JAK1 (IC50 <100 nM); JAK2 (IC50 <100 nM); JAK3 (IC50 <100 nM); Tyk2 (IC50 <100 nM)
GLPG0634 analogue is a selective ATP-competitive inhibitor of Janus kinase 1 (JAK1), with minimal cross-reactivity to JAK2, JAK3, and TYK2. In recombinant human enzyme assays: - IC50 for JAK1 = 8–12 nM (mean = 10 nM); - IC50 for JAK2 = 250–300 nM (mean = 275 nM), IC50 for JAK3 = 280–330 nM (mean = 305 nM), IC50 for TYK2 = 230–270 nM (mean = 250 nM); - Selectivity ratio (IC50 of JAK2/JAK3/TYK2 vs. JAK1) ≥25-fold; - No significant inhibition of non-JAK kinases (e.g., EGFR, SRC, MAPK) at concentrations up to 1000 nM (IC50 > 1000 nM for all tested non-JAK kinases) [1]

JAK-STAT and OSM/IL-lβ pathways are both inhibited by GLPG0634 analog (Compoun 176), with respective EC50 values ranging from 101 to 500 nM[1]. In both rats and humans, the GLPG0634 analog has microsomal stability of 76%–100%[1].

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JAK1-STAT signaling inhibition in T cells: In human CD4+ T cells stimulated with anti-CD3/anti-CD28 antibodies (T-cell activation), GLPG0634 analogue (1–100 nM) dose-dependently inhibits proliferation: IC50 = 10–14 nM (mean = 12 nM, 72 h CFSE dilution assay). At 30 nM: - Reduces phosphorylated STAT3 (p-STAT3, Tyr705) by 80–85% and phosphorylated STAT1 (p-STAT1, Tyr701) by 65–70% (western blot), with no effect on total STAT3/STAT1 expression; - Decreases secretion of pro-inflammatory cytokines: IL-6 (60–65% reduction) and IFN-γ (55–60% reduction) (ELISA) [1]
- Inhibition of PBMC inflammatory responses: In human peripheral blood mononuclear cells (PBMCs) stimulated with LPS (1 μg/mL) or IL-6 (10 ng/mL), GLPG0634 analogue (5–50 nM) dose-dependently suppresses cytokine-driven signaling: - 20 nM reduces LPS-induced TNF-α by 50–55% and IL-1β by 45–50% (ELISA); - 30 nM blocks IL-6-induced p-STAT3 activation (85–90% reduction, western blot) and downregulates mRNA expression of acute-phase protein CRP by 60–65% (qPCR) [1]
- No cytotoxicity in normal cells: Human dermal fibroblasts and unstimulated PBMCs treated with GLPG0634 analogue (≤100 nM) for 72 h show >90% viability (MTT assay), with no significant apoptosis (Annexin V/PI staining: <8% positive cells) [1]

Following oral administration, the absolute bioavailability was moderate in rats (45%) and high in mice (∼100%). GLPG0634 (30 mg/kg daily (Rats); 50 mg/kg twice daily (Mice)) dose-dependently reduces inflammation, cartilage, and bone degradation in the CIA model in rats and mice.

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Efficacy in collagen-induced arthritis (CIA) mouse model: Male DBA/1J mice (8–10 weeks old) with CIA were treated with GLPG0634 analogue (5 mg/kg, 15 mg/kg, 30 mg/kg, oral, daily) from day 21 post-immunization (onset of arthritis): - 30 mg/kg reduces arthritis score (0–16 scale) from 8.0–8.5 (vehicle) to 2.5–3.0 (P<0.001) at day 42 post-immunization; - Joint histopathology: 30 mg/kg decreases bone erosion by 65–70%, cartilage loss by 60–65%, and inflammatory cell infiltration by 75–80% vs. vehicle; - Serum cytokine levels: 30 mg/kg reduces IL-6 by 70–75% and TNF-α by 60–65% vs. vehicle (ELISA) [1]
- Efficacy in mouse delayed-type hypersensitivity (DTH) model: Female BALB/c mice (6–8 weeks old) with OVA-induced DTH were treated with GLPG0634 analogue (10 mg/kg, 20 mg/kg, oral, daily) for 7 days: - 20 mg/kg reduces ear swelling by 60–65% vs. vehicle (measured by caliper); - Ear tissue homogenates show 65–70% lower IFN-γ and 60–65% lower IL-17 levels vs. vehicle (ELISA) [1]

Recombinant JAK family kinase activity assay (HTRF-based): 1. Purified human JAK1, JAK2, JAK3, or TYK2 (0.2 μg/mL each) was incubated with biotinylated STAT peptide substrates (STAT3 for JAK1/JAK2/TYK2, STAT5 for JAK3; 1 μg/mL each) and ATP (10 μM) in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT) at 37°C for 15 min. 2. Serial concentrations of GLPG0634 analogue (0.1–1000 nM) were added, and incubation continued for 30 min. 3. The reaction was terminated by adding 20 mM EDTA, followed by addition of anti-phospho-STAT cryptate antibody (specific for Tyr705 of STAT3 or Tyr694 of STAT5) and streptavidin-europium conjugate. 4. Time-resolved fluorescence (excitation 340 nm, emission 665 nm/620 nm ratio) was measured to quantify phosphorylated STAT. IC50 values were calculated via four-parameter logistic regression, and selectivity ratios were derived by comparing IC50 of JAK2/JAK3/TYK2 to JAK1 [1]

Human CD4+ T-cell proliferation assay (CFSE dilution): 1. Human CD4+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) using magnetic bead separation and labeled with CFSE (5 μM) for 15 min at 37°C. 2. Labeled CD4+ T cells (1×10⁵ cells/well) were plated in 96-well plates, stimulated with anti-CD3 (2 μg/mL) and anti-CD28 (1 μg/mL) antibodies, and treated with serial concentrations of GLPG0634 analogue (1/5/10/30/100 nM). 3. After 72 h of incubation at 37°C (5% CO₂), cell proliferation was analyzed via flow cytometry by measuring CFSE dilution. The percentage of non-proliferating cells was used to calculate the IC50 for proliferation inhibition [1]
- PBMC cytokine secretion assay (ELISA): 1. Human PBMCs (1×10⁶ cells/mL) were seeded in 24-well plates and pre-treated with GLPG0634 analogue (5/10/20/30/50 nM) for 1 h at 37°C (5% CO₂). 2. Cells were stimulated with LPS (1 μg/mL) or IL-6 (10 ng/mL) and incubated for an additional 24 h. 3. Culture supernatants were collected, and concentrations of TNF-α, IL-6, and IL-1β were measured using sandwich ELISA kits. The percentage of cytokine inhibition was calculated by comparing to vehicle-treated (untreated) control cells [1]
- Jurkat cell p-STAT western blot assay: 1. Jurkat T cells (2×10⁵ cells/well) were seeded in 24-well plates and starved in serum-free medium for 4 h at 37°C (5% CO₂). 2. Cells were treated with GLPG0634 analogue (10/20/30 nM) for 1 h, then stimulated with IL-6 (10 ng/mL) for 30 min. 3. Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors, and 30 μg of total protein was separated by 10% SDS-PAGE. 4. Proteins were transferred to PVDF membranes, probed with primary antibodies against p-STAT3 (Tyr705), p-STAT1 (Tyr701), and total STAT3/STAT1 (loading controls) overnight at 4°C, followed by HRP-conjugated secondary antibodies. Bands were visualized via enhanced chemiluminescence (ECL), and densitometry was used to quantify p-STAT levels relative to total STAT [1]

Dissolved in 0.5% (v/v) methylcellulose; 30 mg/kg (Rats) and 50 mg/kg (Mice); Oral administration Rat and mouse CIA model

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CIA mouse model protocol: 1. Male DBA/1J mice (8–10 weeks old, 20–25 g) were acclimated for 7 days before experimentation. On day 0, CIA was induced by subcutaneous injection of 100 μg bovine type II collagen emulsified in adjuvant into the base of the tail. A booster injection of the same collagen-adjuvant emulsion was given on day 21. 2. On day 21 post-immunization (when arthritis symptoms appeared: paw swelling ≥0.5 mm vs. baseline), mice were randomized into 4 groups (n=8/group): - Vehicle group: 0.5% methylcellulose in PBS, oral gavage, once daily; - GLPG0634 analogue 5 mg/kg group: dissolved in 0.5% methylcellulose, oral gavage, once daily; - GLPG0634 analogue 15 mg/kg group: same solvent and administration route as the 5 mg/kg group; - GLPG0634 analogue 30 mg/kg group: same solvent and administration route as the 5 mg/kg group. 3. Treatment continued for 21 days (until day 42 post-immunization). Body weight and arthritis score (0–4 per paw: 0 = normal, 1 = mild swelling, 2 = moderate swelling, 3 = severe swelling, 4 = joint deformation; total score 0–16) were measured daily. 4. On day 42, mice were euthanized. Blood was collected via cardiac puncture to measure serum cytokines (ELISA). Hind joints were harvested, fixed in 10% neutral buffered formalin for 48 h, decalcified in 10% EDTA for 2 weeks, paraffin-embedded, sectioned, and stained with hematoxylin-eosin (HE) for histopathological analysis (bone erosion, cartilage loss, inflammatory infiltration) [1]
- DTH mouse model protocol: 1. Female BALB/c mice (6–8 weeks old, 18–22 g) were acclimated for 7 days. On day 0, mice were sensitized by subcutaneous injection of 100 μg ovalbumin (OVA) emulsified in adjuvant into the dorsal flank. 2. On day 7 post-sensitization, mice were challenged by intradermal injection of 50 μg OVA in 10 μL PBS into the right ear; the left ear was injected with 10 μL PBS as a control. 3. Mice were randomized into 3 groups (n=6/group) and treated with GLPG0634 analogue (10 mg/kg, 20 mg/kg, oral gavage, once daily) or vehicle (0.5% methylcellulose, oral gavage, once daily) from day 0 to day 7. 4. On day 8, ear thickness was measured using a digital caliper (3 measurements per ear), and ear swelling was calculated as (right ear thickness – left ear thickness). Mice were then euthanized, and right ear tissue was homogenized in PBS containing protease inhibitors. The homogenate was centrifuged, and the supernatant was used to measure IFN-γ and IL-17 levels via ELISA [1]

Absorption, Distribution and Excretion
Figotinib is rapidly absorbed after oral administration. The median peak plasma concentration (CMC) of filagtinib occurs 2–3 hours after administration, while the median CMC of GS-829845 occurs 5 hours after administration. Steady-state plasma concentrations are reached for filagtinib within 2–3 days, and for GS-829845 within 4 days. Food appears to have no significant effect on the absorption of filagtinib; therefore, administration of this drug is not affected by food intake. Following repeated oral administration of 200 mg filagtinib, the reported Cmax and AUCτ values were 2.15 μg/mL and 6.77 μg·h/mL, respectively. For the major metabolite GS-829845, the reported Cmax was 4.43 μg/mL and AUCτ was 83.2 μg·h/mL. Approximately 87% of the total administered dose is excreted via the kidneys, and 15% via feces.

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Metabolism/Metabolites
Carboxylesterases are involved in the metabolism of filgotinib. The carboxylesterase 2 (CES2) isoenzyme is primarily responsible for metabolizing filgotinib to its major metabolite GS-829845. Although carboxylesterase 1 (CES1) plays a minor role in the biotransformation of filgotinib, in vitro studies have shown that CES1 can partially compensate when CES2 is saturated. GS-829845 is the only major circulating metabolite identified to date.

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Biological Half-Life
The half-life of filgotinib is estimated to be 7 hours, while the half-life of its active metabolite GS-829845 is estimated to be 19 hours.

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Oral bioavailability in rats: Male Sprague-Dawley rats (250–300 g, n=4 per group) received the GLPG0634 analogue via gavage (10 mg/kg) or intravenous (iv) injection (2 mg/kg): - Oral bioavailability = 58–65% (mean = 62%); - Oral administration: peak plasma concentration (Cmax) = 3.5–4.0 μg/mL, time to peak concentration (Tmax) = 1.2–1.5 h, terminal half-life (t1/2) = 4.0–4.5 h, area under the plasma concentration-time curve (AUC0-24h) = 19.5–21.0 μg·h/mL; - Intravenous administration: Cmax = 9.0–9.8 μg/mL, t1/2 = 3.8–4.2 h, AUC0-∞ = 31.5–33.5 μg·h/mL [1]
- Plasma protein binding: In human plasma, the protein binding of the GLPG0634 analog was 90–94% (mean = 92%), as determined by equilibrium dialysis at 37°C for 4 hours. The proportion of free drug in plasma was 6–10% (mean = 8%) [1]
- Tissue distribution in CIA mice: Female DBA/1J mice (CIA model, n=3 per time point) were given a single oral dose of the GLPG0634 analog (30 mg/kg). Two hours after administration: - Plasma concentration = 3.5–3.8 μg/mL; - Joint tissue concentration = 4.2–4.5 μg/g (1.15–1.2 times the plasma concentration); - Spleen concentration = 4.0–4.3 μg/g (1.1–1.15 times the plasma concentration); - Liver concentration = 5.0–5.5 μg/g (1.35–1.45 times the plasma concentration) [1]

Effects During Pregnancy and Lactation
◉ Overview of Use During Lactation
Figotinib has not been approved by the U.S. Food and Drug Administration (FDA). There is currently no information regarding the clinical use of filgotinib during lactation. The European manufacturer recommends discontinuing breastfeeding during filgotinib treatment.
◉ Effects on Breastfed Infants
No published information found as of the revision date.
◉ Effects on Lactation and Breast Milk
No published information found as of the revision date. Protein Binding
The protein binding rate of filgotinib is approximately 55-59%, while the protein binding rate of its active metabolite GS-829845 is 39-44%.

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28-Day Repeated-Dose Toxicity Study in Rats: Male and female Sprague-Dawley rats (n=4 per sex per group) were administered the GLPG0634 analogue daily by gavage at doses of 5 mg/kg, 30 mg/kg, or 100 mg/kg for 28 days: - No deaths or obvious clinical symptoms were observed. No toxic symptoms (e.g., lethargy, diarrhea, decreased appetite) were observed in any group; - No adverse reaction dose (NOAEL) = 30 mg/kg; - At a dose of 100 mg/kg: Mild, reversible lymphopenia (lymphocyte count decreased by 18-22% compared to the control group) was observed in both male and female mice, with no histopathological changes observed in lymphoid organs (spleen, thymus). Serum ALT, AST (liver function), creatinine, and BUN (kidney function) levels remained within the normal range in all groups [1]
- Safety in mouse models: In CIA and DTH mice treated with GLPG0634 analogues at doses up to 30 mg/kg (orally, once daily for 21 days): - Body weight change ≤4% compared to the vector group; - No significant toxicity was observed (e.g., skin lesions, behavioral abnormalities); - Serum creatinine and urea nitrogen levels were within the normal physiological range [1]

[1]. Novel compounds useful for the treatment of degenerative and inflammatory diseases. WO2010010190A1.

Pharmacodynamics
In addition to targeting and inhibiting Janus kinase (JAK) 1, filgotinib also targets pro-inflammatory cytokine signaling pathways by inhibiting IL-6-induced STAT1 phosphorylation. Serum C-reactive protein levels also decrease after filgotinib administration. Mechanism of Action: GLPG0634 analogs exert their anti-inflammatory effects by selectively inhibiting JAK1 (a key kinase in the JAK-STAT signaling pathway). It competitively binds to the JAK1 kinase domain with ATP, thereby preventing JAK1-mediated phosphorylation of STAT proteins (primarily STAT1 and STAT3). Inhibition of STAT activation can suppress the transcription of pro-inflammatory cytokines (such as IL-6, IFN-γ, TNF-α) and the proliferation of activated T cells, which play a central role in the pathogenesis of inflammatory and degenerative diseases [1]
- Therapeutic indications: Preclinical data support the GLPG0634 analogue as a potential treatment for inflammatory and degenerative diseases, including rheumatoid arthritis (RA), psoriasis, and other JAK1-driven autoimmune diseases. Its high selectivity for JAK1 minimizes off-target effects associated with non-selective JAK inhibitors (such as JAK2-mediated myelosuppression) [1]
- Compound design principles: The GLPG0634 analogue is a structural derivative of GLPG0634 (Filgotinib) that has been optimized to improve chemical stability and water solubility compared to the parent compound while maintaining high JAK1 selectivity. These improvements enhance oral bioavailability and reduce inter-individual pharmacokinetic variability, thus supporting its potential for clinical development [1]

C23H18N6O2

410.43

410.149

C, 67.31; H, 4.42; N, 20.48; O, 7.80

1206101-20-3

Filgotinib;1206161-97-8

49831257

Light yellow to khaki solid powder

1.4±0.1 g/cm3

1.733

2.26

1

6

5

30

715

0

RIJLVEAXPNLDTC-UHFFFAOYSA-N

InChI=1S/C21H23N5O3S/c27-20(17-8-9-17)23-21-22-19-3-1-2-18(26(19)24-21)16-6-4-15(5-7-16)14-25-10-12-30(28,29)13-11-25/h1-7,17H,8-14H2,(H,23,24,27)

N-[5-[4-[(1,1-dioxo-1,4-thiazinan-4-yl)methyl]phenyl]-[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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