HM71224, a novel Bruton's tyrosine kinase inhibitor, suppresses B cell and monocyte activation and ameliorates arthritis in a mouse model: a potential drug for rheumatoid arthritis.
Division of Rheumatology, Department of Internal Medicine, Seoul National University College of Medicine, 101 Daehank-no Chongno-gu, Seoul, 03080, Korea.
Hanmi Research Center, Hanmi Pharm.Co., Ltd., 550 Dongtangiheung-ro, Hwaseong-si, Gyeonggi-do, 18469, Republic of Korea.
Hanmi Pharm.Co., Ltd., 45 Bangi-dong, Songpa, Seoul, Gyeonggi-do, 138-724, Republic of Korea.
Hanmi Research Center, Hanmi Pharm.Co., Ltd., 550 Dongtangiheung-ro, Hwaseong-si, Gyeonggi-do, 18469, Republic of Korea. leeym@hanmi.co.kr.
Division of Rheumatology, Department of Internal Medicine, Seoul National University College of Medicine, 101 Daehank-no Chongno-gu, Seoul, 03080, Korea. leb7616@snu.ac.kr.
Abstract
BACKGROUND Bruton's tyrosine kinase (Btk) is critical for activation of B cells and myeloid cells. This study aimed to characterize the effects of HM71224, a novel Btk inhibitor, both in vitro and in a mouse model of experimental arthritis.
METHODS The kinase inhibition profile of HM71224 was analyzed. The in vitro effects of HM71224 on B cells and monocytes were analyzed by examining phosphorylation of Btk and its downstream signaling molecules, along with cytokine production and osteoclast formation. The in vivo effects of HM71224 were investigated in a mouse model of collagen-induced arthritis (CIA).
RESULTS HM71224 irreversibly bound to and inhibited Btk (IC50 = 1.95 nM). The compound also inhibited the phosphorylation of Btk and its downstream molecules such as PLCγ2, in activated Ramos B lymphoma cells and primary human B cells in a dose-dependent manner. Furthermore, HM71224 effectively inhibited the production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β by human monocytes, and osteoclast formation by human monocytes. Finally, HM71224 improved experimental arthritis and prevented joint destruction in a murine model of CIA.
CONCLUSIONS HM71224 inhibits Btk in B cells and monocytes and ameliorates experimental arthritis in a mouse model. Thus, HM71224 is a potential novel therapeutic agent for rheumatoid arthritis in humans.
Keywords
B cellsBtk inhibitorHM71224InflammationMonocytesOsteoclastRheumatoid arthritis
Figures
Fig. 1Kinase selectivity of the Bruton’s tyrosine kinase (Btk) inhibitor, HM71224. a Kinome Binding Tree Spot analysis of HM71224 based on Scan Max data (DiscoveRx). HM71224 selectively bound to Btk (a TEC family kinase) at a concentration of 2 μM (indicated by red circles). b Selectivity of Btk for a screened panel of kinases. *TEC family kinase (TFK), EGFR epidermal growth factor receptor, JAK Janus kinase, BMX bone marrow kinase on chromsome X, TK tyrosine kinase, TKL tyrosine kinase-like, STE homolog of Sterile, CK1 Casein kinase 1, AGC protein kinase A, G, and C families, CAMK Ca2+/calmodulin-dependent protein kinase, CMGC cyclin-dependent kinases (CDKs), mitogen-activated protein kinases (MAP kinases), glycogen synthase kinases (GSK), CDK-like kinases, BLK B-cell lymphocyte kinase, ITK IL2-Inducible T-Cell Kinase, JAK3 Janus kinase 3, LCK lymphocyte-specific protein tyrosine kinase, CSK C-Src Tyrosine KinaseFig. 2Effect of HM71224 on Bruton’s tyrosine kinase (Btk) signaling. a The inhibitory effects of HM71224 on B cell receptor (BCR) signaling in the Ramos cell line. Ramos cells were stimulated with anti-IgM F(ab’)2 fragments in the presence of the indicated doses of HM71224. b Time-dependent restoration of Btk phosphorylation in Ramos cells after HM71224 washout. c Btk occupancy by HM71224 on Ramos cells. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. untreated control (crtl). ERK extracellular signal-regulated kinaseFig. 3HM71224 inhibits human B cell activation. a Healthy primary human CD19+ B cells (n = 3) were activated with anti-IgM F(ab’)2 fragments in the presence of increasing concentrations of HM71224. Phosphorylation of Bruton’s tyrosine kinase (Btk) and PLCγ2 in human B cells was inhibited in a dose-dependent manner. The immunoblot shown is representative of three independent experiments. b HM71224 inhibits the surface expression of CD40, CD69, and CD86 by activated B cells. c Percentage of Btk sites on human peripheral blood mononuclear cells (PBMCs) occupied by HM71224, as measured by a biotinylated probe. *P < 0.001 vs. 0 nM HM71224. ERK extracellular signal-regulated kinaseFig. 4HM71224 inhibits cytokine production by human peripheral blood monocytes and plasmacytoid dendritic cells. a Human CD14+ monocytes were isolated from healthy donors (n = 3) and stimulated with heat-inactivated immune complexes (ICs) in the presence of HM71224. The levels of the indicated cytokines in the culture supernatant were then measured by ELISA at 5 h post stimulation. HM71224 significantly reduced the levels of secreted cytokines in a dose-dependent manner. b Human peripheral blood dendritic cells were isolated from healthy donors (n = 2) and stimulated with CpG oligodeoxynucleotides (CpG ODN) or lipopolysaccharide (LPS). The levels of the indicated cytokines in the culture supernatant were then measured by ELISA at 48 h post stimulation.*P < 0.05, **P < 0.01, and ***P < 0.001 vs. no treatment. LPS lipopolysaccharideFig. 5HM71224 inhibits osteoclastogenesis. a Representative images of osteoclastogenesis. Human CD14+ monocytes were cultured with the indicated concentrations of HM71224 during osteoclast differentiation (magnification × 100). b Dose-dependent inhibition of tartrate resistant alkaline phosphatase (TRAP)+ cells by HM71224. *P < 0.05 and **P < 0.01 vs. no treatment. MNGs is multinucleated giant cellsFig. 6HM71224 ameliorates symptoms in a mouse model of collagen-induced arthritis (CIA). As described in “Methods”, arthritis was induced by immunization with collagen. From 10 days after the second immunization, mice were treated with oral HM71224, dexamethasone (Dexa), or vehicle (Veh). Compared with vehicle, treatment with HM71224 led to dose-dependent improvements in the clinical arthritis score (a) and body weight (b). c HM71224 reduced serum IL-6 (left), and anti-type II collagen (middle) and total IgG levels (right). d The bone erosion was assessed on a hematoxylin and eosin section. HM71224 treatment reduced the degree of bone erosion. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. CIA mice treated with vehicle. Ctrl (healthy) control
