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Drug transporters emerging technologies.

Drug transporters are increasingly being implicated as a source of drug -- drug interactions (DDIs) and potential organ-specific toxicity in humans and interactions with these membrane bound proteins are becoming increasingly important to assess during drug development as recent regulatory draft guidance reflects. As a result, in vitro technology is rapidly advancing in the assessment of drug transporter interactions and some of the major approaches being adopted are discussed.

In the US, approximately 770,000 people are injured or die each year from adverse drug reactions (ADR) (in the UK the rate has recently been estimated at 50,000 people a year).(1), (2) A drug that is a victim of, or perpetrator of, clinically relevant DDIs in humans risks postmarket withdrawal. The DDI potential for a new drug, therefore, is a crucial parameter to assess during drug development. From a regulatory perspective (FDA and EMA), this assessment requires a full understanding of the clearance mechanism(s) for the drug, which includes both metabolic and transport-mediated processes.(3), (4)

From the metabolic perspective, our understanding of the major drug metabolizing enzymes (DMEs), such as cytochrome P450s (CYP450) and UGTs (UDP-glucurononsyltransferases), allow us to make certain predictions from in vitro data regarding DDI potential in vivo, particularly with respect to CYP450 inhibition and induction. During the past 5 -- 10 years, however, DDIs involving transporter proteins have been increasingly identified and the development of in vitro transporter assays is an area of active research for pharmaceutical DMPK/ADME (drug metabolism and pharmacokinetics/absorption, distribution, metabolism and excretion) groups.

The wide distribution of drug transporters (across the body) may lead to them playing fundamental roles in ADME and organ-specific toxicity. Drug transporter proteins can be embedded in the membranes of physiologically important organs and tissues, such as intestine, kidneys, blood brain barrier and liver. Their function is to either pump drugs out of cells (efflux) or to transport drugs into the cell (uptake).

Regulatory Landscape

In 2007, the International Transporter Consortium (ITC) was formed to collate and clarify current knowledge and key opinion of the role of drug transporters in therapeutic and adverse drug response. The ITC consists of scientists from all sectors of drug discovery, development and regulatory approval, academia, industry and FDA.

The ITC met periodically from 2007 to 2009 and held an open Critical Path Transporter Workshop in October 2008, which resulted in a white paper that proposed that interactions be assessed using in vitro methods for seven specific transporters.5 The ITC recommendations were subsequently endorsed by FDA's Pharmaceutical Science and Clinical Pharmacology Advisory Committee and were largely included in last year's draft guideline on The Investigation of Drug Interactions issued by EMA (their second ITC meeting is schedules for March 2012).(4)

The transporters recommended for initial assessment generally Included those for which A clinical relevance to drug disposition has Been identified, namely, the efflux transporters P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP), and the uptake transporters OCT2, OAT1, OAT3, OATP1B1 and OATP1B3. There was, however, no ITC recommendation for BSEP and OCT1, although they have been included in the EMA 2010 draft guideline on drug interactions.(4).

For each transporter, it is recommended that in vitro testing is conducted to assess whether a new drug is either a substrate or an inhibitor of these transporter proteins. The ITC suggests decision trees for each of the recommended transporters outlining how in vitro data should be viewed and the possible consequences for clinical assessment in vivo.

In Vitro Models

An accurate assessment of transporter interactions must take into account the cellular permeability of the test compound under test, as this will determine the appropriate in vitro model to use (generally, only sufficiently permeable molecules will be subject to efflux unless they undergo active uptake into the cell). Furthermore, compounds that undergo a degree of nonspecific binding may also be unsuitable for testing in cellular systems. A variety of models, therefore, have been developed and are currently used to test for interactions with drug transporters in vitro, which typically involve cellular and/or membrane-based assays. The approaches outlined below focus on the major human efflux transporters.

Cell-Based Model Systems

Monolayer Efflux Assay

Polarized cells, either differentiated (for example, Caco-2 cells) or transfected with the transporters of interest (such as MDCKII, HEK293, LLCPK cells) are often used to characterize efflux drug transporter interactions. Usually, in these models, the cells are cultured as a monolayer and the vectorial transport of the drug is measured (the drug flux is measured in two directions, namely, apical-to-basolateral [AB] and vice versa [BA]). The net flux ratio is then calculated (flux BA/AB) and if Greater than 2 (and if it can be reduced in the presence of chemical inhibitors) then this is generally regarded as positive for active efflux of the test compound. By measuring the flux of probe substrates in the presence of various concentrations of the test drug, inhibitory interactions can also be assessed.

The major issues with these monolayer models are that the physicochemical properties of the compound can affect the results (for example, permeability and nonspecific binding of the test compound) and it is necessary to fully understand the behaviour of the drug in a cellular system to interpret the data meaningfully. The availability of monolayers in a variety of well formats (for example, 12, 48 and 96), however, makes them highly flexible and amenable to automation, which can assist throughput. Furthermore, monolayer models consisting of a double (and triple) transfect are now commercially available; for example, MDCK11 cells transfected with both OATP and BCRP. These double/triple transfected models will help to improve predictions regarding efflux interactions with poorly permeable compounds.

Figure 1 presents some of our recent results obtained using Caco-2 cells in a novel 96-well format using digoxin as the P-gp substrate. The apparent permeability of digoxin is markedly reduced in the presence of P-gp inhibitors indicating that digoxin is subject to P-gp-mediated efflux in Caco-2 cells.
Digoxin Papp values (x[10.sup.-6] cm/s)-in Caco2 monolayer

 AB BA

Solvcnt (DMSO) 0.49 15.59
LY33597 (1 [mirco]M) 2.99 3.30
Vcrapamil (100 [mirco]M) 2.87 3.95
PSC-833 (1 [mirco]M) 1.92 3.48
CsA (20 [mirco]M) 2.25 2.47

Figure 1: Inhibition of P-gp-mediated transport of digoxin
by commonly encoutered diagnostic chemical inhibitors of
P-gp activity in vitro.

Note: Table made from bar graph.


Uptake Assays

Drug uptake can also be measured using cell-based models including the use of both primary cells (for example, hepatocytes and kidney cells) and transfected cell lines (for example CHO and HEK293 cells).

Non-Cell-Based Models

Membrane-Based/Preparations

For membrane-based assays, the transporters of interest are over-expressed in cellular systems (usually eukaryotic, insect or mammalian cells) and then membrane fractions are prepared. Membrane-based assays can be highly sensitive to detecting transporter-mediated interactions because they provide 'clean' test conditions without potentially interfering drug metabolising enzymes or other cellular barriers. They also provide the opportunity to study drug transporters individually.

Vesicular Transport

Inverted plasma membrane vesicles (so called 'inside-out' vesicles) are usually used to study the ATP-dependent ABC efflux transporters such as P-gp, BCRP and MRP-2. With vesicle-based assays drug concentrations present at the active regions of the transporter proteins can be measured/monitored and the permeability of the test drug is not a limiting factor to study interactions.

For all the vesicle assays, rapid filtration of the membrane suspension through a filter retains the membrane vesicles containing the effluxed (trapped) probe substrate, which is then quantified. Generally, these assays are used to indicate that an interaction between the test compound and the transporter has occurred and, in typical format, they cannot distinguish between interactions as a substrate or inhibitor because a probe substrate is used as marker of the interaction.

Once an interaction has been identified, additional investigations (typically using polarized cell monolayers) would be undertaken to look specifically at the type of interaction occurring. Figure 2 presents some of our results obtained using P-gp-containing vesicles using N-methyl quinidine (NMQ) as the P-gp substrate. The potency of PSC-833 and LY335979, two commonly used diagnostic inhibitors of P-gp in vitro, can be clearly seen (complete inhibition of NMQ transport).

[FIGURE 2 OMITTED]

ATPase Assays

In ATPase assays, drug transporter interactions are detected via stimulation of ATPase activity (ATP consumption) in cellular membranes that have been over-expressed with a particular transporter protein. Because ABC transporters are active transporters, they are able to operate against cellular concentration gradients and ATP hydrolysis is used as the energy source. Transported substrates enhance the ATPase activity of the transporter over-expressed in the membranes and this is usually detected as an increase in inorganic phosphate measured by colourimetric analysis. Unlike vesicle-based assays, ATPase assays can be used to differentiate between substrates and inhibitors. Currently, ATPase assays are available for a broad range of human drug transporters including P-gp, BCRP and MRPs. In addition to the membrane-based methods outlined above, other noncell-based models have been used, including nucleotide trapping and liposomal-based approaches, but generally these assays are not widely used.

Summary

It is becoming evident that assessment of transport related DDIs will become an increasingly important part of safety assessment during drug development and will form part of the clinical development strategy. The potential for transporter-mediated DDIs to occur is great, considering, for example, that the widely prescribed statins are substrates for the OATP uptake transporters and metformin (widely prescribed for type 2 diabetes) is a substrate for OCT-mediated secretion in the kidney.(6), (7) Fortunately, drug transporter technology is developing quickly and we now have at our disposal a variety of sophisticated in vitro models to help assay for transporter-related interactions.

References

(1.) D.C. Classen, et al., "Adverse Drug Events in Hospitalized Patients, " JAMA (2774,) 301 -- 306 (1997).

(2.) Tai-Yin Wu, et al., "Ten-Year trends in Hospital Admissions for Adverse Drug Reactions in England (1999) -- 2009," J. R. Soc. Med. 103, 239 -- 250 (2010).

(3.) Center for Drug Evaluation and Research, Food and Drug Administration (Rockville, MD, USA), Drug Interaction Studies -- Study Design, Data Analysis, and Implications for Dosing and Labeling (September 2006).

(4.) Guideline on the Investigation of Drug Interactions, Committee for Human Medicinal Products (CHMP) CPMP/EWP/560/95/Rev. 1 -- Corr.* European Medicines Agency, 22 April 2010.

(5.) K.M. Giacomini, et al., "Membrane Transporters in Drug Development," Nat. Rev. Drug Disc. 9, 215 -- 236 (March 2010).

(6.) W.J. Hua, W.X. Hua and H.J. Fang, "The Role of OATP1B1 and BCRP in Pharmacokinetics and DDI of Novel Statins," Cardiovascular Therapeutics (2011). doi: 10.1111/j.1755-5922.2011.00290

(7.) M.L. Reitman and E.E. Schadt, "Pharmacogenetics of Metformin Response: A Step in the Path Toward Personalized Medicine," J. Clin. Invest. 117(5), 1226-1229 (2007).

For more information

Guy Webber, Msc Chief Scientist(In Vitro Sciences) Quotient Bioresearch Ltd

guy.webber@quotientbioresearch.com
Abbreviations

ATP Adenosine Tri Phosphate
BSEP Bile Salt Export Pump
MDCKII Madin-Darby Canine Kidney Type II
OAT Organic Anion Transporter
OATP Organic Anion Transport Protein
OCT Organic Cation Transporter
MRP Multi Resistance Protein
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Title Annotation:DRUG DEVELOPMENT
Author:Webber, Guy
Publication:Pharma
Date:Nov 1, 2011
Words:1841
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