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Journal of Pharmacological and Toxicological Methods 58 (2008) 77–87

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Journal of Pharmacological and Toxicological Methods

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Appraisal of state-of-the-art

Safety and secondary pharmacology: Successes, threats, challenges and opportunities

Jean-Pierre Valentin ⁎, Tim HammondSafety Assessment UK, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, SK10 4TG, United Kingdom

⁎ Corresponding author.E-mail address: [email protected]

1056-8719/$ – see front matter © 2008 Elsevier Inc. Alldoi:10.1016/j.vascn.2008.05.007

A B S T R A C T
A R T I C L E I N F O
Article history:
This review summarises the Received 19 March 2008Accepted 19 May 2008
Keywords:Safety pharmacologyPharmacological profilingAdverse drug reactionsQT interval prolongation

lecture of Dr Tim Hammond, recipient of the Distinguished Service Award of theSafety Pharmacology Society, given on 20 September 2007 in Edinburgh. The lecture discussed the rationalebehind the need for optimal non-clinical Safety and Secondary Pharmacology testing; the evolution of Safetyand Secondary Pharmacology over the last decade; its impact on drug discovery and development; the valueof adopting an integrated risk assessment approach; the translation of non-clinical findings to humans andfinally the future challenges and opportunities facing these disciplines.

© 2008 Elsevier Inc. All rights reserved.

1. Rationale for a non-clinical safety and secondary pharmacology(SSP) testing

A pharmaceutical industry survey revealed that, over the lastdecade, despite significant R&D investment (+70%), reflecting the in-crease in sales (+120%), the overall drug development time hasincreased (+20%) and, the number of New Chemical Entities (NCEs)being launched has significantly decreased (−30%). Over the sameperiod, both non-clinical and clinical safety has remained the majorcause of drug attrition during clinical development and of withdrawalof marketed drugs; accounting for approximately 35 to 40% of all drugdiscontinuation (Kennedy, 1997; Lasser et al., 2002; Kola & Landis,2004). Recent reviews have explored themain causes for adverse drugreactions (ADRs)-related drugs withdrawals from either the U.S.A. orworldwide market (Fung et al., 2001; Stephens, 2004; Table 1).Cardiovascular, CNS and hepatic-related toxicities ranked top in thesereviews, accounting globally for 9–26% of drug withdrawals. HumanADRs fall into 5 types (A to E), out of which the type A represents ~75%of all cases (Redfern, Wakefield, Prior, Hammond & Valentin, 2002).The type A ADRs are dose-dependent and predictable from primary,secondary and safety pharmacology. A significant proportion of thesetoxicities is functional in nature and, therefore, should be predictablefrom a thorough assessment of SSP. For instance, the prominence ofarrhythmias as a reason for drug discontinuation (approximately halfof the 19% reported by Stephens, 2004) probably reflects the concernfor drug-induced Torsades de Pointes (TdP) type arrhythmias and theeffort, froma safety pharmacology perspective, to detect such an effect.

One of the primary objectives of the non-clinical safety evaluation isto define the starting dose in humans, in relation to the potential

m (J.-P. Valentin).

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therapeutic concentration, and the range of unacceptable toxicological/pharmacological side effects. A report published in 2005 under theauspice of the British Academy of Medical Sciences (Anon, 2005a)showed that healthy volunteer single dose first time in man (FTIM)studies are extremely safe (Table 2). This is related to the fact thatcompounds with potentially dangerous adverse events (AEs) aredetected earlier in non-clinical safety studies and therefore not pro-gressed into clinical development. Therefore, AEs reported during FTIMstudies are usually related to the procedure (e.g., needle puncture,headache secondary to caffeine deprivation) and/or to the primarypharmacology of the drug (i.e., mechanism of action). Interestingly, themain ADRs reported are often not detected by toxicology/pharmacologystudies (e.g., headache, dizziness, nausea). Thus non-clinical safetyevaluation to support Phase I is effective; compounds that do get intomando so safely. However,while this holds true for smallmolecules, theserious AEs observed with the CD28 agonist antibody TGN-1412 in aphase I study in early 2006 (Anon., 2006; Table 2) opens new challengesfor safety pharmacology as it relates to the increasing developmentof biologics and points out how difficult it is to lay out any generalexamples of typical non-clinical safety programs for biologics. Never-theless, recent investigations on the mechanisms of the TGN-1412-mediated “cytokine storm” will enable to develop novel procedures toimprove non-clinical safety testing of immunomodulatory therapeutics(Stebbings et al., 2007). Meanwhile, this serious AE has lead to thedevelopment and implementation of more stringent regulatory guide-lines for high-risk medicinal products (Anon., 2007).

2. Evolution of safety and secondary pharmacology

Over the last decade, reflecting increasing regulatory concerns,there has been an increased number and scope of regulatory guidancedocuments referring entirely or in part to safety pharmacology (Fig.1).One would intuitively expect that scientific and technological

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Table 1Main reasons for drug discontinuation during non-clinical or clinical development, withdrawal or ADRs

Physiological systems References

CVS CNS Hepatic Respiratory GI Renal

Combine non-clinical and clinical (1) 27–34⁎⁎ 14⁎⁎⁎ 15 2–3 3–4$ 2–3 Car B.D. (2006)Incidence of ADRs associated with marketed drugs (2) 35 56 11 32 67 32 Kresja et al. (2003)Incidence of ADRs associated with marketed drugs (3) 15⁎⁎ 14 – 8 14 2–3 Budnitz et al. (2006)Withdrawal (4) 19⁎⁎ 12⁎ 9–26 – – 5 Fung et al. (2001), Stephens (2004)

CVS, Cardiovascular; CNS, Central nervous System; GI, Gastrointestinal. Incidence (i.e., N3%) of Adverse Drug Reactions (ADRs) related to each of the major physiological systems. (1)Data are based on 88 candidates discontinued between 1993 and 2006 in non-clinical or clinical development. (2) Data based on a set of 1138 drugs annotated for human ADRs andextracted from BioPrint®. Not all compound-ADR annotations include incidence data; hence the figures have been corrected to account for missing incidence data. (3) Based onpatients treated in emergency departments in the U.S.A. in 2004 and 2005. (4) Drug withdrawn from the US and worldwide market. ⁎Combining Neurologic, Psychatric and Abuse.⁎⁎Combining Cardiovascular and Haematology. ⁎⁎⁎Combining central and peripheral nervous system and retina. $ Including pancreatic toxicities.

78 J.-P. Valentin, T. Hammond / Journal of Pharmacological and Toxicological Methods 58 (2008) 77–87

advancements should drive and influence the regulatory environment.In fact, looking at the trend in the number of publications on “QT” and“hERG” on one hand and “General Pharmacology” and “SafetyPharmacology” on the other hand, it becomes evident that the rise innumber of publication follows, rather than precede, the emergence ofregulatory documents (i.e., CPMP, Anon., 1997 and ICH S7A, Anon.,2000, respectively; Fig. 2).

During the drug discovery process, 3 types of pharmacologystudies are conducted; primary, secondary and safety pharmacologystudies. Primary pharmacodynamic studies aim at investigating themode of action and/or effects of a substance in relation to its desiredtherapeutic target. Secondary pharmacodynamic studies aimed atinvestigating the mode of action and/or effects of a substance notrelated to its desired therapeutic target, whereas safety pharmacologystudies aimed at investigating the potential undesirable pharmaco-dynamic effects of a substance on physiological functions in relation toexposure in the therapeutic range and above. Safety pharmacody-namic effects may result from activity at the primarymolecular target,secondary targets or non-specific interactions (Bowes, Rolf & Valentin,2006; Fig. 3). Hence, having a good understanding of the overallpharmacology of the drug is important in designing an optimal safetypharmacology programme. As to achieve the main objectives of safetypharmacology, SSP studies can be aligned and applied to the differentphases of drug discovery and development (Anon., 2000; Valentin &Hammond, 2006). More specifically, safety pharmacology studies aim(i) to identify undesirable pharmacodynamic properties of a substancethat may have relevance to its human safety; (ii) to evaluate adversepharmacodynamic and/or pathophysiological effects of a substanceobserved in toxicology and/or clinical studies; (iii) to investigate themechanism of the adverse pharmacodynamic effects observed and/orsuspected. The impact of SSP in relation to these objectives referprimarily to (i) Hazard Identification and Elimination, (ii) RiskAssessment and (iii) Risk Management and Mitigation, respectively(Table 3).

Table 2Risks to healthy volunteers in Phase I clinical research trials

Year Number ofvolunteers

Moderatelysevere AE

Potentially life-threatening AE

Deaths References

1965–77 29,162 58 (0.2%) – 0 Zarafonetis C.J.et al. (1978)

1980 – – – 1 Kolata G.B. (1980)1983 – – – 1 Darragh A. et al.

(1985)1984 – – – 1 Anon. (1985)1986–87 8163 45 (0.55%) 3 (0.04%) 0 Orme M. et al.

(1989)1986–95 1015 43 (3%) 0 0 Sibille M. et al.

(1998)2000 – – – 1 McCarthy M. (2001)2006 6 – 6 (100%) 0 Anon. (2006)

AE, Adverse Event. Adapted from Redfern et al. (2002).

3. Successes of safety and secondary pharmacology

In this section we shall provide examples of application of SSPapproaches to the different phases of drug discovery and developmentand highlight their impact in the discovery/development process.

3.1. Hazard identification and elimination

Although hazard can be identified at any stage of the discovery/development process, during the early drug discovery phases SSP isprimarily applied, to hazard identification and wherever possible tohazard “eradication” or elimination. Approaches that are being usedshould be amenable to the chemistry make-test cycle, by having rapidturn around time and low compound requirements. Consequently, theapproaches used involve primarily in vitro assays. Over recent yearspathway mapping tools have become available to better understand,deconvolute and optimally utilize unstructured data, both proprietaryandpubliclyavailable. For example, one canask thequestiononwhetherthere is a potential link between the primary molecular target and thehERG encoded K channel. If the answer is yes, the project team mayconsider assessing the potential QT-liability of NCEs early in thediscovery process. In silico models are being developed and applied toSSP endpoints. For example, in silico models exist to determine thepotency at the hERG channel enabling to screen millions of virtualcompounds, thus streamlining chemistry to avoid synthesising com-pounds likely to be potent hERG blockers (Lesson & Springthorpe, 2007;Gavaghan, Arnby, Blomberg, Strandlund & Boyer, 2007; Fig. 4B).Correlations between predicted and measured hERG potencies aregood (Fig. 4A). Historically, one of the limitations for developing in silicotools has been the lack of significant and consistent databases. Severalcompanies have expended considerable resources creating proprietarydatabases focusing on different areas of pharmacological profiling sothat in silico tools cannowbeapplied in severalways (Bowes et al., 2006;Hamon, Crawford & Jean, 2006). In vitro assays to assess drug effects onmolecular targets known to be associated with undesirable pharmaco-dynamic properties are used during the lead identification andoptimisation phases in order to establish structure activity relationship(SAR) and therefore influence the medicinal chemistry design andprovide tools for effective decision making. Examples include electro-physiology-based cardiac ion channels assays (hERG: Bridgland-Tayloret al., 2006; INaV: Harmer et al., 2008; Fig. 4C/D), radioligand binding orfunctional assays to measure activity at GPCRs, transporters, andenzymes (Bowes et al., 2006; Hamon et al., 2006).

3.2. Risk assessment

Once a NCE is identified to progress into non-clinical development, acomprehensive set of SSP studies is generated to build an integrated riskassessment on key physiological systems with a focus on detectingadverse effects (e.g., cardiovascular, respiratory and central nervoussystems). The studies are primarily, but not exclusively, conducted inrelevant animal models. The data generated include, but are not limited

Fig. 1. Implementation of regulatory guidance documents referring to safety pharmacology during the last 30 years. Over the last decade there has been an increase in number andscope of regulatory guidance reflecting increasing regulatory concerns.

79J.-P. Valentin, T. Hammond / Journal of Pharmacological and Toxicological Methods 58 (2008) 77–87

to, the “core battery” and any “follow-up” and “supplemental” studies asappropriate (Anon., 2000, 2005b). The data generated by these studiesare used in several ways (Table 3) including: (i) fulfil the regulatoryrequirements; (ii) influence the design of the Phase I clinical trials; (iii)identify clinically relevant safety biomarkers; (iv) contribute to thepatient risk management plan; (v) assess the safety for compoundprogression to man; (vi) assess the risk-benefit for compound progres-sion to man; and (vii) contribute defining the starting dose during thePhase I clinical trials. Fig. 5 summarises thedata set that form the basis ofan integrated risk assessment to assess the liability of a NCE to prolongthe duration of the QT interval (Fig. 5A) and to reduce cardiac force of

Fig. 2. Trends in number of publications referring to “QT”, “hERG” (left panel), “General Pharmnumber of publications in the SP area follows the emergence of regulatory guidance docum

contraction (Fig. 5B) in relation to the expected therapeutic plasmaexposure in man. In the first example, the recommendation was tothoroughly monitor the QT interval duration during the Phase I clinicaltrials, which resulted in the human data included in Fig. 5A, whereas inthe second example (Fig. 5B) it was felt that the benefit-risk for thetargeted patient population did not warrant progression into man.

3.3. Risk management and mitigation

Once a NCE progresses into clinical development, SSP data can beapplied in the context of risk management and risk mitigation. In this

acology” and “Safety Pharmacology” (right panel) over the last few decades. The rise inents (i.e., CPMP, Anon., 1997 and ICH S7A, Anon., 2000).

Fig. 3.Mechanisms of drug action: Beneficial, deleterious or neutral functional effects can be mediated via binding to the desired therapeutic target and/or to other molecular targetssuch as G-protein-coupled receptors (GPCR), ion channels, or transporters on the cell membrane, or to intracellular targets such as enzymes and nuclear hormone receptors.


context, SSP studies can be conducted at any stage during the drugdevelopment process, based on cause for concern that emerges fromclinical and/or toxicological studies, literature information and knowl-edge about the class and/or pharmacology of that specific or similardrugs. The data are used in several ways (Table 3) which include butare not limited to: (i) support regulatory approval; (ii) investigatediscrepancies that may have emerged within and/or between non-clinical and clinical data; (iii) understand the mechanism of anundesirable pharmacodynamic effect; (iv) provide reassurance forprogression into multiple dosing in humans and/or large scale clinicaltrials; (v) assess drug–drug interactions. Based on emerging data, theintegrated risk assessment is reviewed and the benefit-risk for NCEprogression re-assessed. Figs. 6 and 7 provide examples of the impactof safety pharmacology data in support to the safe progression ofcompounds through the clinical development phases.

TheQT-liability of compoundAwas assessed as part of the 1-monthdog toxicology study using a non-invasive jacketed telemetry system(Fig. 6A). Compound A dose- and time-dependently prolonged theduration of the QT interval; the magnitude of increases in QT intervalduration, almost negligible after single dose administration, becamesignificant after several days of treatment. Consequently, appropriateECG monitoring was incorporated into the design of the single andimportantly the multiple ascending dose Phase I studies.

Compound B was suspected to induce fibrodysplasia in rodents.Therefore, motor coordination was measured by assessing the per-formance of Han Wistar rats on a rotarod throughout the duration of a1-month duration toxicology study (Fig. 6B). A deficit in rotarod perfor-mance was detected from day 15 onwards. The deficit preceded theappearanceof adverse signs during clinical observations in rats. Thedatasupported the careful progression to single ascending dose in volunteersproviding appropriate monitoring.

A set of experiments was conducted to test the hypothesis onwhether 2 known hERG blocking and QT-prolonging drugs (ondanse-

Table 3Application and alignment of safety and secondary pharmacology studies to the phases of d

Drug discovery and development phase Lead identification and optimisation Pre-cand

Objectives Hazard identification and elimination Risk asseSSP assays / methods Front-loaded approach: in silico,

in vitro, in vivo assays“Core-ba“supplem

Number compounds testedper project

N106 (in silico) 100–10,000 (in vitro)b100 (in vivo)

b10

Impact •Establish SAR •Fulfil re•Influence chemistry •Influenc•Solve problems •Identify•Provide reassurance to progress •Contribu

tron (Benedict et al., 1996) and compound C; Fig. 6C)) could haveadditive or synergistic effect onQT interval duration. In an anaesthetiseddog model, two successive dose–response curves to ondansetron wereestablished in the presence of compound C or its vehicle. The resultsdemonstrated an absence of additive or synergistic effect of the twocompounds on QT interval duration; these findings were further sup-ported by in vitro evidence showing no additive or synergistic effect onhERG potency. The findings supported, with careful monitoring in place,the proposed clinical combination.

Clinical development of compound D was discontinued for unac-ceptable risk of cardiovascular toxicity suggestive of myocardialinfarction. In non-clinical models, compound D induced a dose-dependent increases in blood pressure and heart rate; such changeswere associatedwithmarked dose-related incidence ofmildmyocardialnecrosis within the left ventricle that were paralleled with increases introponin T plasma levels (Fig. 7). To determine whether the structuralchanges could be the consequences of the haemodynamic changes,studies were conducted in rats where the increases in blood pressureand heart rate were prevented by using a combination of a β-adrenoceptor antagonist (atenolol) and a L-type Ca2+ channel blocker(nifedipine), respectively. In these experimental conditions both theoccurrence of mild myocardial necrosis and the elevation of troponinlevel were fully prevented, thus suggesting that the structural damageswere secondary to the haemodynamic changes. These data provided arationale for the resumption of clinical trials.

4. Integrated risk assessment

The integrated risk assessment is the stepwise and holisticevaluation of non-clinical study results in conjunction with anyother relevant information and should be scientifically based andindividualised for a NCE. Such an assessment should contribute to thedesign of clinical investigations and the interpretation of their

rug discovery and development and impact of their data

idate and candidate drug selection Clinical development and life cycle management

ssment Risk management and mitigationttery”, “follow-up” andental” assays

“Follow-up” and/or “supplemental” assays

1

gulatory requirements •Support regulatory approvale Phase I design •Investigate discrepanciesclinical biomarker •Understand mechanismste to clinical plan •Assess drug–drug interactions

Fig. 4. Correlation between predicted and measured hERG pIC50 values (A). Physico-chemical properties influencing hERG potency (B). Examples of structure activity relationshipshowing increased separation between activities at the primary target and hERG resulting in increased safety index (C/D). Separationwas obtained by reducing hERG potency whilstmaintaining the activity at the primary target (yellow squares; C) or by increasing the activity at the primary target whilst maintaining low hERG potency (grey squares; D).


findings. Risk assessment in terms of protecting Phase I clinical trialparticipants is relatively straightforward, as it does not take intoaccount any consideration of the therapeutic target (unless when thePhase I trials include patients) or the degree of unmet medical need.Therefore, the assessment of the safety pharmacology data has to takeinto consideration the ‘severity’ of the outcome in any given safetypharmacology test (see below — bearing in mind the sensitivity andspecificity and predictive value of the assays), and the plasmaconcentration at which it occurred relative to the expected exposurelevels in the clinical situation. Depending on the stage of drugdevelopment, the integrated risk assessment should consider con-tribution of metabolites as well as metabolic differences betweenhumans and animals.

In terms of risk assessment of NCE viability during the drug dis-covery phases, the situation ismore complex. In an attempt to simplifyand standardise how safety pharmacology data can contribute to earlyrisk assessment of project viability, Redfern et al. (2002) proposed thematrix type approach described below (Table 4). This requires agrading process; each of the factors in the risk assessment can begraded into, for example, three categories — low, medium and high.Starting with the safety pharmacology tests themselves, they can becategorised as follows: i) minor — potentially predicting the presence

of non-serious, reversible ADRs (e.g., certain gastrointestinal or renaleffects); ii) moderate — potentially predicting ADRs that can impairthe ‘quality of life’ (e.g., sedation, motor coordination); iii) major —

predicting potential life-threatening ADRs (e.g., QT interval prolonga-tion, pronounced hypotension, bronchoconstriction). The next stepconsists in grading the therapeutic target according to disease severity:i) ‘minor/moderate’ disease (e.g., eczema, rhinitis, Raynaud's syn-drome); ii) ‘debilitating’ disease (e.g., asthma, epilepsy, Parkinson's,stroke, angina); iii) ‘life-threatening’ disease (e.g., cancer, AIDS, myo-cardial infarction). The third component that can be considered isthe existing therapies to the disease targeted. Theoretically, the NCEshould be anticipated to be superior to existing therapy. Therefore, theexisting therapy cannot be classified as excellent; instead it can berated as i) good; ii) partially effective with side effects; iii) poor/non-existent. Once collected, the set of information can be put together in amatrix that also takes into account the dose level (or concentration) atwhich ADRs were observed in the safety pharmacology tests, incomparison to the expected clinical exposure (total or free plasmaconcentration), as shown in Table 4. Effects well to the left of the line ofcrosses are acceptable without further debate, those well to the rightare unacceptable, and those on or near the line require furtherassessment. An hypothetical example is presented to illustrate the

Fig. 5. Integrated risk assessment to assess the liability of a NCE to prolong the durationof the QT interval (A) and to reduce cardiac force of contraction (B) in relation to theexpected therapeutic concentration in man. In the example A, the recommendationswere to monitor the QT interval duration during the Phase I clinical trials that resultedin the human data included, whereas in the example B, it was felt that the benefit-riskfor the targeted patient population did not warrant progression into man. Note theoverall good concordance across the non-clinical models.

Fig. 6. Examples illustrating the impact of safety pharmacology data in support tothe safe progression of compounds through the early clinical development phases.A: Assessment of the QT-liability of compound A in a 1-month dog toxicology study.B: Assessment of the potential for Compound B to affect motor coordination in rodent asa biomarker of fibrodysplasia. C: Assessment of the potential for drug–drug interactionbetween 2 compounds known to block hERG and prolong the duration of the QTinterval. See text for further details.


usefulness of such amatrix; a NCE targeted at Raynaud's syndrome— a‘minor/moderate’; disease under classification, for which existingtherapy is poor. The NCE is found to block the hERG channel andprolong the QT interval (both considered ADRs of major severity) at arelatively low concentrations (e.g., ~30-fold) above the expectedtherapeutic plasma concentration. According to the matrix analysis,the decision is either to discontinue the progression of the NCE intodevelopment or to accept embarking on an extensive and expensiveclinical programme in compliance with the ICH E14 guidance (Anon.,2005c). To complement this evaluation, other factors should beconsidered, such as (i) the target population: for example, cognitiveimpairment as a side effect may be more problematic in elderly andpaediatric patients; (ii) the ultimate project objective: for example,QT interval prolongation as a side effect may be manageable andacceptable for a NCE aimed at demonstrating proof of mechanism orproof of principle. Overall, the evidence of risk, as part of an integratedrisk assessment, can support the planning and interpretation of sub-sequent clinical studies and should be considered in the decisiontaking process.

5. Predictive value of non-clinical safety and secondarypharmacology testing to humans

In most cases, evidence regarding the predictive value of non-clinical testing is not readily available in the public domain. An effect

of a NCE in a non-clinical test might preclude further clinical develop-ment. The apparent lack of value in communicating this informationresults in the limited public availability of data for the assessment ofthe predictive value of non-clinical safety models. Similarly, if therehas been no effect in a non-clinical test, and likewise no effect on thecorresponding variable in humans, these negative data may have beendeemed not to be of general interest. Consequently, the scientific andmedical community is left only with high profile examples of ADRs inhumans that were apparently not detected in non-clinical safetytesting. One publication attempted to explore the predictive value ofsafety/general pharmacology assays to humans (Igarashi et al., 1995).Some significant correlations were reported; for example, decreasedlocomotor activity, intestinal transit, urinary sodium excretion inrodents correlated with ‘dizziness’ ‘constipation’ and ‘oedema’, re-spectively in humans; decreased blood pressure in dogs with flushes,dizziness, headache and malaise in humans. Rather more bizarrely,the findings of analgesia and hypothermia in rodents and changes inthe pressure reflex to vagal stimulation in dogs correlated with ‘thirst’in humans. These associations indicate the limitations of such analysisand highlight the fact that correlations between events do not neces-sarily imply causal relationships between them. There are, however,numerous examples of drugs that cause ADRs in humans, which

Fig. 7. Contribution of safety pharmacology data to the resumption of clinical trials. Clinical development of compound D was discontinued for unacceptable risk of cardiovasculartoxicity suggestive of myocardial infarction. In rats, compound D induced dose-dependent increases in blood pressure and heart rate associated with marked dose-related incidenceof mild myocardial necrosis within the left ventricle and increases in troponin T plasma levels. Prevention of the increases in blood pressure and heart rate using a Ca++ channelblocker (nifedipine) and a β-adrenoceptor antagonist (atenolol) respectively also prevented the occurrence of mild myocardial necrosis and elevation of troponin T level, thussuggesting that the structural damages were secondary to haemodynamic changes.


would be detectable in SSP evaluation conducted as per the ICH S7Aand S7B directives. For example, i) the sedative effects of clonidine invarious animal species and man; ii) the propensity of cisapride toprolong ventricular repolarization; iii) the respiratory depressanteffects of morphine; iv) the nephrotoxic effect of cyclosporine; v) thegastrointestinal effects of erythromycin (Redfern et al., 2002; Valentin& Hammond 2006). These examples illustrated the good concordanceof effects across species and within a narrow range of doses/concen-trations or exposures (Redfern et al., 2002; Valentin & Hammond2006). In addition, over the last few years, data have been generated toassess the value of non-clinical tests to predict the potential of NCEs toprolong the QT interval of the electrocardiogram and ultimately theproarrhythmic potential of these drugs. The published data convergedin that an integrated risk assessment (see above) based upon data onthe potency against hERG, an in vivo repolarization assay and, ifnecessary, an in vitro repolarization assay are, in a qualitative sense,predictive of the clinical outcome (Ando et al., 2005; Miyazaki et al.,2005; Omata, Kasai, Hashimoto, Hombo, Yamamoto, 2005; Sasaki,

Table 4Safety pharmacology integrated risk assessment matrix

Therapeutictarget

Existing therapy Severity of safety pharmacology outcome

Minor Moderate Major

100× 10× 1× 100× 10× 1× 100× 10× 1×

Minor Good XPartially effective XPoor/none XGood X

Debilitating Partially effective XPoor/none XGood X

Life-threatening

Partially effective XPoor/none X

Outcomes situated to the left of the cross line are acceptable, those outcomes situated tothe right are unacceptable, whereas outcomes on or near the line of identity wouldrequire further discussion and possibly further investigations. Source: Modified fromRedfern et al., (2002). 100×, 10×, 1× represents the exposure levels at which a particularsafety pharmacology outcome is observed, and are fixed at 100, 10 or 1-fold thetherapeutic dose/concentration respectively.

Shimizu, Suganami, & Yamamoto, 2005). These data have been furthersupported by publications suggesting that a 30-fold margin betweenthe highest free plasma concentration of a drug in clinical use and thehERG IC50 could be adequate to ensure an acceptable degree of safetyfrom arrhythmogenesis with a low risk of obtaining false positives (DeBruin, Pettersson, Meyboom, Hoes, Leujkens, 2005; Webster, Leisch-mann,Walker, 2002; Redfern et al., 2003). Published literature lookingat the predictive value of non-clinical safety assays/models to humansis emerging (Table 5). Further work is required to extend thesevalidation set in order to determine both the confidence in themodels,as their capacity to mimic the same physiological/pathophysiologicalmechanisms in humans, and the confidence in their translation tohumans, as predictors of the occurrence of ADRs (Cavero, 2008;Wallis,2007). To achieve this, there is an urgent need to collate, integrate thenon-clinical and clinical data and share the results widely.

However, there are areas that should be carefully considered toensure optimisation of the assays and ultimately increase the predictivevalue of non-clinical testing. These include, but are not limited to:i) species differences in the expression or functionality of themoleculartarget mediating the adverse effects; ii) differences in pharmacokineticproperties between test species and man; iii) sensitivity of the testsystem; iv) optimisation of the test conditions; v) appropriatelystatistically powered study designs; vi) appropriate timing of functionalmeasurements in relation to the time of maximal effect (need forextended PK/PD modelling); vii) delayed/chronic effects of parent drugand/or metabolites; viii) assessment of parameters that are difficultto detect in animals in standard SSP studies (e.g., arrhythmia); ix)assessment, at a pre-clinical level, of sub-optimal surrogate endpointsthat predict with limited degree of confidence the clinical outcome(e.g., QT/QTc interval prolongation as a surrogate of TdP).

6. Current and future challenges

The future of SSP will depend, in part, upon the scientific andtechnological advances and regulatory challenges that envelop thediscovery and development of pharmaceuticals for human use as wellas the availability of trained scientists to design, conduct, interpret andreport studies.

Table 5Sensitivity, specificity and predictability of SSP assays to the clinical outcome

Test system Specie Experimentalconditions

Clinical endpoint Number ofcompounds

Sensitivity % Specificity % Predictability⁎ % References

Optometry Zebrafish In vivo Visual acuity 37 71 78 73 Richards et al. (2008)Speed and distance travelled Zebrafish In vivo Seizure 25 76 63 72 Winter et al. (2008)hERG⁎⁎ Human In vitro QT interval prolongation 19 82 75 79 Wallis R. (2007)QT interval prolongation⁎⁎ Dog In vivo QT interval prolongation 19 83 86 85 Wallis R. (2007)Screenit Rabbit In vitro Torsades de Pointes 64 65 89 75 Lawrence et al. (2006)

Sensitivity=True Positive/True Positive+False Negative. A high sensitivity reflects a low rate of false positives; Specificity=True Negative/True Negative+False Positive. A highspecificity reflects a low rate of false negatives. Predictability=True Positive+True Negative/Total number of compounds. ⁎The assessment was established against the clinicaloutcome in most cases; ⁎⁎within 2-fold clinical exposure.


6.1. New targets and new approaches to treat diseases

Advances in molecular biology and biotechnology allow for theidentification of new molecular targets, leading to the discovery anddevelopment of newer pharmaceutical agents that act at these novelmolecular sites in an attempt to ameliorate the disease condition.Moreover, new therapeutic approaches are being developed (e.g., Genetherapy, Biotech products) that offer new challenges to safety phar-macology. Inherent in the novelty of new targets and new approachesis the risk of unwanted effects that may or may not be detected basedon current scientific knowledge and with current techniques andassays. Over the last decade the sales offixed combinationproducts hasincreased by ~5-fold, representing a significant proportion of theoverall sales of medicinal products, primarily focusing on the combi-nations of 2 establishedproducts. The future lieswith the developmentof (i) an established product with one or more NCEs; (ii) combinationsof a NCE that requires the co-administration of another NCE forefficacy. Some of the key questions that will need to be addressedinclude but arenot limited to: Howmany SSPpackageswill be needed?Howwill a risk assessment be conducted? Howwill the safetymarginsprinciple be applied to combination products? Moreover, a trend forsometime has been to design selective NCEs against a given moleculartarget. However, most pathological conditions are multifactorial andinvolvemultiple, and in some cases redundant, pathways. Therefore, insome cases, such as functional gastrointestinal disorders, there is abelief that poly-pharmacology may provide significant advantage forNCEs. As a consequence, “unselective” NCEs acting simultaneously atmultiple targets might be considered for clinical development, whichwill require a more careful assessment of their potential to induceADRs.

6.2. Science and technology

Safety pharmacology faces significant scientific challenges to keeppace, to adapt, and to incorporate new technologies in the evaluationof NCEs in non-clinical assays and identifying the effects that pose arisk to human volunteers and patients. Recent examples have includedthe use of electrophysiological techniques to evaluate the effects ofNCEs on ionic components of the cardiac action potential (Bridgland-Taylor et al., 2006), and telemetry techniques to permit the chronicmonitoring of physiological functions in unstressed animals (McMahon,Pollard, Hammond, Valentin, 2007). Furthermore, efforts continue toconstruct databases relating to thepredictive valueof non-clinical assaysto man either through retrospective analysis or through purposelydesigned studies (Ando et al., 2005; Lawrence, Pollard, Hammond &Valentin, 2005; Miyazaki et al., 2005; Olson et al., 2000; Omata et al.,2005; Sasaki et al., 2005; Bowes et al., 2006; Hamon et al., 2006).

There are some examples of promising future areas for thedevelopment of safety pharmacology that further illustrate thechallenges in front of us. In silico approaches, using computationalmodels have been developed to predict the hERG potency of NCEs(Egan, Zlokarnik & Grootenhuis 2004). Mathematical models of the

human ventricular action potential are being developed based onactivities at cardiac ion channels. Computer modelling of the heartfrom genes to cells to the whole organ is becoming a reality (Noble,2002; Noble, 2004; Ten Tusscher, Noble, Noble, Panfilov, 2004).In vitro approaches using adult human stem cell-derived cardio-myocytes are being developed. These cells have characteristics ofdifferentiated ventricular cardiomyocyte including a typical actionpotential. The ability to culture and expand human adult stem cellsin vitro provides potential for producing quantities of cardiomyo-cyte-progenitors which could be employed to assess the effects ofNCEs on cardiac ion channels, electrical activity and contractility(Davila et al., 2004). The utilization of semi-automated in vitrohippocampal brain slice assay for assessing the seizure liability ofcompounds is emerging with some promising data (Easter, Sharp,Valentin, Pollard, 2007). In vivo, the organs and tissues of theZebrafish larvae have been demonstrated to present similarities totheir mammalian counterparts at the anatomical, physiological andmolecular levels. This methodology is gaining acceptance in non-clinical safety evaluation, as means of frontloading in vivo studies inthat the assays are amenable to medium/high throughput screening,chronic dosing (i.e., up to several days) and combination therapies(Parng, 2005; Rubinstein, 2003; Spitsbergen & Kent, 2003; Zon &Peterson, 2005). Although, in most instances, it is an in vivophenotype-based (i.e., black box) method of screening, some modelscomply with a conventional strategy of a target-based (i.e., rationaldesign/rational evaluation) approach. Taking this into consideration,models have been developed to assess effects of NCEs on severalphysiological systems (e.g., visual function, seizure liability, gastro-intestinal motility, vasoactive responses; ventricular and bloodpressures; bradycardia and arrhythmia; Richards et al., 2008; Winteret al., 2008; Parng, 2005; Rubinstein, 2003; Spitsbergen & Kent,2003; Zon & Peterson, 2005). Although the preliminary publishedobservations are encouraging, each model would require thorough,extensive validation using a wide range of compounds to ensure itsreliability, robustness, reproducibility and predictive value tomammalian models and ultimately man (see Table 5). One shouldalso consider the inclusion of non-clinical technologies that mayhave a direct translation to the clinical setting, for example use ofechocardiography imaging techniques that enable the measurementof cardiac function in both animals and humans (for review seeHockings, 2006). Example of increased scientific understandinginclude progress made towards (i) analysing of the ECG waveformsand of ECG complexes duration and amplitude and (ii) correcting theduration of the QT interval for changes in heart rate (Fossa et al.,2006; Ollerstam et al., 2007). Another growing area of interest is theintegration of pharmacokinetic/pharmacodynamic analysis into thedesign of SSP studies, in an attempt to understand and predict drugsafety issues arising from patients suffering from pathophysiologicalconditions (i.e., “personalized medicines”), optimise the transla-tional power of non-clinical assays to humans and closely followpertinent technological advances that may help SSP investigators tobetter accomplish their mission (Cavero, 2007, 2008).






The challenge posed by the introduction of new techniques andtechnologies in formulating a risk assessment is to improve andenhance the safe progression of a NCE to the marketplace, whilepreventing unnecessary delays (or discontinuation), based on non-clinical findings that may not be relevant or interpretable in terms ofclinical response or human risk. Ultimately, increases in scientificknowledge and development of new technologies should lead to thedevelopment of new, robust, reliable, reproducible and predictiveassays/models for ADRs that are currently poorly predicted such asarrhythmia or not at all such as headache. In this regard, it would bewise to concentrate efforts on ADRs associated with either highincidence and/or impact. In summary, SSP cannot ignore any scientificand technological innovation that have the potential of improving ourability to detect, predict and eradicate human safety threats.

6.3. Regulatory requirements

The ICH S7B and E14 guidances have been implemented sinceNovember 2005 (Anon., 2005b,c). A guidance focusing on the ‘non-clinical investigation of the dependence potential of medicinalproducts’ has been released that will influence the overall approachto safety pharmacology especially for CNS-targeted NCEs (Anon.,2005d). Moreover, safety pharmacology is considered an importantcomponent to newly emerging regulatory guidance from the US FDAsuch as the ‘safety evaluation of paediatric drug products and non-clinical studies for development of pharmaceutical excipients’. Thediscipline is considered integral to the evolving regulatory strategiesfor safety to contribute to the acceleration of the introduction of NCEsinto clinical phases (c.f., ‘Position paper on non-clinical safety studiesto support clinical trials with a single microdose’; Sarapa, 2003; andthe US FDA, ‘screening investigational new drug application’; Dixit,2004). In its March 2004 Critical Path Report (Anon., 2004), the FDAsuggests that limited exploratory Investigational New Drug (IND)investigations in humans (Phase 0) can be initiated with less, ordifferent, non-clinical support, including the safety pharmacologycomponent, that is required for traditional IND studies becauseexploratory IND (e-IND) studies present fewer potential risks than dotraditional Phase I studies that look for dose-limiting toxicities. In thiscase, the safety pharmacology program should be considered on acase-by-case basis depending on the specific objectives for a given e-IND study. Regulatory interventions in drug safety have become more

Fig. 8. Increasing the extend of safety and secondary pharmacology during the early discov“eradicate” them, therefore leading to the development of safer medicines with fewer ADR

and more stringent as evidenced by the continuous promulgation ofnovel guidelines. Therefore, representatives from the discipline shouldembrace opportunities to shaping the legal regulatory framework ofany safety pharmacology related issues. Representatives from thediscipline should also actively promote a dialogue between key opin-ion leaders and recognised experts from industry, contract researchorganisations, academia and regulatory agencies to foster the integra-tion of suggestions from these partners to develop and adopt opti-mised solutions to recognised safety issues. For instance, scaling downthe usage of animals according to the 3Rs (Reduction, Replacement,Refinement) could profit from such collaboration.

6.4. Training and education

Safety pharmacology also faces the significant challenge ofattracting, training, and certifying investigators in integrativeapproaches to physiology and pharmacology to ensure the future ofthe discipline (Walker & Soh, 2006; Collis, 2006). The paucity oftraining in integrative biomedical sciences has had detrimental longlasting effects such as an impact on i) the development of intactanimalmodels of human function and disease; ii) the skills required toconceptualise biomedical hypothesis and experiments at the level ofthe intact animal; iii) the process of non-clinical and clinical drugdiscovery and development. To be successful and productive anyinitiative will require adequate financial support.

7. Summary and recommendations

SSP are rapidly growing and evolving disciplines that are facingsignificant challenges on scientific, technological, regulatory andhuman fronts. Addressing these challenges will be key in making asignificant impact on reducing the incidence and severity of ADRs inhumans. Although, in the late 1990's, drug-induced QT intervalprolongation and TdP has resuscitated safety pharmacology as astrong, science driven discipline, one should not forget that the “QTinterval is only one element of the ECG, which is only one element ofcardiac function, which is one element of the cardiovascular function,which is only one of the 3 vital functions investigated in safetypharmacology…” (Redfern, W.S., unpublished). One would hope thatincreasing the extend of SSP testing early during the discovery phasesshould enable to identify and eliminate safety hazards, therefore

ery phases should enable to identify safety hazards (red dots) and wherever feasible tos (green dots).


leading to the development of safer medicines with fewer ADRs(Fig. 8). This should be achieved by (i) further developing dynamiccombined and integrated in silico, in vitro and in vivo approaches toSSP; (ii) assessing and integrating new technologies and scientificadvancements and (iii) shaping and implementing regulatory require-ments. Moving forward, there will be a need to (i) carefully designintegrated risk assessments as to rationalise the impact of SSP on drugdevelopment; (ii) evaluate the confidence in the models used in SSPand their translation to humans as to increase the impact of SSP on thedrug development process.

Acknowledgements

The work describe in this review has been made possible throughinteractions and collaborations with many colleagues from AstraZeneca,Academic institutions, Contract Research Organisations and Pharmaceu-tical companies. The authors wish to thank (i) members of the SafetyPharmacology Departments, within Global Safety Assessment for theircontribution over the years; in particular our thanks go to Najah Abi-Gerges, Duncan Armstrong, Russ Bialecki, Joanne Bowes, MatthewBridgeland-Taylor, Alison Easter, Lorna Ewart, Alex Harmer, SilvanaLindgren, Roope Mannikko, Jackie Moors, Karen Philp, Chris Pollard,Helen Prior, Will Redfern, Mike Rolf, Jason Schofield, Matt Skinner,Sharon Storey, Ann Woods; (ii) colleagues within AstraZeneca: ScottBoyer, Leif Carlsson, Jon Curwen, Stewart Davis, Goran Duker, GarethEvans, Chris Lawrence, Karina Meachin, Paul Newbold, Kathryn Owen,Brian Springthorpe, David Roberts, Nick Sanders, Stots Reele, RussWestwood, Roger Yates; (iii) Colleagues in the industry and academicinstitutions: JohnCamm(StGeorges' Hospital), Fred de Clerck (Johnson&Johnson), Sarah Gould (Sanofi-Pasteur), Peter Hoffman (Novartis), MarkHolbrook (Pfizer), Luc Hondeghem (HPC), Dereck Jones (Summit), IanMacKenzie (Covance), Nick McMahon (GSK), Dennis Murphy (GSK),Merle Paule (NCTR), Philip Sager (CardioDX, Inc) Peter Siegl (Independentconsultant), Isobel Strang (Proximagen), Andrew Sullivan (Independentconsultant), Ian Wakefield (UCB), Rob Wallis (Pfizer); and many more…

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