Active Monoepoxide Research Articles

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.

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.

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

Background and ObjectivesTreosulfan 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.MethodsMale 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.ResultsThe 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.ConclusionsTreosulfan 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)<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.

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.

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 7.5years) 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.