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Key Highlights from the New Draft FDA Guidance on DDI Studies– Focus on Transporter Inhibition and Substrate Studies

This is the third blog in the series focusing on the updated draft FDA guidance on in vitro DDI studies.  This week the spotlight is on transporter inhibition and substrate studies.

The key changes are discussed below:

Transporter inhibition studies

  • MATE1 and MATE2-K are now included in the panel of transporters for inhibition assessment due to their important role in the mechanism underlying metformin clinical pharmacokinetic DDIs1. Given that the FDA first mentioned the importance of sponsors having MATE inhibition data at AAPS Transporter Workshops held in 2015 and 2016, the inclusion of these transporters was widely expected.  Indeed, we have been advising our clients to include such studies since early 2015.
  • Another key update for transporter inhibition studies is the requirement for evaluation of time‑dependent inhibition for OATP1B1 and OAPT1B3 by pre-incubating the investigational drug for a specific period of time prior to adding probe substrate. As any observed time-dependency is likely a compound‑related in vitro effect due to the short linear co-incubation times often utilised in SLC transporter inhibition assays, since mid-2016 all Cyprotex SLC inhibition assays have included a 15‑min pre-incubation step with investigational drug as standard.  This ensures clients obtain the appropriate (lowest) IC50 estimate for more accurate DDI risk assessment.  You may be interested in learning our research in this area. Read more
  • As inhibition can be substrate-dependent and in order to extrapolate better to the in vivo situation, a further recommendation is that investigational drug inhibitor evaluation be conducted with in vitro probe substrates that may be used as in vivo probes in later clinical studies. In line with this recommendation our in vitro inhibition assays have always used either a clinically relevant probe substrate (e.g. loperamide for P-gp, metformin for OCT2 and MATE1), a clinically relevant surrogate probe substrate (e.g. estradiol 17β‑glucuronide as a surrogate for statins at OATP1B12, estrone 3‑sulfate as a surrogate for rosuvastatin at BCRP3), or a probe substrate that generates a lower IC50 for known inhibitors (e.g. PAH for OAT1, estrone 3-sulfate for OAT3, TEA for MATE2-K) thereby avoiding any underestimation of interaction potential of the investigational drug.
  • In terms of data interpretation and in recognition that the majority of clinical DDIs mediated via P-gp and BCRP transporters with oral drugs result in increased absorption of the victim drug4,5 only intestinal DDIs are considered for P-gp and BCRP inhibition and systemic interactions (biliary and renal) are no longer considered for these two transporters. Additionally, a more conservative cut-off has been introduced for OATP1B1 and OATP1B3, as well as for MATE1 and MATE2-K compared to the other renal transporters.  This could lead to investigational drugs that passed the criteria previously now being identified as potential inhibitors in vivo.

Transporter substrate studies

  • MATE1 and MATE2K are now included if active renal secretion is greater than or equal to 25% of total clearance.
  • Although it was already common practice based on the 2006 FDA draft guidance to evaluate at least three concentrations of the investigational drug to cover the range of clinically relevant concentrations, this has been reintroduced in the new guidance following its omission from the 2012 guidance.
  • Another point worth noting is that in vitro studies for P-gp and BCRP can be avoided if the investigational drug is BCS class I, unless there is a specific safety concern with distribution into particular tissues where these transporters are expressed.

In terms of the in vitro test systems suggested for transporter studies, in addition to the standard Caco‑2 and transfected cell lines, a much greater range has been highlighted for consideration (should the need arise depending on compound properties) including membrane vesicles, knockout/down cells and hepatocytes.  Despite this, it is likely that, for routine transporter studies, Caco-2 and transfected cell lines will remain the preferred in vitro transporter models as interpreting the data is clearer and more well-defined.

The updated draft 2017 guidance has also reintroduced various recommendations for assay development and validation (e.g. stability, non-specific binding), which were first mentioned in the 2006 guidance for P-gp systems, but omitted from the 2012 guidance.  As such the majority of recommendations are already routinely evaluated either directly (e.g. transport linearity, probe substrate concentration well below Km) or indirectly (e.g. stability and non-specific binding via % recovery in polarised systems) as part of standard transporter assay validations.  However, it is important to also bear in mind that certain recommendations may not need evaluation if deemed inappropriate for the assay design.  For example, it may not be necessary to specifically assess non-specific binding of investigational drug in inhibition assays containing an inhibitor pre-incubation step, as this step may result in saturation of any non-specific binding sites (were it to occur), such that upon incubation with co-administered probe substrate the fresh investigational drug concentrations would be nominal.

To keep you up to date with all the current regulatory guidance for in vitro DDI studies, you may be interested in our popular booklets: ‘Everything you need to know about ADME’ and ‘DDI regulatory guidance – An easy to follow guide’ which you can download from our website.

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1 Elsby et al. (2017) Mechanistic in vitro studies confirm that inhibition of the renal apical efflux transporter multidrug and toxin extrusion (MATE) 1, and not altered absorption, underlies the increased metformin exposure observed in clinical interactions with cimetidine, trimethoprim or pyrimethamine. Pharmacol Res Perspect 5(5).  Learn more

2 Sharma et al. (2012) Prediction of the in vivo OATP1B1-mediated drug-drug interaction potential of an investigational drug against a range of statins. European Journal of Pharmaceutical Sciences 47(1): 244-255.

3 Poster: Estrone 3-sulfate as a surrogate Breast Cancer Resistance Protein (BCRP) in vitro probe substrate for predicting drug-drug Interaction potential with rosuvastatin. Learn more

4 Elsby et al. (2011) The utility of in vitro data in making accurate predictions of human P-glycoprotein mediated drug-drug interactions: A case study for AZD5672. Drug Metab Dispos 39: 275-282.

5 Elsby et al. (2016) Solitary inhibition of the breast cancer resistance protein (BCRP) efflux transporter results in a clinically significant drug-drug interaction with rosuvastatin by causing up to a two-fold increase in statin exposure. Drug Metab Dispos 44: 398-408.