The effect of chemical manipulation on methaemoglobin formation

Abstract

Para‐aminopropiophenone (PAPP) is a newly developed pest control agent for stoats currently being used in Australia and New Zealand. It acts by inducing methaemoglobinaemia and is considered to be more humane than existing chemical methods. PAPP is selective for stoats, and is ineffective against rodents and possums. It is believed however that PAPP can act as a lead compound in developing more potent methaemoglobin (MetHb)‐inducing poisons with activity against other mammalian pests. At present, the exact molecular mechanism of PAPP‐induced MetHb and the roles of metabolites are unclear. This study aimed to identify the main biochemical determinants that influence the ability of PAPP and its analogues to cause MetHb formation, namely metabolism and uptake into the target red blood cell. Firstly, an HPLC assay to quantify PAPP concentrations and its analogues in solution was developed which meet FDA standards (R²>0.99; Precision and accuracy variation <15%). This assay together with mass spectrometry, microsomal and red blood cell uptake studies was then used to show that bioactivation rate, metabolite stability and red blood cell uptake affected the induction of MetHb; These processes were found to be influenced in turn by certain chemical properties of the compounds such as lipophilicity. Results from the alkyl‐derivatives of PAPP, with attached alkyl chains varying from 2‐7 carbons in length, found that CYP 450‐catalysed bioactivation and red blood cell uptake were influenced by lipophilicity. Optimal CYP 450 metabolism and red blood cell uptake was observed for the analogues with log P values between 2‐3. A test of a more complex set of PAPP analogues (DC series) showed a similar trend between log P and bioactivation, but not for red blood cell uptake. While both metabolic stability and red blood cell uptake play critical roles in PAPP‐induced MetHb formation; a clear relationship was not observed between percentage loss of the parent compound (R²= 0.51) or percentage uptake into RBC (R²= 0.62,) and the amount of MetHb formed. This highlights the important point that MetHb formation is not a simple function of RBC uptake rate and metabolism or of molecular size and lipophilicity. Clearly, other factors such as detoxification pathways and the inherent ability of the metabolite to undergo cycling (Kreisprozess cycle) need to be taken into account in the search for more potent MetHb inducers.