Anti-inflammatory compositions and methods of use
a composition and anti-inflammatory technology, applied in the field of pharmaceutical compositions containing active compounds, can solve the problems of inability to meet the needs of human body, etc., and achieve the effect of reducing the risk of inflammatory disease, and reducing the number of side effects
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example 1
Materials and Methods
A. Compound Collections
[0064] The compound collection used herein consists of small molecules obtained from commercial vendors. Source plates contained individual compounds at 1 or 5 mg / ml in dimethyl sulfoxide (DMSO). From these CLIP (compound library in pools) plates were made, where 10 compounds were added per well and diluted to a concentration of 5-50 μg / ml with 20% DMSO. An aliquot of 20 μl of each mixture was put into test plates, which were stored at −20° C. until use.
B. Cells
[0065] CCR1 Transfectants
[0066] CCR1-NSO Cells
[0067] A CCR1 expressing stable transfectant cell line (CCR1-NSO) was cultured in Iscove's Modified Dulbecco's Medium (IMDM) with 4.5 g / L glucose, 5% fetal bovine serum (FBS), 10 mN HCl, 250 μg / L xanthine (from xanthine 100× stock in 1N NaOH), 15 μg / L hypoxanthine (from hypoxanthine 100× stock in 0.1N NaOH), 10 mg / L thymidine (from thymidine 100× stock in H2O), 50 μM β-mercaptoethanol (BME) (from BME 1000× stock in H2O), and 1.5 ...
example 2
Identification of Inhibitors of CCR1 Ligation of MIP-1α
A. Assay
[0078] To identify small organic molecules that prevent the receptor CCR1 from binding ligand, an assay was employed that detected radioactive ligand (MIP-1α) binding to cells expressing exogenous CCR1 on the cell surface. If a compound inhibited binding, whether competitive or not, then fewer radioactive counts would be observed when compared to uninhibited controls.
[0079] A NSO murine myeloma cell line (CCR1-NSO) and a human embryonic kidney (HEK) carcinoma cell line (CCR1-293) were constructed that constitutively expressed human CCR1 on the cell surface; these cells lack other chemokine receptors that bind MIP-1α. Equal numbers of cells were added to each well in the CLIP plate, where each pool contained 10 candidate organic compounds. The cells were then incubated with radio labeled MIP-1α. Unbound ligand was removed by washing the cells, and bound ligand was determined by quantifying radioactive counts. Cells tha...
example 3
Dose Response Curves
[0084] To ascertain a candidate compound's affinity for CCR1 as well as confirm its ability to inhibit ligand binding, inhibitory activity was titered over 1×10−8 to 1×10−4 M range of compound concentration. The assay was essentially the same as for the CLIP screens, except the amount of compound was varied; cell number and ligand concentration were held constant. Only those compounds that were commercially available at the time of the experiments were titered. Of the 22 candidates identified, the following 16 were subjected to dose response experiments: CCX-3343, CCX-1057, CCX-1307, CCX-1513, CCX-238, CCX-3345, CCX-3493, CCX-4425, CCX-4462, CCX-4682, CCX-469, CCX-5062, CCX-5119, CCX-541, CCX-6019 and CCX-6530.
[0085] Tested compounds that failed to inhibit MIP-1α binding in a dose-dependent manner were CCX-238, CCX-4425, and CCX-4462. Compounds with inhibitory activity varied in their affinity for CCR1 as presented in Table 1.
TABLE 1Affinity values for CCR1-M...
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