PERSPECTIVES

clinical setting, a classical definition of the

A G U I D E T O D R U G D I S C O V E RY O P I N I O N
TI is the ratio of the dose of the drug that
causes adverse effects at an incidence and/
The determination and or severity not compatible with the targeted
indication (for example, toxic dose in 50% of
interpretation of the therapeutic subjects; TD50) to the dose that leads to the
desired pharmacological effect (for example,
index in drug development efficacious dose in 50% of subjects; ED50)12.
A high TI is preferable in order for a drug
to be viewed as having a favourable safety
Patrick Y.Muller and Mark N.Milton profile, whereas lower TIs may be acceptable
for the treatment of life-threatening diseases
Abstract | A key part of drug discovery and development is the characterization that have limited treatment options.
and optimization of the safety and efficacy of drug candidates to identify those At earlier stages in the drug discovery
that have an appropriately balanced safetyefficacy profile for a given indication. and development process, the clinical TI of
The therapeutic index (TI) which is typically considered as the ratio of the a drug candidate is unknown. Gaining early
understanding of the likely TI is crucial in
highest exposure to the drug that results in no toxicity to the exposure that
order to initiate mitigation steps to address
produces the desired efficacy is an important parameter in efforts to achieve unfavourable characteristics or to potentially
this balance. Various types of safety and efficacy data are generated invitro and redirect resources to alternative candidates.
invivo (in animals and in humans), and these data can be used to predict the clinical It is also important in avoiding clinical trials
TI of a drug candidate at an early stage. However, approaches to systematically and that could be considered ethically unaccep-
quantitatively compare these types of data and to apply this knowledge more table owing to a low indication-specific TI.
It should be noted, however, that the clas-
effectively are needed. This article critically discusses the various aspects of TI
sical concept of the TI is not applicable to
determination and interpretation in drug development for both small molecule very rare or idiosyncratic adverse drug reac-
drugs and biotherapeutics. tions, which pose an even greater challenge
for detection in the earlier stages of drug
The declining efficiency of pharmaceutical such knowledge about potential toxicities development.
research and development1 is driving the needs to be interpreted in relation to the Despite the widespread use of the concepts
search for improved strategies to identify desired pharmacology of the drug candidate, of TI and safety margins for the benchmark-
unsuitable drug candidates earlier in the as well as the intended clinical indication, ing of drug development candidates and
process and hence reduce the number of the associated unmet medical need and the associated decision-making, there is hardly
expensive failures in late-stage clinical trials. competitive environment. Consequently, on any published literature and no regulatory
Historically, early discovery activities largely the basis of these factors, more or less weight guidance available on the determination and
focused on assessing the invitro and invivo can be given to either the safety or efficacy of interpretation of the TI. In addition, there is
efficacies of lead compounds and drug can- a drug candidate with the aim of achieving a differing understanding of TIs across the
didates. However, safety is increasingly being a well balanced, indication-specific, safety disciplines that are involved in drug develop-
recognized as a key differentiation criterion efficacy profile. ment, and the cross-functional complexities
and competitive advantage for successful A widely used concept in making such of TI calculations are often underestimated.
new drugs due to changing societal2, legal3 decisions is the therapeutic index (TI) This article critically discusses the various
and regulatory4 expectations. Consequently, of drug candidates, which is a quantita- aspects of TI calculation and interpreta-
the industry faces the challenge that tive relationship between their efficacy tion during drug discovery and develop-
improved efficiency in demonstrating (pharmacology) and safety (toxicology) ment for both small-molecule drugs and
drug efficacy may have little impact on the that can be calculated using various pairs biotherapeutics.
time and cost of drug development unless of pharmacological and toxicological end
there is a comparable or better efficiency points. Although the concept seems simple, Exposure-centric TI approach
in demonstrating drug safety5. the determination and interpretation of the There is no universal TI value that could be
In recent years, important advances in TI can be complex, and depends on both considered sufficient or required for a drug
the insilico6,7, genomics8,9, proteomics10 and the stage of development (which affects the candidate. Predictive (translational) surro
biomarker11 fields have enhanced the ability data available) and the characteristics of the gate variables are required in order to estimate
to predict and detect toxicity in preclinical indication for which the drug is being devel- the clinical TI of drug candidates at the
and clinical drug research. Nevertheless, oped. For an approved drug in an established various stages of discovery and development.

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 PERSPECTIVES

O-target Here, we use the term translational TI effects on the selected pharmacodynamic
potency to emphasize the need to understand the end points changes (FIG.2). Additionally,
preliminary TI as early as possible (from multiple TIs may be calculated: one for each
In vitro toxicology/
safety/DDI the lead identification stage onwards) and type of toxicity for which a risk assessment is
Animal Human that it should be understood that there is a performed.
safety safety continuum of TIs that are refined during Various safety and efficacy data are gener-
the process by comparing all the relevant ated invitro, in animals and in humans during
Level of drug exposure

safety data to all the relevant efficacy data the preclinical and clinical evaluation of a
Selectivity

available at that point (FIG.1). drug (FIG.1). On the basis of data generated
TI
In general, the pharmacological and from invivo studies (in animal or in human),
toxicological effects of a drug are determined the TI can be calculated as the ratio of the
by the exposure of a given tissue to the drug highest level of drug exposure that does not
Human (that is, the drug concentration over time) lead to toxicity to the level of drug exposure
Animal PD end rather than the dose of a drug. For example, required to elicit the desired pharmacological
PD end point
In vitro point at the same dose, there may be marked inter- effect. In animal studies, the highest level of
ecacy individual variability in exposure levels to the drug exposure that does not lead to toxicity
Target drug owing to factors such as polymorphisms (termed the no observable adverse effect level;
potency
in genes encoding enzymes that are involved in NOAEL)14 is usually chosen as the reference
Progression through drug development drug metabolism, drugdrug interactions; safety end point instead of the lowest level of
differences in body weight; or to environ drug exposure that leads to toxicity (termed
Figure 1 | An exposure-centric approach to mental or disease factors. the lowest observed adverse effect level;
therapeutic index determination. The extent These variabilities emphasize the LOAEL) in order to be conservative in the
Nature Reviews
of safety data (indicated DrugofDiscovery
by the| size the red importance of using drug exposure rather calculation of the TI. The use of NOAEL is
circles on the red arrow) and extent of efficacy than dose for calculating the TI. In order to particularly important at early preclinical
data (indicated by the size of the green circles
account for delays between drug exposure stages, in which the intervals between the
on the green arrow) increases as a candidate
drug progresses from invitro to animal to human
and the emergence of toxicities that occur exposure levels of the dose groups in animals
studies (indicated from left to right in the figure). after multiple doses, the TI should be may be large. In such situations, using the
Safety margins and therapeutic indices (TIs) are calculated using the exposure to the drug LOAEL exposure may lead to a considerable
calculated by dividing the level of drug expo- at steady state, rather than after adminis overestimation of theTI.
sure (indicated by the position on the vertical tration of a singledose. At the early stages of lead identification,
axis) at which the safety end point occurs by the Toxicity often occurs in tissues other when only invitro data are available, an ini-
level of exposure at which the efficacy end point than those targeted to achieve drug efficacy. tial assessment of the TI of a drug candidate
occurs. Dark grey vertical arrows indicate mar- However, as direct measurements of drug can be obtained on the basis of its on-target
gins involving data of an analogous type. Light exposure in tissues are generally not feasible versus off-target selectivity (BOX3); that is,
grey arrows indicate margins involving data of
(particularly in humans), plasma exposure the off-target IC50 divided by the ontarget
a different type. Solid arrows (light or dark
grey) refer to margins of in vivo data involving
to the drug is usually used as a surrogate IC50 of the drug. There are many examples
both concentration-based (maximum (Cmax) or for tissue exposure (the limitations of using in which an increase in target selectivity
average (Cav)) and/or area (integral) under the exposure data for interpreting the TI are (in particular for targets that have close homo
concentrationtime curve (AUC)-based data. discussed below). logues) leads to an improved invivo TI15.
Dashed arrows (light or dark grey) indicate mar- The most important plasma exposure However, there are also cases in which
gins that are restricted to concentration-based parameters for calculating or predicting the a relatively high target selectivity did not
data as invitro data are compared to invitro or TI of a drug are the maximum concentration prevent a drug from having a challeng-
invivo data. Often, the margins decrease as the (C ), the average concentration acrosstime ing or even unacceptable clinical safety
candidate progresses from invitro to animal to (C max ) and the area (integral) under the con- profile (see the examples in BOX4). In the
av
human studies (see also FIG.2 and the discus-
centrationtime curve (AUC) (see BOX1 for latter case, this is often due to the need for
sion in the main text on animal and human
exposure margins). Invitro efficacy assays and
explanations of the terms). Invivo, it is the certain drugs to have a relatively high level
invitro safety assays consist of a diverse group free drug concentration at the site of action of systemic exposure to drive the pharma-
of functional tests, which are usually conducted that exerts the biological activity (BOX2). cological effect, despite the drug having a
13

in cell-free systems or intact cells by determining By contrast, for invitro studies, it is the con- high affinity (low target IC50) for its phar-
effective concentration (EC) or half maximal centration of a drug that is required for 50% macological target. This further highlights
inhibitory concentration (IC50) values, or mini- inhibition (IC50; BOX1) that is most often the importance of an exposure-centric TI
mally effective or toxic concentrations. Invitro used to quantitatively describe pharmaco- approach. When available at an early stage,
efficacy assays, depending on the drugs indica- logical effects. further (IC50) data from invitro safety and
tion, include growth inhibition of tumour cells, In a translational research setting, the TI efficacy assays should be compared to each
a variety of assays in immune cells, ion channel
can be considered as the quantitative ratio other (FIG.1).
assays using patch-clamp, or enzyme assays.
Invitro safety assays include various off-target
of the exposure level at the chosen safety The comparisons between invitro and/
functional assays, transporter assays, cytotoxicity end point to the exposure level at the chosen or invivo pharmacology assays and in vitro
tests, phototoxicity assays, determination of cyto- efficacy end point. The TI may change dur- and/or invivo toxicology assays based
chrome P450 enzyme inhibition or exvivo assays ing the discovery and development process on the drug exposure levels at which the
in isolated tissues. DDI, drugdrug interaction; of a drug candidate as the understanding end point is reached in each assay that
PD, pharmacodynamic. and expectations of the magnitude of the are needed to derive the TI ratios outlined

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in FIG.1 should be performed as soon as
the data are available in order to inform
decision-making for the next development
stage of a drug.
For most small-molecule drugs, the
preclinical pharmacology model is typically
a different species (often the mouse) than
the species used for preclinical toxicity
studies (most often rats, as well as dogs
or monkeys). A strategy that Novartis is
pursuing during lead optimization and
candidate selection is the addition of safety
end points (for example, clinical pathology,
safety biomarkers, histopathology of target
organs of toxicity and cardiovascular func-
tion parameters) to early preclinical efficacy
studies (for example, multidose pharma
cology studies). This allows the integration
of pharmacology and preliminary safety
evaluations within the same study at several
doses and exposure levels. An advantage of
this approach is that some safety end points
may be determined directly in the disease
models that have been chosen to assess
preclinical efficacy.
Once available, the drug exposure data
obtained in humans is incorporated into
calculations of TIs to compare safety and
efficacy end points, and supersedes any
previous estimates that were based only on
predictive calculations of human exposure.
We suggest using the term TI for exposure
ratios that are derived from data from invivo
safety and invivo efficacy studies in either
animals (preclinical TI) or humans (clinical
TI). When concentration data (IC50) from
invitro safety or efficacy assays are com-
pared to each other or to invivo concentra-
tion data (Cmax or Cav), we consider that the
more general term safety margin is more
appropriate than using the term TI
(discussed further below in the section
about off-target pharmacology).
It should be noted that it may not always
be appropriate to use the exposure at the
therapeutically active dose as the denomi
nator in these calculations. In clinical trials,
in particular in the case of firstinhuman
(FIH) studies, supratherapeutic doses are
often evaluated in order to investigate the
tolerability of the drug or as part of estab-
lishing the doseresponse relationship.
In such trials, it is important to calculate
a predicted safety margin for the human
exposure (that is, exposure multiples) at
each of these doses that will be used16.
Translating the safety margin based on
animal data for each dose level can be
achieved by calculating the ratio of the
exposure at the animal NOAEL dose to the
predicted exposure in humans at that dose.
NATURE REVIEWS | DRUG DISCOVERY VOLUME 11 | O CTOBER 2012 | 753
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Box 1 | Commonly used exposure parameters
IC50
This is the concentration of a drug that is required for 50% inhibition invitro (that is, the half-
maximal inhibitory concentration). IC50 values depend on the conditions under which they are
measured. Depending on the type of inhibition, other factors may influence the IC50; for example,
for ATP-dependent enzymes, the IC50 has an interdependency with the concentration of ATP,
especially for competitive inhibition.
EC50
This represents the plasma concentration of a drug required for obtaining 50% of a maximum
effect (that is, the half-maximal effective concentration).
Cmax
This is the maximum plasma concentration of a drug invivo. The corresponding time-point is Tmax.
AUC
This is the area (integral) under the concentrationtime curve of a drug invivo. AUC values can be
compared between doses to evaluate linearity or between species to understand relative systemic
exposure.
Cav
This is the average concentration of a drug across time invivo. It is calculated by dividing the
AUC by time. The Cav allows direct comparison with benchmark concentrations such as invivo
efficacious plasma concentration or invitro receptor affinity/potency (Kd / IC50). AUC values
contain the component of time and therefore cannot directly be referenced to pharmacological
activities. Conversion of the AUC to the Cav provides a simple procedure to allow such a
comparison by considering the time information imparted by AUC values38.
Limitations of exposure data Before FIH trials of a new drug candi-
There are several limitations to exposure- date, invivo efficacy and safety data from
based TI determinations. First, plasma expo- animals, together with the corresponding
sure is only a surrogate for tissue exposure, exposure levels, is usually extrapolated to
which is actually driving most pharmacologi- predict the efficacious dose or level of
cal and toxicological effects. Using plasma exposure in humans, as well as the level
exposure as a surrogate for tissue exposure of exposure at which dose-limiting toxicity is
assumes that free tissue exposure, in equi- expected to occur. Frequently, the prediction
librium, is similar to free plasma exposure, of an efficacious level of exposure in humans
which is usually the case for drugs that have accounts for known inter-species differences
reasonable membrane permeability (see in the ontarget potency of the candidate
BOX2 for a discussion of the potential limita- drug. Effective translation of an efficacious
tions of these assumptions). Furthermore, in level of exposure from preclinical models
certain cases, plasma levels of a drug may not to humans (with the aim of maximizing the
directly correlate with efficacy. For example, pharmacodynamic response in humans;
for angiotensin-converting enzyme (ACE) FIG.2) depends on the reliability and robust-
inhibitors, this lack of correlation is because ness of the preclinical model used to deter-
of saturable binding to the target ACE pool, mine efficacy (for example, a (humanized)
which is partly circulating (and so contribut- animal disease model or induced-disease
ing to the measured plasma concentration) model compared with a pharmacodynamic
and partly non-circulating (as it is anchored model only).
to the endothelium of blood vessels)17. Therefore, for some drugs the level of
Second, there may be considerable therapeutic exposure required in humans to
differences between species in the levels result in efficacy is higher than the level of
of drug exposure in target tissues and the exposure predicted from preclinical pharma-
pharmacological and/or toxic effects; for codynamic models, which leads to erosion of
example, owing to species-specific active safety margins (see the valdecoxib example
uptake and efflux of the drug (BOX2). in BOX5). The predicted or actually achieved
It should be noted that while it is feasible to efficacious exposure in humans is compared
obtain tissue samples from animals, it would back to exposures achieved in preclinical
not be feasible to do so in most instances in safety studies to calculate animal versus
humans. Therefore, an assumption of the human exposure margins.
tissue:plasma ratio, which may be time- and At later stages in drug development,
concentration-dependent18, will have to be findings from reproductive toxicity19 and
made, which introduces uncertainty into carcinogenicity20 studies in animals provide
the TI calculation. the basis for the respective label sections for
2012 Macmillan Publishers Limited. All rights reserved
PERSPECTIVES

Box 2 | How to consider plasma protein binding and tissue exposure

needs to be considered for calculating the TI,
particularly if the parent:metabolite ratio is
The free (that is, unbound) drug hypothesis states that it is the free drug concentration at the not constant across studies, species and/or
site of action that exerts the biological activity such as ontarget pharmacology, off-target or individual subjects or populations. In cases in
secondary pharmacology and/or toxicity not the total drug concentration or the concentration which human-specific metabolites are highly
of drug that is bound to plasma or tissue proteins13. On the basis of the free drug hypothesis,
abundant, nonclinical characterization is
designing drug candidates to have minimal plasma protein binding (PPB) is not an optimal
approach, for reasons extensively discussed by Smith etal.13.
warranted when the metabolite is observed
For membrane-permeable drugs, free tissue exposure in well-perfused tissues in equilibrium at exposures greater than 10% of total drug-
reflects free plasma exposure, whereas total tissue exposure may be very different from total related exposure and at significantly greater
plasma exposure owing to different binding affinities of the drug to tissue proteins compared levels in humans than the maximum expo-
to plasma proteins. Because free tissue exposure largely reflects free plasma exposure for sure seen in the preclinical toxicity studies21.
membrane-permeable compounds, those compounds do not need to be optimized or selected for It should be noted that the International
high exposure in tissue(s) expressing the pharmacological target or low exposure in target tissue(s) Conference on Harmonisation (ICH) guide-
of toxicity. Moreover, in those instances in which tissue drug levels are measured, usually total drug line Nonclinical Safety Studies for the Conduct
concentrations are measured in these tissues, whereas the free tissue concentration cannot be of Human Clinical Trials and Marketing
directly determined.
Authorization for Pharmaceuticals M3(R2)21
For drugs that have poor membrane permeability, actual free tissue concentrations or gradients
can be influenced by transporter proteins. Tissues with abundant transporter expression include
has been adopted globally, superseding the
brain42, liver58, kidney58, testes43, retina44, as well as tumour cells45. Determination of drug levels in US Food and Drug Administrations (FDAs)
such tissues may need to be considered for drugs known or suspected to be transporter substrates 2008 guidance on Safety Testing of Drug
in order to understand possible discrepancies between plasma and effective tissue concentrations. Metabolites22.
In some cases, the extent of PPB is dependent on the concentration of the drug invivo and Fifth, for feasibility reasons, exposure
therefore needs to be considered accordingly. A high-affinity, but low-capacity, binding process data are most often summarized as the mean
can be responsible for a concentration-dependent PPB42. Some invitro and exvivo assays for each gender and dose group in both
(for example, some cell-based assays, serum supplemented assays or whole-blood exvivo assays) preclinical and clinical studies. However,
may have high protein concentrations and, in such cases, the free drug concentration in these heterogeneity owing to genetic variations in
assays should be considered in addition to the total drug concentration when calculating apparent
metabolizing capacity and environmental
potency. Furthermore, for some invitro pharmacology assays the concentration of serum albumin
affects the potency in binding and functional mode, with the effect of serum albumin being
factors (for example, in nutritional status,
remarkably different for different compounds43. In such cases, careful judgment is required as disease status or existence of comorbidities)
to which experimental conditions should be used to determine the most representative values within and across dose groups leads to vari-
for the concentration of a drug that is required for 50% inhibition (IC50) in vitro or the plasma ability in exposure data (both in animals and
concentration of a drug required for obtaining 50% of a maximum effect (EC50). in humans). Moreover, individual animals
As good practice, we suggest that free drug exposure in addition to total drug exposure is or subjects within the same dose group may
considered when calculating exposure ratios such as therapeutic indices (TIs) or safety margins show considerably different exposure levels,
(see the translational TI heatmap grid in BOX5). In particular, in case there are significant thereby leading to a different magnitude of
inter-species differences in PPB, systemic exposure (that is, the area (integral) under the pharmacological and/or toxicological effects.
concentrationtime curve of a drug invivo (AUC), the average concentration of a drug across time
In such cases, TI assessment should be per-
invivo ( Cav) and maximum plasma concentration of a drug invivo (Cmax)) at steady state should
be corrected for free fraction for inter-species comparison. In rare cases, the extent of potential
formed using a range of exposure levels in
cellular uptake of plasma protein-bound drug into target tissues44,45 (in case it is known) may be an individual animals or humans, if possible.
important factor when deciding to put more emphasis on TIs that are calculated on the basis of Additionally, it is important to understand
free or total drug concentration. We recommend selecting the most appropriate approach based the distribution frequency of the individual
on the mechanism and nature of the pharmacology and toxicities for which the TI is being exposures in order to be able to place the
calculated. Nevertheless, it needs to be noted that regulatory authorities frequently rely on total range of calculated TIs into context.
systemic exposure when considering exposure multiples between clinical exposure in humans Sixth, the dosing regimen and the
and exposures associated with toxicities in preclinical studies (see also discussion on exposure duration of dosing may considerably affect
caps in the main text). the pharmacological and/or toxicological
effects induced by the drug (for example,
an intermittent dosing schedule is sometimes
approved drugs. In this context, the exposure (cell-free) assays and minimal inhibi- used for oncology drugs to decrease toxicity).
multiples of carcinogenicity and reproductive tory concentrations invivo owing to the For some candidate drugs, the envisaged
toxicity findings in relation to human expo- unknown permeability of the drug into clinical dosing regimen is twice a day
sure are a crucial part of the risk assessment target cells invivo. (as opposed to once a day). This is true
underlying the respective label section, and Fourth, exposure-based TI assessments particularly for drugs with (predicted) short
in considering the riskbenefit ratio for the are usually focused on the parent molecule half-lives in humans and oral drugs with
particular indication. as the main compound eliciting the invivo low solubility. A twice a day dosing regimen
Third, for some indications that do not effects. However, pharmacology and toxicity would also be envisaged in cases when
have established preclinical invivo efficacy can be driven by metabolites, although it can lower doses given more frequently are used
models (such as hepatitis C and many other often be difficult to determine whether a par- in an attempt to reduce (expected) Cmax-
virology indications), preclinical efficacy ticular toxicity is caused by the parent drug driven toxicities (for example, drugs known
is based solely on invitro assays. In such and/or a metabolite. If it is suspected that a to inhibit cardiac ion channels, which
situations, there is often a poor correlation metabolite is responsible for a particular tox- might be anticipated to have different Cmax-
between invitro IC50 values in biochemical icity, the level of exposure to the metabolite based TIs compared with AUC-based TIs).

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 PERSPECTIVES

can lead to delayed ventricular repolarization/

Best-in-class
QT prolongation and potentially fatal arrhyth-
mia. Indeed, several drugs have been with-
Registration
end point drawn from the market or limited in their
Magnitude of end point

use owing to off-target hERG inhibition23.

Therapeutic Consequently, regulatory authorities require
eect (PoC)
Unacceptable all candidate drugs to be tested for hERG
Pharmacological toxicity inhibition before entering clinical trials24.
response In a retrospective analysis of marketed
Pharmacodynamic drugs that were found to inhibit hERG,
biomarker Pharmacodynamic end point Redfern etal.23 concluded that a 30fold
Safety end point
safety margin between the hERG IC50 value
Log (exposure)
and free plasma Cmax value in humans pro-
vides a reasonable safety margin regarding
Figure 2 | The therapeutic index in relation to the pharmacodynamic end point. The therapeutic arrhythmogenesis. However, there are also
index (TI) (indicated by the width of the green bars) is determinedNatureby the Reviews
magnitude of the
| Drug effect of
Discovery several examples in which a 30fold clini-
the drug on the selected pharmacodynamic (PD) end points (on the green PD versus logarithm of the cal safety margin was not enough to prevent
exposure curve) in relation to the magnitude of the effect on the end point of toxicity (on the red toxicity
arrhythmogenesis23. Therefore, these authors
versus logarithm of the exposure curve). Generally, the higher the magnitude of the desired PD effect,
the higher the exposure that is required to drive the effect, and consequently the smaller the TI. For recommended aiming for higher preclinical
certain small-molecule drugs and many biotherapeutics, ontarget toxicity is frequently elicited by safety margins, particularly for candidate
exaggerated PD effects, and therefore the red toxicity curve can be considered to be superimposed drugs that are being developed for non-
on the PDexposure curve for such toxicities. For some drugs, the level of exposure required in humans debilitating diseases.
to result in efficacy is higher than the level of exposure predicted from preclinical models (see discussion Similarly, for the cardiac sodium channel
on animal versus human exposure margins in the main text). PoC, proof of concept. Nav1.5, a 30fold safety margin between
the Nav1.5 IC50 value and free plasma Cmax
value in humans seems to confer an accept-
able degree of safety for QRS prolongation
In situations in which different (preclinical) Off-target safety margins and associated arrhythmia25. However, the
dosing frequencies are compared with the In general, drugs can cause toxicity either magnitude of off-target safety margins that
clinical dosing frequency, the same AUC through ontarget pharmacology (effects is required to avoid modulation of a spe-
intervals should be considered for drug mediated through the primary drug cific off-target and, therefore, indicative of
exposure comparisons. target(s)) or through off-target pharma a sufficient TI, depends on many off-target
Seventh, pharmacological and/or cology (effects that are mediated through a and drug-specific factors. Currently, there
toxicological effects elicited in the tissue at known or unknown unintended target(s)) are few data on this relationship, with the
the site of administration are usually not (BOX3). Off-target pharmacology typically exception of hERG and Nav1.5. Based on our
driven by systemic exposure, nor is it usually begins to be considered through invitro experience, a total plasma Cmax that is close
feasible to measure exposure at the site of secondary pharmacology assays during to or above the off-target IC50 (the so-called
injection (for example, in the eye). Such lead identification and optimization. off-target coverage) is indicative of off-target
examples include administration-site effects Off-target safety margins are calculated effects in vivo in many cases. However, free
for topical routes of administration, such as as off-target IC50divided by Cmax, at effica- plasma Cmax can be below the off-target IC50,
dermal, inhalation/intranasal and ocular/ cious exposure, and typically use free plasma as shown for hERG, Nav1.5, 5hydroxy-
intravitreal administration. TI calculations concentrations (BOX2). To err on the side of tryptamine 2B receptor (5HT2b) and bile-
for such local effects should consider dose caution, plasma Cmax should be the preferred salt export pump (BSEP) but still lead to an
per organ volume or weight, or application reference concentration invivo over Cav. This invivo effect (BOX4).
area (dermal) for local effects in addition to is because (repeated) short-term modulation Conceptually, off-target coverage is
exposure-based considerations for systemic of off-targets (in particular around the time highly similar to the quantitative drugdrug
effects, and so may result in two different Cmax is achieved invivo) may lead to (long- interaction (DDI) risk approach using the
TIs (one for local and one for systemic term) invivo effects, depending on the Cmax:Ki ratio, in which Cmax is the maximum
effects). properties of the drug and the off-target. plasma concentration of total drug (bound
Finally, it should be noted that the calcula- Assessment of off-target safety margins plus unbound) and Ki is the inhibition con-
tion of a TI has many challenges in addition should be performed as soon as data on stant for cytochrome P450 (CYP) enzymes.
to those identified above. For example, non- the efficacious plasma concentration from The FDA26 considers CYP inhibition to
clinical studies often involve relatively low preclinical invivo pharmacology studies be likely if this ratio is >0.1 for total drug,
numbers of animals, which potentially may are available. These safety margins should whereas the European Medicines Agency
lead to a less accurate assessment of the mean be recalculated once the efficacious plasma (EMA)27 considers it possible that CYP
exposure to a drug. The determination of concentration (both predicted and actually inhibition will occur if the ratio is >0.02 for
Cmax concentrations rely heavily on the time measured) in humans is known. Strategies Cmax of free drug. Therefore, we recommend
at which the plasma samples are collected that can be used to increase off-target safety calculating DDI margins for both total and
and as such may underestimate the true Cmax. margins are outlined in BOX3. free-drug concentrations (BOX3).
Therefore, Cmax values may be less accurate The best known off-target is the cardiac The level of drug exposure at the tissue(s)
compared to AUCvalues. potassium channel hERG. hERG inhibition or compartment(s) in which the off-target

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Box 3 | On-target versus off-target pharmacology or toxicity

At Novartis, preclinical screens of small-
molecule drug candidates include the use
In general, drugs can cause adverse or toxic effects by acting on the primary pharmacological target of invitro safety pharmacology profiling
to elicit ontarget toxicity (often referred to as mechanistic toxicity) or by acting on a known or panels to assess the activity of the investiga-
unknown off-target (also referred to as an anti-target) to elicit off-target pharmacology or toxicity. tional drug at defined off-target receptors29,30
Recognizing the underlying mechanism of toxicity is essential for any kind of safety-related decision
(BOX3). In general, the following sequence of
that needs to be taken during drug development. Ontarget toxicity is the dominant mode of
toxicity for monoclonal antibody (mAb) therapeutics owing to their high selectivity for the primary
questions should be answered to determine
target31. Ontarget toxicity may include both exaggerated pharmacology in the therapeutic target the potential invivo impact of pharmacologi-
tissue(s) as well as ontarget pharmacology (secondary pharmacology) in tissues other than the cal and toxicological effects that are elicited
therapeutic target tissue(s); the latter is often the case even for highly selective small-molecule by potential activity at an off-target receptor
drugs. By contrast, off-target toxicity is a frequent mode of toxicity for small-molecule drugs, by a drug candidate. First, will invivo expo-
which is often due to their pharmacological promiscuity39 and/or their chemical reactivity with sure at the pharmacologically active dose be
biomolecules40 (either reactivity of the parent drug or its metabolite(s)). high enough to modulate (inhibit or acti-
Ontarget toxicity of drug candidates against (novel) targets can often be predicted on the basis of vate) the off-target? Second, will the magni-
genetic and/or pharmacological knowledge of the target and/or its biological signalling pathway. tude and duration of the off-target activity
Strategies that can be used to improve the therapeutic index (TI) for ontarget toxicities may include
invivo lead to a biologically relevant effect
the following: selection of drug candidates that are partial antagonists or partial agonists of the
target, selection of drug candidates that have more selective distribution into the tissue of intended
(to be assessed in invivo pharmacology and
pharmacological action; or optimizing the dosing regimen. In order to quantitatively assess and toxicology studies by including tailored end
translate TIs pertaining to ontarget pharmacology or toxicity, the relative ontarget potency of the points)? Third, will the off-target-driven
drug candidate needs to be known for both preclinical models and humans. invivo effect negatively affect the safety
The off-target pharmacology or toxicity profile of a compound can be optimized by modifying profile of the drug candidate for the targeted
the chemical structure while retaining its ontarget activity41. Invitro off-target receptor profiling indication?
should be considered for both parent and major metabolites as early as the lead optimization stage The answers to these questions largely
of small-molecule drugs, with hits in radioligand binding assays being confirmed in functional depend on experience with the particular off-
assays to distinguish agonism from antagonism29. An overview on important cardiovascular and target and/or closely related off-targets (BOX4).
central nervous system off-targets that are regularly screened for in invitro preclinical safety
In particular, interpretation of off-target safety
pharmacology profiling panels used by Novartis and validated with regard to human impact
has been published29,30. Quantitative invitro off-target pharmacology data (for example, the
margins needs to consider the available quan-
concentration of a drug that is required for 50% inhibition invitro; IC50) needs to be available titative experience with a particular off-target
before firstinhuman studies. and is largely performed on a casebycase
basis. It is a reasonable strategy to aim for
Strategies to increase off-target safety margins. These can be divided into those based on target-
related parameters and those based on off-target-related parameters, and should be considered
higher preclinical off-target safety margins
for both parent compounds and their metabolites. For target-related parameters, one strategy is to (off-target IC50divided by Cmax, at efficacious
increase the distribution of the drug to the target tissue. Another strategy is to lower the therapeutic exposure) than those accepted clinically.
exposure or the maximum plasma concentration of the drug invivo (Cmax). This can be achieved This is because the therapeutic plasma
by increasing the drugs potency at the primary pharmacological target, by changing the dosing exposure in humans for certain drugs can be
regimen or by changing the formulation. For off-target-related parameters, one strategy is to higher than initially assumed on the basis of
decrease the potency of the drug at the off-target (by increasing the concentration of a drug that preclinical models (see discussion above on
is required for 50% inhibition invitro; IC50). Another strategy is to decrease the distribution of the animal versus human exposure margins).
drug to the off-target tissue (for example, by decreasing the bloodbrain barrier permeability
of the drug for central nervous system-based off-targets).
TI display and decision-making
TI display. The most common visual aid
to display TIs and/or safety margins is the
is expressed relative to the off-target IC50 effects28 (as accepted for many ion socalled exposure thermometer, which is a
(that is, off-target coverage) drives off- channels) one dimensional representation of exposure.
target effects and not just the off-target IC50. Drug-related factors that contribute to Usually, it is done separately for AUC and
Additional off-target-related factors that whether a certain level of drug exposure is Cmax, each for total and free exposure, which
determine whether a certain exposure level actually driving specific off-target effects potentially leads to four different thermo
is actually driving specific off-target effects invivo include the following: meters in which TIs and exposure margins
invivo potentially include the following: High penetration and/or accumulation need to be calculated and depicted manually.
High off-target receptor density invivo into tissues expressing the off-target To introduce a more sophisticated and
(leading to a potential disconnect (for example, owing to active uptake) automated procedure for TI calculation
between invitro and invivo potencies) Cellular permeability of the drug for and visualization, the twodimensional
Low turnover of the off-target receptor intracellular off-targets translational TI heatmap grid (BOX5 and
(that is, slow reconstitution of inactivated Mode of action at the off-target receptor Supplementary informationS1 (table))
off-target receptors) (for example, partial versus full agonists was developed. The concept of the transla-
Comorbidities in tissues expressing the or antagonists; fast versus slow off-rate) tional TI heatmap grid was to compare all
off-target (making it more susceptible to The shape of the Cmax section of the relevant safety exposure data to all relevant
adverse effects) concentrationtime curve invivo (for efficacy exposure data: invitro (binding
The possibility of, for example, the IC10 example, a flat curve versus a sharp peak; and functional) and invivo (in animals and
rather than the IC50 being the appropriate that is, time above a certain concentration in humans). Data can be from real experi-
indicator of off-target pharmacodynamic threshold) ments and studies as well as from modelling

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and simulation data (for example, simulated
human exposure). Frequently, several preclin-
ical pharmacodynamic models are available.
Furthermore, at later stages of development,
data on drug exposure levels from embryo-
fetal developmental and carcinogenicity
studies in animals can be included.
The translational TI heatmap grid is a
technical aid to calculate, visualize and assess
TIs and exposure margins. It is applicable to
small-molecule drugs and their metabolites
as well as, in principle, to biologics. The grid
can be extended to include any number of
assays, studies and dose groups. For topical
drugs, a separate TI heatmap grid could
be produced that is based on dose per
organ volume or weight, or application
area, to account for local effects.
What TI number is sufficient? The inter
pretation of TI numbers and decisions based
on TI numbers depend on many factors
that characterize an acceptable riskbenefit
profile for the targeted indication of the
drug candidate, and are centred around the
following themes:
The drugs intended profile (potentially
compared with competitors), based on
the indication and the patient population
The translatability and relevance of the
pharmacodynamic end points used in
early stage efficacystudies
The translatability and relevance of the
toxicity endpoints
Detailed parameters related to these
themes are listed in BOX6. It is not possible
to state a universal TI number that is con-
sidered sufficient for a drug candidate to
be successfully developed. This complexity
needs to be addressed by dedicated experts
performing an integrated safety assess-
ment that is based on a weight-ofevidence
approach.
Interpretation of TIs and associated
decision-making for a candidate drug may
depend on previous experience with drugs
that have similar indications, efficacy and/
or safety characteristics as the candidate
drug. However, this evaluation may be
biased in several respects. Organizational
experiences with late-stage failures heavily
affect future decisions. Unmet medical need
and competitive landscape (for example,
firstinclass versus followon or backup increased chance of detecting toxicities that
candidate) affect development decisions
related to TI and may change over time.
Furthermore, differentiation versus compet-
itor compounds increasingly drives internal
candidate selection, as does the decreasing
level of acceptance of safety liabilities by
regulatory authorities.
NATURE REVIEWS | DRUG DISCOVERY VOLUME 11 | O CTOBER 2012 | 757
PERSPECTIVES
Box 4 | Invitro target selectivity may not correlate with therapeutic index
The withdrawal in 2007 of the dopamine receptor agonist pergolide, which was indicated for the
treatment of Parkinsons disease, is a highly valuable example of why invitro target selectivity
as an early surrogate for safety should be interpreted with caution. Pergolide was withdrawn
because of the high incidence of valvular heart disease in patients, which is mediated by the
off-target agonist activity of pergolide at the 5hydroxytryptamine 2B (5HT2b) receptor46.
Pergolide is 36times more selective for its main pharmacological target (the dopamine D2
receptor, half maximal effective concentration (EC50) of0.2nM) than for the 5HT2b receptor
(EC50of7.1nM) invitro47. In humans at the therapeutic dose of 1mg three times a day, the total
plasma exposure (Cmax) remained five times below the 5HT2b EC50 and for the free plasma
exposure (10% free fraction), the margin was even higher (47times)48. However, these apparent
safety margins proved to be insufficient to prevent manifestation of valvular heart disease in a
high proportion of subjects treated with pergolide, leading to a therapeutic index (TI) <1 and
ultimately to the market withdrawal of the drug. Such an example provides guidance on what
invivo exposure safety margins may or may not be sufficient, and should be carefully considered
once invivo pharmacology data (animal or human) are available.
Another example is bosentan, an endothelin (ET) receptor antagonist approved for pulmonary
arterial hypertension, the label for which carries a black-box warning for cholestatic
drug-induced liver injury (DILI) due to inhibition of bile-salt export pump (BSEP), which leads to
the accumulation of (toxic) bile salts in hepatocytes49. Bosentan has a >8,000times selectivity
for its main pharmacological target (ETA receptor, half maximal inhibitory concentration (IC50)
of0.0047M)50 over BSEP (IC50of38.1M) invitro51. In humans at the therapeutic dose of 125mg
twice a day, the Cmax (REF.51) remained five-times below the BSEP IC50, with an even higher
margin (>255times) for the free plasma exposure (<2% free fraction). However, these apparent
systemic safety margins are not sufficient to prevent bosentan having a risk of causing DILI
by BSEP inhibition. A likely explanation for this apparent discrepancy when only considering
plasma exposure data might be the active uptake and accumulation of bosentan in hepatocytes52,
thereby increasing its concentration locally in the liver.
In line with the example for bosentan, we calculated systemic safety margins (BSEP IC50divided
byCmax free, at therapeutic dose) on BSEP inhibition of 85 approved, restricted and withdrawn
pharmaceuticals on the basis of data recently published by Dawson etal.51. For drugs showing
inhibition of BSEP, apparent systemic safety margins of >>100 were generally required to avoid
cholestatic/mixed-type DILI. However, to better assess the in vivo potential for inhibition of bile
acid transporters in the liver, the maximum unbound drug concentration at the inlet of the liver
may be the more relevant parameter and is higher than systemic Cmax free for orally administered
drugs53.
At the preclinical stage, findings from unmet medical need for the indication and
invitro and invivo safety studies should the patient population guides development
be used to predict the lowest TI that is decisions based on the TI. The proportion
expected to be dose-limiting in humans, of pharmacological responders to a drug in
and therefore focus candidate optimization the selected population and the magnitude
efforts on mitigating the toxicities that limit of the pharmacodynamic effect will affect
the TI in terms of severity, reversibility, the interpretation of the drugs riskbenefit
monitorability and translatability (BOX 6). ratio.
As drug development progresses, the appar-
ent TI often decreases (FIG.2). In longer-term Special considerations for biotherapeutics.
(in animals and in human) safety studies Ontarget toxicity (BOX3) is the main type of
there is an increased chance of detecting toxicity that is associated with biotherapeu-
toxicities, such as those that develop tics. This is particularly the case for mono-
owing to extended treatment duration, clonal antibodies (mAbs), for which toxicity
or of detecting rarer toxicities that become is usually related to prolonged and/or exag-
apparent as a result of assessing a larger gerated ontarget effects that can often be
number of animals/subjects. There is also an predicted.
It is of great importance that we under-
emerge from assessing an extended battery stand the biology of the target, including,
of safety end points, some of which are not but not limited to, target concentrations,
ethically feasible in humans (for example, target turnover, target distribution and effect
reproductive toxicity, carcinogenicity and of disease on target expression in animals
immunotoxicity). and in humans31. However, biotherapeutics
At the clinical development stage, the differ in several ways from small-molecule
riskbenefit ratio in view of the degree of drugs in terms of efficacy and safety
2012 Macmillan Publishers Limited. All rights reserved
PERSPECTIVES

Box 5 | Example of a translational therapeutic index heatmap grid

The translational therapeutic index (TI) heatmap grid is a two
dimensional quantitative comparison of key safety exposure data
with key efficacy exposure data available at a particular stage of drug
discovery and development (see also Supplementary informationS1
(table)). Safety end points are arranged vertically from top to bottom,
and efficacy end points are arranged horizontally from left to right
in the sequence of invitro (white), animal (light grey) and human
(dark grey) studies. Safety exposure data is divided by efficacy exposure
data and the ratios (that is, exposure multiples) are displayed in the
corresponding cells of the grid. For easier visualization of numbers,
shading of cells is as follows: purple 1, yellow=10, green30, with
continuous colour gradients for numbers in between. The colouring
of the cells itself does not attempt to indicate a positive or negative
aspect of the exposure ratio, and needs to be interpreted in the context
of the study and the respective dose level.
For the purpose of illustration, exposure data available from valdecoxib
is shown in the figure. Valdecoxib, a selective cyclooxygenase 2 (COX2)
inhibitor, is a non-steroidal anti-inflammatory drug (NSAID) that was
formerly approved for the treatment of arthritis54. In 2005, valdecoxib
was withdrawn from the market owing to concerns of an increased risk
of heart attack and stroke in patients taking the drug, which only came
to light post-marketing55. However, the TI calculations for valdecoxib are
not intended to explain these relatively rare adverse events in subjects
that may be predisposed to developing this risk following long-term
treatment, which are thought to be related to selective inhibition of
COX2 versus COX1. Valdecoxib was chosen as an example on the basis
of a publicly available complete data set.
Invitro, animal and human exposure data (mean values; including
plasma protein binding data) as well as data on efficacy and safety
end points were taken from the US Food and Drug Administration
approval package for valdecoxib54. For the purpose of visualization,
the exposure data itself is not shown, but only the ratios (that is,
exposure multiples) of safety exposure data divided by efficacy
exposure data. For comparisons involving both invivo efficacy and
invivo safety data, the exposure multiples in large bold frames consist
of the following four fields: the upper two fields contain total plasma
exposure (Cmax)-related margins, whereas the two lower fields contain
the area (integral) under the concentrationtime curve (AUC)-related
margins (in italics). Margins relating to total exposure are to the left (in
bold) and margins relating to free exposure are to the right. In this way,
one can visually capture potential differential effects of plasma protein
binding (PPB) on Cmax-related and/or AUC-related TIs (for valdecoxib,
the free fraction in dog plasma is twice the free fraction in rat or human
plasma). Numbers 1 indicate subpharmacological exposure in safety
studies (for example, exposure at the first-in-human (FIH) starting dose).
The example of valdecoxib illustrates the interplay of key safety and
efficacy data. The target (COX2) selectivity versus off-target (COX1
in this case, as COX1 inhibition was thought to have a key role in the
gastrointestinal toxicity of non-selective COX inhibitors), both in
binding and whole-blood assays, can be considered as one of the earliest
comparisons of safety versus efficacy data. Inhibition of cytochrome P450
(CYP) enzymes (for valdecoxib, this was the half maximal inhibitory
concentration (IC50) for CYP2C19), another invitro safety end point, can
be compared to Cmax in preclinical and clinical efficacy studies to assess
the risk of drugdrug interactions26,27. Before progression into FIH studies,
invivo safety data in animals (for valdecoxib, this was 4weeks of good
laboratory practice (GLP)-compliant toxicity studies in rats and dogs) is
generated and is compared to preclinical efficacy data (for valdecoxib,
this was in the rat adjuvant-induced arthritis model). In animal studies,
rows containing data for dose or exposure levels that do not lead to
toxicity (the so-called no observed adverse effect level (NOAEL)) are
indicated by green flags in the boxes on the left. These rows represent the
TI numbers. By contrast, rows containing data for dose or exposure levels
that lead to toxicity, the nature of the toxicities occurring beyond the
NOAEL and the associated dose or exposure levels are indicated by red
flags. Furthermore, exposure in animal safety studies should be compared
to human efficacious exposure to determine animal versus human safety
margins. Finally, the efficacious exposure in humans is compared to the
highest safe exposure tested in humans (for valdecoxib, this was in
healthy volunteers). As is the case for valdecoxib, often the therapeutic
exposure in humans needed to drive efficacy is considerably higher than
the efficacious exposure in preclinical pharmacodynamic models.
ED80, effective dose leading to 80% efficiency; qd, quaque die (once a day);
MAD, multiple ascending dose; SAD, single ascending dose.

In vitro Animal Human

pharmacodynamics pharmacodynamics pharmacodynamics
Ecacy COX2 COX2 Rat adjuvant-induced 10 mg qd (dosing
endpoints binding whole arthritis model (ED80 regimen for indication

blood assay of 0.09 mg per kg) osteoarthritis)
IC50 IC50 Cmax, AUC Cmax, AUC
Safety endpoints Total drug Free drug Total drug Free drug
COX1 binding IC50 28,000 1,358 67,917 273 13,659
In vitro
safety

COX1 whole blood assay IC50 91 212 10,624 43 2,137

CYP2C19 inhibition IC50 185 9,259 37 1,862
Dog 4 weeks Rat 4 weeks

Cmax 757 16 37 37 7 7
5 mg per kg per day: NOAEL
AUC 35 35 10 10
Animal safety

10 mg per kg per day: kidney, liver, adrenals as target Cmax 2,379 50 115 115 23 23
organs of toxicity AUC 82 82 23 23
Cmax 215 4 10 21 2 4
1 mg per kg per day: NOAEL
AUC 5 9 1 3
2.5 mg per kg per day: changes in urinary parameters, Cmax 575 12 28 56 6 11
focal degeneration of interstitium in renal papilla AUC 16 32 5 9
1 mg single dose: rst-in-human starting dose in Cmax 8 0.16 0.36 0.36 0.07 0.07
FIH SAD

healthy volunteers AUC 0.26 0.26 0.07 0.07

Human safety

400 mg single dose: maximum tolerated dose in Cmax 1,978 41 96 96 19 19

healthy volunteers AUC 102 102 29 29
Cmax 1,034 22 50 50 10 10
MAD

100 mg per day x 2 weeks: in healthy volunteers

AUC 57 57 16 16

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evaluation. For example, mAbs that bind to
a human protein (target) might not bind as
well, or at all, to the same target in the species
used to assess preclinical effficacy (for example,
mice) owing to species-related differences in
amino acid sequence of the target.
In such circumstances, animal proofof
concept efficacy studies might be conducted
with a surrogate mAb; conducted in an
animal model that either expresses the
human target or to which the human target
is administered; or not conducted at all.
In lieu of efficacy data from an animal model
of the disease, a strong scientific rationale for
the involvement of the target in the disease
and an invitro assessment that the mAb
can bind to the target can be sufficient to
progress to clinical trials.
However, mAbs that target a human
protein often, but not always, bind to the
corresponding target in the cynomolgus
monkey, which is the most commonly used
species used for safety assessment in the
development of biologics. In many cases a
true TI that is based on exposure in an effi-
cacy model cannot be calculated. Instead an
exposure margin can be calculated based
upon the relative exposures in the toxicology
studies and the clinical trials. Invitro data,
coupled with human exposure data (either
predicted or observed) can be used to create a
pharmacokineticpharmacodynamic model
from which a TI can be derived.
Classical off-target pharmacology (toxicity)
evaluation is not usually performed for bio-
therapeutics owing to their target specificity
and the lack of the appropriateness of many
of these assays for a large molecule that
has limited cellular penetration. Although
surrogate molecules (for example, an anti-
body that binds to the target in rodents)
might provide some useful information
in determining the role of the target in an
animal model of disease, the calculation of
a TI based on such data involves so many
assumptions and caveats that it may have
very limited, if any, value.
The presence of anti-drug antibodies
that sometimes arise as a consequence of the
administration of a biotherapeutic may lead
to an increased clearance of the biothera-
peutic (and consequently, decreased level of
exposure)32. If the level of exposure to the
active drug is markedly lower in an animal
or human subject that develops anti-drug
antibodies than the level of exposure to the
drug in other animals or human subjects
in the dose group, that subject should be
excluded from the calculation of the mean
exposure at that dose group in order to
provide a more reliable calculation ofaTI.
NATURE REVIEWS | DRUG DISCOVERY VOLUME 11 | O CTOBER 2012 | 759
PERSPECTIVES
Box 6 | The determination and interpretation of the therapeutic index
Here, we list key properties pertaining to pharmacology and safety that affect the determination
and interpretation of the therapeutic index. Properties that are marked with an asterisk mean that
a higher therapeutic index is necessary, whereas those marked with a double dagger mean that a
lower therapeutic index could be acceptable.
Pharmacology
Indication
Life-threatening versus manageable (less severe) condition*
Low standard of care in relation to high unmet medical need
Highly competitive landscape in indication or target, competitors ahead in the field*
Reliability of underlying preclinical pharmacodynamic (PD) model(s)
Invivo model versus invitro model only* (as with many biologics and antivirals)
Nature of PD model (induced disease model versus PD model only*, humanized model or
xenograft model*)
Definition of PD effect: efficacious dose in 90% of subjects (ED90) versus ED50*; tumour
regression versus tumour stasis*; modulation of invivo biomarker only* (for example, 90%
inhibition of target phosphorylation in tissue); proximal versus distal* target PD biomarker
High variability of magnitude of PD effect*
Low reproducibility of efficacious exposure in invivo PD model*
Low concordance between PD models (in case of multiple models used)*
Poor translatability of PD effect to humans*
Relative ontarget potency between preclinical models and humans
Variability of human pharmacology
Definition of human PD end point
High variability of magnitude of PD effect*
Low sustainability of PD effect (for example, tachyphylaxis or hysteresis)*
High variability of efficacious exposure in humans*
Safety
Indication
Good safety profile of current standard of care in indication*
Increased susceptibility of specific patient populations to toxicities of drug (for example,
those with impaired renal, hepatic or cardiac function, immunocompromised subjects,
paediatric populations)*
Toxicities limiting preclinical noobserved adverse effect level or clinical dose
Nature of toxicity and severity
For toxicities due to exaggerated pharmacology: PD effect possible to antagonize
(for example, warfarin versus factor Xa inhibitors)
Time course of occurrence (single dose* versus chronic dosing)
High incidence*
No or low reversibility*
Challenging monitorability at early stage*; for example, with biomarkers11
Limited translatability to humans (species-specific effect)
High consistency across species*
Ample experience with similar toxicity (in the same or other species or indication)
For ontarget toxicities: possibility to differentiate from ontarget efficacy solely based on
the dosing regimen
Steep exposure or toxicity curve*
Parameters modulating human safety
Acute versus long-term or chronic* intended clinical treatment
Variability of clinical exposure: high impact of drugdrug interactions due to comedication*;
genetic polymorphisms in metabolizing enzymes or transporters*; low or variable bioavailability*;
pronounced dietary effects* (food-effect studies); comorbidities affecting drug disposition*
Oncology indications. Many targeted oncol- pathway targeted (for example, hyperten-
ogy drugs exhibit ontarget toxicity (BOX3). sion and cardiovascular events as the most
This type of toxicity is often predictable prominent toxicities of both mAbs and
based on their mode of action and the tyrosine kinase inhibitors targeting vascular
2012 Macmillan Publishers Limited. All rights reserved
PERSPECTIVES
endothelial growth factor signalling33).
With regard to an evaluation of the TI and
associated decision-making, the same con-
siderations regarding clinical safety and
patient monitoring should be given to both
on- and off-target toxicities.
For oncology indications, the preclinical
and clinical TI are often <1, meaning that
toxicity is evident at subpharmacological
exposure. In oncology indications that have
limited therapeutic options, limited toxici-
ties that are amenable to early and reliable
monitoring may be ethically justified in order
to maximize pharmacological exposure and,
therefore, efficacy of the drug. However,
potential expansion of the indications for
which the drug could be used beyond can-
cer is feasible only for oncology drugs with
reasonable TIs (such as imatinib, which has
also been investigated for pulmonary arterial
hypertension34).
Drug candidates with low TIs. Progression
of drug candidates that have low TIs
through the development process for non-
life-threatening indications may be justified
to explore a new therapeutic mechanism
(so-called proofofmechanism studies).
These studies must use limited exposure
and exposure duration, and ideally use
sensitive biomarkers to monitor target
modulation.
The FDA defines narrow therapeutic
index (NTI) drug products, in the scope
of bioequivalence studies for generics, as
containing certain drug substances subject
to therapeutic drug concentration or phar-
macodynamic monitoring, and/or where
product labelling indicates a narrow thera-
peutic range designation35 (for example,
warfarin and digoxin). Typically, such NTI
drugs have less than a twofold difference in
the minimum toxic concentration and mini-
mum effective concentration in the blood,
and often require blood-level monitoring.
This demonstrates the potential practical
implications of NTI drugs for both patients
and health-care professionals.
FIH clinical trials. The main use of TIs is in
providing the basis for decisions about the
developability of drug candidates, not set-
ting the starting dose in FIH clinical trials.
Indeed, the FIH starting dose is typically
determined primarily by the dose levels at
which toxicity is seen in preclinical species
and the nature of this toxicity in terms of
severity, monitorability, reversibility and
translatability to humans. Furthermore,
the FIH starting dose is generally expected
to lead to subpharmacological exposure36
760 | O CTOBER 2012 | VOLUME 11 www.nature.com/reviews/drugdisc
(compare this to the minimal anticipated
biological effect level (MABEL) concept16).
Exploratory clinical trials paradigms
(for example, exploratory investigational new
drug (eIND) applications and exploratory
clinical trial applications (eCTAs)) provide a
strategy for rapid initial evaluation of investi-
gational drugs in humans. In contrast to
traditional INDs, exploratory clinical studies
typically are not intended to examine clini-
cal tolerability, and involve a small number
of human subjects at limited dose and expo-
sure duration. Early decision data derived
from such clinical studies may include
pharmacokinetics, pharmacodynamics and/
or biomarker-based translational medicine
end points.
Under exploratory FIH clinical trial
paradigms, an exposure cap is set based
on NOAELs in preclinical safety studies37.
Therefore, the TI may affect whether suf-
ficient exposure to achieve the desired
pharmacological effects can be reached
under such an exposure cap. The lower the
(preclinical) TI of a drug, the more limited
exploratory clinical trials are in terms of
clinical dose and exposure duration, and
potentially reaching pharmacologically active
exposure levels. In addition, there are situa-
tions in which exposure caps are increasingly
being applied to FIH studies even under full
(that is, conventional) IND and CTA para-
digms. This can either be decided voluntarily
by the applicant or be imposed by regulatory
authorities, depending on the nature of the
toxicities encountered in preclinical safety
studies and the riskbenefit ratio for the
underlying indication.
Toxicities that are difficult to monitor at
an early stage invivo and/or that are have
limited reversibility or are irreversible in
particular those that have a high likelihood
of being translatable from animals to humans
may call for an exposure cap. Frequently,
such an exposure cap is set at a fraction (for
example, one-tenth) of the NOAEL expo-
sure (usually total drug AUC) of the most
sensitive species. Lifting of an exposure cap
usually requires mechanistic investigations
to show either that the preclinical toxicity
of concern is not relevant to humans or that
it can be successfully managed in clinical
practice. Not being able to lift exposure caps
frequently prevents drug candidates from
being further developed.
Conclusions
The TI is an important parameter for assess-
ing the safety profile of drug candidates
and for aiding associated decision-making.
The calculated TI (or safety margin) must be
2012 Macmillan Publishers Limited. All rights reserved
assessed on the basis of the indication, the
nature of the toxicities observed in animals
and in humans, the relevance of the animal
models, the duration of intended clinical
treatment, the inter- and intra-individual
variability in exposure and the riskbenefit
ratio for the envisaged indication. The TI
calculated during the development of a drug
should only be used in riskbenefit analyses
for those toxicities that have been actually
observed in animals and/or humans, and is
not applicable to rare, but often severe, tox-
icities that can only be detected in very large
patient populations (that is, post-marketing).
All TI calculations that are feasible at
a particular development stage should be
performed as soon as the data are available.
Patrick Y.Muller and Mark N.Milton are at
the Novartis Institutes for BioMedical Research,
250 Massachusetts Avenue, Cambridge,
Massachusetts 02139, USA.
Correspondence to P.Y.M.
email: patrick.mueller@novartis.com
doi:10.1038/nrd3801
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Acknowledgements
The authors would like to thank B. Schmouder, L. Urban, P.
Bouchard, P. Hoffmann, P. Heining and V. Jarugula for their
valuable input and in-depth interdisciplinary discussions on
this topic.
Competing interests statement
The authors declare no competing financial interests.
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