Btk (IC50 < 0.5 nM)

Spebrutinib (CC-292) is a covalent, highly selective, orally active inhibitor of Btk with IC50 value of 0.5 nM. Spebrutinib also has IC50s of 723 nM, 1.729 μM, 2.43 μM, 4.4 μM, and 7.15 μM for Yes, c-Src, Brk, Lyn, and Fyn, respectively, indicating less potent inhibition of these genes. Following a thorough investigation, it was discovered that the cellular EC50 of Btk kinase inhibition with Spebrutinib (EC50=8 nM) and the EC50 of Btk occupancy from a Spebrutinib dose-response in Ramos cells (EC50=6 nM) correlate. Moreover, it has been observed that the concentration of Spebrutinib needed to achieve 90% occupancy of Btk is 39 nM, whereas the concentration at which it inhibits 90% of Btk activity in Ramos cells is 35 nM[1].

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Spebrutinib (CC-292) is an oral Btk inhibitor that is covalent, highly selective, and has an IC50 of 0.5 nM. Moreover, spebrutinib exhibits mild inhibitory effects on Yes, c-Src, Brk, Lyn, and Fyn, with corresponding IC50s of 723 nM, 1.729 μM, 2.43 μM, 4.4 μM, and 7.15 μM. A further investigation revealed a close correlation between the cellular EC50 of Spebrutinib inhibition of Btk kinase (EC50=8 nM) and the EC50 of Btk occupancy in Ramos cells in response to the drug's dosage response (EC50=6 nM). Moreover, 35 nM of spebrutinib is the concentration that suppresses 90% of Btk activity in Ramos cells, whereas 39 nM is the concentration needed for 90% Btk occupancy [1].

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In this study, researchers first investigated the antitumor effects of CC-292 in five MCL cell lines (REC-1, MINO, UPN-1, MAVER-1 and Z138) after 72 h of treatment. CC-292 (10–1000 nM) had a cytostatic effect in a subset of cell lines, with REC-1, MINO and UPN-1 appearing to be the most sensitive, while MAVER-1 and Z138 were the most resistant to CC-292, following a trend similar to that for ibrutinib (Figure 1A,B). CC-292 induced marginal apoptosis (10–15%) in the most sensitive cell lines (UPN-1 and REC-1) (Online Supplementary Figure S1). Identification of Tyr223 pBTK is considered a surrogate marker for kinase activity.6 MCL cell lines pre-incubated with CC-292 were IgM-stimulated to mimic BCR activation. As displayed in Figure 1C, CC-292 significantly reduced both constitutive and IgM-induced BTK phosphorylation at the Y223 residue in MCL cell lines and primary cells, independently of their sensitivity to the inhibitor.[2]

In in vivo studies using the adoptive transfer TCL1 mouse model of CLL, CC-292 reduced tumor load and normalized tumor-associated expansion of T cells and monocytes, while not affecting T cell function. Importantly, the combination of CC-292 and bendamustine impaired CLL cell proliferation in vivo and enhanced the control of CLL progression. Our results demonstrate that CC-292 is a specific BTK inhibitor with promising performance in combination with bendamustine in CLL. Further clinical trials are warranted to investigate the therapeutic efficacy of this combination regimen.[3]

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CC-292/bendamustine treatment effectively controls CLL development in vivo. In order to assess the in vivo activity of CC-292 and to validate the in vitro results obtained from its combination with bendamustine, we used the TCL1 AT mouse model of CLL. Leukemic TCL1 AT splenocytes were transplanted into syngeneic immunocompetent C57BL/6 N mice. After 14 days, mice presented mean TL of 50% (Supporting Information Fig. S5a). They were then randomized in four groups (Supporting Information Fig. S5b) and treated for 11 days (Fig. 4a). During the treatment, absolute lymphocyte counts (ALC) in blood were monitored weekly. In all treatment conditions, a decrease was detected, being this more remarkable in the combination treatment (Supporting Information Fig. S6a). Furthermore, untreated mice exhibited low red blood cell and platelet counts, which were improved after treatment with CC-292, bendamustine, or the combination (Supporting Information Fig. S6b and S6c). Untreated mice showed severe splenomegaly and hepatomegaly (Fig. 4b), which were significantly less severe both in CC-292 and bendamustine-treated animals (spleen weight 2.2-fold lower in CC-292 cohort (p < 0.0001) and 2.5-fold lower in bendamustine cohort (p < 0.0001), liver weight 1.6-fold lower both in CC-292 (p < 0.0001) and bendamustine cohorts (p = 0.0002)). Mice treated with the combination showed a markedly lower spleen weight of up to 5-fold less (p < 0.0001) and a 1.7-fold lower liver weight (p < 0.0001) compared to untreated animals.[3]

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In a collagen-induced arthritis mouse model, AVL-292 (3-30 mg/kg, p.o.) dose-dependently inhibits the clinical signs of inflammatory disease, including reduction in joint and paw swelling and visible redness of the affected paws.

Targeted therapies that suppress B cell receptor (BCR) signaling have emerged as promising agents in autoimmune disease and B cell malignancies. Bruton's tyrosine kinase (Btk) plays a crucial role in B cell development and activation through the BCR signaling pathway and represents a new target for diseases characterized by inappropriate B cell activity. N-(3-(5-fluoro-2-(4-(2-methoxyethoxy)phenylamino)pyrimidin-4-ylamino)phenyl)acrylamide (CC-292) is a highly selective, covalent Btk inhibitor and a sensitive and quantitative assay that measures CC-292-Btk engagement has been developed. This translational pharmacodynamic assay has accompanied CC-292 through each step of drug discovery and development. These studies demonstrate the quantity of Btk bound by CC-292 correlates with the efficacy of CC-292 in vitro.[1]

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ELISA cytokine quantification [2]
CCL3 and CCL4 levels were assessed in duplicate using ELISA kits in supernatants harvested from cells that had been pretreated with 1µM CC-292 at 37ºC for 1h and subsequently stimulated with 10µg/ml of anti-IgM for 24h.

In RPMI media free of serum, cells are cultured for one to one and a half hours. At final concentrations of 0.001, 0.01, 0.1, and 1 μM, isolated human B cells are cultured with spebrutinib. A solution of 0.1 nM–3 μM spebrutinib is added to Ramos cells. Subsequently, the cells are incubated for an hour at 37°C with the compound present. Once the cells have been incubated, they are centrifuged and resuspended in 100 μL of serum-free RPMI. Next, 5 μg/mL of α-human IgM is added to stimulate the BCR. The specimens undergo centrifugation, followed by a PBS wash and lysing in 100 μL of Cell Extraction Buffer supplemented with 1:10 (v/v) PhosSTOP Phosphatase Inhibitor and 1:10 (v/v) Complete Protease Inhibitor. The following antibodies are used in immunoblot analysis: Btk, P-Btk, Tubulin, Syk (2712; CST), P-PLCγ2, PLCγ2 (3871; CST), and Syk (2712; CST). Infrared fluorescence detection is used on a Li-Cor Odyssey scanner to scan membranes[1].

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Cell proliferation assay and apoptosis quantification [2]
MCL cells (5x104 ) were treated with Spebrutinib (CC-292), lenalidomide or NIK inhibitors for the times indicated and 0.5 mg/mL MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) reagent was added for 2–6 additional hrs before spectrophotometric measurement. Each measurement was made in triplicate. Values were represented using untreated control cells as reference. Apoptosis induction was evaluated by flow cytometry in an Attune acoustic focusing cytometer after staining MCL cells with Annexin V-FIT and co-stained with CD19-PE in the case of primary cells.

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Flow cytometry [2]
Cells were pretreated with 1µM Spebrutinib (CC-292) at 37ºC for 1h and subsequently stimulated with 10µg/ml anti-IgM for 24h. Cellular activation was evaluated by co-staining of MCL cells with CD69-PC7/CD86-FITC, including CD19-PE and Annexin V-Pacific Blue in the case of primary cells, followed by cytofluorimetric evaluation in an Attune cytometer.

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Migration assay [2]
SDF-1α/CXCL12-induced migration was evaluated using 24-well chemotaxis chambers containing 5 µm pore size inserts and coated with 1 µg/ml VCAM-1. The lower chamber contained 200ng/ml CXCL12. The cells were pretreated with 1µM Spebrutinib (CC-292) at 37ºC for 1h and deposited in the upper compartment allowing them to migrate for 3h at 37ºC and enumerated by flow cytometry.

TCL1 adoptive transfer (AT) mouse model[3]
Eμ-TCL1 (TCL1) mice on C57BL/6 background were used. In this model, the overexpression of TCL1 in B cells under the VH-promoter-IgH-Eμ-enhancer drives a clonal expansion of CD5+ B cells, representing an aggressive form of CLL.24 For treatment studies, adoptive transfer of TCL1 tumors were performed in C57BL/6 WT mice as described before.25 Briefly, 106 splenocytes with more than 95% of viable CD19+CD5+ cells from leukemic TCL1 mice were transplanted in 3-month-old female C57BL/6N wild-type mice via tail vein injection. Mice were housed in pathogen-free conditions, closely monitored for signs of illness. All experiments were performed according to the University of Barcelona animal experimental ethics committee guidelines. When PB tumor load (TL) reached mean values of 50% of CD19+ CD5+ (out of total CD45+ cells), animals were randomized into 4 groups (Vehicle, CC-292, bendamustine and Combination) with equal mean and standard deviation of TL percentage values. Fifteen milligram/kilogram CC-292 were administered twice daily via oral gavage, whereas 25 mg/kg bendamustine were administered intravenously once weekly. Mice were euthanized after 11 days of treatment and single cell suspensions were obtained from BM, inguinal LN and spleen.[3]

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3-30 mg/kg; p.o

Collagen-induced arthritis (CIA) mouse model

[1]. Inhibition of Btk with CC-292 provides early pharmacodynamic assessment of activity in mice and humans. J Pharmacol Exp Ther. 2013 Aug;346(2):219-28.

[2]. The Bruton tyrosine kinase inhibitor CC-292 shows activity in mantle cell lymphoma and synergizes with lenalidomide and NIK inhibitors depending on nuclear factor-κB mutational status. Haematologica . 2017 Nov;102(11):e447-e451.
[3]. Selective BTK inhibition improves bendamustine therapy response and normalizes immune effector functions in chronic lymphocytic leukemia. Int J Cancer . 2019 Jun 1;144(11):2762-2773.

Spebrutinib Besylate is the besylate salt form of spebrutinib, an orally bioavailable, selective inhibitor of Bruton's agammaglobulinemia tyrosine kinase (BTK), with potential antineoplastic activity. Upon administration, spebrutinib targets and covalently binds to BTK, thereby preventing its activity. By irreversibly inhibiting BTK, administration of this agent may lead to an inhibition of B cell receptor (BCR) signaling and may inhibit cell proliferation of B-cell malignancies. BTK, a cytoplasmic tyrosine kinase and member of the Tec family of kinases, plays an important role in B lymphocyte development, activation, signaling, proliferation and survival.

C28H28FN5O6S

581.615228652954

581.174

C, 57.82; H, 4.85; F, 3.27; N, 12.04; O, 16.50; S, 5.51

1360053-81-1

Spebrutinib;1202757-89-8

74892828

Solid

6.485

4

11

11

41

745

0

COCCOC1=CC=C(NC2=NC=C(F)C(NC3=CC=CC(NC(C=C)=O)=C3)=N2)C=C1.O=S(C4=CC=CC=C4)(O)=O

ABSXPNGWJFAPRT-UHFFFAOYSA-N

InChI=1S/C22H22FN5O3.C6H6O3S/c1-3-20(29)25-16-5-4-6-17(13-16)26-21-19(23)14-24-22(28-21)27-15-7-9-18(10-8-15)31-12-11-30-2;7-10(8,9)6-4-2-1-3-5-6/h3-10,13-14H,1,11-12H2,2H3,(H,25,29)(H2,24,26,27,28);1-5H,(H,7,8,9)

benzenesulfonic acid;N-[3-[[5-fluoro-2-[4-(2-methoxyethoxy)anilino]pyrimidin-4-yl]amino]phenyl]prop-2-enamide

AVL292; AVL-292; AVL 292; CC292; AVL-292 benzenesulfonate; Spebrutinib besylate; AVL-292 (benzenesulfonate); AVL 292 benzenesulfonate; AVL-292 BESYLATE; Spebrutinib besilate; Spebrutinib besylate [USAN]; CC-292; CC 292

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: ≥ 45 mg/mL (~106.3 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.)