Application of monoclonal antibodies to measure metabolism of an anti-trypanosomal compound in vitro and in vivo.

Human African trypanosomiasis (HAT), also called African sleeping sickness, is a neglected tropical parasitic disease indigenous to sub-Saharan Africa. Diamidine compounds, including pentamidine and CPD-0801, are potent anti-trypanosomal molecules. The latter is a potential drug in the development at the UNC based Consortium for Parasitic Drug Development. An orally bioavailable prodrug of CPD-0801, DB868, is metabolized primarily in the liver to the active form. A monoclonal antibody developed against a pentamidine derivative has shown significant reactivity with CPD-0801 (EC(50) 65.1 nM), but not with the prodrug (EC(50)>18,000 nM). An inhibitory enzyme-linked immunosorbent assay (IELISA) has been used to quantitatively monitor prodrug metabolism by detecting the production of the active compound over time in a sandwich culture rat hepatocyte system and in rats. These results were compared with the results of the standard LC/MS/MS assay. Spearman coefficients of 0.96 and 0.933 (in vitro and in vivo, respectively) indicate a high correlation between these two measurement methods. This novel IELISA provides a facile, inexpensive, and accurate method for drug detection that may aide in elucidating the mechanisms of action and toxicity of existing and future diamidine compounds. (c) 2010 Wiley-Liss, Inc.