lines, our transient expression system allows novel transporter assays to be established quickly, and confers unprecedented scalability and unrivaled flexibility in transporter assay development and design.
OPTI-EXPRESSION Technology allows the over-expression of more than one exogenous transporter per cell, while simultaneously enabling siRNA knockdown of specific endogenous transporters. As a result, compared to stable cell lines expressing single exogenous transporters, the cell monolayers produced by our technology more closely mimic the in vivo biology of barrier membranes, and so provide more physiologically-relevant models for studying transporter biology. Although stable cell lines expressing multiple exogenous transporters can be created using conventional methods, they tend to be difficult and much more time consuming to generate. By contrast, multi-transporter models can be generated much more quickly and easily with our OPTI-EXPRESSION Technology.
Multi-Transporter Model Case Study
We used OPTI-EXPRESSION Technology to study creatinine clearance in a kidney cell model. Creatinine is a metabolic waste product that is filtered out of the blood by the kidneys, and excreted in the urine. Most creatinine is passively removed by glomerular filtration; however, approximately 15% is actively excreted from the proximal tubules by transport proteins.
Eleven transport proteins known to be present in kidney cells were selected for the study. To determine which of these transporters were most likely involved in creatinine excretion, we expressed each one individually in polarized MDCK-II monolayers and assayed it. The transporters MATE1, MATE2-K, OCT2, OCT3, and OAT2 were shown to be the main transporters responsible for creatinine transport. Illustrated in the diagram below, these five transporters were then transfected into polarized MDCK-II monolayers in different combinations and assayed to determine the relative contribution of each transporter to creatinine transport.
We used this model in a research collaboration with Incyte Corporation to investigate the interaction of itacitinib (INCB039110), a Janus kinase 1 [JAK1] inhibitor under development, with the five transporters listed above that are likely involved in the renal clearance of creatinine. In this study, the effects of INCB039110 on creatinine transport in cells transfected with individual uptake transporters were compared to the effects in cells transfected with multiple transporters. Results from these studies indicated that data obtained from cells transfected with individual transporters was incomplete compared to the information gained from studying the interactions between the uptake and efflux transporters in the quintuple model.
The study demonstrated the ability of our quintuple transporter model to capture drug-transporter interactions that could not be observed with single-transporter assays. When data from this study was combined with Incyte's clinical studies (done by Incyte) that showed that the small increases in serum creatinine levels caused by INCB039110 were not due to impaired kidney function, it was clear that the increased serum creatinine levels were likely due to transporter inhibition. Our multi-transporter model suggested that transporter inhibition had caused the decreased creatinine secretion, thus predicting the in vivo results showing no kidney damage. In the future, such results could potentially be used to save companies from having to run costly in vivo studies. Study results were published in Y. Zhang, et al., Drug Metab Dispos 43:485–489, April 2015.
To learn more about how BioIVT’s OPTI-EXPRESSION Technology can benefit your drug discovery and development efforts, please contact us.