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Drug Toxi c i t y I

Toxi col ogy:Mol ecul ar Mechani sms

Gar y St ephensRoom 208 Hopki ns

g. j . st ephens@r eadi ng. ac. uk

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Af t er t hi s l ect ur e, and f ur t her r eadi ng as r equi r ed, st udent s wi l l be abl e t o:• expl ai n how dr ugs ar e i mpor t ant agent s f or poi soni ng • descr i be t he mani f est at i ons of t oxi c i t y • out l i ne t he maj or mol ecul ar mechani sms of t oxi c i t y and how dr ug met abol i t es may be t oxi c • expl ai n how t oxi c pot ent i al of a dr ug can be quant i f i ed usi ng a var i et y of met hods i ncl udi ng car ci nogeni ci t y, mut ageni ci t y, t er at ogeni ci t y, al l er gy t est i ng• expl ai n LD50 val ues and t her apeut i c i ndex

• eval uat e t he benef i t s and l i mi t at i ons of ani mal t est i ng t o pr edi ct human t oxi c i t y

Lect ure obj ect i ves

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Pharmacol ogy : t he st udy of t he ef f ect of dr ugs on t he f unct i on of l i v i ng syst ems[ or i gi n: Gk p ha r ma ko n = dr ug]

Toxi col ogy : t he st udy of t he ef f ect of poi sons on t he f unct i on of l i v i ng syst ems

Chemi cal agent s t hat cause t oxi ci t y i ncl ude:• Dr ugs• I nsect i c i des/ her bi c i des • Pl ant t oxi ns • Ani mal t oxi ns • Chemi cal weapons• Radi oact i ve el ement s

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Par acel sus ( 1493- 1541)‘ Gr andf at her of

Toxi col ogy’

“The dose makes t he

poi son”

" Al l t hi ngs ar e poi son and not hi ng i s wi t hout poi son, onl y t he dose per mi t s somet hi ng not

t o be poi sonous. "

t her apeut i cef f ect

t oxi cef f ecti ncr easi ng dose

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Adverse Drugs React i ons ( ADRs)

ADRs ar e noxi ous or uni nt ended r esponses occur r i ng at t herapeut i c doses ( WHO def i ni t i on) ~ 5% of al l acut e hospi t al admi ssi onsType A ( augment ed) ADRs

Ef f ect s ar e:∙ r el at ed t o known phar macol ogy, but undesi r abl e∙ common, dose-r el at ed∙ pr edi ct abl e

Exampl es∙ haemor r hage wi t h ant i coagul ant s∙ r espi r at or y depr essi on wi t h opi oi ds∙ sedat i on wi t h anxi ol yt i c and ol der ant i hi st ami ne dr ugs

Type B ( bi zar r e) ADRs

Ef f ect s ar e:∙ unr el at ed t o known phar macol ogy∙ r ar e∙ unpr edi ct abl e∙ of t en i di osyncr at i c

Exampl es∙ anaphyl axi s wi t h peni ci l l i n∙ al l er gi c l i ver damage by hal ot hane∙ bone mar r ow suppr essi on by chl or ampheni col∙ i ndi v i dual al l er gy/ genet i c basi s

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t he ef f ect s of t he body on t he poi son( r el at es t o Absor pt i on, Di s t r i but i on, Met abol i sm, Excr et i on ( ADME) ) .

Wi t h t hi s i nf or mat i on i t i s possi bl e t o pr edi ct concent r at i on of t ox i n t hat r eaches t he s i t e of i nj ur y and t he r esul t i ng damage.

Absorpt i on i ngest i on mer cur y and di oxi n i n f i shpest i c i des i n pr oducesal monel l a ( di ar y) , bot ul i num ( meat ) t oxi ns

i nhal at i on asbest os, ner ve gases

Di st ri but i on as di scussed f or t her apeut i c dr ugs

Toxi coki net i cs

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Met abol i sm Phase I by cyt ochr ome P450 ( oxi dat i on, r educt i on, hydr ol ysi s)

Phase I I conj ugat i on t o al l ow excr et i on i n ur i ne and bi l e

Det oxi f i cat i on: compound r ender ed l ess t oxi cToxi f i cat i on: r el at i vel y i ner t compound

conver t ed i nt o t oxi n

Excret i on t oxi ns not excr et ed may be st or ed i n: bone ( eg. l ead) f at ( eg. DDE a met abol i t e of t he pest i c i de

DDT di chl or odi phenyl t r i chl or oet hane)

The t oxi n may be r el eased sl owl y i nt o t he body

Toxi coki net i cs

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Mol ecul ar Mechani sms of Toxi col ogy

1. Al l ergi c responsesCommon f or m of ADR, usual l y wi t h a di f f er ent t i me cour se t o phar macol ogi cal ef f ect s

4 basi c cl i ni cal syndr omes – t ypes I , I I , I I I & I V ( Gel l & Combes, 1963)

Type I hypersensi t i vi t y react i on – I gE- medi at ed mast cel l degr anul at i on

Type II ant i body- medi at ed cyt ot oxi c hypersensi t i vi t y- i nvol ve haemat ol ogi cal r eact i ons i . e. t hose per t ai ni ng t o t he bl ood cel l s and bl ood- f or mi ng or gans

Type I I I i mmune compl ex- medi at ed hyper sensi t i v i t y

Type I V del ayed- t ype hyper sensi t i v i t y

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Mol ecul ar Mechani sms of Toxi col ogyType I hypersensi t i vi t y react i ons can t ri gger

anaphyl act i c shock

1 2

l ow MW al l er gen( eg. bee venom,

peanut oi l )

i mmunogeni c conj ugat e

eg. peni c i l l i n 75% of al l

deat hs

hapt en

mast cel l

I gE r ecogni t i on t r i gger s hi st ami ne r el ease

br onchoconst r i ct i onvasodi l at i oni nf l ammat i on

t r eat ed wi t hadr enal i ne

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Mol ecul ar Mechani sms of Toxi col ogyType II hypersensi t i vi t y react i ons depl et e

bl ood cel l t ypes

These r eact i ons can depl et e:Red bl ood cel l s ( haemol yt i c anaemi a) eg. sul f onami desNeut r ophi l es ( agr anul ocyt osi s) eg. cer t ai n NSAI DsPl at el et s ( t hr ombocyt openi a) eg. qui ni ne and hepar i n

T cel l

bl ood cel leg. RBC

ant i gen-bound RBC

cyt ot oxi c T cel l - medi at ed cel l l ysi s

I gG- boundRBC

Cel l l ysi s1.

t oxi nant i gen

2. 3.

compl ement -medi at ed l ysi s

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Mol ecul ar Mechani sms of Toxi col ogy2. Recept or, i on channel and enzyme- medi at ed

t oxi c i t yMol ecul ar drug/t oxi n t arget s

Recept ors ( 4 maj or super f ami l i es)∙ Li gand- gat ed i on channel s i onot r opi c

r ecept or s vol t age- gat ed i on channel s

∙ GPCRs - G prot ei n coupl ed recept ors ( met abot r opi c r ecept or s)

∙ Enzyme- l i nked recept ors ( t yr osi ne ki nase act i v i t y)

∙ Nucl ear recept ors ( r egul at e gene t r anscr i pt i on)

Enzymes met abol i c and cat abol i c pat hways

Carri ers upt ake/ t r anspor t syst ems

Ot hers pr ot ei ns i nvol ved i n vesi c l e r el ease

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Mol ecul ar Mechani sms of Toxi col ogySources of t oxi ns

Source Act i ve agent

Mechani sm of act i on

Pl ant s

Ama ni t a p ha l l o i d e s

α- amani t i n i nhi bi t s RNA pol ymer ase

Di g i t a l i s l a na t a di goxi n/ di gi toxi n

Na+/ K+ ATPase i nhi bi t or

Cal abar ( or deal ) bean

physost i gmi ne ant i chol i nest er ase

At r o p i n e b e l l a d o nna

at r opi ne bl ocks muscar i ni c AChR

Bact eri a

Cl o s t r i d i um b o t ul i num

bot ul i num t oxi n

i nhi bi t s synapt i c pr ot ei n

Cho l e r a v i b r i o chol er a t oxi n act i vat es Gαs pr ot ei ns

Bo r d e t e l l a p e r t us s i s

per t ussi s t oxi n

i nhi bi t s Gαi / o pr ot ei ns

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Mol ecul ar Mechani sms of Toxi col ogyAni mal sources of venoms and t oxi ns

Source Act i ve agent Mechani sm of act i on

Kr ai t s ( el api d snakes)

α- bungar ot oxi n bl ocks ni cot i ni c AChR

Gr een mamba snakes dendr ot oxi ns bl ock K+ channel s

Funnel web spi der ω- agat oxi n bl ocks CaV2. 1 Ca2+ channel s

Coneshel l ω- conot oxi n bl ocks CaV2. 2 Ca2+ channel s

Tar ant ul a spi der SNX- 482 bl ocks CaV2. 3 Ca2+ channel s

Puf f er f i sh t et r odot oxi n bl ocks Na+ channel sFr og ( De nd r o b a t e s ) ski n

car di ac gl ycosi des

Na+/ K+ ATPase i nhi bi t or

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Ani mal t oxi ns bl ock i on- conduct i on

α- bungar ot oxi n on ni cot i ni c acet yl chol i ne r ecept or ( nAChR)

r ecept or gat e ( α hel i ces)

ACh ACh

Na+

Banded kr ai t ( Bung a r us mul t i c i n c t us )

α- bungar ot oxi n

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Vol t age- gat ed K+ channel s are bl ocked by dendrot oxi ns

dendr ot oxi ns

Bl ack mamba ( De nd r o a s p i s p o l y l e p i s )

Gr een mamba ( De nd r o a s p i s a ng us t i c e p s )

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Vol t age- gat ed Ca2+ channel s are i mport ant t oxi n t arget s

Funnel web spi der Coneshel l Tar ant ul a spi der

ω- agat oxi n( CaV2. 1)

ω−conot oxi n( CaV2. 2)

SNX- 482( CaV2. 3)

Ca2+ cur r ent r ecor di ng f r om a sensor y neur on i n pai n pat hway ( Wi l son e t a l . 2001)

ω- conot oxi nω- agat oxi n

SNX- 482

Cu

rrent (p

A)

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Tet rodot oxi n act s on Na+ channel s t o bl ock act i on pot ent i al s

Puf f er f i sh Tet r odot oxi n ( TTX)

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“ You ar e wal ki ng t hr ough a cr owded shoppi ng mal l , when you hear a sof t ‘ pop’ and see smoke comi ng f r om t he ot her end of t he mal l . You i mmedi at el y not i ce di m vi s i on, and your nose begi ns t o r un sever el y. Less t han 1 mi nut e l at er , you not i ce shopper s col l apsi ng t o t he f l oor , br eat hi ng heavi l y, some of t hem l osi ng consci ousness and devel opi ng sei zur e act i v i t y. You not i ce t hat t hat t hei r pupi l s ar e const r i ct ed. You i mmedi at el y gr ab 2 smal l chi l dr en near you, cover your nose and mout h wi t h your j acket , and r un out of t he mal l ”

Col . Jonat han Newmar k, Ar c h Ne ur o l .   2004; 61: 649- 652US Ar my Medi cal Resear ch I nst i t ut e of Chemi cal Def ense

Mol ecul ar Mechani sms of Toxi col ogyEnzyme- medi at ed t oxi col ogy

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Irreversi bl e ant i chol i nest erase eg. parat hi on and sari n

P OR2 R1

X

O

N

N

ser i ne

O

gl ut amat e

COO-

hi st i di ne O

POR2

R1

N

N

ser i ne

HO

gl ut amat e

COO-

hi st i di ne

cat al yt i csi t e

ani onic

si t e

no hydr ol ysi s- d e n o v o synt hesi s needed

enzymeact i ve si t e

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Oxi mes are st rong nucl eophi l es t hat react i vat e AChest erase

N

N

ser i ne

O

gl ut amat e

COO-

hi st i di ne O

POR2

R1HO N

N+

O NN+

P

OR2

R1

O

pr al i doxi me

N

N

ser i ne

HO

gl ut amat e

COO-

hi st i di ne

cat al yt i csi t e

ani onic

si t e

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Fi rst l i ne of def ence agai nst bi ol ogi cal nerve gases :

• At r opi ne- mAChR bl ocker - cent r al r espi r at or y depr essi on• Pr al i doxi me- r eact i vat i on of acet yl chol i nest er ase

React i vat i on of pl asma chol i nest er ase ( ChE) i n a vol unt eer subj ect by i nt r avenous i nj ect i on of pr al i doxi me. ( Si m V M 1965 J Am Med Assoc 192: 404. )

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Mol ecul ar Mechani sms of Toxi col ogy3. Bi ochemi cal pat hways

( i ) Cyani de i nhi bi t s mi t ochondr i al cyt ochrome c oxi dase t o pr event cel l ul ar r espi r at i on

( i i ) Car bon monoxi de: di spl aces oxygen f r om haemogl obi n causi ng hypoxi a

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Mol ecul ar Mechani sms of Toxi col ogy4. Organ- Di rect ed Toxi c i t y

Organs part i cul arl y suscept i bl e t o t oxi n damage are t he l i ver

and ki dney

Hepat ot oxi c i t y(i) hepat i c necrosi s

par acet amol poi soni ng

(i i) hepat i c i nf l ammat i on ( hepat i t i s ) hal ot hane can coval ent l y bi nd t o l i ver pr ot ei ns t o t r i gger an aut oi mmune r eact i on

(i i i) chroni c l i ver damage ( c i rrhosi s) l ong- t er m et hanol abuse causes cel l ul ar t oxi c i t y and i nf l ammat i on and mal nut r i t i on as et hanol becomes a f ood sour ce

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Paracet amol i s a promi nent cause of hepat i c poi soni ng( 48 % of al l poi son admi ssi ons and >200 deat hs/year)

Tr eat ment :Acet y l cyst ei neMet hi oni ne( gl ut at hi one pr ecur sor s)

par acet amol

O

NH

OH+

Phase I Igl ucur oni deor sul phat econj ugat i on ( ~90%)

hepat ot oxi c( bi nds t o pr ot ei n

t hi ol gr oups)

Phase I

O

O

N

N- acet y l - p - benzoqui nonei mi ne ( NAPQI )

( ~10%)( non-t oxi c) gl ut at hi oneconj ugat ion

excr et i on

Phase I I

over dose:( i ) enzymes sat ur at i on( i i ) gl ut at hi one depl et i on

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Mol ecul ar Mechani sms of Toxi col ogyOrgan- Di rect ed Toxi c i t y

Nephrot oxi c i t y

( i ) changes i n gl omerul ar f i l rat i on rat e ( GFR)Lar gel y due t o dr ugs t hat al t er bl ood f l ow :NSAI Ds ( eg. aspi r i n) r educe pr ost agl andi ns whi ch i n t ur n r educes bl ood f l ow/ GFRACE i nhi bi t or s ( eg. r ami pr i l ) i ncr ease bl ood f l ow/ GFR

( i i ) al l ergi c nephri t i s al l er gi c r eact i on t o NSAI Ds ( eg. f enopr of en) and ant i bi ot i cs ( eg. met aci l l i n)

( i i i ) chroni c nephri t i sl ong- t er m NSAI D and par acet amol use

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Mol ecul ar Mechani sms of Toxi col ogy5. Mut agenesi s and carci nogenesi s

Mut agens cause changes t o cel l DNA t hat ar e passed on when cel l di v i des, i f t hi s pr oduces a neopl ast i c cel l t he agent i s t er med a carci nogen.

2 maj or c l asses of gene ar e i nvol ved i n car ci nogenesi s:• Prot o- oncogenes : pr omot e cel l cycl e pr ogr essi on eg. const i t ut i ve act i v i t y of gr owt h f act or t yr osi ne- ki nase r ecept or s can cause neopl ast i c t r ansf or mat i on

• Tumour- suppressor genes : i nhi bi t cel l cycl e pr ogr essi oneg. mut at i ons i n t umour suppr essi on gene pr oduct p53 ( pr eval ent i n smoker s)

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Mol ecul ar Mechani sms of Toxi col ogy6. Terat ogeni ci t y

Thal i domi de( R) - enant i omer

sedat i ve

Thal i domi de( S) - enant i omer

t erat ogen

Terat ogenesi s : t he cr eat i on of bi r t h def ect s dur i ng f et al devel opment Terat ogens : subst ances t hat i nduce bi r t h def ect s

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• 1950’ s- t hal i domi de was synt hesi zed by t he Grünent hal

• Non- t oxi c at hi gh doses i n al l ani mal s speci es t est ed

• 1957 - mar ket ed t hr oughout Eur ope i n as Cont ergan a non- l et hal hypnot i c and sedat i ve, r ecommended as an ant i - emet i c t o t r eat mor ni ng si ckness i n pregnant women

• 1961 - t hal i domi de was t he best - sel l i ng sl eepi ng pi l l i n West Ger many and t he UK

• However , t hal i domi de pr oduced t er at ogeni c ef f ect s i n 100% of f oet uses exposed bet ween 3- 6 weeks gest at i on

The t hal i domi de di sast er heral ded modern t erat ogeni ci t y t est i ng

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• An est i mat ed 8- 12, 000 i nf ant s wer e bor n wi t h def or mi t i es caused by t hal i domi de, and onl y about 5, 000 of t hese sur vi ved beyond chi l dhood

• 1968 - Cont ergan case was br ought t o t r i al

• 1970 - cour t di smi ssed t he case due t o onl y mi nor r esponsi bi l i t y of Grünent hal and " mi nor i mpor t ance t o t he publ i c of t he Feder al Republ i c of Ger many"

• I n f act , t hal i domi de i s a usef ul dr ug, used t oday t o t r eat l eprosy and mul t i pl e myel oma ( pr obabl y due t o i nhi bi t or y act i v i t y on t umour necr osi s f act or ( TNF) - α pr oduct i on)

The t hal i domi de di sast er heral ded modern t erat ogeni ci t y t est i ng

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Drug ef f ect s on f et al devel opment

St age Gest at i on per i od

Cel l ul ar pr ocess

Af f ect ed by

Bl ast ocyt e f or mat i on

0- 16 days Cel l di v i s i on Cyt ot oxi c dr ugsAl cohol

Or ganogenesis

~17- 60 days

Di vi s i on mi gr at i ondi f f er ent i at ion deat h

Ter at ogens( t hal i domi de,r et i noi dsant i epi l et i cswar f ar i n)

Mat ur at i on >60 days As above Al coholNi cot i ne ACE i nhi bi t or s St er oi ds

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Drug Toxi c i t y II

Toxi col ogy:Treat ment and prevent i on

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St ages of drug devel opment

Dr ug di scover y phar macol ogi cal sel ect i onPr ecl i ni cal devel opment t oxi c i t y t est i ng

Phase I t est i n heal t hy ( ~20- 80) vol unt eer s Phase I I smal l scal e t est i n ( ~100- 300) pat i ent sPhase I I I l ar ge scal e ( ~1000- 5000) cont r ol l ed t r i al

Phase I V post - mar ket i ng sur vei l l ance

~50 pr oj ect s

1

12

5

3

1.7

2- 5~2

5- 7

year s

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St ages of drug devel opment

Phase I

100 – 200

Healthy Subjects

• Does it seem safe in humans?

• What does the body do to the drug (pharmacokinetics)?

• What does the drug do to the body (pharmacodynamics)?

• Might it work in patients?

Phase II 200 – 300

Patients

• Does it seem safe in patients?

• Does it seem to work in patients?

Phase III 1,000 – 3,000

Patients

• Does it seem safe in patients?

• Does it really work?

Phase IIIb Hundreds - Thousands

Patients

• Does it seem safe in a different group of patients?

• Does it really work in a different group of patients?

Phase IV Tens to many thousands

Patients

• Is it truly safe?

• How does it compare with similar drugs?

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Precl i ni cal drug devel opment t est i ng

To assess genot oxi c pot ent i al a bat t ery of t est s are used:

i n vi t ro t est s f or mut ageni ci t y eg Ames t est• st r ai ns of Sa l mo ne l l a t y p hi mur i um bact er i a cannot synt hesi s hi st i di ne • mut ant gr own on hi st i di ne- cont ai ni ng medi a • drug and a l i ver mi cr osomal enzyme pr epar at i on ( t o t est f or r eact i ve met abol i t es) added • hi st i di ne becomes depl et ed and onl y back-mut ant s can gr ow• mut at i on r at e measur ed

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Precl i ni cal drug devel opment t est i ng

i n vi t ro cyt ogenet i c eval uat i on of chromosome damage i n r esponse t o dr ug

• carci nogeni ci t y t est i ng: chr oni c dr ug dosi ng; l ook f or t umour s

• reproduct i ve ( t erat ogeni ci t y) t est i ng: pr egnant f emal es f r om one r odent speci es and one non- r odent ( usual l y r abbi t ) speci es dosed wi t h dr ug at di f f er ent or ganogenesi s st ages out l i ned pr evi ousl y; l ook f or bi r t h def ect s

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Prel i mi nary t oxi c i t y t est i ngMaxi mum non- t oxi c dose ( gi ven f or 28 days t o 2 speci es) .

Ani mal s exami ned post - mor t em and t i ssue damaged assessed

Let hal dose LD50 - t he dose of dr ug whi ch ki l l s 50% of t r eat ed ani mal s wi t hi n a speci f i ed shor t amount of t i me

LD50

l og [ dr ug] ( M)

Toxi

c re

spon

se

-9 -8 -7 -6 -5 -4 -30

25

50

75

100

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Prel i mi nary t oxi c i t y t est i ng

NOAEL

LOAEL

NOAEL ( no obser ved adver se ef f ect s l evel )Hi ghest concent r at i on t hat does not a t oxi c r esponse

LOAEL- l owest obser ved adver se ef f ect s l evelLowest concent r at i on t hat pr oduces a t oxi c r esponse

l og [ dr ug] ( M)

Toxi

c re

spon

se

-9 -8 -7 -6 -5 -4 -30

25

50

75

100

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Prel i mi nary t oxi c i t y t est i ng

NOAEL ( no obser ved adver se ef f ect s l evel )Hi ghest concent r at i on t hat does not a t oxi c r esponse

1. Det er mi ne NOAEL

2. Conver t NOAEL t o a ‘ Human Equi val ent Dose’ ( HED)

• Adj ust f or ant i c i pat ed exposur e i n humans

• Adj ust f or i nt er - speci es di f f er ence i n af f i ni t y and pot ency

3. Appl y >10 f ol d saf et y f act or

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Prel i mi nary t oxi c i t y t est i ng

Cal cul at i ng HED ( Human Equi val ent Dose)

NOAEL: dog 50 mg/ kg

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LD50 val ues f or di f f erent t oxi nsToxi ci t y rat i ng Exampl e LD50 ( mg/kg)

Sl i ght l y t oxi c( 5- 15 g/ kg)

Et hanol 8000

Moder at el y t oxi c( 0. 5- 5 g/ kg)

Sodi um chl or i de Par at hi on

40001300

Ver y t oxi c( 50- 500 mg/ kg)

Aspi r i nPar acet amol

300300

Ext r emel y t oxi c( 5- 50 mg/ kg)

Theophyl l i ne Di phenhydr ami ne

5025

Super Toxi c( <5 mg/ kg)

Pot assi um cynani de Di goxi nTet r odot oxi n Bot ul i num t oxi n

30. 20. 010. 00001 ( 10 ng/ kg ! )

4141

Therapeut i c i ndex

The rat i o of t he dose of t he drug t hat produces an unwant ed

( t oxi c ) ef f ect t o t hat produci ng a want ed ( t herapeut i c ) ef f ect

= LD50 / ED50

res

pons

e (%

)

l og [ dr ug] ( M)

Smal l TI: e. g. warf ari n

res

pons

e (%

)

l og [ dr ug] ( M)

Large TI: e. g. peni ci l l i n, aspi ri n

Therapeut i c wi ndow Therapeut i c wi ndow

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Prel i mi nary t oxi c i t y t est i ng

The or al LD50 of a new dr ug was det er mi ned i n r at s.Q. What can t hi s val ue t el l us:

A. Shor t t er m, l et hal ef f ect sB. Long- t er m, l et hal ef f ect s C. Long- t er m, non- l et hal ef f ect sD. Pot ent i al Type B adver se dr ug r eact i ons E. Let hal dosage when i nj ect edF. Toxi c i t y i n young and ol d humans

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The El i xi r Sul f ani l ami de di sast er of 1937 was one of t he most consequent i al mass poi soni ngs of t he 20t h cent ur y.

Sul f ani l ami de was di l ut ed i n di et hyl ene gl ycol t o gi ve a r ed El i x i r Sul f ani l ami de.

One hundr ed and f i ve pat i ent s di ed f r om i t s t her apeut i c use.

Under t he exi st i ng dr ug r egul at i ons, pr emar ket i ng t oxi c i t y t est i ng was not r equi r ed.

I n r eact i on, t he U. S. Congr ess passed t he 1938 Federal Food, Drug and Cosmet i c Act , whi ch r equi r ed pr oof of saf et y bef or e t he r el ease of a new dr ug.

Why do we need toxicity testing……..

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The TGN1412 di sast er has hi ghl i ght ed need f or

accurat e t oxi c i t y t est i ng

• TGN1412 i s a monocl onal ant i body ( MAB) desi gned t o bi nd CD28 pr ot ei n t o act i vat e l eucocyt es

• TGN1412 coul d f i ght l eukaemi a by t r i gger i ng cyt oki ne r el ease

• Ani mal st udi es of TGN1412 i ndi cat ed no t oxi c i t y

• 6 vol unt eer s wer e gi ven 1: 500 di l ut i ons of doses used i n ani mal st udi es at 30 mi nut e i nt er val s accor di ng t o agr eed pr ot ocol s. A f ur t her 2 vol unt eer s r ecei ved a pl acebo

• Wi t hi n mi nut es of t he 6 t h vol unt eer r ecei v i ng t he dose, seri ous si de- ef f ect s occur r ed sever e headache, backache, f ever and pai n l eadi ng t o br i ef coma, k i dney f ai l ur e, head swel l i ng

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Pot ent i al f l aws i n t he TGN1412 st udy

• Lack of bi ol ogi cal knowl edge ( of how CD28 wor ks)

• Use of heal t hy vol unt eer s wi t h i nt act i mmune r esponse coul d t r i gger a ‘ cyt oki ne st or m’

• TGN1412 wor ks di f f er ent l y bet ween speci es ( mai nl y human pr ot ei n)

• Dose r egi me t oo shor t ( i . e gi ven t oo f r equent l y)

• Test i ng shoul d have been st agger ed over sever al days

• Pr obl em wi t h cont ami nant s i n f or mul at i on ( l at er di scount ed)

• Suggest ed i mpr ovement : Bl i st er t est - expose smal l amount of ski n t o dr ug t o check adver se r eact i on pr i or t o whol e body exposur e

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Summary: Treat ment and prevent i on of t oxi c i t y

1. Pr ecl i ni cal t oxi c i t y t est i ng i s a v i t al par t of dr ug devel opment

2. New compounds must be assessed i n par t i cul ar f or mut ageni c, car ci nogeni c and t er at ogeni c pot ent i al

3. Pr el i mi nar y t oxi c i t y t est i ng t ypi cal l y uses LD50 and NOAEL, LOAEL val ues

4. LD50 exper i ment s ar e not per f ect

5. Prevent i on of t oxi c i t y i s based on knowl edge of mol ecul ar mechani sms of t oxi n act i on