JAK1 (IC50 = 10 nM); JAK2 (IC50= 28 nM); Tyk2 (IC50= 116 nM); JAK3 (IC50= 810 nM)

In a dose-dependent manner, filgotinib (maleate) (1–10 μM) suppresses Th1 and Th2 differentiation [1].Th2 cell differentiation mediated by IL-4, a cytokine that signals through JAK1 and JAK3, is dose-dependently inhibited by filgotinib (GLPG0634). Moreover, filgotinib also inhibits Th1 differentiation at 1 μM or less in potency [1]. JAK2 homodimer-mediated signaling generated by PRL or EPO (IC50 > 10 μM) is not inhibited by filgotinib (GLPG0634) [2].

Filgotinib (maleate) (1–10 mg/mL; oral and intravenous; rats with collagen-induced arthritis) has good pharmacokinetic profiles and decreases inflammatory cells, bone and cartilage deterioration, and paw swelling. Factor magnitude [1].

Biochemical assays[1]
IC50 determination.[1]
Recombinant JAK1, TYK2, JAK2, and JAK3 were used to develop activity assays in 50 mM HEPES (pH 7.5), 1 mM EGTA, 10 mM MgCl2, 2 mM DTT, and 0.01% Tween 20. The amount of JAK protein was determined per aliquot, maintaining initial velocity and linearity over time. The ATP concentration was equivalent to 4× the experimental Km value and the substrate concentration (ULight-conjugated JAK-1(Tyr1023) peptide) corresponded to the experimentally determined Km value. After 90 min incubation at room temperature (RT), the amount of phosphorylated substrate was measured by addition of 2 nM europium-anti-phosphotyrosine Ab (PerkinElmer) and 10 mM EDTA in Lance detection buffer. Compound IC50 values were determined by preincubating the enzyme with compound at RT for 60 min, prior to the addition of ATP.

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Kd determination.[1]
Dissociation constants were determined at a CRO company. Proprietary fluorescently labeled ATP mimetics with fast dissociation rates (PRO13, PRO14, and PRO13 for JAK1, JAK2, and JAK3, respectively) were incubated with JH1 domains of purified JAKs in 20 mM MOPS (pH 7.5), 1 mM DTT, 0.01% Tween 20, and 500 mM hydroxyectoine (JAK3 only) for 30 min. Compounds (concentrations ranging from 520 pM to 1.1 μM) were added in 100% DMSO and time dependency of reporter displacement was measured. IC50 values corresponding to 50% probe displacement were obtained and Kd values were calculated according to the Cheng–Prusoff equation.

Cellular assays[1]
STAT6 phosphorylation induced by IL-4.[1]
THP-1 cells (ATCC TIB-202) were preincubated with compound at RT for 1 h, incubated with IL-4 (10 ng/ml) at RT for 60 min, and processed for flow cytometry. Cells were fixed in Cytofix/Cytoperm buffer and permeabilized in Phosflow perm buffer III on ice for 30 min. After blocking (Fc blocking reagent), pSTAT6 was detected with mouse anti-human PE-labeled anti-pSTAT6 Ab.

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STAT5 phosphorylation induced by IL-2, IL-3, and erythropoietin.[1]
NK-92 cells (ATCC CRL-2407) were IL-2 starved overnight, preincubated with compound at 37°C for 1 h, stimulated with IL-2 (1 ng/ml) at RT for 20 min, and processed for AlphaScreen analysis. TF1 cells were starved overnight in RPMI 1640 with 0.1% FBS, preincubated with compound at RT for 1 h, stimulated with IL-3 (30 ng/ml) at RT for 20 min, and processed for AlphaScreen analysis. UT-7-erythropoietin (EPO) cells (EPO-dependent derivative of UT-7; Centocor) were preincubated with compound at RT for 1 h, stimulated with EPO (1 U/ml) for 20 min, and processed for AlphaScreen analysis. pSTAT5 was measured using AlphaScreen technology essentially according to the manufacturer’s protocol.

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STAT1 phosphorylation induced by IFN-α and IFN-γ.[1]
STAT1 U2OS cells (Invitrogen, catalog no. K1469) were preincubated with compound at 37°C for 1 h, treated with 30,000 U/ml IFN-αB2 (PBL IFN source, catalog no. 11115-1) or 20 ng/ml IFN-γ at 37°C for 1 h, lysed (lysis buffer containing 2 nM Tb-Ab) according to manufacturer’s protocol, and incubated at RT for 60 min. pSTAT1 was detected by time-resolved fluorescence resonance energy transfer.

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STAT5 phosphorylation induced by prolactin.[1]
22Rv1 cells (ATCC CW22Rv) were starved overnight, preincubated with compound, triggered with prolactin (PRL; 500 ng/ml human PRL for 20 min), lysed in 10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, 50 mM NaF, 30 mM sodium pyrophosphate, 10% glycerol buffer containing phosphatase/protease inhibitor cocktails, and centrifuged. Cell lysate (180 μg) was used for STAT5 immunoprecipitation (anti-STAT5 polyclonal Abs, C-17; protein A-Sepharose beads). Total and phosphorylated STAT5 were measured by densitometric analysis after Western blotting.

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IL-3/JAK2–induced proliferation of Ba/F3 cells.[1]
Ba/F3 cells (provided by V. Lacronique, Paris, France), which are dependent on IL-3 and JAK2 signaling, were incubated with compound at 37°C for 40 h, after which cell proliferation was analyzed by measuring ATP content.

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Oncostatin M-induced STAT1 reporter assay in HeLa cells[1]
. HeLa cells (ATCC CCL-2) were transfected with a pSTAT1 reporter construct (Panomics, catalog no. LR0127). After transfection for 24 h, cells were incubated for 1 h with compound and triggered with oncostatin M (OSM; 33 ng/ml). After 20 h incubation, the cells were lysed and luciferase activity was determined with the luciferase SteadyLite kit according to the supplier’s recommendations. In parallel, β-galactosidase activity was measured in the presence of 4 mg/ml 2-nitrophenyl β-d-galactopyranoside.

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Knockdown experiments.[1]
HeLa and HCT116 cells obtained from the American Type Culture Collection were transfected with 50 nM ON-TARGETplus SMARTpool small interfering RNA (siRNA) for human JAK1, JAK2, JAK3, or TYK2, or with nontargeting or GAPDHnegative control siRNAs using Lipofectamine RNAiMAX transfection reagent from Invitrogen. Four days after transfection cells were starved overnight and stimulated with IL-6/sIL-6R (both 250 ng/ml) for 20 min and pSTAT1 levels were determined using AlphaScreen technology according to the manufacturer’s protocol.

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T cell differentiation studies.[1]
PBMCs were isolated from buffy coats of healthy donors using density gradient centrifugation on Lymphoprep. Naive CD4+ T cells were further isolated by depletion of non–T helper and memory CD4+ T cells using a naive CD4+ T cell isolation kit II. Isolated naive CD4+ T cells were stimulated with plate-bound anti-CD3 (3 μg/ml) and anti-CD28 (5 μg/ml) Abs in the presence of cytokines that drive differentiation into Th1, Th2, or Th17 Th subsets. For Th1 cell polarization, cells were cultured in the presence of 10 μg/ml anti–IL-4 Ab, 10 ng/ml IL-2, and 10 ng/ml IL-12. For Th2 cell polarization, cells were cultured in the presence of 10 μg/ml anti–IFN-γ Ab (Becton Dickinson), 25 ng/ml IL-4, and 10 ng/ml IL-2. For Th17 cell polarization, a mix of the following cytokines was used: 10 ng/ml IL-6, 10 ng/ml IL-1β, 1 ng/ml TGF-β, and 100 ng/ml IL-23. To monitor effects of compounds on T cell differentiation, compounds were added at indicated concentrations at the start of T cell differentiation. After 5 d, RNA was extracted using an RNeasy Mini kit, reverse transcribed, and the extent of Th subset differentiation was monitored by determining expression of IFN-γ (Th1 marker), IL-13 (Th2 marker), or IL-17F (Th17 marker) using real-time PCR on the ViiA7 thermocycler with predesigned TaqMan Assay-on-Demand gene expression primer/probe sets. Gene expression was normalized to 18S and expressed as ΔCt values, with ΔCt = Ctgene − Ct18S or expressed as relative mRNA level of specific gene expression as obtained using the 2−ΔCt method.

Pharmacokinetics[1]
Formulations.[1]
GLPG0634 was formulated in polyethyleneglycol 200/0.9% NaCl (60/40; v/v) for i.v. administration and in 0.5% (v/v) methylcellulose for oral administration for all in vivo studies described. Compound purity was >95% as measured by HPLC.Animals.[1]
Male Sprague Dawley rats (180–200 g) and CD1 mice (23–25 g) were obtained from Janvier and Harlan, respectively. Two days before administration of compound, rats underwent surgery to place a catheter in the jugular vein under isoflurane anesthesia. Animals were deprived of food for at least 16 h before oral dosing until 4–6 h after. Before oral dosing, animals were deprived of food for at least 12 h before compound administration until 4 h after administration. All in vivo experiments were carried out in a dedicated pathogen-free facility (22°C).

Pharmacokinetic studies.[1]
GLPG0634 was orally dosed as a single esophageal gavage at 5 mg/kg (dosing volume of 5 ml/kg) and i.v. dosed as a bolus via the caudal vein at 1 mg/kg (dosing volume of 5 ml/kg). In the rat study, each group consisted of three rats and blood samples were collected via the jugular vein. In the mouse study, each group consisted of 21 mice (n = 3/time point) and blood samples were collected by intracardiac puncture under isoflurane anesthesia. Lithium heparin was used as anticoagulant and blood was taken at 0.05, 0.25, 0.5, 1, 3, 5, and 8 h (i.v. route) and 0.25, 0.5, 1, 3, 5, 8, and 24 h (by mouth).

GLPG0634 plasma concentrations were determined by liquid chromatography–tandem mass spectrometry with a lower limit of quantification of 2 ng/ml. Pharmacokinetic parameters were calculated by noncompartmental analysis using WinNonlin software.

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In vivo pharmacology[1]
Rodent CIA models.[1]
Animals.[1]
Dark Agouti rats (females, 7–8 wk old) and DBA/1J mice (male, 6 wk old) were obtained from Janvier.

Materials.[1]
CFA and IFA were purchased from Difco (Detroit, MI). Bovine collagen type II (CII) was used. All other reagents used were of reagent grade and all solvents were of analytical grade.

CIA.[1]
One day before the start of the experiment, CII solution (2 mg/ml) was prepared with 0.05 M acetic acid and stored at 4°C. Just before the immunization, equal volumes of IFA and CII were mixed by a homogenizer in a precooled glass bottle in an ice water bath. For rat CIA experiments, the emulsion (0.2 ml) was injected intradermally at the base of the tail at day 1 and again at day 8. This immunization method was modified from published methods. The in vivo efficacy of GLPG0634 was determined after daily oral administration for a period of 14 d after onset of disease (average clinical score at onset, 2.5 ± 0.3; 10 rats/treatment group) over the dose range 0.1–30 mg/kg. The TNF-α blocker etanercept was administered three times per week at 10 mg/kg by i.p. injection. A fully active dose was reported to require repeated dosing in the 3–9 mg/kg range. In our model of Dark Agouti female rats, disease normalization was reached for 10 mg/kg etanercept dosed three times a week i.p. as measured by clinical score, inflammation, bone resorption, pannus, and cartilage damage. At day 7 or 11, 200 μl blood was collected by retro-orbital puncture with lithium heparin as anticoagulant at predose and 1, 3, and 6 h (n = 2 or 3/time point) for steady-state pharmacokinetics analysis. At sacrifice, hind paws were removed for x-ray analysis and histological examination. A Tukey multiple comparison test was used to perform a meta-analysis of three studies carried out for GLPG0634. The score of each rat was divided by the average score obtained for vehicle in the same readout and study and multiplied by 100. Relative scores were averaged per readout for all animals present in all studies that received the same dose. For mouse CIA experiments, the IFA/CII emulsion (0.2 ml) was injected intradermally at the base of the tail at day 1 and again at day 21. This immunization method was modified from published methods. The in vivo efficacy of GLPG0634 was determined after daily oral administration for a period of 14 d after onset of disease (average clinical score at onset, 2.4 ± 0.6; 10 mice/treatment group) over the dose range 50 mg/kg twice daily. Administration of etanercept and pharmacodynamic and pharmacokinetic analyses were essentially carried out as described for the rat CIA model.

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30 mg/kg daily in Rats); 50 mg/kg twice daily in Mice

In the rat model of collagen-induced arthritis (CIA), oral administration of GLPG0634 shows a marked protection from bone damage at dose of 3 mg/kg. It reduces the infiltration of inflammatory cells significantly from 1 mg/kg onward

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

Use 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 filagtinib during lactation. European manufacturers recommend discontinuing breastfeeding during filagtinib treatment.
◉ Effects on breastfed infants
As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding rate
The protein binding rate of filagtinib is approximately 55-59%, while the protein binding rate of its active metabolite GS-829845 is 39-44%.

[1]. Preclinical characterization of GLPG0634, a selective inhibitor of JAK1, for the treatment of inflammatory diseases. J Immunol. 2013 Oct 1;191(7):3568-77.

[2]. Filgotinib for the treatment of Crohn's disease. Expert Opin Investig Drugs. 2018 Mar;27(3):295-300.

Drug Indication
Rheumatoid Arthritis: Jyseleca is indicated for the treatment of adult patients with moderate to severe active rheumatoid arthritis who have not responded to or are intolerant of one or more disease-modifying antirheumatic drugs (DMARDs). Jyseleca can be used as monotherapy or in combination with methotrexate (MTX). Ulcerative Colitis: Jyseleca is indicated for the treatment of adult patients with moderate to severe active ulcerative colitis who have not responded to, lost response to, or are intolerant of conventional therapies or biologics.

C25H27N5O7S

541.5762

541.163

C, 55.44; H, 5.03; N, 12.93; O, 20.68; S, 5.92

1802998-75-9

Filgotinib;1206161-97-8

131801100

White to off-white solid powder

3

10

7

38

834

0

C1CC1C(=O)NC2=NN3C(=N2)C=CC=C3C4=CC=C(C=C4)CN5CCS(=O)(=O)CC5.C(=C\C(=O)O)\C(=O)O

BFENHEAPFWQJFL-BTJKTKAUSA-N

InChI=1S/C21H23N5O3S.C4H4O4/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;5-3(6)1-2-4(7)8/h1-7,17H,8-14H2,(H,23,24,27);1-2H,(H,5,6)(H,7,8)/b;2-1-

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

Filgotinib maleate; 1802998-75-9; Filgotinib (maleate); JG8OB4UL9Y; Filgotinib maleate [USAN]; GS-6034; (Z)-but-2-enedioic acid;N-[5-[4-[(1,1-dioxo-1,4-thiazinan-4-yl)methyl]phenyl]-[1,2,4]triazolo[1,5-a]pyridin-2-yl]cyclopropanecarboxamide; Cyclopropanecarboxamide, N-(5-(4-((1,1-dioxido-4-thiomorpholinyl)methyl)phenyl)(1,2,4)triazolo(1,5-a)pyridin-2-yl)-, (2Z)-2-butenedioate (1:1);

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)