Treosulfan Research Articles

Treosulfan compared to busulfan in allogeneic haematopoietic stem cell transplantation for myelofibrosis: a registry-based study from the Chronic Malignancies Working Party of the EBMT.

We aimed to compare outcomes following treosulfan (TREO) or busulfan (BU) conditioning in a large cohort of myelofibrosis (MF) patients from the EBMT registry. A total of 530 patients were included; 73 received TREO and 457 BU (BU ≤ 6.4 mg/kg in 134, considered RIC, BU > 6.4 mg/kg in 323 considered higher dose (HD)). Groups were compared using adjusted Cox models. Cumulative incidences of engraftment and acute GVHD were similar across the 3 groups. The TREO group had significantly better OS than BU-HD (HR:0.61, 95% CI: 0.39-0.93) and a trend towards better OS over BU-RIC (HR: 0.66, 95% CI: 0.41-1.05). Moreover, the TREO cohort had a significantly better Progression-Free-Survival (PFS) than both the BU-HD (HR: 0.57, 95% CI: 0.38-0.84) and BU-RIC (HR: 0.60, 95% CI: 0.39-0.91) cohorts, which had similar PFS estimates. Non-relapse mortality (NRM) was reduced in the TREO and BU-RIC cohorts (HR: 0.44, 95% CI: 0.24-0.80 TREO vs BU-HD; HR: 0.54, 95% CI: 0.28-1.04 TREO vs BU-RIC). Of note, relapse risk did not significantly differ across the three groups. In summary, within the limits of a registry-based study, TREO conditioning may improve PFS in MF HSCT and have lower NRM than BU-HD with a similar relapse risk to BU-RIC. Prospective studies are needed to confirm these findings.

Comparable Outcome after Busulfan- or Treosulfan-Based Conditioning Regimen in Children Above 4 Years of Age with ALL Undergoing Allogeneic HSCT. Results from the Prospective International Forum-Trial

Purpose: Total body irradiation (TBI) has proved to be the “gold standard” as part of the conditioning regimen before allogeneic hematopoietic stem cell transplantation (HSCT) in pediatric patients with acute lymphoblastic leukemia (ALL). Its superiority over chemo-based conditioning was recently demonstrated in the prospective, international, randomized phase III study, which enrolled 417 patients beyond the age of 4 years transplanted for ALL in complete morphological remission (CR) from either matched sibling (MSD) or matched unrelated donor (MD) (Peters et al, JCO 2021, FORUM study; EudraCT: 2012-003032-22;ClinicalTrials.gov: NCT01949129). The use of either of the two protocol-prespecified chemo-conditioning regimens resulted in significantly worse EFS. Given the unavailability of TBI in some regions/centres and contraindications to TBI in individual patients, we here compare the outcomes of patients who received busulfan (BU) - based regimen vs of those who were given a treosulfan (TREO) - based conditioning in FORUM centres in both randomizing and non-randomizing countries in the years 2013-2018. Patients and methods: Patients ≤ 18 years at diagnosis (median age at HSCT 9.9 years, range 4-19.5), in CR pre-HSCT and with an MSD or MUD were assigned to myeloablative conditioning with fludarabine (FLU), thiotepa (THIO) and either BU or TREO according to country preference. Children transplanted from MSDs received cyclosporine A only as graft-versus-host disease (GvHD) prophylaxis, whereas recipients of MUD HSCT also received short-term methotrexate and anti-thymocyte globulin (ATG). Further details of the transplant procedure have been previously described (Peters C, et al. J Clin Oncol 2021). Results:In addition to the 193 patients from the randomizing countries, the FORUM trial included 115 additional children enrolled in countries where randomization could not occur (for legal or technical reasons). Overall, 180 vs. 128 patients received BU/THIO/FLU vs TREO/THIO/FLU, respectively. There were no differences about the patients' gender, age, and remission status and slight differences regarding donor, stem cell source and MRD status pre-transplant between the two cohorts of patients (see Table 1 for details). Patient's outcomes were updated as of February 20 th, 2023, and median follow-up was 4.2 years (range 0.3 - 9.1). There were neither differences between the 3-year overall survival (OS) (0.71±0.03 for BU/THIO/FLU vs 0.72±0.04 for TREO/THIO/FLU) nor event-free survival (EFS) (0.61±0.04 for BU/THIO/FLU vs 0.55±0.04 for TREO/THIO/FLU, p=NS). Three-year cumulative incidence of relapse (CIR) and non-relapse mortality (NRM) were 0.31±0.03 and 0.08±0.02 following BU/THIO/FLU and 0.36±0.04 and 0.09±0.03 following TREO/THIO/FLU, respectively (p=NS). Only one case of secondary malignancy was observed in the TREO cohort and one case of fatal liver veno-occlusive disease (VOD) in the BU group. No statistical differences were observed regarding the cumulative incidence of both aGvHD and cGvHD between the two groups; 3-year GFRS was almost identical (0.42± 0.04 in the BU/THIO/FLU group and 0.43±0.04 in the TREO/THIO/FLU cohort). There were no differences in OS, EFS, CIR, NRM and GFRS in either CR1 or CR2 patients between the two conditioning regimens. Furthermore, there were no differences in outcomes between countries. Patients given BU/THIO/FLU had a faster leucocyte, neutrophil and platelet recovery as compared to those prepared with TREO/THIO/FLU. Conclusion: Comparable long-term outcomes were observed after BU/THIO/FLU or TREO/THIO/FLU in children with ALL undergoing allogeneic HSCT included in the FORUM trial. Therefore, either of the 2 regimens may be effectively and safely used worldwide in patients > 4 years having a contraindication to or treated in centres/countries unable to deliver TBI.

Long-Term Data Confirm the Superiority of Total Body Irradiation-Containing Conditioning Regimen in Comparison to a Chemotherapy-Based Preparation in Children with Acute Lymphoblastic Leukemia Above the Age of 4 Years Given an Unmanipulated Allograft. Results of the Forum Randomized Clinical Trial

Background Conditioning regimen plays a crucial role in determining the outcomes of patients with hematological malignancy treated with allogeneic hematopoietic stem cell transplantation (HSCT). In a multicenter, international, randomized, phase 3 clinical Trial (FORUM study), we documented (Peters C, et al. J Clin Oncol 2021) that children with acute lymphoblastic leukemia (ALL) given total body irradiation (TBI) in combination with etoposide had a superior probability of 2-year overall (OS) and event-free survival (EFS) in comparison to patients given either one of two myeloablative chemo-conditioning regimens. In this report, we assessed whether the benefit deriving from the use of TBI was sustained over time. Patients and methods Patients <18 years at diagnosis, 4-21 years at HSCT, in complete morphological remission (CR) pre-HSCT, and with an HLA-compatible related or unrelated donor (UD) were randomly assigned 1:1 to a myeloablative conditioning regimen with fractionated TBI and etoposide or fludarabine, thiotepa, and either busulfan (BU) or treosulfan (TREO). Children transplanted from a matched sibling donor (MSD) received cyclosporine A only as graft-versus-host disease (GvHD) prophylaxis, whereas recipients of UD HSCT also received short-term methotrexate and anti-thymocyte globulin (ATG). Further details on transplant procedure have been described elsewhere (Peters C, et al. J Clin Oncol 2021). Results Between 04/2013 and 12/2018, 413 patients were randomly assigned to receive either TBI (212) or a chemotherapy-based conditioning (201). Compliance with random assignment was 92%. Sixty-five percent of patients were male, 72% had B-cell precursor ALL, 73% were transplanted from a UD, and in 82% of patients bone marrow was the stem cell source employed. Fifty-four percent of patients were in CR1 at time of allograft, while 40% and 4% were transplanted in CR2 and CR3, respectively. Patient's outcomes were updated on February 1st, 2022. With a median follow-up of 3.7 years (range, 0.3-7.9), the 3-year probability of OS of patients allocated to TBI/etoposide or to the chemotherapy-based conditioning regimens was 90±2% and 71±3% (p<0.001), respectively, while that of 3-year EFS was 81±3% and 59±4% (p<0.001). OS and EFS remain comparable in patients receiving BU or TREO in combination with fludarabine and thiotepa. In detail, OS and EFS of the 102 patients given BU, fludarabine and thiotepa were 73±5% and 69±5% respectively, while those of the 99 patients treated with TREO, fludarabine and thiotepa were 62±5% and 61±5%. Multivariate analysis confirmed that the use of chemotherapy-based regimens correlated with worse OS [Hazard Ratio, HR, 2.63, 95% confidence interval, CI, (1.61- 4.31), p<0.001] and EFS [HR 2.30, 95%CI (1.58- 3.35) p<0.001]. Transplantation in >CR1 predicted a worse EFS in multivariate analysis, as well [HR 1.67, 95% CI (1.14- 2.45) p=0.009]. Patients given TBI benefited from a lower cumulative incidence of relapse (Figure 1A), as well as from a lower 3-year cumulative incidence of non-relapse mortality (2+1% vs. 10+2% in the chemo arm, p=0.007). The 3-year cumulative incidence of chronic GvHD was comparable between the 2 arms (15+3% vs. 12+2% in the TBI and chemo arm, respectively, p=n.s.). We also analyzed the probability of GvHD/relapse-free survival (GRFS) considering both relapse and chronic GvHD as events; children prepared with TBI had a better 3-year GRFS probability (Figure 1B) as compared to chemo-prepared patients. Five patients (all transplanted from an UD) developed secondary malignancies: 4 of them had received TBI. Conclusions With a significantly longer follow-up, we confirm that the use of TBI during the conditioning regimen is associated with better outcomes in children and adolescents with ALL above the age of 4 years transplanted in CR, mainly due to a lower risk of leukemia recurrence. The number of cases of secondary malignancies, particularly in patients offered radiotherapy, deserves particular attention and underlines the importance of continuous safety monitoring over time to comprehensively evaluate the long-term sequels observed in this population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Relapse Is the Most Common Treatment Failure Post HSCT in Children with ALL below 4 Years of Age Given a Chemo-Based Conditioning Regimen. Results from the Prospective Multinational Forum-Trial

Background: Relapse and non-relapse mortality (NRM) are the major causes of treatment failure in infants and young children with high-risk ALL undergoing HSCT. Conditioning regimens for this high-risk population usually omit total body irradiation (TBI) to avoid acute and long-term side effects that are more pronounced in this age group. The optimal chemotherapeutic approach aiming improved event-free survival (EFS) and low NRM is not yet defined. Methods: The prospective FORUM trial used two myeloablative chemo-conditioning regimens for children <4 years (yrs) of age: fludarabine (FLU), thiotepa (THIO) and either intravenous busulfan (BU) or treosulfan (TREO) as described previously (Peters et al., JCO 2021). This report includes 194/202 patients (pts) (107 male, 87 female) from 26 countries enrolled in FORUM who received HSCT (5 pts received TBI-based conditioning and 3 pts with unknown conditioning were excluded). Median age at HSCT was 2.2 yrs (6 months - 4 yrs; 38 pts 0-1yrs (20%), 53 pts 1-2yrs (27%), 53 pts 2-3yrs (27%), 50 pts 3-4yrs (26%)). Stem cell source was bone marrow in 132 (68%) pts, peripheral blood stem cells in 37 (19%), and cord blood in 24 (13%). Donors were HLA-matched siblings (MSD) in 39 (20%) pts and 9 or 10/10 HLA allele-matched unrelated donors (MUD) in 155 (80%) pts.Clonal genetic abnormalities reported at diagnosis included 9 pts with BCR-ABL, 13 pts with TEL-AML, and 53 pts with KMT2A r-rearrangements; all pts underwent HSCT in complete morphological remission (CR) (142 pts CR1; 50 pts CR2, 2 pts CR3). FLU/THIO/BU and FLU/THIO/TREO were used in 101, and 93 HSCTs, respectively. Graft-versus-host-disease (GvHD)-prophylaxis was Cyclosporin-A-based in 90%; recipients of MUD-grafts also received ATG and 3 doses of methotrexate. At data cut-off, median follow-up (FU) was 3 yrs (range, 3 months - 7.2 yrs). Results: 3-yrs OS was 0.69±0.04, 3-yrs EFS was 0.52±0.04; 3-yrs Cumulative incidence of relapse (CIR) was 0.44+0.04, and 3-yrs NRM was 0.05 ± 0.02. OS and EFS did not differ between pts < 2 and 2-4 yrs of age at HSCT (Table 1). Pts transplanted in CR 1 had significantly better EFS (0.58±0.04) as compared to >CR1pts (0.36±0.07, p=0.01); due to a higher CIR. OS was worse for the 53 pts with KMT2A-rearrangements (3-yrs OS 0.56±0.08 vs. 0.73±0.04, p=0.040). EFS for pts < 1 yr of age at diagnosis was significantly inferior as compared to older pts (3-yrs EFS 0.40±0.05 vs. 0.63±0.05, p=0.002). OS/EFS were not significantly different when donor type, stem cell source, and immunophenotype were compared (Table 1). At 3-yrs, OS was 0.63±0.05 and 0.76±0.05 and EFS was 0.52±0.05 and 0.51±0.06 (p=0.794) for the FLU/THIO/BU and FLU/THIO/TREO-group, respectively (p=0.075) with no significant difference in CIR and TRM. However, pts who relapsed post-HSCT had a better 3-yr post-relapse OS after FLU/THIO/TREO compared to FLU/THIO/BU (0.38±0.11; 0.16±0.07. p = 0.012) respectively. Most relapses occurred prior to 1 yr after HSCT. Acute GVHD occurred in 32 pts (20 pts experienced grade III/IV) with no significant difference between the two conditioning regimen). The 3-yr incidence of chronic GVHD (cGVHD) was comparable between the BU- and -TREO-group (p = 0.943). cGVHD/relapse-free survival at 3 yrs for FLU/THIO/BU was 0.44±0.05 and for FLU/THIO/TREO 0.41±0.06 (p=0.943). In multivariate analysis, remission > CR1 and the presence of KMT2A, negatively influence OS while EFS was worse for patients who underwent HSCT > CR1 or had ALL-diagnosis below 1 yr of age. Donor type, stem cell source, age between 1 and 4 yrs at HSCT, conditioning regimen, and PCR-MRD positivity pre-HSCT did not significantly influence outcome. 1-yr TRM was low for both conditioning regimens (0.06±0.02 for FLU/THIO/BU and 0.03±0.02 after FLU/THIO/TREO (n.s.) Conclusion: Within the FORUM-trial, infants and young children receiving HSCT after conditioning with either BU- or TREO containing regimens have a lower OS and EFS as compared to children above the age of 4 yrs due to a higher CIR. Because of decreased NRM, these results represent an improvement as compared to previously reported series; however, KMT2A-rearrangement continues to be an obstacle to successful HSCT. Pre- and post-transplant immunotherapeutic strategies aimed at reducing relapse-incidence without increasing chemotherapy-associated complications are needed. Figure 1: 3-yrs OS according to Conditioning Flu: fludarabine, Thio: thiotepa, Treo: treosulfan, Bu: busulfan Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Treosulfan-based conditioning is feasible and effective for cord blood recipients: a phase 2 multicenter study

AbstractAlthough the use of treosulfan (TREO) in conventional donor hematopoietic cell transplantation (HCT) has been extensively evaluated, its use in cord blood transplantation (CBT) for hematologic malignancies has not been reported. Between March 2009 and October 2019, 130 CBT recipients were enrolled in this prospective multicenter phase 2 study. The conditioning regimen consisted of TREO, fludarabine, and a single fraction of 2 Gy total-body irradiation. Cyclosporine and mycophenolate mofetil were used for graft-versus-host disease prophylaxis. The primary end point was incidence of graft failure (GF), and based on risk of GF, patients were classified as low risk (arm 1, n = 66) and high risk (arm 2, n = 64). The median age was 45 years (range, 0.6-65 years). Disease status included acute leukemias in first complete remission (CR; n = 56), in ≥2 CRs (n = 46), and myelodysplastic (n = 25) and myeloproliferative syndromes (n = 3). Thirty-five patients (27%) had received a prior HCT. One hundred twenty-three patients (95%) engrafted, with neutrophil recovery occurring at a median of 19 days for patients on arm 1 and 20 days for patients on arm 2. The 3-year overall survival, relapse-free survival (RFS), transplant-related mortality, and relapse for the combined groups were 66%, 57%, 18%, and 24%, respectively. Among patients who had a prior HCT, RFS at 3 years was 48%. No significant differences in clinical outcomes were seen between the 2 arms. Our results demonstrate that TREO-based conditioning for CBT recipients is safe and effective in promoting CB engraftment with favorable clinical outcomes. This trial was registered at www.clinicaltrials.gov as #NCT00796068.

Elimination of Acute GvHD with Three Doses of Abatacept Prophylaxis Combined with Post-Transplant Cyclophosphamide after Myeloablative Treosulfan-Based Conditioning in the HLA Identical Sibling and Unrelated (10-12/12) Peripheral Blood Stem Cell Donor Setting: Improved Safety and Efficacy Compared to Alemtuzumab Prophylaxis

Background. Acute graft versus host disease (GvHD) remains a major cause of treatment failure post allogeneic stem cell transplantation (HSCT). CD28-CD80/CD86 axis T cell co-stimulation blockade with CTLA4-Ig using abatacept (AB) is an emerging acute GvHD prophylaxis strategy1. After myeloablative conditioning (MAC), together with methotrexate and calcineurin inhibition, four doses of AB commencing day (d)-1 is being investigated in the unrelated HLA 7/8 and 8/8 mismatched/matched donor settings, mostly in children2. It is unknown if AB can be de-escalated in adults to three doses for HLA matched sibling (SIB) or unrelated 10-12/12 donors (MUD), or combined with post-transplant cyclophosphamide (PT-CY) GvHD prophylaxis3, or combined with treosulfan (TREO)-based MAC4, which are other emerging transplant strategies. We investigated the safety and efficacy of this combination, and compared outcomes to a standard anti-CD52 directed IgG1 alemtuzumab (CH) prophylaxis regimen5 in a reduced intensity conditioning (RIC). Methods. We report a single centre consecutive cohort study using peripheral blood stem cells from SIB or HLA 10-12/12 MUDs, for HSCT in adults with blood cancer indications (intuitional registration: 1920-31). One group received AB GvHD prophylaxis while the other group received CH. During 2018-2019, the AB cohort received MAC with IV fludarabine 150 mg/m2, IV TREO 42 mg/m2, and 2 Gy total body irradiation (TBI). GvHD prophylaxis comprised three doses of IV AB 10 mg/kg on d+5, +14, and +28. This was combined with IV PT-CY 50 mg/kg on d+3 and +4, IV tacrolimus 0.03 mg/kg/d infusion from d+5, and IV mycophenolate mofetil 15 mg/kg tds from d+53. The MAC AB cohort was compared to a RIC CH cohort, chosen to be biased against AB. During 2017-2018 the CH cohort received RIC with IV fludarabine 150 mg/m2and IV melphalan 140 mg/m2. GvHD prophylaxis comprised IV CH 10 mg on d-7 to d-3, and IV ciclosporin 1.5 mg/kg bd from d-1. The primary outcome measure was the incidence of acute GvHD6 at d100 and 180 post HSCT. Secondary outcomes measures were the incidence of graft failure, mixed donor chimerism, donor lymphocyte infusion (DLI), CMV reactivation, relapse, overall survival, at d100 and 180. The AB and CH cohorts were indirectly compared. Results. A total of 39 patients were included in this study, with 15 consecutive patients in the AB group (Table). The AB group had a median Karnofsky score of 70% and HCT-CI of 6; significant comorbidity greater than other reports1,2. The major cancer indications were acute leukaemia and myelodysplastic syndrome (MDS). The AB and CH groups were largely balanced. There were no AB related infusion reactions. As expected for MAC, the AB group had a numerically but not clinically significantly slower time to engraftment relative to the RIC CH group. There was no AB group graft failure. Remarkably, the AB group did not experience acute GvHD compared to the CH group by d100 (P=0.03) and d180 (P&lt;0.01). In contrast, by d100, 29% in the CH group experienced acute GvHD after a median of 59 days post HSCT. No AB group patients experienced mixed donor chimerism (P&lt;0.01), nor required a DLI (P&lt;0.01) compared to the CH group, where all had &lt;99% T cell or whole blood values at d100. Consequently, 45% of the CH cohort without acute GvHD were treated with DLI, with all patients experiencing acute GvHD at a median of 39 days post DLI (Table). Other secondary outcomes were similar. The AB group experienced a CMV reactivation rate of 67% in IgG seropositive patients. Unfortunately, 93% of CMV IgG positive patients in the CH group experienced CMV reactivation. No relapses were observed by d180 in the AB group, whereas in the CH group, 8% of patients experienced relapse. Mortality at d180 was 20% in the MAC AB group, and 21% in the RIC CH group. Conclusions. Acute GvHD prophylaxis with only three doses of AB and PT-CY post TREO-based MAC appears to be safe and efficacious in the SIB and MUD settings, in a highly co-morbid adult blood cancer population. This AB approach seems favourable compared to CH despite RIC, where acute GvHD without DLI or post DLI remains significant. A prospective multicentre clinical trial with AB in this setting seems warranted to confirm these remarkable findings of patient benefit. References. 1. Biol Blood Marrow Transplant (BBMT) 2013;19:1638-49. 2. NCT01743131. 3. Lancet Haematol 2019;6:e132-43. 4. Cancer 2017;123:2671‐79. 5. BBMT 2017;23:805-12. 6. BBMT 2016;22:4-10. Table Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Abatacept, alemtuzumab, cyclophosphamide all the for the indication of graft versus host disease (GvHD) prophylaxis.

N-7-Guanine Adduct of the Active Monoepoxide of Prodrug Treosulfan: First Synthesis, Characterization, and Decomposition Profile Under Physiological Conditions

(2S,3S)-1,2:3,4-diepoxybutane (DEB) cross-links DNA guanines by forming the intermediate epoxy-adduct ((2′S,3′S)-N-7-(3′,4′-epoxy-2′-hydroxybut-1′-yl)guanine [EHBG]). This process is presently considered a primary mechanism for the action of treosulfan (TREO), the prodrug that transforms to DEB via the monoepoxide intermediate (2S,3S)-1,2-epoxybutane-3,4-diol 4-methanesulfonate (EBDM). In this article, the N-7-guanine adduct of EBDM ((2′S,3′S)-N-7-(2′3′-dihydroxy-4′-methylsulfonyloxybut-1′-yl)guanine [HMSBG]) was synthesized for the first time, and its stability was investigated at physiological in vitro conditions. To synthesize HMSBG, EBDM, formed in-situ from TREO, was treated with guanosine in glacial acetic acid at 60°C followed by ribose cleavage in 1 M HCl at 80°C. HMSBG was stable during the synthesis, which showed that a β-hydroxy group protects the sulfonate moiety against hydrolysis in acid environment. At pH 7.2 and 37°C, HMSBG exclusively underwent first-order epoxidation to EHBG with a half-life of 5.0 h. EHBG further decomposed to trihydroxybutyl-guanine, chlorodihydroxybutyl-guanine (major products), phosphodihydroxy-guanine, and a structural isomer (minor products). The isomeric derivative was identified as guanine with a fused 7-membered ring, which provided a new insight into the EHBG stability. To conclude, the exclusive conversion of HMSBG to EHBG indicates that EBDM might contribute to DNA cross-linking independently from DEB and play a more important role in the TREO action than expected before.

Relationship between exposure to treosulfan and its monoepoxytransformer – An insight from population pharmacokinetic study in pediatric patients before hematopoietic stem cell transplantation

Treosulfan (TREO), a structural analog of busulfan, is currently studied as a myeloablative agent in conditioning regimens before hematopoietic stem cell transplantation in pediatric patients. High exposure to TREO (>1650 mg∗h/mL) might be related to early toxicity, especially skin toxicity and mucositis. The aim of the present study was to investigate a potential relationship between exposure to TREO and its monoepoxytransformer (S,S-EBDM), as well as variability of the pharmacokinetics of these entities by means of a population pharmacokinetic approach with a non-linear mixed-effects analysis.The study included data from 14 children with malignant and non-malignant diseases treated with TREO in daily doses 10–14 g/m2. The parent-metabolite population pharmacokinetic model was developed in NONMEM 7.3 software. Upon the constructed model, an extensive simulation was performed to assess the correlation between exposure to TREO and S,S-EBDM.It was found that TREO and S,S-EBDM pharmacokinetics was best described with 2-compartmental and 1-compartmental linear models, respectively. The vast majority (>65%) of TREO was transformed to S,S-EBDM. Overall, a considerable interpatient variability of pharmacokinetic parameters was observed, especially the clearance of S,S-EBDM. A weak correlation was found between the exposure to TREO and S,S-EBDM (r = 0.1681, p < 0.0001). Also, patients with an exposure to TREO above 1650 mg∗h/mL were most likely to have also a high exposure to S,S-EBDM (35.38 μM∗h vs. 43.14 μM∗h, p < 0.0001).In summary, a parent-metabolite population pharmacokinetic model for TREO and S,S-EBDM was developed for the first time. It was shown that there is a weak correlation between exposure to TREO and S,S-EBDM. Therefore therapeutic drug monitoring of not only prodrug but also its active epoxide might be needed.

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→∞.

Disposition of treosulfan and its active monoepoxide in a bone marrow, liver, lungs, brain, and muscle: Studies in a rat model with clinical relevance

For the recent years, the application of treosulfan (TREO)-based conditioning prior to hematopoietic stem cell transplantation (HSCT) has been increasing as an alternative to busulfan-based therapy, especially for patients presenting high risk of developing hepato-, pulmo-, and neurotoxicity. So far, the penetration of TREO and its epoxy-derivatives into central nervous system and aqueous humor of the eye has been investigated. However, lacking knowledge on the compounds distribution into the other key tissues precludes comprehensive understanding and assessment of TREO clinical efficacy and toxicity. In this paper, the disposition of TREO and its active monoepoxide (S,S-EBDM) in a bone marrow, liver, lungs, brain, and quadriceps femoris was studied in an animal model. Male and female adult Wistar rats (n=48/48) received an intraperitoneal injection of TREO at the dose of 500mg/kg b.w. Concentrations of TREO and S,S-EBDM in tissues were determined with a validated HPLC-MS/MS method. Pharmacokinetic calculations were performed in WinNonlin using a noncompartmental analysis. Mean values of the maximal concentrations of TREO and S,S-EBDM in the organs were sex-independent and ranged from 61 to 1650μM and 25–105μM, respectively. No quantifiable levels of S,S-EBDM were found in the liver. Average tissue/plasma area under the curve (AUC) ratio for unbound TREO increased in the sequence: brain (0.10)<muscle (0.77)<bone marrow=lungs (0.82)<liver (0.96). The tissue/plasma AUC ratio for unbound S,S-EBDM changed as follows: brain (0.35)<lungs (0.50)<bone marrow (0.75)<muscle (1.14). Elimination half-lives of the compounds in plasma and the organs ranged from 0.7h to 2.1h. Scaling of the obtained AUCs of TREO and S,S-EBDM and the literature AUCs of busulfan to concentrations of the drugs in HSCT patients' plasma show that TREO reaches much higher levels in the organs than busulfan. Nonetheless, low S,S-EBDM exposure in a liver, lungs, and brain, even compared with busulfan, may contribute to relatively low organ toxicity of TREO-based conditioning regimens. Similarity of the scaled bone marrow AUCs of S,S-EBDM and busulfan corresponds to comparable myeloablative potency of TREO- and busulfan-based conditioning. The biological half-lives of TREO and S,S-EBDM in plasma and the studied organs indicate that 48h lag time following administration of the last dose of TREO to HSCT patients is sufficient to protect the transplanted stem cells from the compounds' exposure.

Effect of Temperature on the Kinetics of the Activation of Treosulfan and Hydrolytic Decomposition of Its Active Epoxy Derivatives

Treosulfan (TREO) is a prodrug applied in the treatment of ovarian cancer and a myeloablative conditioning prior to stem cell transplantation. A sequential activation of TREO to intermediate monoepoxide (S,S-EBDM) and then to (2S,3S)-1,2:3,4-diepoxybutane (S,S-DEB) involves a nonenzymatic intramolecular nucleophilic substitution. The aim of this study is to determine the effect of temperature on the rate constants (k) for the activation of TREO and the hydrolysis of its epoxy derivatives in a phosphate buffer of pH 7.4 at an ionic strength of 0.16-0.17 M. Over the tested temperature range, the ln of k changed linearly with a reciprocal of absolute temperature. The mean activation energy (Ea) values for the TREO → S,S-EBDM and S,S-EBDM → S,S-DEB reactions were close to each other (122 and 118 kJ/mol, respectively). In opposition, the Ea for the hydrolysis of S,S-EBDM and S,S-DEB differed significantly (140 and 80 kJ/mol, respectively), which indicates that the structure of S,S-EBDM hampers a nucleophilic attack of water on the epoxide ring. The obtained results show that a temperature change by 1°C, from 36.5°C to 37.5°C, entails a 17% increase in the k of TREO decay, which might lead to an increased TREO clearance in vivo.

Determination of prodrug treosulfan and its biologically active monoepoxide in rat plasma, liver, lungs, kidneys, muscle, and brain by HPLC–ESI–MS/MS method

A prodrug treosulfan (TREO) is currently investigated in clinical trials for conditioning prior to hematopoietic stem cell transplantation. Bioanalysis of TREO and its active derivatives, monoepoxide (S,S-EBDM) and diepoxide, in plasma and urine underlay the pharmacokinetic studies of these compounds but cannot explain an organ pharmacological action or toxicity. Recently, distribution of TREO and S,S-EBDM into brain, cerebrospinal fluid, and aqueous humor of the eye has been investigated in animal models and the obtained results presented clinical relevance. In this paper, a selective and rapid HPLC–ESI–MS/MS method was elaborated and validated for the studies of disposition of TREO and S,S-EBDM in rat plasma, liver, lungs, kidneys, muscle, and brain. The two analytes and codeine, internal standard (IS), were isolated from 50μL of plasma and 100μL of supernatants of the tissues homogenates using ultrafiltration Amicon vials. Chromatographic resolution was accomplished on C18 column with isocratic elution. The limits of quantitation of TREO and S,S-EBDM in the studied matrices ranged from 0.11 to 0.93μM. The HPLC–MS/MS method was adequately precise and accurate within and between runs. The IS-normalized matrix effect differed among the tissues and was the most pronounced in a liver homogenate supernatant (approximately 0.55 for TREO and 0.35 for S,S-EBDM). Stability of the analytes in experimental samples was also established. The validated method for the first time enabled determination of TREO and S,S-EBDM in the six life-important tissues in rats following administration of the prodrug.

Ocular disposition of treosulfan and its active epoxy-transformers following intravenous administration in rabbits

Treosulfan (TREO) has an established position in chemotherapy of advanced ovarian cancer but has been also applied in uveal melanoma patients. Moreover, it is used as an orphan drug for a myeloablative conditioning prior to stem cell transplantation. In this paper, biodistribution of prodrug TREO and its active monoepoxide (S,S-EBDM) and diepoxide (S,S-DEB) into aqueous humor of the eye was studied for the first time. For that purpose, alone TREO and the mixture of TREO, S,S-EBDM and S,S-DEB were administered intravenously to New Zealand White rabbits. The three analytes were determined in plasma and aqueous humor by validated HPLC methods and pharmacokinetic calculations were performed in WinNonlin. After the infusion of TREO, the aqueous humor/plasma Cmax ratio and area under the curve ratio amounted 0.04 and 0.10 for TREO, and 1.1 and 2.2 for S,S-EBDM, respectively. Following the bolus injection of the mixture of the prodrug and its epoxides, the aqueous humor/plasma Cmax ratios for TREO, S,S-EBDM and S,S-DEB were 0.05, 0.66, and 4.0, respectively. The presented results indicate a poor penetration of TREO into the eye, which may impair systemic treatment of ocular tumors but is beneficial in terms of a lack of clinically relevant ophthalmic adverse effects.

Formation Rate–Limited Pharmacokinetics of Biologically Active Epoxy Transformers of Prodrug Treosulfan

A prodrug treosulfan (TREO) is being evaluated in clinical trials as a myeloablative agent before hematopoietic stem cell transplantation. The active derivatives of TREO, monoepoxide (EBDM), and diepoxide (DEB) are formed in a pH-dependent nonenzymatic reaction. The aim of the study was to investigate pharmacokinetics of the TREO epoxy transformers in a rabbit model and explain the causes of low plasma concentrations of EBDM and DEB observed in patients receiving high-dose TREO before hematopoietic stem cell transplantation. New Zealand white rabbits (n = 5 per cohort) received an intravenous infusion of TREO (group I), injection of DEB (group II), and injection of a solution containing EBDM (group III). When EBDM and DEB were administered to the rabbits, they underwent a very rapid elimination (half-life 0.069 and 0.046 h) associated with a high systemic clearance (10.0 and 14.0 L h−1 kg−1). After administration of TREO, the t1/2 of EBDM was statistically equal to the t1/2 of the prodrug (1.6 h). To conclude, after administration of TREO, its epoxy transformers demonstrate a formation-limited elimination. Then EBDM and DEB have the same elimination half-life as TREO, but the levels of EBDM and DEB in the body, including plasma, are much lower than TREO on account of their inherently high clearance.

Activation of Prodrug Treosulfan at pH 7.4 and 37°C Accompanied by Hydrolysis of Its Active Epoxides: Kinetic Studies with Clinical Relevance

Treosulfan (TREO), originally registered for treatment of ovarian cancer, is currently being investigated for conditioning prior to hematopoietic stem cell transplantation. TREO is a prodrug, which undergoes a pH- and temperature-dependent two-step conversion to active monoepoxide [S,S-EBDM, (2S,3S)-1,2-epoxybutane-3,4-diol-4-methanesulfonate] and diepoxide [S,S-DEB, (2S,3S)-1,2:3,4-diepoxybutane]. In this paper, the kinetics of the nonenzymatic transformation of TREO at pH 7.4 and 37°C were studied for the first time including the effects of the TREO concentration, buffer concentration, ionic strength, and the presence of NaCl. Transformation of TREO was well described by a kinetic model, which included first-order reactions for TREO activation, that is, TREO → S,S-EBDM → S,S-DEB, and pseudo-first-order reactions for the hydrolytic decomposition of S,S-EBDM and S,S-DEB. In contrast to the two-step activation of TREO, the hydrolysis of epoxides was influenced by electrolytes. In phosphate-buffered saline, decomposition of S,S-EBDM and S,S-DEB (mean half-lives 25.7 and 15.4 h) proceeded much slower than their formation (mean half-lives 1.5 and 3.5 h). In conclusion, the kinetics of the nonenzymatic transformation of TREO in the presence of plasma electrolytes cannot contribute to the very low levels of S,S-EBDM and S,S-DEB observed in patient plasma. The results also indicate that elimination of TREO proceeds primarily via conversion to S,S-EBDM.

Penetration of Treosulfan and its Active Monoepoxide Transformation Product into Central Nervous System of Juvenile and Young Adult Rats.

Treosulfan (TREO) is currently investigated as an alternative treatment of busulfan in conditioning before hematopoietic stem cell transplantation. The knowledge of the blood-brain barrier penetration of the drug is still scarce. In this paper, penetration of TREO and its active monoepoxide (S,S-EBDM) and diepoxide (S,S-DEB) into the CNS was studied in juvenile (JR) and young adult rats (YAR) for the first time. CD rats of both sexes (n = 96) received an intravenous dose of TREO 500 mg/kg b.wt. Concentrations of TREO, S,S-EBDM, and S,S-DEB in rat plasma, brain, and cerebrospinal fluid (CSF, in YAR only) were determined by validated bioanalytical methods. Pharmacokinetic calculations were performed in WinNonlin using a noncompartmental analysis and statistical evaluation was done in Statistica software. In male JR, female JR, male YAR, and female YAR, the brain/plasma area under the curve (AUC) ratio for unbound TREO was 0.14, 0.17, 0.10, and 0.07 and for unbound S,S-EBDM, it was 0.52, 0.48, 0.28, and 0.22, respectively. The CSF/plasma AUC ratio in male and female YAR was 0.12 and 0.11 for TREO and 0.66 and 0.64 for S,S-EBDM, respectively. Elimination rate constants of TREO and S,S-EBDM in all the matrices were sex-independent with a tendency to be lower in the JR. No quantifiable levels of S,S-DEB were found in the studied samples. TREO and S,S-EBDM demonstrated poor and sex-independent penetration into CNS. However, the brain exposure was greater in juvenile rats, so very young children might potentially be more susceptible to high-dose TREO-related CNS exposure than young adults.

Transport of treosulfan and temozolomide across an in-vitro blood-brain barrier model.

In vitro, treosulfan (TREO) has shown high effectiveness against malignant gliomas. However, a first clinical trial for newly diagnosed glioblastoma did not show any positive effect. Even though dosing and timing might have been the reasons for this failure, it might also be that TREO does not reach the brain in sufficient amount. Surprisingly, there are no published data on TREO uptake into the brain of patients, despite extensive research on this compound. An in-vitro blood-brain barrier (BBB) model consisting of primary porcine brain capillary endothelial cells was used to determine the transport of TREO across the cell monolayer. Temozolomide (TMZ), the most widely used cytotoxic drug for malignant gliomas, served as a reference. An HPLC-ESI-MS/MS procedure was developed to detect TREO and TMZ in cell culture medium. Parallel to the experimental approach, the permeability of TREO and the reference substance across the in-vitro BBB was estimated on the basis of their physicochemical properties. The detection limit was 30 nmol/l for TREO and 10 nmol/l for TMZ. Drug transport was measured in two directions: influx, apical-to-basolateral (A-to-B), and efflux, basolateral-to-apical (B-to-A). For TREO, the A-to-B permeability was lower (1.6%) than the B-to-A permeability (3.0%). This was in contrast to TMZ, which had higher A-to-B (13.1%) than B-to-A (7.2%) permeability values. The in-vitro BBB model applied simulated the human BBB properly for TMZ. It is, therefore, reasonable to assume that the values for TREO are also meaningful. Considering the lack of noninvasive, significant alternative methods to study transport across the BBB, the porcine brain capillary endothelial cell model was efficient to collect first data for TREO that explain the disappointing clinical results for this drug against cerebral tumors.

Paediatric reduced intensity conditioning: analysis of centre strategies on regimens and definitions by the EBMT Paediatric Diseases and Complications and Quality of Life WP.

The aim of this analysis was to explore the diversity of reduced intensity conditioning (RIC) in paediatric allo-SCT in daily practice across Europe. Data from the European Group for Blood and Marrow Transplantation (EBMT) Promise database from 1994 to 2008 were supplemented by a survey of EBMT centres performing paediatric allo-SCT on the current policy asking for the underlying diseases and for the drug combinations. Records from 161 centres from 30 countries were analysed and 139 various RIC regimens were reported. More centres applied RIC for malignant rather than for non-malignant diseases. In general, fludarabine (FLU)-based regimens predominated except for BU-based regimens in myeloid malignancies and haemoglobinopathies. Treosulfan (TREO) was mainly applied for unspecified malignant diseases and for haemophagocytic diseases. FLU-based regimens revealed the greatest number of different combinations. Correlating the number of regimens with the number of treating centres revealed the lowest variety in FLU and the highest variety in TBI and TREO. FLU/melphalane and FLU/CY were the most frequent combinations. This extreme heterogeneity in RIC may influence both the efficacy and the safety of the procedures, which requires further investigation. Optimization and standardization of RIC is the final goal to provide a platform for future prospective studies.

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.

Rapid and sensitive liquid chromatography-tandem mass spectrometry method for determination of protein-free pro-drug treosulfan and its biologically active monoepoxy-transformer in plasma and brain tissue

For the first time a high performance liquid chromatography method with tandem mass spectrometry detection (HPLC-MS/MS) was developed for simultaneous determination of a pro-drug treosulfan (TREO) and its active monoepoxide (S,S-EBDM) in biological matrices. Small volumes of rat plasma (50μL) and the brain homogenate supernatant (100μL), equivalent to 0.02g of brain tissue, were required for the analysis. Protein-free TREO, S,S-EBDM and acetaminophen, internal standard (IS), were isolated from the samples by ultrafiltration. Complete resolution of the analytes and the IS was accomplished on Zorbax Eclipse column using an isocratic elution with a mobile phase composed of ammonium formate – formic acid buffer pH 4.0 and acetonitrile. Detection was performed on a triple-quadrupole MS via multiple-reaction-monitoring following electrospray ionization. The developed method was fully validated according to the current guidelines of the European Medicines Agency. Calibration curves were linear in ranges: TREO 0.2–5720μM and S,S-EBDM 0.9–175μM for plasma, and TREO 0.2–29μM and S,S-EBDM 0.4–44μM for the brain homogenate supernatant. The limits of quantitation of TREO and S,S-EBDM in the studied matrices were much lower in comparison to the previously used bioanalytical methods. The HPLC-MS/MS method was adequately precise (coefficient of variation≤12.2%), accurate (relative error≤8.6%), and provided no carry-over, acceptable matrix effect as well as dilution integrity. The analytes were stable in acidified plasma and the brain homogenate supernatant samples for 4h at room temperature, for 4 months at−80°C as well as within two cycles of freezing and thawing, and demonstrated 18–24h autosampler stability. The validated method enabled determination of low concentrations of TREO and S,S-EBDM in incurred brain samples of the rats treated with TREO, which constitutes a novel bioanalytical application.