With the recent issuance of the new FDA draft guidance on drug interaction studies, it came as no surprise that some of the focus at the second International Transporter Consortium workshop (ITCW2) in March was on the guidance itself.
I had assumed that I would hear explanations of the decision trees, the reasoning for particular decisions, etc.; i.e., topics that have been addressed at Drug Metabolism Discussion Groups and other forums over the past several years. But instead of simply providing an overview of the guidance as I had expected, the unthinkable happened – members of the ITC suggested that we should challenge the guidance. Admittedly, the use of the word “challenge” may be overstating it a bit; in reality, what they suggested was for each lab to come up with their own criteria to determine the need for a clinical DDI study based on the results of in vitro testing. But when you are talking about deviating from a document that is basically the 10 Commandments of the transporter world, “challenge” seems appropriate.
Caroline Lee and Lei Zhang, who spoke regarding P-gp and OATP interactions, respectively, both expressed that one-size-fits-all criteria may not be appropriate. They acknowledged that the criteria in the guidance are based on a limited number of in vitro values, and at least in the case of OATP are biased toward false positives. Of course, such a conservative bias is important in terms of the safety of patients, but what’s the point if the result of every in vitro screen is a positive result? Why bother with an in vitro test if it doesn’t discriminate between compounds that precipitate a drug-drug interaction in the clinic and those that don’t?
The solution proposed by both speakers is to calibrate your own test systems. Absorption Systems has already undergone a similar exercise with regard to predicting blood-brain barrier penetration based on in vitro testing. It’s exciting to contemplate a similar approach to validation of our transporter interaction assessments as well. Through rational justification of our own [I]/IC50 and R-value cut-offs, we should not only better understand the behavior of our assays, but also improve their clinical context. After all, the whole point of performing in vitro tests is to predict what will happen in the clinic.
In the world of drug transporters, the emphasis is typically on ‘bench to bedside’ translation. But growing awareness about certain disease conditions that impact transporter function speaks to the need for sometimes going the other way, i.e., ‘bedside to bench.’
Regulation of transporter expression was the focus of one of the breakout sessions at the second International Transporter Consortium Workshop (ITCW2). One interesting aspect of this discussion was the impact of disease state on the regulation of transporters. For example, the up-regulation of BCRP and down-regulation of OATPs in inflammatory diseases such as rheumatoid arthritis is due to the impact of high circulating levels of pro-inflammatory cytokines.
The new FDA draft guidance on drug interaction studies requires evaluation of all NCEs as inhibitors of renal transporters. The importance of renal function in drug clearance is well appreciated. Chronic renal failure and end-stage renal disease can alter drug disposition by reducing the systemic clearance of drugs that are cleared by the kidneys, and by affecting plasma protein and tissue binding of drugs. But now there is evidence that renal failure affects not only the disposition of drugs that are cleared renally, but also the non-renal disposition of drugs that are metabolized by the liver. For example, erythromycin exhibits significantly reduced non-renal clearance in patients with end-stage renal disease. Although reduced metabolic enzyme activity can account for some of the reduced clearance, alterations in drug transporters also appear to be involved in some cases. One example is rosuvastatin, which is not highly metabolized; thus, reduced activity of metabolic enzymes alone cannot explain the observed reduction in non-renal clearance in patients with renal failure
With the development of renal failure, the renal secretion of organic ions mediated by members of the OAT and OCT families is decreased, presumably due at least in part to certain anionic uremic toxins that competitively inhibit OATs. Now it is known that renal failure also impairs the OATP-mediated uptake of drugs and organic anions into the liver, suggesting that these toxins could also inhibit hepatic transporters. An important observation is that most uremic toxins are highly protein bound; thus, the free concentration appears not to predict inhibitory potential, emphasizing the need to use the total (as opposed to unbound or free) Cmax for predicting interactions with hepatic transporters.
The mechanism of the alteration in drug transporter activities with disease state is thus far poorly characterized. Uremic toxins that accumulate in patients with renal failure may directly (i.e., inhibition) and/or indirectly (i.e., regulation of expression) modulate transporter function. Further in vitro and in vivo studies are required to elucidate these potential mechanisms. Going forward, a ‘bedside to bench’ approach may improve our understanding of the impact and consequences of diseases on drug transporters
For years, Absorption Systems has been proactive in developing innovative assay systems to investigate transporter-mediated drug-drug interactions (DDIs). Specifically with regard to MRP2 and multidrug and toxic compound extrusion transporters (MATEs), we use our patented MRP2 knockdown cell line CPT-M1 to identify MRP2 substrates and are developing test systems to investigate MATEs.
Urinary excretion is governed by glomerular filtration, tubular secretion across the proximal tubules, and reabsorption. For organic anions and cations, active transport across the renal proximal tubule followed by elimination via the urine is a major detoxification pathway. Secretion of drugs with anionic or cationic moieties across the renal tubules is achieved by the sequential action of uptake transporters at the basolateral (serosal, blood-facing) membrane and efflux transporters at the brush border membrane facing the lumen of the renal tubules. Whenever two drugs are co-administered, there is the potential for a DDI, possibly resulting in an adverse reaction. Elucidating the molecular mechanism of renal drug transport helps ensure a predictable risk of DDIs for any drug for which the kidney is a major route of elimination.
A number of cell-based and subcellular transporter test systems are available for the study of OCT2 and MATEs in isolation. However, all are less than optimal at predicting in vivo outcomes; therefore, there is an unmet need for more predictive in vitro models. The lack of suitable models for in vitro assessment of renal uptake and efflux transporters, their interplay in drug elimination, and their involvement in DDIs presents a major challenge in drug discovery and development.
Previous studies have demonstrated that MDCK cells can be doubly transfected to express basolateral uptake and apical efflux transporters, e.g., the pairing of OCT1 or OCT2 with MATE11. Thus, it is evident that MDCK cells will form polarized monolayers expressing the transporters of interest at the appropriate physiological membrane, either apical or basolateral. We are using MDCK cells with high transepithelial electrical resistance as hosts for transfection because they are well-characterized, rugged, and form very tight monolayers of polarized cells when cultured on Transwell® plates, thus minimizing paracellular leak, which could compromise the signal-to-noise ratio of bidirectional transport assays.
Our vast experience with Caco-2 and MDR1-MDCK cell monolayers has taught us that modeling the interplay between uptake and efflux transporters in the same cell is important to a complete understanding of the interactions of drugs with either transporter. That perspective is lost when a single transporter is studied in isolation. Doubly transfected cells enable us to examine the overlap of substrate specificities between uptake and efflux transporters, and can help elucidate the rate-limiting process of elimination of a given drug in the kidney. Thus, we expect these test systems to contribute to a fuller understanding of renal drug transport and DDIs.
Stay tuned: MDCK cells doubly transfected with various pairs of basolateral uptake and apical efflux transporters should be available as contract testing platforms soon, exclusively from Absorption Systems.
1König J, et al., Double-transfected MDCK cells expressing human OCT1/MATE1 or OCT2/MATE1: determinants of uptake and transcellular translocation of organic cations. Br J Pharmacol. 2011 Jun;163(3):546-55
At the second International Transporter Consortium Workshop (ITCW2), held on March 12-13 prior to the 2012 American Society for Clinical Pharmacology and Therapeutics (ASCPT) Annual Meeting, the latest information on transporter-mediated drug-drug interactions (DDIs) was presented and discussed.
Shiew-Mei Huang of the FDA predicted that the anion efflux transporter MRP2 would be one of the next transporters recommended for DDI evaluation. (Note: If Shiew-Mei says to keep an eye on MRP2, you’d better keep an eye on MRP2.)
In addition, Kathy Giacomini of UC San Francisco illustrated the importance of another family of efflux transporters, the multidrug and toxic compound extrusion transporters (MATEs), specifically in renal elimination of cationic drugs. Furthermore, she emphasized that DDIs manifesting as interference with renal elimination of a drug can result from inhibition of either OCT2 (uptake from the blood into renal proximal tubule epithelial cells) or MATEs (efflux from renal epithelial cells into the urine). It’s important to know the rate-limiting step, which can be elucidated by determining, in vitro, the inhibitory potency of an interacting drug toward an uptake transporter (e.g., OCT2) and its coupled efflux transporter (e.g., one of the MATEs). MATE(s) may be more important than OCT2 for DDIs and may mediate DDIs that were previously thought to involve OCT2.
Clearly, the investigation of transporter-mediated DDIs continues to influence the drug approval process and post-marketing drug evaluation. Such investigations will be impacted by the importance of evaluating the interplay between uptake and efflux transporters. A specific application of this concept by Absorption Systems, resulting in the creation of yet another innovative test system, will be the subject of a subsequent blog post.
As you probably know by now, the FDA recently (February 17, 2012) published a new draft guidance on drug interaction studies. The guidance covers all sorts of potential pharmacokinetic drug-drug interactions (DDIs), which could be mediated by either drug-metabolizing enzymes or drug transporters.
In terms of drug transporters, specifically hepatic uptake transporters, the FDA recommends that all new drug candidates should be evaluated as inhibitors of OATP1B1 and OATP1B3. In addition, any new drugs for which hepatic pathways contribute at least 25% of the elimination should be evaluated as substrates of these two transporters. Without a doubt, these two members of the OATP family are important in terms of their potential involvement in DDIs.
However, don’t forget about another hepatic uptake transporter, organic cation transporter 1 (OCT1). It may not be on the FDA’s list, but it’s one of nine transporters recommended for evaluation by the European Medicines Agency (EMA). In addition, published reports suggest that OCT1 plays a significant role in the disposition and, as a result, the efficacy of metformin, one of the most widely prescribed treatments for Type 2 diabetes. Furthermore, OCT1 mediates the uptake of some, but not all, tyrosine kinase inhibitors (TKIs) into cancer cells. Since OCT1 expression is down-regulated in some tumors, a higher dose (which might lead to additional side effects and/or toxicity) is required to achieve clinical efficacy for TKIs that are OCT1 substrates, such as imatinib. Other TKIs, such as nilotinib, do not require dose adjustment based on OCT1 expression. According to an EMA representative at the recent ITC2 workshop, this is the reason evaluation of new drug candidates as substrates of OCT1 is recommended by the EMA.