Metabolic and pharmacokinetic study to enhance busulphan therapeutic efficacy

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

Metabolic and pharmacokinetic study to enhance busulphan therapeutic efficacy

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

The effect of N-acetyl-l-cysteine (NAC) on liver toxicity and clinical outcome after hematopoietic stem cell transplantation

Busulphan (Bu) is a myeloablative drug used for conditioning prior to hematopoietic stem cell transplantation. Bu is predominantly metabolized through glutathione conjugation, a reaction that consumes the hepatic glutathione. N-acetyl-l-cysteine (NAC) is a glutathione precursor used in the treatment of acetaminophen hepatotoxicity. NAC does not interfere with the busulphan myeloablative effect. We investigated the effect of NAC concomitant treatment during busulphan conditioning on the liver enzymes as well as the clinical outcome. Prophylactic NAC treatment was given to 54 patients upon the start of busulphan conditioning. These patients were compared with 54 historical matched controls who did not receive NAC treatment. In patients treated with NAC, aspartate transaminase (AST), alanine transaminase (ALT) and alkaline phosphatase (ALP) were significantly (P amp;lt; 0.05) decreased after conditioning compared to their start values. Within the NAC-group, liver enzymes were normalized in those patients (30%) who had significantly high start values. No significant decrease in enzyme levels was observed in the control group. Furthermore, NAC affected neither Bu kinetics nor clinical outcome (sinusoidal obstruction syndrome incidence, graft-versus-host disease and/or graft failure). In conclusion: NAC is a potential prophylactic treatment for hepatotoxicity during busulphan conditioning. NAC therapy did not alter busulphan kinetics or affect clinical outcome.Funding Agencies|Swedish Cancer Society [CAN2011/595, CAN2014/759]; Swedish Childhood Cancer Foundation [PR2017-0083]; Radiumhemmets [161082]</p

Rapid and Robust Quantification of <i>p</i>‑Xyleneselenocyanate in Plasma via Derivatization

<i>p</i>-Xyleneselenocyanate (<i>p</i>-XSC) is one of the most investigated selenium compounds in cancer-prevention and -therapy. Despite the potent anticancer property, there is still no proper method to perform the quantitative analysis of <i>p</i>-XSC in plasma. In this investigation, we aimed at developing a method based on liquid chromatography–mass spectrometry (LC-MS) for the measurement of <i>p</i>-XSC in plasma. Direct deproteinization was first used to extract parent <i>p</i>-XSC from plasma, but failed to achieve high recovery rate (<2%) due to formation of selenium–sulfur bond between <i>p</i>-XSC and plasma protein. To overcome this problem, we modified the extraction method to three steps: (1) break the selenium–sulfur bond by tris­(2-carboxyethyl)­phosphine; (2) stabilize the newly formed intermediate selenol by N-ethylmaleimide; (3) deproteinization. This three-step method efficiently recovered bound <i>p</i>-XSC by more than 75%. In in vivo study, <i>p</i>-XSC was injected intravenously into mice and plasma was collected for LC-MS analysis. Consistently, <i>p</i>-XSC was undetectable in its parent form, whereas the bound form was readily quantified, employing the modified extraction method. In summary, we describe a novel, robust, and sensitive method for quantification of <i>p</i>-XSC in plasma. The present method will enable pharmacokinetic and pharmacodynamic studies of <i>p</i>-XSC in both clinical and preclinical settings