[Methods and Principles in Medicinal Chemistry] Hit and Lead Profiling Volume 43 || In Vitro Safety...

12In Vitro Safety Pharmacology Profiling: an Important Toolto Decrease AttritionJacques Hamon and Steven Whitebread

12.1What is �In Vitro Safety Pharmacology Profiling?�

�Safety pharmacology� is a term which started to be used in the early 1990s,specifically for the in vivo pharmacology assays designed to detect adverse effectsof drugs in preclinical development. At that time, in vitro pharmacology was includedunder the term �general pharmacology�, which encompassed all in vivo and in vitroassays designed to characterize the pharmacology of a clinical candidate, includingboth desired and undesired effects [1–3]. The concept of using general pharmacologyto profile drugs for safety or �pharmacological toxicity� was already well under-stood [4]. In 2001, some guidance for the industry was published (ICH S7A) definingsafety pharmacology studies as those studies that investigate the potential undesir-able pharmacodynamic effects of a substance onphysiological functions in relation toexposure within the therapeutic range and above [5]. While the S7A guidance largelydeals with in vivo safety pharmacology studies, it states that in vitro studies onreceptors, enzymes, transporters and ion channels can also be used as test systemsand data from ligand binding and enzyme assays, suggesting that a potential foradverse effects should be taken into consideration when designing safety pharma-cology studies. We classify these studies as �in vitro safety pharmacology� and theroutine testing of compounds during early drug discovery we call �in vitro safetypharmacology profiling� [6].In vitro safety pharmacology assays have been around formore than 35 years – ever

since the first in vitro pharmacology assays were developed to measure binding oractivity at a specific protein. Initially of course, they were used to discover newmedicines acting through such targets. However, it quickly became clear, especiallyfor those working in the cardiovascular and neuroscience fields, that many of thesetargets were also responsible for unwanted side effects seen in animal experimentsandhumans, and testing (profiling) of newdrug candidates against a number of thesesafety-related targets (also called �antitargets� [7]) was performed.

Hit and Lead Profiling. Edited by Bernard Faller and Laszlo UrbanCopyright � 2009 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimISBN: 978-3-527-32331-9

j273

The best example of a safety-related target is probably the hERG (human ether-a-go-go-related gene) potassium channel. This target is strongly implicated in QTprolongation and can result in the potentially fatal type of arrhythmia, torsades depointes (TdP), which has been one of themain causes of drug withdrawals in recentyears (see Chapter 16). We review more specifically a number of other importanttargets for early in vitro safety pharmacology assessment in the next sections of thischapter.The main aim of in vitro safety pharmacology profiling is to characterize the

secondary pharmacology profile of compounds in discovery, using a core battery ofhuman in vitro assays designed to predict potential adverse drug reactions, theultimate goal being to reduce late stage attrition [8, 9]. In vitro safety pharmacologyprofiling is �nothing new�, but thanks to faster throughput assay technologies, clonedhuman proteins, miniaturization, robotics and a rapidly expanding knowledge base,it can be put tomore efficient usemuch earlier in the drug discovery process to guidemedicinal chemists in the lead selection and optimization phases.We do not address early toxicology profiling in this section, that is, those phenotypic

assays for which molecular targets are hardly known and which attempt to bepredictive of the standard later stage assays, such as those covering genotoxicity,hepatotoxicity or phototoxicity. These are all covered elsewhere in this book.

12.2Examples of Drug Failures Due to Secondary Pharmacology

Of the 16 drugs withdrawn from the market between 1992 and 2002, 15 (94%) werewithdrawn due to toxic events and adverse drug reactions and eight compounds werewithdrawn due to a well defined mechanism of action [10]. As examples, fenflur-amine (Pondimin) and dexfenfluramine (Redux), two appetite-suppressant agents,were withdrawn due to cases of valvular heart disease linked to a secondary activity atthe serotonin 5HT2B receptor. Pergolide (Permax), a drug used for the treatment ofParkinson�s disease, was removed from the market in 2007 for the same adversereaction and was also shown to display secondary 5HT2B agonist activity. Rapacur-onium (Raplon), a rapidly acting, nondepolarizing neuromuscular blocker used inmodern anaesthesia, to aid and enable endotracheal intubations, was withdrawnfrom the United States market by the manufacturer in 2001 [11]. This was due to arisk of fatal bronchospasm linked to amuscarinicM2 antagonist activity. Amineptine(Survector), an atypical tricyclic antidepressant, was withdrawn from the market formultiple adverse effects including acneiform eruptions, hepatotoxicty and addiction.The latter effectwas attributed to its dopaminergic properties. Anolder example is thecase of PCP (Sernyl), introduced as a dissociative anaesthetic agent in 1963, butwithdrawn two years later due to hallucinations experienced by about 30% of thepatients. This adverse effect was the reason for the use of PCP as a drug on the streetin the 1970s under the names �angel dust� or �peace pill�. This effect is linked to itsNMDA antagonist property, but also to its complex pharmacological profile affecting

274j 12 In Vitro Safety Pharmacology Profiling: an Important Tool to Decrease Attrition

monoamine transporters, the cholinergic system, the sigma and opioid receptors andsome ion channels [12].A number of mechanism of actions are known to be linked with serious adverse

effects which prevent some compounds from reaching the market. One example isPDE4 inhibition. Despite the efforts of most major pharmaceutical companies todevelop safe PDE4 inhibitors for the treatment of asthma or COPD, none have so farbeen marketed, due in particular to emetic side effects most probably linked withtheir primary pharmacology. In other cases, drugs were not withdrawn from themarket, but their prescription decreased dramatically following the late characteri-zation of adverse effects. Such is the case with MAO inhibitors. The use of thefirst generation of nonselective monoamine oxidase inhibitors as neuropsychiatricdrugs was seriously limited, mainly because of what became known as the �cheesereaction�. This reaction is due to the presence of tyramine in many fermentedfoodstuffs including cheese that are not deaminated by MAO in the intestine of thepatients treated withMAO inhibitors. The consequence can be a severe hypertensivereaction induced by the absorbed tyramine [13].A different example concerns fialuridine, a uridine analog that was being devel-

oped for the treatment of hepatitis B before it was stopped in Phase II trials, due tofatal mitochondrial hepatotoxicity [14]. This hepatotoxicity was found to be enhancedby filuridine being actively transported into the mitochondria by the humanequilibrative nucleoside transporter (hENT1, adenosine transporter). It was thenshown that, unlike in humans, the mouse ENT1 is not incorporated into themitochondrial membrane [15]. This explains why the toxicity was not picked up inanimal experiments. Although not a true secondary pharmacology, screening ofcompounds in a hENT1 assay might prevent such deaths in future. Speciesdifferences are common and this example demonstrates that human in vitro assayscould be more predictive of human ADRs than animal experiments.It is important to keep in mind that about 30% of the late failures during drug

development occur due to toxicity and safety issues [16]. Also, once a drug reachesthe market, the chance of receiving a black box warning is rather high [17],sometimes dramatically impacting the sales. Furthermore, ADRs are believed tobe a leading cause of death in theUnited States [18]. All of these facts show the needfor an early characterization of the potential adverse effect profiles of new chemicalentities (NCE), starting early in the discovery stage. This need is even reinforced byan apparent increase in regulatory caution by the FDA, which possibly led to adecrease of drug approvals and an increase in the late drug discovery stage attritionrate [19].

12.2.1Components

12.2.1.1 Target SelectionAkey point for the success of a good in vitro safety profile is the selection of the targetsor pathways to include in such a profile in order to cover both a large spectrum of

12.2 Examples of Drug Failures Due to Secondary Pharmacology j275

adverse effects and the pharmacological space. Each target has to be linked to a knownadverse or unwanted effect. It should be noted that a pharmacological effect may bethe �wanted� or primary therapeutic effect in some cases, but in most other cases itwould be �unwanted�. The hypnotic or sedative agent zolpidem (a benzodiazepinereceptor agonist) for instance is useful for its intended clinical use, but such aproperty is not wanted for most medications. So, a first strategy is to start from theknown failures due to safety issues: Serotonin 5HT2B agonism as an example toavoid �fen-phen�-type disasters or PDE4 inhibition to avoid emetic side effects.However, the different types of adverse effects need to be considered.Adverse drug reactions (ADRs) are classified into fivemain types: A–E [20, 21]. The

main type, A, which accounts for about 75%of all ADRs, is caused by dose-dependentprimary or secondary pharmacology. If themolecular target whichmediates the ADRis known, then this type can be predicted by in vitro safety pharmacology profiling.By definition, the idiosyncratic toxicities, or type B, which account for most of theremaining ADRs, cannot be predicted (although this may change as they becomebetter understood). However, some teratogenic effects which are included in type Dcan be predicted, for example, those which are mediated through the endothelin orretinoic acid receptors [22, 23].Table 12.1 shows the most commonly occurring type A ADRs associated with the

clinical use of drugs, sorted by therapeutic areas. These would be the main ADRswhich in vitro safety pharmacology assays should aim to detect. It is clear that,especially at an early stage, an oncology program must consider mainly life-threat-ening adverse effects while a program on a chronic treatment for hypertension or thetreatment of nasal congestion as examples must consider a wider range of potentialadverse effects.Each of these adverse effects is often related to different targets or pathways. For

instance, sedation could be linked to an interaction with the histaminergic, alpha2adrenergic or opioid receptors, but also with GABAergic transmission and manyother targets. The list of potential targets is extensive when considering effects invarious organs.

12.2.1.2 Target AnnotationCritical to any safety prediction based on in vitro safety pharmacology profiling isan accurate and comprehensive knowledge base to enable links to be madebetween activities at individual targets and side effects seen in the clinic. Tradi-tionally, this is done by searching the primary literature for hints from in vitro andin vivo animal pharmacology experiments. This is of course still an essential sourceand can be the only way for those targets where no known ligand has yet beentested in humans. Ideally, however, the annotation should be based on knownhuman clinical evidence, such as the primary and secondary pharmacologies ofknown drugs and phenotypic information from human genetic mutations. Thechallenge is to link data on known side effects to the targets through which they aremediated. This requires an overall assessment of all the available in vitro and in vivodata from animals and humans. One way to do this is to apply in silico predictionmodels (see Chapter 13).


Literature searching has been made much easier and faster with the currentgeneration of search engines, but much of the drug side effect and related data hasnot been published in the primary literature and is only to be found in sources such asunpublished regulatory reports and drug labels. Information on drugs which failedduring development is particularly difficult to find. However, much of this informa-tion has been collated and made available through web-based databases, some ofwhich are freely available, although others are commercial. A list of some of the freelyavailable databases is given in Table 12.2. Some restrictions may apply to their use.Some commercial databases are listed in Table 12.3.

12.2.1.3 Examples of In Vitro Safety Pharmacology Profiling PanelsDifferent panels of assays are most generally used at different stages of the drugdiscovery process covering only themost critical targets for safety (targets linkedwithlife-threatening adverse effects or safety targets known to display a high hit rate) or abroad range of targets potentially involved in many different diseases. Someexamples can be found among the in vitro safety profiles offered by different contract

Table 12.1 Major type A adverse effects associated with the clinical use of drugs.

GI tract Hematology Dermatology CardiovascularHepatitis/hepato-cellular damage

Agranulocytosis Erythemas Arrhythmias

Constipation Hemolytic anemia Hyperpigmentation HypotensionDiarrhea Pancytopenia Photodermatitis HypertensionNausea/vomiting Thrombocytopenia Eczema Congestive

heart failureUlceration Megaloblastic anemia Urticaria Angina/chest painPancreatitis Clotting/bleeding Acne PericarditisDry mouth Eosinophilia Alopecia Cardiomyopathy

Endocrine Respiratory Psychiatric MusculoskeletalThyroid dysfunction Airway obstruction Delirium, confusion Myalgia/myopathySexual dysfunction Pulmonary infiltrates Depression RhabdomyolysisGynecomastia Pulmonary edema Hallucination OsteoporosisAddison syndrome Respiratory

depressionSchizophrenia/paranoia

Galactorrhea Nasal congestion

Metabolic Renal Neurological Ophthalmic/OtologicalHyperglycemia Nephritis Seizures Disturbed color visionHypoglycemia Nephrosis Tremor CataractHyperkalemia Tubular necrosis Sleep disorders Optic neuritisHypokalemia Renal dysfunction Peripheral neuropathy RetinopathyMetabolic acidosis Bladder dysfunction Headache GlaucomaHyponatremia Nephrolythiasis Extrapyramidal effects Corneal opacityHyperuricemia Drowsiness Deafness

Vestibular disorders


Table 12.2 Some freely available web-based databases providing drug annotation.

Name and URL Comment

FDA Center for Drug Evaluation andResearchhttp://www.fda.gov/cder/site/default.htmhttp://www.fda.gov/cder/aers/default.htm

Includes drug information and regulatoryguidance. Useful pages within this site includethe Adverse Event Reporting System (AERS,a web-based reporting system for adverseevents; this also gives drug safety and ADRinformation, including FDA safety alerts),the FDA Orange Book and Drugs@FDA(listed separately).

FDA Electronic Orange Bookhttp://www.fda.gov/cder/ob/default.htm

Up-to-date information on all drug approvalsand withdrawals in the US. Includes appli-cant, dosage form, proprietary name, dateapproved, patent information. Does notinclude drug label information or safetyinformation.

Drugs@FDAhttp://www.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm

Similar to the Orange Book, but drugs listedalphabetically, therefore easier to browse.

RxList The Internet Drug Indexhttp://www.rxlist.com/script/main/hp.asp

Alphabetically index of drugs (trade name only),giving detailed information, including struc-ture, indications, safety. Provides a ranked list ofthe top 200 most prescribed drugs.

MedlinePlus drug informationhttp://www.nlm.nih.gov/medlineplus/druginformation.html

Alphabetical index of drugs, herbs and supple-ments listed separately. Indications, ADRs,but no structure. For herbs and supplementsgrades are given according to whether theclaimed activities are scientifically provenor not.

DailyMed (current medicationinformation)

Alphabetical listing of drugs. Provides FDAapproved drug labels.

http://www.dailymed.nlm.nih.gov/dailymed/about.cfm

DrugDigesthttp://www.drugdigest.org/DD/Home

Similar to DailyMed, but search only, nobrowsing.

Common terminology criteria for adverseevents (cancer therapy evaluation program)

Categorizes ADRs according to MedDRAterminology, including severity grades.

http://resadm.uchc.edu/hspo/ investigators/files/Common%20Toxicity%20Criteria_version%203.0.pdf

PharmGKB (the pharmacogenetics andpharmacogenomics knowledge base)http://www.pharmgkb.org/index.jsp

Free database, but registration requested.Genes, pathways, drugs and diseases database.No structures.


research organizations (CROs), which are heavily used by most of the small andmajor pharmaceutical companies:

. The �general safety profile� from CEREP including 155 in vitro assays specificallydesignedtoidentifypotentialsideeffectsofdrugcandidates(not inaspecificpathology).

Table 12.2 (Continued)


BIDD (BioInformatics and DrugDesign group)

Various databases, including: drug adversereaction target (DART) database.

http://xin.cz3.nus.edu.sg/group/sitemap.htm

Drug Withdrawalshttp://www.ganfyd.org/index.php?title¼Drug_withdrawals

List of UK drug withdrawals and changesin indication for use.

Fact and Comparisonshttp://online.factsandcomparisons.com/index.aspx?

Comprehensive monographs on individualdrugs and drug classes. ADRs given withlevels of incidence. Also available onCD-ROM.

List of bestselling drugs Ranked list of the 200 best selling drugs.http://en.wikipedia.org/wiki/List_of_bestselling_drugs

Online Mendelian Inheritance in Man(OMIM)http://www.ncbi.nlm.nih.gov/sites/entrez?db¼OMIM

Catalog of human genes and genetic disorders.Can provide genetic evidence for linking ADRsto interactions with specific targets.

Gene Cardshttp://www.genecards.org/

Comprehensive genomic and proteomicinformation.

NIMH Psychoactive Drug Screening Pro-gram: Receptor Affinity Database

Receptor affinities of drugs and referencecompounds.

http://kidb.bioc.cwru.edu/pdsp.php

DrugBank [24]http://www.drugbank.ca/

Extensive chemical and pharmacological an-notation of 4800 compounds, including >1480FDA-approved smallmolecule drugs and>3200experimental drugs. Annotation includes drugtarget and indication information, but notADRs.

Matadorhttp://matador.embl.de

Amanually annotated database linking drugs totargets.

UN list of banned, withdrawn,severely restricted or not approvedpharmaceuticals [25]

Comprehensive world list of withdrawn drugsgiving the reasons for withdrawal.

http://www.un.org/esa/coordination/CL12.pdf


. The �adverse reaction enzymes� profile fromMDSPharma including 41 enzymaticassays to predict moderate to serious adverse effects.

. The �LeadProfilingScreen� fromMDS Pharma dedicated also to the adverse effectprediction.

. The �general side effect profile� from Caliper with 65 different targets.

. The �broad safety� and �focused safety� panels of functional GPCR assaysoffered by Millipore.

As opposed to selectivity profiling panels which generally include only relatedtargets from the same family, the in vitro safety pharmacology panels are composed ofa high diversity of targets, including representatives from GPCRs, ion channels,different families of enzymes, transporters and nuclear receptors, the main criteriabeing their link with ADRs. The GPCRs are often the most important target familyrepresented in these panels. It reflects the fact that more than 30% of the marketeddrugs are GPCR modulators and that most diseases can be impacted by someGPCRs [26]. With the increase in kinase drug discovery targets, broad kinaseselectivity profiling has become very important. However, much less is known aboutthe safety relevance of individual kinases than, for instance, for GPCRs. This is inpart due to the fact that there are relatively few drugs for kinase targets in theclinic which could provide the necessary ADR annotation. This is certainly a fieldwhich needs expanding in future. Table 12.4 gives some examples of �safety targets�with the potential consequences of target interaction.

12.3Processes

12.3.1Assay Requirements and Technologies

The first requirement of an in vitro safety profiling assay is to be as predictiveas possible of an adverse event, given all the limitations of in vitro assays and theother important parameters to consider in combination, such as physicochemical

Table 12.3 Some commercial web-based databases providing drug annotation.


GVK Biosciences http://www.gvkbio.com/informatics.htmlProus Science Integrity http://integrity.prous.comPharmaPendium http://www.pharmapendium.comGeneGo http://www.genego.com/Biovista http://www.biovista.com/MedicinesComplete http://www.medicinescomplete.com/mc/Facts and Comparisons http://www.factsandcomparisons.com/


Table12.4

List

oftargetsofteninclud

edin

invitrosafetyph

armacolog

ypa

nelsan

dthepo

tentialm

ajor

consequences

ofreceptor

interaction.

Targets

Possibleconsequences

oftarget

interaction

Serotonin

5-HT1A

receptor

HTR1A

Agonism

:Indu

cesabehaviouralsyn

drom

echaracterizedby

flatbo

dypo

sture

andheadweavingin

rats–

Inhum

ans,5-HT1A

agon

ists,such

asBuspirone,

indu

celig

ht-headedn

ess,miosis,nervousnessor

agitation.T

hey

may

also

indu

cehypothermia,d

ecreasebloodpressure

andheartrate.

Antagonism:N

oside

effectsclearlydefined

–May

havecogn

itionen

han

cingeffectsusefulfor

Alzheimer

disease�s(see

Lecozotan).

Serotonin

5-HT2B

receptor

HTR2B

Agonism

:Cardiac

valvulopathy,fibrob

last

mitog

enesis,h

ypertension

.Antagonism:N

oside

effectsclearlydefined,b

utcardiaceffectscannot

beexcluded,

especially

atem

bryonic

stage.

Adenosine2a

receptor

ADORA2A

Agonism

:Inhibitionof

platelet

aggregation,anti-in

flam

mationan

dneu

roprotective

effects,coronary

vasodilation

,decreased

bloo

dpressure,increasedplasmarenin

activity

andsleepindu

ction.

Antagonism:Increasedplatelet

aggregation,h

ypertension

,nervousness(tremor,agitation

),arou

sal,

insomnia,cerebralan

dcoronaryvasodilation

(inmicrovesselson

ly).

Adenosine3receptor

ADORA3

Agonism

:Im

munosupp

ression,hypoten

sion

,an

ti-ischaemic

(cardiop

rotective),pro-isch

aemic

(cereb

ral),cellnecrosis,cellproliferationan

dan

giog

enesis.

Antagonism:m

ightcause

myocardialischaemia,p

roinflam

matoryeffects,hyperten

sion

andinterfere

withtheregu

lation

ofcellgrow

th.

Adren

ergicAlpha1A

receptor

ADRA1A

Agonism

:Smooth

muscle

contraction

(prostatein

particular,effectson

thelower

urinarytract)an

dcardiacpo

sitive

ionotropy,arrhythmia.

Antagonism:O

rthostatichypoten

sion

andothe

rbloo

dpressure

relatedadverseeffectsan

dim

pact

onvariou

saspectsof

sexual

function.

Adren

ergicAlpha2A

receptor

ADRA2A

Agonism

:Sedation–an

esthetic-sparingeffect

–central

hypoten

sive

andhypo

thermic

action

s,hyperglycem

ia.

Antagonism:M

ayindu

cegastrointestinal

prok

inetic

effects.

(Continu

ed)

12.3 Processes j281

Table12.4

(Contin

ued)

Targets


oftarget

interaction

Adren

ergicBeta1receptor

ADRB1

Agonism

:May

stim

ulate

cardiacmuscle(increase

heartrate

andforceof

contraction

)an

dcontributes

totherelaxation

ofbloodvessels.

Antagonism:M

aystress

cardiovascularperforman

ce.

Dop

amineD1receptor

DRD1

Agonism

:May

indu

cedyskinesia,extremearou

sal,locomotor

activation

,vasod

ilatation

andhypoten

sion

.Antagonism:T

remor.

Dop

amineTran

sporter

SLC6A

3Inhibitorswill

preven

tdo

pamineuptake(cocaine-lik

edrugs).Im

portan

teffectson

locomotor

activity,

motivation,rew

ardan

dcogn

ition,d

opam

inergichyperactivity,A

DHD,d

epression,P

arkinsonism,

psychotic

disorders,seizure,d

ystonia,d

yskinesia.

HistamineH1receptor

HRH1

Agonism

:Allergic

reaction

.Antagonism:S

edation.

Muscarinic

M1receptor

CHRM1

Agonism

:May

increase

bloo

dpressure,h

eartrate

andsympathetic

outflow

–May

beinvolved

inthe

regu

lation

ofcircadianrhythm.

Antagonism:D

isruptionof

cogn

itivefunctionssuch

aslearningan

dmem

ory.

Muscarinic

M2receptor

CHRM2

Agonism

:Vagaleffects(key

role

inthecontrol

ofhe

artrate

andsm

ooth

muscleactivity);Bradycardia.

Antagonism:M

ayindu

cecardiacside

effects(palpitation

s,dysrhythmia)or

periph

eral

edem

a.bron

chocon

strictioncanresultfrom

presyn

apticM2receptor

antago

nism

ifpo

stsynapticM3receptors

arenot

also

blocked.

Opiatemureceptor

OPRM1

Agonism

:Analgesia,

Sedation

,Physical

dependence,B

owel

dysfunction,R

espiratory

depression

,Mod

ulation

ofcough

reflex.

Thrombo

xaneA2receptor

TBXA2R

Agonism

:Vaso-,b

ronchocon

striction,p

lateletaggregation,m

yocardialischem

ia,h

eartfailu

re.

Antagonism:cou

ldcause

bleedingby

inhibitingplatelet

aggregation.


Progesteron

ereceptor

PGR

Agonism

:May

cause

loss

ofbo

nemineral

density,b

leedingdisordersan

dprom

otebreast

cancerin

females,a

ndgestagen

iceffectsin

males.

Antagonism:can

causeexcessivemen

strual

bleeding,

uterinecram

ping,

endo

metrial

hyperplasia;

contraindicatedin

youngfemales.

Nicotinic

receptor

central

CHRNA2

Agonism

:May

play

arole

inthemod

ulationof

anumberof

neu

rotran

smitters

(e.g.,do

paminergic,

serotoninergic,glutamatergic)

witheffectson

cogn

itivean

dmotor

function.T

hey

exhibitan

algesic

activity

andmay

stim

ulate

autonom

iccardiovascular,respiratory

andgastrointestinal

function

(palpitation

/nau

sea).

Antagonism:M

uscle

relaxants

andan

ti-hypertensive

agen

ts.A

numberof

neu

rotoxins

(e.g.,bu

ngarotoxin,con

otoxins)

displayalso

anan

tago

nistaction

ondifferen

tnicotinic

acetylcholine

receptor

subtypes.

PCPreceptor

(NMDAchan

nel)

GRIN

1Agonism

:Anestheticprop

erties,m

ayindu

cepsychosis(schizop

hren

ialik

e),hallucination,delirium

and

disorien

tedbehavior,may

cause

seizures,neu

rotoxicity.

Epiderm

algrow

thfactor

receptor

(HER1)

EGFR

Activation:

Increasedcellproliferation,angiog

enesis,m

etastasisan

ddecreasedapop

tosis.

Inhibition:S

kinrash,can

cermetastasis.

CathepsinD

CTSD

Inhibition:N

eurodegeneration.

Phosph

odiesterase3A

PDE3A

Inhibition:M

ayindu

cepo

sitive

cardiacionotropiceffects.

Phosph

odiesterase4D

PDE4D

Inhibition:E

mesis,A

rteritis.

Mon

oamineOxidase

AMAOA

Inhibition:M

ayindu

cesevere

hypertensive

crisis(know

nas

�theCheese

reaction

�)–Cen

trallymediated

side

effectssuch

astheserotonin

syndrom

e,dizziness,blurred

vision

andweakn

ess.

Cyclooxygen

ase-1

PTGS1

Inhibition:M

aydisrupt

normal

cellu

larho

meostasisan

ddisrupt

theprod

uctionof

prostaglan

dins,

causingelevated

levelsof

gastrointestinal

toxicity,g

astric

bleeding,

pulm

onarybleeding.

Table12

.4(Contin

ued)

Targets


oftarget

interaction

12.3 Processes j283

and ADME properties of the compounds. The assay has to be robust, reproducible,cost-effective, medium-throughput and use a small amount of compound. Therevolution in this field came with the development of high-throughput screening(HTS) technologies. These HTS assays are the starting point of most therapeuticprojects in all major pharmaceutical companies and, despite their limitations,allowed the identification of a number of NCEs [27]. The same assay technologiescan be used for the early assessment of ADRs. Initially, profiling assays were largelybased on radioligand-binding filtration assays, often nonhuman. For the low numberof compounds that were typically tested in the past for safety, these were perfectlyadequate. However, the newer screening technologies for binding or enzymaticinhibition assays, for example, scintillation proximity assay (SPA), fluorescentpolarization (FP) and fluorescence resonance transfer (FRET) and for functionalcell-based assays (e.g.,measuring cAMP, IP, calciumorGTP) allowed safety profilingto be moved earlier in the drug discovery process where many more compounds canbe tested. The required throughput is not so much �high�, but �medium� and �fast�.For this reason, other medium-throughput technologies, such as automated patchclamp systems for ion channels, high content imaging technologies and/or technol-ogies described as more physiologically relevant such as those using impedancemeasurements, find their place in in vitro safety profiling. With the recent accent oncardiosafety profiling, the automated patch clamp systems in particular have becomeroutine technologies to functionally test for ion channel blockers such as hERG,sodium (Nav1.5) and calcium (Cav1.2).

12.3.2Binding and/or Functional Assays

Should a bindingassay or a functional assay beused as theprimaryprofiling assay for agiven target; and which technology is most suitable? Both formats using varioustechnologies are available for most targets from the various commercial providers.Depending on the target and mechanism of action(s) which need to be assessed, oneassay may be better to use than another, that is, more predictive, more robust, or lessexpensive. Radioligand-binding studies were the first to be used on a large scale anddemonstrated their usefulness in in vitro safety pharmacology profiling panels. Theseassays are generally very robust, easy to automate, high-throughput and cost-effective;and their predictivity can be good enough as a primary assay. Indeed, direct correla-tions between some ADRs and ligand-binding activities for some receptors can bedemonstrated. Figure 12.1 shows some of these correlations, established first byCEREP [28], and which we confirmed at Novartis. Such correlations are very usefulbecause they can give an indication as to howpotent a compound has to be at the targetbefore anADRbecomes a possibility (ADMEdata always have to be taken into accountas well). It should however be pointed out that, even though a correlation can bedemonstrated between certain target/ADRpairs, it does not necessarilymean that thatparticular target actually mediates the ADR. Due to similar pharmacophores betweenrelated and even unrelated targets, all might show such a correlation, while actuallyonly one might mediate the effect. The latter might not even be included in the safety


panel andmight not even be known as the mediator of the effect. A typical example isthe similarity between the different dopamine receptors.Many compounds show littleselectivity between all five dopamine receptors; and the correlation between tremorand dopamine D1 receptor binding (shown in Figure 12.1) can also be found with allother dopamine receptors.Binding assays generally require the availability of a high affinity ligand that can be

chemically labeled (e.g., with a radioactive isotope or a fluorescent group) and oftenrequire overexpression of the target of interest in a given cellular system, which is notalways easy to achieve. Another limitation is that a ligand-binding assay cannotusually provide any information on the mechanism of action (agonist/activator,antagonist/inhibitor) and cannot detect an indirect modulator of a given target. ForGPCRs,when theADR is clearly related to one particularmechanismof action, itmaybe of interest to consider the use of a functional assay as a primary assay rather than afollow-up to a binding assay. Especiallywhen looking forGPCRagonism, a functionalassay is often more sensitive than a binding assay and also detects compounds withallosteric effects. A cAMP quantification agonist assay on the histamine H2 receptor

Figure 12.1 Examples of in vitro bindingassays correlating with ADRs. Marketed drugswith known ADR profiles were tested in threedifferent in vitro receptor binding assays andtheir IC50s (concentration required to achieve50% inhibition) were determined. Thepercentage of drugs having (black bars) andnot having (dotted bars) the stated ADR is

plotted for each IC50 bin (X-axis). The receptor/ADR pairs dopamine D1 and tremor (a),histamine H1 and somnolence (b), and hERGand arrhythmia (c) all show a marked increase inthe presence of the ADR in the lower IC50 bins. Asa control, the pair adenosine Ad3 and arrhythmia(d), shows no correlation, with the arrhythmiadrugs evenly distributed across the IC50 bins.

12.3 Processes j285

is much more valuable in an in vitro safety pharmacology profiling panel than abinding assay for the detection of a secondary histamine H2 agonist activity. Thisactivity is known to induce positive inotropic effects on the human ventricle(amthamine is a cardiotonic agent) and potentially to stimulate gastric acid secretion,while H2 antagonists are known to be rather safe – ranitidine (Zantac) is among themost prescribed drugs without major adverse effects, although overdosing can causemuscular tremors, vomiting, dizziness and rapid respiration.Likewise, functional agonist assays for the serotonin 5HT2 receptors (5HT2A,

5HT2B, 5HT2C) are more relevant for safety than binding assays. The latter tend togive a very high hit rate, but most of the binders are antagonists, for which no majorADRs have been reported.Finally, and this could be the future of in vitro safety pharmacology profiling,

new functional technologies, described to be more physiologically relevant, arebeing developed and may give an additional advantage to functional technologiesover bindingassays, especiallywhen associatedwith theuse ofprimary cells.However,there will always be a need for some binding experiments as primary profiling assaysor follow-up assays in order to confirm the interaction with a given target.

12.3.3Processes and Logistics

Even though profiling assay technologies are highly similar to screening assaytechnologies, the process and automation required are completely different andmore complex. Instead of dealing with a very high number of compounds and platesto run in a given assay (as in high-throughput screening), one has to deal with a lowernumber of plates to test in a set of diverse assays. Fully automated systems need to besufficiently flexible to handle assays using different reagents, conditions and tech-nologies within the same run. They have to integrate different readers and requiresophisticated scheduling software. Compoundmanagement can also be complex, asdifferent sets of compounds often need to be tested in different panels or evenindividual assays.A fast turn-around time has to be maintained, as it is part of the project flowchart

and contributes to the optimization cycles of the different chemical scaffolds,together with the physichochemical properties and in vitro ADME data. At Novartis,rather than testing initially at a single concentration, we decided to performdirect fullIC50 determinations in order to ensure not only a good turn-around time, but moreimportantly a good data quality. We thereby avoid the cherry picking and retesting ofactive compounds. The additional consumption of reagents when doing direct IC50

determination is largely compensated by the reduction of compound managementtasks. Also, one can easily differentiate between inactive compounds and low-activecompounds (micromolar range activities) and see the potential solubility issues(compounds showing activity at low concentrations, but not at the highest concen-trations due to precipitation in the incubation medium). Each assay run includes atleast one reference compound which is most generally included in each plate as anintra-plate control. A deviation of no more than threefold is generally accepted with


the average value acquired during the assay validation step. Also, parameters such asthe Z0 value, the signal to background ratio, the percentage of nonspecific signal andother parameters linked with each technologies are systematically calculated forfurther validation.Finally, all the data need to be registered in the company database together with all

the details on how they were obtained. This is important, as it may contribute,sometimes several years later, to a drug data package to be used either inside thecompany for different decision points or externally as part of a regulatory dossier forhealth authorities. Although in vitro safety pharmacology profiling data are notofficially requested by regulatory bodies, it is often one piece of data which helps toprove the good safety and selectivity profile of a drug. Data registration is alsovery important to get the full benefit of profiling activities, as it becomes a very richsource for data mining, allowing the development of in silico tools to drive drugdiscovery (see Chapter 13) or providing starting points for new therapeutic projects.

12.4Application to Drug Discovery

12.4.1How and When to Use In Vitro Safety Pharmacology Profiling

After the hit discovery process (often using high-throughput screening), early drugdiscovery is generally split into a �hit to lead� phase and a �lead optimization� phase,followed by the selection of development candidates (DCs) (Figure 12.2). In vitro

Figure 12.2 The use of in vitro safety pharma-cology profiling during early drug discovery.A smaller (primary) panel of targets is usuallysufficient during the hit to lead phase andlead optimization phases of a drug discoveryprogram to detect promiscuous scaffolds and

to pick up the most commonly occurringliabilities. At each phase transition, it is advisableto test the candidate compounds in a broader(secondary) panel, to detect the less commonlyoccurring liabilities. The broadest panel is used totest the final selectionofdevelopment candidates.

12.4 Application to Drug Discovery j287

safety pharmacology profiling can be applied to the first of these phases with the aimof identifying and avoiding chemical series which are inherently promiscuous.At this stage, the number of compounds that need to be tested can be relativelyhigh, but it is usually sufficient to test them in a relatively small, but diverse (primary)panel of assays, thereby keeping the cost low. Most compounds at this stage have arather low affinity for the primary target, often not very different from any off-targetaffinities. It is often believed that, as the primary target affinity is optimized, the off-target activities are lost. While this can happen, in most cases it does not succeed.At lead selection, after which typically more chemistry effort is invested, the

selected compounds can be profiled in a broader (secondary) panel of assays,hopefully confirming the selective nature of the leads. If this is the case, spotchecking in the primary panel through the optimization phase may be sufficientto ensure selectivity is retained while the required potency at the primary target isachieved. If the selected leads are still rather promiscuous, or certain individualunwanted liabilities remain, these should be monitored by testing in the primarypanel (or in additional individual assays) and improved upon during lead optimiza-tion. The broader panel can then be applied again to the selected developmentcandidates for a final check and these may even be extended further to additionalspecialized panels for added security.At this point in the program, key information fromother in vitro and in vivo studies

become available, such as efficacy, pharmacokinetics, potential drug–drug interac-tions, metabolites and some early toxicology. All of these factors combined enable afirst integrated risk assessment to be made.In vitro safety pharmacology profiling can also be applied to other stages of the drug

discovery process. For instance, a broad profile may discover an unknown target foran orphan drug or during target feasibility studies, before starting a drug discoveryprogram, any known reference or competitor compounds can be tested for an earlyassessment. Using profiling, salvinorin A was found to be the first naturallyoccurring non-nitrogenous opioid receptor subtype-selective agonist [29] and thisresult suggested that kappa opioid receptors may play a prominent role inthe modulation of human perception. A study by Elphick et al. investigating theinhibition of human polyomavirus JCV infection by antipsychotics highlighted theimportance of pharmacological profiling in discovering roles of receptors in dis-eases [30]. It is also by using the profiling of a number of antipsychotics that a linkbetween muscarinic M3 receptor and type 2 diabetes was shown [31]. During later-stage development, new metabolites, especially human, may be discovered whichshould also be tested, plus competitor compounds as they become known. In vitrosafety pharmacology profiling will also be very useful for back-up programs toimprove on earlier compounds which suffer from unfavorable safety profiles.

12.4.2Pharmacological Promiscuity and Its Clinical Interpretation

Most antipsychotic compounds are known to bind to many different receptors,especially those for serotonin,dopamineandhistamine [32, 33]. Suchpharmacological


promiscuity is possibly required for certain central indications, such as psychosis,depression, Alzheimer�s disease [34–37] and possibly also for cancer [38], but it iscertainly also the source of themany known side effects of such drugs [39, 40]. Severalauthors use the term polypharmacology for this phenomenon [41–43], but this termwas introduced for the broad pharmacology obtained by combination therapies,irrespective of the number of targets hit [44, 45]. Due to the higher risk of side effectsoccurring with pharmacologically promiscuous compounds, it makes sense to pro-mote compounds during the research phase which are inherently selective.The target hit rate (THR) was introduced to quantify the phenomenon of pharma-

cological promiscuity [46, 47]. THR is defined as the number of targets bound by adrug at a given concentration, expressed as a percentage of all targets tested, forinstance in a panel of in vitro safety pharmacology assays. THR10 is the THR wherea �hit� is defined as >50% inhibition at 10mM. Compounds with a THR10 of <5%were defined as �selective�, 5–20% as �medium promiscuous� and >20% as�promiscuous�. A similar quantification was used by Leeson and Springthorpe [48],except that they used >30% inhibition at 10mM. The THR is not a constant term,as it depends heavily on the number of targets tested and the degree of target diversity.It can however be used in a standardized profiling panel calibrated against knownpromiscuous compounds. The THR classification given above is used in the Novartisin vitro safety pharmacology profiling panel where >50 targets have been tested.A THR analysis of 293 marketed drugs demonstrated that over 60% were selective

(THR <5%; Figure 12.3). This group included 22 antipsychotics, which were allpromiscuous. A subset of 132 of themost prescribed and best selling drugs, excludingany withdrawn drugs or antipsychotics, had only 5% �promiscuous� but 73%

Figure 12.3 TargetHit Rates (THR) formarketeddrugs andNovartis compounds. A THRof>20%is considered to be �promiscuous� (black),5–20% �medium promiscuous� (hatched) and0–5% �selective� (white). 65% of the �MarketedDrug� set of 293 compounds, includingantipsychotics and withdrawn drugs, wereselective, whereas only 13% were promiscuous.

Promiscuity dropped to 5% in a subset of 132most often prescribed and top selling drugs(�Top selling�). This contrasts with the 31%promiscuity found in the Novartis clinicalcandidates (CCs) which were discontinued. Themost recent Novartis development candidates(DCs) on the other handwere very comparable tothe best selling marketed drug set.

12.4 Application to Drug Discovery j289

�selective�. In contrast, of 26Novartis clinical candidates (CCs)which failed to advanceto human trials between 2001 and 2007, only six were selective. Novartis developmentcandidates (DCs) from 2004 becamemore selective and can now be called �marketeddrug-like�, which suggests that compoundswhichmight previously have failed duringextensive animal toxicology studies are now being selected out during the researchphase and should reduce the attrition rate in development.It is important to point out that when a compound is pharmacologically

promiscuous in a panel of 50 diverse targets, it is highly likely that the compoundalso hits several additional targets which are not included in the panel, therebyfurther increasing the liability risk. The reasons why certain compounds are morepromiscuous than others and how promiscuity can be avoided is discussed inChapter 13.If pharmacological promiscuity is strongly reduced and only very few activities

remain, a risk assessment has to be performed based on the therapeutic index.

12.4.3Relevance of Potency and Therapeutic Index (TI)

In most drug discovery programs, the first goal is to achieve a high potency at theprimary target and also a good selectivity against closely related targets. Optimally, invitro potency correlates perfectly with activity in vivo, in both animals and humans,and the chosen compound is highly bioavailable, allowing a very low maximumfree circulating concentration (Cmax) at the therapeutic dose. Everything beingequal, the fold selectivity against other targets in vitro can then be used to estimatethe safetymargin. Unfortunately there are somany factors working against this idealsituation that the fold selectivity in vitro is rarely equivalent to the actual therapeuticindex (TI) in humans. Amicromolar off-target activity may still be important, even ifthe affinity for the primary target is in the nanomolar range or lower.

Factors which can affect the TI include:. Poor translation of in vitro to in vivo activity (e.g., due to poor accessibility ofthe target, up-/downregulation of target or endogenous ligand or compensatoryeffects).

. Poor translation of in vivo activity in animals to humans (e.g., due to speciesselectivity, different pharmacodynamics).

. Gender and age.

. General health of the patient.

. Circadian variations.

. Active human metabolites with lower TI.

. Drug–drug interactions.

. Accumulation in tissues.

. Low bioavailability.

. High protein binding.

If any of these factors influence the unwanted effects more favorably, then the TIwill drop. This could occur in just a subset of patients, for instance those with


genetic risk factors, and the side effect may therefore be seen only after a drug hasbeen in the clinic for some while [49]. If it is a serious side effect, it could cause thedrug to be withdrawn, or it may receive a black box warning [17]. If there is apotential for the recommended dose being exceeded, a higher therapeutic indexmay have to be applied.Redfern et al. studied the occurrence of QTprolongation and the lethal arrhythmia

torsades de pointes (see Chapter 16) in marketed drugs from the perspective of thetherapeutic index [50]. This group proposed a therapeutic index of 30-fold, calculatedfrom the free Cmax at the therapeutic dose and the in vitro potency in the manualhERGpatch clamp assay. This level, or even higher, seems to be generally followed bythe industry.Unfortunately suchaprecise estimationof theminimumTI isnot available formost

other targets, and each target is different. However, hERG may be considered torepresent the extreme, and acceptable TIs for other targets could be 10-fold or less. Forsome targets, the areaunder the curve (AUC)maybemore appropriate touse for theTIcalculation than the Cmax. For some indications, the medical need may outweigh theside effect potential, in which case a lower TI than usual may be acceptable.Considering that many drugs reach free Cmax values in the micromolar range, off-target affinities in the micromolar range can, and do, result in side effects. As anexample, grepafloxacin (withdrawn from the market in 1999 due to 13 hERG-relatedfatalities) had a hERG IC50 around 30mMand free Cmax values of around 20mM [50].

12.4.4Possible Benefits of Off-Target Effects

While in vitro safety pharmacology profiling is primarily designed to identifypotential liabilities, the off-target data can be used to identify additional beneficialproperties of the drug. These could enhance the efficacy of the drug, complement theintended indication, or allowa better positioning of it against competitor compounds.Activities at other targets could provide repositioning of the drug for new indications.This concept is actively being pursued bymany companies for existing drugs [51, 52].A newer generation of more selective compounds will however be more difficult toreposition and will probably require extensive additional optimization.

12.5Conclusions and Outlook

In vitro safety pharmacology profiling is a very useful tool in the drug discoveryprocess and contributes to the selection of the best chemical scaffolds for leadoptimization. Promiscuous scaffolds and compounds with a high risk of failing canbe avoided and potential development compounds with a lower risk pharmacologicalprofile can be identified. As illustrated by the THR comparison of failed developmentcompounds and the best selling drugs, we believe that this tool will help to reduce latedrug attrition due to safety reasons.However, we need to improve the predictive value

12.5 Conclusions and Outlook j291

of existing assays, expand the number of in vitro assays which predict for ADRs andincrease the number of ADRs which can be predicted.Over the past two or three years, the industry has moved towards more functional

cell-based profiling assays to complement the receptor-binding assays and to godeeper into the characterization of the mechanism of action. The use of imagingtechnologies or technologies described as being more physiological (e.g., cellulardielectric spectroscopy (CDS) [53], assays using primary cells [54, 55], emergingtechnologies for in vivo pharmacological assessment [56, 57]) may be among the nextsteps to explore in order to continue to improve our ability to provide an early safetyassessment with simple, robust, inexpensive and medium-throughput assays.Most importantly, our knowledge and understanding of the links between drug–

protein interactions and adverse drug reactions has to extend into the whole phar-macological space [33]. The number and diversity of targets currently used during invitro safety pharmacology profiling is not very great, considering the huge number ofproteins which could potentially interact with a drug. These are variously estimated atbetween3000and5000, ofwhichabout 800areknown to interactwith smallmoleculesand only about 300 which are targeted by approved drugs [33, 58, 59]. There is atremendous push within the pharmaceutical industry to find novel drug targets anddrugs to treat diseases with unmet medical needs. At the same time, the potential foreach new drug target to alsomediate side effects should be examined. New tools beingdeveloped which could help to address this problem include chemogenomics [33, 60](see also Chapter 13) and gene-expression signatures [61].

References

1 Kinter, L.B., Gossett, K.A. and Kerns,W.D.(1993) Status of safety pharmacology in thepharmaceutical industry, 1993. DrugDevelopment Research, 32 (4), 208–216.

2 Kinter, L.D. and Dixon, L.W. (1995)Safety pharmacology program forpharmaceuticals. Drug DevelopmentResearch, 35 (3), 179–182.

3 Sullivan, A.T. and Kinter, L.B. (1995)Status of safety pharmacology in thepharmaceutical industry-1995. DrugDevelopment Research, 35 (3), 166–172.

4 Williams, P.D. (1990) The role ofpharmacology profiling in safetyassessment. Regulatory Toxicology andPharmacology, 12 (3), 238–252.

5 U.S. Food and Drug Administration(2001) International conference onharmonisation, S7A Safety pharmacologystudies for human pharmaceuticals.

U.S. Food and Drug Administration,Washington.

6 Whitebread, S., Hamon, J., Bojanic, D.and Urban, L. (2005) In vitro safetypharmacology profiling: an essential toolfor successful drug development. DrugDiscovery Today, 10 (21), 1421–1433.

7 Vaz, R.J. and Klabunde, T. (eds) (2008)Antitargets, Wiley-VCH, Weinheim.

8 Wakefield, I.D., Pollard, C., Redfern, W.S.,Hammond, T.G. and Valentin, J.P. (2002)The application of in vitro methods tosafety pharmacology. Fundamental andClinical Pharmacology, 16, 209–218.

9 Kinter, L.B. and Valentin, J.-P. (2002)Safety pharmacology and risk assessment.Fundamental and Clinical Pharmacology,16, 175–182.

10 Schuster, D., Laggner, C. and Langer, T.(2005) Why drugs fail – A study on side


effects in new chemical entities. CurrentPharmaceutical Design, 11, 3545–3559.

11 Jooste, E., Klafter, F., Hirshman, C.A. andEmala, C.W. (2003) A mechanism forrapacuronium-induced bronchospasm:M2 muscarinic receptor antagonism.Anesthesiology, 98 (4), 906–911.

12 Morris, B.J., Cochran, S.M. and Pratt, J.A.(2005) PCP: from pharmacology tomodelling schizophrenia. Current Opinionin Pharmacology, 5 (1), 101–106.

13 Gentili, F., Pizzinat, N., Ordener, C.,Marchal-Victorion, S., Maurel, A.,Hofmann, R., Renard, P., Delagrange, P.et al. (2006) 3-[5-(4,5-dihydro-1H-imidazol-2-yl)-furan-2-yl]phenylamine(Amifuraline), a promising reversible andselective peripheral MAO-A inhibitor.Journal of Medicinal Chemistry, 49,5578–5586.

14 McKenzie, R., Fried, M.W., Sallie, R.,Conjeevaram, H., Di Bisceglie, A.M.,Park, Y., Savarese, B., Kleiner, D. et al.(1995) Hepatic failure and lactic acidosisdue to fialuridine (FIAU), aninvestigational nucleoside analogue forchronic hepatitis B.New England Journal ofMedicine, 333, 1099–1105.

15 Lee, E.-W., Lai, Y., Zhang, H. andUnadkat, J.D. (2006) Identification of themitochondrial targeting signal of thehuman equilibrative nucleosidetransporter 1 (hENT1): Implications forinterspecies differences in mitochondrialtoxicity of fialuridine. The Journal ofBiological Chemistry, 281 (24), 16700–16706.

16 Kola, I. and Landis, J. (2004) Can thepharmaceutical industry reduce attritionrates? Nature Reviews. Drug Discovery, 3,711–715.

17 Lasser, K.E., Allen, P.D.,Woolhandler, S.J.,Himmelstein, D.U., Wolfe, S.M. andBor, D.H. (2002) Timing of new blackbox warnings and withdrawals forprescription medications. JAMA – Journalof the American Medical Association,287 (17), 2215–2220.

18 Lazarou, J., Pomeranz, B.H. and Corey,P.N. (1998) Incidence of adverse drug

reactions in hospitalized patients. JAMA –

Journal of the AmericanMedical Association,279, 1200–1205.

19 Hughes, B. (2008) FDA drug approvals:a year of flux. Nature Reviews. DrugDiscovery, 7 (2), 107–109, http://dx.doi.org/10.1038/nrd2514.

20 Redfern, W.S., Wakefield, I.D., Prior, H.,Pollard, C.E., Hammond, T.G. andValentin, J.-P. (2002) Safety pharma-cology – a progressive approach.Fundamental and Clinical Pharmacology,16, 161–173.

21 Smith, D.A. and Schmid, E.F. (2006)Drug withdrawals and the lessons within.Current Opinion in Drug Discovery andDevelopment, 9 (1), 38–46.

22 Spence, S., Anderson, C., Cukierski, M.and Patrick, D. (1999) Teratogeniceffects of the endothelin receptorantagonist L-753,037 in the rat.Reproductive Toxicology, 13 (12), 15–29.

23 Niederreither, K. and Doll�e, P. (2008)Retinoic acid in development: Towardsan integrated view. Nature Reviews.Genetics, 9, 541–553.

24 Wishart, D.S., Knox, C., Guo, A.C.,Cheng, D., Shrivastava, S., Tzur, D.,Gautam, B. and Hassanali, M. (2008)DrugBank: a knowledgebase for drugs,drug actions anddrug targets.Nucleic AcidsResearch, 36, D901–D906.

25 United Nations (2005) Consolidated list ofproducts whose consumption and/or salehave been banned, withdrawn, severelyrestricted or not approvedby governments.12th issue. UN Office for Economic andSocial Council Support and Coordination,pp. 595.

26 Wise, A., Gearing, K. and Rees, S. (2002)Target validation of G-protein coupledreceptors. Drug Discovery Today, 7 (4),235–246.

27 Fox, S., Farr-Jones, S., Sopchak, L.,Boggs, A., Nicely, H.W., Khoury, R. andBiros, M. (2006) High-throughputscreening: update on practices andsuccess. Journal of Biomolecular Screening,11 (7), 864–869.

References j293

28 Krejsa, C.M., Horvath, D., Rogalski, S.L.,Penzotti, J.E., Mao, B., Barbosa, F.and Migeon, J.C. (2003) PredictingADME properties and side effects: TheBioPrint approach. Current Opinion inDrug Discovery and Development, 6 (4),470–480.

29 Roth, B.L., Baner, K., Westkaemper, R.,Siebert, D., Rice, K.C., Steinberg, S.,Ernsberger, P. and Rothman, R.B. (2002)Salvinorin A: A potent naturally occurringnonnitrogenous k opioid selective agonist.Proceedings of the National Academy ofSciences of the United States of America,99, 11934–11939.

30 Elphick, G.F., Querbes, W., Jordan, J.A.,Gee, G.V., Eash, S., Manley, K., Dugan, A.,Stanifer, M. et al. (2004) The HumanPolyomavirus, JCV Uses SerotoninReceptors to Infect Cells. Science, 306,380–1383.

31 Silvestre, J.S. andProus, J. (2005) Researchon adverse drug events I. Muscarinic M3receptor binding affinity could predict therisk of antipsychotics to induce type 2diabetes. Methods and Findings inExperimental and Clinical Pharmacology,27 (5), 289–304.

32 Yildirim, M.A., Goh, K.-I., Cusick, M.E.,Barabasi, A.-L. and Vidal, M. (2007)Drug—target network. NatureBiotechnology, 25, 1119–1126.

33 Paolini, G.V., Shapland, R.H.B.,Van Hoorn, W.P., Mason, J.S. andHopkins, A.L. (2006) Global mapping ofpharmacological space. NatureBiotechnology, 24, 805–815.

34 DeVane, C.L. and Nemeroff, C.B. (2001)An evaluation of risperidone druginteractions. Journal of ClinicalPsychopharmacology, 21, 408–416.

35 Roth, B.L., Sheffler, D.J. and Kroeze, W.K.(2004) Magic shotguns versus magicbullets: selectively non-selective drugs formood disorders and schizophrenia.NatureReviews. Drug Discovery, 3, 353–359.

36 Youdim,M.B.H. (2006) The path from antiParkinson drug selegiline and rasagiline tomultifunctional neuroprotective anti

Alzheimer drugs ladostigil and M30.Current Alzheimer Research, 3, 541–550.

37 Stephenson, V.C., Heydingb, R.A. andWeaver, D.F. (2005) The �promiscuousdrug concept� with applications toAlzheimer�s disease. FEBS letters, 579 (6),1338–1342.

38 Hampton, T. (2004) �Promiscuous�anticancer drugs that hit multiple targetsmay thwart resistance. The Journal of theAmerican Medical Association, 292 (4),419–422.

39 Rochon, P.A., Normand, S.-L., Gomes, T.,Gill, S.S., Anderson, G.M., Melo, M.,Sykora, K., Lipscombe, L., et al. (2008)Antipsychotic therapy and short-termserious events in older adults withdementia. Archives of Internal Medicine,168 (10), 1090–1096.

40 Whitebread, S., Hamon, J., Scheiber, J.,Fekete, A., Azzaoui, K., Mikhailov, D., Lu,Q. andUrban, L. (2008) Broad-scale in vitropharmacology profiling to predict clinicaladverse effects. American Drug Discovery,3 (2), 32–38.

41 Hopkins, A.L.,Mason, J.S. andOverington,J.P. (2006) Can we rationally designpromiscuous drugs? Current Opinion inStructural Biology, 16 (1), 127–136.

42 Aronov, A.M., McClain, B., Moody, C.S.and Murcko, M.A. (2008) Kinase-likenessand Kinase-Privileged Fragments: TowardVirtual Polypharmacology. Journal ofMedicinal Chemistry, 51, 1214–1222.

43 Whitlock, G.A., Fish, P.V., Fray, M.J.,Stobie, A. andWakenhut, F. (2008) Pyridyl-phenyl ether monoamine reuptakeinhibitors: Impact of lipophilicity on dualSNRI pharmacology and off-targetpromiscuity. Bioorganic and MedicinalChemistry Letters, 18, 2896–2899.

44 Abe, C., Kikukawa, T. and Komatsu, Y.(1995) Combination therapy on murinearthritis - Salazosulfapyridine,bucillamine, and methotrexate.International Journal of Immunotherapy,11, 129–132.

45 Burcoglu-O�Ral, A., Erkan, D. andAsherson, R. (2002) Treatment of


catastrophic antiphospholipid syndromewith defibrotide, a proposed vascularendothelial cell modulator. Journal ofRheumatology, 29, 2006–2011.

46 Hamon, J., Azzaoui, K., Whitebread, S.,Urban, L., Jacoby, E. and Faller, B. (2006)In vitro safety pharmacology profiling.European Pharmaceutical Review, 2006 (1),60–63.

47 Azzaoui, K., Hamon, J., Faller, B.,Whitebread, S., Jacoby, E., Bender, A.,Jenkins, J.L. and Urban, L. (2007)Modeling promiscuity based on in vitrosafety pharmacology profiling data.ChemMedChem, 2, 874–880.

48 Leeson, P.D. and Springthorpe, B. (2007)The influence of drug-like concepts ondecision-making in medicinal chemistry.Nature Reviews. Drug Discovery, 6, 881–890.

49 Wilke, R.A., Lin, W., Roden, D.M.,Watkins, P.B., Flockhart, D., Zineh, I.,Giacomini, K.M. and Krauss, R.M. (2007)Identifying genetic risk factors for seriousadverse drug reactions: current progressand challenges. Nature Reviews. DrugDiscovery, 6, 904–916.

50 Redfern, W.S., Carlsson, L., Davis, A.S.,Lynch,W.G.,MacKenzie, I., Palethorpe, S.,Siegl, P.K.S., Strang, I. et al. (2003)Relationships between preclinical cardiacelectrophysiology, clinical QT intervalprolongation and torsade de pointes for abroad range of drugs: evidence for aprovisional safety margin in drugdevelopment. Cardiovascular Research,58, 32–45.

51 Ashburn, T.T. and Thor, K.B. (2004) Drugrepositioning: identifying and developingnew uses for existing drugs. NatureReviews. Drug Discovery, 3, 673–683.

52 O�Connor, K.A. and Roth, B.L. (2005)Finding new tricks for old drugs: Anefficient route for public-sector drugdiscovery. Nature Reviews. Drug Discovery,4, 1005–1014.

53 Verdonk, E., Johnson, K., McGuinness, R.,Leung, G., Chen, Y.W., Tang, H.R.,Michelotti, J.M. and Liu, V.F. (2006)Cellular dielectric spectroscopy:

A label-free comprehensive platform forfunctional evaluation of endogenousreceptors. Assay and Drug DevelopmentTechnologies, 4 (5), 609–619.

54 Meyer, T., Sartipy, P., Blind, F., Leisgen, C.and Guenther, E. (2007) New cell modelsand assays in cardiac safety profiling.Expert Opinion on Drug Metabolism andToxicology, 3 (4), 507–517.

55 Pouton, C.W. and Haynes, J.M. (2007)Embryonic stem cells as a source ofmodels for drug discovery.Nature Reviews.Drug Discovery, 6 (8), 605–616.

56 Houck, K.A. and Kavlock, R.J. (2008)Understanding mechanisms of toxicity:insights from drug discovery research.Toxicology and Applied Pharmacology,227 (2), 163–178.

57 Barros, T.P., Alderton, W.K.,Reynolds, H.M., Roach, A.G. andBerghmans, S. (2008) Zebrafish: anemerging technology for in vivopharmacological assessment to identifypotential safety liabilities in early drugdiscovery. British Journal of Pharmacology,154, 1400–1413.

58 Imming, P., Sinning, C. and Meyer, A.(2006) Drugs, their targets and thenature and number of drug targets.Nature Reviews. Drug Discovery, 5,821–834.

59 Overington, J.P., Al-Lazikani, B. andHopkins, A.L. (2006) How many drugtargets are there? Nature Reviews. DrugDiscovery, 5, 993–996.

60 Bender, A., Scheiber, J., Glick, M.,Davies, J.W., Azzaoui, K., Hamon, J.,Urban, L., Whitebread, S. and Jenkins, J.L.(2007) Analysis of pharmacology dataand the prediction of adverse drugreactions and off-target effects fromchemical structure. ChemMedChem, 2,861–873.

61 Lamb, J., Crawford, E.D., Peck, D., Modell,J.W., Blat, I.C., Wrobel, M.J., Lerner, J.,Brunet, J.-P. et al. (2006) The ConnectivityMap: Using Gene-Expression Signaturesto connect small molecules, genes, anddisease. Science, 313, 1929–1935.

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