Control of Neutrophil Endothelial Transmigration By EC-18 in Chemotherapy Induced Neutropenia

Chemotherapy-induced neutropenia (CIN) is a common dose-limiting side-effect that often compromises chemotherapy resulting in dose reduction and/or delayed treatment. Chemotherapy-induced myeloid suppression is a prevalent mechanism to describe CIN. However, diapedesis of neutrophils from the blood is also a possible mechanism of CIN. Cytokines, chemokines, and damage- (or danger-) associated molecular pattern molecules (DAMPs) are produced from the damaged tissues by chemotherapy, which accelerates neutrophil transmigration. EC-18 is a synthetic monoacetyldiglyceride (MW=635) currently under development as an oral agent to treat and potentially prevent CIN. Here, we showed that neutrophil transmigration is induced in CIN model, and the efficacy and potential mechanism of EC-18 have been evaluated in mice and various cell models. Gemcitabine (50mg/Kg), cyclophosphamide (100mg/Kg) or tamoxifen (50mg/Kg) was administered daily for 3 days in mice (C57BL/6), followed by oral administrations of EC-18 (50mg/Kg) for 21 days. Blood neutrophils were analyzed by complete blood count (CBC) and fluorescence-activated cell sorting (FACS). All three chemotherapeutic agents significantly reduced absolute neutrophil count (ANC) in the blood; 40.4% decrease in gemcitabine, 19.8% in cyclophosphamide, and 21.8% in tamoxifen, which were recovered by the co-administration of EC-18. Microarray analysis of the lymph nodes from gemcitabine-treated mice revealed that the expression of DAMP molecules, S100A8 and S100A9, was induced by gemcitabine and decreased by the co-treatment of EC-18. Using immune cell lines, the expression of neutrophil transmigratory molecules, such as C3 anaphylatoxin, IL-6 and IL-8, was increased by gemcitabine treatment, and EC-18 blocked the expression of those cytokines in dose-dependent manners. Western blot analysis showed that EC-18 regulated STAT3 and STAT6 signaling pathways to modulate the expression of DAMPs, C3, IL-6, and IL-8. The decrease of C3 and IL-6 levels by EC-18 treatment was also verified in PBMCs from normal adults. In a pilot clinical study (WJON 2015 accepted, ASCO e20697), orally administrated EC-18 (1,000mg/day) alleviated neutropenic phenotypes in patients with pancreatic cancer who had been treated with gemcitabine (1,000mg/m2) and erlotinib (100mg daily). As a supportive oncology agent, EC-18 can maximize the antitumor efficacy of myelo-suppressive chemotherapeutic agents by enabling the treatment to continue or even higher doses to be administered.