Composition and Preliminary Pharmacology Studies with Anvirzelt

Initial identification and characterization of the major biochemical and biological properties of Anvirzelt, a patented hot-water extract of Nerium oleander, were undertaken using HPLC, LC/MS, and in vitro cell growth inhibition assays. Analyses using high pH anion exchange chromatography with electrochemical detection consistently revealed seven major polysaccharide peaks which were subsequently designated as a “carbohydrate fingerprint.” Analyses of monomer sugar composition revealed glucose and galacturonic acid as major carbohydrate residues, while carbohydrate linkage studies identified gluco-pyranosyl and 6-glucopyranosyl as major terminal carbohydrate residues. Nonpolar compounds were separated from polar components through solid phase extraction and analyzed by both reverse phase HPLC and LC/MS methods. Two nonpolar cytotoxic components, oleandrin and its aglycone, oleandrigenin, were detected. Quantitative analysis showed that the oleander extract contained oleandrin and oleandrigenin at concentrations of 2.5 and 4.4 μg/mg extract, respectively. Five proteins with molecular weights of 6, 20, 35, 68, and 150 kD were also identified in the oleander extract although their functions remain unknown. Cytotoxicity studies showed oleandrin to be a potent growth inhibitory compound against human melanoma BRO cells with an IC50 of 4.0 ng/ml. In the same test system, the IC50 values for oleandrigenin and the complete oleander extract against human melanoma cells were 17.0 ng/ml and 1.6 μg/ml, respectively. This initial characterization and pharmacology research has served as a basis for quality control studies for the production and subsequent clinical Phase I trial of Anvirzel t.