Diacetylmorphine degradation to 6-monoacetylmorphine and morphine in cell culture: implications for in vitro studies

Abstract

Diacetylmorphine deacetylates rapidly to 6-monoacetylmorphine and then to morphine. The immunomodulatory effects of diacetylmorphine are under investigation by several groups utilising various methods including in vitro cell culture; however, diacetylmorphine stability under these conditions is unknown. The aim of this study was to quantify diacetylmorphine degradation under cell culture conditions and to determine the mechanism by which this occurs. Diacetylmorphine degradation in a mouse splenocyte mitogenesis assay was investigated. Morphine and 6-monoacetylmorphine were quantified using HPLC with UV detection. After 6 h, approximately 73% of diacetylmorphine had been hydrolysed in the presence of cells. The half-life of diacetylmorphine was 1.4 h in cell media alone and 1.2–2.2 h in incubations containing cells, while the half-life of 6-monoacetylmorphine was 3.1 h in cell media alone and 0.99–1.2 h in incubations containing cells. 6-Monoacetylmorphine and morphine formation were found to be dependent on incubation time and diacetylmorphine concentration, and were not dependent on esterase activity, mitogen concentration, presence of erythrocytes and cell media evaporation. Only morphine formation was dependent on lymphocyte concentration. 6-Monoacetylmorphine formation was independent of cells and appeared to be due to the conditions of the cell culture (pH and temperature), while morphine formation was dependent to a greater extent on cells, but independent of esterase activity. The study highlights the limitations of conclusions made in previous studies which have not recognised diacetylmorphine instability.