A potent and selective cFMS inhibitor regulates the tumor by mikesanye

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									                                       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




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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                  kg



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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                kg



                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              kg
  presence of tartrate-resistant acid phosphatase (TRAP;Sigma). For bone




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                                                                                                                                                Mouse                     6.50                       1.17                     1.00                  87




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                                                                                                                                                                                                                                                                                                                                                                                                                                                                               Ve
  resorption determination, human osteoclast precursors were plated on a human




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                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         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.

								
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