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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.

Park JK, Byun JY, Park JA, Kim YY, Lee YJ, Oh JI, Jang SY, Kim YH, Song YW, Son J, Suh KH, Lee YM, Lee EB - Arthritis Res. Ther. (2016)

Bottom Line: 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.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.Thus, HM71224 is a potential novel therapeutic agent for rheumatoid arthritis in humans.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Department of Internal Medicine, Seoul National University College of Medicine, 101 Daehank-no Chongno-gu, Seoul, 03080, Korea.

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.

No MeSH data available.


Related in: MedlinePlus

HM71224 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 kinase
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Fig3: HM71224 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 kinase

Mentions: We next examined the effects of HM71224 on primary human cells. Upon BCR ligation, phosphorylation of Btk was followed by rapid phosphorylation of PLCγ2. HM71224 effectively inhibited the phosphorylation of Btk and PLCγ2 (Fig. 3a). This inhibitory effect was confirmed by phospho-flow cytometry (Additional file 1: Figure S3). In addition, the surface expression of CD40, CD69, and CD86 by activated primary human B cells was markedly inhibited by HM71224 (IC50 = 4.2 nM for CD40; IC50 = 4.2 nM for CD69; and IC50 = 7.7 nM for CD86) (Fig. 3b). HM71224 occupancy of Btk in primary human cells was dose-dependent: 100 nM HM71224 occupied >70 % of Btk sites (Fig. 3c).Fig. 3


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.

Park JK, Byun JY, Park JA, Kim YY, Lee YJ, Oh JI, Jang SY, Kim YH, Song YW, Son J, Suh KH, Lee YM, Lee EB - Arthritis Res. Ther. (2016)

HM71224 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 kinase
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4835877&req=5

Fig3: HM71224 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 kinase
Mentions: We next examined the effects of HM71224 on primary human cells. Upon BCR ligation, phosphorylation of Btk was followed by rapid phosphorylation of PLCγ2. HM71224 effectively inhibited the phosphorylation of Btk and PLCγ2 (Fig. 3a). This inhibitory effect was confirmed by phospho-flow cytometry (Additional file 1: Figure S3). In addition, the surface expression of CD40, CD69, and CD86 by activated primary human B cells was markedly inhibited by HM71224 (IC50 = 4.2 nM for CD40; IC50 = 4.2 nM for CD69; and IC50 = 7.7 nM for CD86) (Fig. 3b). HM71224 occupancy of Btk in primary human cells was dose-dependent: 100 nM HM71224 occupied >70 % of Btk sites (Fig. 3c).Fig. 3

Bottom Line: 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.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.Thus, HM71224 is a potential novel therapeutic agent for rheumatoid arthritis in humans.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Department of Internal Medicine, Seoul National University College of Medicine, 101 Daehank-no Chongno-gu, Seoul, 03080, Korea.

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.

No MeSH data available.


Related in: MedlinePlus