VIEWS: 105 PAGES: 1 POSTED ON: 4/18/2011
A potent and selective cFMS inhibitor regulates the tumor macrophage microenvironment leading to tumor growth inhibition A. Dale Wright, Anna Gomez, Mark L. Boys, Robert Kirk DeLisle, Laurence E. Burgess, Mark C. Munson, April L. Kennedy, George T. Topalov, Qian Zhao, Martha E. Rodriguez, Bryson Rast, Poster #551 James P. Rizzi, Walter E. DeWolf, Christine Lemieux, Deborah Anderson, Brandon S. Willis, Jason R. Neale, Matthew Martinson, Stefan D. Gross, Michele Callejo, Tyler Risom, Shannon L. Winski and Patrice A. Lee. Available at www.arraybiopharma.com Array BioPharma Inc., Boulder, Colorado, USA Introduction ARRY-382 inhibits human osteoclast ARRY-382 inhibits MCF7-AP1 tumor growth with an ARRY-382 is a highly selective cFMS inhibitor differentiation and bone resorption in vitro and accompanying decrease F4/80 (+)-staining cells Interactions between tumor cells, stromal cells, macrophages blocks TNF-α production in vivo and the extracellular matrix are pivotal to the processes of 1000 tumorigenesis, metastasis, and neovascularization POC < 20 Vehicle 20 < POC < 50 POC > 50 Inhibition of human osteoclast differentiation by ARRY-382 900 10 mg/kg ARRY-382 QD, PO Macrophages within the tumor microenvironment may 180 100 mg/kg ARRY-382 QD, PO facilitate cancer progression, making them intriguing targets number of osteoclast/10 fields 800 160 cFMS IC50 (nM) PDFGR IC50 (nM) cKIT IC50 (nM) for therapy cFMS 140 120 700 100 ARRY-382 9 >10000 >10000 Colony stimulating factor 1 (CSF-1) and its receptor, cFMS or Tumor Volume (mm 3) 80 600 60 CSFR1, play a central role in the development of 40 mononuclear phagocytes, recruitment of macrophages to 20 500 0 tumors, and differentiation and function of osteoclasts In a panel of 257 kinases at 1µM, ARRY-382 undiff Diff 62.5 31.3 15.6 7.8 3.9 1.95 0.98 0.49 400 is highly selective for cFMS and has no ARRY-382 (nM) In this study, we investigate in more detail the molecular cellular activity against PDGFR or cKIT 300 mechanisms involved in cFMS inhibition on modulation of tumor-associated macrophage function and tumor growth Inhibition of human osteoclast bone resorption by ARRY-382 200 500 450 100 Compound administration Materials and Methods 400 ARRY-382 displays favorable pharmacokinetics C -t elo p ep tid e ( n M ) 350 0 300 0 5 10 15 20 25 PK/PD analysis: HEK 293 cells expressing a doxycycline-inducible cFMS construct in preclinical species 250 200 Day of Study were injected subcutaneously into female NCr nu/nu mice. Tumor bearing animals Plasma concentrations time profiles for ARRY-382 150 were randomized into treatment groups and administered 30 mg/kg doxycycline by following a 10 mg/kg oral dose oral gavage 14 hr prior to ARRY-382 treatment. Mice received a single oral dose of 100 Inhibition of F4/80+ staining in MCF7-AP1 tumors by ARRY-382 10 50 Plasma Conc. (µg/mL) either vehicle or ARRY-382 and sacrificed at the indicated time point. Plasma and Monkey 0 tumor drug concentrations were determined by LC-MS, and Western blot analysis undiff Diff 2000 1000 500 250 125 62.5 31.3 15.6 7.8 3.9 1.95 0.98 1 Rat ARRY-382 (nM) was performed to determine phospho-cFMS/total cFMS protein levels. Results are Mouse expressed as mean ± SEM. 0.1 Inhibition of LPS-induced TNF-α production by ARRY-382 In vivo LPS challenge: Male Swiss-Webster mice received either a single, oral dose of ARRY-382 or vehicle 30 min prior to an intraperitoneal injection of LPS (3 0.01 20000 10 ARRY-382 µg/mL in plasma µg/kg), and animals were euthanized 90 min later. Serum samples were evaluated 16000 8 for TNF-α concentrations by ELISA (R&D Systems) and plasma drug 0.001 TNF-α (pg/mL) concentrations were determined by LC-MS. Results are expressed as mean ± 0 10 20 30 40 50 12000 6 SEM. Vehicle ARRY-382 100 mg/kg Time (hr) 8000 4 In vitro osteoclast differentiation and bone resorption analysis: Human osteoclast precursor cells (Lonza) were stimulated with 33 ng/mL M-CSF and 66 ng/mL AUCinf Cmax Tmax F 4000 2 SPECIES RANKL for 7 days to induce differentiation in the presence of increasing (hr-µg/mL) (µg/mL) (hr) (%) 0 0 concentrations of ARRY-382. Osteoclasts were identified by staining for the Summary of Results l kg kg kg kg presence of tartrate-resistant acid phosphatase (TRAP;Sigma). For bone e Mouse 6.50 1.17 1.00 87 ro cl g/ g/ g/ g/ nt hi m m m m Co Ve resorption determination, human osteoclast precursors were plated on a human 3 10 30 0 10 bone plate and allowed to differentiate for 7 days in the presence of M-CSF and Rat 3.02 0.404 1.17 59 TNF-α (pg/mL), (±SEM) RANKL. On day 8, ARRY-382 was added and supernatants were collected on day µg/mL in plasma, (±SEM) 13 to evaluate calcium levels (C-telopeptide). Results are expressed as mean ± ARRY-382 is a potent, selective inhibitor of cFMS (IC50 = 9 nM) Monkey 14.3 2.72 2.72 47 SEM. No significant cellular activity against cKIT and PDGFR. No Labeling and FACS analysis of ID8 ascites: Female CB6F1 mice were inoculated ARRY-382 decreases myeloid/macrophage enzyme activity against >200 other kinases at 1µM IP with 106 ID8 tumor cells. Upon ascites accumulation, animals received daily oral administration of 100 mg/kg ARRY-382 or vehicle for 6 days. Standard volumes of lineages in ID8 tumor ascites ascites fluid were aliquoted, FC-blocked and labeled for 30 minutes with (anti- ARRY-382 inhibition of phospho-cFMS correlates Vehicle ARRY-382 inhibits cFMS phosphorylation, in vivo, in a dose- mouse) CD11b, F4/80, CD115 (e-Bioscience) and CD204 (AbD Serotec). Cells were fixed and RBCs lysed (BD Lyse/Fix Buffer). Samples were washed and then with plasma drug levels CD115 Vehicle CD11b dependent manner (ED50=3 mg/kg) cFMS (CD115) CD11b (myeloid cells) CD11b+ cells in ascites fluid CD115+ cells in ascites fluid a set volume was analyzed on a Canto II (BD Biosciences) flow cytometer. Data 35000 ARRY-382 blocks M-CSF induced differentiation and bone P h o s p h o -c F M S /to ta l c F M S HEK293-cFMS tumors 8000 A R R Y -3 8 2 µ g /m l p la s m a were analyzed with FlowJo software (TreeStar). Results are expressed as group mean (red bar) and individual animals (blue diamond). Statistical significance 120 2.5 7000 6000 30000 25000 resorptive capacity of human osteoclast precursor cells P e rc e n t o f C o n tro l 5000 ARRY-382 ARRY-382 determined by student t-test. 100 ED50= 3 mg/kg 20000 2 4000 3000 CD115 15000 p=0.011 CD11b A single dose of ARRY-382 significantly inhibited LPS-induced In vivo tumor growth inhibition study: Female NCr nu/nu mice were implanted with 80 p=0.017 10000 TNF-α release in mice 1.5 2000 slow release estrogen pellets (Innovative Research of America) then inoculated 5000 60 1000 subcutaneously in the flank with 5x106 MCF7-AP1 mammary adenocarcinoma cells 1 0 0 in 50% matrigel (BD Biosciences). Studies were initiated when tumors reached a 40 Vehicle ARRY-382 Vehicle ARRY-382 In the ID8 tumor ascites model, ARRY-382 decreases tumor 0.5 100 mg/kg 100 mg/kg infiltrating myeloid cells and M2 macrophage subtypes. minimum of 200 mm3 at which time mice were randomized by tumor size and 20 dosed daily by oral gavage. Tumor size (caliper) and body weight were measured Vehicle Vehicle 0 0 F4/80+/CD204+ cells in ascites fluid on the indicated days over the course of each study. ARRY-382 was well tolerated F4/80 (Mø) F4/80 F4/80/CD204 (M2-Mø) Targeting of myeloid/macrophages with ARRY-382 delayed solid V e h ic le 3 m g /k g 1 0 m g /k g 3 0 m g /k g F4/80+ cells in ascites fluid tumor growth of the human MCF7-AP1 tumor line that is F4/80 and daily administration did not result in drug-related body weight loss exceeding 35000 9000 10% or death in any study. Test compound administration are displayed as 30000 8000 7000 accompanied by a substantial loss of F4/80+cells 25000 indicated. Tumor measurements are expressed as group mean ± SEM 6000 20000 CD204 (n=8/group). p=0.03 5000 15000 ARRY-382 4000 ARRY-382 Taken together, these results suggest that ARRY-382 may be a F4/80 3000 Immunohistochemistry: Tumor tissue was harvested and immediately placed in p=0.0008 10000 2000 novel therapy targeting the tumor microenvironment and POC p-cFMS(Y708)/total cFMS (±SEM) F4/80 formalin for preparation of paraffin embedding. F4/80 immunostaining (AbD 5000 1000 Serotec) was performed on FFPE sections with hematoxylin counterstaining. 0 0 supports further evaluation in clinical trials Vehicle ARRY-382 Vehicle ARRY-382 [ARRY-382] in plasma (µg/ml) (±SEM) 100 mg/kg 100 mg/kg CD204 ARRY-382 is currently being evaluated in Phase 1 clinical trials All animal studies were performed in accordance to IACUC guidelines and in harmony with the Guide for Laboratory Animal Care and Use. for safety and pharmacokinetics.
Pages to are hidden for
"A potent and selective cFMS inhibitor regulates the tumor"Please download to view full document