Transporters can have an enormous influence on the pharmacokinetics of drugs that are substrates, either limiting their intestinal absorption, mediating their excretion via bile or urine, or determining how much gets into the brain, cancer cells, or placenta.
Researchers continue to grapple with a lack of definitive tools, unlike drug metabolism, which has a plethora of highly specific pharmacologic reagents and assay systems. Nearly all available transporter probe substrates and inhibitors interact with multiple transporters. In recognition of this, the new FDA Draft Guidance on drug interaction studies is less reliant on chemical inhibitors to identify the transporters mediating drug efflux than was the previous (2006) draft guidance. As a result, the industry is in pursuit of definitive solutions like CellPort Technologies® to make drugs safer by predicting DDIs reliably before they happen.
The evaluation of transporters is of critical importance in drug development, as it is part of the FDA's regulatory review process and Critical Path Initiative as well as the EMA's regulatory review process. The current FDA Draft Guidance on drug interactions recommends reduced reliance on chemical inhibitors to identify the transporters mediating drug efflux.
You need to know what potential drug interactions to watch for and which ones you might have to test for in a clinical trial. For example, any new drug that inhibits members of the OATP uptake transporter family in vitro will probably have to be tested alongside a statin in a clinical trial.
Some transporters have multiple binding sites, which can result in substrate-dependent inhibition as has been reported for drug-metabolizing CYP enzymes and some transporters. Therefore, reliance on probe substrates and inhibitors can potentially lead to false negatives. Without specific probe substrates, you need very definitive assay systems like CellPort Technologies.
Transporter Reference Guide Ebook
Waiving Clinical Studies
Kinetics of Drug Elimination
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Transporter Testing Methods
We have years of experience in evaluating transporter interactions. There are two in vitro cell-based assay formats for determining transporter interactions: bidirectional monolayers and cellular uptake. In addition, Absorption Systems has pioneered a more definitive assay system for efflux transporters which stably knocks down one transporter at a time using our suite of proprietary technology, CellPort.
ExpressPlus BBB Penetration Potential Determined Using MDR1-MDCK Cell Monolayers
ExpressPlus BCRP IC50 Determination Using BCRP-MDCK Cells
ExpressPlus BCRP Inhibition Assessment in BCRP-MDCK Cells
ExpressPlus BCRP Substrate Assessment in BCRP-MDCK Cells (+/- Inhibitor)
ExpressPlus BCRP Substrate Assessment in Caco-2 Cells (+/- Inhibitor)
ExpressPlus Bidirectional Permeability Through Caco-2 Cell Monolayers
ExpressPlus BSEP IC50 Determination Using Vesicles
ExpressPlus BSEP Inhibition Assessment Using Vesicles
ExpressPlus MRP2 Substrate Assessment in Caco-2 Cells (+/- Inhibitor)
ExpressPlus OAT1 IC50 Determination Using Transfected Cells
ExpressPlus OAT1 Inhibition Assessment Using Transfected Cells
ExpressPlus OAT3 IC50 Determination Using Transfected Cells
ExpressPlus OAT3 Inhibition Assessment Using Transfected Cells
ExpressPlus OATP1B1 IC50 Determination Using Transfected Cells
ExpressPlus OATP1B1 Inhibition Assessment Using Transfected Cells
ExpressPlus OATP1B3 IC50 Determination Using Transfected Cells
ExpressPlus OATP1B3 Inhibition Assessment Using Transfected Cells
ExpressPlus OCT1 IC50 Determination Using Transfected Cells
ExpressPlus OCT1 Inhibition Assessment Using Transfected Cells
ExpressPlus OCT2 IC50 Determination Using Transfected Cells
ExpressPlus OCT2 Inhibition Assessment Using Transfected Cells
ExpressPlus P-gp IC50 Determination Using Caco-2 Cells
ExpressPlus P-gp IC50 Determination Using MDR1-MDCK Cells
ExpressPlus P-gp Inhibition Assessment in Caco-2 Cells
ExpressPlus P-gp Inhibition Assessment in MDR1-MDCK Cells
ExpressPlus P-gp Substrate Assessment in Caco-2 Cells (+/- Inhibitor)
ExpressPlus P-gp Substrate Assessment in MDR1-MDCK Cells (+/- Inhibitor)
ExpressPlus PepT1 Substrate Assessment in Caco-2 Cell Monolayers
ExpressPlus Unidirectional Permeability Through Caco-2 Cell Monolayers
BCRP Interaction Assessment in BCRP-transfected MDCK Cells
BCRP Interaction Assessment in Caco-2 Cells
Bidirectional Permeability Through Caco-2 Cell Monolayers
BSEP Interaction Assessment in Vesicles
Human P-gp Substrate Assessment Across MDR1-MDCK Cell Monolayers
Induction of P-gp in a Human Intestinal Cell Line
MATE1 Interaction Assessment in MATE1-transfected HEK293 Cells
MATE2-K Interaction Assessment in MATE2-K-transfected HEK293 Cells
MRP2 Interaction Assessment in Caco-2 Cells
OAT1 Drug Interaction Assessment in OAT1-Transfected Cells
OAT3 Drug Interaction Assessment in OAT3-Transfected Cells
OATP1A2 Drug Interaction Assessment in OATP1A2-Transfected Cells
OATP1B1 Drug Interaction Assessment in OATP1B1-Transfected Cells
OATP1B3 Drug Interaction Assessment in OATP1B3-Transfected Cells
OATP2B1 Drug Interaction Assessment in OATP2B1-Transfected Cells
OCT1 Interaction Assessment in OCT1-transfected HEK293 Cells
OCT2 Interaction Assessment in OCT2-transfected HEK293 Cells
P-gp Interaction Assessment in Caco-2 Cells
P-gp Interaction Assessment in hP-gp-transfected MDCK Cells
Unidirectional Permeability Through Caco-2 Cell Monolayers
EA401 Metabolic Stability in Human Liver Microsomes
EA426 CYP Inhibition in HLM–Single Concentration
Artursson P, et al., Caco-2 monolayers in experimental and theoretical predictions of drug transport. Adv Drug Deliv Rev. 2001 Mar 1; 46(1-3):27-43.
Bhardwaj RK, et al., Delineation of human peptide transporter (hPepT1)-mediated uptake and transport of substrates with varying transporter affinities utilizing stably transfected hPepT1/Madin-Darby Canine Kidney Clones and Cac0-2 Cells. J Pharm Exp Ther. 2005 Sept; 314(3):1093-1100.
Borchardt RT, et al., Are MDCK cells transfected with the human MRP2 gene a good model of the human intestinal mucosa?. Pharm Res. 2002 Jun; 19(6):773-9.
Chen C, et al., P-glycoprotein limits the brain penetration of nonsedating but not sedating H1-antagonists. Drug Metab Dispos. 2003 Mar; 31(3):312-8.
de Vrueh, RLA, et al., Transport of L-Valine-Acyclovir via the oligopeptide transporter in the human intestinal cell line, Caco-2. J Pharm Exp Ther. 1998 Sept; 286(3):1166-70.
Ee, PLR, et al., Modulation of breast cancer resistance protein (BCRP/ABCG2) gene expression using RNA interference. Mol Cancer Ther. 2004 Dec; 13(12):1577-83.
Fabre G, et al., Correlation between oral drug absorption in humans, and apparent drug permeability in TC-7 cells, a human epithelial intestinal cell line: comparison with the parental Caco-2 cell line. Pharm Res. 1998 May;15(5):726-33.
Fenner, KS, et al., Drug–drug interactions mediated through P-glycoprotein: Clinical relevance and in vitro–in vivo correlation using Digoxin as a probe drug. Clin Pharmacol Ther. 2009 Feb; 85(2):173-81.
Hunter J, et al., Functional expression of P-glycoprotein in apical membranes of human intestinal Caco-2 cells. Kinetics of vinblastine secretion and interaction with modulators. J Biol Chem. 1993 Jul 15; 268(20):14991-7.
Lin, JH, Drug-drug interaction mediated by inhibition and induction of P-glycoprotein. Adv Drug Deliv Rev. 2003: 55 (1); 53-81.
Obradovic T, et al., Assessment of the first and second generation antihistamines brain penetration and role of P-glycoprotein. Pharm Res. 2007 Feb; 24(2):318-27.
Polli JW, et al., Rational use of in vitro P-glycoprotein assays in drug discovery. J Pharmacol Exp Ther. 2001 Nov; 299(2):620-8.
Rautio, J, et al., In vitro P-glycoprotein inhibition assays for assessment of clinical drug interaction potential of new drug candidates: A recommendation for probe substrates. Drug Metab Disp. 2006; 34 (5): 786-92.
Summerfield SG, et al., Central nervous system drug disposition: the relationship between in situ brain permeability and brain free fraction. J Pharmacol Exp Ther. 2007 Jul; 322(1):205-13.
Tanaka H, et al., Drug-protein binding and blood-brain barrier permeability. J Pharmacol Exp Ther. 1999 Mar; 288(3):912-8
Taub ME, et al., Functional assessment of multiple P-glycoprotein (P-gp) probe substrates: influence of cell line and modulator concentration on P-gp activity. Drug Metab Dispos. 2005 Nov; 33(11):1679-87. Epub 2005 Aug 10.
Trauner M, et al., Bile salt transporters: molecular characterization, function, and regulation. Physiol Rev. 2003 Apr; 83(2):633-71.
Van Deusen J. et al., Use of a physiologically based pharmacokinetic model to study the time to reach brain equilibrium: an experimental analysis of the role of blood-brain barrier permeability, plasma protein binding, and brain tissue binding. J Pharmacol Exp Ther. 2005 Jun; 313(3):1254-62.
Wang Q, et al., Evaluation of the MDR-MDCK cell line as a permeability screen for the blood-brain barrier. Int J Pharm. 2005 Jan 20; 288(2):349-59.
Zhang, L, et al., Scientific perspectives on drug transporters and their role in drug interactions. Mol Pharm, 2006 Jan; 3 (1): 62-9.
Development of cell-based assays for assessing drug interactions with human renal transporters OCT2, OAT1, and OAT3 (Poster #216, ADMET Europe 2011)
Stably transfected HEK293 cell assays for the human hepatic transporters OATP1B1, OATP1B3, and OCT1 (Poster #215, ADMET Europe 2011)
Development of cell-based assays for assessing drug Interactions with human renal transporters (Poster #W3012, AAPS Drug Transporters Workshop 2011)
Development of a human cell-based BCRP inhibition assay (Poster #W3008, AAPS Drug Transporters Workshop 2011)
Establishment and characterization of an HEK293 cell line with stable expression of human organic cation transporter (Poster #T2024, AAPS Drug Transporters Workshop 2011)
Development of transfected HEK293 cell lines stably expressing individual human organic anion transporters (OATs) (Poster #T2025, AAPS Drug Transporters Workshop 2011)
Use of Caco-2 knockdown cells to investigate transporter-mediated efflux of statin drugs (Poster #T3371, AAPS 2010)
Induction of breast cancer resistance protein (BCRP) and enhanced transport of Estrone-3-sulfate and Pheophorbide A across Caco-2 cell monolayers (Poster #T3408, AAPS 2010)
Determination of substrate specificity and relative activity of uptake and efflux transporters in a new cell line: OATP1B1-transfected MDR1-MDCK cells (Poster #T3407, AAPS 2010)
Determination of BCRP activity and evaluation of potential BCRP-mediated drug-drug interactions in Caco-2 cells using the fluorescent substrate Pheophorbide A (Poster #T2392, AAPS 2010)
Fluorescence assay for evaluation of MRP2 based drug interactions (ISSX 2010)
Evaluation of LS-180 human intestinal cells as an in vitro model of P-gp induction (ISSX 2009)
OATP expression in human intestinal epithelial cells and its influence on the transport of statin drugs across Caco-2 cell monolayers (ISSX 2009)
Generation of Ritonavir phase I metabolites using a biomimetic approach and in vitro investigation of their potential for drug interactions (ISSX 2009)
Applications of BCRP-silenced Caco-2 cells and murine Bcrp-transfected MDCK cells in identifying BCRP substrates in vitro (ISSX 2009)
Investigation of transporters involved in biliary clearance of drugs in the human liver using Caco-2 cells with stable MDR1 and MRP2 knockdown by shRNA (ISSX 2008)
Silencing of multidrug resistance-associated protein 2 (MRP-2) expression and function in Caco-2 cells using Lentiviral shRNA (AAPS 2008)
Functional characterization of RNAi induced BCRP knockdown Caco-2 cells (AAPS 2008)
Silencing of breast cancer resistance protein (BCRP) expression and function in Caco- 2 cells using Lentiviral shRNA-mediated RNA interference (ISSX 2008)
Functional characterization of the MRP2 transporter using the isolated rat liver perfusion model (AAPS 2007)
Silencing of breast cancer resistance protein expression by Lentiviral shRNA transduction particles in Caco-2 models (AAPS 2007)
Utility of the P-gp knockout mouse in situ brain perfusion to determine the brain penetration potential of P-gp substrate drugs (AAPS 2007)
Functional evidence of multiple efflux transport systems in Caco-2 cell model (AAPS 2007)
Why not wait until my drug gets into the clinic to test?
You could miss a DDI even in a clinical trial designed to detect one; test for it early and in vitro with our definitive human test systems. Our assays provide definitive data, quickly and cost-effectively. We use the same validated test systems, with different degrees of rigor, all the way from early screening to an NDA. In vitro drug interaction studies help predict the risk of clinical drug-drug interactions, contribute to the design of appropriate in vivo drug interaction studies (or, if the results are negative, make in vivo studies unnecessary), and support appropriate labeling. Plus, because our Caco-2 cell assay is dually validated for permeability and efflux transporter interactions, you may be able to avoid a clinical transporter interaction study even if your compound is a substrate for P-gp or BCRP.
What is the impact of active transport on PK parameters for compounds that don't undergo metabolism but are substrates of hepatic uptake transporters?
Transporter-mediated uptake can affect the overall rate of elimination. Both clearance (CLin vivo) and volume of distribution (Vss) can be impacted resulting in poor prediction of PK parameters in man.
Does Absorption Systems offer assays for all seven of the ITC transporters?
Yes – and more.
When CLH and CLR are not available, can I use preclinical data to determine them?
You can, but you should certainly be cautious of species differences. Human results would be best.
If CLR is <1% of total clearance, do I still need an inhibition assessment with renal transporters?
Yes, if the compound is expected to be co-administered with drugs that are substrates of renal transporters. Although the compound may not be a substrate of OATs or OCTs, it could be an inhibitor and thereby interfere with the renal clearance of other drugs.
[I]2 doesn't take into account solubility. How do we overcome solubility problems?
Appropriate excipients can be used to enhance solubility so that a broader range of concentrations may be tested. Absorption Systems has evaluated a number of excipients that are ideal for in vitro drug transporter studies: they do not alter monolayer integrity or affect transporter function. We can check if these excipients improve solubility of your test compound(s).
If in vitro experiments for determination of P-gp/BCRP substrate interactions result in an efflux ratio ≥2, do I need to always perform an in vivo DDI study??
No, this may not be necessary if is determined that efflux is not rate limiting because the compound has favorable drug specific-factors, such as high permeability and high solubility. You can determine if your compound has good intestinal absorption using a validated in vitro test system such as Absorption Systems’ Caco-2 monolayer system. See section on waiving clinical studies (under intestinal transporters).
Is the ITC decision tree's use of CLH > 25% based on preclinical data?
No, this is based on human data.
Is it possible to evaluate "sticky" compounds with low recovery using an in vitro test system?
Yes, various techniques may be implemented to alleviate non-specific binding (NSB). As a diagnostic measure, the NSB of the test compound to the experimental apparatus is assessed prior to any permeability experiment. If the recovery is low, then a pre-incubation step is included to saturate binding sites. 1% BSA is included in the receiver compartment to improve recovery. At the end of a permeability experiment, donor and receiver compartments may be rinsed with organic solvent to collect residual test compound. Cellular accumulation may also be measured.
Absorption Systems is recognized by the industry and the FDA as a leader in drug transporter assessment.
Our Chief Scientific Officer, Ismael Hidalgo, was working on drug transporters twenty years ago, and he pioneered the Caco-2 model in the bidirectional monolayer transport format identified by the FDA as definitive for permeability studies.
We have created unique, patented knockdown cell lines for studying interactions of test compounds with the efflux transporters P-gp and BCRP.
We have stably transfected human cells expressing each of the drug uptake transporters required by the FDA and the EMA.
Our Caco-2 cell assay is dually validated for permeability and efflux transporter interactions, you may be able to avoid a clinical transporter interaction study even if your compound is a substrate for P-gp or BCRP.