| In Vitro | In vitro activity: AZD-5069 is a novel and potent CXCR2 chemokine receptor antagonist with potential anticancer and antiinflammatory activities. The CXC chemokine receptor CXCR2 is upregulated in a variety of different tumor cell types and involved in tumor cell proliferation and progression. Inhibition of CXCR2 led to reduced metastasis and decreased tumorigenesis. AZD-5069 inhibits the binding of radiolabeled CXCL8 to human CXCR2 with a pIC50 value of 9.1. AZD5069 as an antagonist of CXCR2 is currently being investigated in phase Ib/II studies in combination with tremelimumab for patients with advanced solid tumors and metastatic squamous cell carcinoma of the head and neck. AZD-5069 has the potential to treat patients with cancer and inflammatory conditions such as COPD.
Kinase Assay: AZD-5069 acts as CXCR2 antagonist by inhibition of radiolabelled [125I]-IL-8 binding to human CXCR2 receptors and as inhibitor of GROα-induced Ca2+ flux in human neutrophils loaded with fluo-3 dye.
Cell Assay: Human embryonic kidney 293 (HEK293) cells, expressing recombinant human CXCR2 or CXCR1, were grown to approximately 80% confluence in Dulbecco’s modified Eagle’s medium–Glutamax medium (Life Technologies Ltd, Paisley, UK) containing 10% (v/v) fetal calf serum and 0.5 mg/ml geneticin in a humidified incubator at 37°C, 5% CO2. Cells were harvested from the flask using Accutase (Sigma-Aldrich Company Ltd., Dorset, UK) at 37°C for 3–5 minutes. |
|---|
| In Vivo | Rat Airway LPS Challenge Model.: Rats in treatment groups (eight per group) were dosed orally 1 hour before LPS challenge with AZD5069, AZD8309, or AZ10397767 or dexamethasone (5.8 μmol/kg). Rats in group 1 were challenged with 0.9% saline. Rats in groups were challenged with 0.1 mg/ml LPS in saline (0.9%) or saline vehicle control. The rats were placed in perspex boxes and challenged with an aerosol generated with two jet nebulizers operated at an airflow rate of 12 l/min for 30 minutes. At 4 hours following LPS challenge, the trachea was cannulated and the airway lavaged using 3 aliquots of 3.3 ml of sterile PBS at room temperature. An aliquot of lavage fluid was removed for cell counting. Cytospin slides were prepared by adding a 100-μl aliquot of lavage fluid into cytospin funnels in a Shandon Cytospin3 (GMI, Inc., Ramsey, MN) operated at 700 rpm for 5 minutes. Slides were stained with Wright-Giemsa stain using a Hema-Tek-2000 automatic slide stainer (Fisher Scientific Ltd, UK, Loughborough, UK), and typically, 200 cells were counted under a microscope. Cells were classified as eosinophils, neutrophils, and mononuclear cells. Mononuclear cells included monocytes, macrophages, and lymphocytes. The number of neutrophils was quantified by expressing the cell number as a percentage of the total count. The number of neutrophils per animal was averaged across each treatment group and the result expressed as the mean ± S.E.M. Results between treatment groups were compared using nonparametric statistics, Mann-Whitney, or Kruskal-Wallis methodology in GraphPad InStat. Rats were anesthetized with isofluorane, and terminal blood samples (0.5 and 2 ml) were taken from the abdominal vena cava of animals and collected. Animals were then terminated with 1.0 ml of pentobarbitone sodium intraperitoneally. One set of blood samples was assessed for differential cell numbers using an Advia Haematology System (Siemens, London, UK). The other set of blood samples (2 ml) was centrifuged at 2800g for 10 minutes at 4°C. The plasma was removed and stored at –20°C for subsequent determination of compound concentration. |
|---|
m.cnreagent.com