Bruton tyrosine kinase (BTK) (IC50 = 1 nM)
The primary molecular target of Remibrutinib is Bruton's tyrosine kinase (BTK), a non-receptor tyrosine kinase that plays a critical role in B cell receptor (BCR) signaling, Fc receptor signaling, and other immune cell functions. Remibrutinib is a covalent irreversible inhibitor that binds to the inactive conformation of BTK, forming a covalent bond with Cys481 in the kinase domain. The compound has an IC50 of 1 nM for BTK inhibition and inhibits BTK activity in blood with an IC50 of 0.023 µM. It selectively binds to BTK over bone marrow tyrosine kinase on chromosome X (BMX) and Tec with Kds of 0.63, 540, and 110 nM, respectively, and shows >10,000 nM affinity for Itk, EGFR, HER2, HER4, and JAK3. By inhibiting BTK, Remibrutinib blocks BCR signaling, reduces B cell activation, and suppresses the production of autoantibodies and inflammatory cytokines.

Remibrutinib (Example 6) inhibited Btk enzyme activity in the biochemical enzyme assay, with an IC50 value of 1 nM [1]. Remibrutinib reduced blood Btk enzyme activity in an in vitro B cell activation test, with an IC50 value of 0.023 μM. Sector Lewis was put under anesthesia, and whole blood was extracted from the abdominal aorta [1].
In vitro studies demonstrate that Remibrutinib is a potent and highly selective BTK inhibitor. The compound has an IC50 of 1 nM for BTK inhibition in biochemical assays and inhibits BTK activity in blood with an IC50 of 0.023 µM. Remibrutinib inhibits anti-IgM/IL-4-induced CD69 expression on human blood B cells with an IC50 of 18 nM, demonstrating functional inhibition of BCR signaling in primary human cells. The compound also inhibits FcγR-induced ILB with an IC50 of 2.5 nM, indicating effects on Fc receptor signaling. Remibrutinib shows high selectivity for BTK over other kinases, with Kds of 0.63 nM for BMX, 540 nM for Tec, 110 nM for Itk, and >10,000 nM for EGFR, HER2, HER4, and JAK3. The compound's covalent binding to the inactive conformation of BTK confers a distinct selectivity profile.

Remibrutinib inhibits Bruton’s tyrosine kinase with high selectivity, potency, and covalent inhibition. In clinical trials, it ameliorated mast cell-mediated inflammatory effects in chronic urticaria. Remibrutinib inhibits mast cell and basophil degranulation, including release of histamine and other proinflammatory mediators, mediated by pathogenic IgE or IgG directed against the FcεR1 or IgE. Remibrutinib is a selective Bruton's tyrosine kinase inhibitor. The FDA approved remibrutinib on September 30, 2025 as the only oral, targeted treatment for chronic spontaneous urticaria. Bruton's tyrosine kinase plays a role in B cell receptor and Fc receptor signalling, releasing histamine and pro-inflammatory mediators. Remibrutinib has also been investigated in other inflammatory conditions, such as autoimmune encephalomyelitis and Sjögren's syndrome.
In vivo studies of Remibrutinib have demonstrated its efficacy in animal models of autoimmune diseases. As an orally active BTK inhibitor, Remibrutinib is suitable for convenient administration. In preclinical studies, the compound has been shown to reduce B cell activation, suppress autoantibody production, and ameliorate disease in models of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and other immune-mediated conditions. In vivo protocols typically involve oral administration of Remibrutinib at doses determined from pharmacokinetic studies. Pharmacodynamic endpoints include assessment of BTK occupancy, B cell activation markers, autoantibody levels, and disease severity. The compound's oral bioavailability and favorable pharmacokinetic profile support once-daily dosing.

For BTK kinase assays, the enzymatic activity of recombinant human BTK is measured using a radiometric or fluorescence-based assay. Purified BTK enzyme is incubated with varying concentrations of Remibrutinib (typically 0.001-100 µM) in reaction buffer containing 50 mM HEPES (pH 7.5), 10 mM MgCl₂, 1 mM DTT, and 0.01% Triton X-100 at 30°C. The reaction is initiated by the addition of ATP and a peptide substrate, and incubated for 30-60 minutes. IC50 values are calculated from dose-response curves. For selectivity profiling, the compound is tested against a panel of kinases, including BMX, Tec, Itk, EGFR, HER2, HER4, and JAK3.

For cellular BTK activity assays, B cells or other BTK-expressing cells are cultured in appropriate medium and treated with Remibrutinib (0.001-10 µM) for 1-4 hours. Cells are stimulated with anti-IgM or other BCR agonists, and BTK phosphorylation (autophosphorylation at Tyr223) and downstream signaling (e.g., PLCγ2 phosphorylation) are assessed by Western blot. For B cell activation assays, human peripheral blood mononuclear cells or purified B cells are treated with Remibrutinib and stimulated with anti-IgM/IL-4. CD69 expression on B cells is measured by flow cytometry.

For in vivo efficacy studies, adult mice (e.g., collagen-induced arthritis model, lupus model) are used. Remibrutinib is formulated in vehicle (e.g., 0.5% methylcellulose or 10% DMSO in PEG400) and administered orally at doses of 1-30 mg/kg, typically once daily for 2-4 weeks. Disease severity is assessed by clinical scores, joint swelling, autoantibody levels (e.g., anti-dsDNA, anti-collagen antibodies), and histopathological examination of affected tissues. BTK occupancy in blood and tissues is measured by a probe-based assay. For pharmacokinetic studies, blood and tissue samples are collected at various time points for compound quantification by LC-MS.

Pharmacokinetic data for Remibrutinib indicate that it is orally bioavailable and has a favorable pharmacokinetic profile. The compound's pharmacokinetic properties support once-daily dosing. Specific pharmacokinetic parameters, including half-life, Cmax, Tmax, and AUC, may be available from published studies but are not extensively reported in the public domain. The compound's favorable pharmacokinetic properties contribute to its potential as a best-in-class BTK inhibitor.

Toxicological data for Remibrutinib are primarily derived from preclinical toxicology studies in animal models. As a covalent BTK inhibitor, the compound is expected to have on-target toxicities related to BTK inhibition, including effects on B cell function and immune responses. In preclinical studies, Remibrutinib has been shown to be well-tolerated at pharmacologically active doses. The compound's selectivity for BTK over other kinases may contribute to a favorable safety profile. No acute toxicity, organ-specific toxicity, or mutagenicity data have been reported in the public domain. As with all research compounds, appropriate safety precautions should be taken when handling Remibrutinib.

[1]. Novel amino pyrimidine derivatives. Patent WO2015079417A1.

[2]. New treatments for chronic urticaria. Ann Allergy Asthma Immunol. 2020 Jan;124(1):2-12.

Drug Indication
Treatment of Multiple Sclerosis Treatment of Chronic Spontaneous Urticaria (CSPI) Remibutinib is a kinase inhibitor. Remibutinib's mechanism of action is as a Bruton's tyrosine kinase inhibitor and a P-glycoprotein inhibitor. Remibutinib is a small molecule drug that has completed Phase III clinical trials (covering all indications) and has six investigational indications. The approval of Remibutinib is a significant advancement in the treatment of chronic spontaneous urticaria. "It rapidly relieves symptoms, helping patients control daily hives and itching," said Giselle Mosnaim, MD, MSc, allergist and immunologist at Endeavor Health, clinical associate professor at the University of Chicago Pritzker School of Medicine, and investigator of the REMIX trial. "This is significant because it goes beyond existing injectable therapies, providing patients with an oral option that can be easily integrated into their daily lives." Purpose: Chronic urticaria (CU) is a common, heterogeneous, and distressing disease. Antihistamines and omalizumab are the main treatment options for chronic urticaria (CU). However, more treatment options are needed. This article reviews off-label use of approved drugs, new therapies currently under development, and promising new targets. Data Sources: We searched the MEDLINE database for recent reports on successful applications of CU treatment and promising new therapies and targets for development. We also searched ClinicalTrials.gov for recent and ongoing randomized clinical trials of CU. Study Selection: We screened and reviewed relevant articles. Results: Omalizumab is the first-line treatment for patients with antihistamine-resistant chronic spontaneous urticaria (CSU) and its use in treating chronic inducible urticaria in children under 12 years of age with CSU should be explored, and higher doses should be considered. Off-label use of dupilumab, relizumab, mepolizumab, and benazepril may be effective for chronic urticaria (CU). Two novel anti-IgE monoclonal antibodies, linfalimumab and UB-221, are currently undergoing clinical trials for CU. Other potential drugs under development for the treatment of chronic urticaria (CU) include: antagonists of chemokine receptor homologs expressed on TH2 cells, anti-Siglec-8 monoclonal antibody (AK002), Bruton's tyrosine kinase inhibitors (fenbubrutinib and Lou064), spleen tyrosine kinase inhibitors, and dupilumab. Potential targets for future treatment include Mas-associated G protein-coupled receptor X2; histamine 4 receptor; C5a and its receptor; inhibitory mast cell receptors other than Siglec-8; interleukin-33, interleukin-25, thymic stromal lymphopoietin, and stem cell factors. Conclusion: There is an urgent need for novel and effective therapies for chronic urticaria (CU). Some drugs have entered clinical trials (e.g., linigzumab), and more drugs should be developed by leveraging the numerous potential targets recently discovered and characterized.
Remibrutinib (LOU064) is a potent, highly selective, orally active, covalent irreversible BTK inhibitor. It has an IC50 of 1 nM for BTK inhibition and inhibits BTK activity in blood with an IC50 of 0.023 µM. The compound binds to the inactive conformation of BTK, conferring a distinct selectivity profile. It selectively binds to BTK over BMX, Tec, Itk, EGFR, HER2, HER4, and JAK3. Remibrutinib inhibits anti-IgM/IL-4-induced CD69 expression on B cells with an IC50 of 18 nM. It is being developed for autoimmune diseases and is considered a potentially best-in-class BTK inhibitor. The compound is strictly for research use only.

C27H27F2N5O3

507.531792879105

507.208

C, 63.90; H, 5.36; F, 7.49; N, 13.80; O, 9.46

1787294-07-8

118107483

White to off-white solid powder

3.9

2

8

9

37

815

0

FC1=C(C(NC2C=C(C=C(C3C(=C(N)N=CN=3)OCCN(C(C=C)=O)C)C=2C)F)=O)C=CC(=C1)C1CC1

CUABMPOJOBCXJI-UHFFFAOYSA-N

InChI=1S/C27H27F2N5O3/c1-4-23(35)34(3)9-10-37-25-24(31-14-32-26(25)30)20-12-18(28)13-22(15(20)2)33-27(36)19-8-7-17(11-21(19)29)16-5-6-16/h4,7-8,11-14,16H,1,5-6,9-10H2,2-3H3,(H,33,36)(H2,30,31,32)

N-(3-{6-amino-5-[2-(N-methylprop-2-enamido)ethoxy]pyrimidin-4-yl}-5-fluoro-2-methylphenyl)-4-cyclopropyl-2-fluorobenzamide

LOU064; LOU-064; Remibrutinib; 1787294-07-8; NVP-LOU064-NXA; LOU064-NXA; LOU 064

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 : ~125 mg/mL (~246.29 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.10 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 20.8 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: ≥ 2.08 mg/mL (4.10 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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 3: ≥ 2.08 mg/mL (4.10 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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