Falling productivity is perhaps the greatest challenge facing the pharmaceutical industry today. Although vast numbers of compounds can be evaluated by drug discovery groups as a result of high-throughput target-based drug design, numerous indicators point to decreased productivity in the pharmaceutical industry despite increased spending1–5. Amid an earlier period of reports of low productivity, high-throughput screening technologies were adopted by the pharmaceutical industry in the 1980s in an effort to increase the number of lead molecules entering into the discovery/development pipeline. Rather than delivering an increase in successful clinical development candidates, the immediate effect was an increase in failures due to poor pharmacokinetics and bioavailability, resulting largely from suboptimal physical and chemical properties6,7. Earlier evaluation of pharmacokinetics and drug metabolism along with increasing throughput and sensitivity of liquid chroma-tography mass spectrometry techniques8 throughout the 1990s reduced attrition due to poor pharmacokinetics, as evidenced in the ‘reasons for attrition’ data reported by Kola and Landis7. However, together with improved systemic exposures came increased observations of development-limiting toxicity. Today, toxicity is a leading cause of attrition at all stages of drug development. As a result, many companies developing small-molecule therapeutics have adopted a strategy that includes the

earlier incorporation of preclinical safety assessment before advancement into regulated preclinical studies9.

Much focus in the industry has been placed on attempts to identify human-specific toxicities, and toxicities for which common preclinical models are poorly predictive. Although such approaches are sure to have an impact in the long term, the earlier identification of dose-limiting and development-limiting preclinical toxicities that are known to be predictive of human toxici-ties10,11 may have a more profound impact on the overall compound attrition, cost and time to first-in-human (FIH) clinical trials. Importantly, most of the safety-related attrition (~70%) occurs preclinically, suggesting that approaches to identify ‘predictable’ preclinical safety liabilities earlier in the process could lead to the design and/or selection of better candidates that have increased probabilities of becoming marketed drugs. It has been estimated that the average cost associated with the discov-ery and preclinical evaluation of a single drug candidate ranges between US$28 million and $38 million12; and that a 10% improvement in predicting failure before the initiation of clinical trials could save $100 million in development costs per drug13,14. Additionally, many dis-covery toxicology efforts have focused on the application of new technologies; although such technologies when separated from more traditional toxicological principles, may do little to advance compounds into development.

*Department of Drug Metabolism and Pharmacokinetics, Lexicon Pharmaceuticals Inc., 8800 Technology Forest Place, The Woodlands, Texas 77381, USA.‡Seventh Wave Laboratories, Suite 209, 743 Spirit 40 Park Drive, Chesterfield, Missouri 63005, USA.§Drug Safety Research and Development, Pfizer Inc., 700 Chesterfield Parkway West T1A, Chesterfield, Missouri 63017, USA.Correspondence to J.A.K. e-mail: jkramer@lexpharma.comdoi:10.1038/nrd2378

Published online 13 July 2007

Development-limiting toxicityA toxicity that is either

irreversible or unmonitorable,

has an unacceptable safety

margin or therapeutic index,

or would negatively affect sales,

patient compliance, competitive

advantage or marketability.

The application of discovery toxicology and pathology towards the design of safer pharmaceutical lead candidatesJeffrey A. Kramer*, John E. Sagartz‡ and Dale L. Morris§

Abstract | Toxicity is a leading cause of attrition at all stages of the drug development

process. The majority of safety-related attrition occurs preclinically, suggesting that

approaches to identify ‘predictable’ preclinical safety liabilities earlier in the drug

development process could lead to the design and/or selection of better drug candidates

that have increased probabilities of becoming marketed drugs. In this Review, we discuss

how the early application of preclinical safety assessment — both new molecular

technologies as well as more established approaches such as standard repeat-dose

rodent toxicology studies — can identify predictable safety issues earlier in the testing

paradigm. The earlier identification of dose-limiting toxicities will provide chemists and

toxicologists the opportunity to characterize the dose-limiting toxicities, determine

structure–toxicity relationships and minimize or circumvent adverse safety liabilities.

A G U I D E TO D R U G D I S C OV E RY

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Structure–toxicity relationship(STR). An assessment of

the structural features

that determine the

occurence and/or severity

of a particular toxicity.

Safety marginA preclinical indication of

the safety of a compound

that represents the ratio of

a maximum safe exposure

divided by an efficacious

exposure.

Dose-limiting toxicity Any toxicity that limits the

ability to continue escalating

the dose.

Signal generationA study intended to identify

the dose-limiting safety liability

of a compound or drug target.

Ames assayA bacterial ‘reverse mutation’

mutagenicity assay that is

designed to identify frame-

shift and base-pair-substitution

point mutations. A good

laboratory practice Ames

assay is required before

first-in-human studies.

Micronucleus assayAn assay that identifies

chromosomal aberrations,

visible as an extra staining

material in metaphase/

anaphase cells. Both an in vitro

and in vivo chromosomal

aberration assay are required

before first-in-human studies.

MutagenicityThis is DNA damage that is

considered to be predictive

of carcinogenicity.

ClastogenicityThis is chromosome breakage,

a form of mutagenesis that is

considered to be predictive of

carcinogenicity.

In this Review, we discuss how the early application of preclinical safety assessment, using both new technolo-gies as well as more established approaches, can identify predictable safety issues earlier in the testing paradigm; providing chemists and toxicologists the opportunity to determine structure–toxicity relationships (STRs) and avoid potentially development-limiting toxicity. This approach allows for the advancement of lead candidates with a better chance of successfully reaching FIH trials, while maximizing safety margins for the dose-limiting toxicities that are likely to be observed in long-term repeat-dose good laboratory practice (GLP) toxicity studies.

The early application of toxicology

A typical testing scheme for a small-molecule therapeutic (outlined in FIG. 1) begins with large numbers of com-pounds and high-throughput assays. As the number of viable leads is reduced, incrementally more predictive but lower throughput assays identify those leads with the most drug-like properties and optimal in vitro and in vivo efficacy. Confirmed hits identified in high-throughput screens are evaluated for potency, selectivity, ADME (absorption, distribution, metabolism and excretion), physical and chemical properties, and activity in relevant animal models (FIG. 1). This testing paradigm typically delivers drug-like compounds that have promising pharmacokinetic parameters and efficacy in preclinical models within a 1–2-year cycle time. Compounds that successfully meet preclinical efficacy, ADME, pharma-cokinetics and safety criteria are nominated as candidates for formal development. Historically, the move from dis-covery to development consisted of a discreet hand-off from the ‘discovery’ organization to the ‘development’ organization, and little preclinical safety assessment was performed on lead molecules beyond a few basic in vitro toxicity assays. As toxicity has continued to be a primary cause for compound attrition and long development cycle times, companies in the past 5–10 years have increasingly integrated safety assessment principles into earlier phases of the drug discovery process.

The early application of preclinical safety assessment includes both high and low-to-intermediate throughput in vitro assays as well as in vivo toxicity studies. In vitro toxicology assays can be divided on the basis of timing and purpose of the application into prospective assays and retrospective assays. Prospective assays predict devel-opment-limiting toxicities that may be missed in short duration in vivo toxicology studies, and retrospective assays are used for issue management and compound prioritization after target organ toxicities are identified in early in vivo signal-generation studies. Optimally applied, this approach is not simply an attempt to move attrition to an earlier stage of the process, but a strategy and an opportunity to enhance small-molecule drug discovery by incorporating an assessment of STRs and toxicity issue management into drug design. In this Review we discuss prospective toxicology assays, in vivo signal generation and discovery pathology, then describe how information gleaned from these investigations can be used in toxi-cology issue management. Many of the assays discussed will not apply to biotherapeutic agents, as the regulatory

preclinical safety requirements for such agents are sig-nificantly different, and toxicities observed with biologics are largely limited to immunological or adverse primary pharmacological effects15,16.

Prospective in vitro toxicology assays

Prospective in vitro toxicology assays are those assays that are conducted before the first in vivo toxicology studies, and attempt to predict toxicities that are devel-opment-limiting (that is, toxicities for which there is no acceptable margin of safety for a given indication) or those that may be commonly missed in early in vivo toxicity assays (that is, toxicities for which there is no histo pathological correlate). These include assays for gen-eral or cell-type-specific cytotoxicity, genotoxicity, hERG (human ether-a-go-go-related; also known as KCNH2) channel block, drug–drug interactions and metabolite-mediated toxicity. Of these, cytotoxicity assays are often among the earliest toxicity assays to be conducted. The predictive ability of in vitro cytotoxicity assays for in vivo target organ toxicity is often questionable at times owing to the limitations of in vitro models (for example, lack of metabolism, differences in sensitivity of cell types, culture conditions and the context of the ultimate target tissue in vivo). However, in vitro cytotox-icity assays can be valuable for interpreting the results of in vitro safety and efficacy assays. Results for many of the in vitro assays detailed below may be confounded by cytotoxicity; indeed the Ames assay and micronucleus

assay include an assessment of cytotoxicity that is critical for the interpretation of the assay results.

Genetic toxicology. Genetic toxicity assays are typically conducted before in vivo toxicity assays and are often associated with a binary outcome (that is, positive or neg-ative), which is useful for decision making. Historically this has included a non-GLP version of the Ames assay for mutagenicity17–19 and an in vitro chromosomal aberra-tion assay such as the micronucleus assay20. These assays identify mechanisms of DNA interaction or damage that predict mutagenicity and therefore potential carcino-genicity21,22, an end point that may not be identified in short-term in vivo toxicity studies typically run before FIH studies. Additionally, there may be no acceptable safety margin for genetic toxicity when seeking drugs for many therapeutic indications. Although these assays have remained essentially unchanged in the decades since they were developed, many of the newer (and in the case of ‘-omics’, more costly) technologies have yet to demonstrate sufficient value to warrant replacing the ‘tried and true’ assays, and so the regulatory agencies universally require these standard assays before FIH studies. However, these standard genetic toxicity assays do not readily lend themselves to high-throughput applications, and there are several higher-throughput alternatives for mutagenicity23–27 and clastogenicity28–32 assessment. Although not intended as regulated studies supporting compound development, these assays can be used to trigger the earlier application of the more defini-tive but lower throughput assays, and they are ideal for building an understanding of STRs. For example,

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High-throughput screening,IC50 determination, hit triage

Selectivity assays, in vitro efficacy assays,Tier I ADME/physical chemistry assays

In vivo efficacy assays (preclinical POC),Tier II ADME/physical chemistry assays

Second species PK, PK/PD modelling, salt-form selection, crystal-form assessment

GLP toxicology studies: genetic toxicology,safety pharmacology, in vivo toxicology in two species

Safety and tolerability in normal healthy volunteers

Safety and tolerability in patients, early clinical POP

Definitive clinical POP

Phase I

Phase II

Phase III

Clin

ical

dev

elop

men

t

High-throughput screening1,000s of compounds

Hit to lead100s of compounds

Lead optimizationDozens of compounds

Candidate seeking1–3 compounds

Preclinicaldevelopment

Preclinicalproof-of-principle

Freedom to operateThe ability to synthesize new

molecular entities in a

chemical space that has not

been previously described in

relevant existing patents.

Patch-clamp assayAn assay that uses a

microelectrode to study

the activity of ion channels

in single cells.

Reactive metaboliteA chemically reactive

metabolite that binds

covalently to cellular proteins.

Albertini et al.33 used a novel DNA-unfolding assay to screen a mutagenicity liability out of a series of 5-hydroxy-tryptamine (serotonin) 5-HT2C receptor antagonists. Additionally, toxicogenomic approaches have been used to discriminate indirect from direct acting genotoxins34,35.

Safety pharmacology. The safety pharmacology studies performed in development include a battery of in vivo and ex vivo assays. Before development however, safety pharmacology assays are typically limited to an assessment of hERG binding and/or hERG blockade. Blocking the hERG potassium channel may be predic-tive for QT interval prolongation and ultimately for the potential to cause Torsades de Pointes, a rare but potentially life threatening drug-induced ventricular tachyarrhythmia36. Although there has been some speculation regarding the application of these assays for drug-induced arrhythmias in humans37, numerous

compounds have been withdrawn from the market because of arrhythmogenic activity38, and the risks of advancing a pro-arrhythmogenic compound are high. Assays that are conducted during the discovery phase are usually either patch-clamp assays that assess hERG channel function in transfected cells or higher through-put hERG binding assays that measure the ability of a test compound to compete with a radiolabelled hERG-binding control compound39,40. Matasi et al.41 used a hERG-inhibition assay to screen this liability out of series of adenosine A2A receptor antagonists. Similarly, a hERG binding assay has been used to identify a phos-phodiesterase 4 (PDE4) inhibitor with a large safety margin relative to QT interval prolongation42.

Drug–drug interactions. Although not often a dose-limiting toxicity in themselves, the potential for other pharma ceutical or dietary agents to affect the metabolism, clearance or safety of a novel drug candidate can be a significant liability. Assays to identify the risk of drug–drug interactions, such as cytochrome P450 inhibition and induction, and P-glycoprotein (P-gp; also known as ABCB1) interac-tion assays, are an integral part of preclinical ADME and safety assessment43,44. Such assays are used to identify the potential for drug–drug interactions early in the testing funnel, and once issues are identified these assays can also be used to screen out such liabilities. In vitro P450-inhibition assays have been used to screen out drug–drug interaction liabilities in programmes seeking sodium-channel blockers45, dopamine D3 receptor antagonists46 and p38 kinase inhibitors47. Wang et al.48 have used a P-gp assay to identify compounds that modulate multidrug-resistance-associated protein 1 (MRP1), a transporter that is structurally and evolutionarily related to P-gp, while sparing P-gp itself.

Metabolite-mediated toxicity. Metabolite-mediated toxicity is an emerging and increasingly important issue for the pharmaceutical industry49. The forma-tion of reactive metabolites or electrophilic metabolites is not uncommon in vivo, and may result in geno toxicity, target organ toxicity and possibly idiosyncratic toxicity. Vignati et al.50 have reported the use of standard cyto-toxicity assay end points together with a xeno biotic metabolism component to identify compounds that may form toxic metabolites, an approach that may improve the predictivity for some in vivo toxicities. Although in vitro genetic toxicity and in vivo toxicology studies can potentially identify many metabolite-mediated toxicities, current hypotheses implicate metabolic activation as an obligate early step in the aetiology of idiosyncratic (for example, liver, skin) adverse drug reactions (ADRs)51,52. Glutathione-binding assays are being incorporated into testing schemes to assess the risk of reactive metabolite formation using human drug-metabolism systems53. Interpretation of the output from these assays is con-founded by the fact that many successfully marketed drugs are positive in glutathione-binding assays, including acetaminophen (paracetamol) and raloxifene (Evista; Eli Lilly)54. There fore, caution must be exercised in depri-oritizing a compound based on a positive result, so that

Figure 1 | The discovery testing scheme. A typical testing scheme for an orally delivered

small-molecule drug discovery programme is shown. The primary goal of the high-

throughput screening stage is to identify chemical hits from which lead series may be

derived. Confirmed hits are then divided into broad chemical series and each series is

evaluated with respect to potency, physical and chemical properties (for example, the

rule of five164), predicted ease of synthesis, cost of goods, scalability, target selectivity,

structural alerts, freedom to operate and the ability to create and protect intellectual

property. The hit to lead stage represents an evaluation of those series that survived

this triage process to identify potential liabilities associated with each series. Focused

libraries may be generated to build a structure–activity relationship (SAR) in target

binding and cell-based target modulation assays. Potent and selective leads are then

evaluated in early ADME (absorption, distribution, metabolism and excretion) assays to

measure properties such as metabolic stability, plasma-protein binding, permeability,

cLogP and solubility. Upon selecting the most promising chemical series, efforts are

made in the lead optimization stage to achieve proof-of-principle in a widely accepted

preclinical model of disease, while optimizing drug properties with additional ADME

assays. In the candidate-seeking stage, safety, ADME and formulation liabilities are

addressed to identify a candidate for formal preclinical development. Note that

toxicology assays have been left out of this figure, their application by stage is discussed

in the main text and in FIG. 3. PK/PD, pharmacokinetic/pharmacodynamic; POC, proof-

of-concept; POP, proof-of-principle.

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Electrophilic metaboliteA reactive metabolite

characterized by an affinity to

form covalent modifications

with endogenous nucleophiles.

Idiosyncratic toxicityA toxicity that occurs rarely

(with a frequency that is

typically less than 1 in 1000)

and unpredictably among the

population.

Therapeutic indexA clinical indication of the

safety of a compound

determined by dividing the

exposure at which dose-limiting

clinical adverse effects are first

observed by the exposure at

which efficacy is achieved.

NOAELNo observable adverse effect

level is the highest exposure

at which no adverse effects

are observed.

Exaggerated pharmacologyToxicity that is due to

excessive modulation of the

activity of the primary

pharmacological target

beyond the point necessary

for efficacy.

the development of a useful and potentially profitable compound won’t be unnecessarily halted. Researchers at Merck53 have proposed go/no-go criteria for individual compounds based on levels of covalent binding of reac-tive metabolites; however, such evaluations require the use of a radiolabelled compound, which is not available in the discovery or early development phases at many organizations. Risk assessment for reactive intermediates is a complex issue and beyond the scope of this Review, although many companies are increasingly making deci-sions on compound advancement based on the results of such findings55.

Early knowledge of the toxicities described above will enable project teams to build customized testing schemes so that the STRs are understood and safety liabilities can be designed out of leads to deliver superior early development candidates. Although there are numerous in vitro assays available that predict various target organ toxicities, such assays are best applied retrospectively after the target organs have been identified in vivo. It is a general principle of toxicology that all compounds are toxic at some dose or exposure level, and an adequate safety margin defined in vivo is key to advancing a compound. If applied prospectively, attempts to predict target organ toxicity in vitro in the absence of a consid-eration of safety margin run the risk of deprioritizing promising leads. As such, target organ toxicity assays are best used for prioritizing compounds within a limited chemical space once the target organ has been shown to be a concern in vivo, whereas prospective assays ought to focus on those that predict development-limiting toxici-ties (for example, genetic toxicity) and/or toxicities for which there is no in vivo histopathological correlate (for example, hERG block, drug–drug interactions).

In vivo signal generation

Among the most common causes of preclinical toxicity-related attrition is target organ toxicity, which, as noted previously, can be difficult to predict accurately in vitro. Even for target organs such as the liver, in which primary cells and tissue slices are available, in vitro assays often can not reliably predict safety margins, as there is no efficacy end point and pharmacokinetic parameters are not faithfully reproduced in vitro. The earliest in vivo toxicology studies serve therefore as signal generation studies, the primary aim of which is to identify dose-limiting target organ toxicity or a lack of detectable toxicity at a limit dose, which can assist in estimating compound requirements for the conduct of subsequent preclinical safety studies. As all compounds are toxic at some dose or exposure level, a key question is whether the dose-limiting toxicity is potentially limiting for further development. Development-limiting toxicity is difficult to define and depends on factors such as the safety margin (therapeutic

index in the clinic); the nature of the toxicity (that is, reversibility, availability of monitorable biomarkers); the route of administration, duration of treatment and intended therapeutic indication being sought (that is, risk to benefit); and the potential for occurrence in the human population at large. There are numerous definitions

for a preclinical safety margin, but one that is com-monly used in the pharmaceutical industry is a ratio of the NOAEL (no observable adverse effect level) divided by the predicted human efficacious exposure level or exposure at the maximum anticipated human dose. As a predicted human efficacious level is difficult to determine prior to human clinical trials or extensive pharmaco-kinetic/pharmacodynamic modelling, exposure at a fully efficacious dose in a widely accepted preclinical model of disease is often used for the denominator. An acceptable preclinical safety margin is key to selecting the most viable development candidates, and this can be best determined by repeat-dose in vivo toxicology studies. Although the therapeutic indication being sought can determine in part what safety margin is acceptable, the dose-limiting toxicity identified preclinically in most cases still must be reversible and monitorable. For example, the ability to manage vasculitis as a dose-limiting toxicity depends on the mechanism and how this relates to the ability to monitor the finding clinically. The dose-limiting adverse event for potent vasodilators such as systemically administered minoxidil is due to exaggerated pharmacology (profound lowering of blood pressure) resulting in haemodynamic-mediated vasculitis56–58. If the vasculitis can be shown to be due to haemodynamic effects, a safety margin of ~10 times may be deemed acceptable, as a suit-able safety biomarker (that is, blood pressure) is in place to monitor the effect clinically59. By contrast, if the finding is not related to haemodynamics there is essentially no acceptable margin of safety.

The early generation of toxicity signals, such as the identification of development-limiting toxicity in a non-regulated (that is, non-GLP) setting before initiation of more costly and time and resource intensive regulatory toxicology studies, is a primary driving force behind the early application of in vivo safety studies. However, this is not the only advantage of running in vivo toxicology studies during the drug discovery process. If more than one compound in a project’s lead chemical series exhib-its the same toxicity, mechanistic efforts are initiated to understand the nature of the toxicity in an attempt to avoid it in future lead candidates. Additional in vivo studies can be performed to adequately characterize the toxicity. This might include studies with active but structurally unrelated compounds, studies with struc-turally related but inactive compounds (such as inac-tive enantiomers), studies in knockout mice in which the pharmacological target is not expressed or studies in a non-rodent species. For example, studies using fibrates in peroxisome proliferator-activated receptor-α (Pparα)-knockout mice demonstrated an absence of the pleiotropic effects common to peroxisome proliferators and a lack of induction of several Pparα target genes, confirming the primary pharmacological nature of these characteristic peroxisome-proliferator-mediated effects60. Even when the dose-limiting toxicity is not develop-ment-limiting, mechanistic information regarding the safety of the compound and/or the target are invalu-able in managing the toxicity, providing guidance to discovery chemistry for designing out the toxicity and improving safety margins in back-up programmes.

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Primary pharmacology Also referred to as target-

based toxicity, this is toxicity

that is caused by a

modulation of the primary

pharmacological target.

Secondary pharmacologyToxicity caused by a lack of

specificity for the primary target

resulting in a molecule crossing

over onto and modulating the

activity of a secondary, often

structurally and/or

evolutionarily related target.

Chemically mediated toxicityA toxicity that is due to

the physical and chemical

properties of a particular

chemical or an entire

chemical class.

Black box warningThe most serious safety

warning required on a

pharmaceutical label, indicative

of a significant risk of a serious

or even life-threatening adverse

drug reaction.

Park et al.61 have classified clinical ADRs into four categories based on the nature of the reaction. These are type A augmented ADRs, which result from the pharma-cology of the drug target; type B idiosyncratic reactions, which are unpredictable from the pharmacology of the drug target, show differences in individual susceptibility and lack dose-dependency; type C chemically mediated ADRs that are predictable from the chemistry of the drug; and type D delayed ADRs (such as carcinogenicity) for which there may be no early indication of risk in short-term studies. Edwards and Aronson62 have described a similar classification system that includes six categories related to dose and time relatedness of clinical ADRs. Preclinical toxicities can similarly be divided into three broad classes on the basis of how they are best managed and/or minimized. These are primary pharmacology, secondary pharmacology and chemically mediated toxicity. Similar to type A clinical ADRs, primary pharmacology or target-mediated toxicity represents toxicity that results from the modulation of the drug target. Secondary phar-macology is the result of an interaction with a second-ary target, often one that is structurally, functionally or evolutionarily related to the primary therapeutic target. Similar to type A ADRs, secondary pharmacology can be predicted from the established pharmacology of the secondary target, provided that the identity of that target is known. Chemically mediated toxicity is independent of the pharmacological target(s) with which a compound interacts, and is similar to type C ADRs described by Park et al.61 Type B idiosyncratic clinical ADRs can not be reli-ably predicted preclinically at this time, although there have been hypotheses implicating reactive metabolites (see discussion of metabolism-mediated toxicity, above) and a role for inflammation and oxidative stress63–65. Efforts to apply new technologies towards the predic-tion of idiosyncratic toxicity may be doomed to failure, as the resulting assays would be virtually impossible to validate and could only serve to generate untestable safety concerns around otherwise promising drug can-didates. If applied, such assays should be positioned to reduce risk, rather than as an attempt to screen out an actual safety liability. As with type B clinical ADRs, many type D delayed ADRs can not be definitively predicted in short-term in vivo studies performed in discovery or early (pre-investigational new drug) development, although efforts have been initiated to define criteria by which some delayed toxicities may be predicted in early preclinical studies66,67. An understanding of the nature of the dose-limiting in vivo toxicity (that is, primary pharmacology, secondary pharmacology or chemically mediated toxicity) is instrumental in efforts to manage the finding, as detailed below.

Primary pharmacology. Adverse primary pharmacology results directly from modulation of the drug target rather than from the physical and chemical properties of the compound (for example, pH, planar aromatic moiety and amphiphilicity). One well-characterized example of adverse primary pharmacology has been observed with angiotensin converting enzyme (ACE) inhibitors. Developmental effects have been observed in humans

and preclinical species that have been dosed with ACE inhibitors during a key period in prenatal develop-ment68,69. Although there are broad chemical similarities among the ACE inhibitors (FIG. 2a), their structures are disparate enough to suggest that the developmental effects are mediated by ACE inhibition, and are not caused by a particular chemical series. Indeed, Ace-knockout mice demonstrate comparable developmental abnormalities70. Although ACE inhibitors can be safely dosed in children and adults, the drug labelling carries a black box warning against its use during the second and third trimesters of pregnancy, and so the patient population has been limited accordingly. Not all adverse pharmacology translates into black box warnings and limited patient populations. In many cases adverse pharmacology is observed only at supra-efficacious exposures. For example, high doses of essentially all antithrombotic agents can result in haem-orrhage and increased bleeding time both clinically and preclinically regardless of the structure of the antithrom-botic agent and even the molecular target71–76. Some such examples of exaggerated pharmacology can be dealt with clinically through careful attention to dosage.

Secondary pharmacology. Toxicities may also be the result of secondary pharmacology, or effects medi-ated by targets other than the primary efficacy target. For example, fenfluramine was taken off the market in 1997 owing to heart-valve disease and pulmonary hypertension77. Fenfluramine was marketed as an appe-tite suppressant for weight control and its efficacy was attributed to the serotonin-receptor agonist activity of its desmethyl metabolite, norfenfluramine78. It has since been suggested that agonism of the serotonin 2C receptor is sufficient for activity, whereas the valvular heart disease and pulmonary hypertension are believed to be mediated by interaction with the serotonin type 2B receptor79,80. Presently, several companies are developing selective serotonin 2C receptor agonists as anti-obesity drugs81. In an attempt to screen out secondary phar-macology, most discovery efforts include selectivity assessment against one or more secondary targets that are closely related to the primary target (FIG. 1). However, secondary pharmacology can be difficult to distinguish from primary pharmacology. For example, for decades the potential for structurally diverse non-steroidal anti-inflammatory drugs (NSAIDs; FIG. 2b) to cause gastrointestinal ulceration was thought to represent primary pharmacology. It has since been demonstrated that the anti-inflammatory efficacy is mediated by cyclooxygenase 2 (COX2) inhibition, whereas the gastrointestinal effects are mediated largely by the inhibition of COX1. Inhibitors that are selective for COX2 (FIG. 2c) maintain the primary pharmacology (anti-inflammatory activity), while sparing many of the gastrointestinal side effects (secondary pharmacology) that are associated with COX1 inhibition82,83.

Chemically mediated toxicity. The third broad category of toxicity that may be identified in in vivo signal-generation studies is chemically mediated toxicity, such as oxidative stress, phospholipidosis or haemolysis. Chemical toxicity

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NH

OH

COOH

HS

N

NH

O

HCOOH

EtOOC

H

H

H

H

COOH

NH

Cl

Cl

COOH

N OO

Cl

COOHO

OH

O

O

HN

O OH

O

COOH

N SN

NH

O

HO

OO

N NCF3

SO

O

H2N

O

O

SO

O

ON

SH2N O

O

O

O

I

I

ON

N

Cl

NHN

F3C

O NH

N

N

Ibuprofen

Lotensin

AltaceCaptopril Aceon

Accupril

Valdecoxib

Imipramine Chloroquine

Fluoxetine Azithromycin Amiodarone

Rofecoxib Celecoxib

Mefenamic acid

Naproxen

Piroxicam

Aspirin

Diclofinac

Indomethacin

b

a

c

d

NN

O H

COOEtCOOH

HCl

H

HN

COOH

O

HN C3H7

COOEt

H2N C(CH3)3

HCl

NO

N

H

HH

O

HO

O

O

O

O

N

O

O

HO

HOOH

O

O

OH

O

OH

N

Figure 2 | Toxicity classification. a | Angiotensin converting enzyme (ACE) inhibitors share a common developmental

toxicology liability independent of their chemical structure. Although there are some shared chemical features in these

examples of ACE inhibitors, Ace-knockout mice show similar developmental pathologies, which demonstrates the

primary pharmacological nature of the toxicity. b | The non-steroidal anti-inflammatory drugs (NSAIDs) represent a

class of compounds that share common secondary pharmacological liabilities despite their structural diversity. Anti-

inflammatory efficacy is mediated by the inhibition of cyclooxygenase 2 (COX2), whereas gastrointestinal ulceration is

largely mediated by COX1 inhibition. c | The COX2-specific inhibitors have a reduced risk for peptic ulceration relative

to equipotent levels of non-selective NSAIDs, demonstrating that the gastrointestinal side effects of non-selective

NSAIDs are largely a secondary pharmacological effect. d | Chemically and pharmacologically diverse compounds may

share common toxicological liabilities as a result of a particular structural motif or physical and chemical property.

Many cationic amphipathic drugs cause phospholipidosis despite a broad range of unrelated primary pharmacological

activities. The examples shown here represent selective serotonin reuptake inhibitors for depression (imipramine and

fluoxetine), an antimalarial (chloroquine), an antibiotic (azithromycin) and an anti-arrhythmic (amiodarone), yet all are

reported to induce phospholipidosis.

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is typically mediated through direct chemical rather than target-mediated effects. For example, phospholipidosis can occur when amphipathic compounds (compounds that have one charged or polar end and one hydrophobic end) incorporate into cell membranes and interfere with phospholipid metabolism84. Phospholipidosis has been observed in vivo with amphipathic amines that have varied pharmacology and are otherwise structurally distinct85 (FIG. 2d). Last, chemically mediated toxicity may be a function of specific functional groups or a liability associated with an entire chemical series. For example, terminal acetylene functionalities are prone to cause mechanism-based inhibition of metabolic enzymes86–88 regardless of the template to which they are attached, whereas polycyclic aromatic hydrocarbons may be prone to DNA intercalation89 and mutagenicity regardless of the chemical moieties attached to the central planar template. When compound-mediated toxicity is identified, it can be designed out of a chemical series using approaches similar to those described for structure–activity relationships90.

It is important to note that the purpose of the pivotal GLP toxicology studies run in early development is not to show that a compound is not toxic, but to identify the dose-limiting toxicity. Similarly, the early applica-tion of in vivo toxicology studies ought to identify safety liabilities earlier, at a time when mechanistic work can be performed to characterize and address the findings, and when synthetic chemistry efforts can be fully engaged to screen out safety liabilities. We have found that a well-executed short-term (1 week) repeat-dose in vivo toxicity study will predict most of the dose-limiting target organ toxicities observed in pivotal 4-week rodent studies. Although some target organs may be missed using this approach — such as musculoskeletal toxicities observed with broad-spectrum matrix metalloproteinase (MMP) inhibitors, which may take 10–14 days to develop in vivo91 — dose-limiting target organ toxicities for most com-pounds (for example, clofibrate, phenobarbital and gen-tamicin) can be identified in 5-day to 7-day studies92–94. The costs associated with routinely running longer in vivo studies before development may not make up for the limited risk of missing an occasional target organ toxicity prior to development. Once dose-limiting toxicities have been identified, efforts can be initiated to characterize the findings, determine the safety margin and assess whether the findings are development limiting. As described in the toxicology issue management section, managing dose-limiting toxicity depends in part on the nature of the finding, that is, whether it is chemically mediated or due to primary or secondary pharmacology.

Discovery pathology

The practice of preclinical safety evaluation earlier in the drug discovery process has resulted in a shift away from toxicological pathology (interpreting results primarily from GLP safety studies) to experimental pathology in which attempts are made to determine the consequences of intended or unintended pharmacologi-cal activity9. In addition, a closer interaction between pathology, pharmacology and chemistry has enabled

discovery pathology to inform the design and interpre-tation of pharmacology models in which there may be anatomical, haematological or biochemical end points indicating efficacy95–98 (FIG. 3).

Evaluation of safety considerations that are associated with intended or unintended pharmacology is greatly enhanced by the use of genetically modified animals in the drug discovery process. Targeted and contextual knockout mouse models are frequently available or are routinely created to determine the consequences of complete abolition of the function of potential phar-macological targets. Evaluation of phenotypic altera-tions that are associated with genetic manipulation of mouse models ideally includes a careful comparison of end points used in traditional safety evaluation between genetically modified animals and their wild-type controls. These end points should include the evaluation of behaviour, body and organ weights, clinical pathology parameters (haematology, clinical chemistry), and full histopathology from a small number of animals of each gender. Results of these studies may not be completely applicable to the interpretation of data from subsequent safety studies for several reasons, includ-ing the potential impact of pharmacokinetics on the manifestations of toxicity, the potential for alternative signalling or adaptive pathways to accommodate for normal phenotypes in genetically modified animals and the potential for absolute loss-of-target gene function to be incompatible with life or normal reproduction and development. However, combining phenotypic data obtained from genetically modified models with target organ toxicity observations from early safety studies can contribute significantly to an understanding of the phar-macological basis of safety findings, as described earlier with Pparα- and Ace-knockout mice60,70.

Several special microscopy and pathology approaches have shown promising application to both preclinical pharmacology and preclinical safety evaluation. Quan - titative microscopy and imaging techniques (for example, morphometry, stereology, magnetic resonance imaging) have been used to quantify efficacy in a number of animal models99,100. For example, evaluation of both the absolute volume and composition of atherosclerotic plaques in various animal models is frequently used to relate changes in dyslipidaemic biomarkers with out-come of disease101,102. Similarly, quantitative evaluation of histopathological changes in the joints of animal models of arthritis can be used to characterize the contributions of different signalling pathways to the development of disease95, and can confirm the therapeutic application of new targets.

Use of comparative molecular pathology techniques in target validation is also enhanced by access to appro-priate human clinical samples. In situ distribution studies of molecular targets (for example, in situ hybridization, immunohistochemistry) within tissue and cell types in human disease tissues provide supportive information with regards to the relevance of a given pathway to the pathogenesis of the natural disease. Similar techniques can be applied in the determination of pharmacology (primary or secondary) to an understanding of drug

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Discovery pathologyModel development,target validation

Prospective toxicity screensCytotoxicity, genetic toxicityhERG binding, drug–drug interactions, metabolism-mediated toxicity

Retrospective toxicity screensCharacterization, mechanisticevaluation, modelling and screeningfor STRs (for example, phospholipidosis, apoptosis, mitochondrial toxicity)

In vivo signal generationIn vivo signal generation and dose-range-finding studies

Targetidentification

PrimaryHTS

Parallelmedicinalchemistry

Optimalpotency/selectivity

Efficacy inpivotal in vivomodels

Preclinical proof-of-principle

Screendevelopment andhigh-throughputscreening

Hit to lead Leadoptimization

Candidateseeking

safety98,103–106. For example, differences in the expression and inducibility of COX2 in the kidney macula densa has been demonstrated between preclinical species107, and it has been speculated that this may explain the species differences in nephrotoxic responses to COX2 inhibition in the dog108. Recent advances in the use of laser capture microdissection have made this approach even more sensitive, often enabling the retrieval of small numbers of cells in a highly localized tissue compartment for the evaluation of gene expression related to pharmacology or toxic injury109. Key to these approaches is the avail-ability of appropriately procured and stored specimens from representative animal models and spontaneous human disease, and, for comparative purposes, their corresponding normal tissues.

Last, veterinary pathology has been more frequently engaged in the development and evaluation of mechanistic models of toxic injury. Evaluation of complex data emerging from toxicogenomic and proteomic studies, as well as the interpretation of tissues from mechanistic toxicology studies requires the application of modern

molecular pathology techniques. In comparative toxico-genomic data sets, the repertoire of gene-expression changes must be related to the ultimate cellular or tissue manifestation of injury. For example, lesions that are primarily proliferative in nature should sensibly be asso-ciated with gene-expression changes in proliferative and cell-death pathways. However, anatomical changes that are associated with toxic injury are seldom mechanisti-cally simple. For example, when considering cellular necrosis as a primary toxic event, corresponding gene-expression changes would vary dramatically relative to the timing of sampling of tissue. In this example, cellular necrosis may subsequently lead to inflammation, regeneration and fibrosis. Depending on the time at which tissues are sampled, the gene-expression changes characterizing the morphological features would be expected to be dramatically different. As such, toxico-genomic evaluation should always be accompanied by a careful evaluation of morphological changes in the target tissue corresponding to the times of sampling for toxicogenomics.

Issue management and retrospective assays

The purpose of conducting early in vivo signal-generation studies is to identify dose-limiting toxicities and to understand the safety liabilities of a target and/or a chemical series. This is ideally performed immediately after achieving preclinical proof-of-principle (FIG. 3). By detecting dose-limiting toxicities early, studies can be initiated to understand the mechanism of the toxicity and to develop and apply retrospective assays for use in improving safety margins or designing out such liabili-ties altogether. It is in the process of addressing in vivo dose-limiting toxicities that the various ‘-omics’ tech-nologies are best applied. Even when the dose-limiting toxicity is not development limiting, the ability to identify safer lead compounds is critical: as the industry faces loss of patent exclusivity for numerous compounds, differen-tiation from marketed compounds is becoming increas-ingly important. Once identified, toxicology issues can be managed by asking a set of questions the answers to which will enable the toxicologist to decide how best to proceed (BOX 1). These issue management questions are:• What is the safety margin?• Is the toxicity reversible?• Is there a biomarker?• What is the mechanism?• What is the relevance of the finding to humans?

The answers to these questions are useful in deter-mining whether the toxicity has the potential to limit or end development. The first two questions are typically answered by standard in vivo toxicology studies. The answer to the third question may also be established on the basis of results from a thorough in vivo study using standard clinical pathology (that is, haematology, clini-cal chemistry, urinalysis, coagulation) end points. The standard clinical pathology parameters that are included in an in vivo study represent biomarkers for many pathological effects, and their translation to the clinic is comparatively well understood. Additionally, there are

Figure 3 | Toxicology profiling in discovery. Investigative toxicology and pathology

can affect drug discovery projects at all stages. Quantitative microscopy (for example,

morphometry, stereology) and molecular pathology techniques (for example, immuno-

histochemistry and in situ hybridization) are crucial for developing and validating

preclinical models of pharmacological effects and of disease, and for evaluating the

druggability of novel drug targets. Prospective in vitro toxicity screens identify toxicities

for which there may be no histopathological correlate in short-term in vivo studies, such

as mutagenicity and safety pharmacology. For many of these toxicities there are high-

throughput assays that can be run early (for example, in the hit to lead stage) to identify

potential liabilities that are associated with a chemical template and to shape the

testing scheme to address those liabilities with more laborious but more predictive

assays at the appropriate stage. The crux of the early application of discovery toxicology

is the early identification of target organs, which allows the creation of a platform of

knowledge regarding the toxicological liabilities of the drug target and the chemical

series. This allows the toxicologist and the synthetic chemist to apply target-organ-

specific in vitro assays retrospectively for lead optimization to understand structure–

toxicity relationships (STRs), screen out development-limiting toxicities and minimize

other adverse findings, thus delivering superior lead candidates into development.

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significant efforts underway to identify novel biomarkers for target-organ-specific toxicities109–114. Although it is possible to predict the risk to human health using in vitro and/or ex vivo assays without an understanding of the mechanism of the toxicology, knowledge of the mechanism is useful in assessing risk, identifying novel biomarkers and developing predictive in vitro assays. For example, there are several well-characterized rodent-specific toxicities that are considered not to be relevant to humans, including PPARα−agonist-mediated liver effects115–118 and UDP-glucuronosyl transferase inducer mediated thyroid findings119–121. Additional efforts to develop retrospective target-organ-specific assays, iden-tify novel mechanistic biomarkers and understand novel mechanisms of toxicity have incorporated new technolo-gies, including investigative pathology, stem cells122 and the various molecular and ‘-omics’ technologies123–129. For example, Gomez-Brouchet et al.130 have applied RNA interference to study the role of sphingosine kinase 1 (SPHK1) in Aβ−peptide-mediated neuroblastoma cell death, and Searfoss et al.114 have used transcription pro-filing to identify a novel biomarker of gastrointestinal toxicity mediated by γ-secretase inhibitors. Additionally, genetically engineered animal models such as human-ized mice, transgenics, knockouts and knock-ins have all been increasingly utilized in mechanistic and predictive toxicology131–136. Minimally, it is important to know if the toxicity represents a primary or secondary phar-macological effect or a chemically mediated toxicity, as these three types of toxicity are dealt with differently, as detailed below. The answer to the pivotal fifth question regarding the relevance of a finding to humans relies on the answers to the first four questions as well as a con-sideration of the intended therapeutic indication, route of administration and duration of treatment.

When managing a dose-limiting toxicity, additional preclinical models and predictive in vitro assays may be used to eliminate or minimize the incidence and severity of the finding in subsequent molecules within a chemical

class. The ability to rapidly screen out the liability depends on whether or not there is a predictive in vitro assay suitable for understanding the STR. For a toxicity that takes 14 days to develop in vivo and for which there is no in vitro assay, a 2-week in vivo assay may be the only screen by which to prioritize leads. A primary function of in vitro toxicology is to develop assays to screen out in vivo toxicities. These assays may then be incorporated into the testing scheme for all new analogues synthesized (FIG. 3). A key advantage to doing this before development is that in the discovery phase the synthetic chemistry effort is fully engaged, enabling the creation of a rich STR. Once a compound is in development, organizations are typically more conservative with respect to the nature of investigative toxicology studies that they will under-take, making it more difficult to generate answers for the questions listed above. Additionally, the medicinal chemistry effort may have been slowed or discontinued as chemists are shifted to different projects, delaying the identification of safer back-up candidates.

Screening out chemically mediated toxicity. Managing dose-limiting toxicity depends on the nature of the find-ing; whether it is chemically mediated or due to primary or secondary pharmacology (FIG. 4). When the dose-limiting toxicity is chemically mediated, in vitro assays can be applied to screen the liability out of a chemical series or to identify other chemical classes that do not have that particular liability. In vitro assays exist for several types of systemic and target-organ-specific toxicities, including steatosis137, phospholipidosis138,139, numerous apoptosis signalling pathways140–143, reactive oxygen species generation144 and mitochondrial toxicities affect-ing oxidative phosphorylation and other mitochondrial functions145. It would be impractical to run all of these assays prospectively, and the data would be difficult to interpret as safety margins determine the significance of many adverse effects. However, used in conjunction with in vivo signal generation studies, these assays are particularly well suited to building STRs and prioritizing compounds within a limited chemical space. For exam-ple, steatosis may be reversible and monitorable, but inadequate margins can be problematic in advancing a compound. However, compounds from a chemical class that is prone to cause steatosis in vivo can be assayed using an in vitro cell-based model146 to identify those with the least likelihood of causing steatosis. Similarly, Sun et al.147 have used an in vitro phospholipidosis assay to minimize this toxicity in a series of H3 antagonists.

Dealing with secondary pharmacology. Dealing with adverse secondary pharmacology may seem straight-forward initially; one simply needs to create an assay for the relevant secondary target. However, it can be difficult to distinguish secondary pharmacology from other types of toxicity, and it is often difficult to iden-tify the actual target responsible for the adverse effects observed in vivo. For example, it took ~50 years to prove that COX1 was responsible for gastrointestinal ulcera-tion and COX2 responsible for the anti-inflammatory effects of NSAIDs148,149. Similarly, it took nearly 50 years

Box 1 | The five questions of toxicology issue management

What is the safety margin? An acceptable safety margin depends on the nature of the dose-limiting adverse event, the therapeutic indication being sought, the intended patient population, the competitive environment and present standard of care, and several other factors.

Is the toxicity reversible? Toxicity that is irreversible is typically unacceptable, as human subjects in clinical trials can not simply be removed from treatment when the adverse event occurs.

Is there a biomarker? Toxicity that can not be monitored may develop into an irreversible toxicity before it is diagnosed.

What is the mechanism? Some mechanisms of preclinical toxicity may be species specific and not relevant to human health.

What is the relevance of the finding to humans? The answers to the above questions, in light of project-specific information such as the nature of the dose-limiting adverse event, the therapeutic indication being sought, the intended patient population, the competitive environment and present standard of care, will allow for an assessment of the risks of continuing to advance a particular compound into first-in-human trials.

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Inactive compounds lack toxicity, safety margins the same regardless of compound potency, toxicity not recreated in the primary target KO.Hypothesis: 1° pharmacology

Mixed toxicities observed with diverse chemotypes, structurally related inactive compounds show similar toxicity, toxicity recreated in KO model.Hypothesis: Chemically mediated toxicity

Toxicity is identified in an in vivo signal generation study

Assess the toxicity of structurally diverse selective inhibitors and structurally related but inactive compounds, and/or assess the toxicity in a KO mouse model.

Similar toxicity observed within and across chemotypes, safety margins vary with potency, toxicity recreated in KO. Hypothesis: 2° pharmacology

Assess species specificity and human relevance of toxicity. Identify compounds with optimal PK profile.

Identify compounds that lack toxicity but retain pharmacological activity.

Identify compounds that spare the secondary target and lack toxicity but retain pharmacological activity.

Evaluate species specificity andhuman relevance, evaluate margins with respect to indication and patient population, optimize PK parameters to minimize toxicity.

In vitro/in vivo modelling focused on physical and chemical properties, structure, metabolites, and potency versus target. Consider advancing a structurally distinct chemical series.

In vitro/in vivo modelling focused on structure and potency againstprimary versus potential secondary target. Consider advancing a structurally distinct chemical series.

to demonstrate that gynaecomastia associated with spironolactone, a mineralocorticoid-receptor agonist, was a secondary pharmacological effect of modulating the activity of a separate nuclear receptor150,151. The most likely targets for secondary pharmacology are typically included in early selectivity screens (FIG. 1), but these are usually limited to a few closely related targets. However, with large gene families or drug targets that interact with common endogenous molecules, the number of poten-tial secondary targets is enormous. For example, there are ~518 kinases in the human genome152 and numerous non-kinase proteins with ATP-binding domains. If seek-ing a small molecule that targets the ATP-binding site of a particular kinase, it would be both expensive and unreasonable to screen against all other ATP-binding proteins prospectively. Furthermore, with closely related targets the structure–activity relationship may be highly conserved for the primary and the secondary target, which may cause researchers to confuse some secondary pharmacological effects with adverse primary pharmacology. In one example, Ridings and Baldwin153 undertook studies with 11 active dopamine mimetics and one structurally related inactive compound to assess the nature of teratogenicity in rats. Although doses were selected to be equipotent in terms of pharmacological activity in the adult, limited pharmacokinetic data and

uncertainty regarding the extent of placental transfer of the compounds confounded efforts to understand the nature of the findings in the developing fetus. Correctly demonstrating whether an effect represents primary or secondary pharmacology is important, as a finding of adverse primary pharmacology can lead researchers to discontinue efforts on what had appeared to be a prom-ising drug target. The use of knockout mouse models can be particularly useful in differentiating secondary pharmacology or compound-mediated toxicity from primary adverse pharmacology. Other novel approaches are being developed that may hold promise in identify-ing secondary pharmacology targets154, and once the other target or targets are identified, in vitro assays can usually be developed to screen out the off-target binding and test the hypothesis that a particular secondary target is causative for the adverse event in vivo.

Managing adverse primary pharmacology. Adverse primary pharmacology can also be problematic. As described above for ACE inhibitors, adverse primary pharmacological effects may in some cases be managed by limiting patient populations. Similarly, exaggerated pharmacology, such as bleeding events with antithrom-botics, can often be managed simply by lowering dosages or decreasing the frequency of treatment. This assumes

Figure 4 | Toxicology issue management decision tree. When a dose-limiting toxicity is identified, mechanistic efforts

can be initiated to understand the nature of the toxicity in an attempt to avoid it in future lead candidates. Such efforts

may include assessing the toxicity of pharmacologically active but structurally diverse compounds and structurally related

but inactive compounds, and/or assessing the toxicity in a knockout (KO) mouse model. The results of such investigative

studies can lead to the generation of a testable hypothesis regarding the nature of the toxicity. The three classes of toxicity

described are then addressed using different strategies. PK, pharmacokinetic.

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that the adverse effects are both monitorable and reversible, allowing the agent to be discontinued before irreversible damage occurs. Different ADME properties such as the distribution of the drug within the body may result in differences in the incidence and severity of adverse pharmacology in specific target organs. For example pharmacokinetic effects may play a role in a superior safety profile of the newer, more potent statins with respect to skeletal muscle effects155–158. Specifically, increased oral bioavailability and resultant systemic plasma concentrations due to low gastrointestinal and hepatic cytochrome P450 3A4 (CYP3A4) activity may increase the risk for myotoxicity with statins whose primary route of clearance is CYP3A4 mediated. In such an example, a compound with greater potency and lower volume of distribution may never achieve sufficient systemic exposures to cause a high incidence of toxicity, but may still mediate efficacy in the liver. However, tissue distribution can be difficult to predict from in vitro assays, making it difficult to identify compounds that have optimal properties without testing multiple candi-dates in vivo. In some cases, the adverse pharmacology can not be overcome, resulting in a once promising drug target being abandoned.

In order to appropriately manage preclinical toxicity, a clear issue management strategy must be in place to determine whether the toxicity represents adverse pri-mary or secondary pharmacology or chemically mediated toxicity (FIG. 4). The issue management decision tree relies on mechanistic toxicology studies to assess the toxicity of structurally diverse selective inhibitors and structur-ally related but inactive compounds. These mechanistic efforts could also include an assessment of the toxicity of the compound in a knockout mouse model. Zambrowicz and Sands159 have described an approach to target iden-tification that involves knocking-out 5,000 genes in the

mouse genome in an effort to identify novel drug targets. This approach will generate knockout mouse models for the genes that represent the ‘druggable genome’160, followed by a rapid phenotype assessment in an effort to identify targets for therapeutic intervention. This effort also holds the promise of identifying potential adverse primary pharmacology and providing knockout mouse models for use in distinguishing compound-mediated effects from primary pharmacology.

Conclusions

Toxicity continues to be a leading cause for drug-candidate attrition at all stages. To overcome these challenges, the pharmaceutical industry is applying innovative approaches to minimize the rate of attrition, to facilitate earlier data-driven decisions to discontinue the develop-ment of drug candidates before entry into more costly later phases of clinical development, and to deliver safer leads into development. Many companies have adopted a strategy that includes the early incorporation of pre-clinical safety assessment before selection of a drug candidate for formal development. These efforts ought to include both novel technologies as well as the earlier application of more traditional toxicological approaches (BOX 2). The earliest in vitro assays predict toxicities that are development limiting (that is, genetic toxicology) or those that may not be readily identified in early in vivo toxicity studies (for example, hERG, drug–drug interac-tions). Ideally, these assays are performed early to build structure–activity relationships and identify liabilities before advancing compounds into in vivo studies. Upon proof-of-concept in preclinical efficacy models, early in vivo toxicology studies are carried out to establish the dose-limiting toxicity, identify target organs and esti-mate safety margins. Findings that are then identified are characterized, managed and/or screened out using directed retrospective in vitro toxicity assays.

Many discovery and investigative toxicology groups rely heavily on new technologies, such as toxicogenomics and proteomics. These have had high visibility and have been reviewed extensively elsewhere122–129. However, new molecular technologies, used in isolation from the more traditional toxicology methodologies, may do little to advance compounds into the clinic. An acceptable pre-clinical safety margin is key to selecting the most viable development candidates, and this can best be determined by repeat-dose in vivo toxicology studies. Once the dose-limiting target organ(s) has been identified, newer tech-nologies can be crucial in understanding and positioning the finding(s). As these technologies continue to mature, great advances in our ability to predict, understand and manage toxicities will result161. However, classical assays such as the repeat-dose in vivo rodent toxicity assay will continue to be a mainstay in any effort to identify and optimize drug candidates.

It is important to note that a focus on rodent toxicity (usually in a single gender) in discovery risks the pos-sibility of missing certain gender- or species-specific toxicities. The rodent, alone or in combination with a second preclinical toxicology species, is an imperfect predictor of clinical safety10, suggesting a significant

Box 2 | Challenging current practices

• Approximately 70% of all toxicity-related failures occur preclinically and are comprised of toxicities for which the preclinical models are predictive of human toxicity.

• Many start-up and small biotech companies perform little safety assessment, and often do it as an afterthought; whereas prospective pharmaceutical partners pay close attention to the safety of potential in-licensed compounds, and safety is the primary focus of preclinical development and early phases of clinical development.

• The proper focus of discovery toxicity should be the earlier identification of ‘predictable’ toxicities before nominating a development candidate.

• Identification of dose-limiting toxicities before development allows for a synthetic chemistry effort toward the goal of minimizing or designing out the liability, resulting in superior safety margins.

• Although new technologies generate a great deal of attention in the investigative toxicology field, a well-designed repeat-dose rodent toxicity study can identify most dose-limiting toxicities more quickly, more cheaply and more reliably.

• New molecular technologies, separated from the more traditional toxicology methodologies, may do little to advance compounds into the clinic.

• Efforts to apply new technologies towards the prediction of idiosyncratic toxicity will be virtually impossible to validate and may only serve to generate untestable safety concerns around otherwise promising drug candidates. If applied, such efforts should be positioned to reduce risk, rather than an attempt to screen out an actual safety liability.

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The earlier identification of development-limiting toxicity saves significant time and resources spent on leads that have little chance of reaching FIH studies or advancing through clinical trials. Investigative pathol-ogy, retrospective in vitro modelling and toxicity issue management, all of which continue to benefit from new technologies, will lead to the identification of develop-ment candidates with superior safety profiles and a better chance of reaching FIH studies and eventually becoming successfully marketed pharmaceutical agents with enhanced benefit to patients.

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Competing interests statementThe authors declare no competing financial interests.

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