Pharmacokinetics Of Treosulfan Research Articles

Efficacy and Safety of High-Dose Chemotherapy with Treosulfan and Melphalan in Multiple Myeloma

(1) Background: Upfront treatment consolidation with high-dose chemotherapy (HDCT) and autologous stem cell transplantation (ASCT) has relevantly contributed to achieving durable remissions following induction treatment in multiple myeloma (MM) patients. The optimization of HDCT regimens can, therefore, essentially contribute to improving the depth and duration of tumor remissions. To date, melphalan at 200 mg/m2 is the standard HDCT regimen for fit MM patients. In our previous work, we showed promising efficacy and safety results for treosulfan (14 g/m2) and melphalan (200 mg/m2) (TreoMel) in acute myeloid leukemia (AML) patients receiving ASCT. Based on these data, TreoMel became the standard of care for fit MM patients at our institution. (2) Methods: We identified 115 consecutive MM patients who underwent consolidation with TreoMel between 01/2020 and 08/2022 at the University Hospital of Bern. We analyzed the safety and efficacy data, as well as the treosulfan pharmacokinetics, correlating them with tumor responses. (3) Results: A complete response (CR) rate of 84% was achieved, which is comparable to the CR rate reported for the quadruplet combination. The median PFS was 30 months (95% CI: 20.4—not reached), and the 31-month OS rate was 83%. The median area under the curve (AUC) for treosulfan was 952.5 mg*h/L (range: 527.4–1781.4), and the median peak level was 332.3 mg/L (range: 168–554). The treosulfan pharmacokinetics showed no significant correlation with MM responses after HDCT and ASCT. However, female patients had a significantly higher AUC (p = 0.007) and peak value (p = 0.001), and the higher values were associated with longer hospitalizations. (4) Conclusions: Treatment consolidation with TreoMel HDCT demonstrated a promising efficacy and safety profile in our cohort of MM patients and deserves further investigation in prospective studies.

Treosulfan Therapeutic Drug Monitoring: Optimizing Conditioning Therapy in Pediatric Hematopoietic Stem Cell Transplantation

One of the major challenges to improving outcomes in hematopoietic stem cell transplantation is to choose the best conditioning regimen to reduce toxicity and Non Relapse Mortality, while maintaining adequate transplant efficacy. The use of Treosulfan, in combination with other chemotherapeutic agents, as part of the conditioning regimen, has progressively increased during the last decade, given its remarkable myeloablative and immunosoppressive properties and more favorable toxicity profile in comparison with Busulfan and Total Body Irradiation. For a therapy to be considered effective and safe, it is necessary to know its pharmacokinetic and pharmacodynamic properties, so that it reaches and maintains adequate concentrations in blood and tissues. Data on the pharmacokinetics of Treosulfan and the relationship between toxicity and efficacy in the pediatric population are still scarce, with reports of high inter-individual variability of Treosulfan concentrations in blood. The aim of this multicenter, prospective study is to develop a therapeutic drug monitoring system for Treosulfan dosing, in children undergoing hematopoietic stem cell transplantation for both malignant and non-malignant disorders, through the investigation of the pharmacokinetics in a large population of children after the first dose of Treosulfan, during the pre-transplant conditioning regimen (proposed cumulative therapeutic target, 'Area Under the Curve' (AUC) 4800 mg*h/L, range 3840 - 6000 mg*h/L). To achieve this aim, we measure the percentage of patients in therapeutic range, make dose adjustments if required, and define the relationship between Treosulfan exposure, early toxicity and efficacy, measured by time to engrafment and donor chimerism percentage after transplant. Of the 24 patients so far enrolled, the first 14 were analysed (Table I). Seven (54%) required a dose adjustment after the first administration of Treosulfan: reduced in three cases (43%) for a cumulative AUC above 6000 mg*h/L, increased in four (57%) for AUC below the therapeutic target, confirming the wide inter-individual variability. Enrollment continues, and follow-up of patients should allow the definition of the relationship between personalized Treosulfan dosing, toxicity and efficacy. Therefore, the development of a drug monitoring system for Treosulfan may be an important tool to optimize outcomes in the pediatric population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pharmacokinetics of Treosulfan in Patients with Thalassemia Major Undergoing Allogeneic Hematopoietic Stem Cell Transplantation — a Comparison of Two Formulations and Correlation with Clinical Outcomes

Introduction: We have shown before that treosulfan based conditioning improves outcome of allogeneic hematopoietic stem cell transplantation (aHSCT) in patients with thalassemia major (TM), particularly in those with class 3 high risk disease. (Mathews et al, PLoS ONE, 2013) Treosulfan is a bi-functional alkylating agent, similar to busulfan except for two additional hydroxyl moieties. Unlike busulfan, treosulfan is a prodrug which non-enzymatically converts into its mono and di-epoxide active derivatives. Treosulfan (Medac, GmbH, Germany) (TreoM) is the only product currently available. Its cost accounts for nearly 20-25% of the total cost of aHSCT in India. There is limited data on the pharmacokinetics (PK) of Treosulfan in thalassemia major or its correlation with clinical outcomes and toxicities. In this analysis, we have evaluated the PK of Treosulfan with two different Treosulfan formulations, TreoM and Treosulfan (Emcure, Pune, India) (TreoE), in patients with TM undergoing aHSCT at our center and correlated them with outcomes.Patients and methods: All patients undergoing aHSCT for TM with the following conditioning regimen - thiotepa 8 mg/kg/day on day -6, fludarabine 40 mg/m2/day on days -5 to -2 and treosulfan 14 G/m2 days -5 to -3, were included in this analysis. While TreoM was in use since 2009, TreoE was evaluated from 2015 as a pivotal drug registration study in India. Both treosulfan formulations were in 5 gm lyophilized vials with similar instructions for reconstitution and administration. Blood samples were obtained for Treo assay just before (0hr), at the end of infusion & 1, 2, 3, 4, 6, 8 & 24 hrs after infusion. Treo levels in plasma were measured using HPLC with refractometric detection. Treo PK (AUC, CL, V) was estimated using nonlinear mixed effects modeling via Monolix 4.3.3. Several covariates were also evaluated. Clinical outcome related to regimen related toxicities (RRT), engraftment, graft versus host disease and rejection were prospectively documented. Chimerism was assessed on day +28 first and thereafter at 3, 6 and 12 months unless otherwise indicated. Both studies were approved by the institutional review board.Results: A total of 87 patients were treated with TreoM and 15 with TreoE. The demographics in the two groups were comparable with median age (range) being 9 years (1-25) and 11 years (2-17), and class 3 high risk being 41% and 47%, respectively (p=ns). Comparison of Treo PK parameters showed high inter-individual variation (IIV) in AUC (TreoM-median:1325, range: 126-4484 vs. TreoE-median:675, range: 125-1367 mg*hr/L; p=0.00033) and Cl (TreoM median:10.83, range: 3-111 vs. TreoE median: 20, range 7.6-111 L/h/m2; p=0.0004) (Figure) The IIV was >30 fold with TreoM and ~10 fold with TreoE for AUC. There were 5 rejections including 2 primary graft failures with TreoM and none with TreoE (p=ns). When rejection was correlated with TreoM AUC values in quartiles, there was no rejection in the lowest quartile, 0/20 vs 5/59 in the higher quartiles (p=ns). AUC values of 13/15 patients receiving TreoE were in the range of the lowest quartile of TreoM. Other major RRT including grade 3 or 4 mucositis (20% vs 33%) and sinusoidal obstruction syndrome (15% vs 13%), respectively, were also similar in both groups.Conclusion: This study reports the comparison of the first generic treosulfan with the original molecule and shows that even though there are major differences in the PK parameters, there was no adverse impact of the clinical outcome. The basis for this difference as well as the lower inter-individual variation of TreoE remains unclear. The data also suggests that as a pro-drug, higher clearance and low AUC of Treosulfan may indicate higher levels of the active metabolites which appears to correlate with better engraftment but without increase in toxicity. Optimal PK parameters for Treosulfan in conditioning for TM remain to be defined. In the meantime, the significantly lower cost of the generic product should improve access to this drug in the world. [Display omitted] DisclosuresSrivastava:Bayer Healthcare, Shire, Novo Nordisk, Roche Genentech, LFB: Other: Educational grants / Advisory Board / Grants Review / Data Monitoring Committee, Research Funding.

Population pharmacokinetic approach for evaluation of treosulfan and its active monoepoxide disposition in plasma and brain on the basis of a rat model

PurposeEfficacy of treosulfan, used in the treatment of marrow disorders, depends on the activity of its monoepoxy—(EBDM) and diepoxy compounds. The study aimed to describe the pharmacokinetics of treosulfan and EBDM in the rat plasma and brain by means of mixed-effects modelling.MethodsThe study had a one-animal-per-sample design and included ninty-six 10-week-old Wistar rats of both sexes. Treosulfan and EBDM concentrations in the brain and plasma were measured by an HPLC–MS/MS method. The population pharmacokinetic model was established in NONMEM software with a first-order estimation method with interaction.ResultsOne-compartment pharmacokinetic model best described changes in the concentrations of treosulfan in plasma, and EBDM concentrations in plasma and in the brain. Treosulfan concentrations in the brain followed a two-compartment model. Both treosulfan and EBDM poorly penetrated the blood–brain barrier (ratio of influx and efflux clearances through the blood–brain barrier was 0.120 and 0.317 for treosulfan and EBDM, respectively). Treosulfan plasma clearance was significantly lower in male rats than in females (0.273 L/h/kg vs 0.419 L/h/kg).ConclusionsThe developed population pharmacokinetic model is the first that allows the prediction of treosulfan and EBDM concentrations in rat plasma and brain. These results provide directions for future studies on treosulfan regarding the contribution of transport proteins or the development of a physiological-based model.

Treosulfan in combination with fludarabine as part of conditioning treatment prior to allogeneic hematopoietic stem cell transplantation

The combination of treosulfan with fludarabine (Treo-Flu) is one of the new megatherapies prior to hematopoietic stem cell transplantation. Preclinical studies have documented the myeloablative and myelosuppressive effects of treosulfan, in addition to its immunosuppressive and antimalignant activities. Treosulfan pharmacokinetics involves nonenzymatic activation and generation of epoxides as active compounds. In the clinical studies, the most common adverse effects of Treo-Flu were grade 4 hematologic toxicities with leukopenia, neutropenia and thrombocytopenia. Among the most common nonhematologic toxicities, grade 2 or lower mucositis was usually reported. One of the advantages of treosulfan-based conditioning regimens is their good safety profile, especially the low risk of hepatic venoocclusive disease. Additional drugs reported in combination with the Treo-Flu backbone are thiotepa and melphalan. Treo-Flu alone and in multiple drug combinations can be successfully and safely combined with posttransplant cyclophosphamide immunosuppression for unma¬nipulated haploidentical transplantations. On the basis of chimerism studies, the Treo-Flu megatherapy lacks full myeloablative potential, but the profound myelosuppression with donor cell-mediated alloreactivity can result in full donor chimerism in the majority of transplant recipients. The clinical studies of allogeneic hematopoietic stem cell transplantation show high heterogeneity, but the safety and feasibility of the Treo-Flu regimen are evident and support its place among reduced-intensity protocols.

Treosulfan Pharmacokinetics and its Variability in Pediatric and Adult Patients Undergoing Conditioning Prior to Hematopoietic Stem Cell Transplantation: Current State of the Art, In-Depth Analysis, and Perspectives.

Treosulfan is a prodrug that undergoes a highly pH- and temperature-dependent nonenzymatic conversion to the monoepoxide {(2S,3S)-1,2-epoxy-3,4-butanediol 4-methanesulfonate [S,S-EBDM]} and diepoxide {(2S,3S)-1,2:3,4-diepoxybutane [S,S-DEB]}. Currently, treosulfan is tested in clinical trials as an alternative to busulfan in conditioning prior to hematopoietic stem cell transplantation (HSCT). Of note, the optimal dosing of the prodrug is still unresolved, especially in infants. In this paper, the pharmacokinetics of treosulfan, together with its biologically active epoxides, is comprehensively reviewed for the first time, with the focus on conditioning prior to HSCT. Most of the insightful data presented in this review comes from studies that have been conducted in the last 3 years. The article widely discusses the volume of distribution and total clearance of treosulfan. In particular, the interindividual variability of these key parameters in infants, children above 1 year of age, and adults is analyzed, including possible covariates. A clinically important aspect of the formation rate-limited elimination of S,S-EBDM and S,S-DEB is described, including the correlation between the exposure of the prodrug and S,S-EBDM in children. The significance of the elimination half-life of treosulfan and its epoxides for successful conditioning prior to HSCT is also raised. Furthermore, the organ disposition of treosulfan and S,S-EBDM in rats is discussed in the context of the clinical toxicity and myeloablative activity of treosulfan versus busulfan. Moreover, perspectives for future therapeutic drug monitoring of treosulfan are presented. The review is intended to be helpful to pharmacists and doctors in the comprehension of the clinical pharmacokinetics of treosulfan.Electronic supplementary materialThe online version of this article (10.1007/s40262-018-0647-4) contains supplementary material, which is available to authorized users.

High interpatient variability of treosulfan exposure is associated with early toxicity in paediatric HSCT: a prospective multicentre study.

Treosulfan-based conditioning is increasingly employed in paediatric haematopoietic stem cell transplantation (HSCT). Data on treosulfan pharmacokinetics in children are scarce, and the relationship between treosulfan exposure, toxicity and clinical outcome is unresolved. In this multicentre prospective observational study, we studied treosulfan pharmacokinetics and the drug's relationship with regimen-related toxicity and early clinical outcome in 77 paediatric patients. Treosulfan dose was 30g/m2 , administered over 3 consecutive days in infants <1year old (n=12) and 42g/m2 in children ≥1year old (n=65). Mean day 1 treosulfan exposure was 1744±795mg*h/l (10g/m2 ) and 1561±511mg*h/l (14g/m2 ), with an inter-individual variability of 56 and 33% in the respective groups. High treosulfan exposure (>1650mg*h/l) was associated with an increased risk of mucosal [Odds ratio (OR) 4·40; 95% confidence interval (CI) 1·19-16·28, P=0·026] and skin toxicity (OR 4·51; 95% CI 1·07-18·93, P=0·040). No correlation was found between treosulfan exposure and the early clinical outcome parameters: engraftment, acute graft-versus-host disease and donor chimerism. Our study provides the first evidence in a large cohort of paediatric patients of high variability in treosulfan pharmacokinetics and an association between treosulfan exposure and early toxicity. Ongoing studies will reveal whether treosulfan exposure is related to long-term disease-specific outcome and late treatment-related toxicity.

Population pharmacokinetics of treosulfan and development of a limited sampling strategy in children prior to hematopoietic stem cell transplantation

PurposeThere is an increasing interest in use of treosulfan (TREO), a structural analogue of busulfan, as an agent in conditioning regimens prior to hematopoietic stem cell transplantation (HSCT), both in pediatric and adult populations. The aim of this study was to develop a population pharmacokinetic model and to establish limited sampling strategies (LSSs) enabling accurate estimation of exposure to this drug.MethodsThe study included 15 pediatric patients with malignant and non-malignant diseases, undergoing conditioning regimens prior to HSCT including TREO administered as a 1 h or 2 h infusion at daily doses of 10, 12, or 14 g/m2. A population pharmacokinetic model was developed by means of non-linear mixed-effect modeling approach in Monolix® software. Multivariate regression analysis and Bayesian method were used to develop 2- and 3-point strategies for estimation of exposure to TREO.ResultsPharmacokinetics of TREO was best described with a two-compartmental linear model with proportional residual error. Following sampling schedules allowed accurate estimation of exposure to TREO: 1 h and 6 h or 1 h, 2 h, and 6 h for a TREO dose 12 g/m2 in a 1 h infusion, or at 2 h and 6 h or 2 h, 4 h, and 8 h for a TREO dose of 12 g/m2 and 14 g/m2 in a 2 h infusion.ConclusionsA two-compartmental population pharmacokinetic model of TREO was developed and successfully used to establish 2- and 3-point LSSs for accurate and precise estimation of TREO AUC0→∞.

Population Pharmacokinetics of Fludarabine and Treosulfan in Patients with Thalassemia Undergoing Hematopoietic Stem Cell Transplantation

A toxicity reduced conditioning regimen containing Treosulfan (Treo), fludarabine (Flu), thiotepa for high risk Thal Major (TM) has been used since 2009 at our centre that has significantly improved transplant outcomes of these patients compared to the historical cohort of patients receiving busulfan/ cyclophosphamide based myeloablative regimen (Mathews et al, 2013). Limited knowledge is available on the pharmacokinetics (PK), pharmacogenetics (PG) and pharmacodynamics of fludarabine and treosulfan, especially in non-malignant hematological disorders like TM. We describe here the PK of Flu and Treo in patients with TM undergoing HSCT, the factors influencing the inter-individual variability in PK and the role of these factors on HSCT outcome.Seventy one patients diagnosed with TM undergoing HSCT with Flu/Treo based conditioning regimen between January 2012 and January 2015 were included (Table: Patient demographics). Selected functional polymorphisms in the NT5E, DCK, hENT1 and GST genes that are involved in fludarabine or treosulfan metabolism were screened. All patients received Flu 40mg/m2/day x 4 days as an 1hr infusion on days 1 and 4 and Treo as 14g/m2/day x 3 days at the rate 5g/hr. Plasma was separated from the peripheral blood collected at predetermined time points after the infusion of Flu and Treo PK analysis. Plasma Flu was analyzed using a LC-MS/MS method and the concentration was expressed as mMole/ml while Treo was analyzed using a HPLC-RI method and concentration was expressed as mg/L. Flu and Treo PK was estimated using nonlinear mixed effects modeling via Monolix 4.3.3. The covariates tested for both PK were: age, sex, body weight, BSA, ferritin, and polymorphisms in NT5E, hENT1, dCK and GST genes. The PK parameters AUC, CL, V and k were estimated on day 1 for Treo and on day 1 and day 4 for Flu (Table). The influence of Flu and Treo PK and PG on graft rejection, early transplant related mortality (TRM) & chimerism status was estimated using logistic regression analysis.Wide inter-individual variation in Flu and Treo PK was noted (7 and 9 fold Vs 5 and 8 fold respectively for Day 1 & 4 Flu AUC & Cl; 33 & 31 fold variation in Treo AUC and Cl) (Table). Flu CL was significantly higher on day 4 compared to day1 (Figure A). The variation in Flu PK was explained by genetic variants in NT5E and dCK. Patients having variant genotype for the SNPs in NT5E (rs2295890) and dCK (rs11544786) showed significantly lower plasma Flu clearance compared to those with wild type genotype (p=0.006 & p=0.05 respectively) (Figure B). This is consistent with our previous report in patients with aplastic anemia undergoing HSCT (Mohanan et al. 2014; Blood: 124 (21)). None of the genetic variants in the GST genes explained the variation in Treo PK.Day21 mortality was seen in 6/71 patients (8.5%) and graft rejection in 3/66 evaluable patients (4.5%). Analysis of the influence of PK and PG variables on transplant outcome showed significantly high first dose Flu AUC to be associated with D21 mortality upon Univariate analysis (median 42.5, range 32.1-63.7 compared to 31.8, range 15.2-111 mMole*h/mL, in those with and without TRM respectively; p=0.043); none of these parameters were significantly associated with graft rejection or mixed chimerism. There was no association between Treo PK parameters and graft rejection or TRM. The influence of Flu and Treo PK on regimen related toxicity is yet to be evaluated. The lack of the influence of PK on transplant outcome could be due to lower incidence of rejection and TRM in this cohort. Further analysis in a larger cohort of patients will be done once we enroll more patients for PK analysis.Our results demonstrate that Flu PK is influenced by genetic variants in NT5E and dCK, the enzymes involved in Flu biotransformation. The relationship between high-plasma Flu exposure and TRM and given the fact that multiple factors influence TRM, we can extrapolate that the plasma Flu AUC may be a surrogate marker of overall preparative regimen intensity as reported previously (Long-Boyle et al, Bone Marrow Transplant, 2011). The lack of association of genetic variants in GST genes in explaining the inter-patient variability in treosulfan exposure suggests the involvement of other drug metabolizing genes on treosulfan PK. We are currently evaluating the role of genetic variants in a large panel of drug metabolizing genes on explaining this inter-individual variability in Treo PK. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pharmacokinetics of treosulfan and its active monoepoxide in pediatric patients after intravenous infusion of high-dose treosulfan prior to HSCT

Pro-drug treosulfan (TREO) is currently evaluated in randomized phase III clinical trials as a conditioning agent prior to HSCT. In the present paper pharmacokinetics of both TREO and its biologically active monoepoxide (S,S-EBDM) was investigated in pediatric patients for the first time. The studies were carried out in 16 children (median age 7.5years) undergoing TREO-based preparative regimen prior to HSCT, who received 10, 12 or 14g/m2 of the drug as a 1h or 2h intravenous infusion. Plasma concentrations of TREO as well as S,S-EBDM were determined using the validated HPLC–MS/MS method. The changes in S,S-EBDM concentration over time followed TREO levels. The area under the curve (AUC) of TREO was 100-fold higher than AUC of S,S-EBDM. No statistically significant dependency of the dose-normalized AUC of either TREO or S,S-EBDM on the patients’ age and body surface area was stated. Moreover, plasma Cmax as well as AUC of S,S-EBDM demonstrated linear correlation with the Cmax and AUC of TREO, respectively. The biological half-lives of TREO and S,S-EBDM were similar. This indicates that S,S-EBDM was completely eliminated from the patients’ blood within relatively short time, comparable to TREO.

Pharmacokinetics of Treosulfan in Pediatric Patients Undergoing Hematopoietic Stem Cell Transplantation

High-dose treosulfan is used in conditioning regimens before hematopoietic stem cell transplantation in children. Pharmacokinetic data to optimize treosulfan dosing are scarce in this patient population. The aims of this study were the development and validation of an analytical method for treosulfan in human serum and the development of a pharmacokinetic model for treosulfan in pediatric patients. Furthermore, we aimed to develop a limited sampling strategy to estimate treosulfan systemic exposure with a minimum of inconvenience and risk for the patient. A reversed phase high-performance liquid chromatography method using ultraviolet detection to determine treosulfan in human serum samples was developed and validated according to food and drug administration guidelines. Serum pharmacokinetics after the first treosulfan administration was investigated in 20 children using nonlinear mixed-effect modeling, and a limited sampling strategy was developed and validated. The assay was validated in a 10-500 mg/L concentration range with a lower limit of quantification of 10 mg/L. Accuracies were within the 90%-110% limit. The coefficients of variation of the within-day imprecision and between-days imprecision were less than 5%. Pharmacokinetics was adequately described with a 1-compartment model. The population estimates for clearance (CL) and volume of distribution were 6.85 L/h and 13.2 L for a typical patient of 20 kg, respectively. Treosulfan exposure could be adequately quantified with 2 samples, at 4 and 7 hours after the start of a 3-hour treosulfan infusion, with a mean deviation of 3% of individual CL and area under the curve based on limited sampling in comparison with the full data set in a total cohort. In this study, a bioanalytical method, PK model, and limited sampling model were developed and validated. Furthermore, PK parameters of 20 pediatric patients were analyzed, demonstrating an interpatient variability in area under the curve of 14.5%. This study demonstrates the essential developments in the optimization of treosulfan therapy based on PK data.

Pharmacokinetics of high-dose i.v. treosulfan in children undergoing treosulfan-based preparative regimen for allogeneic haematopoietic SCT

Pharmacokinetic studies of high-dose treosulfan were carried out in seven paediatric patients (age range: 2-15 years) undergoing treosulfan-based conditioning regimen prior to allogeneic haematopoietic SCT. Treosulfan was administered intravenously in a daily dose of 10, 12 or 14 g/m(2) within 2 h. Five out of seven patients received 12 g/m(2). The plasma concentrations of treosulfan and its quantity eliminated with urine were determined using a validated HPLC method with refractometric detection. Pharmacokinetic parameters were evaluated following first dose using a two-compartment disposition model. These studies demonstrated a dose-dependent increase of area under the concentration (AUC) and maximum concentrationplasma (C(max)), but there was variability of these parameters. Rapid clearance of tresoulfan was observed, especially in 10 and 12 g/m(2) doses. Terminal half-life (t(0.5)) of treosulfan was in the range of 1.71-2.15 h, but the mean percent of parent drug eliminated with urine was 30%, range 16.3-45.4% of the total dose eliminated during the first 12 h after administration. The results of this study confirmed the linear pharmacokinetics of treosulfan, as used in children. However, variability of pharmacokinetic results observed in children studied demonstrates the need for pharmacokinetic evaluation in each paediatric patient undergoing the treosulfan-based preparative regimen, including those using different doses. This approach could enable further reduction of the risk of early and late organ toxicity related to high-dose treosulfan in paediatric patients.

Preclinical studies of treosulfan demonstrate potent activity in Ewing’s sarcoma

High-dose chemotherapy with the alkylating agent busulfan has been widely used in the treatment of patients with high-risk Ewing's sarcoma. Because of risks for toxicity, busulfan and radiotherapy can not be applied together, leading to the omission of one effective therapy component. Treosulfan is a derivative of busulfan which has a lower side effect profile than busulfan and which can be used together with radiotherapy. We investigated the effect of treosulfan in a panel of Ewing's sarcoma cell lines on cell survival, cell cycle and apoptosis in vitro and compared it to busulfan. Furthermore, the anti-tumor effect of treosulfan was studied in an orthotopic Ewing's sarcoma mouse xenograft model. Cell survival was measured by MTT assay and cell cycle analysis by flow cytometry. Apoptosis was analyzed via detection of DNA fragmentation, Hoechst 33258 staining, Annexin V, and cleavage of caspases-3 and 9. The effect of treosulfan and busulfan on primary tumor growth was assessed in Ewing's sarcoma xenografts in NOD/SCID mice (10 mice per group), pharmacokinetics of treosulfan were analyzed in nude mice. Treosulfan inhibited cell growth to at least 70% in all cell lines at concentrations achievable in vivo. Treosulfan had a greater effect on the inhibition of cell growth at equivalent concentrations compared to busulfan. The growth inhibitory effect of treosulfan at low concentrations was mainly due to a G2 cell cycle arrest, whereas at higher concentrations it was due to apoptosis. Apoptosis was induced at lower concentrations compared to busulfan. In contrast to busulfan, treosulfan induced cell death in an apoptosis-deficient cell line at concentrations achievable in vivo. In mice, treosulfan suppressed tumor growth at dosages of 2,500 and 3,000 mg/kg. Pharmacokinetic exposures of treosulfan in mice were similar to previous reports in human patients. At maximal tolerated dosages treosulfan had a higher anti-tumor activity than busulfan. Our results suggest that treosulfan has efficacy against Ewing's sarcoma cells in vitro and in mice. Therefore, controlled trials examining the role of treosulfan in patients with Ewing's sarcoma are warranted.

Determination of treosulfan in plasma and urine by HPLC with refractometric detection; pharmacokinetic studies in children undergoing myeloablative treatment prior to haematopoietic stem cell transplantation

A direct and selective HPLC method with refractometric detection was worked out for determination of treosulfan in plasma and urine of children. Before injection onto reverse phase column plasma samples with treosulfan and barbital (I.S.) were clarified using filtration. The mobile phase was composed of phosphate buffer, pH 5 and acetonitrile. The linear range of the standard curve of treosulfan spanned concentrations of 10.0–2000.0 μg/ml and 50.0–10000.0 μg/ml in plasma and urine, respectively, and covered the levels found in biological fluids after infusion of the drug. The limit of detection amounted to 5 μg/ml for plasma and 25 μg/ml for urine. Intra- and inter-day precision and accuracy of the measurement fulfilled analytical criteria accepted in pharmacokinetic studies. Recovery of treosulfan as well as stability in biological fluids was also calculated. The validated method was successfully applied in pharmacokinetic studies of treosulfan administered to children prior to haematopoietic stem cell transplantation. Differences between pharmacokinetics of treosulfan in children and adults were also studied.

Pharmacokinetics of treosulfan in a myeloablative combination with cyclophosphamide prior to allogeneic hematopoietic stem cell transplantation

Pharmacokinetics of treosulfan in a myeloablative combination with cyclophosphamide prior to allogeneic hematopoietic stem cell transplantation