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1 Table S1 Selected small-molecule combinations tested clinically for fibrotic diseases No. and name of combination, if available Molecules in combination Molecular targets or mechanisms and selected in vitro pharmacology Route of delivery Preclinical or clinical evidence for anti-fibrotic activity*; (trial name) Clinical trials ; indication (status); identifier (trial name) 3 Prednisone Metabolized to the active in vivo; GR K i (prednisolone) 2.4 nm and MR K i (prednisolone) 37 nm 1 Azathioprine Multiple targets; azathioprine is a prodrug metabolized to purine antagonists 2 ; it incorporates into replicating DNA and blocks purine synthesis 3 NAC Multiple activities 4,5 : ROS scavenger; increases cellular glutathione levels; reducing agent NAC plus prednisone plus azathioprine preserves vital capacity and D Lco in patients with IPF, better than standard care does 6 High-dose NAC in combination with prednisone plus azathioprine had small positive effects in patients with IPF (IFIGENIA trial) 7 No significant benefit of NAC in patients with IPF with mild to moderate impairment in lung function 8 Prednisone plus azathioprine plus NAC had increased risks of death and hospitalization in patients with IPF 9 IPF (azathioprine plus prednisone); NCT IPF (Phase III, NCT (IFIGENIA) 6 Losartan AT1 receptor antagonist: losartan AT1 (IC nm) 10 ; the major active metabolite (EXP 3174) is fold more potent in vivo Sildenafil PDE5 inhibitor (IC nm) 11 Sildenafil did not cause a significant difference in the proportion of patients with an improvement of 20% or more in the 6-minute walk distance at 12 weeks (the primary outcome) but had small benefits in some secondary outcomes, including the degree of dyspnoea and quality of life 12 IPF (Phase II/III); NCT Prednisone Metabolized to the active Colchicine Blocks cell division by disrupting microtubules and at 50 nm blocks almost all the cells at mitosis 13 Neither colchicine nor D-penicillamine modified the progressive course of prednisone-treated IPF 15 Neither prednisone nor colchicine resulted in objective improvement in IPF; the disease continued to progress 16 No difference in survival of patients with IPF treated with colchicine or prednisone and those No current or recent clinical trials

2 D-penicillamine Copper chelator 14 on no therapy Prednisone Metabolized to the active Cyclophosphamide Inactive in vivo until it is metabolized by cytochrome P450 to yield phosphoramide mustard and acrolein, which alkylate DNA and proteins 18 Intravenous Corticosteroid plus cyclophosphamide therapy had no impact on survival in patients with IPF 19 Cyclophosphamide plus low-dose prednisolone is at least as effective as a course of high-dose corticosteroids followed by an alternate-day low-dose regimen 20 IPF (Phase II, NCT SSc-ILD (Phase III); NCT (SCLEROCYC) 16 PTL-202 NAC Multiple activities 4,5 : ROS scavenger; increases cellular glutathione levels; reducing agent (IC μm) 21 ; inhibits TNFα 22 A synergistic relationship was observed, resulting in an increase in PTL-202 active ingredients in the blood and an increase in known therapeutic effects without any new side effects (see Pacific Therapeutics Ltd. company website) plus NAC resulted in super-additive and synergistic effect in a mouse model of pulmonary fibrosis 23 IPF (Phase II trials planned) Budesonide directly modulates 17 Budesonide (budenofalk) GR agonist (IC 50 (rat GR) 2.9 nm) 24 contraction of collagen gels and can decrease collagen degradation under inflammatory conditions Ursodeoxycholic acid Weak FXR agonist 25 Regular treatment with ursodeoxycholic acid reduced rates of PBC-induced mortality 27 PBC (Phase III); NCT (IC μm) 21 ; inhibits TNFα 22 α-tocopherol (vitamin E) Multiple activities 28 Anti-fibrotic effect mediated at least in part by inhibition of the TGFβ1 cascade, supporting pentoxifylline vitamin E synergy and its use as a first-line treatment of RIF 29 Reduces superficial RIF. Synergism is likely, as treatment with each drug alone is ineffective 30,31 NASH (Phase III); NCT NASH; NCT RIF (Phase II, NCT

3 Prednisolone (active metabolite of prednisone) GR (K i (prednisolone) 2.4 nm) and MCR (K i (prednisolone) 37 nm) 1 No additional survival advantage with corticosteroids plus pentoxifylline compared with corticosteroids alone in patients with severe alcoholic hepatitis 32 Alcoholic hepatitis (Phase III); NCT (IC μm) 21 ; inhibits TNFα 22 Prednisone Metabolized to the active Zileuton prevented lung inflammation induced by bleomycin administration 34 IPF (Phase II, NCT Azathioprine Multiple targets; a prodrug metabolized to purine antagonists 2 ; incorporates into replicating DNA and blocks purine synthesis 3 Zileuton 5LO inhibition (IC μm); inhibits biosynthesis of LTB4 in human whole blood 33 INV-144 Losartan AT1 receptor antagonist: losartan AT1 (IC nm) 10. Major active metabolite (EXP 3174) is fold more potent in vivo ALA inhibits hepatic PAI1 expression through inhibition of TGFβ-mediated molecular mediators and prevents the development of BDL-induced hepatic fibrosis 36 Hypertension and type 2 diabetes with nephropathy (Phase II, NCT ALA Antioxidant and metal chelator 35 5LO, 5-lipoxygenase; ALA, alpha lipoic acid; AT1, angiotensin II receptor type 1; BDL, bile duct ligation; D Lco, diffusing capacity of lung for carbon monoxide; FXR, farnesoid X receptor; GR, glucocorticoid receptor; IC 50, half-maximal inhibitory concentration; ILD, interstitial lung diseases; IPF, idiopathic pulmonary fibrosis; K i, inhibition constant; LTB4, leukotriene B4; MR, mineralocorticoid receptor; NAC, N-acetylcysteine; NASH, non-alcoholic steatohepatitis; PAI1, plasminogen activator inhibitor 1; PBC, primary biliary cirrhosis; PDE, phosphodiesterase; RIF, radiation-induced fibrosis; ROS, reactive oxygen species; TGFβ, transforming growth factor-β; TNFα, tumour necrosis factor-α. *For the combination where possible, otherwise data for the individual components reported. Selected clinical trials for fibrosis indications (ongoing, completed, suspended or terminated). Clinical trials found at 1. Coghlan, M. J. et al. Synthesis and characterization of non-steroidal ligands for the glucocorticoid receptor: selective quinoline derivatives with pred-

4 nisolone-equivalent functional activity. J. Med. Chem. 44, (2001). 2. Hoffmann, M., Rychlewski, J., Chrzanowska, M. & Hermann, T. Mechanism of activation of an immunosuppressive drug: azathioprine. Quantum chemical study on the reaction of azathioprine with cysteine. J. Am. Chem. Soc. 123, (2001). 3. Maltzman, J. S. & Koretzky, G. A. Azathioprine: old drug, new actions. Clin. Invest. 111, (2003). 4. Samuni, Y., Goldstein, S., Dean, O. M. & Berk, M. The chemistry and biological activities of N-acetylcysteine. Biochimica et Biophysica Acta 1830, (2013). 5. Zafarullaha, M., Li, W. Q., Sylvestera, J. & Ahmad, M. Molecular mechanisms of N-acetylcysteine actions. Cell. Mol. Life Sci. 60, 6-20 (2003). 6. Demedts, M. et al. High-dose acetylcysteine in idiopathic pulmonary fibrosis. N. Engl. J. Med. 353, (2005). 7. Behr, J. et al. Lung function in idiopathic pulmonary fibrosis - extended analyses of the IFIGENIA trial. Respiratory Research 10, 101 (2009). 8. 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Biol. 25, (2005). 26. Fang, Q. et al. Effect of budesonide on fibroblast-mediated collagen gel contraction and degradation. Journal of Inflammation Research 6, (2013). 27. Jackson, H., Solaymani-Dodaran, M., Card, T.R., Aithal, G.P., Logan, R. et al. Influence of ursodeoxycholic acid on the mortality and malignancy associated with primary biliary cirrhosis: a population-based cohort study. Hepatology 46: (2007) 28. Marquardt, D. et al. Tocopherol activity correlates with its location in a membrane: a new perspective on the antioxidant vitamin E. Journal of the American Chemical Society 135, (2013). 29. Hamama, S., Gilbert-Sirieix, M., Vozenin, M-C. & Delanian, S. Radiation-induced enteropathy: molecular basis of pentoxifylline-vitamin E anti-fibrotic effect involved TGF-b1 cascade inhibition. Radiotherapy and Oncology 105, (2012). 30. Chiao, T. B. & Lee, A. J. Role of pentoxifylline and vitamin E in attenuation of radiation-induced fibrosis. Annals of Pharmacotherapy 39, (2005). 31. Delanian, S., Porcher, R., Balla-Mekias, S. & Lefaix, J-L. Randomized, placebo-controlled trial of combined pentoxifylline and tocopherol for regression of superficial radiation-induced fibrosis. Journal of Clinical Oncology 21, (2003). 32. Sidhu, S.S., Goyal, O., Singla, P., Gupta, D., Sood, A. et al. Corticosteroid plus pentoxifylline is not better than corticosteroid alone for improving survival in severe alcoholic hepatitis (COPE Trial). Digestive Diseases and Sciences 57: (2012) 33. Boschi, D. et al. Multitarget Drugs: Synthesis and preliminary pharmacological characterization of zileuton analogues endowed with dual 5-LO inhibitor and NO-dependent activities. ChemMedChem. 5, (2010). 34. Failla, M. et al. Pharmacological inhibition of leukotrienes in an animal model of bleomycin-induced acute lung injury. Respiratory Research 7, 137

5 (2006). 35. Guillonneau, C. et al. Synthesis and pharmacological evaluation of new 1,2-dithiolane based antioxidants. European Journal of Medicinal Chemistry 38, 1-11 (2003). 36. Min, A-K. et al. Alpha-lipoic acid inhibits hepatic PAI-1 expression and fibrosis by inhibiting the TGF-β signaling pathway. Biochemical and Biophysical Research Communications 393, (2010). Pacific Therapeutics Ltd. Company website;