1. TREATMENT OF RA 2014 The 1rst Kuwait-North American Update in Internal Medicine Conference 8-9 February 2014 Henri A. Ménard, MD, FRCP (C) Professor of Medicine McGill University McGill University Health Center

2. Sequence of Events in RA Immune responsePathologic inflammatory response T 100 LymphomaCVD Destruction Amyloidosis Vasculitis T0T0 Pre-Early-Established- RA Genes Environment Anti-CCP RF Break of tolerance RA Onset Anti-Sa

3. Smoking Habit Ulnar Drift Rheumatoid Hand

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. 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

7. 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. Personalizing Is Challenging

8. Contribution of Cytokines to RA Clinical Manifestations IL-6 LiverAPR, anemia TNF, IL-17, IL-6, IL-1Bone Erosion Joint IL-1, IL-17Cartilage DegradationMMP TNF, IL-17, IL-6, IL-1 Leukocyte Chemotaxis TNF, IL-6Angiogenesis Colmegna et al. Clin Pharmacol Ther 2012;91:607-20

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

10. Effect Of MTX On Citrullination In 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. MTX (nM) % Inhibition PAD2 CMC 0 1 10 50 100 113 92 53 113 92 MTX (nM) Lora M et al (Ménard HA) ACR 2005

11. 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 A Scientific Basis For A Century Of Empiricism In Treating RA: All DMARDs Downregulate The Production Of Citrullinated Proteins/Antigens In Vitro. INTRODUCTION MATERIALS AND METHODS RATIONALE AND HYPOTHESIS CONCLUSIONS ACKNOWLEDGEMEN T RESULTS RA patients have IgG auto-antibodies against citrullinated (cit-)epitopes. RESULTS 113 92 53 Anti-CMC Anti-Sa Sa 113 92 53 - + Ca 2+ - + Sa 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). 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. 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. UMR106 cells have PAD activity at confluence only. ECV304 cells have PAD activity at both subconfluence and confluence. 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). At the same dosage, MTX had no effect on PAD activity of confluent UMR106 or subconfluent and confluent ECV304 Methotrexate (MTX) Clinical concentration: 25 nM Sulphasalazine (SSZ) Clinical concentration 50 µM Azathioprine (AZA) Clinical concentration 0.5 to 10µM Hydroxychloroquine (HCQ) Clinical concentration 1 µM Prednisone (Pred) Clinical concentration 0.1 µM WB with anti-PAD2 MTX treatment showed no decrease in PAD-2 protein H N O H NH H2N+H2N+ NH 2 + H 2 O N O NH ONH 2 + NH 3 + H + PeptidylArginine PeptidylCitrulline PADs Ca ++ CITRULLINATION is the conversion of an arginine within a peptidic link to a citrulline in an enzymatic process carried out by PeptidylArginine Deiminases. 115 96 51 CTRL25125250µM WB with anti-CMC SSZ treated subconfluent UMR 106 cells. SSZ at 250 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. 115 96 51 CTRL5001000µM 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. The Abs are present before or at disease onset at 40-80% sensitivity with >95% specificity. WB with anti-PAD2 SSZ treated subconfluent ECV304 cells. SSZ at 750 µM significantly decreased PAD2 protein. CTRL1050200µM100 193 115 97 53 WB with anti-CMC AZA treated confluent UMR106 cells. AZA at 200 µM decreased PAD activity. WB with anti-CMC AZA treated subconfluent UMR106 cells. AZA at 50 µM to 200 µM significantly decreased PAD activity. 193 115 97 53 CTRL1050200µM100 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. CTRL50200µM100 193 115 97 53 10 WB with anti-CMC HCQ treated subconfluent & confluent UMR106 cells. HCQ at 50 µM to 100 µM significantly decreased PAD activity. 193 115 97 53 CTRL50µM100 Sub-confluentConfluent 10CTRL5010010 µMCTRL5010010 WB with anti-CMC HCQ treated subconfluent ECV304 cells. HCQ at 50 and 100 µM decreased PAD activity. 115 97 53 97 53 115 193 µMCTRL5010010 WB with anti-CMC HCQ treated confluent ECV304 cells. HCQ at 50 and 100 µM decreased PAD activity. CTRL 100 µM WB with anti-PAD2 HCQ treated subconfluent ECV304 cells. 115 97 53 193 37 CTRL1.0 µM 10 UMR106 ECV304 0.1CTRL1.0100.1 µM WB with anti-CMC Pred. treated subconfluent UMR106 & ECV304 cells. Pred. had no effect on PAD activity. 115 97 53 193 CTRL1.0100.1 µM WB with anti-CMC Pred. treated confluent UMR106 cells. Pred. at 10µM might be increasing PAD activity. 115 97 53 193 37 WB with anti-CMC Pred. treated confluent ECV304 cells. Pred. at 10 µM may actually increase PAD activity. CTRL1.0100.1 µM 115 96 51 37 CTRL2505001000 µM CTRL750 µM PAD2 CTRL MTX Folinic NS 193 115 97 53 115 97 PAD2 CRA Kanaskis 2009 and ACR Philadelphia 2009 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 possibility. 2.HCQ, AZT and, SSZ decrease the quantity of PADIs in resting and proliferating cells in vitro. 3.Corticosteroids have no direct effect on citrullination. 4.DMARDs decrease the afferent antigenic input while Prednisone and the Biologicals suppress the efferent mechanisms of the immune synapse involving citrullinated epitopes.

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 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.

13. 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

14. 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

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

16. HA MÉNARD, Jan 2013 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

17. SHUKRAN ALA ALDAWAH QUESTIONS? COMMENTS?