Unique Cytokine Profile Generated by Synergistic Interaction Between Prednisolone and Dipyridamole, the Components of Crx-102

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Unique Cytokine Profile Generated by Synergistic Interaction between Prednisolone and Dipyridamole, the Components of CRx-102

#193 PS3 Christopher C. Fraser, Yanjun Wang, Alyce Finelli, Grant R. Zimmermann. CombinatoRx, Incorporated, Cambridge, MA 02142, USA

Introduction Glucocorticoids synergize selectively with PDE4 inhibitors Synergy between Dipyridamole and Prednisolone Dipyridamole and Prednisolone (CRx-102) synergize to A synergistic interaction of glucocorticoids with PDE4 inhibitors to suppress production of TNFα from stimulated PBMCs is independent of PKA kinase activity inhibit multiple inflammatory targets. including a combination of prednisolone (Pd) and dipyridamole (Dp) Inhibition of TNF-α from LPS-stimulated PBMCs MCP-1 MDC w/o Pd Inhibition (%) Inhibition (%) Inhibition (%) No synergy SynergymMDC w Pd 0.03 μM Synergy between the components of CRx-102 produces a DT-1042399 DT- 1 04 2 3 98 DT-1042396 (CRx-102) was discovered. 75 75 75 77 76 72 68 75 73 70 66 76 76 78 84 84 89 92 80 77 78 78 80 79 83 82 84 4500 PKA dependent 600 PKA independent response that is distinct from the individual agents 74 70 66 69 71 58 66 71 65 62 68 72 74 74 83 87 92 91 75 74 71 72 74 76 83 76 79 66 62 65 68 63 64 64 60 66 62 67 67 74 76 79 85 88 92 70 68 72 70 74 73 76 77 79 (blocked by H89) 500 (not blocked by H89) TNF-α() IL-6 RANTES() MDC CRx-102 has demonstrated significant clinical activity in phase 2a 58 58 54 50 52 56 56 59 58 53 62 62 66 65 78 82 89 89 63 60 62 57 66 64 63 69 73 3600 M)M)

48 44 52 48 47 41 30 45 49 43 53 53 52 63 67 79 84 87 43 45 41 49 54 55 55 60 62 μμ 51 66 80 80 30 66 79 85 17 39 61 71 400 * P<0.05 74 86 94 96 Macrophage synergy trials for the treatment of hand osteoarthritis and rheumatoid arthritis. 32 22 21 29 26 24 29 27 29 34 36 37 43 58 66 69 81 84 22 30 29 29 32 39 42 52 49 2700 compared to 46 58 74 80 51 65 75 78 6 17 51 64 54 78 88 92 11 14 15 7.3 12 -.6 2.6 5.4 6.8 14 21 28 31 42 54 67 76 82 17 8.9 17 10 19 18 15 30 36 300 The synergistic interaction between the very low dose Pd and Dp is 5.1 .8 8.1 8 1 -7 -3.7 -18 -4.5 10 22 16 32 44 52 64 72 80 -3.8 7.1 -5.8 3 3.5 3.2 23 28 34 DP or Pd alone 38 45 52 72 26 45 57 75 21 5.1 10 58 44 74 72 90 Cytokines: pg/ml Prednisolone (uM) Prednisolone (uM) Prednisolone Prednisolone (uM) Prednisolone 1800 11 05.3 .0160 4.9 .062-.4 .25-13 -7.2 1 -1.8 -9.8 -15 04 .0166.8 14 .06220 .2537 45 1 63 74 78 06.4 -4.4 .0164.9 .062-3.3 .25-9.5 9.4 .99 13 22 42 *

N=3-5 N=3-5 N=1-2 200 01310 01310 0 14 36 62 69 0 24 21 67 58 0 1.1 12 42 37 0 52 44 82 76 IL-6, TNF-α, IL-12 (p40),

* NN

thought to amplify the anti-inflammatory activity of the steroid without DipyridamoleDipyridamole ( ( 0 .65 2.6 10 41 0 .016 .063 .25 1 0.41.66.425

Dose Response Matrix IL-1. Cilostazol (uM) Rolipram (uM) Tadalafil (uM) 900 100 * * 0.01.03.31 0.01.03.31 0.01.03.31 0.01.03.31 * P<0.05 * Prednisolone (μM) affecting steroid-mediated side effects. Isobologram Isobologram Isobologram DT-1042399 DT-1042398 DT-1042397 compared to 0 Chemokines: y 0 y y LPS k RANTES, MDC, MIP-1α, m S m M M im S m m M M Experiments were performed to help identify the cellular and dditivit i sk dditiv it dditiv it t u r t A s LP 1u 3u 0u MIP-1β A A s LP 3u 0u - 1 - 1 Fors k 10u Synergy No on DP 1umDP P 9 10uM + Fo DP DP 89 s molecular mechanism of the anti-inflammatory response. N D 8 9 Non DP 10uM H H or Synergy + Forsk 10H8 F H89 + Proteases:

No =60 =60 =60 ) ) ) um + MMP-9 % % % ( 10 Synergy ( ( 10um Isobologram MMP-2 DP DP 012 0.51 0.631.3 Prednisolone / .2uM / Prednisolone Prednisolone / .12uM Prednisolone Dipyridamole does not Inhibition Inhibition Prednisolone / .12uM / Prednisolone Discovery of CRx-102 Inhibition Inhibition Synergy of dipyridamole, ibudilast C57/BL6 mouse bone marrow cells, cultured with DMEM+10%FBS for 48h and non-adherent cells collected and plated in 012 increase cAMP levels in Dipyridamole synergy with 50ng/ml m-CSF for 7 days. Cells were stimulated with LPS +/- Dp or Pd and media analyzed 20h later for cytokine levels. 0.51 0 .63 1.3 and rolipram with prednisolone is Cilostazol / 41uM Rolipram / .21uM Tadalafil / 25uM macrophages. Ibudilast and Rolipram suggests not blocked by the PKA inhibitor CRx-102 synergistically suppresses TNFα production in vitro 8 involvement of targets in addition PDE-3: Cilostazol PDE-4: Rolipram PDE-5: Tadalafil H89 suggesting that synergy is to PDE. Combining Dp and Pd results in a synergistic multi-target independent of PKA kinase activity. 7 Prednisolone synergizes with PDE-4 but not PDE-3 & -5 mechanism that inhibits key effectors underlying OA and RA 1250 6 TNF-α inhibitors to suppress production of TNFα. MDC *Simulated human peripheral 2500 Venn diagram indicating in vitro LPS- -H89 5 CRx-102 blood mononuclear cells 1000 2.5 6 +H89* 2000 responsive factors inhibited synergistically (PBMC). 4 LPS PI Dp sensitive TNFα Low dose Synergy PDE specificities 750 1500 by CRx-102 or by the components (Dp (3- Synergy 5 3 MIP-1α 2 Dipyridamole multiple factors Pd sensitive 10 uM) or Pd (30 nM)) alone. Ibudilast multiple 500 1000 (3-10 uM) RANTES factors

Inhibition % = 50 = % Inhibition 2 Inhibition % = 70 = % Inhibition 4 Rolipram PDE4 MDC cAMPcAMP level levelfold)fold) ( ( 500 JE (MCP-1) (30 nM) 1.5 250 1 * Based on a human PK study, clinical doses of Cilostazol PDE3 MCP-5 IL-6 3 Sildenafil PDE5 0 CRx-102 are predicted to result in serum levels of LPS Stimulated PBMCs PMA/Ca2+ Stimulated PBMCs 0 MMP-9 Vardenafil PDE5 0 T 3 .3 N .0 0.3 dipyridamole and prednisolone of 0.3-6 uM and 30- 1 LPS 0 l 0.3 l 0 0 Dp 10 u o o

Dose-response combination matrices detected by ELISA measuring TNFα secretion from PBMCs after Tadalafil PDE5 NT rd Ib R 2 P 200 nM respectively. LPS + Ibu 1 stimulation with LPS or PMA / Ionomycin 1 Rol Dp 10 Dp 10 + R .3 Dp+Pd

Ibu+Pd 0.3 + Prd Rol+Pd Pd 0.03 Dp 10 + Ibu 0.3 DPDP 10uM 10uM RoliRoli 1uM 1uM Dp u 0.5 Synergy scores are calculated 1uM 1uM Ibud Ibud Dp 10 + Prd 0.03 DPDP + +ForskForsk

1 forsk forsk + + Roli Roli Non-treated Non-treated

* Also seen with PKA inhibitor 14-22 + +Forsk Forsk Ibud Ibud

from a dose response matrix Dp+Pd+H89 10 + Rol 0 Synergy Score Score Synergy Prednisolone with combination In Ibu+Pd+H89 Rol+Pd+H89 p using the Loewe additivity Dp 10D + Ib 0 ForskolineForskoline 10uM 10uM Conclusion CRx-102 was shown to be clinically active in treating 0 model. The score is based on Synergy of dipyridamole, ibudilast and rolipram with prednisolone is independent of PKA kinase activity. the excess volume between the Dipyridamole was not observed to increase cAMP levels in macrophages, in contrast to data from human PBMCs, Rheumatoid Arthritis (RA) and Osteoarthritis (OA) experimentally observed and and synergizes with ibudilast and rolipram suggesting a molecular mechanism distinct from the PDE-4 inhibitors. Ibudilast

Tadalafil CRx-102 is a novel synergistic combination drug candidate that has shown Rolipram modeled surfaces. A larger Sildenafil Cilostazol Tadalafil Ibudilast

Vardenafil C57/BL6 mouse bone marrow cells, cultured with DMEM+10%FBS for 48h and non-adherent cells collected and plated in 50ng/ml m-CSF Rolipram Sildenafil Cilostazol Vardenafil score indicates more synergy. for 7 days. Cells were stimulated with LPS +/- Dp or Pd and media analyzed 20h later for cytokine levels. CRx-102 is a synergistic combination of a very low dose of Dipyridamole significant anti-inflammatory effects in OA and RA clinical studies and is being Dipyridamole Prednisolone and Dipyridamole developed for the treatment of multiple immuno-inflammatory diseases. Dipyridamole increases cAMP in human PBMCs and is an inhibitor of Molecular model for the anti-inflammatory effect of CRx-102 RA: Improved ACR20 OA: Improved AUSCAN Pain phosphodiesterases (PDEs) including PDE-4 The components of CRx-102 (Dp and Pd) modulate converging intracellular 120 signaling pathways to synergistically inhibit key inflammatory modulators (TNFα, 80% p=0.006 Dipyridamole synergizes with rolipram suggesting a Day 42: p=0.025 100 distinct mechanism. RANTES, MDC, MIP-1α, IL-6) that control disease progression and mediators p=0.023 Cell-free PDE Assay cAMP Inhibition (%) Inhibition (%) Inhibition (%) 60% DT-1024938 DT - 1 0 2 4 9 39 DT-1024940 (MMP-9) that contribute directly to disease pathology. The inhibitory profile 80 CRx-102 80 82 82 84 82 83 84 87 91 87 86 87 88 89 90 90 91 92 80 81 80 80 80 81 81 84 86 p=0.010 4 CRx-102 80 71 71 71 72 74 76 78 84 88 79 80 81 82 83 86 87 88 90 73 74 73 71 73 75 77 77 81 observed in combination is distinct from that observed for the individual agents at 60 56 57 59 59 61 64 72 79 87 66 71 72 76 80 82 85 87 90 60 59 63 65 66 70 69 78 80 40% 38 42 35 44 44 48 61 74 83 49 57 60 67 71 74 80 84 88 48 46 50 54 54 60 68 74 79 equivalent doses. 70 28 22 32 30 35 43 56 71 84 32 53 51 59 65 69 78 83 89 42 41 47 42 49 52 61 72 81 3 40 9.8 16 18 28 24 38 47 72 82 19 36 49 51 54 65 75 84 87 26 26 33 40 42 53 55 68 79 p=0.191 -.5 14 16 21 24 38 54 72 84 10 37 40 48 52 65 72 81 85 21 26 30 31 41 48 57 73 80 60 Rolipram (uM)Rolipram

20% AUSCAN pain (mm) Placebo (NSS) -2.8 7.6 5.1 9.5 21 27 51 69 83 7 25 36 38 48 63 69 78 86 13 20 18 32 38 50 58 69 76 Synergy of dipyridamole, ibudilast and rolipram with prednisolone is not blocked Dipyridamole (uM) Dipyridamole (uM) Dipyridamole 0.31.24.919 0.31.24.919 20 Placebo -13 6.7 1.7 19 22 29 47 70 82 -.6 23 25 28 38 49 63 72 81 0-3.9 .01620 26 .06329 .2534 45 1 56 69 79 N=3-4 N=4 N=4 50 2 0.31.24.919 0 .016 .063 .25 1 0 .016 .063 .25 1 by the PKA inhibitor H89 suggesting that synergy is independent of the kinase DipyridamoleIsobologram (uM) RolipramIsobologram (uM) RolipramIsobologram (uM) 0% 0 DT-1024938 DT-1024939 DT-1024940 Mean improvement from baseline in in baseline from improvement Mean activity of PKA.

% Patients of Achieving ACR20 Response 0 7 14 21 28 35 42 0 7 14 21 28 35 42 40 Fold Increase Additivity Additivity 1 Additivity Day of Study Day of Study Additivity Synergy Additivity

Percent Inhibition Percent 30 Dipyridamole synergizes with both ibudilast and rolipram suggesting that Dp inhibits a set of targets and has a molecular mechanism that is distinct from that RA: Phase 2a; 59 subjects with moderate-severe RA; 6 centers; blinded, placebo-controlled, randomized, six 20 0 123457 891011 0.01 0.1 1 10 100 weeks in duration; received either CRx-102 (prednisolone, dipyridamole) or placebo on top of stable DMARD. .29uM / Rolipram Dipyridamole / 4.7uM

Dipyridamole / 6.7uM of these PDE inhibitors. 0.51 0 .58 1.2 0.611.2 Inhibition (%)=65 Inhibition Inhibition (%)=65 Inhibition Primary Endpoint: Reduction in CRP. Secondary Endpoints: ACR20, DAS28, inflammatory cytokines Phosphodiesterase Isotype Dipyridamole [ uM ] (%)=65 Inhibition 0 .58 1.2 0.51 0.611.2 Dipyridamole / 8.4uM Rolipram / .29uM Rolipram / .4uM OA: Phase 2a; 83 subjects enrolled with hand OA; Blinded, placebo-controlled, randomized, six weeks in Dipyridamole (10 uM) inhibits Dipyridamole increases Combining Dp and Pd results in a synergistic multi-target mechanism that duration; Enrollment Criteria: ≥1 swollen and ≥1 tender joint, and self-described hand pain PDE isotypes 2, 4, 5, 8, 10, and cAMP in PBMCs by three- Both DP and the PDE-4 specific inhibitor rolipram inhibit TNFα secretion from inhibits key effectors underlying OA and RA and suggests that CRx-102 may be ≥30 mm on AUSCAN scale, and ≥2 score on the Kellgren-Lawrence x-ray scoring system; Randomized (1:1 11 in cell-free enzyme fold after stimulation with PBMCs. Self-crosses of rolipram and DP display the expected additive interactions. ratio) to receive either CRx-102 or placebo inhibition assays. forskolin. Rolipram and DP combine synergistically to suppress TNFα, and therefore act a valuable new therapeutic agent to treat immuno-inflammatory diseases. through non-redundant molecular targets.