Clinical Drug 
Interaction Studies   
Cytochrome P450 
Enzyme- and 
Transporter-Mediated 
Drug Interactions 
Guidance for Industry 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
January 2020 
Clinical Pharmacology 
 
    
Clinical Drug 
Interaction Studies   
Cytochrome P450 
Enzyme- and 
Transporter-Mediated 
Drug Interactions 
Guidance for Industry 
 
Additional copies are available from: 
Office of Communications, Division of Drug Information  
Center for Drug Evaluation and Research 
Food and Drug Administration 
10001 New Hampshire Ave., Hillandale Bldg., 4th Floor  
Silver Spring, MD 20993-0002  
Phone: 855-543-3784 or 301-796-3400; Fax: 301-431-6353 
Email: druginfo@fda.hhs.gov  
https://www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
January 2020 
Clinical Pharmacology 
 
  Contains Nonbinding Recommendations 
 
TABLE OF CONTENTS 
 
 
 
I.  INTRODUCTION ...................................................................................................... 1 
II.  BACKGROUND ........................................................................................................ 2 
III.  TIMING OF CLINICAL DDI STUDIES................................................................... 2 
IV.  DESIGN AND CONDUCT OF CLINICAL DDI STUDIES ...................................... 3 
A.  Types of DDI Studies..................................................................................................... 3 
B.  Study Planning and Considerations for Stand-Alone Prospective DDI Studies ................... 6 
C.  Study Planning and Considerations for Prospective Nested DDI Studies ........................... 9 
D.  Specific Considerations for CYP-Mediated Interactions..................................................10 
E.  Specific Considerations for Transporter-Mediated Interactions ......................................12 
F.  Cocktail Approaches ....................................................................................................14 
G.  Other Considerations ...................................................................................................15 
V.  REPORTING AND INTERPRETING STUDY RESULTS..................................... 16 
A.  Study Results Reporting ...............................................................................................16 
B.  Interpreting DDI Studies ..............................................................................................17 
VI.  LABELING RECOMMENDATIONS ..................................................................... 21 
VII.  ABBREVIATIONS .................................................................................................. 22 
VIII.  DEFINITIONS ......................................................................................................... 23 
i  Contains Nonbinding Recommendations 
 
Clinical  Drug Interaction Studies   
Cytochrome P450 Enzyme- and Transporter-Mediated  Drug 
Interactions 
Guidance for Industry1 
 
 
This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) on 
this topic.  It does not establish any rights for any person and is not binding on FDA or the public.  You 
can use an alternative approach if it satisfies the requirements of the applicable statutes and regulations.  
To discuss an alternative approach, contact the FDA office responsible for this guidance as listed on the 
title page.   
 
 
 
 
I.  INTRODUCTION  
 
This final guidance helps sponsors of investigational  new drug applications  and applicants of 
new drug applications  evaluate drug-drug interactions (DDIs) during drug development and 
determine essential information  to communicate in labeling.2    
 
This final guidance describes clinical  studies to evaluate the DDI potential of an investigational 
drug, including:   (1) the timing  and design of the clinical  studies; (2) the interpretation  of the 
study results; and (3) the options for managing DDIs in patients.  Specifically,  this guidance 
provides considerations  for evaluating pharmacokinetic  cytochrome P450 (CYP) enzyme- or 
transporter-mediated interactions.   
 
A related final January 2020 FDA guidance for industry entitled In Vitro Drug Interaction 
Studies  Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions3 focuses on 
how to assess the DDI potential of a drug in vitro and how to use the results from those 
assessments to inform clinical  DDI studies.  Together, these two final guidances on DDIs 
describe a systematic, risk-based approach for evaluating DDIs and determining  essential 
information  to communicate in labeling.      
 
In general, FDAs guidance documents do not establish legally  enforceable responsibilities.   
Instead, guidances describe the Agencys current thinking  on a topic and should be viewed only 
                                              
1 This guidance has been prepared by the Office of Clinical Pharmacology, Office of Translational Sciences in the 
Center for Drug Evaluation and Research at the Food and Drug Administration.  
 
2 This guidance does not discuss DDIs involving therapeutic proteins, protein displacement, modulation of Phase II 
metabolic enzymes, or other mechanisms that do not involve cytochrome P450 enzymes or transporters (e.g. gastric 
pH change, complexation). 
 
3 We update guidances periodically.  For the most recent version of a guidance, check the FDA guidance web page 
at https://www.fda.gov/RegulatoryInformation/Guidances/default.htm. 
  1  Contains Nonbinding Recommendations 
 
as recommendations,  unless specific regulatory or statutory requirements are cited.  The use of 
the word should in Agency guidances means that something  is suggested or recommended, but 
not required.  
 
 
II.  BACKGROUND 
 
Patients frequently use more than one medication at a time.  Unanticipated,  unrecognized,  or 
mismanaged DDIs are an important cause of morbidity  and mortality  associated with 
prescription drug use and have occasionally  caused the withdrawal of approved drugs from the 
market.  In some instances, understanding  how to safely manage a DDI may allow the FDA to 
approve a drug that would otherwise have an unacceptable level of risk.  Clinically  relevant 
DDIs between an investigational  drug and other drugs should therefore be:  (1) defined during 
drug development as part of the sponsors assessment of the investigational  drugs benefits and 
risks; (2) understood via nonclinical  and clinical  assessment at the time of the investigational 
drugs approval; (3) monitored after approval; and (4) communicated in the labeling.     
 
The goals of studies that investigate CYP enzyme- and transporter-mediated DDIs are to: 
 
  Determine whether the investigational  drug alters the pharmacokinetics of other drugs 
 
  Determine whether other drugs alter the pharmacokinetics  of the investigational  drug 
 
  Determine the magnitude of changes in pharmacokinetic  parameters 
 
  Determine the clinical  significance of the observed or expected DDIs 
 
  Inform the appropriate management and prevention strategies for clinically  significant 
DDIs  
 
 
III.  TIMING OF CLINICAL DDI STUDIES 
 
After conducting in vitro drug metabolism and drug transporter studies, sponsors should 
determine the need for and timing of clinical  DDI studies with respect to other studies in their 
clinical  development program.  Sponsors should assess the DDI potential before the product is 
administered to patients who are likely  to take concomitant medications that could interact with 
the investigational  drug.  Furthermore, sponsors should collect enough DDI information  to 
prevent patients from being unnecessarily excluded from any clinical  study because of their 
concomitant medication use.  Unnecessary restrictions on patient enrollment can result in clinical 
study populations  that are not representative of the indicated patient population.   Inadequate 
studies of DDIs can hinder the FDAs ability  to determine the benefits and risks of an 
investigational  drug and could result in restrictive labeling,  postmarketing requirements or 
commitments,  and/or delayed approval until sufficient information  on DDIs is available.      
 
  2  Contains Nonbinding Recommendations 
 
Sponsors should summarize their DDI program at milestone meetings with the FDA.  Potential 
discussion topics at these meetings include the planning,  timing,  and evaluation of studies to 
determine the DDI potential of the investigational  drug.   
 
 
IV.  DESIGN AND CONDUCT OF CLINICAL DDI STUDIES 
 
Clinical  DDI studies compare substrate concentrations in the absence and presence of a 
perpetrator drug in vivo.  For the purposes of this guidance, the terms substrate and victim are 
used interchangeably  to refer to the drug whose exposure may or may not be changed by a 
perpetrator drug.  The term perpetrator refers to the drug that causes an effect on the substrate 
drug by inhibiting  or inducing  enzymes or transporters.  Index perpetrators are drugs that inhibit 
or induce a given metabolic pathway by a defined magnitude when administered with a sensitive 
substrate and are commonly used in prospective DDI studies.  See section VIII for definitions  of 
key terms used in this guidance. 
 
A.  Types of DDI Studies 
   
1.  Prospective Studies and Retrospective Evaluations  
 
Clinical  DDIs can be evaluated in prospective studies and retrospective evaluations.  Regulatory 
decision-making  generally requires prospective studies specifically  designed for this purpose.  
Retrospective evaluation of drug concentrations from studies not designed to evaluate DDIs 
rarely includes sufficient precision to provide an adequate assessment (see section V.B.2 for 
more details).  
 
Protocols for prospective clinical DDI studies are specifically  designed to detect DDIs as a major 
objective, and the data analysis method and study design elements (e.g., the pharmacokinetic 
sampling  plan and the timing  of concomitant medication administration)  are prespecified.  
Prospective DDI studies are often stand-alone studies.  However, a prespecified subgroup 
analysis within a larger study (e.g., a phase 3 study) may qualify as a prospective DDI study if it 
includes certain factors common to prospective studies (see section IV.C).  Sponsors should 
contact the appropriate OND prescription  drug review division  regarding prospective DDI 
studies that are nested within a larger study whose primary objective is not to assess DDIs, if 
such a design was not previously  discussed at a milestone meeting. 
 
2.  DDI Studies With Index Perpetrators and Index Substrates:  Index Studies 
 
To test whether an investigational  drug is a victim  of DDIs, sponsors should use index 
perpetrators.  Index perpetrators predictably inhibit  or induce drug metabolism by a given 
pathway and are commonly  used in prospective DDI studies.  The magnitude of inhibition  or 
induction  (i.e., strong or moderate) caused by index perpetrators is described in section V.B.3.  
Strong index perpetrators cause DDIs of the greatest magnitude when co-administered with the 
investigational  drug (as a substrate) by altering the function of a given metabolic pathway.  
Results provide essential information  about the DDI potential of an investigational  drug and can 
inform future DDI studies. 
  3  Contains Nonbinding Recommendations 
 
 
To test whether the investigational  drug is a perpetrator, sponsors should use index substrates, 
which have defined changes in systemic exposure when administered with a strong inhibitor  for 
a specific drug elimination  pathway.  Sensitive index substrates are drugs whose area under the 
concentration-time  curve (AUC) values increase 5-fold or more when co-administered with a 
known index inhibitor  for a particular pathway, or whose AUC ratio in poor metabolizers for a 
specific enzyme is greater than or equal to 5-fold compared to extensive metabolizers.   
Moderately sensitive index substrates are drugs whose AUC values increase 2- to <5-fold when 
co-administered with a known strong index inhibitor  or whose AUC values increase 2- to <5-fold 
in individuals  with certain genetic polymorphisms  of a specific enzyme.  Studies with sensitive 
index substrates determine the maximum decrease or increase in substrate exposure resulting 
from the investigational  drugs induction  or inhibition  of enzymes, respectively.  Moderately 
sensitive index substrates can be used if a sensitive index substrate is not available for an enzyme 
(e.g., CYP2C9).    
 
A list of currently recommended index drugs for specific CYP pathways (either as substrates, 
inhibitors,  or inducers) is maintained on the FDAs Web site for Drug Development and Drug 
Interactions.4  The magnitude of DDIs from studies with index inhibitors  or inducers is typically 
representative of the magnitude of the interaction for other drugs with the same level of 
inhibition  or induction  (i.e., strong or moderate).  Similarly,  the effect of the investigational  drug 
on index substrates is representative of the effect on other sensitive substrates for that metabolic 
pathway. 
 
Most of the drugs listed on the FDAs Web site for Drug Development and Drug Interactions as 
transporter substrates, inducers, or inhibitors  cannot be considered as index drugs for prospective 
DDI studies because they lack specificity  for one transporter.  However, clinical  interaction 
studies conducted with these drugs can provide useful information  about potential DDIs with 
concomitant drugs.  See sections IV.A.3 and IV.E for considerations for transporter-mediated 
DDI studies.    
 
Evaluating the effect of an investigational  drug on an endogenous substrate (e.g., 4-
hydroxycholesterol5)  can provide information  about its effect on a metabolic pathway (e.g., 
induction  of CYP3A-mediated metabolism).  However, we generally do not recommend using 
the endogenous substrate for the index studies because it is difficult  to consistently extrapolate 
the effect on an endogenous substrate to other substrates for the same enzyme or transporter. 
 
3.  DDI Studies With Expected Concomitant Drugs:  Concomitant-Use Studies 
 
Index substrates and perpetrators are not chosen based on their use in the investigational  drugs 
target population,  but rather because of their well-defined interaction effects that provide 
information  about the DDI potential of the investigational  drug.  Therefore, the results from DDI 
                                              
4 FDAs Web site on Drug Development and Drug Interactions can be found at 
http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DevelopmentResources/DrugInteractionsLabeling/ucm080499.htm   
 
5 Mao J, I Martin, J McLeod, G Nolan, R van Horn, M Vourvahis, and YS Lin, 2017, 4-Hydroxycholesterol as an 
Emerging Biomarker of Hepatic CYP3A, Drug Metab Rev, 49(1):18-34. 
  4  Contains Nonbinding Recommendations 
 
studies with index perpetrators or substrates are used to either extrapolate findings  to 
concomitant medications sharing the same DDI properties or to help design DDI studies with 
commonly  used concomitant medications in the investigational  drugs target population.   In 
contrast to DDI studies with index drugs, results from a concomitant-use study with a non-index 
drug can be difficult  to extrapolate to other drugs. 
 
The relevant concomitant medications for study include those used to treat the same condition 
for which the investigational  drug is being studied or those used to treat common co-morbidities 
in the patient population.   Sponsors should evaluate concomitant medications that are likely  to 
interact with the investigational  drug in the clinical  practice setting (e.g., add-on drug therapies 
or treatments for common co-morbidities)  using a risk-based approach that considers the drug 
interaction mechanisms and the clinical  significance of any changes in the drugs exposure.  
Examples and classifications  of drugs for individual  elimination  pathways  either as substrates, 
inhibitors,  or inducers  are maintained on the FDAs Web site for Drug Development and 
Drug Interactions.6 
 
Currently, substrates or perpetrators of transporters fulfilling  the criteria of an index drug have 
not been identified  (see section IV.A.2).  The choice of victim  or perpetrator drug for transporter 
studies should be based primarily  on the likelihood  of concomitant use of the two drugs.  Results 
from DDI studies that investigate transporter-mediated interactions are most relevant to the 
studied drugs; extrapolation  of study results to other drugs is limited.   Thus, most clinical  DDI 
studies that investigate the effects of transporter interactions are considered concomitant-use 
studies.  See section IV.E for considerations  when investigating  transporter-mediated 
interactions.       
 
4.  In Silico DDI Studies 
 
Physiologically  based pharmacokinetic  (PBPK) models can be used in lieu of some prospective 
DDI studies.  For example, PBPK models have predicted the impact of weak and moderate 
inhibitors  on the substrates of some CYP isoforms (e.g., CYP2D6, CYP3A) as well as the impact 
of weak and moderate inducers on CYP3A substrates.7,8,9  These predictions were made after 
prospective clinical  trials showed a significant  DDI between the investigational  drug and strong 
                                              
6 FDAs Web site on Drug Development and Drug Interactions can be found at 
http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DevelopmentResources/DrugInteractionsLabeling/ucm080499.htm   
 
7 Wagner C, P Zhao, Y Pan, V Hsu, J Grillo, SM Huang, and V Sinha, 2015, Application of Physiologically Based 
Pharmacokinetic (PBPK) Modeling to Support Dose Selection:  Report of an FDA Public Workshop on PBPK, CPT:  
Pharmacometrics & Systems Pharmacology, 4(4):226-230. 
 
8 Vieira, MD, MJ Kim, S Apparaju, V Sinha, I Zineh, SM Huang, P Zhao, 2014, PBPK Model Describes the Effects 
of Co-Medication and Genetic Polymorphism on Systemic Exposure of Drugs that Undergo Multiple Clearance 
Pathways, Clinical Pharmacol Ther, 95(5):550-557. 
 
9 Wagner, C, Y Pan, V Hsu, JA Grillo, L Zhang, KS Reynolds, V Sinha, P Zhao, 2015, Predicting the Effect of 
CYP3A Inducers on the Pharmacokinetics of Substrate Drugs Using Physiologically Based Pharmacokinetic 
(PBPK) Modeling:  An Analysis of PBPK Submissions to the US FDA, Clinical Pharmacokinet, 54(1):117-127. 
 
  5  Contains Nonbinding Recommendations 
 
index inhibitors  or inducers.  Before using a PBPK modeling  approach to predict the effects of 
moderate or weak perpetrator drugs on the exposure of an investigational  drug, the sponsor 
should verify the models using human pharmacokinetic  data and information  from DDI studies 
that used strong index perpetrators.  Suggestions for how sponsors should conduct PBPK 
analyses and present results for intended purposes are available in the January 2020 FDA 
guidance for industry In Vitro Drug Interaction Studies  Cytochrome P450 Enzyme- and 
Transporter-Mediated Drug Interactions and the 2018 FDA guidance for industry 
Physiologically Based Pharmacokinetic Analyses Format and Content.  Because of evolving 
science, new uses of in silico methods to predict DDIs in lieu of clinical  DDI studies are 
continuously  being considered by the FDA.10  We encourage sponsors to discuss issues and 
considerations related to the use of in silico models with the FDA. 
 
B.  Study Planning and Considerations for Stand-Alone Prospective DDI Studies  
 
Protocol development11 and study design can vary depending on factors including: 
 
  Whether the victim  and/or perpetrator drugs are used acutely or chronically 
 
  Whether there are exposure-related safety concerns with the substrate 
 
  The pharmacokinetic  and pharmacodynamic  characteristics of the substrate and 
perpetrator drugs 
 
  Whether both induction  and inhibition  will be assessed  
 
  The mechanism of the DDI (e.g., time-dependent inhibition) 
 
  Whether the persistence of inhibition  or induction  after withdrawal of the perpetrator drug 
will be assessed    
 
The above factors can influence study design elements, including  the number of experimental 
allocations  (e.g., two-way versus three-way cross-over), the duration of exposure to the 
perpetrator, the substrate pharmacokinetic  sampling  strategy, and the study design (e.g., single-
dose or steady-state design).  The purpose of most DDI studies is to determine the ratio of a 
measure of substrate drug exposure (e.g., AUC ratio) in the presence and absence of a perpetrator 
drug.  The following  considerations are important when designing  prospective clinical  DDI 
studies to unambiguously  determine this ratio. 
 
1.  Study Population and Number of Subjects 
 
                                              
10 Wagner C, P Zhao, Y Pan, V Hsu, J Grillo, SM Huang, and V Sinha, 2015, Application of Physiologically Based 
Pharmacokinetic (PBPK) Modeling to Support Dose Selection:  Report of an FDA Public Workshop on PBPK, CPT:  
Pharmacometrics & Systems Pharmacology, 4(4):226-230. 
 
11 Unless otherwise noted, the information below applies to both index studies and concomitant-use studies. 
  6  Contains Nonbinding Recommendations 
 
Most clinical DDI studies can be conducted using healthy subjects, assuming that findings  in 
healthy subjects can be used to predict findings in the intended patient population.   Safety 
considerations can prevent the use of healthy subjects in studies of certain drugs.  Use of the 
intended patient population  allows the researcher to study pharmacodynamic endpoints  that 
cannot be studied in healthy subjects.    
 
The number of subjects included in a DDI study should be sufficient to provide a reliable 
estimate of the magnitude and variability  of the interaction.   
 
2.  Dose  
 
The doses of the perpetrator drug used in DDI studies should maximize the possibility  of 
identifying  a DDI.  Thus, the sponsor should use the maximum  dose and the shortest dosing 
interval of the perpetrator under the intended conditions  of use or as labeled.   
 
If the substrate drug has dose-proportional  pharmacokinetics,  the sponsor can use any dose in the 
range where exposure to the drug increases in a dose-proportional  manner.  If the substrate drug 
has dose-dependent pharmacokinetics,  the sponsor should use the therapeutic dose most likely to 
demonstrate a DDI.  When there are safety concerns in the aforementioned scenarios, the sponsor 
can use lower doses of the substrate.  A PBPK model verified for the mechanism of dose-
dependent pharmacokinetics  of the substrate can be used to support dose selection.    
 
3.  Single or Multiple Doses 
 
Single-dose  administration  of the perpetrator should be done only if the perpetrator is not a 
potential inducer or time-dependent inhibitor.     
 
An inhibitor  can be administered as a single dose if it is justified  that single-dose administration 
of the inhibitor  has a similar effect on the enzyme or transporter of interest to that after multiple 
dosing.  For substrates with long half-lives it may be necessary to administer a perpetrator 
multiple  times to cover the full time-course of the substrate exposure. 
 
The sponsor should administer inducers as multiple  doses to ensure the maximal  induction  of a 
specific pathway.  It may take about 2 weeks of daily drug administration  to achieve the 
maximum  level of induction  in a specific pathway.  When there are multiple  mechanisms of 
interactions for a specific perpetrator, single-dose administration  may be appropriate in certain 
situations (e.g., evaluation  of rifampin as an inhibitor  of organic anion transporting polypeptide 
1B1 (OATP1B1)), while multiple-dose  administration  may be appropriate in other situations 
(e.g., evaluation of rifampin as a CYP3A inducer). 
 
Single-dose  administration  of the substrate is acceptable if the substrate does not show time-
dependent pharmacokinetics  (e.g., auto-inhibition  or auto-induction).   In those situations,  the 
observed magnitude increase in exposure in single-dose studies can be extrapolated to steady-
state conditions.   Multiple-dose  administration  of the substrate and a perpetrator should be 
studied (in vivo or in silico based on in vivo single dose administration)  if the substrate 
demonstrates time-dependent pharmacokinetics.       
  7  Contains Nonbinding Recommendations 
 
 
4.  Route of Administration 
 
For in vivo DDI studies, the route of administration  of the investigational  drug should generally 
be the one planned for routine clinical  use.  When multiple  routes of administration  are 
developed for clinical  use, the route of drug administration  for DDI studies should be selected 
based on the expected mechanisms of the DDIs and the similarity  of the corresponding 
concentration-time  profiles for the parent drug and metabolites after different routes of 
administration.    
 
5.  Parallel Versus Crossover Studies 
 
A study can use a randomized crossover (e.g., S followed by S+I, S+I followed by S, where S 
refers to a substrate and I designates an inhibitor  or inducer), one-sequence crossover (e.g., S 
followed by S+I), or a parallel (e.g., S in one group of subjects and S+I in another group) design.  
 
Crossover studies (one-sequence or randomized) are preferred over parallel study designs in 
order to reduce inter-subject variability.   The sponsor should base the duration of the washout 
period on the known pharmacokinetics  of the substrate and the perpetrator, the anticipated 
impact on the substrates half-life,  and the duration necessary for enzyme activity to return to 
baseline or for potential  pharmacodynamic effects to return to pre-treatment levels (if 
pharmacodynamic  effects are also assessed).  Typically,  the two experimental periods evaluate 
the substrate alone and the concomitant use of the substrate and perpetrator.  In some situations, 
a third crossover period may be informative  (e.g., to evaluate the time it takes for the enzymes 
activity to return to normal following  removal of the investigational  drug when it is an inducer or 
time-dependent inhibitor,  or to evaluate a pair of drugs when each drug can be the perpetrator or 
the substrate).    
 
Parallel, two-arm studies can be appropriate when a crossover study design is not feasible.  
Typically,  parallel-design  studies require larger sample sizes than crossover studies. 
 
6.  Timing of Drug Administration 
 
In most cases, the perpetrator and substrate drugs can be administered at the same time.  
However, the timing of administration  of the perpetrator is critical if it is both an inhibitor  and an 
inducer.  For example, if the investigational  drug is a substrate for CYP enzymes and OATP1B, 
and rifampin  is used as an enzyme inducer, the simultaneous  administration  of the drug with 
rifampin  which is an OATP1B inhibitor   may not accurately capture the effects of enzyme 
induction.   In such cases, delayed administration  of the substrate is recommended. 
 
Sometimes multiple  drug-dosing  schedules can be studied (in vivo or in silico) to understand 
whether staggered dosing is a viable mitigation  strategy for the DDI. 
 
When evaluating the interaction between drugs that require different food conditions  for optimal 
absorption,  the sponsor should adjust the timing  of drug administration  to maximize  the potential 
to detect an interaction (i.e., index studies) and/or to reflect the clinically  relevant conditions 
  8  Contains Nonbinding Recommendations 
 
(i.e., concomitant-use studies). 
 
7.   Co-Medications and Other Extrinsic Factors Affecting DDIs 
 
To reduce variability  in the magnitude of DDIs, the sponsor should exclude and/or account for 
the use of prescription or over-the-counter medications,  dietary/nutritional  supplements,  tobacco, 
alcohol,  foods, and fruit juices that may affect the expression or function of enzymes and 
transporters for a sufficient time before subject enrollment.   The sponsor should exclude these 
items for a longer period of time if the DDI mechanism is induction or time-dependent 
inhibition.     
 
8.  Sample and Data Collection 
 
Pharmacokinetic sampling times should be sufficient to characterize the AUC  (for single-
0-INF
dose studies) or the AUC (for multiple-dose  studies) and the maximum concentration (C ) 
0-TAU  max
of the substrate drug administered alone and under conditions  of the anticipated interaction.  
Sponsors should collect data on additional  pharmacokinetic  parameters based upon the 
pharmacokinetic  or pharmacological  relevance for the proposed indication  (e.g., the minimal 
concentration (C ), partial AUC).  The sampling  times for single-dose studies should be 
min
planned so that the mean difference between the AUC  and the AUC  is less than 20 
0-t 0-INF
percent.  Sponsors should collect samples that contain the moieties needed to interpret study 
results; in most cases, the moiety needed to interpret results will be the parent drug.  The sponsor 
should determine metabolite  concentrations if the results provide information  about the effect of 
a DDI on the investigational  drugs safety or efficacy, or if the data inform the mechanism of the 
drug interaction.   
 
All studies should collect relevant safety information  based on the knowledge of existing safety 
concerns with the administered drugs.    
 
9.  Pharmacodynamic Endpoints 
 
In some situations,  pharmacodynamic endpoints indicate changes in efficacy or toxicity  that 
systemic drug exposures do not predict.  One possible scenario is when transporter inhibition 
alters access of the drug to specific organs or tissues.  In such scenarios, clinical  consequences 
such as altered efficacy or increased toxicity resulting  from altered tissue distribution  of a 
substrate drug can be measured as pharmacodynamic  endpoints,  and in vitro evidence of a drugs 
interaction potential can support data interpretation. 
 
When in vitro data provide a plausible  DDI mechanism that cannot be evaluated with systemic 
drug exposure, sponsors can collect and analyze pharmacodynamic  endpoint data. The sponsor 
should consult with the FDA regarding pharmacodynamic  endpoint evaluation. 
 
C.  Study Planning and Considerations for Prospective Nested DDI Studies 
 
Prospective nested DDI studies should be carefully designed.  Stand-alone studies typically 
include a large number of pharmacokinetic  samples per subject, resulting in a rich sampling 
  9  Contains Nonbinding Recommendations 
 
strategy.  In contrast, DDI studies that are part of another study (e.g., large phase 2 or phase 3 
studies) often rely on sparse pharmacokinetic  sampling with fewer samples per subject. 
 
Population  pharmacokinetic analyses of data obtained from large-scale clinical  studies can help 
characterize the clinical  impact of known or newly identified  interactions and determine 
recommendations for dosage modifications  when the investigational  drug is a substrate.  The 
results of such analyses can be informative,  and sometimes conclusive,  when the clinical  studies 
are adequately designed to detect significant  changes in drug exposure due to DDIs.  Normally, 
the exposure of co-administered  drugs is not determined; therefore, it is not possible to use the 
population  pharmacokinetic  method to evaluate the investigational  drug as a perpetrator.  
However, if the sponsor prospectively plans and collects the necessary data to support the 
evaluation of targeted, concomitant drugs, population  pharmacokinetic  analyses can be useful for 
evaluating the investigational  drug as a perpetrator.     
 
To be optimally  informative,  population  pharmacokinetic  analysis for prospective DDI 
evaluation should have carefully designed study procedures and sample collection  protocols.  
The sponsor can simulate various DDI scenarios using available pharmacokinetic models (e.g., 
PBPK models, population  pharmacokinetic  models) to optimize  study sampling (e.g., sampling 
times, number of subjects) and data collection.   Sponsors should document detailed information 
on the dose given, the time of drug administration,  and the time of drug discontinuation  for both 
the investigational  and co-administered drugs.  If food affects the exposure of the investigational 
drug or co-administered  drug, the sponsor should also document the time and type of food 
consumed.  Analyses should focus on detecting a specific clinically  meaningful  change in drug 
exposure.  The sponsor should prespecify the population  pharmacokinetic  DDI assessment 
before conducting the prospective nested DDI study to increase confidence in the studys results.    
 
D.  Specific Considerations for CYP-Mediated Interactions  
 
1.  The Investigational Drug as a Substrate for CYP Enzymes 
 
When evaluating the investigational  drug as a substrate, clinical  DDI studies should start with a 
strong index inhibitor  and a strong index inducer.  Moderate index inhibitors  or inducers are 
acceptable if strong index inhibitors  or inducers are not available for a particular enzyme.   
These index inhibitors  and inducers are preferred because there is a large body of information 
about:  (1) their defined effects on specific CYP pathways; (2) their appropriate dosing regimens; 
(3) their safety profiles; and (4) their anticipated effects on their respective sensitive substrates.  
Some of these inhibitors  and inducers can also affect other metabolism  and/or transporter 
pathways.  When selecting index inhibitors  and inducers for prospective DDI studies, the 
sponsor should consider the elimination  pathways of the investigational  drug as a substrate.  
Other strong inhibitors  and inducers of CYP enzymes can also be appropriate.  Examples of 
inhibitors  or inducers are available on the FDAs Web site on Drug Development and Drug 
Interactions.12   
 
                                              
12 FDAs Web site on Drug Development and Drug Interactions can be found at 
http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DevelopmentResources/DrugInteractionsLabeling/ucm080499.htm 
.   
  10  Contains Nonbinding Recommendations 
 
If a DDI study with a strong index inducer or inhibitor  indicates that no DDI is present, 
additional  clinical studies with other inhibitors  or inducers of the same enzyme are not needed.  
If a DDI study with strong index inhibitors  or inducers indicates that there is a clinically 
significant  interaction,  the Agency recommends evaluating the impact of other moderate 
inhibitors  or inducers to gain a full understanding  of the investigational  drugs DDI potential.  
The effect of the additional  inhibitors  and inducers can be evaluated in a clinical  interaction 
study or through modeling  and simulation  approaches, such as PBPK modeling  with a verified 
perpetrator (inhibitor  or inducer) and substrate models.  DDI studies with index substrates and 
perpetrators can be used to inform potential future concomitant-use studies.     
 
If the investigational  drug is subject to significant  metabolism by a genetically polymorphic 
enzyme (e.g., fraction of metabolism,  f , accounted by the enzyme  80 percent) for which a 
m
well-defined poor metabolizer (PM) phenotype exists (e.g., for CYP2D6 and CYP2C19), a 
comparison of the pharmacokinetic  parameters of the drug in individuals  with the PM phenotype 
versus those with an extensive metabolizer (EM) phenotype can substitute for an interaction 
study for that particular pathway (see section IV.G.1).  The effect of a PM phenotype is expected 
to be similar  to the effect of a strong inhibitor  of that pathway.  If this comparison reveals a 
clinically  significant  difference in exposure between individuals  with the PM and EM 
phenotypes, the sponsor should evaluate the potential for DDIs with moderate inhibitors  or 
inducers of the enzymes as described above.     
 
2.  The Investigational Drug as an Inhibitor or an Inducer of CYP Enzymes 
 
When studying an investigational  drug as a potential inhibitor  or inducer of a CYP enzyme, the 
index substrate selected for the initial  clinical  studies should be sensitive to changes in activity or 
amount of the CYP enzyme being evaluated.  These sensitive index substrates are preferred 
because there is a large body of information  about:  (1) the relative contribution  of specific CYP 
pathways on their overall elimination;  (2) their appropriate dosing regimens; (3) their safety 
profiles; and (4) their anticipated interaction effects when co-administered with strong index 
inhibitors  and inducers.  When determining  which index substrates to use for prospective DDI 
studies, the sponsor should consider the inhibition  and/or induction  properties of the 
investigational  drug.  Other CYP enzyme substrates can also be appropriate.  Examples of 
sensitive substrates are listed on the FDAs Web site on Drug Development and Drug 
Interactions.13,14   
 
If an initial  study determines that an investigational  drug either inhibits  or induces the 
metabolism  of sensitive index substrates, further studies using other substrates (e.g., relevant co-
medications) can be useful.  The sponsor should consider additional  studies, depending  on the 
magnitude of the effect of the investigational  drug on the sensitive index substrate and the 
                                              
13 FDAs Web site on Drug Development and Drug Interactions can be found at 
http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DevelopmentResources/DrugInteractionsLabeling/ucm08
0499.htm 
 
14 CYP2B6 is not listed because we currently do not have index substrates for this enzyme.  CYP2B6 is the principal 
enzyme involved in the formation of hydroxybupropion from bupropion which may be used as a victim drug in 
clinical DDI studies with measurement of hydroxybupropion concentrations to evaluate the potential inhibitory 
effect of an investigational drug on CYP2B6. 
  11  Contains Nonbinding Recommendations 
 
potential for concomitant use with other drugs that are substrates of the same enzyme.  If the 
initial  study with the most sensitive index substrates is negative, the sponsor can presume that 
less sensitive substrates will also be unaffected.    
 
Some substrate drugs that are typically  used in DDI studies are not specific for one CYP enzyme; 
furthermore, some of these drugs are also substrates for transporters.  When conducting  a DDI 
study, using a substrate metabolized  by more than one enzyme is only appropriate if the 
investigational  drug is a selective inhibitor  or inducer of the substrates primary CYP 
metabolizing  enzyme.  For example, dextromethorphan  elimination  is carried out primarily  by 
CYP2D6, with minor contributions  from other enzymes; therefore, dextromethorphan  would be 
an appropriate substrate for an investigational  drug that is suspected to be a selective inhibitor  of 
CYP2D6.  If the substrate drug is metabolized  by more than one enzyme, measuring the 
metabolites can help the sponsor interpret study results. 
 
If the investigational  drug is both an inducer and an inhibitor  of an enzyme, the net effect of the 
drug on enzyme function may be time dependent.  The timing  of pharmacokinetic  endpoints 
should permit an understanding of the changes in effects over time (see section IV.B.6). 
 
E.  Specific Considerations for Transporter-Mediated Interactions 
 
1.  The Investigational Drug as a Substrate of Transporters 
 
If in vitro studies, as described in the January 2020 FDA guidance for industry In Vitro Drug 
Interaction Studies  Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions, 
indicate that the investigational  drug is a transporter substrate, the need for clinical DDI studies 
is determined based on the drugs putative site of action, route of elimination,  likely  concomitant 
drugs, and safety considerations.15  The following  general guidelines  help to determine when a 
sponsor should perform a clinical  DDI study for investigational  drugs that are transporter 
substrates in vitro: 
 
  P-glycoprotein  (P-gp)- and breast cancer resistance protein (BCRP)-mediated DDIs:  
 
-  When intestinal absorption,  biliary  excretion, or renal active secretion is likely to be a 
major cause of the variability  in a drug pharmacokinetics and response 
 
  OATP1B1- and OATP1B3-mediated DDIs:  
 
-  When hepatic/biliary  elimination  is a significant  clearance pathway for the 
investigational  drug, and the drugs properties (e.g., low passive membrane 
permeability,  high hepatic concentrations relative to other tissues) support the 
importance of active uptake of the drug into the liver 
 
                                              
15 Giacomini KM, SM Huang, DJ Tweedie, LZ Benet, KLR Brouwer, X Chu, A Dahli, R Evers, V Fischer, KM Hillgren, 
KA Hoffmaster, T Ishikawa, D Keppler, RB Kim, CA Lee, M Niemi, JW Polli, Y Sugiyama, PW Swaan, JA Ware, SH 
Wright, SW Yee, MJ Zamek-Gliszczynski, and L Zhang, 2010, Membrane Transporters in Drug Development, Nat Rev Drug 
Discov, 9(3):215-236. 
  12  Contains Nonbinding Recommendations 
 
  Organic anion transporter 1 and 3 (OAT1 and OAT3)-, organic cation transporter 2 
(OCT2)-, and multidrug  and toxin extrusion proteins (MATE)-mediated DDIs:  
 
-  When the investigational  drug undergoes significant active renal secretion (i.e., 
accounting for  25 percent of systemic clearance) or there are concerns about renal 
toxicity 
 
When testing an investigational  drug as a substrate in transporter-mediated DDIs, the selected 
perpetrator drug should be a known inhibitor  of the transporter under investigation.   The sponsor 
can select perpetrators for the DDI study based on the goal of the study (e.g., if the goal of the 
study is to gain mechanistic understanding  or to conduct a clinical  assessment).   
 
Because of a general lack of index perpetrators for transporter-mediated pathways, the choice of 
transporter perpetrators is typically  based on the likelihood  of concomitant use (e.g., to obtain 
clinically  relevant DDI information  that can inform labeling  regarding the management of a 
DDI).     
 
A few transporter perpetrators can also be used to understand the underlying  mechanisms of 
transporter-mediated DDIs or to study the worst-case DDI scenario.  For example, to understand 
the transporter-mediated DDI of the largest possible magnitude for an investigational  drug that is 
a substrate for multiple  transporters, an inhibitor  of several transporters (e.g., cyclosporine, 
which inhibits  intestinal  P-gp and BCRP and hepatic OATPs) can be used as the inhibitor  in the 
DDI study.  Negative results from this kind of study can rule out the need to further evaluate the 
drug as a substrate for any of the individual  transporters.  If the study result is positive,  additional 
studies with more selective inhibitors  of specific transporter pathways can help determine the 
relative contribution  of each transporter to the disposition  of the substrate drug.  The same 
experimental paradigm can apply to an investigational  drug that is a substrate for both 
transporters and metabolic enzymes (e.g., CYP3A and P-gp).    
 
If the goal of the study is to determine the role of a specific pathway in the pharmacokinetics  of a 
substrate drug, then the sponsor should use a more selective inhibitor  for that transporter.  A few 
inhibitors  block specific transporter pathways in a relatively selective manner.  For example, a 
single dose of rifampin inhibits  the hepatic transporter OATPs, and probenecid inhibits  the renal 
transporters OAT1 and OAT3.  Use of these inhibitors  in vivo can provide a mechanistic 
understanding of transporter-mediated DDIs.  In addition,  the investigational  drug can be a 
substrate of a genetically polymorphic  transporter (e.g., OATP1B1 and BCRP are encoded by the 
genetically  polymorphic  genes SLCO1B1 and ABCG2, respectively) for which phenotypes with 
reduced functionality  exist.  Similar  to drugs that are substrates of CYPs encoded by 
polymorphic  genes, the relative contribution  of a specific transporter to the disposition  of the 
investigational  drug can be evaluated in subjects with different transporter genotypes (see section 
IV.G.1).    
 
  13  Contains Nonbinding Recommendations 
 
Examples of transporter inhibitors  are listed on the FDAs Web site on Drug Development and 
Drug Interactions.16  Many of them not only inhibit  the specified transporters but also can inhibit 
some other transporters and/or CYP enzymes.  Thus, results from most transporter inhibition 
studies are not easily extrapolated to other drugs (see sections IV.A.2 and IV.A.3).  Interpretation 
of the study results requires knowledge of the transport and metabolic pathways for the 
investigational  drug.   
 
2.  The Investigational Drug as an Inhibitor or an Inducer of Transporters 
 
If in vitro studies, as described in the FDA guidance for industry In Vitro Drug Interaction 
Studies  Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions, indicate 
that the investigational  drug is a transporter inhibitor,  the sponsor should consider a clinical  drug 
interaction study based on likely  concomitant drugs and safety considerations.17  When studying 
the investigational  drugs potential to act as a perpetrator drug for a transporter, the sponsor 
should select a substrate whose pharmacokinetic  profile is markedly altered by coadministration 
of known inhibitors  of that transporter and is also a likely  concomitant drug.  Examples of 
transporter substrates that can be used in drug interaction studies are listed on the FDAs Web 
site on Drug Development and Drug Interactions.  Many drugs are substrates of multiple 
transporters and/or enzymes.  The observed clinical  interactions can be a result of the inhibition 
of multiple  pathways if the investigational  drug is also an inhibitor  for the same pathways.  
Results from these studies are thus not easily extrapolated to other drugs (see sections IV.A.2 
and IV.A.3).  The choice of substrates can be determined by the therapeutic area of the 
investigational  drug and the probable concomitant drugs that are known substrates of the 
transporters.   
 
The sponsor should consult with the FDA to determine whether to evaluate the investigational 
drugs ability  to induce transporters.  Some drugs can induce P-gp; however, there is no 
validated in vitro system to study P-gp induction.   Therefore, determining  a drugs potential to 
induce P-gp should be based on clinical  studies.  Because of similarities  in the mechanisms of 
CYP3A and P-gp induction,  results from CYP3A induction  studies can inform decisions about 
whether to investigate the induction  of P-gp.  If a study indicates that an investigational  drug 
does not induce CYP3A, it is not necessary to evaluate the drugs potential to induce P-gp.  If the 
clinical  CYP3A induction  test is positive,  then the sponsor should consider an additional  study of 
the investigational  drugs effect on a known P-gp substrate.  If the drug also inhibits  P-gp, then 
an induction  study can be combined with the inhibitor  study using a multiple-dose  design. 
 
F.  Cocktail Approaches 
 
                                              
16 FDAs Web site on Drug Development and Drug Interactions can be found at 
http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DevelopmentResources/DrugInteractionsLabeling/ucm08
0499.htm.   
 
17 Giacomini KM, SM Huang, DJ Tweedie, LZ Benet, KLR Brouwer, X Chu, A Dahli, R Evers, V Fischer, KM Hillgren, 
KA Hoffmaster, T Ishikawa, D Keppler, RB Kim, CA Lee, M Niemi, JW Polli, Y Sugiyama, PW Swaan, JA Ware, SH 
Wright, SW Yee, MJ Zamek-Gliszczynski, and L Zhang, 2010, Membrane Transporters in Drug Development, Nat Rev Drug 
Discov, 9(3):215-236. 
 
  14  Contains Nonbinding Recommendations 
 
A cocktail study includes the simultaneous  administration  of substrates of multiple  CYP 
enzymes and/or transporters to study subjects.  A cocktail approach can simultaneously  evaluate 
a drugs inhibition  or induction  potential for multiple  CYPs and transporters as long as the study 
is properly designed, and the following  conditions  are satisfied: (1) the substrates are specific for 
individual  CYP enzymes or transporters; (2) there are no interactions among the substrates; and 
(3) the study is conducted with a sufficient number of subjects.  Negative results from a well-
conducted cocktail study can eliminate  further evaluation of particular CYP enzymes or 
transporters.  Positive results from a well-conducted  cocktail study that includes all elements of a 
prospective DDI study can be interpreted and presented in labeling  the same way as positive 
results from any other well-conducted drug interaction study.  It should be noted the findings 
obtained with a microdose of a substrate may not always be extrapolated to a therapeutic dose of 
that substrate. 
 
G.  Other Considerations 
 
1.  Genetics 
 
If a drug is a substrate for a polymorphic  enzyme or transporter, a subjects genotype for a 
specific enzyme or transporter affects the extent of drug induction  or drug inhibition.   When a 
DDI study uses an index inhibitor  or substrate (e.g., omeprazole for CYP2C19) to evaluate 
pharmacokinetic  changes, individuals  who have no functional  enzyme activity should typically 
be excluded, or the study should be sufficiently powered to evaluate DDIs in subjects with 
functional  enzymes.  In cases where study enrollment is not based on the genotype of a 
polymorphic  enzyme or transporter, sponsors should still routinely  collect DNA from all subjects 
for retrospective analysis of the enzymes or transporters of interest to characterize differences in 
the magnitude of the DDI across genotype groups and to understand why some subjects have 
unusual increases or decreases in drug concentrations (see the 2013 FDA guidance for industry 
entitled Clinical Pharmacogenomics: Premarket Evaluation in Early-Phase Clinical Studies and 
Recommendations for Labeling). 
 
The combined effects of different genotypes of polymorphic  enzymes and transporters can also 
be explored in a drug interaction study.  For example, if a drug is metabolized  by both CYP3A 
and CYP2C19, examining  the effect of CYP3A inhibition  in CYP2C19 poor metabolizers  may 
help uncover the consequences of losing  compensatory pathways.  This kind of study may be 
accomplished by prospective enrichment of poor metabolizers or through retrospective analysis, 
provided that a sufficient number of poor metabolizers  are enrolled. 
 
In some instances, a gene-drug interaction study may substitute for a prospective DDI study and 
vice versa.  Suitable substrates for these studies have a high fraction of metabolism  (e.g., f  80 
m 
percent) by a single CYP enzyme that has loss-of-function  alleles (see section IV.D.1.).   
 
Comparing the pharmacokinetics  of an investigational  drug in subjects with different genotypes 
of specific transporters (e.g., OATP1B1, BCRP) can help determine the importance of a specific 
transporter in the drugs clearance pathway.     
 
  15  Contains Nonbinding Recommendations 
 
2.  Smokers 
 
Smoking  induces CYP1A2 activity.  If an investigational  drug is a CYP1A2 substrate, the 
sponsor should consider conducting a study in smokers based on the intended patient population 
and the effect of CYP1A2 induction  on the drugs exposure.  The study arms for a smoking study 
include nonsmokers (e.g., have not smoked in the past 6 months) in the control arm and current 
smokers in the investigational  arm.  Data collected in the smoking  study should include the 
number of cigarettes smoked per day and, when feasible, plasma nicotine levels in both smokers 
and nonsmokers.  The sponsor should evaluate the effects of different levels of smoking if it 
considers the information  important for the patient population. 
 
3.  Complex Drug Interactions 
 
When there are multiple  factors that affect the absorption and disposition  of an investigational 
drug as well as multiple  mechanisms of DDIs (e.g., multiple  CYP enzymes and/or transporters), 
the sponsor should evaluate the investigational  drugs DDI potential by integrating knowledge 
from multiple  in vitro and clinical studies.  PBPK models may be useful to:  (1) integrate the 
information  from multiple  studies; (2) determine whether a clinical  study is appropriate; and (3) 
inform the design of clinical  studies.  See the January 2020 FDA guidance for industry entitled 
In Vitro Drug Interaction Studies  Cytochrome P450 Enzyme- and Transporter-Mediated Drug 
Interactions for more information.   Sponsors are encouraged to discuss the strategies to study 
complex DDIs with the FDA. 
 
 
V.  REPORTING AND INTERPRETING STUDY RESULTS 
 
A DDI study report should include and justify the study design and the data analysis method 
based on what is known about the mechanism of the DDI and the pharmacokinetic  properties of 
the perpetrator and victim drugs.   
 
A.  Study Results Reporting 
 
Typical pharmacokinetic endpoints for DDI studies include changes in drug exposure parameters 
such as AUC  and C .  Sponsors should report the pharmacokinetic  results of DDI studies 
0-INF max
as the geometric mean ratio of the observed pharmacokinetic exposure measures with and 
without the perpetrator drug and include the associated 90 percent confidence interval.  Sponsors 
should also report measures of the observed variability  of the interaction when applicable.    
 
The sponsor should summarize all information  on pharmacodynamic endpoints.  If the 
pharmacodynamic  endpoint is a continuous  response (e.g., certain blood biomarkers), the 
sponsor can analyze the data and report the results in the same manner as for pharmacokinetic 
endpoints.  If the pharmacodynamic  endpoint is not a continuous  response, the sponsor should 
consult with the FDA to determine an appropriate data analysis method.    
 
  16  Contains Nonbinding Recommendations 
 
When possible, the sponsor should specify the criteria for defining  outliers in the protocol and 
make a distinction  between outlying  individuals  versus outlying  data points.  In general, sponsors 
should report results with and without suspected outliers.   
 
The sponsor should report AUC values for all individuals  and include the percentage of 
0-INF 
extrapolation.   Sponsors should highlight  individuals  with more than 20 percent extrapolated 
AUC report the results with and without those subjects, and discuss the potential impact on 
0-INF, 
the interpretation  of the findings.   
 
1.  Non-Compartmental Analysis 
 
The sponsor should report substrate exposure measures for all subjects, for example, the AUC
0-
 the AUC , the percentage extrapolated AUC  the C , and T .  For multiple-dose 
INF, 0-t 0-INF, max max
studies, sponsors should also report the C  and the AUC  at steady-state.  Sponsors should 
min 0-TAU
report additional  pharmacokinetic  parameters such as the clearance, the volume of distribution, 
and the half-life  if they help interpret the pharmacokinetic  results.  The sponsor should also 
consider collecting and reporting pharmacokinetic parameters that are relevant to the clinical 
significance of the interaction.  Measuring metabolite levels can help confirm the mechanism of 
an interaction or differentiate the effect of inhibitors  or inducers on pathways mediated by 
different CYP enzymes.    
 
2.  Population Pharmacokinetic Analysis 
 
When relevant, population  pharmacokinetic  analysis should derive pharmacokinetic  exposure 
parameters, such as AUC , AUC  C , and T .  For multiple-dose  studies, sponsors 
0-INF 0-t, max max
should also report the C and the AUC at the steady-state.  Sponsors should investigate the 
min  0-TAU 
DDI using all plausible  structural elements of the pharmacokinetic  model (e.g., clearance (CL/F), 
relative bioavailability,  rate of absorption).  Further considerations for population 
pharmacokinetic  analysis are available in the 1999 FDA guidance for industry entitled 
Population Pharmacokinetics.  In certain cases, traditional  pharmacokinetic  data analysis using 
non-compartmental analysis methods may not be adequate.  For example, it may be difficult  to 
design a study for drugs with a long half-life that would allow AUC  to be estimated with less 
0-INF
than 20 percent extrapolation  from AUC .  Such studies could be analyzed with population 
0-t
pharmacokinetic  methods in addition to non-compartmental  analysis.18   
  
B.  Interpreting DDI Studies 
 
The goal of a DDI study with pharmacokinetic  endpoints is to inform management and 
prevention strategies by determining  whether there is a clinically  significant  increase or decrease 
in exposure to the substrate in the presence of the perpetrator.  The results of a DDI study are 
interpreted based on the no-effect boundaries for the substrate drug.  No-effect boundaries 
represent the interval within which a change in a systemic exposure measure is considered not 
significant  enough to warrant clinical  action (e.g., dose or schedule adjustment, or additional 
therapeutic monitoring). 
                                              
18 Svensson EM, C Acharya, B Clauson, KE Dooley, and MO Karlsson, 2016, Pharmacokinetic Interactions for 
Drugs With a Long Half-Life  Evidence for the Need of Model-Based Analysis, AAPS J, 18(1):171-179. 
  17  Contains Nonbinding Recommendations 
 
 
1.  Approaches for Determining No-Effect Boundaries 
 
There are two approaches to determining no-effect boundaries: 
 
  Approach 1 (Preferred)  When possible,  no-effect boundaries can be based on 
concentration-response relationships  derived from pharmacokinetic  and 
pharmacodynamic  analyses, as well as other available information  for the substrate drug 
(e.g., the maximum-tolerated  dose).  A good understanding  of dose-concentration and/or 
concentration-response relationships  for desirable and undesirable drug effects, as well as 
knowledge of the variability  of exposures in the indicated population,  can facilitate data 
interpretation.  The FDAs 2003 guidance for industry entitled Exposure-Response 
Relationships  Study Design, Data Analysis, and Regulatory Applications provides 
further considerations for exposure-response analysis.   
 
For example, if the 90 percent confidence interval for the measured changes in systemic 
exposures in the DDI study falls completely  within these no-effect boundaries,  no 
clinically  significant  DDI is present.  The percentile method to determine the proportion 
of subjects that extend beyond the no-effect boundary can be more appropriate in some 
instances.   
 
  Approach 2 (In the absence of no-effect boundaries defined in Approach 1 or when the 
aim of the study is to determine whether a drug is a perpetrator or not when using index 
substrates)  The sponsor can use a default no-effect boundary of 80 to 125 percent in 
these instances.  When the 90 percent confidence intervals for systemic exposure ratios 
fall entirely within the equivalence range of 80 to 125 percent, the FDA concludes that 
there is no clinically  significant  DDI.   
 
The 80 to 125 percent boundaries represent a very conservative standard for drugs that 
have wide safety margins, so Approach 1 is preferred for evaluating  the impact of DDI on 
the safety and efficacy of the substrate drug.  In the absence of a clearly defined 
exposure-response relationship,  the totality of evidence should be taken into 
consideration  when determining  the clinical  impact of the DDI on the substrate drug. 
 
2.  Interpreting Results From Retrospective DDI Evaluations 
 
Retrospective DDI evaluations can be useful to identify DDIs that were unanticipated at the start 
of clinical  development.  Sponsors should confirm results from retrospective DDI studies that 
suggest clinical  management or prevention strategies are warranted with a prospective DDI 
study.  Negative findings  from retrospective studies generally do not provide useful information 
to include in labeling. 
 
3.  Classifying the Investigational Drug as an Inhibitor or Inducer 
 
  18  Contains Nonbinding Recommendations 
 
If an investigational  drug is a CYP inhibitor,  it can be classified as a strong, moderate, or weak 
inhibitor  based on its effect on an index CYP substrate.  The convention  is to categorize CYP 
inhibition  in the following  way: 
 
  A strong inhibitor  increases the AUC of a sensitive index CYP substrate  5-fold.   
 
  A moderate inhibitor  increases the AUC of a sensitive index CYP substrate by  2- to < 
5-fold. 
 
  A weak inhibitor  increases the AUC of a sensitive index CYP substrate by  1.25- to < 2-
fold. 
   
These categories typically  describe the effect of the investigational  drug when given at the 
highest clinical  dose and the shortest dosing interval within  its therapeutic dose range/dosing 
regimen.    
 
If an investigational  drug is a CYP inducer, it can be classified as a strong, moderate, or weak 
inducer based on its effect on an index CYP substrate.  The convention  is to categorize CYP 
induction  in the following  ways: 
 
  A strong inducer decreases the AUC of a sensitive index CYP substrate by  80 percent.  
 
  A moderate inducer decreases the AUC of a sensitive index CYP substrate by  50 to < 
80 percent. 
 
  A weak inducer decreases the AUC of a sensitive index CYP substrate by  20 to < 50 
percent. 
 
This classification  information  helps to determine whether other drugs that have not been 
investigated in a DDI study have clinically  significant  DDIs with the investigational  drug and 
therefore need to be mentioned in labeling.   For example, if an investigational  drug is a strong 
CYP3A inhibitor,  its potential  to interact with drugs that have clinically  significant  interactions 
with other strong CYP3A inhibitors  should be considered, and the sponsor should add 
appropriate language regarding these additional  interactions to the investigational  drugs 
labeling. 
 
Currently, there is no standardized classification system for transporter and phase II metabolizing 
enzyme inducers or inhibitors.   
 
4.  Development of DDI Clinical Management and Prevention Strategies 
 
The FDA recommends developing  DDI management and prevention strategies when a clinically 
significant  DDI is identified.   An interaction is clinically  significant  if concomitant use of the 
drugs leads to safety, efficacy, or tolerability  concerns greater than those present when the drugs 
are administered alone.    
 
  19  Contains Nonbinding Recommendations 
 
In general, DDI clinical  management and prevention strategies should result in drug 
concentrations of the victim drug that are within the no-effect boundaries.  In addition, such 
strategies should consider several factors, including,  but not limited  to:  
 
  The exposure-response relationships  for safety and efficacy 
 
  The variability  of the observed DDI data, if available  
 
  The expected duration of concomitant drug use (e.g., acute, short-term, or chronic use of 
one or both drugs) 
 
  The timing  of the introduction  of the concomitant medication (e.g., will the new drug be 
given to a patient already taking a concomitant medication or will the concomitant 
medication be given to a patient already taking the new drug) 
 
  The mechanism of the DDI (e.g., competitive,  noncompetitive  or time-dependent 
inhibition,  induction,  combined inhibition  and induction) 
 
  The availability  of monitoring  parameters (e.g., therapeutic drug monitoring,  laboratory 
tests) 
 
  The medical need for the new drug, the ability  to interrupt concomitant interacting 
medications,  and the availability  of other therapeutic choices in patients with potentially 
clinically  important interactions with the new drug. 
 
With the above considerations,  DDI management and prevention strategies may include: 
 
  Contraindicating  concomitant use 
 
  Avoiding  concomitant use 
 
  Temporarily  discontinuing  one of the interacting drugs 
 
  Modifying  the dosage of the new drug 
  
  Staggering drug administration  (e.g., administer the new drug at a different time than an 
acid-reducing agent) 
 
  Implementing  specific monitoring  strategies (e.g., therapeutic drug monitoring, 
laboratory testing)   
 
5.  Extrapolating Study Results  
 
Clinical  evaluation of all possible combinations  of drugs is not feasible.  When possible,  results 
from DDI studies should be extrapolated to other drugs and clinical  situations.  Results from DDI 
  20  Contains Nonbinding Recommendations 
 
studies with index drugs are generally relevant to other drugs and may represent a worst-case 
scenario for other drugs (see section IV.A.2).  For example, if there is no effect on the exposure 
of an investigational  drug when co-administered with a strong CYP3A4 index inhibitor,  then one 
can generally assume that there is no effect when other strong, moderate, or weak CYP3A4 
inhibitors  are co-administered  with the investigational  drug.  If a strong CYP2D6 index inhibitor 
results in a significant  increase in exposure of the investigational  drug, these results can be 
directly extrapolated to other strong CYP2D6 inhibitors.   Extrapolation  of positive findings  to 
moderate and weak inhibitors  is not always possible (see section IV.A.4).  In cases where 
extrapolation  is not possible, the FDA may recommend a dedicated clinical  DDI study.   
 
Concomitant-use DDI studies can be warranted in cases when extrapolation  is not feasible and 
drugs with DDI potential are likely  to be co-administered.   Although  concomitant-use studies 
have limited  potential  for extrapolation  to other drugs, they may have great relevance to 
practitioners and patients.   
   
Because of the lack of specific transporter substrates and inhibitors  and possible interplay with 
metabolism,  it is generally challenging  to extrapolate results from DDI studies evaluating 
transporter-mediated DDIs or transporter-metabolism  interactions from one drug to other drugs 
(see section IV.E).    
 
 
VI.  LABELING RECOMMENDATIONS 
 
The Prescribing Information should include a summary of essential DDI information  that is 
needed for the safe and effective use of the drug by the health care provider.  The DRUG 
INTERACTIONS and CLINICAL PHARMACOLOGY sections of drug labeling include the 
majority  of the DDI information.   When DDI information  has direct implications  for the safe and 
effective use of the drug, this information is often included in varying levels of detail in other 
sections of the labeling (e.g., BOXED WARNING, DOSAGE AND ADMINISTRATION, 
CONTRAINDICATIONS, and/or WARNINGS AND PRECAUTIONS sections), and must be 
discussed in more detail in the DRUG INTERACTIONS section ( 201.57(c)(8)(i)).  For more 
specific recommendations  on content for these labeling sections, refer to the following  FDA 
guidances for industry:   
 
  Clinical Pharmacology Section of Labeling for Human Prescription Drug and Biological 
Products  Content and Format (December 2016) 
 
  Content and Format of the Dosage and Administration Section of Labeling for Human 
Prescription Drug and Biological Products (March 2010) 
 
  Warnings and Precautions, Contraindications, and Boxed Warning Sections of Labeling 
for Prescription Drug and Biological Products  Content and Format (October 2011) 
 
  Patient Counseling Information Section of Labeling for Human Prescription Drug and 
Biological Products  Content and Format (December 2014) 
 
  21  Contains Nonbinding Recommendations 
 
 
VII.  ABBREVIATIONS 
 
AUC   Area under the plasma concentration-time  curve integrated from time of 
0-t
administration  (0) to time of last quantifiable  observation (t)  
 
AUC   Area under the plasma concentration-time  curve from time of administration 
0-INF
extrapolated to infinity  from AUC
0-t 
 
AUC   Area under the plasma concentration-time  curve integrated across the dosing 
0- TAU
interval 
 
BCRP  Breast cancer resistance protein 
   
C   Maximum concentration 
max
   
C   Minimum  concentration 
min
 
CYP  Cytochrome P450 
 
DDI  Drug-drug interaction 
 
EM  Extensive metabolizers 
 
MATE  Multidrug  and toxin extrusion protein 
 
OAT   Organic anion transporter 
 
OATP  Organic anion transporting polypeptide 
 
OCT  Organic cation transporter 
 
PBPK  Physiologically  based pharmacokinetic 
 
P-gp  P-glycoprotein 
 
PLR  Physician Labeling Rule 
   
PM  Poor metabolizers 
 
TDI  Time-dependent inhibition 
   
T   Time to C  
max max
 
 
 
  22  Contains Nonbinding Recommendations 
 
VIII.  DEFINITIONS 
 
Cocktail studies  Evaluates an investigational  drug as a potential inducer or inhibitor  of 
multiple  enzymes and/or transporters and includes the simultaneous 
administration  of multiple  substrates for multiple  CYP enzymes and/or 
transporters to study subjects. 
Concomitant-use  Clinical  DDI studies that investigate DDIs between drugs likely  to be 
studies  used by the target population  under clinically  relevant scenarios.   
In silico DDI  Simulation  studies conducted with adequately validated computer models.   
studies 
Index perpetrator  Drugs recommended for use in prospective clinical  DDI studies because 
they have well-established potency and selectivity profiles that cause a 
defined degree of inhibition  or induction  of a given elimination  pathway 
when administered with a sensitive and specific substrate of that pathway.   
Index substrate  Drugs recommended for use in prospective clinical  DDI studies as 
substrates because they have well-established  sensitivity  and specificity 
profiles that demonstrate a defined degree of change in exposures when 
administered with a strong inhibitor  or inducer for that specific 
elimination  pathway.   
Moderate inducer  Drugs that decrease the AUC of sensitive index substrates of a given 
metabolic pathway by  50 percent to < 80 percent.   
Moderate  Drugs that increase the AUC of sensitive index substrates of a given 
inhibitor  metabolic pathway by  2- to < 5-fold.   
Moderately  Drugs whose AUC values increase  2- to < 5-fold with strong index 
sensitive substrate  inhibitors  of a given metabolic pathway in clinical  DDI studies, or whose 
AUC values increase   2- to < 5-fold in poor metabolizers for a specific 
enzyme compared to extensive metabolizers.  
No-effect  Interval within which a change in a systemic exposure measure is 
boundaries  considered not significant  enough to warrant clinical action (e.g., dose or 
schedule adjustment,  or additional  therapeutic monitoring) 
Perpetrator  Moiety that can induce or inhibit  an enzyme or a transporter.   
 
Prospective  Clinical  DDI investigations  that are part of trials with a primary endpoint 
nested DDI  different than investigation  of DDIs.  However, these trials are adequately 
studies   designed to prospectively  investigate DDIs and define DDIs as one of the 
endpoints.   
Prospective stand- Separate clinical  trials prospectively  designed to investigate a DDI as the 
alone DDI studies  primary endpoint. 
Sensitive  Drugs whose AUC values increase  5-fold with strong index inhibitors 
substrate  of a given metabolic pathway in clinical DDI studies, or whose AUC ratio 
in poor metabolizers for a specific enzyme increase  5-fold compared to 
extensive metabolizers.    
  23  Contains Nonbinding Recommendations 
 
Strong inducer  Drug that decreases the AUC of sensitive index substrates of a given 
metabolic pathway by  80 percent.   
Strong inhibitor  Drug that increase the AUC of sensitive index substrates of a given 
metabolic pathway  5-fold. 
Substrate  Used interchangeably with victim (see definition  for victim).   
Retrospective  Clinical  evaluations that have not been prospectively and adequately 
DDI evaluations  designed to investigate DDIs. 
Victim  Substrate whose exposure changes due to inhibition  or induction  of an 
enzyme or transporter by a perpetrating moiety.   
Weak inducer  Drug that decreases the AUC of sensitive index substrates of a given 
metabolic pathway by  20 percent to < 50 percent.   
Weak inhibitor  Drug that increases the AUC of sensitive index substrates of a given 
metabolic pathway by  1.25- to < 2-fold.   
 
 
 
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