BTK (IC50 = 37.9 nM)

Evobrutinib is a novel, highly selective, irreversible BTK inhibitor that effectively blocks signaling mediated by Fc and BCR receptors. Evobrutinib has the ability to block BTK activity and stop the BCR signaling pathway from being activated. It is broken down by GSH conjugation, O-dealkylation, hydroxylation, hydrolysis, and glucuronidation[2].

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Evobrutinib is a novel, highly selective, irreversible BTK inhibitor that potently inhibits BCR- and Fc receptor–mediated signaling and, thus, subsequent activation and function of human B cells and innate immune cells such as monocytes and basophils.[3]

In this study, researchers evaluated evobrutinib in preclinical models of RA and SLE and characterized the relationship between BTK occupancy and inhibition of disease activity. In mouse models of RA and SLE, orally administered evobrutinib displayed robust efficacy, as demonstrated by reduction of disease severity and histological damage. In the SLE model, evobrutinib inhibited B cell activation, reduced autoantibody production and plasma cell numbers, and normalized B and T cell subsets. In the RA model, efficacy was achieved despite failure to reduce autoantibodies. Pharmacokinetic/pharmacodynamic modeling showed that mean BTK occupancy in blood cells of 80% was linked to near-complete disease inhibition in both RA and SLE mouse models. In addition, evobrutinib inhibited mast cell activation in a passive cutaneous anaphylaxis model. Thus, evobrutinib achieves efficacy by acting both on B cells and innate immune cells. Taken together, our data show that evobrutinib is a promising molecule for the chronic treatment of B cell-driven autoimmune disorders.

Kinase assays[3]
The potency of evobrutinib against BTK was determined using purified, full-length recombinant BTK. The BTK protein was diluted in buffer to a final concentration of 0.05 ng/μl with 75 μM ATP and 1 μM KinKDR peptide FITC-AHA-EEPLYWSFPAKKK-NH2. Various concentrations of evobrutinib were also included. Reactions were performed at 25°C for 90 min and halted by addition of stop solution containing 0.5 M EDTA. Plates were then read on the Caliper LabChip 3000, and the data were loaded into Genedata Screener for generation of IC50 curves. For the comparison of inhibition of wild-type (WT) BTK versus C481S BTK by evobrutinib or ibrutinib, recombinant proteins covering the kinase domain (BTK WT 328-659 or BTK C481S 328-659) were used. For the jump dilution assay, 20 μl of assay buffer containing 100-fold standard biochemical assay concentration of BTK (0.63 nM) was added to 200 nl of evobrutinib or RN486 at a final concentration of 10-fold IC50 or to negative control (DMSO). After incubation at room temperature for 90 min, 1 μl of the mix solution was diluted into 99 μl of assay buffer containing substrate peptide (sequence FITC-AHA-EEPLYWSFPAKKK-NH2, 1 μM) and ATP (75 μM). The microplate was placed in the Caliper Life Sciences LabChip 3000, and wells were repeatedly sampled for 112 min. Kinase selectivity for evobrutinib and ibrutinib was determined in the KinaseProfiler screening panel (EMD Millipore, Billerica, MA) that tested the inhibitory activity of the compounds at 1 μM against 267 kinases.[3]

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BTK phosphorylation in Ramos cells[3]
The effect of evobrutinib on BTK phosphorylation after BCR activation was determined in Ramos B cells. The Ramos Burkitt lymphoma cell line was procured from the American Type Culture Collection and maintained in RPMI 1640 media containing penicillin/streptomycin, 2 mM l-glutamine, and 10% FBS. Ramos cells were seeded into 96-well tissue culture plates at 8 × 106 cells per well. Cells were pretreated with the BTK inhibitor evobrutinib dissolved in DMSO for 30 min at 37°C. After compound treatment, the cells were stimulated with an anti-IgM F(ab′)2 Ab (SouthernBiotech, Birmingham, AL) at a concentration of 5 μg/ml to activate the BCR. The cells were incubated with the anti-IgM for 5 min at 37°C. After treatment, the cells were collected by centrifugation at 500 × g for 5 min. The medium was aspirated, and 150 μl of ice-cold Thermo Scientific Pierce M-PER lysis buffer containing Thermo Scientific Halt protease/phosphatase inhibitor mixture was added to the cells. The cells were resuspended in the lysis buffer, and the lysates were frozen to −80°C for subsequent measurement of BTK phosphorylation. Analysis of BTK phosphorylation was performed by Western blotting using the automated Wes instrument according to the manufacturer’s instructions. For the Western blot assay, 6 μl of lysate was used, and a 1:3000 dilution of primary anti-BTK p-Y551 or 1:50 anti-BTK p-Y223 was used for detection of phosphorylated BTK.

B cell activation in PBMCs and whole blood[3]
The ability of evobrutinib to block BCR signaling was determined using B cells in whole blood or purified PBMCs. Human blood was collected with citrate as an anticoagulant. For the PBMC assay, PBMCs were isolated over a Ficoll gradient, and 2.5 × 105 cells per well were seeded into a 96-well plate. For the whole blood assay, 90 μl of blood per well was directly transferred to 96-well plates. Cells were pretreated for 60 min at 37°C with dilutions of evobrutinib before activation with goat anti-human IgM F(ab′)2 (Dianova), added to a final concentration of 20 μg/ml, and incubated at 37°C overnight. After activation, cells were stained for 45 min with anti-CD69–allophycocyanin and anti-CD19–PerCP-Cy5.5 and then lysed using FACS lysis solution and resuspended in PBS prior to FACS analysis. FACS analysis was performed on the FACSCanto II instrument. Cells were first gated on CD19, and the percentage of CD19+ cells that were also positive for CD69 was determined.

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B cell proliferation, cytokine release, and plasmablast differentiation assays[3]
CD19+ B cells were isolated from PBMCs of healthy volunteers by negative selection using the B cell purification kit II following the manufacturer’s instructions. Purified B cells were incubated with evobrutinib for 1 h and stimulated with 10 μg/ml goat F(ab′)2 anti-IgM and 10 ng/ml recombinant human IL-4 for 4 d. One microcurie of [3H]thymidine was added for the last 18 h of culture. Proliferation was measured using a multiplate β counter. For the cytokine release assay, CD19+ B cells isolated from PBMCs of healthy volunteers were incubated with evobrutinib for 1 h and stimulated with 10 μg/ml rabbit anti-human IgA + IgG + IgM (H+L), 3 μg/ml CpG oligodeoxynucleotide 2006, and 8000 IU/ml recombinant human IFN-α for 48 h. Cytokines in the supernatants were measured with Cytometric Bead Array kits. For Ig production, isolated B cells were stimulated with 20 U/ml IL-2, 100 ng/ml IL-10, 10 μg/ml inactivated Staphylococcus aureus Cowan, and various concentrations of evobrutinib. After 10 d of culture, IgG and IgM levels in the supernatant were measured by ELISA.

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Inhibition of FcγR signaling[3]
U937 NF-κB–Luc reporter cells were maintained as an adherent culture at 37°C in a CO2-regulated tissue culture incubator. The day of the experiment, cells were collected, counted, and plated in a 96-well tissue plate. Evobrutinib was added at concentrations ranging from 5 nM to 10 μM. Cells were incubated with evobrutinib for 30 min in a 37°C tissue culture incubator. The cells were then transferred to fresh plates coated with anti-CD64 and stimulated for 4 h. Luciferase activity in cell lysates was measured using an EnVision plate reader.

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Basophil inhibition assay[3]
The ability of evobrutinib to block Fc receptor signaling was determined using basophils in whole blood. Human blood was collected with citrate as an anticoagulant and transferred to 96-well plates. Blood was pretreated for 30 min at 37°C with dilutions of evobrutinib before activation with anti-IgE, added to a final concentration of 2 μg/ml, and incubated at 37°C for 5 min. After activation, cells were stained for 15 min with anti-CD63–FITC, and then PBS–EDTA (20 mM) was added, followed by fixative/lysing buffer. Cells were fixed in formaldehyde prior to FACS analysis. The mean fluorescence intensity (MFI) for CD63 expression was determined after first gating for CD123+HLA-DR− cells using the FACSCanto II instrument.[3]

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BioMAP profiling[3]
The biological selectivity of evobrutinib and ibrutinib was assessed in vitro using primary human cells with BioMAP profiling by BioSeek. The activity of the compound was assessed using a concentration range of 370 nM–10 μM in 12 different primary cell coculture assay systems according to previously published detailed methods

DBA/1J female mice
12 mg/kg
o.g.

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Ex vivo B cell stimulation in mouse whole blood[3]
Evobrutinib was administered by oral gavage to female C57BL/6 mice (five per group) at indicated doses and time points before heparinized whole blood was obtained and divided into two aliquots. One aliquot was incubated with anti-IgD as stimulation and another with PBS as basal control. CD69 MFI on the B cell surface was measured by flow cytometry. The difference between stimulated and basal levels of MFI was calculated and expressed as Δ MFI. Percent inhibition was calculated according to the following formula: percent inhibition = (1− [Δ MFIevobrutinib/Δ MFIvehicle]) × 100.[3]
PK/PD model and occupancy assay for whole blood[3]
To build a PK/PD relationship for evobrutinib, DBA/1J female mice aged 11–12 wk were dosed with the compound. BTK occupancy in the blood and plasma concentrations were measured over time. Evobrutinib was formulated in a vehicle solution of 20% kleptose and 50 mM citrate, pH 3, and mice were administered the compound via oral gavage. At various time points after dosing, mice were euthanized, and blood was collected into heparinized tubes via the vena cava. For determination of BTK occupancy, a previously described method that uses a biotinylated BTK-binding probe and a streptavidin-capture ELISA was used. Plasma samples were analyzed by liquid chromatography–tandem mass spectrometry for determination of evobrutinib concentrations.[3]
Passive cutaneous anaphylaxis in mice[3]
C57BL/6 mice were sensitized intradermally in the back with 250 ng of anti-DNP IgE or anti-OVA IgE. Twenty-four hours later, all mice were challenged i.v. with 0.5 mg of DNP–human serum albumin in the presence of 0.5% Evans blue. Mice were sacrificed 30 min after challenge, back skin was harvested, and the Evans blue was extracted in formamide for 24 h at 55°C. OD was measured at 620 nm and compared with a standard curve of known Evans blue concentrations. The results are expressed as nanograms of dye per milligram of tissue.[3]
Collagen-induced arthritis in mice[3]
All mouse collagen-induced arthritis (CIA) studies were performed at Bolder BioPATH. DBA/1OlaHsd mice (12–15 animals per group) were anesthetized with isoflurane, shaved at the base of the tail, and injected intradermally with 150 μl of CFA (Sigma) containing bovine type II collagen (2 mg/ml) at the base of the tail on day 0 and again on day 21. On study day 18, mice were randomized by body weight into treatment groups. Treatment was initiated after enrollment and continued daily (once daily at 24-h intervals) through study day 33. Animals were dosed by the oral route with vehicle (20% hydroxy-propyl-β cyclodextrin in H2O) or evobrutinib at various doses or the reference compound methotrexate (MTX; 0.5 mg/kg). On study day 34, the studies were terminated. Daily clinical scores were given for each of the paws (right front, left front, right rear, and left rear) on arthritis days 18–34 using the following criteria: 0 = normal; 1 = one hind or forepaw joint affected or minimal diffuse erythema and swelling; 2 = two hind or forepaw joints affected or mild diffuse erythema and swelling; 3 = three hind or forepaw joints affected or moderate diffuse erythema and swelling; 4 = four hind or forepaw joints affected or marked diffuse erythema and swelling; and 5 = entire paw affected, severe diffuse erythema and severe swelling, and unable to flex digits. Histopathological scoring was performed on forepaws, hind paws, and knees from mice. Inflammation, pannus formation, cartilage damage, and bone resorption were scored separately. Scores for all four parameters were added for each individual ankle or knee. Mean scores for all six tissues were calculated for each animal, and mean scores for each group are shown.[3]
SLE model[3]
Ten-week-old female NZB/W F1 mice were given two i.v. injections on day 0 and day 1 of 1 × 108 IU/100 μl of adenovirus with mmIfna5_v1 insert in saline or left untreated (sham). Drug treatments were initiated at 2 wk after delivery of adenovirus with mmIfna5_v1 insert and continued until the end of the experiment (at 10 wk). Mice (10 per group) were treated once daily with evobrutinib at indicated doses or mycophenolate mofetil at 300 mg/kg by oral gavage. Serum and urine samples were collected for anti-dsDNA Ab determination (by ELISA) and urinary protein creatinine ratio (UPCR; measured on ADVIA 1800) determination, respectively, on the days indicated. Proteinuria was defined as UPCR > 3. In addition, serum was analyzed for clinical chemistry parameters including urea nitrogen, albumin, total protein, and cholesterol on an ADVIA1800 chemistry analyzer on the final day of the experiment. Hematological analysis was performed on whole blood on the final day using a Sysmex XT-2000iV analyzer. Spleen cells were analyzed for B and T cell subsets on the final day using flow cytometry. The gating strategy is shown in Fig. 9. BTK occupancy in splenocytes was measured as described by Honigberg and colleagues

PK/PD modeling of BTK occupancy in mice[3]
In this study, researchers established a relationship between exposure, target occupancy, and efficacy in preclinical disease models. PK parameters were estimated from a two-compartment model with first-order absorption for male DBA/1OlaHsd and for female NZB/W mice, which were used in the RA and SLE models, respectively. Strain-specific PK parameters may be found in Supplemental Table II. The corresponding exposure data are shown in Supplemental Fig. 1F and 1G.
Once evobrutinib has engaged BTK, the PD effect depends largely on the resynthesis and degradation rates of BTK in vivo rather than the systemic exposure of evobrutinib. This is due to the covalent binding of evobrutinib to BTK and illustrated in Fig. 12A. BTK occupancy PK/PD modeling was performed for male DBA/1OlaHsd mice using the BTK occupancy and exposure data shown in Fig. 4C and ​and4D.4D. PK parameters from this strain were used to parameterize the second-order rate constant kirrev, which describes binding of evobrutinib to BTK. The plasma concentration and BTK occupancy time course following oral administration of evobrutinib were fit to a PK/PD model adapted from Abelö et al. and shown schematically in Fig. 12A. The selected PD model consisted of an indirect response model that describes both the second-order rate constant describing the irreversible binding of evobrutinib to BTK (kirrev) and the mouse degradation rate of the BTK protein (kdeg). The estimated PK/PD parameters in mouse WBCs are reported in Supplemental Table II, and the occupancy dose–response time course is shown in Fig. 12B. These drug (kirrev) and system (kdeg) parameters were integrated into a model describing fluctuation of BTK occupancy at steady state after daily dosing. As shown in Fig. 12C, this model predicts that daily dosing with 1 mg/kg of evobrutinib in mice results in a maximum BTK occupancy of 70% and a minimum occupancy of 50% at steady state. A daily dose of 5 mg/kg results in maximum and minimum BTK occupancy of 97 and 70%, respectively. Higher doses of evobrutinib are predicted to have a marginal effect on the BTK occupancy time course in mice. This is in line with the PD effect being entirely driven by the turnover of BTK protein in vivo rather than exposure with evobrutinib.

[1]. J Med Chem . 2018 Mar 22;61(6):2227-2245.

[2]. Drug Test Anal. 2018, doi: 10.1002/dta.2477.

[3]. J Immunol . 2019 May 15;202(10):2888-2906.

Evobrutinib is under investigation in clinical trial NCT03934502 (Effect of Meal Composition and Timing on Evobrutinib Bioavailability).
Evobrutinib is an inhibitor of Bruton's tyrosine kinase (BTK) with potential antineoplastic activity. Upon administration, evobrutinib inhibits the activity of BTK and prevents the activation of the B-cell antigen receptor (BCR) signaling pathway. This prevents both B-cell activation and BTK-mediated activation of downstream survival pathways, which leads to the inhibition of the growth of malignant B-cells that overexpress BTK. BTK, a member of the Src-related BTK/Tec family of cytoplasmic tyrosine kinases, is overexpressed in B-cell malignancies; it plays an important role in B-lymphocyte development, activation, signaling, proliferation and survival.

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Drug Indication
Treatment of multiple sclerosis

C25H27N5O2

429.5142

429.216

C, 69.91; H, 6.34; N, 16.31; O, 7.45

1415823-73-2

1415823-73-2

71479709

White to off-white solid powder

1.2±0.1 g/cm3

683.4±55.0 °C at 760 mmHg

367.1±31.5 °C

0.0±2.1 mmHg at 25°C

1.637

3.19

2

6

7

32

595

0

O=C(C([H])=C([H])[H])N1C([H])([H])C([H])([H])C([H])(C([H])([H])N([H])C2C(=C(N([H])[H])N=C([H])N=2)C2C([H])=C([H])C(=C([H])C=2[H])OC2C([H])=C([H])C([H])=C([H])C=2[H])C([H])([H])C1([H])[H]

QUIWHXQETADMGN-UHFFFAOYSA-N

InChI=1S/C25H27N5O2/c1-2-22(31)30-14-12-18(13-15-30)16-27-25-23(24(26)28-17-29-25)19-8-10-21(11-9-19)32-20-6-4-3-5-7-20/h2-11,17-18H,1,12-16H2,(H3,26,27,28,29)

1-[4-[[[6-amino-5-(4-phenoxyphenyl)pyrimidin-4-yl]amino]methyl]piperidin-1-yl]prop-2-en-1-one

MSC-2364447-C; MSC-2364447 C; Evobrutinib; 1415823-73-2; 1-(4-(((6-amino-5-(4-phenoxyphenyl)pyrimidin-4-yl)amino)methyl)piperidin-1-yl)prop-2-en-1-one; Evobrutinib [INN]; M-2951; M 2951; M2951; MSC-2364447C; MSC 2364447C; MSC2364447C

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: ~86 mg/mL (~200.2 mM)
Ethanol: ~10 mg/mL (~23.3 mM)

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.)