αvβ1-integrin (IC50 = 1.3 nM)

GLPG0187 exhibits selectivity for multiple RGD integrin receptors in solid-phase analysis, as demonstrated by its IC50 values of 1.3, 3.7, 2.0, 1.4, 1.2, 7.7 nM 1, αvβ3, αvβ5, αvβ6, αvβ8, and α5β1, in that order. A powerful model of osteoclastic bone resorption and angiogenesis is GLPG0187. A more epithelial, sessile phenotype is adopted by cells treated with GLPG0187 at doses that increase the E-calcin adhesion/morphogenic protein component. Mold acetaldehyde dehydrogenase shrinks in size in a dose-dependent way when exposed to GLPG0187 [1]. Cells under GLPG0187 treatment clumped and rounded. A notable dose-dependent decrease in tumor cell migration was shown by GLPG0187. All GLPG0187 concentrations considerably decreased cell swelling [2].

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GLPG0187 is the inhibitor of integrin α5β1, which is essential for migrasome biogenesis. GLPG0187 inhibited migrasome biogenesis in a concentration-dependent manner without cytotoxicity [3].

The growth of the metastatic tumor was greatly inhibited when GLPG0187 blocked αv-integrin. Both the burden of bone tumors and the quantity of bone metastases/mouse were markedly lowered. During treatment, there was a considerable inhibition of both the growth of existing bone metastases and the creation of new ones [1].

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In line with our in vitro observations, administration of GLPG0187 resulted in a significant delay in both invasion of the caudal vertebrae by osteoclasts and blood vessels (at V20 [capillaries] and V21 [osteoclasts], respectively, under control conditions and at V14 [capillaries] and V15 [osteoclasts], respectively, in the GLPG0187-treated animals) (Figure 2, E–G). Taken together, our data show that GLPG0187 is a potent inhibitor of angiogenesis both in vitro and in vivo.[1]

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PC3-GFP cell homing to bone in vivo in the presence of GLPG0187 [2]
The metatarsal-DSC model was then used to investigate any modulation of PC3-GFP cells homing to bone, in the presence of the αν integrin antagonist. GLPG0187 significantly (p < 0.05) reduced the number of tumour cells present in the implanted metatarsal from day 17 onward (Fig. 3d). Tumour cell numbers remained significantly lower (p < 0.05) in treatment groups compared to controls throughout the duration of the experiment. MF analysis confirmed the number of PC3-GFP cells within the metatarsal ex vivo. Whole body imaging of the mouse during the experimental period did not detect tumour cells in either treated or control groups, in the skeleton or organs. However, at post mortem following removal of the muscle from the bone, small tumours were visible in the tibia of control animals but this did not occur in GLPG0187-treated mice.

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Implanted bone responses to GLPG0187 [2]
Metatarsal trabecular volume and number were compared in GLPG0187-treated and vehicle control mice using microCT, with no significant difference between the two groups observed at 25 days (treatment vs. control; trabecular volume 9.1 ± 1.3% vs. 9.8 ± 1.9%; trabecular number 3.5 ± 1.4 mm−1 vs. 3.9 ± 1.6 mm−1). Cortical volume and total bone volume demonstrated no significant difference between the two groups (cortical volume 2.6 ± 0.4mm3 vs. 1.9 ± 0.5 mm3; total bone volume 27.0 ± 2.9% vs. 30.6 ± 3.1%). However, the bone cortex appeared smoother and thicker in treatment groups compared to the control. Quantification of osteoclast numbers assessed using TRAP staining, revealed a nonsignificant decrease in osteoclast numbers in treated mice compared to vehicle-control group (6.2 ± 1.2 mm−1 vs. 5.0 ± 0.7 mm−1; Fig. 4a). In addition, there was a significant (p < 0.05) increase in osteoblast numbers in GLPG0187-treated mice compared to the vehicle-control group (10.5 ± 1.1 mm−1 vs. 15.0 ± 1.1 mm−1, Fig. 4b). Bone viability assessed at the end of the study by calcein deposition, demonstrated viability in both treatment and control groups, with no visible difference between the two groups (data not shown).

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Vascular responses to GLPG0187 in implanted bone [2]
The vascular density supplying the implanted metatarsal increased over the course of the experiment. However, administration of GLPG0187 commencing at day 7 resulted in a small but significant reduction in vascular density (p < 0.05) from day 15 onward, when compared to the vehicle-control treated group; this remained stable for the experimental duration (Fig. 5a). MVD of the metatarsal bone cortex, quantified using CD31 stained sections also demonstrated a significant reduction (p < 0.05) in GLPG0187-treated animals compared to the control group at the end of the study (Figs. 5b).

Migration Assay [1]
Migration was performed in 8-µm Transwell migration chambers. A total of 6 x 104 prestarved (0.1% fetal calf serum) PC-3M-Pro4/luc cells were seeded in the upper chamber containing either vehicle or GLPG0187 compound and allowed to migrate toward serum-containing medium in the lower chamber. Cells were fixed after 6 hours with 4% paraformaldehyde and stained with 0.1% crystal violet (2 mg/ml). Three random fields were counted for each well, and mean numbers of migrated cells/field were calculated.

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Angiogenesis Assays [1]
Metatarsal angiogenesis assay. Seventeen-day-old fetuses were removed from pregnant Swiss albino mice, and metatarsals were dissected as described previously. The isolated metatarsals were cultured for 10 days in 24-well plates in the presence of vehicle (1:1 dimethyl sulfoxide/PBS) or different concentrations of GLPG0187. Subsequently, the metatarsals were fixed and stained for platelet/endothelial cell adhesion molecule 1 (CD31) as described previously. The experiment was performed three times with eight cultures per condition. Cell viability [2]
Cell viability was measured using propidium iodide (PI). Cells were seeded at a density of 300,000 in T25 flasks and treated with varying concentrations of GLPG0187 (0.5, 5 and 50 ng/ml) and vehicle control (2% DMSO in PBS). After 24 hr, cells were harvested and stained with PI. In brief, PI 50 µg/ml was added to the cells, incubated for 2 min, then analysed using FACSCalibur™ . The assay was repeated at 48 and 72 hr.

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Proliferation assay [2]
Tumour cell proliferation was determined using the MTS assay. PC3 cells were seeded at 10,000 cells/well in 96 well plates containing either GLPG0187 (0.5, 5, or 50 ng/ml), vehicle or media control, then cultured in 100 µl medium for 24 hr. Cell proliferation was analysed using 20 µl MTS dye incubated for 3 hr at 37°C in the dark. Absorbance from each well (6/treatment) was quantified using the FLUOstar Galaxy plate reader at 490 nm and the mean fluorescence calculated. The assay was repeated at 48, 72 and 96 hr, on three independent occasions.

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Transwell migration assay [2]
Cell migration was quantified in transwell chambers containing membranes with 8 µm pores. PC3 cells (prestarved) were seeded in the upper chamber at 1 × 105 in 200 µl DMEM medium in the presence and absence of GLPG0187 (0.5, 5, or 50 ng/ml), vehicle control, or media control. DMEM containing 5% serum (300 µl) was added to the lower chamber. Cells were allowed to migrate for 16 hr at 37°C, then inserts were removed and the cells fixed with 4% PBS/formalin, rinsed with PBS and incubated with 100% methanol followed by another rinse in PBS. Inserts were stained with Gills Haematoxylin then rinsed with tap water. Nonmigrated cells on the inner surface of the transwell were removed. Three random fields were counted for each well (6/treatment), and mean numbers of migrated cells/field were calculated. This was repeated on three separate occasions.

Neonatal Tail Angiogenesis Assay [1]
The neonatal mouse tail provides a suitable model to simultaneously assess bone angiogenesis and osteoclastogenesis in vivo. Two-day-old neonatal Swiss albino mice were subcutaneously treated with GLPG0187 (30 mg/kg per day) or vehicle (1:1 dimethyl sulfoxide in PBS) for four consecutive days, starting at the second day after birth (n = 4). After 4 days of treatment the animals were killed, and the tails were stained for lectin and for osteoclasts (TRAcP staining)

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GLPG0187 treatment in vivo [2]
Animals were administered GLPG0187 daily for 17 days (i.p 100 mg/kg; n = 20, dissolved in 2% DMSO in PBS) or vehicle control (2% DMSO in PBS n = 20), commencing on the same day as tumour-cell injection (day 7). At the end of treatment, animals were culled (day 25).

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Bone morphology and immunohistochemistry [2]
Half of the intact resected metatarsals from GLPG0187-treated and vehicle-control groups including age matched controls were fixed in 10% neutral-buffered formaldehyde for 48 hr, followed by decalcification in 0.5 M EDTA for 4 days at 37°C. Serial sections (5 µm) were stained with haematoxylin and eosin (H & E). In brief, sections were dewaxed, rehydrated, then incubated in Gills II haematoxylin and washed. Sections were then incubated in 1% aqueous eosin (with 1% calcium carbonate), washed in water, dehydrated and slide mounted. Osteoblast numbers were counted and scored per millimetre of trabecular bone surface. Osteoclast activity was assessed by Tartrate Resistant Acid Phosphatase (TRAP) staining using standard procedures. In brief, sections were dewaxed, rehydrated, placed in acetate buffer at 37°C, then incubated in Napthol/dimethylformamide buffer, followed by sodium nitrite at 37°C solution. Sections were rinsed, counter-stained with Gill's haematoxylin, dehydrated and mounted.

[1]. Targeting of α(v)-integrins in stem/progenitor cells and supportive microenvironment impairs bone metastasis in human prostate cancer. Neoplasia. 2011 Jun;13(6):516-25.

[2]. Prostate cancer cells home to bone using a novel in vivo model: modulation by the integrin antagonist GLPG0187. Int J Cancer. 2015 Apr 1;136(7):1731-40.

[3]. Chemical screening identifies ROCK1 as a regulator of migrasome formation. Cell Discov. 2020 Aug 4;6(1):51.

GLPG0187 has been used in clinical trials for the treatment of solid tumors. GLPG0187, a small molecule integrin receptor antagonist (IRA), possesses potential antitumor activity. After administration, GLPG0187 binds to and blocks the activity of five RGD integrin receptor subtypes, including αvβ1, αvβ3, αvβ5, αvβ6, and α5β1. This may lead to inhibition of endothelial cell-cell interactions and endothelial cell-matrix interactions, and prevent angiogenesis and metastasis in tumor cells expressing these integrin receptors. Integrin receptors are transmembrane glycoproteins expressed on the surface of tumor vascular endothelial cells and certain types of cancer cells, playing a crucial role in endothelial cell adhesion and migration. Acquiring an aggressive phenotype in cancer cells is essential for bone metastasis. Transformed epithelial cells can transition to a motile mesenchymal phenotype through epithelial-mesenchymal transition (EMT). Recent studies have shown a functional association between EMT and prostate cancer stem cells, which play a crucial role not only in maintaining prostate cancer survival but also in bone metastasis. We found that treatment with the non-peptide α(v)-integrin antagonist GLPG0187 dose-dependently increased the E-cadherin/vimentin ratio, resulting in a more typical epithelial cell phenotype and some cell adhesion. Furthermore, GLPG0187 dose-dependently reduced the number of aldehyde dehydrogenase-overexpressing subsets in prostate cancer cells, indicating that α(v)-integrin plays an important role in maintaining the prostate cancer stem cell/progenitor cell pool. Our data demonstrate that GLPG0187 is a potent inhibitor of osteoclast-mediated bone resorption and angiogenesis in vitro and in vivo. In a preclinical model of prostate cancer, real-time bioluminescence imaging showed that blocking α(v)-integrin with GLPG0187 significantly reduced metastatic tumor growth, depending on the prophylaxis and treatment regimens. In the prophylaxis regimen, bone tumor burden was significantly reduced. In addition, the number of bone metastases per mouse was also significantly reduced. In the treatment regimen, the progression of bone metastases and the formation of new bone metastases were significantly inhibited during treatment. In summary, we demonstrated that GLPG0187 targeting integrins can inhibit the formation and progression of bone metastases in prostate cancer through anti-tumor mechanisms (including inhibition of EMT and reduction of prostate cancer stem cell numbers), anti-bone resorption and anti-angiogenic mechanisms. [1]

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Micrometastases are a barrier to developing effective treatments for bone metastases in prostate cancer. The mechanisms are not fully elucidated, mainly due to the inability to adequately monitor initial metastatic events in vivo. This study aimed to establish a new model for tracking the process of prostate cancer cells homing to the bone and to further evaluate the efficacy of treatment regulation using the integrin antagonist GLPG0187. Single mouse metatarsals were transplanted into the skin fold cavity on the back of SCID mice. Fluorescently labeled human prostate cancer cells (PC3-GFP) or oral cancer cells (SCC4-GFP) were administered intracardiacly (ic) and GLPG0187 or a vector control (100 mg/kg/day) were administered intraperitoneally daily throughout the experimental period. Metatarsal status was recorded every 48 hours for 4 weeks. Tissues were collected and subjected to micro-CT, multiphoton analysis, histological and immunohistochemical analysis. Quantitative analysis of cell viability, proliferation and migration in vitro was also performed after GLPG0187 treatment. Metatarsal revascularization was rapidly achieved through anastomosis with host vessels (days 5–7). PC3-GFP cells adhered to the microvascular endothelium and/or metatarsal matrix 3 days after administration and remained adherent throughout the experimental period. GLPG0187 treatment significantly reduced the number of PC3 cells in the metatarsal in vivo (p < 0.05) and reduced cell migration (p < 0.05) and proliferation (p < 0.05) in vitro, but did not affect cell viability. This novel model was able to assess early events of tumor cell homing and localization to the bone microenvironment and determine the response to therapeutic intervention. [2]

C29H37N7O5S

595.7130

595.257

C, 58.47; H, 6.26; N, 16.46; O, 13.43; S, 5.38

1320346-97-1

53340771

White to off-white solid powder

1.3±0.1 g/cm3

872.8±75.0 °C at 760 mmHg

481.6±37.1 °C

0.0±0.3 mmHg at 25°C

1.621

4.94

4

12

10

42

983

1

CC1=C(N=C(N=C1N2CCC(CC2)C3=NC4=C(CCCN4)C=C3)C)NC[C@@H](C(=O)O)NS(=O)(=O)C5=CC=C(C=C5)OC

CXHCNOMGODVIKB-VWLOTQADSA-N

InChI=1S/C29H37N7O5S/c1-18-26(31-17-25(29(37)38)35-42(39,40)23-9-7-22(41-3)8-10-23)32-19(2)33-28(18)36-15-12-20(13-16-36)24-11-6-21-5-4-14-30-27(21)34-24/h6-11,20,25,35H,4-5,12-17H2,1-3H3,(H,30,34)(H,37,38)(H,31,32,33)/t25-/m0/s1

(S)-3-((2,5-dimethyl-6-(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)piperidin-1-yl)pyrimidin-4-yl)amino)-2-((4-methoxyphenyl)sulfonamido)propanoic acid.

GLPG0187; GLPG 0187; UNII-43A5P87Z4T; 43A5P87Z4T; L-Alanine, 3-((2,5-dimethyl-6-(4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)-1-piperidinyl)-4-pyrimidinyl)amino)-N-((4-methoxyphenyl)sulfonyl); (2S)-3-[[2,5-dimethyl-6-[4-(5,6,7,8-tetrahydro-1,8-naphthyridin-2-yl)piperidin-1-yl]pyrimidin-4-yl]amino]-2-[(4-methoxyphenyl)sulfonylamino]propanoic acid; (2S)-3-({2,5-DIMETHYL-6-[4-(5,6,7,8-TETRAHYDRO-1,8-NAPHTHYRIDIN-2-YL)PIPERIDIN-1-YL]PYRIMIDIN-4-YL}AMINO)-2-(4-METHOXYBENZENESULFONAMIDO)PROPANOIC ACID; GLPG-0187

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~12.5 mg/mL (~20.98 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 1.25 mg/mL (2.10 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.25 mg/mL (2.10 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: ≥ 0.89 mg/mL (1.49 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 4: ≥ 0.89 mg/mL (1.49 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 5: 10 mg/mL (16.79 mM) in 20% HP-β-CD in Saline (add these co-solvents sequentially from left to right, and one by one), Suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)