B-CLEAR®:An In Vitro Model of In Vivo Biliary Excretion

B-CLEAR® combines in vivo-relevant uptake, metabolism, and efflux in a single, patented system that allows researchers to assess and predict biliary excretion and transporter interactions.

The B-CLEAR® model has been extensively published and utilized by academic researchers as well as regulatory and pharmaceutical scientists to answer many types of transporter questions. In addition, data from B-CLEAR® studies have been included in regulatory submissions to the FDA and EMA.

What is the B-CLEAR® Technology?

The B-CLEAR® technology refers to a patented methodology that, when applied to properly functioning hepatocytes in sandwich culture, opens the bile pockets (analogous to bile canaliculi in vivo), allowing the measurement of material that has been transported from inside the cell. Measurement of biliary efflux allows us to exclusively evaluate biliary clearance and biliary transporter interactions, as well as assess uptake, intracellular concentration, and metabolism. There are three patent platforms (including US Patent 6,780,580) that cover over 45 countries around the world.

Figure 1. Potential clearance mechanisms within a hepatocytes. In the case of a circulating drug, it is removed from the blood by uptake transporters. The drug can be effluxed into the bile or back into the blood. In addition, the drug may be metabolized and metabolites may undergo these same efflux processes. The patented B-CLEAR® technology refers to methodologies that assess the amount of efflux into the bile pockets.

How Do I Use The Model?

There are 3 ways researchers and customers have principally used the biliary excretion (BE) model (Figure 1):
1) Species Selection
    Compare biliary clearance between rat, dog, monkey, and human
2) Phase I Clinical Support
    Answer mechanistic clearance questions during clinical work
3) Drug Interaction Studies
    Predict drug interactions with common co-medications

Figure 2. A condensed version of the drug development continuum highlighting where B-CLEAR® technology has helped to support drug development strategies.

What Kind of Data Will I Obtain?

1. Species Selection
Hepatobiliary disposition of a proprietary compound evaluated in multiple species at in vivo-relevant concentrations using B-CLEAR® technology. The goal is to evaluate appropriate animal species for preclinical testing or to predict human drug clearance based on animal data.

Figure 3. A B-CLEAR® study comparing Sprague-Dawley rat, beagle dog, cynomolgus monkey, and human. This study assessed the mass of parent compound taken up in to the hepatocyte (Uptake), the percentage of compound effluxed in to the bile pocket (Biliary Excretion), and total in vitro biliary clearance (Clearance). This type of study data enables both a species selection strategy as well as species-specific mechanistic information. For example, expensive monkey studies could be eliminated given their similarity to dog, and dog in vivo data could be used to better predict human in vivo results.

2. Phase I Clinical Support
Compounds can be assessed for their hepatic uptake, biliary excretion, and clearance. The goal of these studies is to predict the extent of biliary excretion prior to in vivo clinical studies, to retroactively determine if bile/feces was a probable route of excretion for "missing" compound in mass balance studies.

Figure 4. Data from example studies that assessed hepatic clearance mechanisms for three well known, clinically-relevant drugs. Using the B-CLEAR® technology and Transporter Certified™ human hepatocytes, the extent of uptake, biliary efflux and clearance was estimated with these three drugs. While the amount of biliary excretion was similar in all three cases, the extent of uptake was markedly different. Rosuvastatin maintained high levels of uptake and pravastatin was significantly lower. The low level of pravastatin uptake indicates lower intracellular concentrations and very little biliary clearance.

3. Drug Interaction Studies
B-CLEAR® can be used to assess drug-transporter interactions by monitoring clinically-relevant probe substrates co-incubated with test compounds. In addition, commonly prescribed probe substrates can be used in these studies to assess potential drug interaction liabilities, thereby avoiding potentially dangerous and expensive human clinical studies. Some common probes include: digoxin (P-gp), rosuvastatin (OATPs, MRP2), nitrofurantoin (BCRP), and taurocholic acid (NTCP, BSEP).

Figure 5. An in vitro-in vivo drug interactions study was published evaluating the potential drug interaction of ritonavir on the hepatobiliary clearance of mebrofenin. In vivo studies showed that ritonavir significantly increased plasma concentrations of mebrofenin in human subjects without affecting biliary clearance. In order to better understand the mechanism of increased systemic exposure by ritonavir, a B-CLEAR® study with human hepatocytes was performed to elucidate mechanisms of inhibition. Results indicated that ritonavir caused a significant decrease in the uptake of mebrofenin (red bars), with no change in the efflux (BEI; blue bars). In vivo PKPB modeling confirmed that the uptake clearance was decreased by 44% with no change in efflux clearance. So in this case, ritonavir, a common antiviral drug, increased systemic exposure in part by inhibition of uptake, most likely through OATP1B1 and OATP1B3.

Relevant References
1) ND Pfeifer, SL Goss, B Swift, G Ghibellini, M Ivanovic, WD Heizer, LM Gangarosa and KLR Brouwer, Effect of Ritonavir on 99mTechnetium-Mebrofenin Disposition in Humans: A Semi-PBPK Modeling and In Vitro Approach to Predict Transporter-Mediated DDIs. CPT: Pharmacometrics & Systems Pharmacology (2013)
2) Ghibellini, G., Leslie, E.M., Pollack, G.M. & Brouwer, K.L.R. Use of tc-99m mebrofenin as a clinical probe to assess altered hepatobiliary transport: integration of in vitro, pharmacokinetic modeling, and simulation studies. Pharm. Res. 25, 1851-1860 (2008).
3) Ghibellini, G., Johnson, B.M., Kowalsky, R.J., Heizer, W.D. & Brouwer, K.L.R. A novelmethod for the determination of biliary clearance in humans. AAPS J. 6, e33 (2004).

How Do I Get These Tools?

Services

Contract research work is the easiest way to get started and engage our scientists to help solve your problems. We not only put our scientists on the job but also take advantage of our decade of history with the B-CLEAR® model and experience with hepatocytes.

We offer the three versions outlined in Figure 2 but also allow for customized study designs in order to make sure we answer the clearance question you need answered. Click here for more details.

Kits

Order our easy to use B-CLEAR® kits to do your own species or biliary clearance testing. We offer several strains for both mouse and rat, as well as dog, monkey, and human.

Each fresh kit comes with plated hepatocytes in 6- or 24-well formats, protocol, a use-license, and all of the media you need to get started. If you prefer to use cryopreserved hepatocytes, you can purchase Transporter Certified™ hepatocytes from your supplier of choice and use them for B-CLEAR® work as well as any other hepatocyte study and know they are fully functional with respect to metabolism and drug transporters.

In addition, we offer consulting time with our scientists to help you design and run your experiments, and can also help put your results into context as compared to our extensive historical database. Click here for more details.

Copyright 2011-2012 Qualyst Transporter Solutions, LLC. All Rights Reserved. B-CLEAR is a registered trademark of Qualyst Transporter Solutions, LLC.
*B-CLEAR® is covered by US Patent 6,780,580 and other worldwide patents issued & pending.