Metabolic and pharmacokinetic study to enhance busulphan therapeutic efficacy

Abstract: Busulphan (Bu) is an alkylating agent used in high-doses as part of the conditioning regimen prior to hematopoietic stem cell transplantation (HSCT). HSCT is a curative treatment for hematological malignancies and other disorders. Bu can be administered by oral or intravenous route. The intravenous formulation contains high concentrations of dimethylacetamide (DMA), a potent solvent, but both hepatotoxic and neurotoxic. High-dose Bu treatment was shown to induce hepatotoxicity, which can develop to sinusoidal obstruction syndrome (SOS). Another side effect of high-dose Bu treatment is convulsions or generalized tonic-clonic seizures. The general aim of this thesis was to investigate the pharmacokinetics of Bu and DMA including their metabolites and understand the mechanisms underlying their toxicities. In Study I. A liquid chromatography-mass spectrometry (LC-MS) based method was developed to quantify DMA and its primary metabolite methylacetamide (MMA) in human plasma. By following the FDA guidelines for bioanalytical method validation, we established a robust, selective, reproducible, and sensitive method for quantification of DMA and MMA. The calibration curves were linear between 1 and 4000 μM, and the limits of quantification were 1.8 and 8.6 μM for DMA and MMA, respectively. The developed method can be used for occupational exposure studies as well as monitoring both compounds in patients receiving drugs where DMA is present as an excipient. In Study II. The pharmacokinetics of DMA and MMA was investigated in 18 pediatric patients receiving high-dose Bu treatment prior to HSCT. We used the LC-MS method developed in study I to quantify DMA and MMA in patients’ plasma. The results have shown accumulation of MMA throughout the treatment accompanied by an increase in the DMA clearance after the first dose administration of Bu. MMA had a slow elimination, and its half-life was calculated as 12.7h. In patients also, alanine transaminase (ALT) levels increased in more than 60% of the patients during conditioning with intravenous Bu. In vitro, cytotoxic tests on the hepatic cell line Huh 7 showed that the combination of Bu and MMA was more toxic than each compound separately. These findings imply that MMA might enhance Bu-induced hepatotoxicity. In Study III. We investigated Bu-induced seizures, a neurotoxicity associated with high-dose Bu treatment. We quantified Bu and its four metabolites, tetrahydrothiophene (THT), THT 1-oxide, sulfolane, and 3-OH sulfolane in 18 patients receiving Bu prior to HSCT. The results showed that sulfolane, and to a less extend 3-OH sulfolane, were accumulated in patients throughout treatment with Bu. Sulfolane remained detectable up to 60h after last Bu administration. In mice, generalized seizures occurred in the group administered with sulfolane. Furthermore, pharmacokinetic studies in mice organs and plasma showed a high distribution of sulfolane into the brain where the ratio AUC brain/AUC plasma was the highest and calculated as 1.45. Neurotransmitters analysis in mouse brain showed that GABA and calbindin 28k levels were decreased in the group of mice injected with sulfolane. The levels of sulfolane in patients might have a role in potentiating Bu-induced seizures. In Study IV. The role of N-acetylcysteine (NAC), a precursor of glutathione, on Bu-induced hepatotoxicity and the clinical outcome of HSCT was investigated. We evaluated the liver values, transplantation/conditioning complications and clinical outcome in two groups of patients receiving Bu (with and without NAC prophylaxis). The liver enzymes ALT, aspartate transaminase (AST) and alkaline phosphatase (ALP) were significantly decreased in the group of patients receiving Bu with NAC prophylaxis compared to the control group. Those levels were normalized even in patients who had high levels of liver enzymes before the start of Bu treatment. These observations suggest that NAC prophylaxis can potentially reduce Bu-induced hepatotoxicity without negatively affecting the clinical outcome of HSCT.