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Journal of Clinical Oncology, Vol 25, No 13 (May 1), 2007: pp. 1772-1778 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2006.08.8807 Cytotoxicity of Dimethylacetamide and Pharmacokinetics in Children Receiving Intravenous Busulfan
From the Klinik und Poliklinik für Kinder und Jugendmedizin, Pädiatrische Hämatologie/Onkologie; Institut für Pharmazeutische and Medizinische Chemie der Universität Münster; Koordinierungszentrum Klinische Studien, Münster; Klinik für Kinderheilkunde III, Pädiatrische Hämatologie und Onkologie, Klinikum der Johann-Wolfgang-Goethe-Universität, Frankfurt; Universitätsklinikum Jena, Klinik für Kinder und Jugendmedizin, Allgemeine Pädiatrie, Hämatologie, Onkologie und Immunologie, Jena, Germany; and Newcastle University, Northern Institute for Cancer Research, Newcastle upon Tyne, United Kingdom Address reprint requests to Georg Hempel, PD Dr.rer.nat, Institut für Pharmazeutische und Medizinische Chemie der Universität Münster, Klinische Pharmazie-Hittorfstr. 58-62, 48149 Muenster, Germany; e-mail: hempege{at}uni-muenster.de
Purpose To assess the cytotoxicity and the exposure of N,N-dimethylacetamide (DMA) in children during high-dose therapy with an intravenous (IV) formulation of busulfan containing the potentially hepatotoxic and neurotoxic DMA as a solvent. Patients and Methods Eighteen children aged 0.9 to 17.3 years (median age, 4.0 years) received IV busulfan in 15 doses of 0.7 to 1.0 mg/kg busulfan containing overall DMA amounts of between 5 mmol (437 mg) and 70.5 mmol (6,142 mg) per dose. Plasma concentrations of DMA and busulfan were quantified and analyzed using nonlinear mixed-effects modeling. Four different leukemic cell lines were incubated with DMA, and cytotoxicity was assessed in comparison with busulfan as well as in a combination reflecting the ratio in the formulation. Results Maximal plasma concentrations of DMA up to 3.09 mmol/L were observed. No accumulation of the solvent occurred. Instead, the trough levels decreased over the 4 treatment days. The population pharmacokinetic analysis revealed a clearance of 86.9 mL h–1 kg–1 ± 27% that increased to 298 mL h–1 kg–1 on the fourth day and a volume of distribution of 469 mL kg ± 22% (population mean ± interindividual variability). DMA volume of distribution correlated with the volume of distribution of busulfan. The cytotoxicity of DMA in vitro was 3 orders of magnitude lower than that of busulfan. No synergism was observed. Conclusion The lack of accumulation of DMA confirms that there is no safety concern related to the DMA content in this IV busulfan formulation. The contribution of DMA to the antileukemic effect of the formulation seems to be limited.
High-dose busulfan is an important element of many conditioning regimens administered before stem-cell transplantation in adults and children for hematologic malignancies and certain disorders.1 The high interindividual variability in systemic exposure after administration of oral busulfan in children2,3 is problematic, with a substantial portion of patients experiencing reduced systemic exposure with the risk of graft rejection.4 However, with higher systemic exposure, the risk of veno-occlusive disease (VOD) is increased. Therefore, therapeutic drug monitoring is recommended with oral busulfan.5 The introduction of busulfan for intravenous (IV) infusion relieved pediatric patients from the difficulty of swallowing.6 In addition, because of direct systemic application by infusion, the variability of systemic exposure, measured as area under the curve, could be reduced.7 We and others have recently used IV busulfan containing N,N-dimethylacetamide (DMA) as a solvent in conditioning regimens before stem-cell transplantation.6-8 However, as reported from animal models and older studies in humans, DMA displays neurotoxic and hepatotoxic effects.9-11 These toxic effects of DMA are concerning because children receive high cumulative doses of DMA in combination with busulfan. The US Food and Drug Administration pharmacology review of the approval mentions that DMA is applied in a dose that may cause hepatotoxicity and neurotoxicity and that the proposed daily dose is 42% of the maximum-tolerated dose from the phase I study in adults11 (http://www.fda.gov/cder/foi/nda/99/20954_phrmr_P2.pdf). In vitro models indicate that the metabolism of DMA to monomethylacetamide is saturable.12 Thus, there is a need to investigate the pharmacokinetics of DMA in humans because data are completely lacking. In the context of our recent clinical study on IV busulfan in children, we analyzed the pharmacokinetics of DMA in children with the aim to assess the contribution of DMA to the toxicity of the formulation. In additional in vitro experiments, we measured the cytotoxicity of DMA alone and in combination with busulfan in leukemic cell lines to assess whether DMA contributes to the cytotoxicity of the formulation.
This multicenter study was approved by the local ethics committee. From March 2001 to September 2002, pediatric patients whose disease-specific treatment protocol included busulfan were enrolled onto the study. The patients were treated according to the respective disease-specific protocols of the German Society of Pediatric Hematology and Oncology. Written informed consent had to be given by all patients or their parents. Details are described elsewhere.8 IV busulfan was administered on 4 consecutive days. Patients received a total of 15 doses of IV busulfan (three doses on day 1 and four doses each on days 2 to 4). The first infusion was administered as a double dose over 4 hours (eg, 60 mg/m2 or 1.6 mg/kg), followed by the second dose 12 hours later to assess the elimination of busulfan over a longer time period. The second and all following doses (30 mg/m2 or 0.8 mg/kg) were administered as 2-hour infusions every 6 hours. Premedication included anticonvulsive prophylaxis with phenytoin (patients 8, 15, 18, and 19) or clonazepam, antimycotics (fluconazole or amphotericin B), antiviral drugs (acyclovir or ganciclovir), and different antibiotics depending on the local guidelines and the disease-specific study protocol.
Cell Culture
Cell Viability Assay The drug concentration capable of 50% growth inhibition relative to untreated controls (GI50) at the time points of 48 and 72 hours was calculated with the following equation: {[% viable cells (> 50%)] – 50}/{[% viable cells (> 50%)] – [% viable cells (< 50%)]} x (drug concentration > 50% viable cells – drug concentration < 50% viable cells) + (drug concentration < 50% viable cells). Each experiment was carried out in duplicate at different times.
Blood Sampling and Drug Analysis Samples were analyzed for DMA using a recently developed liquid chromatography-mass spectrometry method with a limit of quantification of 2.9 µmol/L.15 Inter- and intraday precision and accuracy were better than 7.7% (coefficient of variation) over the whole concentration range. Busulfan was analyzed using a liquid chromatography-mass spectrometry method developed by Murdter et al16 with a limit of quantification of 5 µg/L.
Pharmacokinetic Analysis and Statistics The influence of the following covariates was investigated consecutively: age, weight, height, sex, body-surface area (BSA), body mass index, serum creatinine, and serum bilirubin. Interoccasion variability (ie, variability of the pharmacokinetic parameters in a patient from one day of busulfan treatment to another day of treatment) was introduced into the model as proposed by Karlsson and Sheiner.17 To select the final models, the change in the objective function as a goodness of fit parameter was used by comparing values before and after adding a covariate to the model. A decrease in the objective function of more than 3.84 or 6.63 (log likelihood ratio test) was considered a significant improvement of the model (P < .05 or P < .01). Parameters are reported as geometric mean ± geometric coefficient of variation assuming log-normal distribution of the parameters.18 Statistical calculations were performed using Microsoft Excel 2003 (Microsoft, Redmond, WA) and SigmaStat 3.1 (Systat Software Inc, San Jose, CA). Comparisons between groups were performed by analyzing the log-transformed parameters.
The patient demographics and diagnoses are listed in Table 1. The ages ranged from 0.9 to 17.3 years, and body weight, height, and BSA showed a wide distribution typical for a pediatric population. None of the patients had elevated serum creatinine or bilirubin values.
Analysis of the DMA Plasma Concentrations The geometric mean of the maximum serum concentration (Cmax) after the first dose was 1.55 mmol/L ± 34.5% (geometric coefficient of variation), with the highest observed plasma concentration being 3.09 mmol/L. No accumulation of the solvent was observed over the 4 days of administration. A one-compartment model with first-order kinetics described the decline in the plasma concentration sufficiently. Adding a second compartment to the model or Michaelis-Menten models did not improve the fit (data not shown). The model development is summarized in Appendix Table A1 (online only). Weight was found to be a better predictor for clearance and volume of distribution than BSA. Introducing a parameter for variability from day to day on clearance clearly improved the fit. Inspection of the plasma concentrations showed that there was no accumulation in the plasma concentrations of DMA. Instead, the Cmax decreased during the 15 administrations in 15 of 18 patients. Statistically significant differences between dose 4 and dose 8 and dose 4 and the last administration were found (t test of the log-transformed Cmax, P = .003 and .002, respectively). This was taken into account when refining the one-compartment model. There was an increase in clearance from the first to the 15th dose, which could be best described by clearance increasing with time. However, introducing the cumulative dose per square meter as a factor for clearance gave only slightly worse results. Laboratory parameters like serum bilirubin and serum creatinine did not show any effect on the pharmacokinetic parameters. Also, anticonvulsive prophylaxis with either phenytoin or clonazepam did not change the pharmacokinetics of DMA in this patient group, although with only four patients receiving phenytoin, an effect cannot be excluded. Thus, the only parameter identified to influence DMA was weight. The final parameter estimates and their SEs are listed in Table 2. The mean initial half-life was 3.74 hours, which decreased to 0.829 hours after 96 hours.
Figure 1A shows the measured plasma concentrations and the model for a representative patient showing the apparent decrease in the trough concentrations. It can be seen that the model describes the plasma concentrations over time with a sufficient precision. The goodness of fit plots showed a good predictive performance of the model (Appendix Fig A1, online only).
Busulfan Plasma Pharmacokinetics The busulfan data analyzed by individual compartmental analysis in our previous report8 were reanalyzed using population pharmacokinetic methods. The best model was a one-compartment model with weight as a covariate for clearance and volume of distribution, a parameter for interoccasion variability on clearance, and residual variability modeled using a proportional model. The final parameter estimates are listed in Table 2, and the concentration measurements versus the model in one patient are shown in Figure 1B. Figure 2 compares the pharmacokinetic parameters of busulfan and DMA. A correlation between the volume of distribution of both substances was found (P = .008; r = 0.726, Pearson correlation), whereas no correlation between the clearance of both compounds was observed.
Cytotoxicity of DMA DMA and DMSO reduced cell viability in a dose-dependent manner. Overall, busulfan was more than 1,000 times more cytotoxic than DMSO after 48 hours and approximately 3,000 times more cytotoxic than DMSO after 72 hours. DMA was approximately 7 times more cytotoxic than DMSO (Fig 3), with GI50 values between 14.4 and 71.6 mmol/L in the four cell lines after exposure for 48 hours. Busulfan cytotoxicity showed a logarithmic increase in proportion to dose in all cell lines studied. The concentrations of busulfan necessary to reduce cell viability by 50% compared with untreated controls were comparable for busulfan dissolved in DMSO and for busulfan dissolved in DMA/polyethylenglycol 400 (Busulfex; Fig 3). GI50 values of busulfan ranged between 60 and 650 µmol/L after 48 hours and 30 and 640 µmol/L after 72 hours. Figure 4 represents the dose-response curves of busulfan dissolved in DMSO, the Busulfex formulation, or DMA alone for the most sensitive cell line MOLT-4.
This is the first investigation reporting the pharmacokinetics of DMA in man. In the literature, pharmacokinetic data of DMA were only available from rat experiments.19,20 From these findings, we expected the volume of distribution to be lower and found the peak plasma concentrations higher than expected from animal experiments. Because there are very limited data in the literature describing the toxicity and pharmacokinetics of DMA, we were concerned about the reported liver toxicity of this solvent and the saturable metabolism reported from rat experiments.21 However, our results show that DMA does not accumulate in children. Instead, a reduction in the trough levels over the 15 administrations was observed, which could be best described with clearance increasing with time. The initial half-life of 3.74 hours is longer than reported in rats, where values between 0.6 and 1.5 hours were reported,19 but subsequent clearance increases resulted in a half-life of 0.829 hours after 96 hours. We hypothesized that clearance may depend on the total dose per square meter or weight applied. However, in the population pharmacokinetic analysis, the model using time instead of dose was slightly better in this patient group. A limitation of this finding is that all patients received the same schedule with the same increments in the cumulative dose. Therefore, the model needs to be tested in other schedules. A dependence of the applied dose on the clearance of DMA cannot be excluded from our results, although it is obvious that a classical Michaelis-Menten model is not suitable to describe the behavior of DMA. Saturable metabolism of DMA is described in an isolated perfused rat liver model at a concentration of 36 µmol/L.12 The authors predicted a saturable metabolism of DMA greater than a threshold concentration of 18 ppm (0.21 mmol/L) in humans. This concentration was exceeded in plasma of the patients reported here (Cmax = 1.55 mmol/L ± 34.5% after the first dose) without observing saturation. One may think that enzyme induction either by DMA itself or the comedication is the reason for the increasing clearance with time.22 Anticonvulsive prophylaxis included phenytoin, a known inducer of P450, in four of 18 patients. Therefore, it is unlikely that the increase in clearance observed in all patients is a result of the comedication. Although earlier reports using rat hepatocytes showed that substrates of P450 2E1, the isoenzyme mainly responsible for the metabolism of DMA, stabilize the enzyme for at least 3 days,23 experiments conducted with rat liver microsomes indicated that DMA does not induce its own metabolism.24 Other investigators found that cimetidine, an inhibitor of P450, does not influence the metabolism of DMA.12 Substantial species differences between rats and humans in the metabolism by P450 2E1 were reported for the butadiene metabolism, making the interpretation of the animal results difficult.25 Therefore, it is unlikely that the increased clearance of DMA is a result of induction of the metabolism. An explanation for the increasing clearance can be an enhanced permeability of the kidney to DMA or its metabolite monomethylacetamide as a result of the exposure with the solvent. Unfortunately, urine samples of the patients were not available. However, there are currently no in vitro experiments supporting the hypothesis of increased renal permeability. The population pharmacokinetic model for busulfan was developed based on our recent results on oral busulfan.3 Although BSA was the better predictor for clearance and volume of distribution than body weight in 48 children aged between 0.4 and 18.1 years, Nguyen et al7 suggested a dosing scheme based on body weight, with higher dosing for children between 9 and 16 kg. However, for the population investigated here, weight correlated better than BSA to the pharmacokinetic parameters, although this difference is small and not clinically relevant. No systematic increase or decrease in clearance over time was observed in the patients. The correlation between the volumes of distribution of the two compounds indicates that the compounds have a similar pattern of distribution in the patients (Fig 2). In contrast, clearance does not correlate between the two compounds as a result of different modes of elimination; although busulfan is mainly eliminated by conjugation with glutathione via glutathione-S-transferase 1,26 renal elimination and metabolism via P450 is responsible for DMA elimination. Therefore, it is very unlikely that the two compounds influence each other in their elimination. In our previous publication, we reported that no signs of VOD occurred in 19 patients. Two of the patients developed signs of neurotoxicity, and three patients died within 1 year after therapy as a result of sepsis or disease progression.8 The patients with toxicity did not differ in their pharmacokinetic parameters from the patients without toxicity. In addition, Andersson et al6 reported no specific toxicity related to the IV formulation in a phase I study in adults. In another study in 70 adults aged up to 71 years, one case of neurotoxicity occurred using a once-daily schedule of IV busulfan 3.2 mg/kg. They reported signs of VOD in one patient and transient elevations in ALT levels in 74% of the patients. In 12 infants, Dalle et al27 reported severe VOD in one patient and no other specific toxicity using the DMA-containing formulation. In summary, one can conclude that, up to now, no additional toxicity besides the toxicity of busulfan has occurred. In the experiments with leukemic cell lines, the GI50 values determined for DMSO and DMA were within the millimolar range, whereas the GI50 values of busulfan were in the micromolar range. Although the different solvents had no influence on the cytotoxicity of busulfan, the higher toxicity of DMA compared with DMSO might impact other organs and tissues. The Cmax of busulfan in plasma in the 18 patients was 6.55 µmol/L ± 37%,8 which is 1 order of magnitude lower than the lowest GI50 of 60 µmol/L observed in cell culture. Comparing cell culture data and plasma concentrations is difficult with effects like plasma protein binding or concentration of drugs in certain compartments present. However, with the Cmax observed of 3.09 mmol/L and the lowest GI50 of 14.4 mmol/L over 48 hours, it seems that DMA does not contribute substantially to the antileukemic effect of the IV busulfan formulation. This can also be seen from Figure 4, in which the curves of busulfan in DMSO and busulfan in DMA only differ slightly. In conclusion, the pharmacokinetics of DMA in children and our in vitro experiments demonstrate no safety concern regarding this solvent. When applying once-daily or continuous-infusion schedules, DMA should be monitored in plasma to confirm these results.
The authors indicated no potential conflicts of interest.
Conception and design: Georg Hempel, Joachim Boos Administrative support: Joachim Boos Provision of study materials or patients: Claudia Lanvers-Kaminsky, Thomas Klingebiel, Josef Vormoor, Bernd Gruhn Collection and assembly of data: Doris Oechtering, Claudia Lanvers-Kaminsky Data analysis and interpretation: Georg Hempel, Doris Oechtering, Claudia Lanvers-Kaminsky Manuscript writing: Georg Hempel, Claudia Lanvers-Kaminsky Final approval of manuscript: Georg Hempel, Doris Oechtering, Claudia Lanvers-Kaminsky, Josef Vormoor, Joachim Boos
Supported in part by Grants No. 01EC9801 and 01GG9846 from the German Federal Department of Research and Technology. Elternverein Viersen provided financial support for the liquid chromatography-mass spectrometry instrument. Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article.
1. Hassan M: The role of busulfan in bone marrow transplantation. Med Oncol 16:166-176, 1999[Medline] 2. Vassal G, Koscielny S, Challine D, et al: Busulfan disposition and hepatic veno-occlusive disease in children undergoing bone marrow transplantation. Cancer Chemother Pharmacol 37:247-253, 1996[CrossRef][Medline] 3. Schiltmeyer B, Klingebiel T, Schwab M, et al: Population pharmacokinetics of oral busulfan in children. Cancer Chemother Pharmacol 52:209-216, 2003[CrossRef][Medline] 4. Vassal G: Pharmacologically-guided dose adjustment of busulfan in high-dose chemotherapy regimens: Rationale and pitfalls. Anticancer Res 14:2363-2370, 1994[Medline] 5. Bleyzac N, Souillet G, Magron P, et al: Improved clinical outcome of paediatric bone marrow recipients using a test dose and Bayesian pharmacokinetic individualization of busulfan dosage regimens. Bone Marrow Transplant 28:743-751, 2001[CrossRef][Medline] 6. Andersson BS, Madden T, Tran HT, et al: Acute safety and pharmacokinetics of intravenous busulfan when used with oral busulfan and cyclophosphamide as pretransplantation conditioning therapy: A phase I study. Biol Blood Marrow Transplant 6:548-554, 2000[CrossRef][Medline] 7. Nguyen L, Fuller D, Lennon S, et al: IV busulfan in pediatrics: A novel dosing to improve safety/efficacy for hematopoietic progenitor cell transplantation recipients. Bone Marrow Transplant 33:979-987, 2004[CrossRef][Medline] 8. Oechtering D, Schiltmeyer B, Hempel G, et al: Toxicity and pharmacokinetics of IV busulfan in children before stem cell transplantation. Anticancer Drugs 16:337-344, 2005[CrossRef][Medline] 9. Kennedy GL Jr, Sherman H: Acute and subchronic toxicity of dimethylformamide and dimethylacetamide following various routes of administration. Drug Chem Toxicol 9:147-170, 1986[Medline] 10. Horn HJ: Toxicology of dimethylacetamide. Toxicol Appl Pharmacol 3:12-24, 1961[CrossRef][Medline] 11. Weiss AJ, Jackson LG, Carabasi RA, et al: A phase I study of dimethylacetamide. Cancer Chemother Rep 16:477-485, 1962[Medline] 12. Palmen NG, Evelo CT, Borm PJ, et al: Toxicokinetics of dimethylacetamide (DMAc) in rat isolated perfused liver. Hum Exp Toxicol 12:127-133, 1993[Medline] 13. Mosmann T: Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 65:55-63, 1983[CrossRef][Medline] 14. Tada H, Shiho O, Kuroshima K, et al: An improved colorimetric assay for interleukin 2. J Immunol Methods 93:157-165, 1986[CrossRef][Medline] 15. Oechtering D, Boos J, Hempel G: Monitoring of N,N-dimethylacetamide in children during IV busulfan therapy by liquid chromatography-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 838:129-134, 2006[CrossRef][Medline] 16. Murdter TE, Coller J, Claviez A, et al: Sensitive and rapid quantification of busulfan in small plasma volumes by liquid chromatography-electrospray mass spectrometry. Clin Chem 47:1437-1442, 2001 17. Karlsson MO, Sheiner LB: The importance of modeling interoccasion variability in population pharmacokinetic analyses. J Pharmacokinet Biopharm 21:735-750, 1993[CrossRef][Medline] 18. Lacey LF, Keene ON, Pritchard JF, et al: Common noncompartmental pharmacokinetic variables: Are they normally or log-normally distributed? J Biopharm Stat 7:171-178, 1997[Medline] 19. Hundley SG, Lieder PH, Valentine R, et al: Dimethylacetamide pharmacokinetics following inhalation exposures to rats and mice. Toxicol Lett 73:213-225, 1994[CrossRef][Medline] 20. Lindstrom B, Sjoberg P, Floberg S: Gas chromatographic-mass spectrometric method for the determination of dimethylacetamide and metabolites in whole blood. J Chromatogr 428:156-159, 1988[CrossRef][Medline] 21. Baum SL, Suruda AJ: Toxic hepatitis from dimethylacetamide. Int J Occup Environ Health 3:1-4, 1997[Medline] 22. Barnes JR, Ranta KE: The metabolism of dimethylformamide and dimethylacetamide. Toxicol Appl Pharmacol 23:271-276, 1972[CrossRef][Medline] 23. Eliasson E, Johansson I, Ingelman-Sundberg M: Ligand-dependent maintenance of ethanol-inducible cytochrome P-450 in primary rat hepatocyte cell cultures. Biochem Biophys Res Commun 150:436-443, 1988[CrossRef][Medline] 24. Silvia M, Vincenzo L, Arturo M, et al: Microsomal metabolism of N,N-diethylacetamide and N,N-dimethylacetamide and their effects on drug-metabolizing enzymes of rat liver. Biochem Pharmacol 48:717-726, 1994[CrossRef][Medline] 25. Lewis DF, Bird MG, Parke DV: Molecular modelling of CYP2E1 enzymes from rat, mouse and man: An explanation for species differences in butadiene metabolism and potential carcinogenicity, and rationalization of CYP2E substrate specificity. Toxicology 118:93-113, 1997[CrossRef][Medline] 26. Hassan M, Ehrsson H: Metabolism of 14C-busulfan in isolated perfused rat liver. Eur J Drug Metab Pharmacokinet 12:71-76, 1987[Medline] 27. Dalle JH, Wall D, Theoret Y, et al: Intravenous busulfan for allogeneic hematopoietic stem cell transplantation in infants: Clinical and pharmacokinetic results. Bone Marrow Transplant 32:647-651, 2003[CrossRef][Medline] Submitted August 23, 2006; accepted January 31, 2007.
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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
