The new draft guidance on in vitro metabolism and transporter studies

November 03, 2017

The new FDA Draft guideline on In Vitro Transporter- Mediated Drug-Drug Interaction Studies – What does it mean for you?

On October 25th the FDA released its new draft guidance on in vitro metabolism and transporter studies. In this newsletter we collected the take-home messages for you and explain how SOLVO can help you in meeting the newest requirements for transporter studies:

1. MATE1 and MATE2-K should now be studied

It was widely anticipated that these transporters would be included in the new guideline. The importance of MATE1 and MATE2-K was already emphasized by a working group from the International Transporter Consortium in 2013 (Hillgren et al., 2013). As a matter of fact, based on scientific evidence we have recommended including MATE1 and MATE2-K in regulatory studies for our clients over the past 1-2 years, and have had assays for these transporters in our portfolio since 2012.

Inhibition of MATE1 and MATE2-K should be studied for all new Investigational Drugs whereas substrate studies are required if 25% of the total clearance is due to active renal secretion, whereby active secretion is defined as CLr – (fu,p x GFR).

2. Time Dependent Inhibition

A relatively new development in the transporter field is the question of time dependence of transporter inhibition. This phenomenon is well known for CYP enzymes and protocols to study time dependent inhibition (TDI) have been well established. In contrast, for transporters this is not the case. TDI of transporters is not yet fully understood and has only been described for a few transporters, and as such established protocols are missing. The FDA recommends considering a 30 minute pre-incubation step to test for the worst-case scenario for OATP1B1 and OATP1B3 (pre-incubation tends to decrease IC50 values). At SOLVO we have established a full protocol to study TDI of your Investigational Drug in more detail and we work on in-house studies to better understand and determine the potential for TDI for all FDA recommended transporters.

Contact us to find out more about our protocol for TDI studies!

Demonstration and characterization of time-dependent transporter inhibition. OATP1B1 was preincubated with cyclosporin A or DMSO prior to conducting an E2-17βG uptake inhibition assay (1 µM total substrate concentration, incubation for 3 min at 37°C) with the same inhibitor. The degree of inhibition enhancement correlated positively with the duration of preincubation (a, IC50 curves; b, IC50 shift values: fold difference of IC50 values obtained without and with preincubation). The effect of preincubation was durable: enhancement of inhibition over the non-preincubation condition was observed even when a 1-h or 3-h washout period was inserted between a 30-min preincubation and the assay (c, IC50 curves; d, IC50 shift values).

3. Calibration of in vitro systems to generate cut-off values for inhibition studies

The most debated parts of the 2012 FDA draft guidance as well as the 2013 EMA guidelines on transporter studies have been the cut-off values that are used to determine whether an in vitro inhibition will likely cause in vivo Drug-Drug Interactions. Part of the discussion is that the cut-offs are often based on a limited amount of data, but more importantly these values are ambiguous due to the enormous lab-to-lab variability of IC50 values for transporters (see Bentz et al., 2013). In the new guideline the FDA suggests to calibrate in vitro systems in order to generate in-house cut-off values that are specific for the lab and test system used. This approach was first suggested by Roche (Poirier et al., 2014)

SOLVO recently published a poster on the calibration of its HEK293-VT-MDR1 assay, which was carried out in collaboration with Roche. Click here to view the poster.

Similar calibration experiments for inhibition assays involving BCRP, OATPs, OATs, OCTs and MATEs are currently underway at SOLVO and we will keep you posted on our progress.

4. More emphasis on study design of in vitro transporter studies

The 2017 guidance puts much more emphasis on the design of in vitro transporter studies than previous guidances. We welcome this trend as we firmly believe in properly designed studies and experiments.

The FDA recommends taking into consideration the following factors when designing an in vitro transporter study:

  • Solubility limits of the test article

Solubility studies are a standard part of every study we run. The solubility of each test article is tested in all the buffers that are applied during the study.

 

  • Non-specific binding of the test article

SOLVO offers standard protocols for non-specific binding and dosing concentration studies. When substantial non-specific binding is observed we can adjust the study protocol and/or use real concentrations rather than nominal concentrations in the calculation of kinetic parameters (e.g. IC50). We have seen a strong increase in the demand for these additional experiments over the past 12-18 months.

 

  • Probe substrate concentrations should be at or below its KM values

At SOLVO we have thoroughly validated all our assays, including the optimal probe substrate concentrations. Characterization data files are available upon request.

In addition we have developed a protocol for measuring Ki values, which are recommended by the EMA.

  • Choice of probe substrate

Transporter inhibition can be substrate dependent. For this reason the FDA recommends to use a probe substrate that may also be used in later clinical studies. Alternatively, the FDA recommends to use a probe substrate that usuallygenerates a lower IC50 for known inhbitors.

SOLVO offers multiple probe substrates for almost all its assays. In the past few years we have put extra emphasis on developing clinically relevant probe substrates, such as metformin (OCTs, MATEs), tenofovir (OAT1), Methotrexate (OAT3), Dabigatran-Etexilate (MDR1, see poster) and statins (OATPs, see report).

Altogether SOLVO welcomes the new recommendations in the 2017 FDA draft guidance and we are well prepared to help you in your next in vitro transporter study for regulatory submission. We look forward to working with you!

The SOLVO Team


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