Markus Neurath

Azathioprine: Mechanism of action

IBD - Pathogenesis

Geneticpredisposition

BacterialAntigens

Activation of the MucosalImmuneSystem

Crohn´sdisease

Th1Colitis-associated colon carcinoma

chromoendoscopy

Ulcerativecolitis

Th2 NK T

CD11cT cells

T cells are key effector cells in IBD

TT

T

Crohn´sdisease

Ulcerativecolitis

T helper 1

TT

T

T helper 2 (?)

Low IL-12High EBI3

BacterialAntigens

DendriticCells

T

High IL-12Low EBI3

T cell apoptosis and IBD-In the antigen-rich environment of the gut T cell expansion is

controlled by apoptosis

- LP T cells exhibit increased susceptibility to Fas-mediated apoptosis compared to blood T cells

- IBD T cells exhibit reduced apoptosis induced by IL-2 deprivation and Fas/FasL activation Boirivant Gastro. 1999, Ina J. Immunol. 1999

- This T cell resistance to apoptosis contributes to T cell expansion and disease perpetuation in IBD

- Antibodies to proinflammatory cytokines appear to suppress intestinal inflammation by the induction of T cell apoptosis Fuss Gastro. 1999, Atreya et al. Nature Med. 2000, Ten Hove Gut 2002,

Blockade of the IL-6/ sIL6R system induces apoptosis in Crohn´s disease

Atreya Nat. Med. 2000

TNFTNFSignaling Signaling

AndAndApoptosisAnti-TNFAntibodiesinduce apoptosisof LP CD4+ Tcells in CDTenHove et al.Gut 2002Van den Brande et al.Gastro 2003

Azathioprine/ 6-Mercaptopurine

6-mercaptopurine6-MP

azathioprine

Gertrude B. Elion(*1918-1999)

Discovered 6-mercaptopurine, thioguanine (JBC 1951) and azathioprine (Cancer Res. 1963)

Screening of 100 anti-metabolites for growthsuppression of Lactobacillus casei

1988 Nobel Price in Physiology or Medicine

Azathioprine/ 6-MP in IBD1962 First case report on 6-MP in IBD by R.H.D. Bean

38 year old patient with chronic active UC

300mg 6-MP: reduction of blood transfusions, X-ray: return of haustration, endoscopy: healingof colonic ulcers

50 mg 6-MP: return of clinical symptoms

300 mg followed by 100 mg 6-MP:Long-term remission over 80 weeks

The Medical Journal of Australia

Induction of remission by azathioprine/ 6-MPin Crohn´s disease

AzathioprineCandy (1995)Ewe, Meyer zum Büschenfelde (1993)Klein (1974)Rhodes (1971)Summers (1979)Willoughby (1971)

All azathioprine trials

6-MercaptopurineOren (1997)Present (1980)

All 6-MP trials

All trials

2,06 [1,25; 3,39]

3,34 [1,67; 6,66]

2,43 [1,62; 3,64]0,1 1 10 100 1000

Odds Ratio [95 % CI]

Pearson et al. 1995, Stange et al. 2002

a z a t h i o p r i n e

6 - m e r c a p t o p u r i n e ( 6 - M P )

6 - t h i o u r i c a c i d 6 - m e t h y l - M P 6 - t h i o g u a n i n e ( 6 - T G )

x a n t h i n �o x i d a s e H P R T

T P M T

H o w d o e s a z a t h i o p r i n e w o r k ?

6 - t h i o g u a n i n e - �n u c l e o t i d e s ( 6 - T G N )

D N A �R N A

i n c o r p o r a t i o n

S u p p r e s s i o n o f p r o l i f e r a t i o n

This mechanism of action in IBDwould lead to:- random efficacy - predominant effects in rapidly dividing cells (e.g. epithelial cells)

- random side effects

The antiproliferative effect requires high dosages of 6-MP

Quemeneur et al. J. Immunol. 2003

Relative cell number/ CFSE labeling

PBL culturedfor 4 days

T cell apoptosis

Azathioprine/ Rac1

Anti-TNF antibodies

Clinical therapy of IBD and T cell apoptosis

Induction of T cell apoptosis(cell death)

rapid

delayed

Holtmann et al. 2002, Ten Hove et al. 2002, Tiede et al. 2003

TNF-R2

Azathioprine induces T cell apoptosis

untreatedCD45RA cells

azathioprinepr

opid

ium

iodi

de 6-MP

untreated azathioprine

prop

idiu

m io

dide 6-MP

CD45RO cells

29%100

1

0110

2

103

10

4

100 101 102 103

100

1

0110

2

103

10

4

35%100 101 102 103

54%100

1

0110

2

103

10

4

100 101 102 103

18%100

1

0110

2

10

3

104

100 101 102 103

34%100 101 102 103

100

1

0110

2

10

3

104

41%100 101 102 103

100

1

0110

2

10

3

104

annexin V annexin V annexin V

annexin Vannexin V annexin V

22% 25% 30%

17% 38% 41%

indu

ctio

nof

ap

opto

sis

[%]

indu

ctio

nof

ap

opto

sis

[%]

-5

0

510

15

20

25

0.5 5azathioprine

5 µmol6-MP

0

10

20

30

40

50

0.5 5azathioprine

5 µmol6-MP

**

**

****

Dose-dependent effects of azathioprine

6-TG levels: 31.5 168 pmol/mg DNALevels in IBD patients under long-term therapy: 100-2305 pmol/mg DNA (Cuffari 1996)

6-MPeffectsinIBDpatients

apop. cells (%)

- + - + culture - - + + therapy

6-MP

IBD no 6-MP IBD 6-MP IBD 6-MP NR

apop

tosi

s[%

]in

duct

ion

of

annexin V100 101 102 103 104

annexin V100 101 102 103 104

annexin V100 101 102 103 104

prop

idiu

m io

dide

100

101

102

10

3 1

04

100

101

102

10

3 1

04

100

101

102

10

3 1

04

untreated 6-MP 6-TG

6-MP 6-TG

17% 37% 49%

30

0

Effects of 6-thioguanine on apoptosis

6-MP-TUNEL

IL2 R

0 5 µmol 0 5 µmol

Bcl-xL

Bcl-xL

40

50

30

20

100

100 10 102 103 104

coun

ts

untreated

Bcl-xL

40

50

30

20

100

100 10 102 103 104co

unts

6-MP

73%

41%

6-MP

GAPDH

TACI

Bcl-xL

azathioprine

-10-505

1015

6-MP

apop

tosi

s[%

]in

duct

ion

+ - + -- + - +

anti-CD3/28

anti-CD3

NFκB

Selectivity of azathioprine for CD28 signaling

6-MP

casp

ase-

9

0

500

1000

1500

0

500

1000

1500

0 5 0 5 µmol6-MP

CD45RA CD45RO

rela

tive

light

uni

ts

100 101 102 103 104

05

1525

membrane potential

coun

tsFC

CP

azat

hiop

rine

6-MP induces a mitochondrial pathway of apoptosis

05

1525

coun

ts

100 101 102 103 104

membrane potential0 5 0 5 µmol6-MP

CD45RA CD45ROca

spas

e-8

rela

tive

light

uni

ts

untreated 6-MP

Ac-LEHD CHO

prop

idiu

m io

dide

6-MP +Ac-IETD CHO

6-MP +

prop

idiu

m io

dide

annexin V

annexin V100 101 102 103 104

100

101

102

103

104

prop

idiu

m io

dide

prop

idiu

m io

dide

100

101

102

103

104

18.3%10.8%

9.8% 21.1%

6-MP induced apoptosis requires caspase-9

100

101

102

103

104

100

101

102

103

104

100 101 102 103 104

annexin V100 101 102 103 104

annexin V100 101 102 103 104

+6-MP

SP1

anti-p65anti-p50 -Jurkat______________

NF-κB

CD3 CD3/28 __________________

CD3/28

p50/65

Azathioprine suppresses NF-kappaB activation

CD4+ T cells

+6-MPJurkat CD3 CD3/28

CD4+ T cells

______________

EMSA: NF-kappaB

EMSA: SP1

annexin V100 101 102 103 104 100 101 102 103 104 100 101 102 103 104

annexin V annexin V

100

1

0110

2

103

104

100

1

0110

2

103

104

100

1

0110

2

103

104ph

osph

o-M

EK

phos

pho-

ME

K

phos

pho-

ME

K

anti-CD3 anti-CD3/28 anti-CD3/28 + 6-TG10% 12% 29% 7% 10% 13%

Azathioprine controls the Rac1-MEK pathway

0 5 µmol

p MEK

p IκB

6-MP

42 kDa

actin

42 kDa

45 kDa

45 kDa

IκB

MEK

ERK2

prine- 6-MPazathio-

- 6-MPazathio-

25 kDa

95 kDa

42 kDa

Rac

Vav

ERK2

prine

Azathioprine suppresses the vav/Rac1 pathway

25 kD

anti-CD3

anti-CD3/28

anti-CD3/28___________

_azathio-prine 6-MP

__________anti-CD3/28

_6-MP

Rac1-GTP

Rac1-GTP

Stat 3

-

prineazathio-

Vav Rac 1

control

6-MP

Azathioprine suppresses the vav/Rac1 pathway

The 6-Thio-GTP hypothesis

6-Thio-Guanine (6-TG)

6-Thio-Guanosine-monophosphate (6-Thio-GMP)

6-Thio-Guanosine-diphosphate (6-Thio-GDP)

6-Thio-Guanosine-triphosphate (6-Thio-GTP)

Evidence: Detection of 6-TG in Rac1 immunoprecipitates from 6-MP treated T cells

50040030020010000

20

40

60

80

100

120

Thio-GTP (µM)

Rel

ativ

e G

TP-

bind

ing

(% o

f con

trol

)

Rac 1

Ras

6-thioguanine-triphosphate (6-Thio-GTP)

radiolabelled GTP

Measure bound radiolabelled GTP

Specificity of 6-ThioGTP binding to Rac1

recombinant Rac1Versus Ras

Specificity of 6-ThioGTP for Rac1

CD

3/28

+ A

za

CD

3/28

+ 6

-TG

CD

3/28

+ 6

-MP

CD

3/28

CD

3

25 KD

CD

3

CD

3/28

CD

3/28

+ 6

-MPRas

CD

3/28

+ 6

-TG

25 KDC

D3/

28 +

Aza

Rac1

Densitometrie Ras

0

20

40

60

80

100

120

CD3 CD3/28 CD3/28 +Aza

CD3/28 +6-MP

CD3/28 +6-TG

%

Densitometrie Rac1

0

20

40

60

80

100

120

CD3 CD3/28 CD3/28 +Aza

CD3/28 +6-MP

CD3/28 +6-TG

%

Densitometry Ras

Densitometry Rac1

Specificity of 6-ThioGTP for Rac1

CD

3/28

CD

3/28

+ 6

-MPRhoA

CD

3/28

+ 6

-TG

CD

3/28

+ A

za

Cdc42

CD

3

25 KD

Densitometrie RhoA

0

2040

6080

100120

140

CD3 CD3/28 CD3/28 +Aza

CD3/28 +6-MP

CD3/28 +6-TG

%

Densitometry RhoA

CD

3/28

CD

3/28

+ 6

-MP

CD

3/28

+ 6

-TG

CD

3/28

+ A

za

CD

3

25 KD

Densitometrie Cdc42

0

2040

6080

100120

140

CD3 CD3/28 CD3/28 +Aza

CD3/28 +6-MP

CD3/28 +6-TG

%

Densitometry Cdc42

6-Thio-GTP interaction with Rac1

The specificity of azathioprine-induced blockade ofRac1 function as compared to other GTPases(e.g. Ras) is due to the steric interaction between thesulfur atom and the Ala159 residue

Ras-GTP

CD3anti-

anti-CD3/28

6-MP 6-TG__

6-ThioGTP blocks vav exchange activity on Rac1

0

20

40

60

80

100

120

No Va

v3No

Vav3

+Inh

Vav3

Vav3

+Inh

0

10

20

30

40

50

60

70

80

90

0 15 30 45

TIME (min

No Vav3

NoVav3+InhiVav3

Vav3+Inhi

- - + +- + - +

Vav6-ThioGTP

No vav

No vavSGTPvav

vav SGTP

Time (minutes)

Rac1-6ThioGTP-vav model

• Cyan = switch 1 region of Rac1 (residues 27-35) • Magenta = switch 2 region of Rac1 (residues 59-71)• Yellow = contact area of vav1 with Rac1

a z a t h i o p r i n e

6 - m e r c a p t o p u r i n e ( 6 - M P )

6 - t h i o u r i c a c i d 6 - m e t h y l - M P 6 - t h i o g u a n i n e ( 6 - T G )

x a n t h i n �o x i d a s e H P R T

T P M T

A n e w m o d e l f o r a z a t h i o p r i n e a c t i o n

6 - t h i o g u a n i n e - �t r i p h o s p h a t e ( 6 - t h i o - G T P )

R a c 1

i n c o r p o r a t i o n

a p o p t o s i s o f �a c t i v a t e d T c e l l s

A model for azathioprine action

M E K K 1 ( M A P 3 K )

M E K 1 ( M A P K K )

I K K αI K K β

I K K γ

I k B α N F - κ B �p 5 0 / p 6 5 b c l - x L

a n t i a p o p t o t i c s i g n a l

C D 2 8

V a v ( G E F )

R a c 1 R a c 1t h i o G D P t h i o G T P

6 - t h i o G T PG D P

P

S T A T - 3

p S T A T - 3

a z a t h i o p r i n e

6 - M P

6 - T G

Response Flares/a TGDP TGTPTGN>100 0.42 1.0 29 118TGN<100 0.16 2.1 6 54

Clinical study using 6-ThioGTP levels

Design: prospective study in 50 Crohn´s patientsReceiving AZA Therapy

Response Flares/a TGDP TGTP>100 Q>90 0.71 0.21 4 155>100 Q<90 0.25 1.7 45 93

<100 Q>90 0.1 1.1 4 68<100 Q<90 0.3 2.6 8 38

Teichgräber et al. 2005TGN - Thioguanine Nucleotides

Status:12 analogues have been identified

6 analogues have been synthesized

Steric Modelling of6-Thio-GTP Analogues

Summary• Azathioprine specifically blocks vav exchange activity on Rac1 in

T cells by incorporation of 6-Thio-GTP• Azathioprine-induced blockade of Rac1 activation causes

suppression of NF-kappaB and STAT3 activation leading to a mitochondrial pathway of apoptosis

• Azathioprine appears to mediate immunosuppression in IBD by inducing T cell apoptosis via 6ThioGTP

• 6ThioGTP levels in erythrocytes correlate with clinical responses to azathioprine

• Selective targeting of apoptosis may be important for designing new therapies for IBD

• Designing of 6-ThioGTP analogues with higher affinity to Rac1 may lead to novel therapies for IBD

Ralf Kiesslich, Martin HoltmannJonas Mudter, Raja AtreyaKai Hildner, Thanka Nadar.Con Schneider, Jürgen Siebler

Jan Schmidt Guido SchürmannStefan Rose-JohnSusanne StrandHans A. LehrHenning WalczakRichard BlumbergReza AhmadianGerhard FritzXose Bustelo

CollaborationsClinic

Lab

Imke Tiede, Christoph BeckerDaniela Poppe, Stefan WirtzBenno Weigmann, Alexej NikolaevBrigitte Bartsch, Massimo Fantini

Thanks