RO9021 was administered orally, randomized into different groups, every day for 14 days starting on the day after second immunization. Clinical arthritis scores of individual paws were assessed and the arth ritic index for each mouse was determined by adding the individual scores of all four paws. The level of cyto kines in serum was determined by Luminex analysis. Histopathological analysis Hind paws from CIA mice were collected into 10% neu tral buffered formalin. After decalcification in 10% for mic acid, paws were embedded in paraffin, sectioned at 8 um and stained with toluidine blue. Inflammation, pannus, cartilage damage, and bone resorption were scored in a double blinded fashion by a board certified pathologist at Boulder BioPATH, Inc. using standard cri teria, with 0 being normal and 5 being the most severe.

Half maximal inhibitory concentration determination and statistical analysis Half maximal inhibitory concentration values and dose/concentration response curves were determined by sigmoidal dose response curve fitting using XLFit or Prism. In most studies, the IC50 values reported were the average from at least two studies conducted with samples in replicate. For in vivo studies, one factor and two factor comparisons were performed, respectively, using one way or two way analysis of variance plus Dunnetts post test. Results Biochemical characterization of RO9021, a potent and selective SYK inhibitor RO9021 was identified following extensive medicinal chemistry optimization of a lead identified from high throughput screening of Roches proprietary chemical compounds library.

In a SYK kinase enzymatic assay, RO9021 potently inhibited SYK kinase activity with an average IC50 of 5. 6 nM. Selectivity of RO9021 against a panel of 451 wild type and mutant protein kinases was assessed using an ATP binding site competition assay developed by KINOMEscan Inc. As shown in the dendrogram depicting a qualitative overall impression of kinase selectivity, RO9021 was highly selective for SYK enzyme at 1 uM concentration. The selectivity of RO9021 was quantitatively expressed as a selective score, which was calculated by dividing the number of RO9021 bound kinases by the total Dacomitinib number of wild type protein kinases tested, excluding mutant variants. The S score is an unbiased measure that enables quantitative comparisons between com pounds.

A lower S score means higher selectivity. As shown in Figure 1D, RO9021 is a highly selective SYK inhibitor with low S scores of 0. 003 for S and 0. 015 for S, indicating that SYK is the only kinase with 99% competition with RO9021 in a total of 392 tested kinases. There were only a total of seven kinases, including SYK, having more than 90% competition with RO9021. The expected binding mode of RO9021 was confirmed by the determination of the co crystal structure of RO9021 and the SYK protein kinase domain.