Background
TAK-779 is an antagonist of chemokine receptor 5 (CCR5), CCR2b, and CXC chemokine receptor 3 (CXCR3).1,2 It inhibits CCR5 and CXCR3 (IC50s = 236 and 369 nM, respectively, for mouse recombinant receptors expressed in 2B4 T cells) and CCR5 and CCR2b (IC50s = 1.4 and 27 nM, respectively, for human recombinant receptors expressed in CHO cells). TAK-779 inhibits the replication of clinical isolates of R5, but not X4, HIV-1 in human peripheral blood mononuclear cells (PBMCs; EC50s = 1.6-3.5 and >20,000 nM, respectively).1 TAK-779 (250 mg/animal per day) inhibits ovalbumin-induced increases in CCR5, CXCR3, IFN-γ, and TNF-α expression in mouse lung, as well as the number of total cells, lymphocytes, and eosinophils in bronchoalveolar lavage fluid (BALF), in a mouse model of asthma.3 It also increases intestinal allograft survival in a rat model of small intestine transplantation when administered at a dose of 10 mg/kg per day.4
1.Baba, M., Nishimura, O., Kanzaki, N., et al.A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective anti-HIV-1 activityProc. Natl. Acad. Sci. USA96(10)5698-5703(1999)
2.Gao, P., Zhou, X.-Y., Yashiro-Ohtani, Y., et al.The unique target specificity of a nonpeptide chemokine receptor antagonist: Selective blockade of two Th1 chemokine receptors CCR5 and CXCR3J. Leukoc. Biol.73(2)273-280(2003)
3.Suzaki, Y., Hamada, K., Nomi, T., et al.A small-molecule compound targeting CCR5 and CXCR3 prevents airway hyperresponsiveness and inflammationEur. J. Respir. J.31(4)783-789(2008)
4.Takama, Y., Miyagawa, S., Yamamoto, A., et al.Effects of a calcineurin inhibitor, FK506, and a CCR5/CXCR3 antagonist, TAK-779, in a rat small intestinal transplantation modelTranspl. Immunol.25(1)49-55(2011)
Protocol
Cell experiment: | The anti-HIV-1 activities of the test compounds (TAK-779, etc.) are based on the inhibition of virus-induced infectious focus formation in MAGI-CCR5 cells and the reduction of p24 antigen production in PBMCs. In brief, MAGI-CCR5 cells (1 × 104 cells per well) are cultured in a microtiter tray. After a 24-h incubation at 37°C, the culture supernatants are replaced with fresh culture media containing the virus (≈300 focus forming units per well) and various concentrations of the test compounds (TAK-779, etc.). After a 2-day incubation, the cells are fixed and stained with 5-bromo-4-chloro-3-indolyl-β-d-galactosidase. The number of infected (blue) cells is counted microscopically. For the PBMC assays, phytohemagglutinin-stimulated PBMCs (2.5 × 105 cells per 500 μl) are infected with HIV-1 in the presence of various concentrations of the test compounds (TAK-779, etc.). The amounts of the virus used for infection are, depending on the replicability of each strain, generally 1-10 ng of p24 per 2.5 × 105 cells. After an overnight incubation at 37°C, the cells are washed extensively to remove unadsorbed viral particles and are incubated further with culture media containing the same concentrations of the compounds as those used during viral adsorption. On day 6 after viral infection, the culture supernatants are collected and determined for their p24 antigen levels with a sandwich ELISA kit. The cytotoxicities of the compounds are evaluated in parallel with their antiviral activities. They are based on the viability and proliferation of mock-infected cells[1]. |
Animal experiment: | Mice[3]The mice are immunized with MOG and are treated s.c. with TAK-779 or vehicle. The mice (N= 10) are injected s.c. with 150 μg TAK-779 (dissolved in 5% mannitol solution) in a volume of 100 μL, once daily after MOG immunization. TAK-779 injection is started from day 0 after immunization and continued once daily for 22 days. The dose of 150 μg is determined based on the observations in prior experiments that the dose of 50 μg per mouse can not produce inhibition, and a dose of more than 100 μg per mouse is required to produce significant inhibition. The dose of 150 μg per mouse has also been used in other mouse experimental models, and approximately the same dose is used in allograft rejection and asthma models. As a control, an equal volume of PBS containing 5% mannitol is injected daily in the control mice (N= 10)[3]. |
参考文献: [1]. Baba M, et al. A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective anti-HIV-1 activity. Proc Natl Acad Sci U S A. 1999 May 11;96(10):5698-703.
[2]. Takama Y, et al. Effects of a calcineurin inhibitor, FK506, and a CCR5/CXCR3 antagonist, TAK-779, in a rat small intestinal transplantation model. Transpl Immunol. 2011 Jul;25(1):49-55.
[3]. Ni J, et al. The chemokine receptor antagonist, TAK-779, decreased experimental autoimmune encephalomyelitis by reducing inflammatory cell migration into the central nervous system, without affecting T cell function. Br J Pharmacol. 2009 Dec;158(8):2046-56.
[4]. Gao P, et al. The unique target specificity of a nonpeptide chemokine receptor antagonist: selective blockade of two Th1 chemokine receptors CCR5 and CXCR3. J Leukoc Biol. 2003 Feb;73(2):273-80. |
没有评价数据