Delivery of pharmacologically active dexamethasone into activated endothelial cells by dexamethasone-anti-E-selectin immunoconjugate

To deliver selectively anti-inflammatory agents into activated endothelial cells, drug-targeting conjugates were developed. Dexamethasone (Dexa) was covalently linked to a monoclonal antibody specifically recognizing E-selectin, which is strongly upregulated in endothelial cells at inflammatory sites. In the present study, the pharmacological effects of this Dexa-mouse antihuman E-selectin antibody (H18/7) (Ab(hEsel)) conjugate were investigated and compared to the effects obtained by free Dexa in human umbilical vein endothelial cells. Flow cytometry and ELISA were performed to analyze the levels of cell adhesion molecules (ICAM-1 and VCAM-1) and secreted cytokines (IL-6 and IL-8). The studies were extended by analysis of a complex gene expression pattern, using a cDNA expression array containing 268 genes encoding human cytokines/cytokine-receptors. Fifty genes and 28 genes were upregulated (ratio> or =2) upon incubation of human umbilical vein endothelial cells with TNFalpha for 6 and 24hr, respectively. This gene expression profile was markedly altered when cells were activated with TNFalpha in the presence of Dexa (100 nM) or Dexa-Ab(hEsel) conjugate (10 micro g/mL conjugate corresponding to 100 nM Dexa). Relative and competitive RT-PCR analysis verified downregulation of TNFalpha-mediated expression of CD40L and IL-8 by Dexa and Dexa-Ab(hEsel), respectively. These results indicated a successful internalization and processing of Dexa-Ab(hEsel) in activated endothelial cells, allowing the intracellularly delivered Dexa to exert its pleiotropic anti-inflammatory activity.