It has been reported that thrombomodulin (TM) expression in endothelial cells is modulated by various agents. We investigated cellular regulatory mechanisms for TM expression in human umbilical vein endothelial cells (HUVECs), incubated with agents, by measuring the time course changes in surface TM activity, total TM antigen in cell lysates, and TM mRNA levels. While dibutyryl cAMP (3 mM) increased TM mRNA levels in HUVECs and was followed by increased TM activity, dibutyryl cGMP had no effect on TM activity. Phorbol myristate acetate (PMA) induced rapid loss of surface TM activity (approximately 8 h) and later increased TM mRNA levels between 4 h and 40 h (maximum at 24 h), resulting in biphasic effects on TM activity. Tumor necrosis factor or interleukin-1 beta suppressed surface TM activity and TM mRNA levels. Internalization/degradation of TM in HUVECs incubated with PMA or cytokines was suggested by co-culture with chloroquine. The decrease in surface TM activity observed was not caused by the release of TM molecules from the cells into the conditioned media. These results suggest that TM activity in HUVECs is modulated by independent mechanisms involving cytoplasmic TM mRNA levels and internalization/degradation of TM molecules. These regulatory mechanisms may involve protein kinase A and protein kinase C-dependent mechanisms but are independent of protein kinase G.

Previous studies have shown that thrombomodulin (TM) on endothelial cells is down-regulated by endotoxin, interleukin-1 beta (IL-1 beta), and tumor necrosis factor (TNF). This loss of anti-coagulant potential is thought to be related to the hypercoagulable state in sepsis, inflammation, and cancer. The current studies describe up-regulation of TM in human umbilical vein endothelial cells (HUVECs) by several compounds as judged by increased surface cofactor activity, surface TM antigen, and TM mRNA levels. Surface TM activity was increased by active phorbol esters (10(-8) M, 24-48 h), analogs of cAMP (1-10 mM, 4 h), and forskolin (10(-5) M, 24-48 h). Up-regulation of TM in HUVECs by 4 beta-phorbol 12-myristate 13-acetate (PMA) and dibutyryl cAMP (dBcAMP) was due to de novo synthesis of TM protein resulting from increased TM mRNA levels. The results suggest that protein kinase C and protein kinase A may be involved in cellular regulatory mechanisms for TM expression. In addition, PMA effects on surface TM activity are biphasic, with an initial reduction followed by a significant enhancement. Hence, we propose that compounds capable of increasing intracellular cAMP concentrations in HUVECs may be useful in preventing thrombosis by increasing the anti-thrombotic properties of endothelial cells.

Thrombomodulin, a glycoprotein expressed in endothelial cells, has an important role in the blood coagulation system as a modulator. Functional characterization of the 5'-regulatory region of the human thrombomodulin gene was carried out to identify elements necessary for its expression. We used a series of dissected gene constructs containing the bacterial chloramphenicol acetyltransferase gene in transient transfection assays on human umbilical vein endothelial cells. The region extending from -290 to -33 of the 5' end flanking sequence is required for the full expression of this gene. Within this region, four potential Sp1 sites were found, and the sequences of Sp1 sites were mutated to identify their role in the promoter activity of the gene, showing that the two Sp1 sites at -207 and -141 are important for the full activity of the thrombomodulin promoter. Site-directed mutation analysis identified sequence elements GCAATC at -110 as a functioning CAAT box. Another three regions, -290 to -223, -99 to -68, and -67 to -33 have unidentified positively and negatively acting elements. A silencer element was located in the region spanning from -947 to -772 bases of the 5' end flanking region. These data indicate that the expression of the thrombomodulin gene is regulated by various elements which act positively or negatively.

Previous reports demonstrated that the expression of thrombomodulin (TM) in endothelial cells was modulated by various agents. Although TM was down-regulated by endotoxin or cytokines, up-regulation of TM was accomplished when endothelial cells were stimulated with unphysiologically high concentrations of cyclic AMP derivatives or tumour-promoting phorbol esters. We investigated the expression of TM in human umbilical-vein endothelial cells (HUVECs) by physiological substances that can be released into the bloodstream. Histamine (0.1-10 microM, 1-48 h) increased TM activity, TM antigen in cell lysates and TM mRNA levels, but 5-hydroxytryptamine and bradykinin had no effect. Enhancement of TM activity by histamine was completely blocked by the H1-selective antagonist pyrilamine, whereas the H2-antagonist cimetidine had no effect, showing that histamine up-regulates TM activity via H1-receptors on HUVECs. Enhanced TM activity by histamine and the resultant increase in protein C activation might play a role in a feedback regulation for prevention of vascular thrombosis.

We would like to introduce Daiichi Sankyo's approach to developing cancer targeted medicines with special reference to the drug discovery strategy, global discovery activities and external research collaboration leading to generation of innovative drugs for cancer patients. We are developing 14 clinical projects for cancer treatment and three of them have been previously approved. These are mostly targeted for growth and survival signals of cancer cells. To overcome the drug resistance mechanism derived from the heterogeneous nature of cancer, we are developing selective inhibitors in three major clusters of signal pathways which may allow future rational combinations of oncology products. In addition to the main research facility in Japan, research sites in the EU and the USA provide us with different technical expertise and diversified ideas of drug discovery. To access novel drug targets, we are facilitating research collaboration with leading academia and successful cancer research scientists. In conclusion, we intend to focus more on developing innovative personalized medicines for better treatment of cancer. Key words: drug discovery -targeted medicine -Daiichi Sankyo DISCOVERY FOCUS IN ONCOLOGYIn 2009, we restructured and more narrowed focused therapeutic disease areas in drug discovery based upon the future trend of unmet medical needs and in-house research capability in drug discovery (Fig. 1). One of the two newly focused disease areas was oncology where better treatment is still highly demanded (1). By selecting more limited disease areas, we intended to accelerate the creation of innovative drugs without delaying novel scientific findings emerging in tumor biology.We previously developed topoisomerase-I inhibitor Irinotecan (CPT-11) with Yakult Co. in Japan and with Pfizer in the USA. The drug has meaningfully contributed to the treatment of colorectal cancer especially in the West. However, the successful R&D for cancer targeted medicine today requires largely different knowledge and technology from that utilized for chemotherapeutic agents in the past.All 14 drugs in the pipeline, both marketed and under development, are molecular targeted medicines (Table 1). Denosumab is an anti-RANKL monoclonal antibody that inhibits osteoclast activation in bone. In addition to the treatment of osteoporosis, Denosumab prevents skeletal-related events and potentially represents a novel treatment option in men with bone metastases from castration-resistant prostate cancer (2,3). Vemrafenib is a mutant BRAF kinase inhibitor approved for the first-line treatment of advanced melanoma. We thus have supported treatment of colorectal cancer, melanoma and bone metastasis with these three approved products.It has been shown that drug discovery in oncology from compound selection through drug approval is least successful according to a survey in major therapeutic disease areas, and only 2% of the research programs can get approval for commercialization (4). To reduce such a high attrition rate in drug development, we firs...