In January 2020, the US FDA finalised its 2017 draft regulatory guidance for industry on in vitro DDI studies by publishing the In Vitro Drug Interactions Studies – Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions Guidance for Industry. This document outlines how experimental in vitro studies should be carried out and provides instruction on how results should be used to determine potential clinical DDI risk.
Functionally, very little has changed between the draft guidance of 2017 and this final version, mainly consisting of clarifications with very few additional requirements. In this blog, we take a closer look at the small differences there are and explain how they impact on data interpretation for in vitro regulatory DDI studies.
In covering the differences from the draft version of the guidance, we will explore metabolism-mediated drug interactions including CYP inhibition, CYP induction and substrate identification (reaction phenotyping) studies, and then discuss transporter inhibition and transporter substrate studies.
Metabolism Mediated Drug Interactions
Initially, the guidance now promotes the investigation of in vitro metabolic studies before conducting first-in-human studies to better inform the necessity and design of clinical PK studies. Further clarifications are focussed on CYP inhibition, induction and enzyme substrate identification.
CYP Inhibition: One of the changes in terms of metabolism mediated drug interactions relates to CYP inhibition where the FDA has indicated that IC50/2 can be used as an estimate of Ki for reversible inhibitors if the probe substrate concentration used is at the Km for the CYP enzyme. This suggests that assessing Ki may no longer be necessary under these conditions – saving both time and cost in DDI assessment. Of further note is that the Ki and KI have been corrected to Ki,u and KI,u to highlight the fact that the unbound values should be used in the calculations.
CYP Induction: The assay design remains the same but the FDA has provided more detail on the data interpretation for CYP induction studies, in particular, for the fold change method. For example, CYP induction is presumed if a concentration-dependent increase in CYP mRNA is observed which is ≥ 2-fold the vehicle control at the expected hepatic concentrations of the drug. The guidance proposes 30-fold of mean unbound maximal steady state plasma concentration at the therapeutic dose as an estimation of the expected hepatic drug concentration. It also recommends, that even if the induction is less than 2-fold, induction potential cannot be ruled out if the increase in CYP mRNA is greater than 20% of the positive control. The alternative methods for calculating CYP induction risk (basic kinetic R3 model and correlation methods) have remained similar to the previous draft guidance.
Enzyme Substrate Identification: In terms of substrate studies, very little has changed, the guidance has extended the panel of possible non-CYP enzymes to consider if CYPs are not found to contribute towards metabolism. These additional enzymes include aldehyde oxidases (AO), carboxylesterases (CES) and sulfotransferases (SULTs) in addition to other non-CYP Phase I and Phase II enzymes.
Transporter Mediated Drug Interactions
Transporter Inhibition: The main change to highlight relates to the basic static equation cut-off for the MATE transporters which has now increased to 0.1 and falls in line with the existing cut-off for the other renal transporters (OATs and OCT2). This amended cut-off is more relaxed than the previous guidance and will reduce the likelihood of investigational drugs being flagged as potential in vivo MATE inhibitors. Another welcome change is the removal of the suggested 30 min pre-incubation time for the duration of the inhibitor pre-incubation step for OATP transporters. This omission recognises research performed by Cyprotex demonstrating that a shorter inhibitor pre incubation time of 15 min is adequate for assessing correct OATP inhibition as no statistically significant difference in IC50 existed following a 15 min or 30 min inhibitor pre-incubation step. Finally, one notable addition to the P-gp and BCRP inhibition guidance is the specific consideration of investigational drugs administered through the parenteral route or of systemically circulating metabolites acting as inhibitors. In these circumstances, a different basic static equation is used to determine DDI risk, namely I1/IC50 (or Ki) ≥ 0.1, where I1 is the total Cmax of the inhibitor drug or metabolite.
Transporter Substrate Identification: Last but not least, both assay design and data interpretation remain relatively unchanged from the draft guidance for transporter substrate studies.
In summary, the majority of the amendments to the draft 2017 guidance focus on the data analysis stage rather than assay design stage. The main changes affect transporter inhibition studies where new cut-offs have been specified for the MATE transporters. One unexpected amendment relates to the use of IC50/2 as an estimate of Ki for reversible CYP inhibitors, potentially reducing the reliance on Ki studies.
With over 20 years’ experience conducting DDI studies, Cyprotex have a team of experts who can guide you through the process and assist in the planning, execution and interpretation of these regulatory studies. Our popular ADME and DDI guides have also been updated to reflect the 2020 guidance and help you further understand why, when, and how to perform these studies.
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