UPLC-Tandem Mass Spectrometry for Quantification of Busulfan in Human Plasma: Application to Therapeutic Drug Monitoring.

Busulfan (Bu) is an alkylating agent commonly used in preparative regimens for hematologic malignant and non-malignant patients undergoing hematopoietic stem cell transplantation (HSCT). The objective of the present study was to develop an UPLC-MS/MS method for quantification of Bu in human plasma. A total of 55 patients with hematologic malignancies (n = 34) and non- malignancies (n = 21) received myeloablative Bu therapy prior to HSCT. A tandem mass spectrometric method was developed and validated to quantify Bu levels in these patients. The method was fully validated over the concentration range of 25-2000 ng/mL (r > 0.99). The assay method demonstrated good precision and accuracy. Stability studies indicated that the drug was stable in various conditions. Incurred sample reanalysis findings were within acceptable ranges (<15% of the nominal concentration). Based on the 1st dose AUC results, one third of hematologic malignant patients and half of non-malignant patients needed dose adjustment. However, in subsequent doses (5th, 9th, and 13th), 77%, 82% and 82%, respectively, of hematologic malignant patients and 71%, 67% and 86%, respectively, of non-malignant patients achieved the target range of Bu AUC. The suitability of the developed method for routine TDM of Bu in HSCT patients was demonstrated. The study suggests that the pharmacokinetic profile of Bu varies in both groups.

Analysis of Busulfan in Plasma by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

Bone marrow transplantation is used to treat particular types of cancers such as lymphoma, leukemia, and multiple myeloma. Appropriate dosing of busulfan during the preparative phase is critical for a successful allograft; if blood concentrations get too high significant liver toxicity can occur, if blood concentrations are too low, then graft-versus-host disease (GVHD) can develop. Busulfan monitoring in blood allows hospitals with the opportunity to provide individualized medicine to patients and improve overall patient outcome. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) is an important analytical method for quantification of busulfan in plasma in order to optimize the dose. © 2020 Wiley Periodicals LLC. Basic Protocol: Analysis of busulfan by liquid chromatography/mass spectrometry.

Novel method for fast trabectedin quantification using hydrophilic interaction liquid chromatography and tandem mass spectrometry for human pharmacokinetic studies.

Few time-consuming bioanalytical methods are currently available for trabectedin quantification in clinical investigations. Here we present a novel, fast and sensitive method for trabectedin determination in human plasma based on hydrophilic interaction liquid chromatography and tandem mass spectrometry (HILIC-MS/MS). Plasma samples are treated with acetonitrile-0.1 % formic acid and the solvent extract is directly injected into an Acquity BEH Amide column (2.1 × 100 mm, 1.7 µm) operating in HILIC mode at 0.2 mL/min with 80:20 acetonitrile-0.1 % formic acid in water. The analyte is separated by an organic solvent gradient and quantified by an Agilent Ultivo triple quadrupole mass spectrometer operating in multiple reaction monitoring (MRM) mode. The quantitative MRM transitions were m/z 762→234 and m/z 765→234 for trabectedin and its d3-labeled derivative, respectively. The lower limit of quantification (LLOQ) was 0.01 ng/mL and the assay was linear up to 2.5 ng/mL. The intra- and inter-day relative error ranged from 1.19 % to 8.52 %, while the relative standard deviation was less than 12.35 %. The method was used to determine the pharmacokinetic profiles of trabectedin in 26 patients with soft tissue sarcoma, showing that this new HILIC-MS/MS method is suitable for use in clinical research.

Comparing Dried Blood Spots and Plasma Concentrations for Busulfan Therapeutic Drug Monitoring in Children.

BACKGROUND: Busulfan (Bu) is one of the conditioning regimen components for pediatric hematopoietic stem cell transplantation. Bu therapeutic drug monitoring (TDM) is essential for a successful treatment outcome and toxicity evasion. Dried blood spot (DBS) sampling is a rapid and simple method for Bu TDM, compared with conventional plasma sampling. This study evaluated the feasibility of using the DBS method for Bu TDM. The hematocrit (Hct) and conditioning day were also examined for their impact on the DBS method's performance. METHODS: Venous blood collected from 6 healthy volunteers was diluted, using their plasma into 4 samples of varying Hct values. Each sample was spiked with Bu calibrators (300, 600, and 1400 ng/mL), prepared using DBS and dried plasma spot (DPS) sampling and analyzed using a validated liquid-chromatography tandem-mass spectrometry method. Clinical blood samples (n = 153) from pediatric patients (n = 15) treated with Bu (mainly from doses 1, 2, 5, and 9) were used to prepare paired volumetric DBS and DPS samples. A Bland-Altman plot and Deming regression were used to define the agreement between the paired DBS and DPS measurements. Passing-Bablok regression analyses investigated the effects of Hct and conditioning day on the linearity between both methods. RESULTS: In vitro analyses showed good agreement between DBS and DPS measurements, with a mean difference of -5.4% and a 95% confidence interval on the limits of agreement of -15.3% to 4.6%. Clinical samples showed good correlation (Pearson correlation coefficient = 0.96; slope = 1.00) between the DBS and DPS methods. The DBS method met the clinical acceptance limits for clinical samples, with a bias <±20%. Bland-Altman plots showed good agreement, with only 5.8% of paired measurements exceeding the limits of agreement (±1.96 SD), although within its 95% confidence interval. Hct observations ranged from 21.7% to 34.7% and did not affect Bu concentrations measured from DBS in either the in vitro or in vivo studies. CONCLUSIONS: These results show that DBS is a useful method for Bu TDM, provided samples are analyzed on the collection day. DBS sampling offers advantages over traditional plasma sampling in infants and younger children because only small volumes of blood are required.

CNS Penetration of Cyclophosphamide and Metabolites in Mice Bearing Group 3 Medulloblastoma and Non-Tumor Bearing Mice.

PURPOSE: Cyclophosphamide is widely used to treat children with medulloblastoma; however, little is known about its brain penetration. We performed cerebral microdialysis to characterize the brain penetration of cyclophosphamide (130 mg/kg, IP) and its metabolites [4-hydroxy-cyclophosphamide (4OH-CTX) and carboxyethylphosphoramide mustard (CEPM)] in non-tumor bearing mice and mice bearing orthotopic Group 3 medulloblastoma. METHODS: A plasma pharmacokinetic study was performed in non-tumor-bearing CD1- nude mice, and four cerebral microdialysis studies were performed in non-tumor-bearing (M1 and M3) and tumor- bearing mice (M2 and M4). Plasma samples were collected up to 6-hours post-dose, and extracellular fluid (ECF) samples were collected over 60-minute intervals for 24-hours post-dose. To stabilize and quantify 4OH-CTX, a derivatizing solution was added in blood after collection, and either directly in the microdialysis perfusate (M1 and M2) or in ECF collection tubes (M3 and M4). Plasma/ECF cyclophosphamide and CEPM, and 4OH-CTX concentrations were separately measured using different LC-MS/MS methods. RESULTS: All plasma/ECF concentrations were described using a population-based pharmacokinetic model. Plasma exposures of cyclophosphamide, 4OH-CTX, and CEPM were similar across studies (mean AUC=112.6, 45.6, and 80.8 µmol∙hr/L). Hemorrhage was observed in brain tissue when the derivatizing solution was in perfusate compared with none when in collection tubes, which suggested potential sample contamination in studies M1 and M2. Model-derived unbound ECF to plasma partition coefficients (Kp,uu) were calculated to reflect CNS penetration of the compounds. Lower cyclophosphamide Kp,uu was obtained in tumor-bearing mice versus non-tumor bearing mice (mean 0.15 versus 0.22, p=0.019). No differences in Kp,uu were observed between these groups for 4OH- CTX and CEPM (overall mean 0.10 and 0.07). CONCLUSIONS: Future studies will explore potential mechanisms at the brain-tumor barrier to explain lower cyclophosphamide brain penetration in tumor-bearing mice. These results will be used to further investigate exposure-response relationships in medulloblastoma xenograft models.

Photochemically stabilized formulation of dacarbazine with reduced production of algogenic photodegradants.

The present study aimed to develop a photochemically stabilized formulation of dacarbazine [5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide; DTIC] for reducing the production of algogenic photodegradant (5-diazoimidazole-4-carboxamide; Diazo-IC). Photochemical properties of DTIC were characterized by UV-visible light spectral analysis, reactive oxygen species (ROS) assay, and photostability testing. A pharmacokinetic study was conducted after intravenous administration of DTIC formulations (1 mg-DTIC/kg) to rats. DTIC exhibited strong absorption in the UVA range, and photoirradiated DTIC exhibited marked ROS generation. Thus, DTIC had high photoreactive potential. After exposure of DTIC (1 mM) to simulated sunlight (250 W/m2) for 3 min, remaining DTIC and yielded Diazo-IC were estimated to be ca. 230 µM and 600 µM, respectively. The addition of radical scavenger (1 mM), including l-ascorbic acid, l-cysteine (Cys), l-histidine, D-mannitol, l-tryptophan, or l-tyrosine, to DTIC (1 mM) could attenuate DTIC photoreactions, and in particular, the addition of Cys to DTIC brought ca. 34% and 86% inhibition of DTIC photodegradation and Diazo-IC photogeneration, respectively. There were no significant differences in the calculated pharmacokinetic parameters of DTIC between DTIC and DTIC with Cys (0.67 mg/kg). From these findings, the supplementary use of Cys would be an effective approach to improve the photostability of DTIC with less production of Diazo-IC.

Determination of melphalan in human plasma by UPLC-UV method.

It is desirable to develop a fast method for quantification of melphalan due to its instability. Here we report a method for quantification of melphalan (MPL) in human plasma using a UPLC-PDA system. Briefly, 50 µL plasma sample was mixed with 25 µL internal standard (2500 ng/mL acetylmelphalan in methanol) and 25 µL 20% trichloroacetic acid, and centrifuged at 21,000 g (15,000 rpm) at 4 °C for 3 min. The supernatant (5 µL) was injected onto an Acquity™ BEH C18 LC column (2.1 × 50 mm, 1.7 µm) and eluted with 25 mM NH4AC (pH 4.7)-acetonitrile in a gradient mode at a flow rate of 0.6 mL/min. The column kept at 40 ± 5 °C and the autosampler kept at 4 ± 5 °C. The detector set at 261 nm, and sampling rate was 40points/sec. The retention times were typically 2.11 min for melphalan and 2.38 min for the internal standard. Total run time is 4 min per sample. Calibration range was 100-40,000 ng/mL. The lower limit of quantification was 100 ng/mL. The method was validated based on the FDA guidelines, and applied to a clinical pharmacokinetic study in pediatric patients.

Pharmacokinetics: Unique Challenges in Blood Monitoring for Oncology Nurses.

BACKGROUND: Pharmacokinetics (PK) is the study of the absorption, distribution, metabolism, and excretion of drugs. Many chemotherapeutic agents have a sensitive PK index, in which a small margin in blood concentrations is the difference between nontherapeutic, therapeutic, and adverse outcomes. OBJECTIVES: This article will provide an overview of evidence-based approaches to the collection of PK samples, monitoring of PK levels, and the resulting management of patients undergoing PK testing. METHODS: A case study involving busulfan, an alkylating agent used in the pre-stem cell transplantation setting, will highlight the cross-contamination of samples while a drug is being infused through a central venous catheter with PK sample collection from a proximal peripherally inserted central catheter. The influence of false elevations in drug concentrations on PK-guided dose adjustments will also be emphasized. FINDINGS: Imprecise blood collections or cross-contamination of samples may lead to inaccurate drug concentration results and, subsequently, undesired low or high drug dosage calculations.

Development and validation of simple step protein precipitation UHPLC-MS/MS methods for quantitation of temozolomide in cancer patient plasma samples.

Temozolomide (TEMODAL™) (TMZ) is an antineoplastic agent that is primarily used for the treatment of glioblastoma and anaplastic gliomas, two aggressive forms of brain cancer. Due to the poor prognosis of brain tumour patients, there is an increasing body of research into improving the stability and delivery of TMZ past the blood brain barrier using carrier molecules. These require accurate determination of TMZ levels for biodistribution and pharmacokinetic evaluation. Unfortunately, current methodologies for the determination of TMZ in human plasma suffer from low reproducibility, recovery, sensitivity or cost ineffective procedures associated with extensive sample cleaning. To surpass these disadvantages, we developed two bioanalytical methods with high sensitivity and excellent recovery for the determination of TMZ in human plasma at minimum cost. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used and both methods were validated under US Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) guidelines. The two methods had minor differences in the sample pre-treatment and each method was developed and applied in separate laboratories. Theophylline was selected as internal standard (IS). Calibration curves were linear over the range of 10-500 ng/mL with extraction recovery ranging from 77.3 to 97.3% while all validation parameters met the acceptance criteria and proved the methods' reliability. The validated methods were successfully applied to plasma samples donated from cancer patient following treatment with temozolomide.

In vitro effect of chlorambucil on human glioma cell lines (SF767 and U87-MG), and human microvascular endothelial cell (HMVEC) and endothelial progenitor cells (ECFCs), in the context of plasma chlorambucil concentrations in tumor-bearing dogs.

The objective of this study was to investigate a possible mechanism of action of metronomic chlorambucil on glioma by studying the in vitro cytotoxicity and anti-angiogenic effects on glioma and endothelial cells, respectively. The in vitro LD50 and IC50 of chlorambucil were determined using human SF767 and U87-MG glioma cell lines, human microvascular endothelial cells (HMVECs) and human endothelial colony forming cells (ECFCs). Results were analyzed in the context of chlorambucil concentrations measured in the plasma of tumor-bearing dogs receiving 4 mg m-2 metronomic chlorambucil. The LD50 and IC50 of chlorambucil were 270 µM and 114 µM for SF767, and 390 µM and 96 µM for U87-MG, respectively. The IC50 of chlorambucil was 0.53 µM and 145 µM for the HMVECs and ECFCs, respectively. In pharmacokinetic studies, the mean plasma Cmax of chlorambucil was 0.06 µM. Results suggest that metronomic chlorambucil in dogs does not achieve plasma concentrations high enough to cause direct cytotoxic or growth inhibitory effects on either glioma or endothelial cells.

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.

A rapid and simple LC-MS/MS method for personalized busulfan dosing in pediatric patients undergoing hematopoietic stem cell transplantation (HSCT).

BACKGROUND: Busulfan is commonly used as a conditioning regimen before hematopoietic stem cell transplantation (HSCT). There is a big inter-individual variability in busulfan exposure and the narrow therapeutic index, especially in pediatric population. Therefore, to achieve therapeutic efficacy and safety concurrently, personalized busulfan dosing, guided by pharmacokinetic study with serial plasma samples, is needed a few hours afterwards. METHODS: A fast, sensitive, and accurate method for busulfan measurement was developed, validated, and implemented with liquid-chromatography-mass spectrometry (HPLC-MS/MS). The sample preparation procedure involves only protein precipitation and dilution, and the HPLC-MS/MS method takes 3 min/sample. The assay was linear from 10 ng/ml to 7500 ng/ml (R2 = 0.99). Recoveries were above 90%. The precision was determined at 3 levels (30, 300 and 4000 ng/ml): the intra-day variability (%CV) ranged from 1.4% to 2.5% (n = 20); the inter-day variability ranged from 2.2% to 5.5% (n = 20). The accuracy of the HPLC-MS/MS test was evaluated with an old HPLC-fluorescence method (n = 84), and a Correlation Coefficient (R) of 0.99 was observed. CONCLUSIONS: The analytical performance of the assay allows for timely dose adjustment and has been implemented in clinical service for better clinical outcome.

Pharmacokinetics of dacarbazine (DTIC) in pregnancy.

PURPOSE: The purpose of this report is to describe, for the first time, the pharmacokinetics of dacarbazine (DTIC) and its metabolites [5-[3-methyl-triazen-1-yl]-imidazole-4-carboxamide (MTIC), 5-[3-hydroxymethyl-3-methyl-triazen-1-yl]-imidazole-4-carboxamide (HMMTIC) and 5-aminoimidazole-4-carboxamide (AIC)] during pregnancy (n = 2) and postpartum (n = 1). METHODS: Non-compartmental DTIC, MTIC, HMMTIC, and AIC pharmacokinetics (PK) were estimated in one case at 29 week gestation and 18 day postpartum and a second case at 32 week gestation, in women receiving DTIC in combination with doxorubicin, bleomycin, and vinblastine for treatment of Hodgkin's lymphoma. Drug concentrations were measured by HPLC. RESULTS: In the subject who completed both pregnancy and postpartum study days, DTIC area under the concentration-time curve (AUC) was 27% higher and metabolite AUCs were lower by 27% for HMMTIC, 38% for MTIC, and 83% of AIC during pregnancy compared to postpartum. At 7 and 9 year follow-up, both subjects were in remission of their Hodgkin's lymphoma. CONCLUSIONS: Based on these two case reports, pregnancy appears to decrease the metabolism of the pro-drug dacarbazine, likely through inhibition of CYP1A2 activity. Lower concentrations of active metabolites and decreased efficacy may result, although both these subjects experienced long-term remission of their Hodgkin's lymphoma.

Development and characterization of an organic solvent free, proliposomal formulation of Busulfan using quality by design approach.

Parenteral administration of Busulfan (BU) conquers the bioavailability and biovariability related issues of oral BU by maintaining the plasma drug concentration in therapeutic range with minimal fluctuations thereby significantly reducing the side effects. Busulfex® is the only commercially available parenteral formulation of BU composed of organic solvents N, N-dimethylacetamide and polyethylene glycol 400. Since, BU is highly susceptible to hydrolytic degradation; Busulfex® has poor physical and chemical stability in IV fluids. It is quintessential to develop organic solvent free formulation of BU using parenterally acceptable excipients to enhance its solubility and stability in IV fluids. The Proliposomal formulation of BU was prepared by adsorption-sonicaton method using egg phosphotidylcholine, cholesterol, tween 80 and mannitol. Vesicle size and entrapment efficiency were optimized using 24 full factorial design and characterized by DSC, PXRD and TEM. Optimized formulation spontaneously forms 74.0 ± 1.7 nm sized nanovesicles with 72.9 ± 1.5 % entrapment efficiency. DSC and PXRD studies revealed that BU was present in phospholipid bilayer in amorphized form and TEM images confirmed the multi lamellar vesicular structure. Physicochemical stability of BU was significantly enhanced with proliposomal formulation. In-vivo studies in Sprague Dawley rats showed proliposomal formulation has comparable immunosuppression activity and 110.62 % relative bioavailability as compared to marketed Busulfan formulation i.e. Busulfex®.

High interpatient variability of treosulfan exposure is associated with early toxicity in paediatric HSCT: a prospective multicentre study.

Treosulfan-based conditioning is increasingly employed in paediatric haematopoietic stem cell transplantation (HSCT). Data on treosulfan pharmacokinetics in children are scarce, and the relationship between treosulfan exposure, toxicity and clinical outcome is unresolved. In this multicentre prospective observational study, we studied treosulfan pharmacokinetics and the drug's relationship with regimen-related toxicity and early clinical outcome in 77 paediatric patients. Treosulfan dose was 30 g/m2 , administered over 3 consecutive days in infants <1 year old (n = 12) and 42 g/m2 in children ≥1 year old (n = 65). Mean day 1 treosulfan exposure was 1744 ± 795 mg*h/l (10 g/m2 ) and 1561 ± 511 mg*h/l (14 g/m2 ), with an inter-individual variability of 56 and 33% in the respective groups. High treosulfan exposure (>1650 mg*h/l) was associated with an increased risk of mucosal [Odds ratio (OR) 4·40; 95% confidence interval (CI) 1·19-16·28, P = 0·026] and skin toxicity (OR 4·51; 95% CI 1·07-18·93, P = 0·040). No correlation was found between treosulfan exposure and the early clinical outcome parameters: engraftment, acute graft-versus-host disease and donor chimerism. Our study provides the first evidence in a large cohort of paediatric patients of high variability in treosulfan pharmacokinetics and an association between treosulfan exposure and early toxicity. Ongoing studies will reveal whether treosulfan exposure is related to long-term disease-specific outcome and late treatment-related toxicity.

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)

Surgical versus percutaneous isolated pelvic perfusion (IPP) for advanced melanoma: comparison in terms of melphalan pharmacokinetic pelvic bio-availability.

BACKGROUND: Isolated pelvic perfusion (IPP) can be used to treat unresectable melanoma metastases of the pelvis. IPP can be performed either by surgical or percutaneous approaches, using different balloon catheters. The aim of this study was to examine whether the surgical and percutaneous approaches were comparable with respect to tumor drug exposure in the pelvis. METHODS: A pharmacokinetic study was performed in 5 melanoma patients treated with surgical IPP and five with percutaneous IPP. Both groups received melphalan at the dose of 30 mg/m2. Melphalan pharmacokinetic analyses were performed and the main parameter used to evaluate pelvic tumor drug-exposure was the ratio of areas under the melphalan plasma concentration curves in the pelvis and the systemic compartment, during the perfusion time (AUC0 to 20). Non-parametric Mann-Whitney tests were employed for statistical comparisons. RESULTS: The median and interquartile range (IQR) values of the ratios between melphalan AUC0 to 20 in pelvic and systemic compartments were 7.9 (IQR 7.2 to 9.9) and 5 (IQR 4 to 7.9) for surgical and percutaneous IPPs, respectively (p = 0.209). CONCLUSIONS: Tumor exposure to drug using these two methods did not statistically differ and both methods, therefore, can be adopted interchangeably, utilizing a perfusion blood flow rate of approximately 120 ml/min. The small sample size is a limitation of this study but our preliminary results can be used to calculate the effect size of a larger trial. Trial Registration Clinical Trials.gov Identifier NCT01920516; date of trial registration: August 6, 2013.

Sensitive LC-MS/MS Method for the Simultaneous Determination of Bendamustine and its Active Metabolite, γ-Hydroxybendamustine in Small Volume Mice and Dog Plasma and its Application to a Pharmacokinetic Study in Mice and Dogs.

A highly sensitive, specific and rapid LC-ESI-MS/MS method has been developed and validated for the simultaneous quantification of bendamustine (BM) and γ-hydroxybendamustine (HBM) in small volume (20 µL) mice and dog plasma using phenacetin as an internal standard (IS) as per regulatory guidelines. Both the analytes and IS were extracted from mice and dog plasma using a liquid-liquid extraction method. Chromatography was achieved on Atlantis dC18 column using an isocratic mobile phase (0.2% formic acid:acetonitrile, 25:75) at a flow rate of 0.40 mL/min. The total chromatographic run time was 3.0 min and the elution of BM, HBM and IS occurred at ~1.2, 1.2 and 2.0 min, respectively. A linear response function was established 0.11-518 ng/mL for both the analytes in mice and dog plasma. The intra- and inter-day accuracy and precisions were in the range of 3.46-12.9 and 3.63-8.23%; 1.15-9.00 and 7.86-9.49% for BM and HBM, respectively in mice plasma and 2.15-6.49 and 1.73-13.1%; 4.35-13.9 and 4.33-10.5% for BM and HBM, respectively in dog plasma. This novel method has been applied to a pharmacokinetic study in mice and dogs.

Temozolomide analogs with improved brain/plasma ratios - Exploring the possibility of enhancing the therapeutic index of temozolomide.

Temozolomide is a chemotherapeutic agent that is used in the treatment of glioblastoma and other malignant gliomas. It acts through DNA alkylation, but treatment is limited by its systemic toxicity and neutralization of DNA alkylation by upregulation of the O6-methylguanine-DNA methyltransferase gene. Both of these limiting factors can be addressed by achieving higher concentrations of TMZ in the brain. Our research has led to the discovery of new analogs of temozolomide with improved brain:plasma ratios when dosed in vivo in rats. These compounds are imidazotetrazine analogs, expected to act through the same mechanism as temozolomide. With reduced systemic exposure, these new agents have the potential to improve efficacy and therapeutic index in the treatment of glioblastoma.

Cyclophosphamide pharmacokinetics and pharmacogenetics in children with B-cell non-Hodgkin's lymphoma.

INTRODUCTION: Variation in cyclophosphamide pharmacokinetics and metabolism has been highlighted as a factor that may impact on clinical outcome in various tumour types. The current study in children with B-cell non-Hodgkin's lymphoma (NHL) was designed to corroborate previous findings in a large prospective study incorporating genotype for common polymorphisms known to influence cyclophosphamide pharmacology. METHODS: A total of 644 plasma samples collected over a 5 year period, from 49 B-cell NHL patients ≤ 18 years receiving cyclophosphamide (250 mg/m(2)), were used to characterise a population pharmacokinetic model. Polymorphisms in genes including CYP2B6 and CYP2C19 were analysed. RESULTS: A two-compartment model provided the best fit of the population analysis. The mean cyclophosphamide clearance value following dose 1 was significantly lower than following dose 5 (1.83 ± 1.07 versus 3.68 ± 1.43 L/h/m(2), respectively; mean ± standard deviation from empirical Bayes estimates; P < 0.001). The presence of at least one CYP2B6*6 variant allele was associated with a lower cyclophosphamide clearance following both dose 1 (1.54 ± 0.11 L/h/m(2) versus 2.20 ± 0.31 L/h/m(2), P = 0.033) and dose 5 (3.12 ± 0.17 L/h/m(2) versus 4.35 ± 0.37 L/h/m(2), P = 0.0028), as compared to homozygous wild-type patients. No pharmacokinetic parameters investigated were shown to have a significant influence on progression free survival. CONCLUSION: The results do not support previous findings of a link between cyclophosphamide pharmacokinetics or metabolism and disease recurrence in childhood B-cell NHL. While CYP2B6 genotype was shown to influence pharmacokinetics, there was no clear impact on clinical outcome.