The Influence of Paracetamol on the Penetration of Sorafenib and Sorafenib N-Oxide Through the Blood-Brain Barrier in Rats.

BACKGROUND AND OBJECTIVE: Sorafenib is an oral, multikinase inhibitor with established single-agent activity in several tumor types. Sorafenib was moderately transported by P-glycoprotein (P-gp) and more efficiently by breast cancer resistance protein. The constitutive androstane receptor (CAR) is a ligand-activated transcription factor involved in P-gp regulation in the brain microvasculature. Paracetamol is a CAR activator. The purpose of this study was to investigate the effect of paracetamol on the brain uptake of sorafenib and sorafenib N-oxide. METHODS: The rats were assigned to two groups-rats receiving oral paracetamol 100 mg/kg and sorafenib 100 mg/kg (n = 42, ISR+PA) and rats receiving oral vehicle and sorafenib 100 mg/kg (n = 42, IISR). The sorafenib and sorafenib N-oxide concentrations in blood plasma and brain tissue were determined by a high-performance liquid chromatography method with ultraviolet detection. Brain-to-plasma partition coefficient (Kp) was calculated as a ratio of the area under the curve from zero to 24 h (AUC) in the brain and plasma. A drug targeting index (DTI) was estimated as the group ISR+PA Kp to group IISR Kp ratio. RESULTS: Pharmacokinetic analysis revealed increased brain exposure to sorafenib and sorafenib N-oxide after co-administration of paracetamol. The brain maximum concentration (Cmax) and the AUC of the parent drug in the ISR+PA group compared with the IISR group were greater by 49.5 and 77.8%, respectively, and the same parameters for the metabolite were higher by 51.4 and 50.9%. However, the Kp values of sorafenib and sorafenib N-oxide did not differ significantly between the two animal groups and the DTI values were close to 1. CONCLUSION: Paracetamol increases exposure to sorafenib and sorafenib N-oxide in the brain, likely due to increased exposure in plasma.

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

PURPOSE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

In Vitro Study of the Enzymatic and Nonenzymatic Conjugation of Treosulfan with Glutathione.

BACKGROUND AND OBJECTIVES: Treosulfan (dihydroxybusulfan), licensed for the treatment of ovarian carcinoma, is investigated in clinical trials as a myeloablative agent for conditioning prior to hematopoietic stem cell transplantation. Clinical experience shows that treosulfan exhibits lower organ toxicity than busulfan, including hepatotoxicity. Elimination of busulfan primarily via enzymatic conjugation with glutathione (GSH) in the liver is considered to be the main cause of the drug's hepatotoxicity and interpatient clearance variability. It is believed that treosulfan undergoes no hepatic metabolism but empirical evidence is lacking. The aim of this kinetic study was to verify if treosulfan is capable of conjugating with GSH. METHODS: Treosulfan (200 µM) was incubated at pH 7.2 and 37 °C with 5 mM GSH in the presence or absence of human liver cytosol, the main store of glutathione S-transferase in the body. Concentrations of treosulfan were determined using liquid chromatography-tandem mass spectrometry and then subjected to kinetic analysis. RESULTS: The decay of treosulfan in the solution followed a one-exponential model in the presence of either GSH or liver cytosol and GSH. The first-order reaction rate constants (0.25 h-1) did not differ statistically from those found for treosulfan conversion in pH 7.2 buffer only. CONCLUSION: Treosulfan does not undergo either spontaneous or enzymatic conjugation with GSH at a noticeable rate. The result indicates that the clearance of treosulfan is independent of glutathione S-transferase activity, GSH stores, and co-administration of drugs utilizing the GSH metabolic pathway.

Kinetics of in Vitro Guanine- N7-Alkylation in Calf Thymus DNA by (2 S,3 S)-1,2-Epoxybutane-3,4-diol 4-methanesulfonate and (2 S,3 S)-1,2:3,4-Diepoxybutane: Revision of the Mechanism of DNA Cross-Linking by the Prodrug Treosulfan.

Prodrug treosulfan, originally registered for treatment of ovarian cancer, has gained a use in conditioning prior to hematopoietic stem cell transplantation. Treosulfan converts nonenzymatically to the monoepoxide intermediate (EBDM), and then to (2 S,3 S)-1,2:3,4-diepoxybutane (DEB). The latter alkylates DNA forming mainly (2' S,3' S)- N-7-(2',3',4'-trihydroxybut-1'-yl)guanine (THBG) and (2 S,3 S)-1,4-bis(guan-7'-yl)butane-2,3-diol cross-link (bis-N7G-BD) via the intermediate epoxide adduct (EHBG). It is believed that DNA cross-linking by DEB is a primary mechanism for the anticancer and myeloablative properties of treosulfan, but clear evidence is lacking. Recently, we have proved that EBDM alkylates DNA producing (2' S,3' S)- N-7-(2',3'-dihydroxy-4'-methylsulfonyloxybut-1'-yl)-guanine (HMSBG) and that free HMSBG converts to EHBG. In this paper, we investigated the kinetics of HMSBG, bis-N7G-BD, and THBG in DNA in vitro to elucidate the contribution of EBDM and DEB to treosulfan-dependent DNA-DNA cross-linking. Calf thymus DNA was exposed to ( A) 100 µM treosulfan, ( B) 200 µM treosulfan, and ( C) DEB at a concentration 100 µM, exceeding that produced by 200 µM treosulfan. Following mild acid thermal hydrolysis of DNA, ultrafiltration, and off-line HPLC purification, the guanine adducts were quantified by LC-MS/MS. Both bis-N7G-BD and THBG reached highest concentrations in the DNA in experiment B. Ratios of the maximal concentration of bis-N7G-BD and THBG to DEB (adduct Cmax/DEB Cmax) in experiments A and B were 1.7-3.0-times greater than in experiment C. EHBG converted to the bis-N7G-BD cross-link at a much higher rate constant (0.20 h-1) than EBDM and DEB initially alkylated the DNA (1.8-3.4 × 10-5 h-1), giving rise to HMSBG and EHBG, respectively. HMSBG decayed unexpectedly slowly (0.022 h-1) compared with the previously reported behavior of the free adduct (0.14 h-1), which revealed the inhibitory effect of the DNA environment on the adduct epoxidation to EHBG. A kinetic simulation based on the obtained results and the literature pharmacokinetic parameters of treosulfan, EBDM, and DEB suggested that in patients treated with the prodrug, EBDM could produce the vast majority of EHBG and bis-N7G-BD via HMSBG. In conclusion, EBDM can produce DNA-DNA lesions independently of DEB, and likely plays a greater role in DNA cross-linking after in vivo administration of treosulfan than DEB. These findings compel revision of the previously proposed mechanism of the pharmacological action of treosulfan and contribute to better understanding of the importance of EBDM for biological effects.

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 ß-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.

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.

Clinical bioanalysis of treosulfan and its epoxides: The importance of collected blood processing for valid pharmacokinetic results.

Currently, there is an urgent need to establish the optimal dosing of TREO in conditioning prior to hematopoietic stem cell transplantation, especially in children. For that purpose, pharmacokinetic analyses are ongoing within clinical phase II and III trials. In this paper, HPLC methods for determination of prodrug treosulfan and/or its biologically active epoxides in human plasma or serum are reviewed for the first time, including the spectrum of analytes being quantified, detection type, and derivatization methodology. The major focus is addressed to the stability of TREO and its monoepoxide related with different strategies of patients' blood processing, e.g. blood pH lowering to different values, no pH adjustment; centrifugation of blood immediately after collection or within a few hours later. This issue is crucially important for the robust bioanalysis because the epoxytransformation of TREO is a nonenzymatic, highly pH and temperature-dependent reaction. In-depth analysis of the literature results demonstrates that some methodologies of blood treatment could produce the systematic underestimation of TREO concentrations. Consequently, the drug clearance and volume of distribution will be overestimated, which might false the association of the drug exposure with the regimen-related toxicity and clinical outcomes. The paper indicates the deficiencies of the blood processing strategies and offers hints for their refinement. The provided information ought to be important in the current investigations of the personalized TREO pharmacokinetics.

In Vivo Red Blood Cells/Plasma Partition Coefficient of Treosulfan and Its Active Monoepoxide in Rats.

BACKGROUND AND OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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)

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.

Kinetic and Mechanistic Study of the pH-Dependent Activation (Epoxidation) of Prodrug Treosulfan Including the Reaction Inhibition in a Borate Buffer.

A prodrug treosulfan (T) undergoes a pH-dependent activation to epoxide derivatives. The process seems to involve an intramolecular Williamson reaction (IWR) but clear kinetic evidence is lacking. Moreover, a cis-diol system present in the T structure is expected to promote complexation with boric acid. As a result, the prodrug epoxidation would be inhibited; however, this phenomenon has not been investigated. In this article, the effect of pH on the kinetics of T conversion to its monoepoxide was studied from a mechanistic point of view. Also, the influence of boric acid on the reaction kinetics was examined. The rate constants observed for the activation of T (kobs) in acetate, phosphate, and carbonate buffers satisfied the equation logkobs = -7.48 + 0.96 pH. The reaction was inhibited in the excess of boric acid over T, and the kobs decreased with increasing borate buffer concentration. The experimental results were consistent with the inhibition model that included the formation of a tetrahedral, anionic T-boric acid monoester. To conclude, in nonborate buffers, the T activation to (2S,3S)-1,2-epoxybutane-3,4-diol 4-methanesulfonate follows IWR mechanism. A borate buffer changes the reaction kinetics and complicates kinetic analysis.

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.

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 7.5 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.

Determination of partition coefficients n-octanol/water for treosulfan and its epoxy-transformers: an example of a negative correlation between lipophilicity of unionized compounds and their retention in reversed-phase chromatography.

For the last decade an alkylating agent treosulfan (TREO) has been successfully applied in clinical trials in conditioning prior to hematopoietic stem cell transplantation. Pharmacological activity of the pro-drug depends on its epoxy-transformers, monoepoxide (S,S-EBDM) and diepoxide (S,S-DEB), which are formed in a non-enzymatic consecutive reaction accompanied by a release of methanesulfonic acid. In the present study partition coefficient n-octanol/water (POW) of TREO as well as its biologically active epoxy-transformers was determined empirically (applying a classical shake-flask method) and in silico for the first time. In vitro the partition was investigated at 37°C in the system composed of the pre-saturated n-octanol and 0.05 M acetate buffer pH 4.4 adjusted with sodium and potassium chloride to ionic strength of 0.16 M. Concentration of the analytes was quantified by reversed-phase high performance liquid chromatography (RP-HPLC) method in which retention time increased from S,S-DEB to TREO. It was shown that neither association nor dissociation of the tested compounds in the applied phases occurred. Calculated logPOW (TREO: -1.58±0.04, S,S-EBDM: -1.18±0.02, S,S-DEB: -0.40±0.03) indicate the hydrophilic character of the all three entities, corresponding to its pharmacokinetic parameters described in the literature. Experimentally determined logPOW of the compounds were best comparable to the values predicted by algorithm ALOGPs. Interestingly, the POW values determined in vitro as well as in silico were inversely correlated with the retention times observed in the endcapped RP-HPLC column. It might be explained by the fact that a cleavage of methansulfonic acid from a small molecule of TREO generates significant changes in the molecular structure. Consequently, despite the common chemical origin, TREO, S,S-EBDM and S,S-DEB do not constitute a 'congeneric' series of compounds. We concluded that this might occur in other low-weight species, therefore measurement of their POW by RP-HPLC had to be applied with a special care.

Direct high-performance liquid chromatography method with refractometric detection designed for stability studies of treosulfan and its biologically active epoxy-transformers.

Treosulfan (TREO) is an alkylating agent registered for treatment of advanced platin-resistant ovarian carcinoma. Nowadays, TREO is increasingly applied iv in high doses as a promising myeloablative agent with low organ toxicity in children. Under physiological conditions it undergoes pH-dependent transformation into epoxy-transformers (S,S-EBDM and S,S-DEB). The mechanism of this reaction is generally known, but not its kinetic details. In order to investigate kinetics of TREO transformation, HPLC method with refractometric detection for simultaneous determination of the three analytes in one analytical run has been developed for the first time. The samples containing TREO, S,S-EBDM, S,S-DEB and acetaminophen (internal standard) were directly injected onto the reversed phase column. To assure stability of the analytes and obtain their complete resolution, mobile phase composed of acetate buffer pH 4.5 and acetonitrile was applied. The linear range of the calibration curves of TREO, S,S-EBDM and S,S-DEB spanned concentrations of 20-6000, 34-8600 and 50-6000 µM, respectively. Intra- and interday precision and accuracy of the developed method fulfilled analytical criteria. The stability of the analytes in experimental samples was also established. The validated HPLC method was successfully applied to the investigation of the kinetics of TREO activation to S,S-EBDM and S,S-DEB. At pH 7.4 and 37 °C the transformation of TREO followed first-order kinetics with a half-life 1.5h.

HPLC method for determination of biologically active epoxy-transformers of treosulfan in human plasma: pharmacokinetic application.

Clinical trials demonstrated treosulfan (TREO) as a promising myeloablative agent prior to hematopoietic stem cell transplantation (HSCT). TREO is a specific pro-drug from which biologically active mono- (S,S-EBDM) and diepoxybutane (S,S-DEB) derivatives are formed in vitro or in vivo by a non-enzymatic pH and temperature-dependent intramolecular nucleophilic substitution. Following extraction of the plasma samples with a mixture of dichloromethane and acetonitrile, S,S-EBDM and S,S-DEB were derivatized with 3-nitrobenzenesulfonic acid (3-NBS) to UV-absorbing esters. Optimal temperature and time of derivatization as well as extraction method and also the effect of pH on TREO stability in plasma were established. Identity of the synthesized derivatives was confirmed by mass spectrometry. The post-derivatization mixture was purified from the excess of unreacted 3-NBS by extraction with water. The derivatization products and 2,2'-dinitrobiphenyl (internal standard) were separated on Nucleosil 100 C18 column using a mobile phase consisted of acetonitrile and water. The developed method was validated and demonstrated adequate accuracy and precision. Limit of quantification for both S,S-EBDM and S,S-DEB amounted to 2.5 µM. The method was applied in clinical conditions to quantify the levels of TREO activation products in plasma of children undergoing HSCT. The methodology for simultaneous determination of TREO epoxy-transformers in human plasma is described for the first time.