Recent Advances of Colony-Stimulating Factor-1 Receptor (CSF-1R

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Recent advances of colony-stimulating factor-1 receptor (CSF-1R) kinase and its inhibitors Mohammed Ibrahim El-Gamal, Shahad K. Al-Ameen, Dania M. AlKoumi, Mawadda G. Hamad, Nouran A. Jalal, and Chang-Hyun Oh J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00873 • Publication Date (Web): 02 Jan 2018 Downloaded from http://pubs.acs.org on January 7, 2018

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Recent advances of colony-stimulating factor-1 receptor (CSF-1R) kinase and its inhibitors Mohammed I. El-Gamal

1,2

, Shahad K. Al-Ameen 1, Dania M. Al-Koumi 1,

Mawadda G. Hamad 1, Nouran A. Jalal 1, and Chang-Hyun Oh 3,4,* 1

College of Pharmacy, University of Sharjah, Sharjah 27272, United Arab Emirates.

2

Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura

35516, Egypt. 3

Center for Biomaterials, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea.

4

Department of Biomolecular Science, University of Science and Technology, 113 Gwahangno,

Yuseong-gu, Daejeon 305-333, Republic of Korea. *

E-mail address of the corresponding author: [email protected] [Chang-Hyun Oh].

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Abstract: Colony stimulation factor-1 receptor (CSF-1R), which is also known as FMS kinase, plays an important role in initiating inflammatory, cancer, and bone disorders when it is overstimulated by its ligand, CSF-1. Innate immunity, as well as macrophages differentiation and survival, are regulated by the stimulation of the CSF-1R. Another ligand, interlukin-34 (IL-34) was recently reported to activate the CSF-1R receptor in a different manner. The relationship between CSF-1R and microglia has been reviewed. Both CSF-1 antibodies and small molecule CSF-1R kinase inhibitors have now been tested in animal models and in humans. In this perspective, we discuss the role of CSF-1 and IL-34 in producing cancer, bone disorders, and inflammation. We also review the newly-discovered and improved small molecule kinase inhibitors and monoclonal antibodies that have shown potent activity toward CSF-1R, reported from 2012 till 2017. Key words: CSF-1; CSF-1R; FMS; IL-34; Kinase Inhibitors; Monoclonal Antibodies.

1. Introduction Receptor tyrosine kinases (RTKs) play a major role in maintaining homeostasis as they provide important pathways for signal transduction that allow cell communication, maintain normal cellular processes, and provide surface receptors for many hormones and growth factors. These trans-membrane proteins contain extracellular domains to which ligands bind and initiate intracellular signaling via activation of their attached TK moieties.1 The human genome includes 58 RTKs including including fibroblast growth factor receptor (FGFRs), epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth Page 1 of 62 ACS Paragon Plus Environment

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factor receptor (PDGFR), and others, along with 32 non-receptor TKs, many of which are tightly linked to receptors. RTKs are classified into different groups depending on the homology of their active sites, and the similarity of their ligands.2 They contribute to the initiation, development, and progression of several types of cancers, inflammations, and bone osteolysis, due to the effects of mutations on receptor function, or to receptor/ or ligand over-expression, so they are very promising therapeutic targets. One of the most important receptors among type III RTKs is CSF-1R (FMS, c-FMS, CD115, or M-CSFR) kinase, a member of the PDGFR class of RTKs that was reported initially to be an oncogene for Feline McDonough Sarcoma.3 CSF-1R is expressed on hematopoietic stem cells (HSCs) at low levels, and at higher levels on monocytes, macrophages, myeloid dendritic cells, microglia, and osteoclasts. It plays an important role in regulating their development. CSF-1R is also found in other cells such as oocytes (immature egg cell), pre-implantation embryos, epithelial cells, and colonic epithelial cells. It binds to macrophage colony-stimulating factor (M-CSF or CSF-1) and interleukin-34 (IL-34). IL-34 has generally similar biological properties to CSF-1, with the main differences arising from their different spatiotemporal regulation, and the fact that CSF-1 acts both in an autocrine and paracrine fashion, whereas IL-34 acts purely locally, Activation of CSF- 1R promotes the survival, differentiation, and proliferation of macrophage and monocyte lineages, yet their overexpression is associated with disorders.4 As a result, inhibitors that regulate their functions are of high interest. We have previously reviewed the biology of CSF-1R kinase and its related diseases, as well as the CSF-1R kinase inhibitors and antibodies published in the literature till the end of 2011.5 Herein, we reviewed the role of CSF-1R in cancers, bone osteolysis, and inflammatory

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disorders, as well as IL-34 and its role as a second ligand for the CSF-1 receptor, and potential inhibitors or antibodies targeting CSF-1R. This article covers the period from 2012 until 2017.

2. CSF-1 and bone osteolysis Bone cells can be divided into three main types: osteoclasts (OCs), osteoblasts (OBs) and osteocytes (OSs). Each one of them is responsible for a particular function in maintaining the bone structure. OBs form the bone while OCs destroy it, and osteocytes keep the balance between OBs and OCs.6 Bone tissues are continuously going through a process called bone remodeling or coupling in which they are destroyed and rebuilt again over time. This process is regulated by cross talk between OBs and OCs.7 OBs are cells with a single nucleus that arise from a mesenchymal precursor,8 and they can be differentiated in vitro with the addition of ascorbic acid (vitamin C), dexamethasone, or beta-glycerol phosphate.6 OBs cells are responsible for initial bone buildup and for later remodeling.7 Osteoclasts, the cells responsible for bone destruction, are multinucleated cells that are developed with the help of CSF-1 and receptoractivated nuclear factor kappa-B (RANK) ligand (RANKL) from osteoclast progenitor cells (OPs) which in turn derive from hematopoietic stem cells (HSCs), or blood stem cells.6,9,10 The ability of bones to renew themselves gives this type of tissues a unique dynamic structure that can adapt to the surrounding environment. Any abnormality in the process of bone remodeling results in different disease conditions that affect bone density and structure such as osteoporosis.6 Mice lacking CSF-1 (op/op) were found to be underweight and toothless and to have a weak skeletal structure8 and a defect in osteocyte proliferation.11 Until recently it was believed that OBs controlled OCs, but recently mutual cross-regulation of the two has been demonstrated. OC formation in the body occurs through a particular

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pathway. First, CSF-1 is produced in the bone marrow by OB cells and binds to CSF-1R located on the surface of osteoclast precursors (OCPs). After that, RANKL expression is stimulated in response to CSF-1R signals. Lastly, OCPs fuse together to form multinucleated OC cells.10 The relationship between CSF-1 and OC cells was demonstrated by a study done using the Meox2Cre line, to generate mice lacking CSF-1 (CSF-KO). This type of mouse showed a defect in osteocyte survival and function which resulted in poor bone structure.11 Some growth factors such as insulin-like growth factor (IGF) and bone morphogenetic protein (BMP), which are released during bone resorption can stimulate OB differentiation and bone buildup. These growth factors are secreted in the presence of an acid generated by osteoclasts.10 CSF-1 maintains appropriate levels of OCPs in the bone marrow. It also increases RANK expression in OCPs.12 Polarization of hematopoietic stem cells (HSCs) occurs in the presence of CSF-1, while RANKL is present in the differentiation process.6 Mice lacking RANK-L or CSF-1 are unable to generate OB cells, which leads to low bone density and osteoporosis.12 When CSF-1 is administered to op/op mice, the bone marrow cavity starts to form again and that is an important step in curing osteoporosis.7

3. CSF-1 and cancer Macrophages play an essential role in innate defense mechanism. Macrophages arise from monocytes, which normally differentiate into M1 proinflammatory macrophages, but under certain circumstances, such as in many tumor microenvironments, they can differentiate into antiinflammatory M2 macrophages, also called tumor-associated macrophages (TAMs), that allow

for

cancer

cell

growth,

metastasis,

angiogenesis

and

especially

localized

immunosuppression. Overexpression of CSF-1, which is a monocyte chemoattractant, by tumor

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cells has been associated with their recruitment and differentiation into TAMs (M2) in various kinds of cancers.13 TAMs are related to macrophages and are derived from monocytes that circulate in the blood stream and situate themselves at the tumor site.14 Tumors release CSF-1, which is a cytokine responsible for the proliferation, survival, and differentiation of the macrophages, after it binds to the CSF-1R. Over-expression of CSF-1 has been associated with activation and recruitment of TAMs in various types of cancers.15 TAMs also tend to accumulate in low oxygen conditions and allow the expression of other growth factors, such as VEGF. VEGF stimulates the formation of blood vessels and endothelial cell proliferation.8 High CSF-1 levels were reported in breast, prostate, pancreas, renal, ovary, and many other types of cancer. Overexpression of CSF-1 and its receptor, CSF-1R, in tumors has been linked to poor prognosis.8 CSF-1/CSF-1R inhibitors are clinically developed to control protein over-expression and are classified as either: 1- kinase domain inhibitors. 2- protein‐protein interaction inhibitors (protein‐ligand or receptor dimerization inhibitors).

3.1. CSF-1 and breast cancer CSF-1 and CSF-1R are normally expressed in breast tissue during puberty, pregnancy, and lactation. However, their over-expression or the repeated stimulation of CSF-1R by released CSF-1, is exhibited in several cancers including breast cancer. Additionally, the co-expression of both CSF-1 and CSF-1R in neoplastic epithelial cells of breast cancer patients correlates with poor prognosis and predicts ipsilateral recurrence. A study was carried out on patients with breast cancer that showed a link between high levels of CSF-1 and tumor progression. 16 Page 5 of 62 ACS Paragon Plus Environment

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Another study on macrophage-deficient (MMTV-PYT) mice lacking the CSF-1 gene was conducted to observe the role of CSF-1 in metastasis, as it is regulated by TAMs. The results of this study showed that TAMs were incapable of accumulation in the tumor, and they reduced cancer growth and lung metastasis. Since the tumor will attract TAMs by the secreted CSF-1, this cytokine migrates with the collagen fibers and invades locally or moves to reach the blood vessels, causing metastasis. Inhibiting TAMs by targeting the CSF-1/CSF-1R may be one of the most effective ways to stop tumor progression and improve cancer outcomes. Such inhibition can decrease metastasis to organs such as the lungs and bones, and the inhibition of TAMs increases neutrophil numbers at some sites.17,18

3.2. CSF-1 and renal cell carcinoma Renal cell carcinoma (RCC) is the seventh most common cancer in men and the ninth most common in women. It appears mostly in patients with end stage renal failure, acquired renal cystic disease, obesity, and tuberous sclerosis.19 After a renal injury (transient renal ischemia/reperfusion), CSF-1/CSF-1R are coexpressed on tubular epithelial cells (TECs), resulting in high proliferative and anti-apoptotic behavior to replenish the injured TECs and promote healing. CSF-1R is expressed in epithelial cells in some cancers, such as prostate and breast cancer, which makes it likely that CSF-1 and CSF-1R are co-expressed in RCC. To test this hypothesis several studies have been done to determine whether CSF-1R is limited only to RCC; by observing TECs near to and away from cancerous cells, that detected a high expression of CSF-1R close to the RCC, CSF-1R expression decreased with increasing distance from RCC. Since CSF-1 and CSF-1R are mostly expressed on cells adjacent to the tumor, this explains the co-expression of CSF-1 and CSF-1R on TECs near the RCC and their role in tumor propagation and proliferation.20 Page 6 of 62 ACS Paragon Plus Environment

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CSF-1-induced activation of M2 macrophages contributes to the immune dysfunction evident in RCC. CSF-1 regulates TAM production in the RCC microenvironment, encouraging cellular migration and tumor growth. TAMs also play a role in the epithelial-mesenchymal transition of RCC and its resistance to inhibitors. Suppression of TAM recruitment is an important target since the presence of TAMs indicates poor prognosis in patients with RCC.21

3.3. CSF-1 and pancreatic cancer Pancreatic cancer (PC) is an aggressive type of cancer with a high death rate due to the late diagnosis of the disease. Its overall survival rate in patients with metastasis is approximately 12 months, while in patients with local advanced PC, it is 24 months.22 The main cause of the tumor microenvironment is the contribution of TAMs, granulocytic myeloid-derived suppressor cells (GMDSC) and myeloid-derived suppressor cells (MDSCs) as they promote the proliferation of tumor cells, ease tumor cells metastasis, and cause cytotoxic resistance which leads to poor prognosis.23 MDSCs, which express two markers CD11b and Gr-1, are precursors to macrophages, granulocytes, and dendritic cells. Their high expression and accumulation in tumor cells is driven by GM-CSF which is a type of colony stimulating factor. Consequently, this leads to an inhibition of the anti-tumor function of CD8+ T-cells.24 Furthermore, CSF-1 is highly expressed in the epithelial cells of pancreatic ductal adenocarcinoma (PDAC) which promotes the production of cytokines by M1 and M2 macrophages, allowing tumor migration.25 In animal tumor models, blockage of CSF-1R enhances antitumor activity only in tumors with high levels of MDSCs, which makes it a pretreatment biomarker for pancreatic cancer patients.26

3.4. CSF-1 and lung cancer

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Lung cancer is one of the most fatal cancers. It has high mortality and metastasis rates; 60% to 70% of patients experience bone metastases.27 Late diagnosis in advanced stage IV lung cancer means that 5 year survival is only 16%. Most of these patients experience chest pain, coughing, dyspnea, weight loss, and chronic obstructive pulmonary disease (COPD).28 There are two major types of lung cancer, non-small cell lung cancer (NSCLC) that affects 85% of patients and small cell lung cancer (SCLC) which accounts for the rest (15%).29 CSF-1 secretion, which is localized in lung cancer, contributes to a poor prognosis. Moreover, CSF-1 stimulates TAMs that exhibit tumorous effects by enhancing tumor expansion and angiogenesis and supporting the production of pro-angiogenic factors such as VEGF, IL-10, TNF-α, or others.30 In the A549 adenocarcinoma cell line model of NSCLC, CSF-1/CSF-1R are expressed and associated with high proliferation, lung cancer bone metastasis, and promotion of chemotherapy resistance.27 Treatment A549 cells with a CSF-1 inhibitor reduces tumor prevalence and bone metastases. Inhibition of CSF-1/CSF-1R signaling causes a rapid reduction in TAMs within 48 h.31

4. CSF-1 and inflammation Rheumatoid arthritis (RA) is a systemic chronic inflammatory condition that results in joint destruction, scars, and deformity. One percent of all age groups globally are affected by RA. This autoimmune disease is caused by a disruption of bone homeostasis which is regulated by the balance between osteoclast and osteoblast.32,33 Manifestations of RA, such as bone loss, are mediated by an immune response which involves pro-inflammatory cytokines including TNF-α and several other immune cells.32 TNF-α, which is secreted from activated macrophages, helps regulate the immune response and the production of RANKL and CSF-1 from osteoblasts.32,33 The involvement of immune cells in inflammation and bone loss was studied to

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examine the effect of inhibiting CSF-1R and its ligands (CSF-1 and IL- 34) in RA patients and models of RA.32 The level of synovial macrophages and their release of inflammatory cytokines were found to affect the progression and the activity of RA. Compounds that are able to inhibit the survival, activation, or differentiation of macrophages through inhibiting CSF-1R or its ligands may have a therapeutic efficacy in treating RA.34,35 CSF-1R is expressed in different cells involved in RA progression such as tissue macrophages, immature dendritic cells (DCs), OCs, and OBs. CSF-1 and IL-34 play a major role in osteoclastogenesis, and are expressed in synovial endothelial cells and are highly expressed in the serum and synovial fluid of RA patients. In vitro data showed that CSF-1 can also be produced by synovial fibroblasts and chondrocytes after the induction effect of TNF-α and IL-1β.34-36 CSF-1 signaling was found to increase the numbers of DCs, and also it is required for their differentiation.34 Animal studies on mice have shown that the administration of CSF-1 increased the severity of collagen-induced arthritis (CIA), in contrast to mice deficient in CSF-1R that had less disease severity. Pharmacological inhibition of CSF-1R, administration of antibodies against CSF-1 or genetical deletion of CSF-1 reduced synovial inflammation and joint erosion in animal models of RA.34-36 Clinical studies found an elevation of CSF-1 levels in the synovium of patients with RA while in healthy individuals no CSF-1 over-expression was noticed.35,36 Using anti-human CSF-1R antibody (huAb1), which inhibits the binding of CSF-1R to its ligands, reduced the production of different chemokines and IL-6 in the synovial explant. In vivo studies on murine models with CIA showed that the administration of anti-mouse CSF-1R antibody (muAb5) reduced the joint deformity in models with RA compared to control.34

5. Interleukin 34 Page 9 of 62 ACS Paragon Plus Environment

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Interleukin-34 (IL-34) was found to signal through CSF-1R along with CSF-1 on cells of mononuclear phagocyte system after examination using a human monocyte proliferation screening assay.37,38 This signaling causes receptor dimerization, auto-phosphorylation, activation of extracellular-signal-regulated kinase 1/2 (ERK1/2) signaling, and expression of genes that play a role in the mononuclear phagocyte cells viability.39 This explains the more severe phenotype of mice deficient in CSF-1R (CSF-1R-/-) compared to those deficient in CSF-1 (CSF-1op/op).38 CSF-1op/op mice experienced a severe loss of bone osteoclasts, reduced levels of macrophages (which partially increased as the mice aged) in some tissues, a moderate reduction of microglia and a relatively normal level of Langerhans cell development in the skin. In contrast, CSF-1R-/- mice are profoundly depleted of macrophages and deficient in osteoclasts, microglia, and Langerhans cells throughout their lifetimes. This suggests that CSF-1R is activated by IL-34 independently of CSF-1. When IL-34 was tested using different assays, it showed a high specificity to monocytes and macrophages, which was supported by the lack of binding of IL-34 to 28 other cell types and the lack of significant sequence similarity between it and any other cytokine or protein.40 Despite the functional similarity between IL-34 and CSF-1, there are some notable differences between them. Cell culture studies show that both ligands are equally important for primary human monocytes viability and for the formation of macrophage colonies from human bone marrow. CSF-1 can be replaced by IL-34 to promote RANKLinduced osteoclastogenesis.37,40 The helical cytokine fold in the crystal structure of dimeric IL-34 is similar to that in CSF-1; both of them are short-chain, helical cytokines and belong to the same class.37 Moreover, IL-34 has more restricted expression than CSF-1, and upon binding CSF-1R produces more efficient receptor activation and internalization.39,40 In addition, IL-34 showed a more potent binding and a stronger activation of CSF-1R than CSF-1 but a more transient

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tyrosine phosphorylation of the receptor. One of the most observed differences between IL-34 and CSF-1 is their expression in embryonic and adult tissues leading to a complementary, but not redundant, activation of CSF-1R.37,40 A research group tested several biopsies from osteosarcoma patients and observed high IL-34 expression levels.41 This increases the progression of osteosarcoma and facilitates lung metastases. IL-34 mRNA could be observed in non-stimulated cells, as could dose-dependent upregulation of IL-34 mRNA expression by TNF-α and IL-1β; this result was confirmed using confocal microscopy analysis, showing an increase of IL-34 in osteosarcoma cells treated with TNF-α and IL-1β, in contrast to no such upregulation by the cytokines in normal cells. Immunohistochemical analysis of human osteosarcoma biopsies revealed that most osteosarcoma cells showed positive nuclear and/or cytoplasmic immunostaining for IL-34. The involvement of IL-34 in the progression of osteosarcoma was studied by modifying the human osteosarcoma cell line (HOS), which normally expresses a small amount of IL-34, to over-express IL-34 and by then implanting in nude mice to observe the tumor progression. Over-expression of IL-34 significantly increased (by 57%) tumor volume and the establishment of lung metastases compared to the control group.41 The role of IL-34 and disease progression was investigated in patients with RA.42 The results of this study showed production of IL-34 in the synovium, synovial fluid and fibroblastlike synovial cells of patients with RA in similar levels as those for CSF-1 suggesting a role of IL-34 in RA pathogenesis.42 The formation of OCs in mouse models is stimulated by IL-34 and RANKL in a dosedependent fashion. Systemic IL-34 administration to the mouse blood stream increases the

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number of CD11b+ cells and decreases bone density, while in humans, IL-34 and RANKL regulate OC differentiation.43

6. CSF-1R and microglia Development of CNS microglia depends on several growth factors including CSF-1R kinase. And CSF-1R is expressed by microglia.44 CSF-1R is very essential for microglia cell viability, and it was reported that mice with knocked out CSF-1R had no microglia.45,46 CSF-1R kinase inhibitors such as PLX3397 inhibited EOC 20 microglial cell growth in vitro, and eliminated almost all microglia from the adult CNS with no negative effect on cognition or behavior. So microglia are physiologically dependent on CSF-1R signaling, and they are not necessary for cognitive or behavioral tasks. When the inhibitor was withdrawn, rapid repopulation occurred, and new cells differentiated into microglia in mice in a week. Elimination of the microglia did not compromise the BBB integrity.47 On the other hand, mice with engineered over-expression of CSF-1R in astrocytes exhibit increased microglial proliferation.48 Microglia are the first cells that respond to intracerebral hemorrhage. As CSF-1R inhibition has a negative impact on microglia, PLX3397 attenuated neurodeficits and brain edema in vivo in intracerebral hemorrhage models. In these models, intracerebral hemorrhage was induced by injection of collagenase or autologous blood. CSF-1R inhibition ablates microglia and helps protect the brain through reduction of leukocyte infiltration in brain and improvement of BBB integrity. So CSF-1R inhibition helps attenuate brain injury after intracerebral hemorrhage.49 It was reported that microglial loss due to CSF-1R inhibition disrupted brain development and caused olfactory deficits.50 It was reported also that CSF-1R gene mutations caused adultPage 12 of 62 ACS Paragon Plus Environment

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onset leukoencephalopathy, which is an autosomal-dominant neurodegenerative disease. Many neurological disorders such as Alzheimer’s disease, Krabbe’s disease, Charcot-Marie-Tooth disease, glioma, and multiple sclerosis can be modulated by CSF-1R ligands and inhibitors.51

7. CSF-1 and CSF-1R antibodies Monoclonal antibodies (mAb) have been developed to block the CSF-1/CSF-1R pathway, inhibit CSF-1 dependent colony formation, and prevent the activities of CSF-1 and CSF-1R. Several studies have backed this method of inhibition. To determine CSF-1R expression in RA patients and the effectiveness of an anti-CSF-1R antibody in different animals with RA, the expression of CSF-1R was inspected in the synovium, bone, and blood samples from RA, osteoarthritis (OA), and healthy humans. CSF-1R expression was increased in the synovium of RA patients compared with OA patients and healthy people. In mice, blockage of CSF-1R stopped cartilage damage, systemic bone loss, and bone erosion. This was associated with reduction of osteoclast in both models.35 Another study was performed to determine the influence of lipopolysaccharide (LPS), an effective inflammatory inducer of bone loss or bone resorption and an anti-CSF-1R antibody on osteoclastogenesis. Mice injected with a combination of LPS and anti-CSF-1R antibody had greater decrease in osteoclast number than mice injected with only LPS.52 Anti-CSF-1R antibodies also stopped further bone destruction which aids in cancer-induced weight loss in a breast cancer model of bone metastasis.53 In addition, mice with unilateral knee arthritis induced by complete Freund’s adjuvant (CFA) injections, the administration of an intra-articular injection of monoclonal antibody. Recombinant human CSF-1 therapy, alone or in combination with other cytokines, successfully increases the number and viability of monocytes/macrophages, improves antibody-dependent Page 13 of 62 ACS Paragon Plus Environment

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cellular cytotoxicity (ADCC), enhances antimicrobial responses, decreased platelet and cholesterol levels and is a therapeutic target in LPS-induced osteoclastogenesis as it inhibits inflammation-induced bone erosion. Therefore, CSF-1 and the inhibition of its receptor is a promising target and therapeutic alternative for different conditions.36,54

7.1. H27K15 H27K15 is a new monoclonal antibody that has been developed to overcome the issue of increased CSF-1 expression caused by treatment with mAb. Blockage of the formation of CSF1/CSF-1R complex results in a rebound effect on treated patients due to disturbance of CSF-1 degradation and internalization.47 H27K15 is a non-ligand competitive anti-CSF-1 mAb that exerts its activity by affecting signal transduction and partially inhibiting CSF-1R. This antibody prevents the differentiation of monocytes into CD163+CD64+ M2-polarized macrophages and moves them toward CD14−CD1a+ dendritic cells. It inhibits the secretion of monocyte chemotactic protein-1 and reduces interleukin-6 production which are involved in M2 macrophage recruitment. H27K15 is derived from an IgG2a antibody called CXIIG6 which is an anti-human CSF-1R mAb that stains a CSF-1R-transfected NIH/3T3 cell line, but not untransfected cells. The mAb recognizes an epitope on the N-terminal domain of the protein to induce a dose-dependent decrease in MMP-9 production by monocyte-derived osteoclasts and a decrease in dependent CSF-1 phosphorylation of the CSF-1R intracellular tyrosine (Tyr 708) in NIH/3T3-CSF-1R cells. H27K15 was generated to be evaluated as a cancer immunotherapy. It exhibits a similar effect to CXIIG6, which decreases CSF-1-dependent phosphorylation of CSF1R. ELISA data showed that H27K15 targets human CSF-1R and has no cross reactions with

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other tyrosine kinases.55 H27K15 is a promising drug candidate for cancer immunotherapy that could help in preventing rebound effects and toxicity in treated patients.56 7.2. RG7155 Emactuzumab (RG7155) is a recombinant, humanized monoclonal antibody that exhibits its activity directly against the CSF-1R expressed on macrophages by binding to the receptor surface and blocking its activity. It decreases TAMs, induced by cancer, which suppress the anticancer immune response in the body.57,58 The RG7155 monoclonal antibody inhibits CSF-1R (IC50 = 0.3 nM) causing cell death and inhibiting CD68/CD163 macrophages. RG7155 is selective for CSF-1R, inhibits dimerization of the receptor and prevents formation of the ligand/receptor complex. Consequently, the antibody inhibits CSF-1R and blocks liganddependent and ligand-independent receptor activation. Inhibition of CSF-1R by the mAb resulted in a reduction of F4/80+ TAMs and an increase in the CD8/CD4 ratio in tumor cells across various solid malignancies. This was reflected by RG7155 treatment where T cell infiltrate dominated by CD4 T cells was switched to CD8 predominated lymphocyte infiltrate.57 RG7155 activity and safety in patients with advanced local diffuse-type, tenosynovial giant cell tumor (Dt-GCT) was supported preclinically by animal models and patients.58 Dt-GCT is a tumor characterized by cytogenetic abnormalities of a chromosomal translocation in the gene encoding CSF-1, and this results in an over expression of CSF-1 and activation of CSF-1R. This allows the recruitment of macrophages that associated with the tumor mass in Dt-GCT. Targeting CSF-1R with Emactuzumab is highly effective in patients with Dt-GCT even when used as a short course therapy in patients who cannot undergo surgery.58

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In conclusion, RG7155 shows many promising activities in treating Dt-GCT, as it is not only limited to TAMs but also increases tumor immune effector cells, as reflected by the CD4\CD8 Tcell ratio.57 7.3. FPA008 Cabiralizumab (FPA008) is a monoclonal antibody developed by the FivePrime Company. FPA008 blocks the binding of both CSF-1 and the IL-34 ligand to CSF-1R in patients with RA, as shown in preclinical models of RA, which results in a decrease in CD14+/CD16++ nonclassical monocytes.59,60 Inhibition of CSF-1R resulted in a dose-dependent increase in the levels of CSF-1R and IL34. FPA008 safety profiling shows that a dose of 1 or 3 mg/kg has adverse effects such as facial swelling, headache, pruritus, fatigue, and eyelid edema; those adverse events are self-limited.59,60 FPA008 is currently being evaluated in phase Ia/Ib of a clinical trial, and targets immune cells, macrophages and monocytes in many advanced solid tumors and RA that are activated in the diseases sittings. FPA008 blocks the binding of CSF-1R to CSF-1 and IL-34, thus inhibiting the survival and activity of these cells.61 7.4. TG3003 TG3003 acts against CD115, a CSF-1 cell surface receptor. TG3003 does not block CSF1/CSF-1R binding, but down modulates CD115 signalling. Compared to other anti-CSF-1 antibodies, TG3003 does not show any cytotoxic activity in normal myeloid cells. In in vitro studies, TG3003 deprives monocyte differentiation from M2-type macrophages to CD14- CDa+ cells, dendritic cells that are able to stimulate T cell responses. Since it inhibits M2-TAMs, it will enhance a patient’s immune response and affect tumor progression.62 It also inhibits the secretion of MCP-1/CCL2 and the production of IL-6, which are associated with M2-macrophage Page 16 of 62 ACS Paragon Plus Environment

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polarization. Moreover, TG3003 maintains CSF-1 circulatory levels as it does not inhibit the physiological pathway of its degradation, in contrast to other mAbs, which helps prevent the toxic and rebound effect in treated individuals.62

8. CSF-1R Kinase Inhibitors 8.1. Cyanoimidazolecarboxamides 8.1.1. JNJ-40346527 JNJ-40346527 (1, Fig. 1) is an oral CSF-1R kinase inhibitor (IC50 = 3.2 nM in cell-free kinase assay) that selectively binds to CSF-1R in RA patients. A phase II clinical study on patients with active RA was carried out based on a phase I study that found high efficacy of compound 1 on inhibition of CSF-1 and expected a more than 90% decrease in CSF-1R activity, suggesting compound 1 as a possible treatment of RA. The phase II study demonstrated no significant difference in efficacy between the compound 1-treated group and the placebo-treated group. Higher levels of CSF-1 and lower levels of CD16+ monocytes indicates that compound 1 binds to the CSF-1 receptor and inhibits the response of macrophages to CSF-1 at a concentration of less than 10 nM. Despite the effective binding of the drug to CSF-1R, the drug was not effective in treating DMARD-refractory RA patients.63 Adverse effects were related to its mechanism of action; inhibition of liver-derived macrophages, called Kupffer cells, which account for approximately 90% of tissue macrophages in the body and acts to clear liver enzymes, and resulted in an increase of liver enzymes.63,64 Other preclinical studies showed that compound 1 could be used in the treatment of relapsed or refractory classical Hodgkin lymphoma (cHL).65

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Compound 1 was found to inhibit macrophages in animal models and to decrease proliferation of Hodgkin lymphoma (HL) cell lines in vitro. This compound affects cHL by two pathways, targeting TAMs and regulating tumor cells in Hodgkin lymphoma.66 CSF-1R bound to the ligand was found in samples taken from patients with cHL. A reduction of HL cell viability was observed following treatment with the CSF-1R inhibitor, suggesting a role for the CSF-1R pathway in the activity of the disease. A phase I/II, multicenter study showed that compound 1 is relatively safe and well-tolerated by cHL patients but had a limited activity as a monotherapy for cHL treatment. Absorption of compound 1 was fast according to a single-dose phase I study where doses ranged from 10 mg daily to 450 mg twice daily with a tmax of 1 to 3.5 h and a Cmax increasing with the dose up to 450 mg daily. A multiple-dose study of doses ranging from 50 mg daily to 300 mg twice daily for 14 successive days showed a proportional increase between the dose and Cmax following daily dosing. Plasma CSF-1R levels 4 h post-dose were more than 80% to 90% inhibited in most patients and all treatment schedules confirmed the binding of compound 1 to the receptor. Twenty-four hour post-dosing analysis showed a decrease in the inhibition of plasma CSF-1R for the daily doses, while the 150 mg twice daily dose showed a sustained inhibition (peak and trough analysis) making it a more suitable dose for the phase II study according to the PK and PD analyses. One patient with multiply-relapsed disease had the best overall response, which is a complete remission after a treatment with 150 mg daily for approximately one year. Other patients showed a stable or progressive disease state. In vivo pharmacological activity showing an inhibition of the phosphorylation of CSF-1R by 95% was observed following a dose of 450 mg in the single-dose study and a dose of 150 mg daily in the multiple-dose study. Dose ranges from 150 to 600 mg/day showed acceptable safety and tolerability in healthy volunteers. Although the percentage of CSF-1R inhibition and targeting in

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the tumor cells was high, this phase II study reported no noticeable efficacy in patients with late stage cHL who had tried other treatments before trying compound 1. This could be due to the ability of HL cells to differentiate and activate other signaling mechanisms or messengers in the tumor cells of these patients making it difficult for single-target molecules to be effective on them and leading to resistance of these molecules. Further assessment of these single-target molecules in combination with other agents is required for optimal cancer treatment.65

8.1.2. Other cyanoimidazolecarboxamide and cyanofurancarboxamide derivatives Numerous arylamides are CSF-1R inhibitors. The main core structure, 1,2,4phenylenetriamine exhibits excellent potency and good efficacy in vivo. However, it may form reactive quinone diimine metabolites that cause tissue damage due to glutathione depletion and protein toxicity. That is why the structure has had several modifications made to it, especially to the C-2 and C-4 located in the aryl group as they have an impact on the activity. The best activity occurred when the methylpiperidine at the C-2 position was replaced with a cyclohexene ring. Replacing piperazine with a piperidine group on C-4 showed potency retention (compounds 3 and 4, Fig. 1).67 They demonstrated good activity against CSF-1R as well as CSF-1 in a cellbased assay. However, they had low efficacy in a pharmacodynamics assay and poor pharmacokinetics properties that produced disappointing results in vivo. In case of more basic piperazine derivative (compound 3), this compound showed the cons of off-target activity and bonded strongly to sodium and calcium channels, which led to increased risk of adverse cardiovascular effects. As a result of these modification, a new compound JNJ-28312141 (5, Fig. 1) with reduced basicity compared to compound 2, an acceptable solubility, better PD and PK properties, and great efficacy in a rodent model of RA progressed to clinical trials. Compound 5 Page 19 of 62 ACS Paragon Plus Environment

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exhibited reasonable selectivity against four kinases (FLT3, AXL, KIT, and TRKA) in addition to CSF-1R. Further testing of this compound against these kinases in cell-based assays resulted in a 16-fold selectivity for KIT over CSF-1R, which appeared to cause bone marrow suppression issue. Despite the fact that KIT inhibition can be beneficial in inflammation as it suppresses the function of dendritic and mast cells, other evidence shows that KIT inhibition may cause bone marrow suppression, anemia, hypospermia, and pigment loss. In vivo studies of compound 5 and its analogs show a reduction in reticulocyte counts and hypocellularity of the bone marrow, which relates its inhibition to unwanted adverse effect. Consequently, additional optimization is underway to improve the selectivity of compound 5 and its analogs to CSF-1R instead of KIT. Modeling shows small differences between CSF-1R and KIT close to the C-2 position that may enhance selectivity toward CSF-1R, while the difference near the C-4 position was less defined due to low resolution in the CSF-1R area. However, since it is close to solvent, this allowed for high flexibility in the variation of the structure without affecting the potency of the compound toward CSF-1R, allowing for optimization in these regions. The addition of 4,4-dimethyl groups to cyclohexene enhanced selectivity of these compounds toward CSF-1R. In the ATP binding site of CSF-1R, compound 5 and its analogs extended from the hinge zone and bound at the ribose pocket, since Phe797 is shifted away from the region in CSF-1R, whereas in KIT, Phe is already forced into that space, preventing the dimethylcyclohexene from binding and causing a steric clash, leading to low potency as determined in a Mo7e cell assay (this assay was utilized here to investigate the inhibitory effect of the compound at cellular level). This also enhanced the potency of the compounds against CSF-1R by 1.8-fold compared with KIT increasing selectivity towards CSF-1R. Although compound 5 showed good potency, it showed limited stability in human, mouse and rat liver microsomes, due to oxidation of the 4-position benzylic methine.

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Methyl substituents there increase metabolic stability, but only compound 6 (Fig. 1) possessing 4-(HO (CH2)2)-piperazin-1-yl moiety exhibited a good in vivo PD activity and reasonable potency against CSF-1R (IC50 = 0.7 nM in cell-free kinase assay), BMDM assay (IC50 = 6.1 nM) and Mo7e (IC50 = 13 nM), allowing it to be considered for further evaluation. The PK properties of the compound 6 hydrochloride salt were evaluated in rats. After injection with 1 mg/kg iv of compound 6 mixed with 20% 2-hydroxypropyl-β-cyclodextrin (HPbCD), the volume of distribution was 7.6 L/kg. The systemic clearance of compound 6 was 55 ml/min/kg, which is moderate; the mean Cmax after 5 mg/kg oral dose values was 88 ng/ml (210 nM) and the elimination t1/2 was 6.5 h. Furthermore, it showed acceptable systemic exposure (F = 31%, AUC = 690 ng*hr/mL). These results encouraged conducting further in vivo studies. Compound 6 was tested in a streptococcal cell wall (SCW)-induced arthritis model of RA and proven to be efficacious in fully and partly inhibiting the progression of ankle swelling progression at doses of 40 mg/kg and 10 mg/kg, respectively.68

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N

H N

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N H

O

N H 2 1 N

O

O

N 4

O

O

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3, R = H, IC50 = 1.1 nM 4, R = Ac, IC50 = 2.4 nM

2 IC50 = 1.0 nM

JNJ-40346527 (1) IC50 = 3.2 nM

N

H N

CN

O

N H

CN

O 6 IC50 = 0.7 nM

5 IC50 = 0.69 nM

Figure 1. Cyanoimidazolecarboxamide and cyanofurancarboxamide CSF-1R kinase inhibitors.

8.2. Aminopyrimidine derivatives 8.2.1. ENMD-2076 ENMD-2076 (7, Fig. 2) is an orally active multi-target kinase inhibitor developed by CASI Pharmaceuticals.69,70 In addition to CSF-1R inhibition, it inhibits several other kinases including Aurora A and B, angiogenesis enzymes (VEGFR and FGFR) and growth factors (CSF1R, FIT-3 , KIT). ENMD-2076 activity has been tested on MV4-11 cells that express CSF-1R and results in the inhibition of CSF-1 signals stimulating CSF-1R and reduction in mammary gland cancer growth (IC50 = 600 nM), which shows that it has an anti-angiogenic and antiproliferative profile that make it a potential therapeutic candidate for treating human cancers.69 Page 22 of 62 ACS Paragon Plus Environment

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Compound 7 has been evaluated in phase I clinical trials in different types of tumors including liver and breast tumors and leukemia, and in phase II clinical trials in ovarian cancer.62 It will be tested in phase 2 clinical trials for treatment of ovarian clear cell carcinoma. O

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O N N

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O

SB1578 (8) IC50 = 69 nM

ENMD-2076 (7)

O

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GW 2580 (9) IC50 = 60 nM

Figure 2. Aminopyrimidine CSF-1R kinase inhibitors. 8.2.2. SB1578 SB1578 (8, Fig. 2) is a macrocyclic JAK2 inhibitor that also has potent activity against the CSF-1R kinase. It plays an essential role in the initiation and pathogenesis of RA. It also has useful effects on bone and cartilage damage. Compound 8 possesses unique activities such as stopping the inflammatory response by preventing the diffusion of neutrophils and macrophages to affected joints, which reflects its great role in managing autoimmune diseases. This compound was discovered through a kinase inhibitor program by the S*BIO company, and it was produced from a pyrimidine-containing, low molecular weight series. It is a potent multi-kinase inhibitor, with IC50 values of 69 nM for CSF-1R, 62 nM for FLT3, 46 nM for JAK2, and 230 nM for TYK2 in cell-free kinase assays. In monocytes isolated from peripheral blood, SB1578 has an IC50 of 277 nM. It also has an inhibitory effect on THP-1 cells, which are stimulated with CSF-1 to induce autophosphorylation of CSF-1R.71

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Compound 8 was also tested using a Micro-Equilibrium Dialyzer (Harvard Apparatus) to measure the amount of plasma protein binding to compound 8 in plasma obtained from five different species (human, rat, mouse, dog, and monkey). A high amount of SB1578 bound to human plasma (99%) and monkey plasma (98.7%) proteins, but lower amounts of compound 8 were bound by plasma proteins in the other species (87.3-87.5%).72

8.2.3. GW 2580 GW 2580 (9, Fig. 2) is an oral bioavailable diaminopyrimidine derivative and a selective CSF-1R inhibitor in vitro with an IC50 of 60 nM in cell-free kinase assay. Compound 9 completely inhibited the growth of CSF-1R dependent M-NFS-60 tumor cells and human monocytes in the peritoneal cavity. It also inhibited joint connective tissue formation and bone destruction in human osteoclast cultures. It is as effective as imatinib in reducing the severity of arthritis in CIA. After oral administation of the drug, the ability of CSF-1R to increase LPSinduced IL-6 production in mice was blocked by compound 9. In addition, it inhibited the growth of M-NFS-60 mouse myeloid cells expressing CSF-1R kinase.73 One study was carried out to determine the role of M2 macrophage using a specific CSF1R signaling inhibition approach to inhibit these macrophages; the results showed that the administration of compound 9 to M2 macrophages for one week decreased the numbers of M2 macrophages in the heart and decreased expression of cardiac arginase-1 and CD206, genes indicative of M2 macrophage activity.74

8.3. Pyridines

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8.3.1. BLZ945 BLZ945 (10, Fig. 3) is a CSF-1R kinase inhibitor that has a great impact on OCs function and macrophage precursors and can be used in preventing some diseases associated with abnormalities in these cells.75,76 Some research has been done on compound 10 and its active metabolite (11, Fig. 3) to determine their effectiveness. The active metabolite which is a structural isomer of the parent compound, is formed via oxidation by P450 followed by reduction of the ketone group. This metabolite is 4 times less potent than its parent drug. Using the equilibrium dialysis method, the amounts of plasma protein binding for compound 10 (96.3%±0.18) was slightly lower than that of its metabolite (96.9%±0.24).75 An in vitro kinase assay used to determine pharmacological activity with a murine AML cell line showed that their proliferation depended on CSF-1; the test was used to determine proliferation and viability due to blockage of CSF-1R phosphorylation. The IC50 values of BLZ945 and its metabolite against CSF-1R kinase in cell-free kinase assay were 1.2 nM and 5.5 nM, respectively.75 Compound 10 was more than 3200 times more selective against CSF-1R than the other types of tyrosine kinase receptors.77 The antiproliferative activity of BLZ945 and its metabolite were significant against the CSF-1-expressing cell line M-NFS-60 (EC50 of 71 nM and 140 nM, respectively). Compound 10 inhibition of CSF-1R reduced the TAM turnover rate and increased the number of cytotoxic T cells in some types of cancer, such as cervical and breast cancer. Moreover, a study on keratin 14-expressing human papillomavirus type 16 (K14-HPV-16) cells showed that compound 10 prevented tumor development. Another study was done on mouse

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mammary tumor virus driven by the polyomavirus middle T antigen (MMTV-PyMT) and showed that compound 10 decreased mutant cell growth.76 O O

HN

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N

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NH

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N BLZ945 (10) IC50 = 1.2 nM

HO S N

BLZ945 metabolite (11) IC50 = 5.5 nM

Figure 3. Structures of BLZ945 and its active metabolite. 8.3.2. PLX647 PLX647 (14, Fig. 4) is a selective and potent CSF-1R kinase inhibitor (IC50 = 28 nM in cell-free kinase assay) that also inhibits KIT (IC50 = 16 nM in cell-free kinase assay) and FLT3ITD in vitro. 7-Azaindole (12) was identified as a kinase inhibitor scaffold in the scaffold-based discovery of PLX647. Introducing a 3-methoxy substituent onto 3-benzylpyrrolo[2,3-b]pyridine gave PLX070 (13, Fig. 4). Replacing the methoxyphenyl moiety of PLX070 with (trifluoromethyl)benzylaminopyridyl led to PLX-647 (14, R = H, Fig. 4). Addition of a 5methoxy gave 15 (R = OMe, Fig. 4), which is more polar and more soluble in aqueous solutions (molar solubility = 77 µM) than compound 14 (molar solubility = 14 µM), according to a turbidity-based assay, but it is a less potent CSF-1R inhibitor (IC50 = 62 nM in cell-free kinase assay) than PLX647. PLX647 was efficacious in animal models and in the inhibition of macrophages, osteoclasts and mast cells stimulated by CSF-1R and KIT.78,79 Compound 14 controls inflammation, cancer progression and bone osteolysis by inhibiting the CSF-1R and KIT kinases. The effect of CSF-1R inhibition was determined by LPS-induced cytokine release, mast cell activation, kidney inflammation, collagen-induced arthritis, cancer-induced bone pain, and osteolysis. The preclinical results were used to Page 26 of 62 ACS Paragon Plus Environment

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characterize compound 14 pharmacology. In vivo, compound 14 was used to determine the effect of CSF-1R inhibition on LPS-induced cytokine stimulation. Injecting mice with LPS increases the levels of the inflammatory cytokines. Mice given an oral dose of compound 14 before an LPS injection had an 85% reduced serum TNF-α level and a 75% inhibition in IL-6 release. Compound 14 also inhibits mast cell activation in the skin by inhibiting the PCA reaction, which is a result of the inhibition of KIT. In mice with renal inflammation induced by unilateral ureter obstruction (UUO), PLX647 reduced the levels of F4/80+ macrophages by 77%, suggesting that compound 14 is sufficiently effective in preventing the accumulation of macrophages in renal inflammation caused by UUO. In a CIA mouse model, compound 14 decreased the signs of arthritis after 31 days. Currently, compound 14 is in clinical trials to reprogram the immune microenvironment in tumors for the improvement of anticancer treatment.78 H N

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7-Azaindole core (12)

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N H R = H, PLX647 (14), IC50 = 28 nM R = OMe, PLX647-OMe (15), IC50 = 62 nM N

H N N

Cl

CF3

CF3 N

N H Pexidartinib (PLX3397, 16) IC50 = 13 nM

N

Figure 4. Structures of the 7-azaindole-containing pyridine analogues.

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PLX3397 (16, Fig. 4) is an orally-active and potent CSF-1R inhibitor (IC50 = 13 nM in cell-free kinase assay) that exhibits an activity on other kinases such as KIT and FLT3.80 compound 16 was developed based on the structure of compound 14. The phenyl ring in the (trifluoromethyl)phenyl tail of compound 14 was replaced with pyridine to enhance its polarity. In addition, a chloro group was introduced to position 5 of the azaindole scaffold. In its autoinhibited state, part of the juxtamembrane of CSF-1R binds into its ATP-binding site. Compound 16 binds in the ATP-binding site of CSF-1R, but has interactions with residues in the juxtamembrane inhibitory loop, which prevents the loop from exiting the ATP-binding cleft upon ligand binding. It makes hydrogen bonds with tyrosine residue 546 (Tyr546) and tryptophan residue 550 (Trp550) in the juxtamembrane region of the kinase. In the presence of water, in addition to pi stacking between pyridine and Trp550, the pyridine nitrogen atom is involved in a network of hydrogen bonds that keep the juxtamembrane domain in its inactive state (Fig. 5). When testing compounds 14 and 16 on cells that depend on CSF-1R for their growth, compound 16 showed a significantly higher potency than compound 14. Preclinical studies in animals showed promising PK properties including enhanced oral bioavailability and a proportional relationship between the effect of compound 16 and its dose. Compound 16 was tested on various animals to evaluate its efficacy. Administration of compound 16 in a CIA mouse model improved the condition of the joints. In patients with tenosynovial giant-cell tumors, 52% of those treated with compound 16 responded significantly to the treatment with more than 8 months of disease control. These results were better than those obtained from imatinib. In most patients, the administration of compound 16 showed a reduction in tumor volume for longer periods.81,82

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Compound 16 was also used on glioblastoma (GB), a deadly cancer that is usually treated by surgical removal followed by radiation therapy in addition to other chemotherapies (temozolomide) with a survival time of approximately 14 months. compound 16 prolonged the survival time and slowed the disease progression rate. Microglia, macrophages, and other tumor microenvironments, which affect the biology of GB, are regulated by CSF-1R and KIT. This makes compound 16 a possible treatment option for GB because of the relationship between high levels of microglia and low survival rates in GB patients. In addition, GB patient samples had high levels of CSF-1R and KIT ligands that activate surrounding microglia, the resident macrophages of the central nervous system (CNS), leading to increased tumor invasion. Another reason for testing compound 16 activity is the slowing of tumor progression after depletion of microglia in orthotopic GB models.80 An in vitro study using normal microglia obtained from C57Bl/6J mice to examine the effect of microglia on glioma 261 (GL261) glioblastoma cell invasion found an 8-fold increase in GL261 glioblastoma cell invasion. The same study showed that the inhibition of CSF-1R signaling inhibited the invasion of breast cancer cells, which are stimulated by macrophages both in vitro and in vivo. For this reason, CSF-1R kinase domain ATP binding site inhibitors were tested on GB.83 In a Matrigel invasion assay on mouse GL261 GB cells cultured with microglia, a dose of 1 µM of 16 caused a significant inhibition of metastasis. compound 16 crosses the normal blood-brain barrier (BBB) readily and reaches the CNS in rats without being actively transported which indicates that compound 16 can reach GB and stromal tumor tissues.80 In wild-type C57 mice injected with GL261 cells, the cells showed extensive CSF-1 expression and infiltration of microglia. Administration of compound 16 resulted in reduced microglia/macrophages numbers and significantly inhibited the invasiveness of the tumor cells.83 Studies on PK/PD models which were obtained from tumor tissue samples

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taken from patients who took compound 16 for 7 days, showed that the concentration achieved by compound 16 in half of the tumor tissue samples was equal to or more than 100 nM, and that was sufficient to inhibit CSF-1R. Phase I studies analyzed CSF-1 plasma levels and confirmed the inhibition of CSF-1R. These data made compound 16 a good candidate for phase II clinical trials. A phase II study was conducted on patients with recurrent GB by giving them a daily dose of 1000 mg of compound 16, which was well-tolerated and resulted in an increase of CSF-1 and a decrease in the number of monocytes of the CD14dim/ CD16+ subset in plasma, compared with pretreatment values. In addition, compound 16 sufficiently penetrated tumor tissues leading to an inhibition of CSF-1R and a decrease in the number of TAMs. A median drug level of 5500 ng/g was detected in the tumor cells after 7 days of administering compound 16. Despite these significant effects, compound 16 showed an increase in CSF-1 and reduced CD14(dim)/CD16+ monocytes in plasma, compared with their values before treatment. So the compound is well tolerated and is able to cross the blood-cancer barrier but no efficacy was seen in this heavily pretreated population.80

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Figure 5. Conformation-specific inhibition of CSF-1R kinase by PLX3397 (16).81 From [Tap et al., Structure-guided blockade of CSF1R kinase in tenosynovial giant-cell tumor, 373, 428-437, 2015, N. Eng. J. Med.]. Reprinted with permission from Massachusetts Medical Society.

8.4. Quinoline and Cinnoline derivatives AZ683 and AZD7505 AZ683 (17, Fig. 6) is a compound that belongs to the 3-amido-4-anilinoquinoline class. It is a potent and highly selective CSF-1R inhibitor, allowing a reduction of TAMs and inhibiting tumor growth. It has a good oral bioavailability, an IC50 of 230 nM in vitro in cell-free kinase assay, and promising biological properties as it was tested on 85 kinases and found to inhibit two specific receptors by more than 20% (CSF-1R 96% and ARK5 51%, at 1 µM concentration).84,85 This compound was linked with serious cardiovascular side effects when tested in dog cardiomyocytes, as it produced a dose-dependent reduction on the action potential (10 and 30 µM) and inhibited cardiac Nav1 channels in humans (IC50 = 14 µM). As a result, compound 17 did not proceed to further development phases. A new class of highly related cinnolines, 3amido-4-anilinocinnolines (compounds 18 and 19, Fig. 6) were discovered to be selective CSF-1 inhibitors with excellent pharmacokinetic properties and their IC50 values in 3T3 cell-based assay (stimulated with CSF-1 and engineered to express CSF-1R kinase) were 13 and 25 nM, respectively. However, compound 18 still showed a dose-dependent prolonging of AV conduction and the QRS duration, similar to AZ683. Therefore, further modifications were made to both classes of compounds, and the aniline substituent and 7-alkoxy groups were combined to produce a new potential CSF-1R inhibitor with a lesser cardiotoxic effect.85 The new compound AZD7507 (19) (32 nM cell activity), in which the N-methylpiperazine at position 6 was replaced Page 31 of 62 ACS Paragon Plus Environment

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with a hydroxyethylpiperazine, had a big impact as it produced a 7 ml/min/kg in vivo clearance, a bioavailability of 42%, and an IC50>20 µM in a canine L-type Ca channel assay in contrast with AZ683 (3.5 µM). In addition, dog myocytes treated with 30 µM AZD7507 had no significant effects, and the maximum dose had no effect on the AV conduction time or QRS duration. In epithelial cells with cholangiocarcinoma (CC), compound 19 treatment significantly reduced the number of CD68+ macrophages and minimized tumor volume and mass.85,86 In conclusion, AZD7507 (19) is considered a promising compound as it retained the desired potency and oral pharmacokinetic properties of AZ683. Moreover, in a telemetry study in dogs given a single, oral dose, there was no cardiovascular toxicity, no elevation of troponin related to the treatment, and no alteration in the ECG.85 It has not been investigated in clinical trials yet. F

F

N

NH N

F O

N NH2

O

N

AZ683 (17)

F

NH N

HO

O NH2

O

N

N

18

N

NH N O

O NH2

N

N

AZD7507 (19)

Figure 6. Structures of the quinoline and cinnoline derivatives reported as CSF-1R kinase inhibitors.

8.5. Miscellaneous 8.5.1. AC708

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AC708 (structure not disclosed) is a potent compound that inhibits CSF-1R and targets TAMs as it prevents CSF-1-mediated phosphorylation of CSF-1R.87 It shows significant selectivity for CSF-1R compared with other protein kinases. In cell-based assays, the cells were stimulated with either CSF-1 or IL-34 resulting in IC50 values of 26 and 33 nM, respectively. In cultured, growth factor-dependent cells, it has an IC50 = 38 nM for CSF-1 and an IC50 = 40 nM for IL-34, and it prevented the differentiation and survival of human OCs mediated by CSF-1 (IC50 = 15 nM). Further studies were done to assess the compound’s efficacy on different types of cells. AC708 prevented the growth of M-NFS-60 cells intraperitoneally in a dose-dependent manner; a 100 mg/kg dose of AC708 produced a more than 80% reduction in cell number, which is a similar effect to that of Ki-20227. In human monocytes stimulated by both ligands, AC708 caused inhibition of MCP-1 release with CSF-1 (IC50 = 93 nM) and IL-34 (IC50 = 88 nM), which are lower IC50 values compared with those obtained from compound 9, a benchmark compound [CSF-1 (IC50 = 148 nM) and IL-34 (IC50 = 140 nM)].87

In vivo studies have been done to test the efficacy of AC708 on immune-competent and nude mice with the use of the F4/80 macrophage marker. The animals were injected with an antiVEGF antibody repeatedly to develop resistance, followed by full immune profiling. The results showed a significant increase of macrophage infiltration in tumor with anti-VEGF antibody resistance in contrast to the tumors of anti-VEGF antibody-sensitive mice (p>0.0001).88 Another study shows the effect of the compound on anti-VEGF antibody-resistant mice when it is combined with another chemotherapy (paclitaxel). The results showed a restoration of the angiogenic therapy, which caused a reduction of 82% in the tumor load and a decrease in macrophages infiltration compared to when paclitaxel was administered alone.88

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Two models were developed to assess the ability of the compound to control endogenous CSF-1R. In the first model, recombinant parathyroid hormone-related protein (PTHrP) was injected, and plasma levels of tartrate-resistant acid phosphatase (TRAP5b) were measured. PTHrP-induced elevation of plasma TRAP5b was decreased in a dose-dependent manner when a 100 mg/kg dose of AC708 was administered. In the second model, CSF-1-mediated MCP-1 release stimulation was inhibited by 60% after a 100 mg/kg dose of AC708 was administered.87

8.5.2. ARRY-382 ARRY-382 is a CSF-1R kinase inhibitor developed by ARRAY Biopharma, and its chemical structure has not been disclosed yet. The compound is still in phase I clinical trials.89 It is a potent and selective inhibitor of CSF-1R (IC50 = 9 nM in cell-free kinase assay). In vitro tests of ARRY-382 prove that it has a potent inhibitory effect on OC differentiation (IC50 = 4 nM) and bone resorption (IC50 = 58 nM). In an HEK-293 cell-induced tumor model in mice, its ED50 value was 3 mg/kg. The safety of ARRY-382 was tested in 26 patients with advanced or metastatic cancers at doses of 25 to 500 mg and no significant major side effects were reported. The maximum tolerated dose with biological effect was 400 mg QD.90

8.5.3. PLX 5622 PLX 5622 (structure not disclosed) is a CSF-1R inhibitor with potent activity (Ki = 5.9 nM) and high selectivity (50-fold for four tyrosine kinases and over 100-fold for 230 different kinases). A study carried out on wild-type mice to test the inhibitory effect of PLX5622 on microglia in adult human brains, mice were tested with two different doses, 300 mg/kg or 1200 Page 34 of 62 ACS Paragon Plus Environment

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mg/kg. The results showed that microglia were eliminated by inhibiting CSF-1R, and that a higher dose of PLX5622 (1200 mg/kg) resulted in nearly complete elimination (98%) of microglia.91,92 In another study on inflammatory monocytes (Ly6C-high) and patrolling monocytes (Ly6C-low), both high and low doses of PLX5622 resulted in the depletion of Ly6C-high within the blood and an unchanged effect on the number of Ly6C-low.93

8.5.4. DCC-3014 DCC-3014 (structure not disclosed) is a potent inhibitor of CSF-1R (IC50 = 5 nM in cellfree kinase assay). It is a Type 2 DFG loop-out kinase inhibitor with the typical slow binding kinetics of that class. with a small molecular weight that was tested in over 300 human kinases including CSF-1R and kinases related to it, such as FLT3 and KIT. DCC-3014 inhibited CSF-1R by more than 100 fold compared with its inhibitory effects on FLT3, KIT, PDGFRα, and PDGFRβ, and by more than 1000-fold compared with other kinases. DCC-3014 is a highly specific inhibitor of CSF-1R and keeps the receptor in an inactivated conformation.94 Preclinical studies showed promising pharmacokinetic and optimized biopharmaceutical properties of DCC3014. In vivo, DCC-3014 was tested in the murine cFOS PK/PD model and resulted in a more than 90% inhibition of CSF-1R at 15 mg/kg 24 h after the dose. A dose of 3 mg/kg administered for 6 days until it reached a steady state resulted in a strong inhibition of CSF-1R. In the MC38 colorectal cancer model, which has a high level of infiltrating TAMs, a dose of 10 mg/kg of DCC-3014 resulted in an inhibition of TAMs, repolarization of the adaptive immune cells (converting them to anti-tumor cells) and depletion of circulating CD16+ monocytes. DCC-3014 was also tested in THP-1 and MNFS-60 monocyte cell lines, a human whole blood assay and in a Page 35 of 62 ACS Paragon Plus Environment

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human osteoclast TRAP assay. The results of these studies showed that the inhibition of CSF-1R in THP-1 monocytes (IC50 = 11 nM), MNFS-60 monocytes (IC50 = 4 nM), human osteoclasts (IC50 = 9 nM), and in a human whole blood monocyte/pERK assay (IC50 = 260 nM) was resilient to 10-1000 ng/ml CSF-1 because DCC-3014 binds CSF-1R for a long time and keeps it in its inactive conformation. The combination of DCC-3014 and the murine anti-PD1 antibody in the MC38 colorectal cancer model showed an improvement in the inhibitory effect and a synergistic immunomodulatory activity by the added blocking of macrophage immune checkpoints compared with other treatment alone.93 DCC-3014 is currently in phase I clinical trials in patients with advanced malignancies to assess its safety, PK, and PD using different doses.94-96

8.5.5. 4-Arylamido-5-methylisoxazole derivatives Compound 20a (Fig. 7), is a 4-arylamido-5-methylisoxazole derivative recently discovered as a potent inhibitor of CSF-1R and other kinases. 20a is a derivative of a highly potent and selective CSF-1R inhibitor.3 While screening a library of protein kinase inhibitors in U937 (human histiocytic lymphoma) and A375P (human melanoma) cells, the methylisoxazole group was discovered to be a hinge binder that exhibited high activity toward U937 cells. Data showed that 2-methyl-N-(5-methylisoxazol-4-yl)benzamide (21, Fig. 7) was selective for CSF-1R (IC50 = 216 nM in cell-free kinase assay). The structure-activity relationship showed that, in U937 cells, the amide linker was better for activity than the urea analogs. The middle phenyl ring with its substituents also has a great impact on the CSF-1R activity, where the number, position, and type of the substituents affect their antiproliferative effect. The tested compounds were more potent against U937 cells than A375P cells. Based on the IC50 values, only four of the compounds demonstrated high inhibitory activity 20a-d (Fig. 7) with GI50 values against U937 Page 36 of 62 ACS Paragon Plus Environment

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hematopoietic cell line as follows: 16, 19, 26, and 49 nM, respectively. An introduction of disubstituted benzamide at positions 3 and 5 increases the activity, especially if the substituent is alkylpiperazine, alkylimidazole, or morpholine. On the other hand, the substitution of a bulky aromatic acid lowers inhibitor activity. When comparing the activity of the four potent compounds in U937 cells, compound 20a, with trifluoromethyl and methylpiperazinyl groups at positions 3 and 5, exhibited the most potent activity. To determine the selectivity index, the compounds were tested against HS27 cells (human normal cell line). The IC50 ranges were > 30 µM. All the tested derivatives displayed weak cytotoxicity against HS27, and great selectivity indexes towards cancer cells rather than normal cells. After determining 20a as the most potent antiproliferative compound, it was screened against a selected panel of kinases to determine its kinase inhibitory profile. It showed an excellent inhibitory effect on CSF-1R (IC50 = 9.95 nM in cell-free kinase assay), and a less potent inhibitory effect against B-RAF, B-RAF/V600E, Lyn, and C-RAF with IC50 values of 5590 nM, 668 nM, 65.9 nM, and 44.8 nM, respectively. To understand how compound 20a binds to CSF-1R, a molecular docking study was done. The docking binding mode of compound 20a to the ATP-binding site of CSF-1R is shown in Fig. 8.3

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CF3

CF3 O N

H N

O N

H N

N

O

O

H N

N

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O 20b

20a IC50 = 9.95 nM CF3 O N

H N

CF3 O N

H N

N

H N

H N

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O

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20c

N

O

O 20d

H N O

O N

21 IC50 = 216 nM

Figure 7. Structures of the 4-arylamido-5-methylisoxazole analogs reported as CSF-1R kinase inhibitors.

Figure 8. Binding mode of compound 20a docked into CSF-1R (PDB ID: 3LCO).3 From [Im et al., Discovery of 4-arylamido 3-methyl isoxazole derivatives as novel FMS kinase inhibitors,

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102, 600-610, 2015, Eur. J. Med. Chem.]. Reprinted with permission from ELSEVIER Publisher.

9. Conclusion In this article, we reviewed different CSF-1R inhibitors that were recently reported in the literature reported from 2012 until the present. Most of the reviewed inhibitors are potent inhibitors of CSF-1R and its ligands (CSF-1 and/or IL-34). Anti-CSF-1 monoclonal antibodies are currently under investigation to be used in macrophage-committed progenitor cells purification in mice and help in identification of human blood monocytes. Moreover, their functions can be studied in human or animals as their activity depends on CSF-1 signaling. Here, we reviewed several monoclonal antibodies with different properties and characteristics that make them unique as chemotherapies. H27K1 was developed to overcome the increase in CSF-1 caused by other mAbs. Emactuzumab was effective in treating patients with Dt-GCT cancer which makes it a promising drug candidate. Another antibody, FPA008, was generated to target immune cells, macrophages and monocytes in many tumors. Lastly, TG3003 showed great activity in preventing cytotoxicity compared with the other mAbs. The main advantage of cancer immunotherapy over kinase inhibitors is higher selectivity and safety. The antibody binds to a specific antigen on the cancer cell. But kinase inhibitory chemotherapeutic agents most probably inhibit several kinases, especially if they mimic the ATP structure and bind to the kinase hinge region. Conjugated mAbs (antibody-drug conjugates) can be a very useful approach to target the chemotherapeutic agent to the cancer cell using a specific mAb that interacts selectively with a specific antigen on the cancer cell. Future research should focus on this approach to maximize the therapeutic effects and minimize the side effects. Page 39 of 62 ACS Paragon Plus Environment

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Oral CSF-1R kinase inhibitors such as JNJ-40346527 showed a significant effect in treating patients with RA (passed phase 2) or in cHL (passed phase 1). ENMD-2076 is effective in some types of cancer such as ovarian cancer, fallopian cancer, peritoneal cancer, multiple myeloma, and triple-negative breast cancer (passed phase 2 studies). GW 2580 can be a promising anticancer candidate (in preclinical studies). It is also effective in reducing collageninduced arthritis, and a new study showed that it may play a role in lowering the levels of M2 within the heart. BLZ945 is also effective in preclinical studies against cancer because it decreases the TAM turnover rate and the growth of mutant cells. ARRY-382 has a great effect on osteoclast cell differentiation, and in patients with metastatic cancer, a high dose showed 28fold increase in activity compared with lower dose. AZD7505 has the same potency as AZ683. AZ683 produces cardiovascular side effects that disappear when using the modified compound, AZD7505. PLX 5622 is another CSF-1R inhibitor that resulted in elimination of microglia from the brain. In addition, it was tested in phase 1b clinical trials in RA patients taking methotrexate (https://clinicaltrials.gov/ct2/show/NCT01329991?term=PLX+5622&rank=2, accessed Dec. 18, 2017). The structures of some of these inhibitors are still not disclosed, and some have recently entered clinical trials. Targeting CSF-1R is a fast-growing field. These trials will be closely watched for their outcomes. Combinations of monoclonal antibodies and CSF-1R inhibitors could produce synergistic therapeutic effects and may be a successful avenue for more efficient treatment of cancer and inflammatory disorders. According to clinicaltrials.gov website, clinical trials will be conducted for emactuzumab in combination with paclitaxel or atezolimumab for advanced

solid

tumors

(https://clinicaltrials.gov/ct2/results?cond=&term=emactuzumab&cntry1=&state1=&recrs=, Page 40 of 62 ACS Paragon Plus Environment

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accessed Dec. 18, 2017). Cabiralizumab will be tested also in combination with nivolumab for treatment

of

advanced

cancers

(https://clinicaltrials.gov/ct2/results?cond=&term=Cabiralizumab&cntry1=&state1=&recrs=, accessed Dec. 18, 2017). And ARRY-382 will be tested in combination with pembrolizumab in phases

1

and

2

for

treatment

of

advanced

solid

tumors

(https://clinicaltrials.gov/ct2/show/NCT02880371?term=pembrolizumab+AND+arry&rank=1, accessed Dec. 18, 2017).

Biographies Mohammed I. El-Gamal earned the Bachelor Degree of Pharmaceutical Sciences from Faculty of Pharmacy, University of Mansoura, Egypt, in 2003 as the 1st achiever (excellent with honor). In January 2008, he earned his M.Sc. degree in Medicinal Chemistry from the same University, Department of Medicinal Chemistry. He received his Ph.D. degree in 2012 at the University of Science and Technology, Korea Institute of Science and Technology, Republic of Korea. He is currently an assistant professor of medicinal chemistry. He has reported and published some articles related to CSF-1R kinase and its inhibitors. His research interests involve kinase inhibitors, other anticancer agents, antiinflammatory agents, radiolabeled imaging agents, prodrugs, antibiotics, and pharmacokinetic properties optimization. Shahad K. Al-Ameen, Dania M. Al-Koumi, Mawadda G. Hamad, and Nouran A. Jalal are recently graduated pharmacists. They were graduated in June 2017 at the College of Pharmacy, University of Sharjah, United Arab Emirates. They were among the best students at their college. They participated in this article to fulfill their graduation requirements. Page 41 of 62 ACS Paragon Plus Environment

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Chang-Hyun Oh received his Ph.D. degree in medicinal chemistry from Hanyang University, Republic of Korea, in 1994. He is working as a principal researcher of medicinal chemistry at Korea Institute of Science and Technology, Republic of Korea. His research interests focused on the development of novel therapeutic agents for the treatment of cancer, prodrugs, carbapenem antibiotics, and radiolabeled imaging agents. He has been interested in the field of kinase inhibitors, and has published several articles related to kinase inhibitors such as CSF-1R kinase inhibitors.

Corresponding author (Chang-Hyun Oh): phone number: +82 2 958 5160; e-mail address: [email protected]. List of abbreviations: ADCC: Antibody-Dependent Cellular Cytotoxicity; AML: Acute Myeloid Leukemia; Anti-PD: AntiProgrammed Death; AV: Atrioventricular Node; BMDM: Bone Marrow-Derived Macrophages; BMP: Bone morphogenetic protein; CC: Cholangiocarcinoma; CFA: Complete Freund’s adjuvant; CHL: Classical Hodgkin Lymphoma; CIA: Collagen-Induced Arthritis; CML: Chronic Myeloid Leukemia; COPD: Chronic Obstructive Pulmonary Disease; CSF-1: Colony-Stimulating Factor-1; CSF-1R: ColonyStimulating Factor-1 Receptor; DC: Dendritic Cells; DMARD: Disease-Modifying Anti-Rheumatic Drug; Dt-GCT: Diffuse-type Tenosynovial Giant Cell Tumor; EGF: Epidermal Growth Factor; ERK1/2: Extracellular Signal-Regulated Kinase-1 and -2; FGFR: Fibroblast Growth Factor Receptor; FLT3: FMSLike Tyrosine Kinase 3; FMS: kinase first discovered as the oncogene responsible for Feline McDonough Sarcoma; GMDSC: Granulocytic Myeloid-Derived Suppressor Cells; GM-CSF: Granulocyte Macrophage Colony-Stimulating Factor; HEK: Human Embryonic Kidney; HOS: Human Osteosarcoma; HPV: Human Papilloma Virus; HSC: Hematopoietic Stem Cell; HuAB: Human Antibody; IGF: Insulin-like Page 42 of 62 ACS Paragon Plus Environment

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Growth Factor; IL: Interleukin; ITD: Internal Tandem Duplication; JAK: Janus kinase; LPS: Lipopolysaccharide; M-CSF: Macrophage or Monocyte Colony-Stimulating Factor; M-CSFR: Macrophage Colony-Stimulating Factor Receptor; MCP: Male Chauvinist Pig; MDSCs: Myeloid-Derived Suppressor Cells; MMP: Matrix Metalloproteinase; MMTV-PyMT: Mouse Mammary Tumor Virus driven Polyomavirus middle T antigen; MuAb: Murine Antibody; mRNA: Messenger Ribonucleic Acid; NaV1: Sodium Channel, Voltage-gated, Type I; NSCLC: Non-small Cell Lung Cancer; OB: Osteoblast; OC: Osteoclast; OCPs: Osteoclast Precursors; OP: Osteoclast Progenitor; OS: Osteocyte; PC: Pancreatic Cancer; PD: Pharmacodynamics; PDAK: Pancreatic Ductal Adenocarcinoma; PDB: Protein Data Bank; PDGFR: Platelet-Derived Growth Factor Receptor; pERK: Phospho-Extracellular signal-Related Kinase; PTHrp: Parathyroid Hormone-Related Protein; PK: Pharmacokinetics; RA: Rheumatoid Arthritis; RANKL: Receptor Activator of Nuclear factor Kappa B Ligand; RCC: Renal Cell Carcinoma; RTKs: Receptor Tyrosine Kinases; SAR: Structure-Activity Relationship; SCLC: Small Cell Lung Cancer; SCW: Streptococcal Cell Wall; TAM: Tumor-Associated Macrophages; TBW: Total Body Washout; TEC: Tubular Epithelial Cell; TNF: Tumor Necrosis Factor; TRAP: Tartrate-Resistant Acid Phosphatase; TRKA: Tropomyosin Receptor Kinase A; TYK2: Tyrosine Kinase 2; UUO: Unilateral Ureter Obstruction; VEGF: Vascular Endothelial Growth Factor; VEGFR: Vascular Endothelial Growth Factor Receptor.

The authors have declared no conflicts of interest

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85) Scotta, D. A.; Dakina, L. A.; Daly, K.; Del Valle, D. J.; Diebold, R. B.; Drew, L.; Ezhuthachan, J.; Gero, T. W.; Ogoe, C. A.; Omer, C. A.; Redmond, S. P.; Repik, G.; Thakur, K.; Ye, Q.; Zheng, X. Mitigation of cardiovascular toxicity in a series of CSF-1R inhibitors, and the identification of AZD7507. Bioorg. Med. Chem. Lett. 2013, 23, 4591-4596. 86) Boulter, L.; Guest, R. V.; Kendall, T. J.; Wilson, D. H.; Wojtacha, D.; Robson, A. J.; Ridgway, R. A.; Samuel, K.; Van Rooijen, N.; Barry, S. T.; Wigmore, S. J.; Sansom, O. J.; Forbes, S. J. WNT signaling drives cholangiocarcinoma growth and can be pharmacologically inhibited. J. Clin. Invest. 2015, 125, 1269-1285. 87) Armstrong, R. C.; Belli, B.; Rowbottom, M. W.; Nepomuceno, R. R.; Dao, A. Q.; Rooks, A. M.; Brigham, D.; McMannus, C. W.; Hocker, M. D.; Holladay, M. W.; Liu, G. Abstract 903: AC708 is a potent and selective inhibitor of CSF1R and reduces tumor associated macrophage infiltration in a breast tumor model. Cancer Res. 2013, 73(8 Supplement), 903. 88) Lyons, Y. A.; Pradeep, S.; Hansen, J. M.; Wagner, M. J.; Dood, R. L.; Wu, S. Y. Previs RA, Hu W, Coleman RL, Sood AK. Less is more: macrophage depletion via CSF1/CSF1R pathway improves anti-VEGF therapy after adaptive resistance. In: The AACR Annual Meeting: Tumor biology; 2017 April 1-5; Washington, US. CTI Meeting Technology.

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