Enantioselectivity in the Metabolism of Cyclophosphamide in Patients With Multiple or Systemic Sclerosis.

The aim of this study was to evaluate the enantioselective pharmacokinetics of cyclophosphamide and its metabolites 4-hydroxycyclophosphamide and carboxyethylphosphoramide mustard in patients with systemic or multiple sclerosis. Patients with systemic sclerosis (n = 10) or multiple sclerosis (n = 10), genotyped for the allelic variants of CYP2C9*2 and CYP2C9*3 and of the CYP2B6 G516T polymorphism, were treated with 50 mg cyclophosphamide/kg daily for 4 days. Serial blood samples were collected up to 24 hours after administration of the last cyclophosphamide dose. Cyclophosphamide, 4-hydroxycyclophosphamide, and carboxyethylphosphoramide enantiomers were analyzed in plasma samples using liquid chromatography-tandem mass spectrometry coupled to chiral column Chiralcel OD-R or Chiralpak AD-RH. Cytokines IL-2, IL-4, IL-6, IL-8, IL-10, IL- 12p70, IL-17, TNF-α, and INT-δ in the plasma samples collected before cyclophosphamide infusion were analyzed by Milliplex MAP human cytokine/chemokine. Pharmacokinetic parameters showed higher plasma concentrations of (S)-(-)-cyclophosphamide (AUC 215.0 vs 186.2 µg·h/mL for multiple sclerosis patients and 219.1 vs 179.2 µg·h/mL for systemic sclerosis patients) and (R)-4-hydroxycyclophosphamide (AUC 5.6 vs 3.7 µg·h/mL for multiple sclerosis patients and 6.3 vs 5.6 µg·h/mL for systemic sclerosis patients) when compared to their enantiomers in both groups of patients, whereas the pharmacokinetics of the carboxyethylphosphoramide metabolite was not enantioselective. Cytokines' plasma concentrations were similar between multiple and systemic sclerosis groups. The pharmacokinetics of cyclophosphamide is enantioselective in patients with systemic sclerosis and multiple sclerosis, with higher plasma concentrations of the (S)-(-)-cyclophosphamide enantiomer due to the preferential formation of the (R)-4-hydroxycyclophosphamide metabolite.

Enantioselective analysis of 4-hydroxycyclophosphamide in human plasma with application to a clinical pharmacokinetic study.

Cyclophosphamide (CY) is one of the most common immunosuppressive agents used in autologous hematopoietic stem cell transplantation. CY is a prodrug and is metabolized to active 4-hydroxycyclophosphamide (HCY). Many authors have suggested an association between enantioselectivity in CY metabolism and treatment efficacy and/or complications. This study describes the development and validation of an analytical method of HCY enantiomers in human plasma by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) that can be applied to pharmacokinetic studies, filling this gap in the literature. HCY enantiomers previously derivatized with phenylhydrazine were extracted from 200-µL plasma aliquots spiked with antipyrine as internal standard and a mixture of hexane and dichloromethane (80:20, v/v) was used as the extraction solvent. The derivatized HCY enantiomers were resolved on a Chiracel(®) OD-R column using water:acetonitrile:formic acid (55:45:0.2, v/v) as the mobile phase. No matrix effect was observed and the analysis of HCY enantiomers was linear for plasma concentrations of 5-5000ng of each enantiomer/mL plasma. The coefficients of variation and inaccuracy calculated in precision and accuracy assessments were less than 15%. HCY was stable in human plasma after three successive freeze/thaw cycles, during 3h at room temperature, and in the autosampler at 4°C for 24h after processing, with deviation values less than 15%. The method was applied to evaluate the kinetic disposition of HCY in a patient with multiple sclerosis who was pretreated with intravenous racemic CY for stem cell transplantation. The clinical study showed enantioselectivity in the pharmacokinetics of HCY.

Use of an Oral Busulfan Test Dose in Patients Undergoing Hematopoietic Stem Cell Transplantation Treated With or Without Fludarabine.

This study investigated the importance of an oral test dose for busulfan (BU) dose adjustment before a conditioning regimen for hematopoietic stem-cell transplantation (HSCT) and the effect of fludarabine (FLU) on the oral BU pharmacokinetics evaluated after the fifth treatment dose (first BU dose on day 2 of treatment). Twenty-eight patients treated with oral BU (1 mg/kg every 6 hours for 4 days) were divided into 2 groups according to the concomitant administration of FLU (n = 15; 30 mg/m2 for 5 days) or subsequent administration of cyclophosphamide (CY) (n = 13; 60 mg/kg for 2 days). On the day prior to the beginning of the conditioning regimen, blood samples were collected (0-6 hours) after administration of an oral BU test dose of 0.25 mg/kg. Busulfan was quantified in plasma samples by LC-MS/MS, and the pharmacokinetic parameters were calculated using WinNonlin software. Blood samples were collected between the fifth and sixth treatment dose to confirm the mean plasma steady-state concentration (Css ) of BU. The AUC0-6 and apparent clearance of BU did not differ (P < .05) between the groups receiving FLU and CY. In 81% of the patients who received BU doses adjusted based on the test dose (n = 21), the Css was within the target range of 600-900 ng/mL. No association was observed between BU AUC0-6 and clinical outcome in the study group (n = 28). The results suggest that in concomitant administration of FLU and BU during conditioning regimens for HSCT, changes in BU dose should be considered only after the administration of the fifth BU dose.

Influence of fludarabine on the pharmacokinetics of oral busulfan during pretransplant conditioning for hematopoietic stem cell transplantation.

This study evaluated the influence of fludarabine on the pharmacokinetics of busulfan administered orally to patients receiving a conditioning regimen for hematopoietic allogeneic stem cell transplantation (HSCT). Twenty-six patients treated with oral busulfan (1 mg/kg/6 h for 4 days) were divided into two groups according to the concomitant administration of fludarabine (n = 11; 30 mg/m(2) for 5 days) or subsequent administration of cyclophosphamide (n = 15; 60 mg/kg for 2 days). Serial blood samples were collected on Day 4 of busulfan administration. Plasma busulfan concentrations were determined by HPLC-UV and the pharmacokinetic parameters were calculated using the WinNonlin program. Patients concomitantly treated with fludarabine showed reduced apparent clearance of busulfan (110.5 mL/h/kg vs. 157.4 mL/h/kg) and higher AUC0-6 (area under the plasma concentrations vs. time curve) than patients subsequently treated with cyclophosphamide (7.9 µg h/mL vs. 5.7 µg h/mL). No association was observed between busulfan AUC0-6 and clinical evolution of the patients. Although plasma busulfan concentrations were higher in patients receiving concomitant fludarabine, myelosuppression-related toxicity was less frequent than in patients treated with busulfan and cyclophosphamide. The results suggest that patients treated with fludarabine should receive 30% lower busulfan doses during conditioning protocols for HSCT.