(292)1516 1. We have developed a technology to systematically and efficiently find synergistic combinations that target multiple biological pathways and that can be developed into new immuno-modulatory therapeutics. 2. A number of novel combination drug candidates have emerged from this approach and we have advanced them into clinical trials in immuno-inflammatory disease settings. 3. The platform has also revealed novel, synergistic pathway interactions that form the basis for future development of multi-targeted therapeutics. cHTS Systematic discovery of novel anti-inflammatory combination therapeutics E. Roydon Price, Palaniyandi Manivasakam, Grant Zimmermann, Joseph Léhar, Alexis A. Borisy, Curtis T. Keith. CombinatoRx Inc., Boston, MA 02118, USA Introduction cHTS Screening Platform Steroid Dissociation through Combinations Translates in Animal Models Summary ACR, San Diego, CA, USA, 2005 Master X Plate Conc: 1000 units Master Y Plate Conc: 1000 units + Combined and Diluted Combination Dilution Plate Conc: 10 units Approved drugs and target probes in pair- wise combination QC Analyze Screen Explore Combine low-dose steroid with an enhancer • Low-dose steroid lower effect and lower side effect • Enhancer selectively synergize on efficacy targets no synergy on side effect targets SSA Example: CRx-119 Synergies between established anti-inflammatories such as cyclosporine and glucocorticoids are observed when each target separate parallel pathways. Compound Library • 2000 FDA and late-stage compounds • ADME/Tox and targets are often known Cell Based Assay • Primary human lymphocytes • Many induction protocols • LPS, PMA/Ionomycin, CD3/CD28, etc. • Many downstream endpoints • TNFα, IL1, IL2, IFNγ, etc. • Can focus on a particular cell type or pathway Synergy Analysis • Proprietary tools to classify and measure combination effect • An ideal combination has unknown ratio/concentration • Both potency shifts and effect boosts possible • Therefore one needs to cover all possibilities with a Dose Response Matrix CombinatoRx has developed a platform to systematically screen for synergistic interactions between any two drugs, not limited to existing anti-inflammatory therapies. In addition, we have discovered compounds which when combined with effective but dose limited anti-inflammatory therapies (e.g. glucocorticoids or calcineurin inhibi- tors) allow dose reduction but maintain anti-inflammatory effect. Approach Selective Steroid Amplifiers (SSA): Distinct Cytokine Profiles CRx-119: a non-obvious in-vitro synergy Prednisolone with Amoxapine Amoxapine – Tricyclic Antidepressant – Not used as an anti-inflammatory Dissociated target and side effect profiles – Synergistic anti-TNFα effect Positive isobologram – Amoxapine does not potentiate glucocorticoid’s ability to transactivate GRE containing promoters – Amoxapine does not potentiate prednisolone’s ability to translocate the glucocorticoid receptor Prednisolone Amoxapine Amoxapine does not potentiate GRE dependent transactivation Amoxapine does not potentiate Glucocorticoid Receptor translocation Good Efficacy: TNFα inhibition GR translocation Rat Adjuvant Model of Arthritis Freund’s adjuvant injected in tail of Lewis rats (male/female) on Day 0, 48. Double placebo animals received mineral oil injections at the base of the tail. Between Days 10 and 20, the animals were treated daily via gastric gavage with compound(s). After Day 10, joint diameters were assessed every other day. Also active in endotoxemia Rat Safety Study Eight Lewis rats per arm were dosed p.o. daily for seven days. Fasting blood glucose levels were measured along with body weight, and serum corticosterone TNFα GR GC CsA IKK NFκB IkB CN P Ca ++ JNK p38 ERK MKK3/6 MKK4/7 MEK1/2 MLK MEKK4 Raf NFAT Ras PKC P ATF2 AP1 P Prednisolone CP Rheumatological diseases are mediated by the intersection of multiple biological pathways and networks. Consequently, a multi-targeted therapeutic approach may provide superior clinical benefit. Historically, combination treatments have been discovered through trial and error using drugs with proven disease modifying effect. We have developed a technology to systematically and efficiently find synergistic combinations of compounds that target multiple pathways underlying disease biology and that can be developed into new anti-inflammatory therapeutics. Pred High Amoxapine Pred Low 60 70 80 90 100 110 120 * # # # Normalized Blood Glucose (mg/dL/ 200 grams) Active in adjuvant models GC toxicity not enhanced No synergy observed with prednisolone for blood glucose, body weight or serum corticosterone T cell Monocyte TNFα PMA/Iono PMA/ Iono LPS ~2,000 approved drugs ~2,000 approved drugs ~10 ~10 ~2 million possible pair wise combos ~2,000 approved drugs ~2,000 approved drugs ~10 ~10 Dose Matrix 100 0 Effect 100 0 Effect 100 0 Effect 100 0 Effect 100 0 Effect CRx -102 CRx -119 CRx -139 CRx -160 CRx -163 CRx -164 CRx-119 CRx-102 TNF-PI IL2-PI IFN-PI TNF-LPS Synergy Synergy Synergy Prednisolone (uM) Prednisolone (uM) Prednisolone (uM) Prednisolone (uM) Prednisolone (uM) Prednisolone (uM) Prednisolone (uM) Prednisolone (uM) Synergy Synergy Synergy Pro-inflammatory genes GC side effect genes GC High Dose GC Low Dose Reduced GC side effect Combination Therapy allows reduction of GC dose and side effects but maintains control over pro-inflammatory gene expression Enhancer GR GR GRE GR Pro-Inflammatory Genes Toxicity-Associated Genes TNFα Robust Pipeline of Anti-Inflammatory Therapies TM Effect Concentration Drug B Effect Drug A Response Surface 1:1? 10:1? 1:10? Drug A Drug B 1:1? 10:1? 1:10? OH HO O OH O H H H N N O Cl NH Control Prednisolone 3.0 mg/Kg CRx-119 Adjuvant Alone Prednisolone 0.1 mg/Kg Amoxapine 3.0 mg/Kg Dipyridamole (uM) Dipyridamole (uM) Dipyridamole (uM) Dipyridamole (uM) Amoxapine (uM) Amoxapine (uM) Amoxapine (uM) Amoxapine (uM) Prednisolone Amoxapine Enhancer 2 Cyclosporine Prednisolone Pred/Amox Pred/Enh2 Cyclosporine Control Pred 10nM Pred 100nM Amoxapine Pred 10nM + Amoxapine Pred 100nM + Amoxapine [Drug A] [Drug B] Immunofluorescence for GR-alpha in Hela, 10um Amoxapine Prednisolone (uM) Amoxapine (uM) Prednisolone (uM) Amoxapine (uM)