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Prodrug treosulfan undergoes a pH and temperature-dependent activation to the monoepoxide intermediate (EBDM) and (2S,3S)-1,2:3,4-diepoxybutane (DEB). The latter DNA cross-linker is presently believed to mainly account for the pharmacological action of treosulfan. However, neither respective monoadducts nor cross-links have been isolated from treosulfan-treated DNA, and the exact alkylation mechanism of the treosulfan epoxides is unclear. In this paper, liquid chromatography method with tandem mass spectrometry detection (LC-MS/MS) for simultaneous determination of the N-7-guanine adducts of EBDM and DEB − (2′S,3′S)-N-7-(2′3′-dihydroxy-4′-methylsulfonyloxybut-1′-yl)guanine (HMSBG), N-7-(2′,3′,4′-trihydroxybut-1′-yl)guanine (THBG), and 1,4-bis(N-7-guanyl)butane-2,3-diol cross-link (bis-N7G-BD) − in calf-thymus DNA has been developed and validated for the first time. The mixture of drug-free nucleic acid with the analytes and 15N-isotope labeled internal standards underwent a mild acid thermal hydrolysis and ultrafiltration (cut-off 10 kDa). Following offline LC purification, the analytes and internal standards were determined in the LC-MS/MS system with an electrospray interface. Complete resolution of THBG, HMSBG, and bis-N7G-BD was accomplished on a Zorbax Eclipse C18 column using gradient elution with a mobile phase composed of 0.1% formic acid and acetonitrile. Calibration curves were linear in the ranges: THBG 0.2–200 pmol, HMSBG 0.2–20 pmol, and bis-N7G-BD 0.4–40 pmol. The limits of quantitation allowed to determine the adducts at concentration of 330 or 660 per 109 DNA nucleotides. The LC-MS/MS method was adequately precise (coefficient of variation ≤ 16.7%) and accurate (relative error ≤ 17.7%). Calibration standards were stable for 14 days at –25 °C. The validated method enabled determination of THBG, HMSBG, and bis-N7G-BD in calf thymus DNA treated with treosulfan at pH 7.2 and 37 °C, which constitutes a novel bioanalytical application. To the authors’ best knowledge, the quantification of THBG and bis-N7G-BD in one analytical run is also reported for the first time.

Treosulfan is a prodrug applied in the treatment of ovarian cancer and conditioning prior to stem cell transplantation. So far, the bioanalysis of treosulfan in either whole blood or red blood cells (RBC) has not been carried out. In this work, the RBC/plasma partition coefficient (Ke/p) of treosulfan and its active monoepoxide was determined for the first time. Male and female 10-week-old Wistar rats (n = 6/6) received an intraperitoneal injection of treosulfan at the dose of 500 mg/kg body weight. The concentrations of treosulfan and its monoepoxide in plasma (Cp) and RBC were analyzed with a validated HPLC–MS/MS method. The mean Ke/p of treosulfan and its monoepoxide were 0.74 and 0.60, respectively, corresponding to the blood/plasma partition coefficient of 0.88 and 0.82. The Spearman test demonstrated that the Ke/p of the prodrug correlated with its Cp, but no correlation between the Ke/p and Cp of the active monoepoxide was observed. Treosulfan and its monoepoxide achieve higher concentrations in plasma than in RBC; therefore, the choice of plasma for bioanalysis is rational as compared to whole blood. The distribution of treosulfan into RBC may be a saturable process at therapeutic concentrations.

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.

Efficacy 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.The 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.One-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).The 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.

(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.

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 7.5 years) undergoing TREO-based preparative regimen prior to HSCT, who received 10, 12 or 14 g/m(2) 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 C(max) as well as AUC of S,S-EBDM demonstrated linear correlation with the C(max) 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.

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 (50 μL) and the brain homogenate supernatant (100 μL), equivalent to 0.02 g 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 0.2–5720 μM and S,S-EBDM 0.9–175 μM for plasma, and TREO 0.2–29 μM and S,S-EBDM 0.4–44 μM 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 4 h at room temperature, for 4 months at−80 °C as well as within two cycles of freezing and thawing, and demonstrated 18–24 h 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.