Enzyme Inhibition

Btk activity was quantified by determining phosphorylation of an exogenous peptide product in a Lanthascreen assay. In a final reaction volume of 25 μl, Btk [human, full-length, C-terminal V5–6× His, expressed in Sf9 cells; 0.075 ng/25 μl reaction) was incubated with 50 mM HEPES, pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 2 mM dithiothreitol, 0.2 mM NaVO4, 0.01% casein, 0.01% Triton X-100, 2.5% glycerol, and 0.4 μM fluorescein poly-Glu/Ala/Tyr. The reaction was initiated by the addition of ATP to 25 μM (Km of ATP). After incubation for 60 min at room temperature, the reaction was stopped by the addition of a final concentration of 2 nM Tb-PY20 detection antibody in 60 mM EDTA for 30 min at room temperature. Detection was determined on a PerkinElmer Envision with 340-nM excitation and emission at 495 and 520 nm.

Cell lines

Rat splenocytes

Preparation method

This compound is soluble in DMSO. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reacting condition

Five hundred microliters of cell suspension (1×107 cells/ml) was incubated with GDC-0834 for 1 h at 37℃, followed by stimulation with 3 μg/ml αIgM (final concentration) for 3 min.

Applications

In the rat splenocytes, GDC-0834 suppressed pBTK activity with an IC50 value of 6.4 ± 1.6 nM with Hill slope value of 0.76 ± 0.19.

Animal models

Female arthritis Lewis rats

Dosage form

Oral dosing of GDC-0834 [vehicle, 1, 3, 10, 30, and 100 mg/kg b.i.d. (twice daily) at 12-h intervals, vehicle, 10, 30, 100 mg/kg once daily (QD) at 24-h intervals, and 100 mg/kg every other day (Q2D) at 48 intervals] was initiated on day 0 of the study and continued through day 16.

Application

Treatment with GDC-0834 resulted in a significant reduction of the area under the ankle diameter-time curve in arthritis rat model.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

GDC-0834 is a potent and selective Bruton's tyrosine kinase (Btk) inhibitor, The IC50 values in biochemical and cellular assays is 5.9 and 6.4 nM. The in vivo IC50 values in mouse and rat are 1.1 and 5.6 μM, respectively [1]. B cell antigen receptor (BCR) stimulation phosphorylated and activated Btk. Btk gene defects result in a disease named as X-linked agammaglobulinemia which profoundly decreased mature B cells due to a block in B cell development [2].

In vivo: In a rat collagen-induced arthritis (CIA) model, administration of GDC-0834 (30-100 mg/kg) model resulted in a dose-dependent decrease of ankle swelling and reduction of morphologic pathology [1].In PXB chimeric mice with humanized liver, GDC-0834 exhibited low clearance. in most samples from the cohorts dosed orally at 35 and 105 mg GDC-0834, plasma concentrations of GDC-0834 in humans were below the limit of quantitation (<1 ng/ml). High interest in a BTK inhibitor for clinical evaluation and uncertainty in human pharmacokinetic prediction prompted an investigational new drug strategy, in which GDC-0834 was rapidly advanced to a single-dose human clinical trial [3].

References:
[1]. Liu L, Di Paolo J, Barbosa J, et al. Antiarthritis effect of a novel Bruton's tyrosine kinase (BTK) inhibitor in rat collagen-induced arthritis and mechanism-based pharmacokinetic/pharmacodynamic modeling: relationships between inhibition of BTK phosphorylation and efficacy[J]. Journal of Pharmacology and Experimental Therapeutics, 2011, 338(1): 154-163.
[2]. Takata M, Kurosaki T. A role for Bruton's tyrosine kinase in B cell antigen receptor-mediated activation of phospholipase C-gamma 2[J]. The Journal of experimental medicine, 1996, 184(1): 31-40.
[3]. Liu L, Halladay J S, Shin Y, et al. Significant species difference in amide hydrolysis of GDC-0834, a novel potent and selective Bruton's tyrosine kinase inhibitor[J]. Drug Metabolism and Disposition, 2011, 39(10): 1840-1849.