A c-fms tyrosine kinase inhibitor, Ki20227, suppresses osteoclast differentiation and osteolytic bone destruction in a bone metastasis model
Abstract
Background
In the complex pathophysiology of bone metastatic lesions, osteoclasts, which are specialized bone-resorbing cells, play an exceptionally critical and central role in initiating and driving the development of osteolysis, a process characterized by progressive bone destruction. Prior comprehensive investigations have unequivocally demonstrated the indispensable importance of macrophage colony-stimulating factor (M-CSF) in orchestrating the intricate processes of osteoclast differentiation, proliferation, and survival. Given this crucial dependency, the present study embarked upon a detailed investigation to ascertain whether the targeted inhibition of the M-CSF receptor, known as c-Fms, could effectively suppress osteoclast-dependent osteolysis specifically within the challenging context of bone metastatic lesions. Our strategic approach involved the innovative development of novel small molecule inhibitors meticulously designed to block the ligand-dependent phosphorylation of c-Fms, which is a key step in its activation pathway. Following their synthesis, we proceeded to rigorously examine the therapeutic effects of these lead compounds on osteolytic bone destruction in a well-established preclinical model of bone metastasis, aiming to translate our mechanistic understanding into potential clinical benefit.
Methods
Through a meticulous and iterative process of chemical synthesis and biological screening, we successfully identified and characterized a groundbreaking novel quinoline-urea derivative, specifically termed Ki20227. This compound, chemically defined as N-{4-[(6,7-dimethoxy-4-quinolyl)oxy]-2-methoxyphenyl}-N’-[1-(1,3-thiazole-2-yl)ethyl]urea, was definitively established as a potent and selective tyrosine kinase inhibitor targeting c-Fms. To ascertain its selectivity profile, the inhibitory concentrations (IC50s) of Ki20227 were precisely determined against a panel of relevant tyrosine kinases. Remarkably, Ki20227 demonstrated exceptional potency against c-Fms, with an IC50 of just 2 nmol/L. Its inhibitory activity against vascular endothelial growth factor receptor-2 (KDR), another critical kinase involved in angiogenesis, was found to be 12 nmol/L. While still showing some activity, its potency was significantly lower against stem cell factor receptor (c-Kit) and platelet-derived growth factor receptor beta, with IC50s of 451 nmol/L and 217 nmol/L, respectively. Crucially, Ki20227 exhibited no inhibitory activity against other important kinases that were tested, including fms-like tyrosine kinase-3, epidermal growth factor receptor, or c-Src (a proto-oncogene product), affirming its remarkable selectivity for c-Fms and a limited panel of related receptor tyrosine kinases.
Beyond its direct kinase inhibition, Ki20227 was also evaluated for its biological effects in cellular models. It was found to potently inhibit the M-CSF-dependent growth of M-NFS-60 cells, a myeloid cell line, consistent with its c-Fms inhibitory activity. Importantly, Ki20227 did not affect the M-CSF-independent growth of A375 human melanoma cells *in vitro*, highlighting its specificity for M-CSF signaling pathways. Furthermore, in a highly relevant osteoclast-like cell formation assay, utilizing primary mouse bone marrow cells, Ki20227 significantly and dose-dependently inhibited the development of tartrate-resistant acid phosphatase-positive (TRAP-positive) osteoclast-like cells, which are the hallmark of mature bone-resorbing osteoclasts. These *in vitro* findings provided compelling evidence of Ki20227′s targeted action against osteoclast differentiation.
In the subsequent *in vivo* studies, the therapeutic potential of Ki20227 was rigorously assessed in relevant animal models of bone disease. Oral administration of Ki20227 in nude rats, following intracardiac injection of A375 human melanoma cells to establish a bone metastasis model, effectively suppressed both the accumulation of osteoclast-like cells and the bone resorption induced by the metastatic tumor cells. This demonstrated Ki20227′s ability to modulate the tumor-bone microenvironment. Moreover, in a model of pathological bone loss, Ki20227 administration significantly decreased the number of tartrate-resistant acid phosphatase-positive osteoclast-like cells found on bone surfaces in ovariectomized (OVX) rats, a well-established model of osteoporosis and increased osteoclast activity.
Conclusion
The cumulative findings of this comprehensive study strongly suggest that Ki20227 effectively inhibits osteolytic bone destruction primarily through the suppression of M-CSF-induced osteoclast accumulation and activity *in vivo*. This targeted mechanism of action positions Ki20227 as a highly promising therapeutic agent. Therefore, Ki20227 holds substantial potential as a valuable treatment option for osteolytic diseases specifically associated with bone metastasis, where excessive osteoclast activity drives destructive bone lesions, as well as for other bone disorders characterized by pathological bone resorption.
Introduction
The phenomenon of bone metastasis, wherein tumor cells disseminate from their primary site and establish secondary neoplastic growths within skeletal tissue, represents a significant and frequently encountered clinical complication in the progression of various aggressive cancers, most notably breast, lung, and prostate carcinomas. Within the complex milieu of the bone microenvironment, metastatic tumor cells actively engage in a multifaceted interplay with resident bone cells, leading to a profound dysregulation of normal bone remodeling processes. A key aspect of this interaction is the enhancement of osteolysis, a pathological process characterized by excessive bone destruction. This osteolysis is primarily driven by the tumor cells’ ability to induce and significantly activate osteoclastic bone resorption. This activation occurs through several critical signaling pathways, predominantly mediated by parathyroid hormone-related peptide (PTHrP) and through the crucial receptor activator of nuclear factor κB ligand (RANKL).
Furthermore, scientific investigations have revealed an intricate feedback loop involving transforming growth factor-beta (TGF-β). This potent cytokine, which is abundantly stored within the bone matrix, is released in active form during osteoclastic bone resorption. Once liberated, TGF-β, in turn, stimulates an increased production of PTHrP by the tumor cells residing in the bone. This augmented production of PTHrP further accelerates the rate of bone resorption, thereby creating more available space within the bone matrix that is conducive to increased tumor cell proliferation and expansion. Given this destructive cycle, the strategic suppression of osteoclastic bone resorption emerges as a highly promising therapeutic avenue for effectively combating bone metastasis and its associated complications.
In line with this therapeutic strategy, numerous studies utilizing various rodent bone metastasis models have consistently demonstrated that several pharmacological agents can successfully suppress osteolytic bone metastasis. These agents include bisphosphonates, osteoprotegerin, anti-PTHrP-neutralizing antibodies, tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), and angiogenesis inhibitors. Bisphosphonates, a well-established class of drugs, primarily exert their inhibitory action on mature osteoclasts. They effectively suppress the enhanced bone resorption driven by metastatic tumor cells by inducing apoptosis (programmed cell death) in osteoclasts. Osteoprotegerin, a member of the tumor necrosis factor receptor family, acts as a molecular decoy. It effectively antagonizes the bone-resorbing capabilities of RANKL by competitively suppressing RANKL’s binding to its cognate receptor, RANK, which is expressed on osteoclast precursors. The clinical administration of either osteoprotegerin or bisphosphonates has been shown to be effective in suppressing osteolytic bone metastasis, underscoring the therapeutic relevance of targeting osteoclast activity.
Osteoclasts originate from monocytic progenitor cells, a lineage within the hematopoietic system. Experimentally, these cells can be induced to differentiate from spleen and bone marrow cells in *in vitro* culture systems when exposed to the simultaneous presence of both macrophage colony-stimulating factor (M-CSF) and RANKL. The indispensable role of M-CSF in osteoclastogenesis has been profoundly highlighted by studies on M-CSF-deficient osteopetrotic (op/op) mice. These genetically modified mice exhibit severe osteopetrosis, a condition characterized by abnormally dense bones due to a profound depletion of functional osteoclasts. Crucially, this pathological condition in op/op mice can be significantly ameliorated by the exogenous administration of M-CSF, directly demonstrating its critical role in restoring osteoclast populations and normal bone resorption. A more recent report focusing on M-CSF receptor (c-Fms)-null mice further corroborated these findings, revealing a severe deficiency of osteoclasts and marked abnormalities in skeletal development, remarkably similar to those observed in op/op mice. These compelling lines of evidence strongly suggest that M-CSF is not merely involved but is absolutely essential for the physiological development and maintenance of osteoclasts *in vivo*. Consequently, this critical dependency implies that the targeted suppression of the M-CSF/c-Fms signaling pathway holds substantial promise as an effective therapeutic strategy against bone metastasis and other osteolytic conditions. In the present study, building upon this strong rationale, we embarked upon a detailed investigation into the inhibitory effects of Ki20227, a newly identified c-Fms inhibitor, on both the development of tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells and the overall osteolytic bone destruction induced by the aggressive A375 human melanoma cell line in preclinical models.
Materials and Methods
Ki20227
Ki20227, formally known as N-{4-[(6,7-dimethoxy-4-quinolyl)oxy]-2-methoxyphenyl}-N’-[1-(1,3-thiazole-2-yl)ethyl]urea, was synthesized as a racemic mixture within the Kirin Pharmaceutical Research Laboratories located in Gunma, Japan. In addition to the racemic form, its individual enantiomers were also synthesized and designated as (R)-Ki20227 and (S)-Ki20227. For all *in vitro* experimental applications, the compounds were initially dissolved in dimethyl sulfoxide (DMSO) to create stock solutions. These stock solutions were then immediately diluted in the appropriate growth medium just prior to use. As a standard procedural rule, the final concentration of DMSO in all *in vitro* assays was meticulously maintained at 0.5% to minimize any potential solvent effects on cellular processes. For *in vivo* studies, Ki20227 was prepared as a suspension in a vehicle consisting of 0.5% methyl cellulose in distilled water, ensuring optimal delivery and bioavailability through oral administration.
Cell Lines and Cultures
A diverse panel of cell lines was utilized to comprehensively evaluate the effects of Ki20227. The RAW264.7 cell line, a well-characterized mouse macrophage cell line, and the THP-1 cell line, a human monocytic cell line, were procured from Dainippon Pharmaceutical Co., Ltd. (Osaka, Japan). The M-NFS-60 mouse myelogenous leukemia cell line was obtained from the American Type Culture Collection (Manassas, VA), and human umbilical vein endothelial cells (HUVEC) were sourced from Cambrex (Walkersville, MD). RAW264.7 and A375 cells were routinely maintained in Dulbecco’s Modified Eagle Medium (DMEM) obtained from Invitrogen Corp. (Carlsbad, CA), supplemented with 10% Fetal Bovine Serum (FCS). These cells were cultured at 37 degrees Celsius in a humidified atmosphere containing 5% CO2. THP-1 cells were maintained in RPMI 1640 medium (Invitrogen) also supplemented with 10% FCS. M-NFS-60 cells, a cell line known to be dependent on M-CSF for growth, were maintained in RPMI 1640 medium supplemented with 10% FCS and, crucially, in the continuous presence of 50 ng/mL of recombinant mouse M-CSF, procured from R&D Systems, Inc. (Minneapolis, MN). HUVEC cells, utilized for angiogenesis-related assays, were cultured in specialized EGM-2 medium supplied by Cambrex. All cell lines were routinely monitored for mycoplasma contamination and authenticated prior to use.
Animals
All *in vivo* experiments were conducted in strict adherence to the ethical guidelines established by the Kirin Animal Care and Use Committee, ensuring the humane treatment and welfare of all research animals. All animals were housed within a meticulously controlled barrier facility, maintaining a consistent 12-hour light/dark cycle. They were provided with sterilized food and water *ad libitum*, ensuring unrestricted access to nourishment and hydration. For the osteoclast-like cell formation assay, ddY mice were obtained from Japan SLC, Inc. (Hamamatsu, Japan). Athymic rats (F344/NJcl-rnu), essential for the establishment of the bone metastasis model due to their immunodeficient status, were procured from CLEA Japan, Inc. (Tokyo, Japan). For the ovariectomized (OVX) rat model, which serves as a common model for osteoporosis and estrogen deficiency-induced bone loss, Sprague-Dawley rats were utilized, also obtained from Japan SLC.
Inhibitory Effects of Ki20227 against Protein Kinases
To precisely quantify the inhibitory effects of Ki20227, as well as its individual enantiomers (R)-Ki20227 and (S)-Ki20227, against a panel of various protein kinases, the IC50 values were determined using the specialized IC50 profiler Express service provided by Upstate Ltd. (Dundee, United Kingdom). A comprehensive range of cell-free kinase inhibition assays was performed, targeting several key human-derived kinases. These included c-Fms, Bruton’s tyrosine kinase, vascular endothelial growth factor receptor-2 (KDR), stem cell factor receptor (c-Kit), platelet-derived growth factor receptor beta, fms-like tyrosine kinase-3, c-Src, Fyn, epidermal growth factor receptor, basic fibroblast growth factor receptor 2, hepatocyte growth factor/scatter factor receptor (c-Met), protein kinase A, and protein kinase C alpha. This broad panel allowed for a detailed assessment of Ki20227′s specificity and selectivity among different kinase families.
Western Blotting
To investigate the effect of Ki20227 on the phosphorylation of c-Fms, RAW264.7 cells, a macrophage cell line known to express c-Fms, were initially serum-starved for 12 hours in DMEM containing 0.1% FCS to reduce basal kinase activity. Following serum starvation, serial dilutions of Ki20227 were introduced into the cell cultures, and the cells were incubated for 1 hour to allow for drug uptake and interaction with its target. Subsequently, RAW264.7 cells were acutely stimulated with 50 ng/mL of recombinant mouse M-CSF for a brief period of 4 minutes to induce c-Fms phosphorylation. Whole cell lysates were then prepared using an ice-cold lysis buffer, precisely formulated to preserve protein integrity and phosphorylation states. This buffer contained 50 mmol/L Tris/HCl (pH 7.4), 150 mmol/L NaCl, 1.0 mmol/L NaF, 0.1% sodium deoxycholate, 4 mmol/L EDTA, 1.0 mmol/L Na3VO4, 1 mmol/L phenylmethylsulfonyl fluoride, 1 µg/mL aprotinin, and 1% NP40. After lysis, the c-Fms protein within the RAW264.7 cell lysates was selectively immunoprecipitated using a rabbit polyclonal antibody directed against c-Fms (C-20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The immunoprecipitated proteins were then resolved by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and subsequently transferred onto a polyvinylidene fluoride (PVDF) microporous membrane. The membrane was initially probed with a highly specific phosphotyrosine antibody, PY20 (Transduction Laboratories, Lexington, KY), to detect phosphorylated c-Fms, and the phosphorylation signal was visualized using a peroxidase-conjugated anti-immunoglobulin G secondary antibody (Amersham Biosciences, Inc., Piscataway, NJ). Following the detection of phosphotyrosine, the PY20 antibody was carefully stripped from the membrane. The same membrane was then re-probed with the anti-c-Fms antibody (C-20) following the identical protocol, allowing for the quantification of total c-Fms protein as a loading control. This dual-probing approach allowed for a direct assessment of Ki20227′s ability to inhibit M-CSF-induced c-Fms phosphorylation without affecting total protein levels.
In Vitro Growth Inhibition Assay
To assess the *in vitro* growth inhibitory effects of Ki20227 on different cell types, M-NFS-60, HUVEC, and A375 cells were initially seeded onto 96-well culture plates and allowed to adhere and proliferate for 24 hours. Following this initial incubation period, the culture mediums were carefully replaced, and the cells were subsequently incubated for an additional 72 hours in the presence or absence of varying concentrations of the test compounds, ranging from 0.1 to 3,000 nmol/L. The specific culture conditions for each cell line during the assay were as follows: M-NFS-60 cells were seeded at a density of 5.0 × 10^3 cells per well in DMEM supplemented with 10% FCS and 50 ng/mL recombinant mouse M-CSF. After 24 hours, the medium was changed to DMEM supplemented with 3% FCS and 50 ng/mL recombinant mouse M-CSF to maintain M-CSF dependency. HUVEC cells were plated at 2.0 × 10^3 cells per well in EGM-2. Their culture medium was then changed to EBM-2 (Cambrex) supplemented with 3% FCS and 20 ng/mL recombinant human vascular endothelial growth factor (VEGF; Peprotech EC, Ltd., London, United Kingdom) to stimulate their growth. A375 cells were seeded at a density of 2.0 × 10^3 cells per well in DMEM supplemented with only 10% FCS, and their medium was subsequently changed to DMEM supplemented with 3% FCS for the growth inhibition assay, as their growth is M-CSF-independent. After the 72-hour incubation period with the test compounds, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent (Promega Corp., Madison, WI) was added to each well. The plates were then incubated at 37 degrees Celsius for 2 hours, allowing metabolically active cells to convert the MTS tetrazolium compound into a colored formazan product. The extent of growth inhibition was then quantitatively determined by measuring the absorbance at 490 nm using a microplate reader, with lower absorbance indicating greater growth inhibition.
Osteoclast-Like Cell Formation Assay
The osteoclast-like cell formation assay was conducted following a methodology previously reported by Kobayashi et al., with some critical modifications to optimize for the present study. Mouse femoral bone marrow cells, obtained from 4-week-old male ddY mice, were carefully isolated and seeded into 48-well culture plates at a density of 1.5 × 10^5 cells per well. These cells were initially cultured for 72 hours in αMEM medium supplemented with 10% FCS and 100 ng/mL recombinant mouse M-CSF, in the presence or absence of varying concentrations of the test compounds (Ki20227). Following this initial differentiation phase, the culture medium was replaced with fresh αMEM supplemented with 10% FCS, 20 ng/mL recombinant mouse M-CSF, and, crucially, 100 ng/mL recombinant mouse soluble RANKL (sRANKL; Peprotech). The cells were then cultured for an additional 72 hours, again with or without the continuous presence of the test compounds, to promote the full maturation and fusion of osteoclast precursors. Upon termination of the culture, the cells were fixed, and tartrate-resistant acid phosphatase (TRAP) staining was performed using a commercially available staining kit (acid phosphatase, leukocyte; Sigma Chemical Co., St. Louis, MO). TRAP is a specific marker for mature osteoclasts. Under microscopic observation, TRAP-positive cells containing two or more nuclei were definitively identified and counted as osteoclast-like cells, indicating successful differentiation and fusion. The inhibitory effect of Ki20227 on osteoclast development was quantitatively assessed by evaluating the reduction in the number of these TRAP-positive, multinucleated cells.
Intracardiac Injections of A375 Cells in Nude Rats
To establish a relevant *in vivo* model of bone metastasis, A375 human melanoma cells were utilized. A total of 5.0 × 10^5 A375 cells were suspended in 0.1 mL of phosphate-buffered saline (PBS) and meticulously inoculated into the left cardiac ventricle of 4-week-old, male F344/NJcl-rnu athymic rats. This intracardiac injection technique facilitates systemic dissemination of tumor cells, leading to robust bone metastasis formation. The injections were performed under anesthesia (induced with 75 mg/kg ketamine and 0.5 mg/kg medetomidine) using a fine 26-gauge needle. Each experimental group consisted of 8 animals. As a crucial control, sham-operated rats (n = 3) received an equivalent volume of PBS only injected into the cardiac ventricle. Beginning on the day immediately following tumor cell inoculation, Ki20227 was administered orally once per day for a duration of 20 days at three different dose levels: 10, 20, and 50 mg/kg/d. Control groups received an equivalent volume of the vehicle (0.5% CMC-Na) orally once per day.
X-ray Analysis of Bone Metastasis
Twenty-one days after the intracardiac injection of A375 cells, the extent of bone metastasis formation and associated osteolytic lesions was thoroughly examined using soft X-ray imaging. The rats were deeply anesthetized and positioned prone against an imaging plate (Fuji Photo Film Co., Ltd., Tokyo, Japan). Soft X-rays were then exposed at 40 kV for 10 seconds using an AFX-1000 system (Fuji). The resulting radiographs were subsequently scanned with a BAS-2500 IP Reader (Fuji), digitizing the images for quantitative analysis. The total osteolytic lesion areas and the total number of distinct lesions in the femurs and tibias of each animal were meticulously measured using specialized ImageGauge digital image analysis software (Fuji). This quantitative assessment allowed for a precise evaluation of the severity of bone destruction induced by the metastatic tumor cells and the inhibitory effects of Ki20227.
Ovariectomized Rat Model
To investigate the effects of Ki20227 on osteoclast activity in a model of pathological bone loss distinct from cancer, an ovariectomized (OVX) rat model was employed. Ki20227 was administered orally once per day at a dose of 20 mg/kg/d to 6-week-old female Sprague-Dawley rats. Each experimental group consisted of 6 animals. The oral administration of Ki20227 commenced 7 days prior to the surgical procedure. Seven days after the first administration of Ki20227 (on day 7 of the treatment), the rats underwent either ovariectomy (OVX group) or a sham operation (sham-operated control group) under sterile conditions. The vehicle-treated OVX-operated and sham-operated control groups received an equivalent volume of 0.5% CMC-Na orally. Treatment with Ki20227 or vehicle continued for a total of 28 days. At 21 days post-surgery (corresponding to day 28 of the overall study), the rats were humanely sacrificed, and their tibias were carefully harvested for further analysis. The successful induction of ovariectomy was biochemically confirmed by measuring the weight of the uterus during dissection on day 28.
Histologic and Histochemical Examination
For detailed microscopic analysis, the hind limbs of both the bone metastasis and ovariectomized rats were meticulously harvested and fixed in 10% neutral phosphate-buffered formalin. Following an appropriate fixation period, the specimens underwent a decalcification process using a 10% EDTA solution for 2 weeks, which is essential to soften the bone tissue for sectioning. After decalcification, the specimens were embedded in paraffin blocks. Thin sections, 5 micrometers in thickness, were then cut from these paraffin-embedded specimens. These sections were subsequently stained using conventional methods, including Hematoxylin and Eosin (H&E) for general histological evaluation of tissue morphology and tumor burden. In addition, histochemical examination for tartrate-resistant acid phosphatase (TRAP) was performed following standard protocols using Naphthol AS-MX phosphoric acid as the substrate. TRAP staining selectively identifies active osteoclasts, allowing for their enumeration and assessment of their distribution on bone surfaces.
Histomorphometric Analysis
In the context of the bone metastasis model, quantitative histomorphometric analysis was meticulously performed to assess tumor burden within both tibias. This was achieved by examining longitudinal sections stained with H&E under 40x magnification. The tissue area occupied by metastatic tumor cells within these sections was precisely measured using an integrated digital camera system coupled with microscopy (DP70; Olympus Corp., Tokyo, Japan) and specialized image analyzing software (WinROOF, version 5.5; Mitani Corp., Fukui, Japan). For the ovariectomized rat model, the number of TRAP-positive, multinucleated cells (which are definitive osteoclasts) located at the tumor-bone interface in the metastatic bone of the proximal tibial metaphyses and at the bone surface in the primary spongiosa of both tibias were meticulously counted. This enumeration was performed across five distinct fields for each section under a higher magnification of 400x. This detailed histomorphometric approach provided quantitative data on both tumor progression and osteoclast activity in response to the experimental interventions.
Bone Resorption Marker
To quantitatively assess the extent of osteoclastic bone resorption in the bone metastasis rat model, a specific bone resorption marker, tartrate-resistant acid phosphatase 5b (TRAP-5b), was measured in the serum of the experimental animals. This measurement was performed at the conclusion of the experimental period. Serum TRAP-5b levels, which serve as a reliable indicator of osteoclast activity and bone degradation, were determined using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (RatTRAP Assay; Suomen Bioanalytiikka Oy SBA Science, Turku, Finland). The assay was conducted strictly in accordance with the manufacturer’s instructions to ensure accuracy and consistency. Each rat serum sample was tested in duplicate to enhance the reliability of the measurements, providing a robust indication of systemic bone resorption.
Analysis of c-Fms Expression
To investigate the expression patterns of c-Fms, particularly within the context of metastasized tumor cells and their surrounding bone microenvironment, a detailed molecular analysis was performed. To prepare RNA specifically from the metastasized tumor, both the left and right tibiae of the rats were carefully isolated. The metaphysial portions of these bones, which encompass both the tumor tissue and the associated bone marrow, were precisely excised and immediately frozen to preserve RNA integrity. The frozen bone tissue, along with its embedded tumor and bone marrow, was then mechanically crushed and thoroughly homogenized. Total RNA was subsequently purified from these homogenized samples using the RNeasy Mini kit (Qiagen, Valencia, CA). For comparative purposes, corresponding bone portions from normal, non-tumor-bearing rats were similarly isolated, and their total RNA was prepared using the identical protocol. Furthermore, to provide cellular controls, cultured A375 human melanoma cells and THP-1 human monocytic cells were lysed, and their respective total RNA was extracted. All purified total RNA samples underwent an essential DNase treatment (Qiagen) to effectively eliminate any contaminating genomic DNA, ensuring that subsequent amplification would only target messenger RNA. Following DNase treatment, first-strand cDNA was synthesized from the purified RNA using a random hexamer primer and the SuperScript III First Strand Synthesis System (Invitrogen). The resulting first-strand cDNAs were then subjected to real-time quantitative PCR (qPCR) analysis on an ABI 7900 system (Applied Biosystems, Inc., Foster City, CA), utilizing the QuantiTect SYBR Green PCR kit (Qiagen) for fluorescence detection. The primer pairs employed for specific gene amplification were commercially available Perfect Real-time Primers (TAKARA Bio, Inc., Shiga, Japan). Their specific ID numbers were: HA040814 for human c-Fms, HA036137 for human MHC, class I, A (HLA-A), and HA031582 for human β-actin (serving as a reference gene). This comprehensive molecular analysis aimed to determine the expression levels of c-Fms in both the metastatic tumor cells *in vivo* and in control cell lines, providing crucial insights into the target engagement of Ki20227.
Statistical Analysis
All collected experimental data underwent rigorous statistical analysis to ensure the reliability and validity of the findings. The Dunnett’s test, a commonly used multiple comparison procedure, was employed for statistical comparisons, utilizing StatLight statistical software (Yukms Co., Ltd., Tokyo, Japan). This test is particularly suitable for comparing multiple treatment groups to a single control group. All quantitative data throughout the study are consistently presented as the mean value plus or minus the standard error (mean ± SE), providing a clear indication of both the central tendency and the variability within each experimental group.
Results
Inhibitory Activity of Ki20227 against Protein Kinases
The novel compound Ki20227 was specifically designed and synthesized with the intention of developing a potent inhibitor targeting c-Fms tyrosine kinase. Its chemical structure is depicted in Figure 1. To thoroughly characterize its inhibitory profile and selectivity, the inhibitory concentrations (IC50s) of Ki20227 were precisely determined against a panel of various protein kinases. The results revealed a remarkable potency against its primary target, c-Fms, with an IC50 of just 2 nmol/L. Ki20227 also exhibited strong inhibitory activity against KDR (vascular endothelial growth factor receptor-2) with an IC50 of 12 nmol/L. Its inhibitory effect against c-Kit and platelet-derived growth factor receptor beta (PDGFRβ) was considerably lower, with IC50 values of 451 nmol/L and 217 nmol/L, respectively. These findings indicate a degree of selectivity, demonstrating higher potency against c-Fms and KDR compared to c-Kit and PDGFRβ.
In stark contrast to its potent activity against c-Fms and KDR, Ki20227 displayed minimal to no inhibitory activity against a broader panel of other kinases that were tested. Specifically, the IC50s for fms-like tyrosine kinase-3 (Flt3), c-Src, Fyn, epidermal growth factor receptor (EGFR), fibroblast growth factor receptor 2 (FGFR2), Met, Bruton’s tyrosine kinase (BTK), protein kinase A (PKA), and protein kinase C alpha (PKCα) were all determined to be greater than 1,000 nmol/L (Table 1). This comprehensive kinase profiling unequivocally demonstrates a high degree of selectivity for Ki20227 towards c-Fms and KDR within the tested panel. Furthermore, the IC50 profiles of both the (R)-Ki20227 and (S)-Ki20227 enantiomers against these protein kinases exhibited similar trends in inhibitory activity to the racemic Ki20227, suggesting that the stereochemistry at the chiral center does not significantly alter the basic kinase inhibition profile.
Effect of Ki20227 in Cell-Based Assays
To further evaluate the biological efficacy and inhibitory activity of Ki20227, particularly in response to M-CSF, a series of cell-based assays were performed, including Western blotting analysis and cell growth determinations. Western blotting analysis provided direct evidence that Ki20227 could effectively inhibit M-CSF-dependent c-Fms phosphorylation in a clear dose-dependent manner. This was observed in RAW264.7 cells cultured in a medium supplemented with a low concentration of FCS (0.1%), as illustrated in Figure 2A. This result directly confirmed that Ki20227 functions by inhibiting the M-CSF-induced activation of its receptor.
The inhibitory activity of Ki20227 against M-CSF-dependent cell growth and VEGF-dependent cell growth was then examined using M-NFS-60 cells and HUVEC cells, respectively, as shown in Figure 2B. It is important to note that the addition of M-CSF and VEGF to a medium supplemented with 3% FCS is an essential requirement for the sustained growth of M-NFS-60 cells and HUVEC cells, respectively, underscoring their dependence on these growth factors. Treatment with 100 nmol/L of Ki20227 resulted in almost complete suppression of the growth of M-NFS-60 cells, which are M-CSF-dependent. In contrast, the suppression of HUVEC cell growth, which is VEGF-dependent, required a significantly higher concentration of Ki20227, specifically 1,000 nmol/L. The calculated IC50 values for Ki20227 in these cell-based assays were approximately 14 nmol/L for M-NFS-60 cells and 500 nmol/L for HUVEC cells.
It is noteworthy that these inhibitory effects observed in cell-based assays were significantly different from the IC50s obtained in the cell-free kinase inhibition assays (where c-Fms inhibition was 2 nmol/L and KDR inhibition was 12 nmol/L). This discrepancy can be largely attributed to the fact that Ki20227 is a compound with high protein-binding affinity, as determined by Biacore analysis. Human and rat albumin-binding ratios for Ki20227 were found to be no less than 95%, indicating substantial non-specific serum protein binding. This extensive protein binding likely reduces the concentration of free, active drug available to inhibit intracellular targets in cell-based assays, thereby accounting for the observed differences in activity between cell-free and cell-based systems. An alternative or additional explanation for this phenomenon might be a direct growth stimulation provided by the FCS present in these cell cultures. In this context, M-CSF and VEGF may act more as modulators of cell proliferation rather than absolute drivers in these assays. Consequently, the proliferation of these cells might represent an indirect index of ligand action, necessitating a higher concentration of Ki20227 to effectively suppress M-NFS-60 and HUVEC cell growth in these culture conditions.
Furthermore, the inhibitory activities of both (R)-Ki20227 and (S)-Ki20227 against the growth of M-NFS-60, HUVEC, and A375 cells were found to be quite similar to those observed for the racemic Ki20227, as also shown in Figure 2B. These collective data robustly demonstrate the inhibitory effects of these compounds against target kinases in both cell-based and cell-free assay formats, providing a comprehensive understanding of their pharmacological profile.
Suppression of the Development of TRAP-Positive Osteoclast-Like Cells
To directly assess whether the inhibition of c-Fms kinase activity by Ki20227 translates into a suppression of osteoclast formation, a well-established mouse bone marrow culture system was utilized, employing both M-CSF and soluble RANKL (sRANKL) to induce osteoclast development. Mouse bone marrow cells were cultured for a period of 6 days in the presence of M-CSF and sRANKL, along with various doses of the test compounds: Ki20227, (R)-Ki20227, and (S)-Ki20227. The results clearly demonstrated that all three compounds effectively suppressed the development of tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells in a distinct dose-dependent manner, as visually represented in Figure 3A and B. This osteoclast-like cell formation was almost completely suppressed when treated with a concentration of 100 nmol/L for each compound. The half-maximal inhibitory concentration (IC50), calculated based on the number of TRAP-positive osteoclast-like cells observed in cultures on day 6, was determined to be approximately 40 nmol/L for all three compounds. These findings provide strong *in vitro* evidence that Ki20227 and its enantiomers are potent inhibitors of osteoclast differentiation.
Inhibitory Effects of Ki20227 on Osteolytic Bone Metastasis in Nude Rats
Based on the highly consistent *in vitro* data indicating that the biological activities of Ki20227, (R)-Ki20227, and (S)-Ki20227 were nearly identical, the racemic Ki20227 was selected for all subsequent *in vivo* studies to simplify the experimental design. To establish a relevant preclinical model of osteolytic bone metastasis, A375 human melanoma cells were intracardially injected into nude rats. Twenty-one days following this injection, the extent of osteolytic lesions was meticulously analyzed using X-ray imaging. Radiographic analyses clearly demonstrated that the A375 tumor cells had aggressively metastasized to various bone sites, including femurs, tibias, jawbones, and pelvises, in all A375-injected animals that received vehicle control (Figure 4A, a). However, a striking and significant reduction in the osteolytic lesion areas was observed in animals that received daily oral administration of 50 mg/kg/d of Ki20227 for 20 days (Figure 4A, b).
Quantitative measurements of the total osteolytic lesion areas in the femurs and tibias confirmed that treatment with 50 mg/kg/d of Ki20227 significantly decreased these lesion areas. Notably, lower doses of 5 or 20 mg/kg/d of Ki20227 did not yield a statistically significant reduction in lesion areas (Figure 4B, a). Similarly, the total number of osteolytic lesions identified in the femurs and tibias was also significantly suppressed by treatment with 50 mg/kg/d Ki20227 but not by the lower doses (Figure 4B, b). These X-ray findings unequivocally demonstrate the *in vivo* efficacy of Ki20227 in suppressing tumor-induced osteolysis.
Further detailed histological examination of the tibias from these animals provided crucial insights into the cellular mechanisms underlying the observed effects. In vehicle-treated rats, the bone marrow cavity was almost entirely replaced by metastatic A375 cells (labeled as T), and a high number of TRAP-positive cells, indicative of active osteoclasts, were prominently observed at the interface between the metastatic tumor and the bone surface (Figure 5A, a). In stark contrast, in Ki20227-treated rats, a remarkable reduction in the number of TRAP-positive osteoclastic cells was noted, and the delicate structure of the cancellous bone in the proximal tibias and epiphysis was largely preserved (Figure 5A, b). Specifically, in the 50 mg/kg/d Ki20227-treated group, while an A375 tumor burden was still evident, the extent of colonization by metastatic A375 cells within the bone marrow cavity was significantly decreased, and, critically, the number of TRAP-positive cells was also significantly reduced (Figure 5B and C).
Complementing these histological findings, serum TRAP-5b levels, a systemic biomarker for bone resorption, were also assessed. In the vehicle-treated A375 tumor-bearing rats, serum TRAP-5b levels were markedly elevated compared to levels in the sham non-tumor-bearing control group, indicating significant osteoclast-mediated bone resorption. However, this increased TRAP-5b level was significantly decreased in the 50 mg/kg/d Ki20227-treated tumor-bearing group (Figure 5D), directly correlating with the reduced osteoclast numbers observed histologically. It is important to note that these suppressive effects (on tumor colonization, osteoclast number, and TRAP-5b level) were not observed in rats treated with either the 5 or 20 mg/kg/d doses of Ki20227. Collectively, these comprehensive data strongly suggest that the osteoclast activity, which drives osteolysis in this bone metastasis model, is effectively diminished by treatment with 50 mg/kg/d of Ki20227.
PCR Analysis of the Expression of c-Fms in Metastatic Tumor Cells
Previous *in vitro* studies showed that Ki20227 effectively suppressed the M-CSF-dependent growth of M-NFS-60 cells, while the proliferation of A375 human melanoma cells, which are considered M-CSF-independent *in vitro*, was not inhibited. However, it was hypothesized that the behavior of metastatic A375 cells within the complex bone microenvironment might differ significantly from their growth characteristics in isolated *in vitro* cultures. Specifically, if A375 cells acquire increased expression of c-Fms when metastasized to bone compared to their levels in standard culture, then Ki20227 might exert a more direct antiproliferative effect on these cells in the bone, thereby contributing to osteolysis suppression not only through osteoclast inhibition but also through a direct effect on the tumor cells themselves.
To address this critical question, we conducted real-time quantitative PCR analysis to determine the expression levels of human c-Fms in metastatic A375 cells residing within rat bone. This analysis aimed to ascertain whether c-Fms expression was maintained or even up-regulated in the *in vivo* metastatic setting. The results indicated that high expression of human c-Fms was detectable only in THP-1 cells cultured *in vitro*, which served as a positive control (Figure 6A). In stark contrast, both the cultured A375 cells and the metastatic A375 cells retrieved from rat bone exhibited barely detectable levels of c-Fms. Furthermore, no PCR product for human c-Fms was detected in normal rat bone samples, confirming species specificity. To ensure the successful isolation of RNA from metastatic A375 cells within the rat bone tissue, human HLA-A expression was also measured, serving as a reliable marker for the presence of human cells. Human HLA-A expression was indeed detectable in all RNA samples, except for those derived from normal rat bone (Figure 6B), confirming that we were successfully analyzing RNA from the metastatic A375 cells embedded within the rat bone. Notably, the expression of HLA-A was found to be higher in metastatic A375 cells within bone compared to cultured A375 cells, potentially indicating phenotypic changes or increased cellularity in the bone microenvironment. However, and crucially, c-Fms expression was not found to be up-regulated in metastatic A375 cells in bone when compared to A375 cells in culture. Therefore, based on these molecular findings, we conclusively infer that the observed inhibitory effects of Ki20227 upon osteolysis, which is induced by these metastatic tumor cells, are not mediated through a direct antiproliferative effect on the metastatic A375 cells themselves. Instead, its action is primarily attributed to its inhibitory effect on osteoclast activity.
Osteoclastic Cells in Bones from Ovariectomized Rats
Building upon the robust findings from the bone metastasis rat model, which strongly indicated that Ki20227 inhibits osteolysis primarily through the suppression of osteoclast development induced by tumor cells, we sought to further confirm the direct inhibitory effect of Ki20227 against osteoclast development in a non-tumor-bearing rodent model. For this purpose, we investigated the effects of Ki20227 in a rat ovariectomy (OVX) model, a well-established experimental system for studying estrogen deficiency-induced bone loss and increased osteoclastogenesis. Ki20227 or its vehicle was administered orally once per day for a total duration of 28 days, with the treatment commencing 7 days prior to the surgical procedure (ovariectomy or sham operation).
At the end of the study, the numbers of tartrate-resistant acid phosphatase (TRAP)-positive cells, which represent active osteoclasts, on the bone surface were meticulously determined in both the vehicle-treated group (Figure 7A, a) and the Ki20227-treated group (Figure 7A, b). The results clearly demonstrated that once-daily oral administration of Ki20227 at a dose of 20 mg/kg significantly decreased the number of TRAP-positive osteoclastic cells on the bone surface (OcN/mm bone surface) in the primary spongiosa compared with that observed in the vehicle-treated group (Figure 7B). These compelling data unequivocally indicate that Ki20227 effectively suppresses TRAP-positive osteoclast-like cell development in a rodent model of pathological bone loss that is not driven by the presence of tumor cells. This further corroborates Ki20227′s direct role in inhibiting osteoclast activity, independent of the complex tumor-bone interactions, and expands its potential therapeutic utility to a broader range of osteolytic bone diseases.
Discussion
The scientific landscape has witnessed the emergence of numerous small molecule inhibitors targeting various tyrosine kinases, including those associated with epidermal growth factor, vascular endothelial growth factor (VEGF), and platelet-derived growth factor receptors. Many of these inhibitors have successfully progressed into advanced clinical trials, with some even securing market approval for therapeutic use. In this comprehensive study, we introduce and characterize a novel c-Fms inhibitor, Ki20227, and provide compelling evidence that this compound effectively suppresses osteoclast development, both in isolated cellular systems *in vitro* and within living organisms *in vivo*. Furthermore, and critically, our findings demonstrate its capacity to attenuate osteolytic bone destruction specifically induced by metastatic tumor cells.
Macrophage colony-stimulating factor (M-CSF) is a pivotal cytokine that exerts profound regulatory control over the monocytic lineage, a cell population that includes the precursors of osteoclasts, throughout the body *in vivo*. Beyond its role in progenitor development, M-CSF has been shown to significantly enhance the survival of isolated osteoclasts *in vitro* and to actively induce the differentiation of bone marrow cells into mature osteoclasts when co-cultured with RANKL. The indispensable nature of M-CSF in osteoclastogenesis is strikingly underscored by *in vivo* studies involving osteopetrotic (op/op) mice. These mice develop a severe form of osteopetrosis, a condition characterized by excessively dense bones, directly attributable to a profound deficiency of functional osteoclasts resulting from the absence of functional M-CSF. Crucially, the administration of exogenous M-CSF has been consistently shown to significantly alleviate this pathological condition by promoting a compensatory increase in osteoclast numbers. A more recent report further reinforced this understanding, demonstrating that c-Fms-null mice, lacking a functional M-CSF receptor, exhibit an identical severe osteopetrotic phenotype. These collective data provide unequivocal evidence that M-CSF plays an essential and non-redundant role in osteoclast development and function, both *in vivo* and *in vitro*. In the present study, our experimental results align perfectly with this established knowledge. We demonstrate that Ki20227, along with its enantiomeric forms (R)-Ki20227 and (S)-Ki20227, effectively suppress the formation of TRAP-positive osteoclast-like cells in a dose-dependent manner within a mouse bone marrow culture system stimulated with M-CSF and soluble RANKL. The observed IC50 values for this inhibitory effect were consistently around 40 nmol/L. Notably, this concentration range is similar to the doses required for Ki20227 to exert its inhibitory effects against M-CSF-dependent growth of M-NFS-60 cells in a cell-based assay. This striking concordance suggests that the observed inhibitory effect on osteoclast formation within this bone marrow culture system, which relies on M-CSF and sRANKL, is highly specific and is most likely not attributable to generalized cytotoxicity.
Bone metastasis is a frequent and devastating complication observed in advanced breast, prostate, and lung cancers. In the intricate process of bone metastasis, metastatic tumor cells actively promote osteolysis, the destructive breakdown of bone tissue, through the secretion and modulation of various potent factors, including parathyroid hormone-related peptide (PTHrP) and transforming growth factor-beta (TGF-β). For instance, in the well-studied human breast cancer cell line MDA-MB-231, it has been reported that PTHrP production by these metastatic cells within bone is significantly augmented by TGF-β. This TGF-β is itself released from the bone matrix by the very activity of osteoclasts. The resultant increase in PTHrP secretion then triggers even greater osteoclast activation and accelerated bone destruction, creating a detrimental “vicious cycle” involving PTHrP and TGF-β that relentlessly drives bone destruction and concurrently promotes tumor growth within the bone microenvironment.
Previous research has also highlighted the significant role of M-CSF in this pathological process. Specifically, studies have shown that the administration of M-CSF antiserum can effectively suppress both osteoclast development and the osteoclastic bone resorption induced by PTHrP. Furthermore, it has been observed that M-CSF expression by MDA-MB-231 cells is considerably higher in bone metastatic sites compared to soft tissue metastatic sites. These collective data strongly indicate that the M-CSF/c-Fms signaling pathway plays a crucial and integral role in the establishment and perpetuation of this detrimental cycle that involves PTHrP-mediated bone destruction.
The A375 human melanoma cell line is another well-characterized model system, akin to MDA-MB-231 cells, for establishing tumors in rodent bone metastasis models. A375 cells are known to induce bone resorption through the secretion of various osteotropic cytokines, including interleukin-6, prostaglandin E2, and TGF-α. Additionally, they have been reported to produce PTHrP, indicating that A375 cells possess a diverse array of capabilities for enhancing osteoclast development and bone resorption. While the precise extent of M-CSF’s direct involvement in the bone metastasis induced by A375 cells remains to be fully elucidated, the fact that A375 cells produce PTHrP, in a manner similar to MDA-MB-231 cells, strongly suggests that PTHrP likely functions in conjunction with M-CSF in driving osteoclast accumulation induced by metastatic A375 cells. Therefore, it is logically anticipated that M-CSF antagonists would be effective in suppressing these pathological osteoclast accumulations.
In the present study, our central objective was to rigorously investigate whether the c-Fms inhibitor Ki20227 could effectively suppress both osteoclast accumulation and the associated osteolytic bone destruction induced by A375 cells in an *in vivo* model. Our comprehensive *in vitro* data, encompassing both cell-free kinase assays and various cell-based assays, consistently indicated that the biological activities of Ki20227, (R)-Ki20227, and (S)-Ki20227 were nearly identical across the tested parameters. This consistent activity profile allowed us to confidently focus our subsequent *in vivo* studies exclusively on the racemic Ki20227.
Our *in vivo* experiments confirmed the aggressive nature of A375 cell metastasis. In untreated control animals, severe bone metastasis by A375 cells was observed in the hind limbs within a mere 3 weeks following intracardiac injection. However, a remarkable therapeutic effect was observed with Ki20227. X-ray analysis clearly demonstrated that once-daily oral administration of 50 mg/kg of Ki20227 for 20 days significantly reduced both the total osteolytic lesion area and the number of individual osteolytic lesions in the femurs and tibias of the treated animals. Further detailed histological examination of the tibias revealed profound pathological changes in the vehicle-treated group: the presence of numerous osteoclasts and severe bone resorption were evident in close proximity to the metastatic tumor cells, and extensive tumor colonization was observed within the bone cavity. In contrast, our findings demonstrated that Ki20227 significantly suppressed the accumulation of osteoclasts induced by metastatic tumor cells in the bone. Furthermore, it was observed that Ki20227 also suppressed tumor colonization, albeit to a lesser extent. This suggests a nuanced mechanism where tumor growth within the bone cavity may, to some degree, be independent of osteoclast activity. On a systemic level, the serum concentration of TRAP-5b, a widely recognized bone resorption marker, was also notably decreased in the Ki20227-treated group, directly corroborating the reduced osteoclast activity. Based on these cumulative data, it is strongly inferred that the observed inhibitory effects of Ki20227 on osteolysis and, to a lesser extent, on tumor colonization, are primarily mediated by the suppression of the development and activity of TRAP-positive osteoclast-like cells. However, it was also considered a possibility that Ki20227 might directly inhibit the growth of metastatic A375 cells within the unique bone microenvironment.
To investigate this possibility, we considered that if A375 cells, upon metastasizing to bone, expressed c-Fms at higher levels than in standard culture, then the growth of these A375 cells within bone metastatic sites might be more effectively suppressed by Ki20227, similar to its effect on M-CSF-dependent M-NFS-60 cell growth. It is well-documented that metastatic tumor cells often exhibit phenotypic changes, including alterations in growth factor production, within the context of the bone microenvironment. Our quantitative PCR analysis did reveal differences in human HLA-A expression levels between metastatic A375 cells in bone and those in culture, hinting at such phenotypic adaptations. However, and critically, our analysis showed that c-Fms expression was not up-regulated in metastatic A375 cells in bone when compared to A375 cells in culture. Furthermore, our *in vitro* cell-based assays consistently demonstrated that Ki20227 did not directly suppress the growth of A375 cells. Therefore, based on this combined evidence, we definitively conclude that the primary inhibitory effects of Ki20227 against osteolytic bone destruction and the reduction in tumor colonization in bone are not mediated through a direct antiproliferative effect on metastatic A375 cells. On an alternative note, it is acknowledged that Ki20227 also exhibits inhibitory activity against KDR (VEGF receptor-2). Given that angiogenesis inhibitors have also been reported to suppress osteolytic bone metastasis, it is plausible that this KDR inhibition might contribute, at least partially, to the observed suppression of tumor colonization within the bone.
To further solidify the understanding of Ki20227′s inhibitory activity specifically against osteoclast development, independent of tumor presence, we investigated its effects in a rat ovariectomy (OVX) model. This model reliably induces increased osteoclastogenesis and bone loss in the absence of tumor cells. In this model, treatment with Ki20227 at a dose of 20 mg/kg/d for 28 days significantly decreased the number of TRAP-positive osteoclastic cells on the bone surface in the primary spongiosa. While it was noted that this 20 mg/kg/d dose of Ki20227 did not significantly suppress osteolysis induced by A375 in the rat bone metastasis model, this discrepancy might be due to the specific dose-response in different models, and it is plausible that higher doses of Ki20227 would further reduce osteoclastic cell numbers in the OVX model. Previous research by Kimble et al. reported that stromal cells from ovariectomized mice produced larger amounts of M-CSF than those from estrogen-replete mice, and Cenci et al. found that anti-M-CSF-neutralizing antibody completely suppressed OVX-induced bone loss. These findings provide strong support for our hypothesis that Ki20227, by targeting M-CSF signaling, possesses the intrinsic ability to suppress osteoclast development caused by ovariectomy *in vivo*. Thus, we firmly believe that the potent inhibitory effects of Ki20227 on osteoclast development constitute a substantial and primary component of its overall efficacy against the osteolytic bone resorption induced by metastatic A375 cells.
Beyond its crucial role in osteoclast development and bone metastasis, accumulating evidence indicates that M-CSF also plays a significant and multifaceted role in various aspects of tumor biology, including promoting tumor growth, tissue invasion, and overall malignancy. For instance, marked elevations in serum M-CSF levels have been consistently observed in patients diagnosed with endometrial or ovarian cancer. Additionally, it has been reported that M-CSF actively promotes the tissue invasion capabilities of lung cancer cells by enhancing their production of matrix metalloproteinase-2. Further demonstrating its systemic involvement in cancer progression, functional M-CSF deficiency has been shown to delay mammary tumor progression and metastasis in op/op mice, while, conversely, the overexpression of both M-CSF and its receptor c-Fms has been linked to the induction of hyperplasia and tumor formation within mammary glands. In the realm of therapeutic interventions, studies using M-CSF antisense oligonucleotides have successfully suppressed the growth of colon and mammary cancers in rodent xenograft models, and small interfering RNAs (siRNAs) targeting M-CSF have inhibited mammary cancer growth in similar models. These findings strongly support the overarching concept that blockade of M-CSF signaling pathways holds significant promise as an effective strategy against various tumor growths and malignancies, highlighting the broader therapeutic potential of inhibitors like Ki20227.
In conclusion, our comprehensive study has unequivocally demonstrated that Ki20227, a novel c-Fms inhibitor, effectively decreases the number of osteoclasts, both in controlled *in vitro* cellular systems and within complex *in vivo* animal models. Crucially, Ki20227 was shown to suppress the severe osteolysis induced by metastatic tumor cells, highlighting its potential in cancer-related bone disease. Given its ability to inhibit osteoclast activity, Ki20227 emerges as a promising potential anti-osteolytic agent for therapeutic application in conditions characterized by excessive bone destruction, such as metastatic disease, where tumor cells drive bone resorption, or in cases of bone loss associated with diminished gonadal function, such as osteoporosis.