TREATMENT OF RA 2014

1. 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

2. Anti-Sa Anti-CCP RF Amyloidosis Vasculitis Destruction Break of tolerance RA Onset CVD Lymphoma Environment Genes Sequence of Events in RA Pre- Early- Established- RA Immune response Pathologic inflammatory response T0 T100

3. Rheumatoid Hand Smoking Habit Ulnar Drift

4. 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)

5. 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.

6. 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 vshumoral • Evolutive disease = Δphysiopathical pattern

7. 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.

8. 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 IL-1, IL-17 Bone Erosion TNF, IL-17, IL-6, IL-1 Joint Colmegna et al.ClinPharmacolTher 2012;91:607-20

9. Do Our Treatments Regulate Citrullinated Ags – ACPAs? Anti-CP Abs

10. MTX (nM) MTX (nM) 10 100 50 0 1 113 92 CMC % Inhibition 53 113 PAD2 92 Effect Of MTX On CitrullinationIn UMR 106 Cells 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

11. CTRL 10 50 100 200 µM MTX Folinic UMR106 ECV304 CTRL MTX Folinic H O H µM O µM CTRL 0.1 1.0 10 CTRL 0.1 1.0 10 193 N N 193 193 115 PADs 97 + NH3 + H+ 115 + H2O 115 97 Ca++ 97 NH NH 53 O NH2 H2N+ NH2 53 PeptidylArginine PeptidylCitrulline CTRL 10 50 100 200 µM NS 53 115 193 37 97 115 PAD2 97 53 CTRL 10 50 100 200 µM 193 CTRL 25 125 250 µM 115 97 115 96 53 51 CTRL 500 1000 µM Sub-confluent Confluent CTRL 10 50 100 CTRL 10 50 100 µM 193 115 115 96 97 53 51 µM CTRL 250 500 1000 115 96 Anti-Sa CTRL 100 µM Anti-CMC - - + + Ca2+ 51 113 CTRL 10 50 100 µM 92 37 193 Sa 53 CTRL 750 µM 115 PAD2 113 92 Sa 53 A Scientific Basis For A Century Of Empiricism In Treating RA: All DMARDs Downregulate The Production Of Citrullinated Proteins/Antigens In Vitro. • CRA Kanaskis 2009 and ACR Philadelphia 2009 • MTX, blocks PADI-2 activity in proliferating cellswithout affecting the quantity of enzyme. It does so via folate-dependent and adenosine receptor-independent pathways. The induction of a PAD Inhibitor is a possibility. • HCQ, AZT and, SSZ decrease the quantity of PADIs in resting and proliferating cells in vitro. • Corticosteroids have no direct effect on citrullination . • DMARDs decrease the afferent antigenic input while Prednisone and the Biologicals suppress the efferent mechanisms of the immune synapse involving citrullinated epitopes. 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 RESULTS INTRODUCTION RESULTS RESULTS Azathioprine (AZA) Clinical concentration 0.5 to 10µM Prednisone (Pred) Clinical concentration 0.1 µM 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. WB with anti-CMC AZA treated subconfluent UMR106 cells. AZA at 50 µM to 200 µM significantly decreased PAD activity. 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). WB with anti-CMC Pred. treated subconfluent UMR106 & ECV304 cells. Pred. had no effect on PAD activity. WB with anti-PAD2 MTX treatment showed no decrease in PAD-2 protein WB with anti-CMC AZA treated confluent UMR106 cells. AZA at 200 µM decreased PAD activity. RA patients have IgG auto-antibodies against citrullinated (cit-)epitopes. CTRL 0.1 1.0 10 µM At the same dosage, MTX had no effect on PAD activity of confluent UMR106 or subconfluent and confluent ECV304 193 WB with anti-CMC Pred. treated confluent UMR106 cells. Pred. at 10µM might be increasing PAD activity. 115 The Abs are present before or at disease onset at 40-80% sensitivity with >95% specificity. 97 Sulphasalazine (SSZ) Clinical concentration 50 µM 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 RATIONALE AND HYPOTHESIS CTRL 0.1 1.0 10 µM 193 Factual commonality 1: pharmacologically unrelated drugs, the 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 auto-Abs 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. WB with anti-CMC SSZ treated subconfluent UMR 106 cells. SSZ at 250 µM significantly decreased PAD activity. 115 WB with anti-CMC Pred. treated confluent ECV304 cells. Pred. at 10 µM may actually increase PAD activity. 97 Hydroxychloroquine (HCQ) Clinical concentration 1 µM 53 37 CONCLUSIONS WB with anti-CMC HCQ treated subconfluent & confluent UMR106 cells. HCQ at 50 µM to 100 µM significantly decreased PAD activity. WB with anti-CMC SSZ treated confluent UMR 106 cells. SSZ at 500 and 1000 µM significantly decreased PAD activity. 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. 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. 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). CTRL 10 50 100 µM WB with anti-CMC HCQ treated subconfluent ECV304 cells. HCQ at 50 and 100 µM decreased PAD activity. 115 97 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. 53 WB with anti-PAD2 HCQ treated subconfluent ECV304 cells. UMR106 cells have PAD activity at confluence only. WB with anti-CMC HCQ treated confluent ECV304 cells. HCQ at 50 and 100 µM decreased PAD activity. 97 ECV304 cells have PAD activity at both subconfluence and confluence. WB with anti-PAD2 SSZ treated subconfluent ECV304 cells. SSZ at 750 µM significantly decreased PAD2 protein. ACKNOWLEDGEMENT 53

12. 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 • Individually, with MTX being the best at it; • In combination with each other, providing a variety of not mutually exclusive inhibition modalities; • Essential to use with biologicals as they are unique in decreasing the afferent arm of the immune process; • Especially relevant when ACPAs (anti-Sa) drive the disease.

13. Take Home MessageOnAnti-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

14. Take Home MessageOn 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

15. ABATACEPT ANTI-CYTOKINES RITUXIMAB TREATMENT IMPLICATIONS PKIs ? MTX, HCQ, SSZ,AZT= PADIBs PKIs ? PKIs ? STEROIDS ACPAs PKIs ? PKIs ?

16. 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

17. SHUKRANALA ALDAWAH QUESTIONS? COMMENTS?