As the daughter of two avid Rolling Stones fans (and subsequently a fan in my own right), I’ve enjoyed some of my most memorable moments at rock ’n roll concerts. Yes – this blog is about drug transporters.
Quite unexpectedly, last week’s AAPS Transporter Workshop in ADME: From the Bench to the Bedside brought me back to the front row at Madison Square Garden, anticipating the moment when the Stones would take the stage - starting, of course, with one of Keith Richards’ unmistakable riffs.
Maybe it’s because I woke up early in the morning, hopped in a minivan with my colleagues, and headed toward a nearby city. Maybe it’s because everyone was wearing a lanyard, reminiscent of concert VIP passes. But the main commonality was throngs of people from around the world – people with a similar passion, gathering to share their experiences. The crowd consisted of both newcomers (including a large showing of students) and ‘diehard fans’ (those well-versed and immersed in the still-emerging transporter field).
Before the transporter ‘rock stars’ took the stage, there was palpable excitement and anticipation. Much has transpired in transporter research in recent months, and this group was ready to share and learn. The ‘set list’ included both ‘classic tunes’ (a comprehensive review of transporters and their role in ADME) and ‘new hits’ (including special focus on renal transporters of emerging importance).
Speaking of renal transporters, Steve Wright (University of Arizona) started the workshop with a great review of these, highlighting the increasing importance of MATE transporters in renal function. More specifically, MATE1 and MATE2-K possess ‘multispecificity’ (resulting from a large receptor surface with multiple binding sites, similar to the multidrug resistance transporters such as P-gp), making them principal sites for unwanted drug-drug interactions (DDIs). Substrate-dependent ligand interactions (and differential selectivity vs. OCT2) will influence prediction of such DDIs, necessitating more advanced models.
There is also a need for better understanding of renal transporters in the clinical setting. Alex Sparreboom (St. Jude Children’s Research Hospital) delineated how inhibition of the renal secretion of OCT2 substrates may potentially impair the usefulness of creatinine as a marker for glomerular filtration rate. Daniel Antoine (University of Liverpool) described the current panel of seven urinary biomarkers (including KIM-1) used to assess renal injury, in addition to more classic markers like serum creatinine and blood urea nitrogen, and limitations related to sensitivity and specificity. The ‘next generation’ for evaluating drug-induced acute kidney injury will likely include microRNA biomarkers.
Following the discussion on kidney transporters, the focus shifted to the liver. Curt Klaassen (Kansas University) and Gian Camensich (Novartis) delivered interesting presentations on prediction of hepatotoxicity and hepatic clearance from in vitro metabolism and transporter data. It was fitting to connect this information to the Biopharmaceutics Drug Disposition Classification System (BDDCS). As one may expect, in vitro metabolism data is most predictive for highly permeable compounds (BDDCS 1 and 2), where metabolism is rate-limiting. For poorly permeable compounds (BDDCS 3 and 4), hepatic uptake is most predictive, as cellular uptake is rate-limiting in overall organ clearance. This approach of incorporating BCS or BDDCS classification in early assessment of DDI potential enables clearance estimates based on an established hepatobiliary model, allowing for more predictive PK, PK/PD, and DDI models.
Extending the clinical implications, the impact of transporter expression and function in disease states was reviewed, with focus on Alzheimer’s disease and non-alcoholic fatty liver disease. Jash Unadkat (University of Washington) commented on the role of P-gp in the pathophysiology of neurological diseases, while Mikko Niemi delivered an interesting presentation on pharmacogenetics and clinical applications. I enjoyed Cuiping Chen’s (Depomed) case study, which highlighted the importance of intestinal transporters in the development of a gastroretentive formulation. Coupling site-specific absorption data from an in situ intestinal perfusion model with information on L-amino acid transporters enabled development of a mucoadhesive formulation to overcome the physiological turnover of mucus in the GI tract, exploiting gabapentin’s preferential absorption in the upper GI tract.
The session which resonated the most with me featured the ‘current state of the art’ of transporter assays. Matt Soars (Bristol-Myers Squibb) commented on the progress with development of transporter tools and their applications, highlighting the importance of robust characterization and understanding test system limitations. He also described a practical approach to utilize current analytical techniques in order to produce high-throughput transporter inhibition screens.
Donna Volpe of the FDA provided a survey of transporter studies, noting that 74 of the 183 package inserts from 2003-2011 included the names of specific transporter(s). We have performed a similar review of the approved labels from 2012: 18 of the 24 small-molecule oral/IV drugs included transporter information on their labels and Absorption Systems contributed to 6 of the 18 labels. Donna also highlighted common assay issues, including solubility, stability, choice of cell line, transporter expression, substrate/inhibitor specificity, interspecies differences, and data analysis. Absorption Systems similarly recognizes the importance of these factors, and incorporates appropriate suitability testing to ensure maximum translatability. See our companion guides related to test system selection and test compound characteristics.
Absorption Systems’ Chief Operating Officer, Dr. Sid Bhoopathy, discussed a novel and nuanced approach to in vitro test system selection for substrate assays. We’ve always understood that you can obtain different results with different test systems – but we now appreciate that these differences may be complementary and not necessarily conflicting. Using decision trees related to intrinsic membrane permeability and clearance pathways, Sid highlighted a rational approach to guide substrate evaluation for maximum clinical relevance. Test systems that are relevant to the site of interaction may provide relevant information on both intestinal absorption and systemic drug disposition, for example. This, of course, embodies the Stones’ words of wisdom: “You can’t always get what you want. But if you try sometimes, you’ll find, you get what you need.”
But, like a rock concert, the experience consists not only of the show itself, but the common bond shared amongst the audience members. During the break sessions, I participated in great discussions on common experimental issues in transporter assays. There was also great discussion around measurement of intracellular concentrations of drug and metabolites. Kim Brouwer (University of North Carolina) discussed an elegantly simple technique employing subcellular fractionation and equilibrium dialysis to quantify unbound drug concentration in sandwich-cultured hepatocytes. It is generally accepted that systemic concentrations may not reflect hepatocellular drug concentrations and that hepatocyte distribution and intracellular unbound drug concentrations may be important determinants of hepatic efflux, DDIs, and drug–induced liver injury (DILI). Absorption Systems’ current collaboration with Temple University addresses some of these important questions.
The ‘grand finale’ of the workshop included presentations from Lei Zhang (FDA) and Eva Gil Berglund (EMA), reviewing current regulatory perspectives. Highlights were:
- the inclusion of MATE1 and MATE2-K on the FDA’s recommended list of transporters for prospective evaluation (as well as BSEP and MRP2 retrospectively, depending on liver toxicity data)
- the importance of test system calibration and in vitro ‘benchmarking’
- the FDA’s addition of a ‘methodology’ appendix to its draft DDI guidance in coming months
- the EMA’s emphasis on including rationale for experimental design choices to assist with identification of sources of variability and standardization of systems
- the importance of evaluating multiple test systems (depending on the site of interaction, e.g., cell line with endogenous expression, knockdown (CellPort Technologies), over-expression, vesicles, or hepatocytes)
- selection of appropriate probes/inhibitors and relevant concentrations (e.g., P-gp and BCRP should be evaluated at both systemic and intestinal concentrations).
Overall, the worskhop provided a great forum for information exchange and discussion. As Mick Jagger might say, had he attended the AAPS workshop – “I know it’s only transporters – but I like it.”