The combination of dissolution testing with permeability and solubility testing provides a way to obtain biowaivers for generic drugs and bypass expensive and time-consuming human bioequivalence (BE) studies. The recent Disso India 2013 conference held in Mumbai, India on May 3^rd and 4^th attracted over 500 delegates and renowned speakers from all over the world. The success of this conference was testimony to the growing importance of in vitro tests that often serve as a better way to establish therapeutic equivalence, especially for cytotoxic and highly variable drugs.

The Biopharmaceutics Classification System (BCS) provides the scientific basis that supports in vivo bioavailability and bioequivalence waivers for immediate-release solid dosage form drugs that have high solubility, high intestinal permeability, and rapid dissolution. In vitro permeability assays provide direct assessment of absorption potential, whereas in vivo BE studies assess drug absorption indirectly via evaluation of a pharmacokinetic profile. With the latter, post-absorption events such as metabolism and enterohepatic recycling can result in increased variability and make it difficult to quantify the fraction absorbed. Highly variable drugs (HVDs) often require a greater number of subjects to minimize the chance of an erroneous conclusion of inequivalence when, in fact, two drug products are therapeutically equivalent. For an HVD that exhibits consistently inconsistent PK due to extensive first-pass metabolism, human mass balance and bioavailability studies may result in ambiguous or incorrect BCS classification because the variability inherent in human studies is compounded by the intrinsic properties of the drug substance. In such cases, in vitro testing provides consistent and accurate classification as the variability associated with permeability and solubility measurements is less dependent on the factors that magnify the variability of human testing.

In a conference filled with talks on dissolution, Absorption Systems provided the only presentation on permeability testing. As the world leader in BCS, our participation in this conference highlights our commitment to work with companies in India and other emerging markets around the globe. Our focus is to apply the BCS and BCS-based biowaivers to avoid clinical bioequivalence testing through more ethical and definitive in vitro tests whenever possible. Many patients go untreated or are undertreated because bioequivalence for highly variable drugs can be challenging to establish, resulting in many medicines being unavailable. While there is much institutional knowledge and expertise in the areas of formulation development and dissolution technologies in a country like India which is well accepted as the generic hub of the world, an improved awareness and understanding of the in vitro approach will help Indian pharmaceutical companies bring better-quality, lower-cost generic medicines to the Indian market and the world.

The path forward requires a global effort, since most major products are registered worldwide. While the FDA has been clear about their acceptance of the BCS-based biowaiver approach through their BCS Guidance (2000), multiple publications, and webinars, drug regulatory agencies in other countries have yet to follow suit. As long as there is a lack of harmonized acceptance, in vivo testing will always be performed, even if in vitro tests are the better and more ethical option. The universal acceptance and utility of in vitro permeability, solubility, and dissolution testing is therefore necessary and needs to become an integral part of quality-by-design (QbD) efforts during product development.

As part of the Affordable Care Act of 2010, the impetus for the FDA to set up a defined pathway for biosimilars was set forth, and followed up with a trio of draft guidances first available in early 2012. However, the pathway is still not nearly as clear as it is for small molecules. Many generic companies want to get into the game as they have for small molecules. However, to prove similarity, all in vitro and in vivo studies of the approved reference molecule and the biosimilar candidate must be run in parallel. Additionally, the realization that the set-up and manufacturing costs can be 10 to 20 times more than that of small molecules may lead to many companies deciding to drop biosimilar projects due to lack of both adequate funding and a clear path forward.

There is a need for good quality biosimilars for the developed and developing world. Currently, in the EU, several biosimilar products have made it to market and are routinely used in lieu of the respective innovator molecules. This is true for many other regions of the world as well. However, currently in the US, the pathway is not clear. For biologics, “the process is the product,” so how similar do biosimilars need to be? How easy will it be for a pharmacist to switch out the biosimilar for the innovator? According to innovator companies, it shouldn’t be that simple. To them, if “the process is the product,” then “similar” is not ”identical”; even the current, vague FDA draft guidances acknowledge “biosimilarity” does not equate to “interchangeability.” Beyond the federal position on testing and approval, state governments have been and are being lobbied on behalf of innovator companies, and state laws have been penned requiring that the pharmacist, prescriber, and patient all be made aware of the differences.

While it is clear that extensive biochemical and biophysical characterization and comparison are necessary and that similarity can be demonstrated empirically at the molecular level, there can still be differences at the level of the bioassay or clinical results. Can all of these aspects be proven to be similar? Or is the new product superior, based on structural properties, efficacy, potency, safety, or manufacturing procedure? Several companies eyeing the biosimilar market in the US market have opted to file a BLA (i.e., as an innovator product) and/or target approval as a biobetter, as opposed to a biosimilar. And why not, when the cost of developing a biosimilar could be nearly as high as that of a new product, with the additional risk of an uncertain regulatory pathway.

With several biosimilars currently approved and on the market in the EU, one would assume the filing process would be streamlined for the US market. However, this is not the case. It is important to note that for potential biosimilars, the reference comparator for all in vitro and in vivo studies must be the relevant approved product in that country or region. That is to say, there may be subtle differences between the same innovator molecule approved in the two regions. This may be the case even if they are produced by the same company (e.g., due to different manufacturing  sites). Thus, a company wishing to apply for a biosimilar in the US, even if it has one approved in the EU, may be required to repeat all in vitro and in vivo studies to demonstrate biosimilarity. This is another factor that will tend to push companies toward filing a BLA rather than taking the biosimilar route.

Biologics have grown and will continue to grow in importance in the pharmaceutical market. Biosimilars are expected to contribute to that growth. Though the pathway to biosimilars is not currently clear for the US, defined processes have been established in other markets and, as the field matures, the FDA will further define these processes as they have done with small molecules. With governmental, payer, and patient pressures mounting, it is only a matter of time before the first biosimilar is approved in the US. The path to a biosimilar for the US market may currently be hazy, but the clouds are beginning to lift.

At AAPS Workshop on Drug Transporters in ADME: From the Bench to the Bedside, held on March 17^th to 20^th, 2013, the latest information on transporter-mediated drug-drug interactions (DDI) was presented and discussed.

One of the hottest topics was still the “new” transporter family, Multidrug and Toxin Extrusion (MATE) including MATE1 and MATE2-K, which have not been officially listed on the 2012 FDA Guidance for Industry[1] and 2012 EMA Guideline on the Investigation of Drug Interactions[2]. Back in 2012, at the second International Transporter Consortium Workshop (ITCW2), Dr. Shiew-Mei Huang, Deputy Director of the Office of Clinical Pharmacology (OCP) in the U.S. Food and Drug Administration (FDA)’s Center for Drug Evaluation and Research (CDER) stated that OCP reviewers would expect sponsors to evaluate proactively new drug candidates as substrates and/or inhibitors of MATE1 and MATE2-K. During this workshop, Dr. Lei Zhang, Special Assistant to Office Director in the Office of Clinical Pharmacology, Office of Translational Sciences, the Center for Drug Evaluation and Research, FDA (OCP/OTS/CDER/FDA) stated that 1.4% of FDA approved New Molecular Entities (NMEs) from 2003 to 2011 has MATE information on their labeling.

As the first presenter, Prof. Steven Wright, talked about the role of MATE transporters in renal function. He emphasized their “multispecificity” which makes them principal sites for unwanted DDIs and makes them a focus of efforts to develop models capable of predicting and ideally, pre-empting unwanted DDIs (italic statements cited from the speaker abstract). Further, the impact of the interplay of the uptake transporter (e.g., Organic Cation Transporter, OCT2) and the coupled efflux transporter (e.g. MATE1 and/or MATE2-K) and the rated-limit step in the drug elimination across both basolateral and apical membrane barriers has also been widely discussed.  Take a newly published paper as an example, Sugiyama and his colleagues reported that the inhibition potency of cimetidine towards OCT2 (K_i = 124 µM) is much lower than that towards MATE2-K (K_i = 6.9 µM)[3]. Considering the clinically reported plasma unbound concentrations (3.6-7.8 µM) of cimetidine, the competitive inhibition of MATE2-K appears to be the mechanism underlying the DDI caused by cimetidine in the renal elimination of cationic drugs. Please note that this phenomenon is also applicable for other coupled transporters, for instance, if the drug elimination is involved in both uptake transporter Na⁺-taurocholate Cotransporting Polypeptide (NTCP) and efflux transporter Bile Salt Export Pump (BSEP) in the hepatocyte.

Currently, the regulatory agencies, the pharmaceutical industry and the academic world have recognized the importance of MATE transporters, which indicates that they are likely to be incorporated in the transporter list in the future versions of FDA guidance and/or EMA guideline.  Absorption Systems had the vision to proactively develop test systems for these “new” transporters, including MATE1 and MATE2-K. We can now help our customers investigate the substrate and inhibitor potential of the drug candidates towards MATEs. In doing so, our mission is to work closely with our customers just like their internal R&D group. We utlilize our knowledge and expertise in analyzing the in vitro data and related other information to accurately predict the in vivo outcome.

[1] FDA Guidance for Industry, Drug Interaction Studies — Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations, 2012
[2] EMA Guideline on the Investigation of Drug Interaction, 2012
[3] Sumito Ito, et al. Competitive inhibition of the luminal efflux by multidrug and toxin extrusions, but not basolateral uptake by organic cation transporter 2, is the likely mechanism underlying the pharmacokinetic drug-drug interactions caused by cimetidine in the kidney. J Pharmacol Exp Ther 340:393-403. 2012

In the first three parts of this series, I have covered the basic principles of the BCS, the fact that it remains under-utilized even in the U.S., and some ethical and geopolitical implications. Now we will discuss the simple fact that the in vitro approach to the BCS, when performed correctly, is a reliable, scientifically proven approach and has been utilized for over thirteen years.

1. It eliminates unnecessary human testing
2. It shortens development time and therefore overall product costs
3. It is an inherently conservative, scientific approach
4. When applicable, it is the BEST approach

Jim Polli of the University of Maryland argues that in vitro BCS testing is actually better than a clinical bioequivalence (BE) study (Polli JE, AAPS J 2008 Jun;10(2):289-299). Besides lower drug development costs and ethical considerations (addressed in prior posts in this series), there are two other important points. In vitro permeability studies assess drug absorption more directly than human BE studies, for which the approach is indirect (one needs to find and account for metabolites as well as the parent drug) and the interpretation is often complicated (e.g., did that compound in feces result from biliary excretion of absorbed drug, or was it never absorbed?). Furthermore, in vitro permeability data is inherently more precise than clinical data due to intra- and inter-individual variability in the latter, particularly for highly variable drugs.

Three simple laboratory tests, two on the active pharmaceutical ingredient (API) − solubility and permeability − and a third on the finished dosage form − dissolution. If the API and the finished dosage form meet the established criteria as outlined in the FDA Guidance, then we know unequivocally that the test product will be bioequivalent to the marketed (“approved” or “reference”) product. This has been proven scientifically over the past 13 years. And, should any of the in vitro test results be ambiguous or fail to meet the Guidance criteria, the manufacturer can simply proceed to human BE testing.

So why has adoption of the BCS been slow in many countries outside the US? To quote Harry Truman, “The only thing new is the history we do not know.” For artisans in the field of in vitro BCS measurements, the history is known and is being told, slowly, from a grassroots perspective. Our challenge is for governments and regulatory authorities around the world to take advantage of what we already know, what makes sense scientifically, and what works. What are your thoughts on the BCS?

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.”