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Introduction Systemic sclerosis (SSc) is a severe, potentially lethal, chronic autoimmune disorder of unknown aetiology involving the skin and visceral organs. The early stage of SSc is characterised by endothelial cell injury, perivascular infiltration by inflammatory cells and a decrease in capillary density. An excessive accumulation of extracellular matrix (ECM) components, produced by activated fibroblasts with an antiapoptotic phenotype and myofibroblast differentiation is the hallmark of the later stages of SSc. Growth factors, such as TGF-β (transforming growth factor beta), PDGF (platelet-derived growth factor) and CTGF (connective tissue growth factor), are believed to play the key role in the synthesis of ECM. To date, there is no effective established treatment of SSc. Imatinib, a multi-targeted small molecule tyrosine kinase inhibitor of c-abl (a new mediator of TGF-β signaling), c-kit and PDGFR, has become a therapy of interest for the treatment of SSc due to its ability to inhibit c-Abl and PDGFR, tyrosine kinases involved in profibrotic pathways. Preclinical data using in vitro and murine models of fibrosis have demonstrated the antifibrotic properties of imatinib. Imatinib is currently used widely in the treatment of chronic myelogenous leukemia, gastrointestinal stromal tumors, and other conditions, with a large amount of available information regarding the safety of the medication in these patient populations. However, due to severe adverse events in recent clinical trials in patients with SSc the effective doses could not be achieved to confirm the findings from pre-clinical studies. Thus, new alternatives are being searched to provide a highly selective inhibition of key targets involved in profibrotic pathways with minimum side effects. ARRY-768 is a highly selective, orally active, small molecule inhibitor of PDGF receptors (PDGFR), which targets PDGFR more potently (IC 50 of 3 vs. 69 nM, respectively) than imatinib. The aim of this study was to investigate the efficacy of ARRY-768 in prevention of dermal fibrosis and treatment of pre-established dermal fibrosis induced by bleomycin. Material and Methods Bleomycin-induced dermal fibrosis: The model of bleomycin-induced dermal fibrosis was used to investigate the effects of ARRY-768 on prevention of experimental fibrosis (Table: A). A modified model of bleomycin-induced fibrosis was used to investigate the potential of ARRY-768 to induce regression of pre-established fibrosis (Table: B). The efficacy of ARRY-768 was compared to that of imatinib mesylate. Bleomycin dissolved in 0.9 % sodium chloride at a concentration of 0.5 mg/ml was administered by subcutaneous injections in defined areas of 1 cm2 at the upper back every other day for several weeks. Subcutaneous injections of 100 μl 0.9 % sodium chloride were used as controls. A total of 56 mice were examined at week 6, 7, 8, 9 (Design A) or 6, 9, 10, 11 and 12 (Design B) for weight, activity and the texture of the fur. After treatment, mice were sacrificed by cervical dislocation and the skin was analysed for dermal thickness, numbers of myofibroblasts, quantification of collagen content and further immunohistochemical analysis. Selective inhibition of PDGFR by ARRY-768: ARRY-768 was kindly provided by Array BioPharma Inc. (Boulder, CO, USA). Extensive biochemical screening against the Upstate Enzyme Kinase panel proved ARRY-768 to be a potent PDGFR/Flt-3 inhibitor with cellular IC50s of 3 and 2 nM, respectively, and suggested its potential activity (<20 POC at 100 nM) against CaMKIIδ, CaMKIIγ, ARK5, Flt-1 (IC50=20 nM) and Flt-4 (IC50= 4 nM). ARRY-768 showed selectivity over VEGFR2 (IC50=97 nM), Kit (IC50=577 nM) and Abl (IC50=4913 nM) indicating that these related kinases are not significantly targeted by ARRY in pharmacologically relevant doses in vivo. ARRY-768 has good PK and in vivo activity at well-tolerated doses. Immunohistochemical analysis of the skin sections for phosphorylated PDGFR-βI and c-Abl: Paraffin embedded skin sections were deparaffinized, incubated with 3 % H 2 O 2 followed by serum blocking with 5 % horse serum. Phospho PDGFR-βI (Y751) and phospho c-Abl (Y412) were detected by incubating with polyclonal rabbit-anti-mouse phospho PDGFR-βI and polyclonal rabbit-anti-mouse phospho c-Abl antibodies (Abcam, Cambridge, UK), respectively, overnight at 4 °C. Isotype antibodies in the same concentration were used for controls (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Polyclonal goat anti-rabbit antibodies (Dako) labelled with horseradish peroxidase were used as secondary antibodies. Staining was visualized with DAB (Sigma-Aldrich) peroxidase substrate solution. Statistics: Data are expressed as the mean ± SEM. Mann-Whitney-U-test was used for statistical analyses. Experimental design and dosage regimens: Summary and Conclusion • ARRY-768 is a highly potent, orally active PDGFR inhibitor • ARRY-768 shows more potent inhibition of PDGFR compared to imatinib • ARRY-768 shows significant selectivity over VEGFR-2, Kit and Abl which may give it a competitive advantage over other multikinase inhibitors that also target PDGFR and thus should not be dose limited in humans by off-target kinase toxicities. • Treatment with ARRY-768 not only prevented induction of dermal fibrosis but also induced regression of pre-established dermal fibrosis induced by bleomycin to under pre-treatment levels. ARRY-768 was well tolerated at all doses and showed no signs of toxicity. • Control treatment with imatinib showed no superior anti-fibrotic effects than ARRY-768 suggesting that the effects of imatinib might be mediated primarily via inhibition of PDGFR, whereas inhibitory effects on c-abl, a downstream mediator of TGF-β, seem to be less relevant. PDGFR inhibitor ARRY-768 prevents experimental dermal fibrosis and induces regression of pre-established dermal fibrosis Michal Tomcik 1,2 , Nicole Reich 1 , Katrin Palumbo 1 , Pawel Zerr 1 , Jérôme Avouac 3 , Alfiya Akhmetshina 1 , John Robinson 4 , Clara Dees 1 ,Christian Beyer 1 , Radim Becvar 2 , Ladislav Senolt 2 , Oliver Distler 5 , Georg Schett 1 , Jörg H.W. Distler 1 1 Department of Internal Medicine 3, University Erlangen-Nuremberg, Erlangen, Germany; 2 Institute of Rheumatology, Department of Clinical and Experimental Rheumatology, 1 st Faculty of Medicine, Charles University in Prague, Czech Republic; 3 Paris Descartes University, Rheumatology A department, Cochin Hospital, Paris, France; 4 Array BioPharma, Inc., Boulder Colorado, USA 5 Center of Experimental Rheumatology, Univ. Hospital Zurich, Zurich, Switzerland Acknowledgements This study was supported by Array (compound) & Celgene (financial support). The research stay of the presenting author in Erlangen was supported by: EULAR Research Training Bursary, Mobility Fund of the Charles University in Prague, Abbott, and OPPA - European Social Fund Prague & EU: Supporting Your Future (CZ.2.17/1.1.00/32698). OPPA also supported the attendance and presentation of this poster at the ACR Annual Meeting 2011 in Chicago. Results (A) model of prevention of bleomycin-induced dermal fibrosis group week 1-3 number of mice control group I sodium chloride / vehicle (water) p.o., BID n = 8 control group II bleomycin / vehicle (water) p.o., BID n = 8 treatment group I bleomycin / ARRY-768 50 mg/kg p.o., BID n = 8 treatment group II bleomycin / imatinib mesylate 50 mg/kg i.p. , BID n = 8 treatment group III bleomycin / imatinib mesylate 200 mg/kg i.p., BID n = 8 (B) model of regression of bleomycin-induced pre-established dermal fibrosis group week 1 - 3 week 4 – 6 number of mice control group I sodium chloride s.c. sodium chloride / vehicle (water) p.o., BID n = 8 control group II bleomycin s.c. bleomycin / vehicle (water) p.o., BID n = 8 control group III bleomycin s.c. sodium chloride / vehicle (water) p.o., BID n = 8 treatment group I bleomycin s.c. bleomycin / ARRY-768 30 mg/kg p.o., BID n = 8 treatment group II bleomycin s.c. bleomycin / ARRY-768 100 mg/kg p.o., BID n = 8 treatment group III bleomycin s.c. bleomycin / imatinib mesylate 50 mg/kg i.p. , BID n = 8 treatment group IV bleomycin s.c. bleomycin / imatinib mesylate 200 mg/kg i.p., BID n = 8 phospho PDGFR-βI (Y751) phospho c-Abl (Y412) phospho PDGFR-βI (Y751) phospho c-Abl (Y412) NaCl 6 weeks bleomycin 6 weeks bleomycin 3 3 weeks + NaCl 3 weeks isotype control bleomycin 6w ARRY-768 30 mg/kg 400-fold bleomycin 6w ARRY-768 100 mg/kg bleomycin 6w imatinib 50 mg/kg bleomycin 6w imatinib 200 mg/kg NaCl (3w) bleomycin (3w) bleomycin (3w) + ARRY-768 50mg/kg bleomycin (3w) + imatinib 50mg/kg bleomycin (3w) + imatinib 200mg/kg NaCl (6w) bleomycin (6w) bleomycin (3w) + NaCl (3w) bleomycin (6w) + imatinib 50 mg/kg bleomycin (6w) + imatinib 200 mg/kg bleomycin (6w) + ARRY-768 30 mg/kg bleomycin (6w) + ARRY-768 100 mg/kg ARRY-768 IC 50 = 3 ± 1 nM ARRY-768 Cellular Activity ARRY-768 shows potent inhibition of PDGF induced phosphorylation of PDGFR in cells ARRY-768 is a potent, selective PDGFR/Flt-3 inhibitor compared to competitor molecules in human cells Flt3: FL-induced Flt-3 phosphorylation in MEF-Flt-3-wt cells (Proqinase) or RS4;11 (wt) human leukemia cells PDGFR: PDGF-induced PDGFR phosphorylation in HS27 human fibroblasts VEGFR-2: VEGF-A-induced VEGFR2 phosphorylation in primary HUVECs cKit: SCF-induced cKit phosphorylation in M-07e human leukemia cells Abl: Bcr-Abl driven cell proliferation in K562 human leukemia cells ARRY-768 structure Cellular Activity, IC 50 s in nM (data previously published: 2008 EORTC, abstract 576) ARRY-768 In-Vitro Activity ARRY-768 shows superior inhibition of PDGFR compared to imatinib mesylate in Nu/nu athymic mice bearing C6 glioma tumors In-Vivo activity data previously published: 2008 EORTC, abstract 576 ARRY-768 selectively reduces the expression of activated (phosphorylated) PDGFR-βI in the skin challenged with bleomycin Inhibition of PDGFR via ARRY-768 prevents induction of dermal fibrosis by bleomycin Treatment with ARRY-768 at the dosis of 50 mg/kg decreased dermal thickening by 67 ± 2% (p<0.001), hydroxyproline content by 23 ± 6% (p=0.642) and myofibroblast counts by 47 ± 5% (p<0.001). Control treatment with imatinib demonstrated comparable reduction of dermal fibrosis. Inhibition of PDGFR via ARRY-768 induced regression of pre-established bleomycin-induced dermal fibrosis Treatment with ARRY-768 at the doses of 30 and 100 mg/kg decreased dermal thickening by 45 ± 2% (p<0.01) and 56 ± 1% (p<0.01), hydroxyproline content by 52 ± 4% (p<0.001) and 55 ± 5% (p<0.001), and myofibroblast counts by 75 ± 6% (p<0.001) and 88 ± 8% (p<0.001), respectively. Control treatment with imatinib showed no superior regression of pre-established dermal fibrosis. The treatment with ARRY-768 was well tolerated for 3 weeks at all dosing regimens (30, 50 or 100 mg/kg) and no signs of toxicity such as weight loss, decreased activity or changes in the texture of the fur were observed ARRY-768 Pharmacokinetics In the experimental design of regression of pre-established bleomycin-induced dermal fibrosis, plasma concentration for ARRY-768 2 hours post final dose was 0.774 ± 0.22 μg/ml for the dose of 30 mg/kg and 9.293 ± 1.80 μg/ml for the dose of 100 mg/kg.
