RESEARCH SEMINAR13 Nov 03; 5:30-

6:30pmUCLan

1) Nitric oxide congeners modify the gastrotoxicity of

NSAIDs

2) but retain the ability to alter thymic function:

Nitrergic system of the thymus and autoimmune susceptibility

Dr James DowningLiverpool John Moores

University,School of Pharmacy & Chemistry

1

“Nitrergic” systems:

Expression ofNitric Oxide Synthase

(NOS).

1

Three Organ Systemsuse NOS to make NO

1

NERVES

3

VASCULATURE

2

IMMUNE SYS

(Inflammation;

incl Gut epithelium)

2

AbbreviationsN.O = Nitric Oxide.N.O.S = Nitric Oxide Synthase.

Enzyme activityConvert L-Arginine to L-Citrulline and N.O.

Isoforms First located (named) Regulation

NOS1 Neural/Brain (n/bNOS) Constitutive (Ca-

dept).

NOS2 'mac'/inducible (iNOS) Inducible (Ca-indept).

NOS3 endothelial (eNOS) Constitutive

(Ca-dept).

AssaysElectrochemical meter.E.S.R (using a "spin trap" eg. MGD).Histology: NADPH-diaphorase / Immunocytochemistry.Citrulline production (from 3H-Arginine).Colourimetric Greiss assay (NOx).

NITRERGIC Enzymes and Assays

3

“Nitrergic” system of THYMUS:

Expression of inducible Nitric Oxide Synthase,

iNOS (NOS2).

• Maintains "Deletional" Immune Tolerance of Self

• BioAssay for toxicological risk of autoimmunity.

4

5

Cortex“outer”

Lobule

Cross-section (120um) through thymic lobule

Nitrergic cells - by diaphorase activityin paraformaldehyde-fixed tissue

Medulla“inner”

Immunological Tolerance• T-cells are produced within the thymus.

• There is an "education" or "selection" process to ensure : - T-cells can recognise antigen (have TCR) and - T-cells are not "auto-aggressive".

• SIMPLIFICATION: - T-cellularity (relative size of the thymic cortex)

= Index of "Production". - Abundance of "Nitrergic" cells (expressing iNOS)

= Index of "Quality Control" (Deletional Tolerance).

• HYPOTHESIS: - A quantitative "balance" between "production" of T-cells and their "Quality control" within the thymus. - May be disturbed by genetic or pharmacological factors.

6

Thymic "Education" of T-LymphocytesCortex: Phase 1 - Positive selection

Progenitor T-cell

TCR binds MHCPositive selection

not bind MHCDeath by neglect

Medulla: Phase 2 - Negative selection

TCR hi avidityDeleted

TCR mod avidity

Allowed

7

COLOCALIZATION OF NADPHd AND TUNEL (APOPTOSIS)

Thymic sections from naïve rat stained for NADPH diaphorase (Tetrazolium - blue) followed by the TUNEL reaction to visualise apoptotic cells (brown). Arrows

indicate apoptotic cells adjacent to NADPHd+ cells.

(400x, scale bar = 20 um).

murderous "Quality Control"of T-cell Production

8

1) Nitric oxide congeners modify the gastrotoxicity of

NSAIDs

2) but retain the ability to alter thymic function:

Nitrergic system of the thymus and autoimmune susceptibility

Dr James DowningLiverpool John Moores

University,School of Pharmacy & Chemistry

1

OUTLINE OF SEMINAR

1) The problem with NSAIDs: Inhibition of COX; gastrotox'

2) Proposed new pro-drugs: NO-glycerol-NSAID

3) Study 1 to test the gastro-protective effects of NO-NSAIDs ; possible involvement of an effect on local synthesis of NO.

4) Study 2 to test for potential immunological side effects of these anti-inflammatory / immunosuppressant drugs.

5) Consistent with an increased risk of autoimmune disorder following damage to thymic mech' for immune tolerance:

- Thymic damage (to NOS) from use/withdrawal from CsA.

- Lewis rats: AI-susceptibility may be due to deficient NOS

- Dpp4 inhibitors trailed for MS; Dpp4 mutant Fischer rats can show disordered iNOS.

2

1) The problem with NSAIDs.

• NSAIDs inhibit COX: • Inhibiting cyclooxygenase COX-1 maybe "all bad" - necessary to control gastric mucus and bicarbonate secretion. • Inhibiting COX-2 originally thought to be the source of inflammatory damage; Reconsideration "not all bad" may also be required for reparative processes of wound healing.

• Systemic levels of NSAIDs may be responsible for gastro toxicity through non-selective inhibition of COX1 and COX2.

2) Proposed new pro-drugs: NO-glycerol-NSAID

• May slow the release of NSAIDs - reducing local and systemic levels.• Provides local NO to increase enteric vascular perfusion and inhibit immune responses.

3

NO-indomethacin

N

O

Cl

CH3

OO

CH3O

OH

O NO2

• NSAIDs modified with a NO donor group attached by a linker molecule.

• NO-NSAID metabolised by esterase to release NO.

• NO has ‘gastroprotective’ effects.

NO-NSAIDs4

OH

O

O

OO

ONO2

O

Ibuprofen

NO-Ibuprofen

5

STUDY 1:Reducing

gastro toxicity(side effects)of NSAIDs

6

Acute effects of ibuprofen and its nitric oxide releasing prodrug on ulceration,

erosion and nitric oxide synthase expression in rat gastric mucosa

C. Wilson, J. Southall, M. Ingram*,C. Rostron, J. Duffy, J. Downing

School of Pharmacy & Chemistry, Liverpool John Moores University, Liverpool L3 3AF,

*School of Pharmacy and Biomolecular Sciences, University of Brighton, East Sussex BN2 4GJ

Corresponding author: j.downing@livjm.ac.uk

7

GASTROTOXICITY OF NSAIDs - NO CONGENORS

• Non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen are effective in the treatment of pain and inflammation but are associated with gastrotoxicity.

• Efficacies of NSAIDs may relate to their ability to inhibit prostaglandin synthesis by alternative cyclooxygenase enzymes, which may compromise mucosal resistance to erosive effects of stomach acid.

• Modifications of NSAIDs to incorporate releasable nitric oxide (NO) are being explored (Wallace et al 1997).

• We examined ibuprofen and its NO-prodrug (Ingram et al 2001) for gastrotoxic effects and association with endogenous levels of nitric oxide synthase (NOS).

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TREATMENTS

• Single oral treatments of 4hr duration were made with either ibuprofen or ibuprofen-NO (ea. 1.33x10-4mol/kg) and compared with vehicle (aqueous Tween 80) using 16-hour fasted male Wistar rats (~250g; n=5 per treatment).

AIMS

• Determine the effects of acute oral administration of ibuprofen and its nitric oxide (NO) congener on signs of gastric damage (mucosal ulceration and thinning).

• Establish the effects of treatments on nitric oxide synthase (NOS) activity by Nitro Blue Tetrazolium-based diaphorase histochemistry.

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Thickness

Length

*

• No OF ULCERS (*) by two alternative methods:

(a) Visual examination of stomachs to produce weighted scores (Duffy et al J. Pharmacy & Pharmacology 2001, 53: 1505-1514).

(b) Microscopic counts of ulcers/cm; 195 sections observed from 15 rats.

• NOS EXPRESSION: % perimeter length stained by diaphorase.

• MUCOSAL THICKNESS: 3775 random transects; 195 sections; 15 rats.

MEASUREMENTS:Ulceration, Thickness & NOS Expression

Fig 1

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Gastric Erosion:Effect of Treatment (vehicle, ibuprofen or NO-ibuprofen) on

the Average Mucosal Thickness ( Mean +/- SEM)

0

10

20

30

40

50

60

70

80

90

Vehicle Ibuprofen NO-Ibuprofen

Treatment

Av

era

ge

Mu

cosa

l T

hic

kn

ess

(Mic

ron

s)

• Analysis of variance (ANOVA) P value < 0.001.• Tukeys follow up test shows all the results to be

significantly different from each other.• NO-ibuprofen caused significantly less erosion than

ibuprofen.

RESULTS: Erosion of mucosal thickness

Fig 2

11

Gastric Ulceration:Effect of treatment (vehicle, ibuprofen or NO-Ibuprofen)

on the average number of ulcers per cm of Gastric Mucosa (Mean +/- SEM)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Vehicle Ibuprofen NO-IbuprofenTreatment

Mea

n N

um

ber

of

Ulc

ers

per

Cen

tim

etre

RESULTS: Ulceration by microscopic sampling

• Analysis of variance (ANOVA) P value = 0.639.• Tukeys test shows that no treatments are

statistically different from one another.• Number of ulcers per centimetre were not

statistically significant between treatments.

Fig 3

12

Ulceration and Irritation Visible to the EyeData produced by visible examination of the Rat Stomachs

0123456789

10

Vehicle Ibuprofen NO-Ibuprofen

Treatment

Irrit

ati

on

Sca

led

Sco

re

RESULTS: Ulceration by visual score

• Results based on a scaled score, Vehicle = 1.• Compare data with the number of ulcers per

centimetre sampled microscopically.• Suggests a trend towards NO-ibuprofen causing

less ulceration.

Fig 4

13

NOS EXPRESSION (BY DIAPHORASE)

• Three isoforms of nitric oxide synthase (NOS) can be identified in paraformaldehyde-fixed tissue by NADPH-diaphorase histochemistry.

• High levels of NO produced by the inducible NOS may contribute to tissue damage at the site of inflammation.

• Lower levels of NO expected from constitutive (neural and endothelial) isoforms may support gastro-protective functions, such as the inhibition of platelet aggregation and increased vascular perfusion (Nathan 1997).

• NSAIDs may modify endogenous NO synthesis and NO-conjugated prodrugs may substitute for the effects of endogenous NO.

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DEGREE OF STAINING BY DIAPHORASE (NOS EXPRESSION):Mean percent length of the gastric mucosa positivley stained by NADPH

Diaphorase (a marker of nitric oxide synthase)

0

5

10

15

20

25

30

35

Vehicle Ibuprofen NO-Ibuprofen

Treatment

Av

e %

le

ng

th s

tain

edRESULTS: NOS expression by diaphorase labelling

• Analysis of variance (ANOVA) P value = 0.001.• Tukeys test shows difference between ibuprofen

and vehicle to be insignificant.• NOS expression is significantly increased by

NO-ibuprofen, but not ibuprofen.

Fig 5

15

CONCLUSIONS• Effects of NO-ibuprofen reported here:

- Reduced mucosal erosion significantly.- Appeared to reduce the number of ulcers / cm (shown by scaled score but not statistically resolved by microscopic sampling).- Increased the expression of diaphorase (NOS).

• Complexing of NO with ibuprofen reduces gastro-toxicity when compared to ibuprofen and this effect is associated with higher levels of expression of mucosal diaphorase, likely to be NOS, indicating a possible mechanism for gastroprotection.

Acknowledgements Thanks to Francis Essuman.

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STUDY 2:Altered thymic function:Risk of additional side effects

to autoimmune susceptibility

17

Acute reduction of nitric oxide synthase (NOS) in rat thymus without change in

T-cellularity following oral non-steroidalanti-inflammatory drug (NSAID) treatments

R. Clark, M. Whitty, M. Ingram*,C. Rostron, J. Duffy, J. Downing

School of Pharmacy & Chemistry, Liverpool John Moores University, Liverpool L3 3AF,

*School of Pharmacy and Biomolecular Sciences, University of Brighton, East Sussex BN2 4GJ

Corresponding author: j.downing@livjm.ac.uk

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INTRODUCTION

• Conjugation of NSAIDs with esterase-releasable nitric oxide (NO) is being explored for its potential to reduce gastrotoxicity (Ingram et al 2001).

• Additional side effects are reported to include disruption of T-cell development within the thymus (Xu et al 2001) and the appearance of over active, possibly autoreactive, T-cells in the periphery (Yamamura et al 1996).

• Pharmacological inhibition of a thymic mechanism using nitric oxide synthase (NOS) and possibly underlying central immune tolerance has also been reported (Kosaka et al 1990).

• The effect of acute oral dosing with NSAIDs and their NO-congeners was therefore investigated for possible disturbance of thymic organisation.

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METHODS

• Single 4hr duration oral treatments with either indomethacin, indomethacin-NO (ea. 2x10-5 mol/kg), ibuprofen or ibuprofen-NO (ea. 1.33x10-4 mol/kg) were compared with vehicle (aqueous Tween 80) using 16-hour fasted male Wistar rats (~250g; n=4).

• Paraformaldehyde-fixed thymi were stored frozen (-20oC) before parallel processing for enzyme histochemistry.

• Two parameters were measured from 100 micron sections:

(i) Size of cortex relative to total surface area gave an index of T-cell production or “positive selection“, T-cellularity, by imaging auto-fluorescent cells (AFC) at the cortico-medullary junction.

(ii) Abundance of medullary nitrergic cells (counts/mm2) stained positive by NADPH-diaphorase, a marker of NOS.

20

30 μm

150 μmcortex

medulla

A) Oil Red O

D) nNOS +ve control

200 μmcortex

medulla

AFCs

B) Auto Fluorescent Cells

150 μm

cortex

medulla

debris

Nitrergic cells

C) Diaphorase

Fig. 1

Illustration of methods to observe T-cellularity and Nitrergic cells

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HYPOTHESIS - Central Immunological Tolerance

• Negative selection (deletion) of potentially autoreactiveT-cells is proposed to involve induction of NOS within thymic medulla and may reflect "quality control" of T-cells.

• Relatively low levels of nitrergic cell expression in the presence of high T-cellularity have been associated with autoimmune susceptibility of the Lewis rat compared to Fischer strain (Downing et al 1998).

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RESULTS

• Possible effects of NSAIDs on thymic organisation were compared using analysis of variance followed by Tukey's test and expressed as mean +/- SEM.

• Both ibuprofen (57.08 +/- 5.19; p<0.001) and indomethacin (62.88 +/- 4.84, p<0.001) reduced nitrergic cell abundance compared to control (94.79 +/- 6.70) in the absence of significant changes in T-cellularity.

• NO-conjugated drugs also suppressed the levels of NOS (NO-ibuprofen, 66.77 +/- 5.23, p<0.001; NO-indomethacin, 65.37 +/- 6.55, p<0.001) and also occurred in the absence of significant changes in T-cellularity.

23

0

0.2

0.4

0.6

0.8

1

Control Indomethacin NO-Indomethacin

ctx

:me

du

lla a

rea

Fig. 2a: Effects of Indomethacin / NO on T-cellularity.• ANOVA P-value = 0.357 (NS).

0

20

40

60

80

100

120

control indomethacin NO-indomethacin

Me

an

ce

ll c

ou

nt

Fig. 2b: Effects of Indomethacin / NO on Nitrergic cell count. • ANOVA P-value < 0.001; No difference in treatments by Tukeys.

Me

an

ce

ll c

ou

nt

Ra

tio

ctx

:me

d a

rea

24

T-Cellularity(mean +/- SEM)

0

0.25

0.5

0.75

Control Ibuprofen NO-Ibuprofen

T-C

ellu

lari

ty

Fig. 3a: Effects of Ibuprofen / NO on T-cellularity.• ANOVA P-value = 0.095 (NS).

020406080

100120

Control Ibuprofen NO-IbuprofenCell

co

un

t /

Med

ull

ary

A

rea

Fig. 3b: Effects of Ibuprofen / NO on Nitrergic cell count. • ANOVA P-value < 0.001; No difference in treatments by Tukeys.

Me

an

ce

ll c

ou

nt

Ra

tio

ctx

:me

d a

rea

25

REFERENCES

Ingram, M.J. et al (2001) J. Pharmacy and Pharmacology 53: 345-350.

Xu, H. et al (2001) Cellular Immunology 214: 184-193.

Yamamura, S. et al (1996) Cellular Immunology 173: 303-311.

Downing, J.E.G. et al (1998) Immunology 95: 148-155.

Kosaka, H. et al (1990) J. Exp. Med. 172: 395.

CONCLUSIONS

• It is proposed that if the effects of acute oral treatment with either NO-conjugated or parent forms of ibuprofen or indomethacin are maintained over chronic periods they may pose a risk to effective immune tolerance and lead to the escape of autoreactive T-cells.

• A comparable autoimmune adverse drug reaction has been identified following withdrawal of the anti-inflammatory drug, cyclosporin A, which is also associated with inhibition of thymic nitric oxide synthesis (Kosaka et al 1990).

26

5) OTHER EVIDENCE

Consistent with an increased risk of autoimmune disorder following damage to a thymic mechanism for immune tolerance:

- Thymic damage (to NOS) from use/withdrawal from CsA.

- Lewis rats: AI-susceptibility may be due to deficient NOS

- Dpp4 inhibitors trailed for Multiple Sclerosis (MS); Dpp4 mutant Fischer rats can show disordered iNOS.

27