The 1rst Kuwait-North American
Update in Internal Medicine Conference
8-9 February 2014
TREATMENT OF RA 2014
Henri A. Ménard, MD, FRCP (C)
Professor of Medicine
McGill University
McGill University Health Center
Sequence of Events in RA
Pre-
Early-
Immune response
Anti-CCP
RF
Established- RA
Pathologic inflammatory response
Anti-Sa
Destruction
Amyloidosis
Vasculitis
T0
T100
Break of
tolerance
RA Onset
CVD
Environment
Genes
Lymphoma
Rheumatoid Hand
Smoking Habit
Ulnar Drift
Oral health: Parodontitis
• Chronic Inflammatory condition.
• Erosive disease
• Associated with RF, HLA DR4 and Coronary Artery Disease.
• Intriguing because associated with Porphyromonas gingivalis. The
bacterial PADI and its products are very different from that of the
mammalian PADIs’. Break of tolerance???
• Circumstantial evidence only. No hard data available.
(Ménard HA Dresden Symposium on Autoimmunity 2007)
Principles of Patient Centered
RA Treatment in 2014
• DO MORE THAN LESS clinical observation and biological
documentation of the disease to pinpoint the particular
context of your patient where N=1.
• EXPLAIN AND REASSURE the patient and the family;
• INSIST ON LIFESTYLE ISSUES: smoking, oral hygiene (flossing)
and beware of the associated obesity and metabolic
syndrome: PREHABILITATION is better than REHABILITATION;
• TREAT EARLY AND AGGRESSIVELY with full DMARDs combos
and use biologicals when needed;
• USE TOOLS BORROWED FROM THE BUSINESS WORLD to
contract with the patient short term and long term objectives
with periodic timely deliverables (Treat-to-Target approach);
• ADAPT AND ADJUST as the disease evolves and changes.
Personalizing Is Challenging
To treat moving targets in vivo we need to develop
HUMAN BIOMARKER(S)
Clinical : intra vs extra-articular features
Serologic : anti-Sa For Prognosis and Monitoring
Genomic
immune response genes (SE and non-SE),
pharmacogenomics (drug metabolism),
innate immunity genes (cytokine SNPs)
Immunopathologic : Cell mediated vs humoral
Evolutive disease = Δ physiopathical pattern
Personalizing Is Challenging
To chose the best drug for the right patient at the
right time, we need to STOP EMPIRICISM i.e.
 Stop making real world medical decisions and
using guidelines based on trial data;
 Start dissecting each individual patient as a N=1
trial, not as a member of poorly characterized
cohorts of N=1000.
 Know why & when one starts & stops a drug
 Know why & when one needs to change/switch
 Know why & when to reassess.
Contribution of Cytokines to RA
Clinical Manifestations
APR, anemia
Liver
IL-6
Leukocyte Chemotaxis
TNF, IL-17, IL-6, IL-1
Angiogenesis
TNF, IL-6
Cartilage Degradation
MMP
Bone Erosion
IL-1, IL-17
TNF, IL-17, IL-6, IL-1
Joint
Colmegna et al. Clin Pharmacol Ther
2012;91:607-20
Do Our Treatments Regulate
Citrullinated Ags – ACPAs?
Anti-CP Abs
Effect Of MTX On Citrullination
In UMR 106 Cells
MTX (nM)
MTX (nM)
113
92
PAD2
53
% Inhibition
113
92
CMC
100
90
80
70
60
50
40
30
20
10
0
0
1
10
50
100
Dose-response curve of in vitro MTX treatment of UMR 106 cells
at 10 µg of total proteins/lane at in vivo therapeutic concentrations.
Lora M et al (Ménard HA) ACR 2005
A Scientific Basis For A Century Of Empiricism In
Treating RA: All DMARDs Downregulate The Production
Of Citrullinated Proteins/Antigens In Vitro.
Henri-André Ménard MD & Maximilien Lora PhD
MSK Research Axis 0f The McGill University Health Center At The Royal Victoria Hospital, Montreal (QC), CANADA H3A 1A1
INTRODUCTION
RESULTS
RESULTS
CRA Kanaskis
2009 and ACR Philadelphia
2009
H
N
H
O
Azathioprine (AZA)
Clinical concentration 0.5 to 10µM
Methotrexate (MTX)
Clinical concentration: 25 nM
CITRULLINATION
is the conversion of an arginine within a peptidic link to a
citrulline in an enzymatic process carried out by
PeptidylArginine Deiminases.
CTRL
193
WB with anti-Sa
MTX (100 nM) treatment of UMR106 at
subconfluence showed a decrease in
PAD activity. This MTX effect was
prevented by folinic acid (20 µM).
O
N
115
97
10
50
100
200
Prednisone (Pred)
Clinical concentration 0.1 µM
µM
UMR106
CTRL 0.1 1.0
193
WB with anti-CMC
AZA treated subconfluent
UMR106 cells.
AZA at 50 µM to 200 µM
significantly decreased PAD
activity.
RESULTS
10 µM
ECV304
CTRL 0.1 1.0
10 µM
193
115
97
115
97
WB with anti-CMC
Pred. treated
subconfluent UMR106 &
ECV304 cells.
Pred. had no effect on
PAD activity.
1. MTX, blocks PADI-2 activity in proliferating cells without affecting the
quantity of enzyme. It does so via folate-dependent and adenosine
receptor-independent pathways. The induction of a PAD Inhibitor is a
Sulphasalazine (SSZ)
Clinical concentration 50 µM
possibility.
RATIONALE AND HYPOTHESIS
+ H2O
NH
H2N+
PADs
+ NH3 + H+
Ca++
O
NH2
PeptidylArginine
53
53
NH
CTRL
NS
NH2
RA patients have IgG auto-antibodies
PAD2
At the same dosage, MTX had no effect on PAD activity of
confluent UMR106 or subconfluent and confluent ECV304
against citrullinated (cit-)epitopes.
100
200 µM
WB with anti-CMC
AZA treated confluent UMR106
cells.
AZA at 200 µM decreased PAD
activity.
25
125 250 µM
WB with anti-CMC
SSZ treated subconfluent UMR 106 cells.
SSZ at 250 µM significantly decreased PAD
activity.
37
115
97
CTRL 0.1
1.0
10 µM
193
53
CTRL
CTRL
53
193
The Abs are present before or at disease onset at
40-80% sensitivity with >95% specificity.
Factual commonality 1: pharmacologically unrelated drugs, the
50
115
97
WB with anti-PAD2
MTX treatment showed no decrease in
PAD-2 protein
PeptidylCitrulline
10
10
50
100
WB with anti-CMC
Pred. treated confluent UMR106 cells.
Pred. at 10µM might be increasing PAD
activity.
200 µM
115
97
193
WB with anti-CMC
AZA treated subconfluent
ECV304 cells.
AZA decreased PAD activity at all
concentrations tested.
At the same dosage, AZA had
little effect on ECV304 confluent
assays.
53
115
97
CTRL 0.1
1.0
10 µM
193
2. HCQ, AZT and, SSZ decrease the quantity of PADIs in resting and
Hydroxychloroquine (HCQ)
Clinical concentration 1 µM
proliferating cells in vitro.
DMARDs have survived empirically as good treatment for RA;
Factual commonality 2: cit-proteins (non-specific products of
inflammation), have a central role in RA as they induce a specific
autoimmune response that drives the disease;
Factual commonality 3: biologicals target effector mechanisms,
downstream from the immunological synapse with little effect on autoAbs and they all work most efficiently when combined with DMARDs ;
Hypothetical commonality 4: DMARDs have a common mode
of action complementary to biologicals via inhibition of citrullination, an
event upstream from the immunological synapse.
115
96
53
WB with anti-CMC
Pred. treated confluent ECV304 cells.
Pred. at 10 µM may actually increase PAD
activity.
51
CTRL
500
1000 µM
Sub-confluent
CTRL 10 50 100
115
96
WB with anti-CMC
SSZ treated confluent UMR 106 cells.
SSZ at 500 and 1000 µM significantly
decreased PAD activity.
Confluent
CTRL 10 50 100 µM
193
WB with anti-CMC
HCQ treated subconfluent &
confluent UMR106 cells.
HCQ at 50 µM to 100 µM
significantly decreased PAD
activity.
115
97
53
CTRL
UMR106 cells have
PAD activity at
confluence only.
113
92
53
CTRL 250 500 1000 µM
115
WB with anti-CMC
SSZ treated subconfluent ECV304 cells.
SSZ at 1000 µM significantly decreased
PAD activity.
At the same dosage, SSZ had no effect on
ECV304 confluent assays.
Anti-Sa
Ca2+
10
50
100 µM
115
97
51
53
WB with anti-PAD2
HCQ treated
CTRL
subconfluent
ECV304 cells.
All DMARDs downregulate the production
of cit-proteins/antigens in vitro.
- MTX blocks PAD-activity in proliferating cells via a folate
dependent pathway. The effect is independent of adenosine receptors
(not shown) and the quantity of PAD-protein is unchanged.
- AZT, SSZ and HCQ decrease the quantity of PADs in various
conditions either in resting or dividing cells.
- Prednisone has no effect on citrullination in dividing cells but
has an unexpected upregulating effect at high dose in resting cells.
100 µM
CTRL
Sa
113
92
53
WB with anti-CMC
HCQ treated subconfluent
ECV304 cells.
HCQ at 50 and 100 µM
decreased PAD activity.
96
37
CTRL
ECV304 cells have
PAD activity at both
subconfluence and
confluence.
Sa
37
51
4. DMARDs decrease the afferent antigenic input while Prednisone and
- +
- +
the Biologicals
suppress the efferent mechanisms of the immune
synapse involving citrullinated epitopes.
Anti-CMC
53
CONCLUSIONS
3. Corticosteroids
have no direct effect on citrullination .
MATERIALS AND METHODS
UMR106 and ECV304 cell lines were treated for 4 days with increasing
doses of methotrexate (MTX), sulphasalazine (SSZ), azathioprine
(AZT), hydroxychloroquine (HCQ) or Prednisone (Pred) at doses
corresponding to those obtained in vivo during RA treatment. We
estimated semi-quantitatively by western blot (WB) on cell extracts,
their effect on the production of all cit-proteins (detected by a rabbit
anti-CMC serum) and cit-antigens (detected by anti-Sa RA sera).
115
97
WB with anti-PAD2
SSZ treated subconfluent ECV304 cells.
SSZ at 750 µM significantly decreased
PAD2 protein.
750 µM
PAD2
10
50
100 µM
193
WB with anti-CMC
HCQ treated confluent
ECV304 cells.
HCQ at 50 and 100 µM
decreased PAD activity.
115
97
53
Those data support our hypothesis that DMARDs all work by decreasing
the cit-Ag load in vivo. In patients with autoAbs to a cit-protein Ag like
cit-vimentin/Sa, DMARDs may influence the putative ongoing auto-Ab
response to cit-vimentin (anti-Sa), thus acting on the two major elements
of the autoimmune amplification loop responsible for chronicity.
ACKNOWLEDGEMEN
T
Conclusions on Citrullination in RA
Those in vitro data provide an explanation for why the
pharmacologically diverse DMARDs are successful in RA :
they are PADIBs
1. Individually, with MTX being the best at it;
2. In combination with each other, providing a variety of
not mutually exclusive inhibition modalities;
3. Essential to use with biologicals as they are unique in
decreasing the afferent arm of the immune process;
4. Especially relevant when ACPAs (anti-Sa) drive the
disease.
Take Home Message On Anti-CCP
• They relate to immune response genes specifically
predisposing to RA but are not always associated with
severe or even actual disease. In the context of N = 1,
• most useful when negative to rule out RA;
• low titers may lead to circular clinical reasoning;
• high titres most useful to rule in RA.
• Their prognostic value is based on longitudinal and
transversal testing of RA COHORTS. They are less
reliable at disease onset in personalized N=1 medicine.
• Normalization is unusual and, as currently tested for,
unchanging titers do not allow immune monitoring.
Ménard HA, Editorial. J Rheumatology 2009
Take Home Message On Anti-Sa
• strictly linked to RA disease:
DIAGNOSIS
• closely linked with the more severe erosive long
term phenotype:
PROGNOSIS
• titers vary with activity as pathogenic antibodies do:
MONITORING
• normalization is achievable and may turn out to be a
robust marker of remission maintenance at T0/S0:
REMISSION
Ménard HA, Editorial J Rheumatology 2009
TREATMENT IMPLICATIONS
PKIs ?
S T E R O I D S
MTX, HCQ, SSZ,AZT= PADIBs
ABATACEPT
PKIs ?
PKIs ?
ACPAs
PKIs ?
PKIs ?
ANTI-CYTOKINES
RITUXIMAB
FORGET THIS OSLER’S QUOTATION
"When a patient with arthritis comes through
the front door,
I want to leave by the back door".
Times are changing
HA MÉNARD, Jan 2013
SHUKRAN
ALA ALDAWAH
QUESTIONS?
COMMENTS?