UCLan Seminar NO NSAIDs 131103c

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Dr James Downing Liverpool John Moores
University, School of Pharmacy Chemistry
RESEARCH SEMINAR 13 Nov 03 530-630pm UCLan
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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
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Nitrergic systems Expression of Nitric Oxide
Synthase (NOS).
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Three Organ Systems use NOS to make NO
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NITRERGIC Enzymes and Assays
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Abbreviations N.O Nitric Oxide. N.O.S
Nitric Oxide Synthase. Enzyme activity Convert
L-Arginine to L-Citrulline and N.O. Isoforms Firs
t located (named) Regulation NOS1 Neural/Brain
(n/bNOS) Constitutive (Ca-dept). NOS2 'mac'/induc
ible (iNOS) Inducible (Ca-indept). NOS3 endotheli
al (eNOS) Constitutive (Ca-dept). Assays Electr
ochemical meter. E.S.R (using a "spin trap" eg.
MGD). Histology NADPH-diaphorase /
Immunocytochemistry. Citrulline production (from
3H-Arginine). Colourimetric Greiss assay (NOx).
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Nitrergic system of THYMUS Expression of
inducible Nitric Oxide Synthase, iNOS (NOS2).
Maintains "Deletional" Immune Tolerance of
Self BioAssay for toxicological risk of
autoimmunity.
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Nitrergic cells - by diaphorase activity in
paraformaldehyde-fixed tissue
Cross-section (120um) through thymic lobule
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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.
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Thymic "Education" of T-Lymphocytes
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COLOCALIZATION OF NADPHd AND TUNEL (APOPTOSIS)
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murderous "Quality Control" of T-cell Production
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).
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Dr James Downing Liverpool John Moores
University, School of Pharmacy Chemistry
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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
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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.
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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.
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NO-NSAIDs
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-indomethacin
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Ibuprofen
NO-Ibuprofen
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STUDY 1 Reducing gastro toxicity (side
effects) of NSAIDs
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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_at_livjm.ac.uk
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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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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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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 n5 per
treatment).
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MEASUREMENTSUlceration, Thickness NOS
Expression
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Fig 1

• 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.

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RESULTS Erosion of mucosal thickness
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Fig 2

• Analysis of variance (ANOVA) P value lt 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.

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RESULTS Ulceration by microscopic sampling
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Fig 3

• 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.

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RESULTS Ulceration by visual score
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Fig 4

• 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.

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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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RESULTS NOS expression by diaphorase labelling
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Fig 5

• 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.

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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.
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• Acknowledgements
• Thanks to Francis Essuman.

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STUDY 2 Altered thymic function Risk of
additional side effects to autoimmune
susceptibility
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Acute reduction of nitric oxide synthase (NOS) in
rat thymus without change in T-cellularity
following oral non-steroidal anti-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_at_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 n4). Paraformaldehyde-fixe
d 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.
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Illustration of methods to observe T-cellularity
and Nitrergic cells
A) Oil Red O
C) Diaphorase
D) nNOS ve control
B) Auto Fluorescent Cells
200 µm
cortex
AFCs
medulla
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Fig. 1
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HYPOTHESIS - Central Immunological Tolerance
Negative selection (deletion) of potentially
autoreactive T-cells is proposed to involve
induction of NOS within thymic medulla and may
reflect "quality control" of T-cells.
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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 plt0.001) and indomethacin (62.88 /- 4.84,
plt0.001) 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, plt0.001 NO-indomethacin, 65.37 /-
6.55, plt0.001) and also occurred in the absence
of significant changes in T-cellularity.
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Fig. 2a Effects of Indomethacin / NO on
T-cellularity. ANOVA P-value 0.357 (NS).
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Ratio ctxmed area
Fig. 2b Effects of Indomethacin / NO on
Nitrergic cell count. ANOVA P-value lt 0.001
No difference in treatments by Tukeys.
Mean cell count
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Fig. 3a Effects of Ibuprofen / NO on
T-cellularity. ANOVA P-value 0.095 (NS).
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Ratio ctxmed area
Fig. 3b Effects of Ibuprofen / NO on Nitrergic
cell count. ANOVA P-value lt 0.001 No
difference in treatments by Tukeys.
Mean cell count
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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).
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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.
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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.