p38 MAP kinase inhibitors: many are made, but few are chosen.

The mitogen-activated protein kinase (MAPK) p38 is a Ser/Thr kinase, originally isolated from lipopolysaccharide-stimulated monocytes. There are four isoforms of the enzyme (p38alpha, p38beta, p38gamma and p38delta), which differ in tissue distribution, regulation of kinase activation and subsequent phosphorylation of downstream substrates. These enzymes also differ in sensitivity to p38 MAPK inhibitors. The most thoroughly studied isoform is p38alpha, for which activation has been observed in many hematopoietic and non-hematopoietic cell types upon appropriate stimuli. p38alpha kinase is involved in the biosynthesis of the cytokines tumor necrosis factor-alpha and interleukin-1beta at the translational and transcriptional level. MAPK p38alpha represents a point of convergence for multiple signaling processes that are activated during inflammation, making it a key potential target for the modulation of cytokine production. The discovery and publication of p38alpha and a pyridinyl-imidazole-based p38alpha inhibitor initiated a huge effort by many companies to develop p38alpha inhibitors as potential treatments for inflammatory diseases. Herein, a brief overview is provided of the discovery and development of AMG-548 (Amgen Inc), a selective and efficacious p38alpha inhibitor, and its pharmacodynamic effects in a first-in-human study. Data from a phase I multidose clinical trial are also included. In addition, other p38alpha inhibitors that have advanced to clinical trials over the last three years are discussed, such as BIRB-796 (Boehringer Ingelheim Pharmaceuticals Inc), SCIO-469 and SCIO-323 (Scios Inc), and VX-702 (Vertex Pharmaceuticals Inc/Kissei Pharmaceutical Co).

Characterization of bispecific T-cell Engager (BiTE) antibodies with a high-capacity T-cell dependent cellular cytotoxicity (TDCC) assay.

The Bispecific T-cell Engager (BiTE) antibody modality is a clinically validated immunotherapeutic approach for targeting tumors. Using T-cell dependent cellular cytotoxicity (TDCC) assays, we measure the percentage of specific cytotoxicity induced when a BiTE molecule engages T-cells, redirects T-cell mediated cytolysis, and ultimately kills target cells. We establish a novel luminescence-based TDCC assay quantified by measuring cell viability via constitutive expression of luciferase. The luciferase-based TDCC assay performance is valid and comparable to an adenosine triphosphate (ATP)-based detection method. We demonstrate that the luciferase-based TDCC assay is an efficient homogeneous assay format that is amenable to both suspension and adherent target cells. The luciferase-based TDCC assay eliminates the need for plate-washing protocols, allowing for higher-throughput screening of BiTE antibodies and better data quality. Assay capacity is also improved by performing serial dilutions of BiTE antibodies in 384-well format with an automated liquid handler. We describe here a robust, homogeneous TDCC assay platform with capacity for in vitro assessment of BiTE antibody potency and efficacy using multiple tumor cell lines and T-cell donors.

3-amino-7-phthalazinylbenzoisoxazoles as a novel class of potent, selective, and orally available inhibitors of p38alpha mitogen-activated protein kinase.

The p38 mitogen-activated protein kinase (MAPK) is a central signaling molecule in many proinflammatory pathways, regulating the cellular response to a multitude of external stimuli including heat, ultraviolet radiation, osmotic shock, and a variety of cytokines especially interleukin-1beta and tumor necrosis factor alpha. Thus, inhibitors of this enzyme are postulated to have significant therapeutic potential for the treatment of rheumatoid arthritis, inflammatory bowel disease, osteoporosis, and many other diseases where aberrant cytokine signaling is the driver of disease. Herein, we describe a novel class of 3-amino-7-phthalazinylbenzoisoxazole-based inhibitors. With relatively low molecular weight, these compounds are highly potent in enzyme and cell-based assays, with minimal protein shift in 50% human whole blood. Compound 3c was efficacious (ED 50 = 0.05 mg/kg) in the rat collagen induced arthritis (CIA) model.