Comparison of Two Analytical Methods for Busulfan Therapeutic Drug Monitoring.

BACKGROUND AND OBJECTIVES: Busulfan (Bu) is an old drug, but is still well recommended as an alkylating agent during conditioning therapy, before hematopoietic stem cell transplantation. Although its dose administration is standardized and based on patient weight, therapeutic drug monitoring is required in order to maintain its exposure [as area under the concentration-time curve (AUC) from 0 to infinity AUC0-∞] within a narrow therapeutic range and, if necessary, to adjust the dose with as short a lead time as possible. The aim of the study is to evaluate the agreement (as calculated AUC) between a gold standard analytical method and a new one that is faster and easier. METHODS: We analyzed 221 plasma samples from 37 children (0.25-16 years; 4-62.5 kg) and 11 adults (21-59 years; 45-80 kg), corresponding to 52 AUC values (ng h/mL). The drug exposure was calculated, simultaneously, by two validated analytical methods. The reference method was a high-performance liquid chromatography (HPLC) assay combined with an ultraviolet detector (UV). The test method had a triple quadrupole mass spectrometer (MS) as detector; the clean-up procedures of the samples were different and faster. RESULTS: The agreement between the two methods (reference and test) was evaluated in terms of Bu exposure differences based on Lin's concordance correlation coefficient (CCC) and represented by the Bland-Altman plot. The CCC between the AUC of the two methods was excellent (0.868; 95% CI: 0.802-0.935). The precision of the measures (expressed by Pearson's italic "r") was 0.872, and the accuracy (accounted by the bias correction factor) was 0.996. CONCLUSIONS: We can conclude that the HPLC-MS/MS assay represents a very good alternative to the reference.

Measurement of the DNA alkylating agents busulfan and melphalan in human plasma by mass spectrometry.

Busulfan and melphalan are cytotoxic DNA alkylating agents that are used in many hematopoietic stem cell transplantation (HCT) conditioning regimens. We report the development of an assay using turbulent flow liquid chromatography (TFLC) and tandem mass spectrometry to simultaneously measure the concentration of busulfan (Bu) and melphalan (Mel) in human plasma. The method involves precipitating proteins in the plasma specimen with an organic solvent containing deuterated internal standards of both compounds. Following centrifugation, an aliquot of the supernatant was injected into the TFLC mass spectrometry system operated in the positive ion mode. The analytical measurement range for both compounds was 10-5000 ng/mL, and with validated dilutions the reportable range was extended to 25,000 ng/mL. Intra-day and inter-day (n = 20 day) precision studies showed a coefficient of variation (CV) of <7% at several concentrations across the measurement range. To determine accuracy recovery studies were performed at several concentrations spanning the measurement range. Recoveries for both compounds were between 98 and 103%. Additionally, busulfan was compared with an existing assay and showed excellent correlation. Experiments were conducted to rule out matrix effects, carryover and interference from endogenous substances. The validated clinically reportable range (CRR) and assay precision will allow this assay to be used clinically to monitor and adjust Mel and Bu levels to ensure better therapeutic outcomes and also to support clinical trials aimed at better defining therapeutic ranges.

Systemic Absorption of Mitomycin-C When Used in Pterygium Surgery.

PURPOSE: To determine whether scleral topical application of mitomycin-C (MMC) results in measurable plasma levels of systemic absorption. METHODS: The study comprised 27 patients who were treated with MMC 0.2 mg/mL (0.02%) for 60 seconds during pterygium surgery. Blood samples were taken 30 minutes after surgery and evaluated by the human plasma liquid chromatography tandem-mass spectrometry method to determine the presence of MMC. RESULTS: The amount of MMC in 27 samples tested was determined as below 0.25 ppb (ng/mL). CONCLUSIONS: In this study of 27 patients treated with topical application of MMC for pterygium surgery, there was no measurable evidence of systemic drug absorption. Although systemic absorption has been found with the use of larger quantities of MMC, there is an extremely low likelihood of systemic absorption or toxicity of MMC after pterygium surgery.

Therapeutic Drug Monitoring of Busulfan for the Management of Pediatric Patients: Cross-Validation of Methods and Long-Term Performance.

BACKGROUND: Busulfan (Bu) is an alkylating agent used as part of the conditioning regimen in pediatric patients before hematopoietic stem cell transplantation. Despite intravenous (IV) administration and dosing recommendations based on age and weight, reports have revealed interindividual variability in Bu pharmacokinetics and the outcomes of hematopoietic stem cell transplantation. In this context, adjusting doses to Bu's narrow therapeutic window is advised. We aimed to assess the utility of therapeutic drug monitoring (TDM) of Bu in children, the reliability of Bu quantification methods, and its stability in plasma when stored for up to 5 years. METHODS: Eighteen patients from our TDM center (252 samples) were included. All of them received a 2-hour Bu IV infusion 4 times daily for a total of 16 doses. The first dose of Bu was age/weight-based, and the subsequent doses were adjusted from third or fifth dose onward based on the estimated first dose pharmacokinetic parameters to target steady-state concentrations (Css) of 600-900 ng/mL. The performance of our unit's high-performance liquid chromatography with tandem mass spectrometry method was assessed using a quality control (QC, 35 series) chart. International, multicenter, cross-validation test (n = 21) was conducted to validate different analytical methods. To assess Bu stability, regression analyses and Bland-Altman plots were performed on measurements at repeated time points on samples stored at -80°C for up to 5 years. RESULTS: We observed a 4.2-fold interindividual variability in Bu Css after the first dose, with only 28% of children having a Css within the target range. During the 4 days of conditioning, 83% of children had their doses modified according to TDM recommendations. This achieved a Css within the target range in 75% of the children. Routine QC measurements were generally within the ±15% range around theoretical values, showing the optimal robustness of our center's analytical method. Two of the 21 Bu TDM centers returned inadequate results during cross-validation testing; both used a UV detection method. Storage at -80°C led to a fall in Bu content of 14.9% ± 13.4% at 2-4 years and of 20% ± 5% by 5 years (roverall = 0.92). CONCLUSIONS: We conclude that TDM is an effective method of achieving targeted Bu levels in children. QC programs are crucial to monitoring and maintaining the quality of an analytical method.

An HPLC-UV method for determining plasma dimethylacetamide concentrations in patients receiving intravenous busulfan.

Dimethylacetamide (DMA) is a solvent used in the preparation of intravenous busulfan, an alkylating agent used in blood or marrow transplantation. DMA may contribute to hepatic toxicity, so it is important to monitor its clearance. The aim of this study was to develop an HPLC-UV assay for measurement of DMA in human plasma. After precipitation of plasma proteins with acetonitrile followed by dilution (1:4) with water, the extract was injected onto the HPLC and detected at 195 nm. Separation was performed using a Cogent-HPS 5 µm C18 column (250 × 4.6 mm) preceded by a Brownlee 7 µm RP18 , pre-column (1.5 cm × 3.2 mm). The mobile phase was 25 mm sodium phosphate buffer (pH 3), containing 2.5% (v/v) acetonitrile and 0.0005% (v/v) sodium-octyl-sulfonate. Using a flow rate of 1 mL/min, the retention times of DMA and the internal standard (IS), 2-chloroacetamide, were 9.5 and 3.5 min, respectively. Peak area ratio (DMA:IS) was a linear function of concentration from 1 to 1000 µg/mL. There was excellent intraday precision (<5% for 5-700 µg/mL DMA), accuracy (<3% deviation from the true concentration) and recovery (74-98%). The limits of detection and quantification were 1 and 5 µg/mL, respectively. In eight children who received intravenous busulfan, DMA concentrations ranged from 110 to 438 µg/mL.

High-Throughput Validated Method for the Quantitation of Busulfan in Plasma Using Ultrafast SPE-MS/MS.

BACKGROUND: Busulfan is an alkylating agent used to ablate bone marrow cells before hematopoietic stem cell transplantation. Because of its highly variable pharmacokinetics, studies have shown that therapeutic drug monitoring is clinically useful for patients undergoing bone marrow transplant so that toxic effects associated with high drug exposure could be reduced and improve clinical outcomes. Current methods for assaying busulfan include the use of gas chromatography mass spectrometry (GC/MS), high-performance liquid chromatography, and liquid chromatography mass spectrometry. The clinical need for faster turnaround times and increased testing volumes has required laboratories to develop faster methods of analysis for higher throughput of samples. Therefore, we present a method for the quantification of busulfan in plasma using an ultrafast solid-phase extraction/tandem mass spectrometry, which has much faster sample cycle times and similar analytical results to GC/MS. METHOD: Calibration standards, quality controls, and patient samples after addition of busulfan-d4 internal standard were extracted into n-butyl chloride from plasma. The organic fraction was dried and reconstituted in 600 µL of water containing ammonium acetate, trifluoroacetic acid, and formic acid. Sample analysis was performed at a rate of less than 20 seconds per sample using a Rapidfire 300 system coupled to an Agilent 6490 MS/MS using electrospray ionization in positive ion mode. Concentrations were calculated based on a 5-point calibration curve using a 1/x linear curve fit. RESULTS: The analytical method shows excellent precision, sensitivity, and specificity. Minimal ion suppression or enhancement due to the matrix effect was observed. No significant carryover was seen following a sample containing 15,000 ng/mL of busulfan. Seventy-two patient samples were cross-validated with a current GC/MS method. All patient results throughout the analytical range correlated within the acceptance criteria of ±20%. The linear regression demonstrated the following: slope = 1.0067, r = 0.9964, and intercept = -6.2. CONCLUSIONS: A simple, fast, and robust method was developed for the quantitation of busulfan in plasma with solid-phase extraction/tandem mass spectrometry cycle times of <20 seconds per sample.

Predictive performance of a physiologically based pharmacokinetic model of busulfan in children.

A physiologically based pharmacokinetic (PBPK) model of the DNA-alkylating agent busulfan was slightly modified and scaled from adults to children in order to predict the systemic busulfan drug exposure in children. Capitalizing on the recent major software release of PK-Sim®, we refined our PBPK model by implementing glutathione S transferase (GST) in 11 organs using the software integrated enzyme expression database. In addition, two irreversible binding processes (i.e., DNA and plasma protein binding) were applied by using Koff and KD values. The model was scaled from adults to children. Simulations were computed and compared to concentration-time data after intravenous (i.v.) busulfan administration to 36 children. Based on the results, an age-dependent enzyme activity and maturation ratio was tailored and evaluated with an external dataset consisting of 23 children. Initial adult to children scaling indicated lower clearance values for children in comparison to adults. Subsequent age-dependent maturation ratio resulted in three different age groups: Activity of busulfan-glutathione conjugate formation was 80%, 61%, and 89% in comparison to adults for children with an age of up to 2 years, > 2-6 years, and > 6-18 years, respectively. Patients of the evaluation dataset were simulated with a mean percentage error (MPE) for all patients of 3.9% with 3/23 children demonstrating a MPE of > ±30%. The PBPK model parameterization sufficiently described the observed concentration-time data of the validation dataset while showing an adequate predictive performance. This PBPK model could be helpful to determine the first dose of busulfan in children.

Development and validation of a liquid chromatography-tandem mass spectrometry assay to quantify plasma busulfan.

BACKGROUND: Busulfan is an anti-leukemic, DNA alkylating agent that is used in conditioning regimens for patients undergoing hematopoietic stem cell transplantation. Because of the large intraindividual and interindividual variations seen in busulfan pharmacokinetics, therapeutic drug monitoring is necessary. Currently at the authors' institution, plasma busulfan in adults is measured by gas chromatography-mass spectrometry (GC-MS) at a reference laboratory, whereas pediatric specimens are sent to a different reference laboratory also for GC-MS analysis. As the result turnaround time is not optimal and this practice is of significant cost, a liquid chromatography-tandem mass spectrometry assay to quantify plasma busulfan was developed. METHODS: Protein precipitation with D8-busulfan (deuterated internal standard) in acetonitrile was carried out on 50 µL of heparinized plasma. Gradient elution with ammonium acetate, formic acid, water, and methanol at 0.6 mL/min had a 4-minute run time. Multiple reaction monitoring was employed using Q1/Q3 transitions of 264/151 and 264/55 for busulfan and 272/159 and 272/62 for D8-busulfan. RESULTS: Sample preparation took ∼30 minutes for 6 patient samples. Six calibrators were used (0-2000 ng/mL) with 3 quality controls (means of 12, 356, and 1535 ng/mL). The limits of detection and quantitation were 1 and 6 ng/mL, respectively. Extraction recovery was ∼77% and ion suppression ∼5%. Within-run and between-run precision studies yielded <15% coefficient of variation at the limit of quantitation and <6% coefficient of variation through the rest of the linear range. Method comparisons between this assay and 2 GC-MS assays revealed mean biases of 7% and 1%. CONCLUSIONS: An accurate, rapid, and sensitive liquid chromatography-tandem mass spectrometry assay for quantification of plasma busulfan was developed. This assay reduces current specimen volume requirements, reduces result turnaround time for patients and clinicians, and additionally saves institutional funds.

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.

Variation in prescribing patterns and therapeutic drug monitoring of intravenous busulfan in pediatric hematopoietic cell transplant recipients.

Personalizing intravenous (IV) busulfan doses in children using therapeutic drug monitoring (TDM) is an integral component of hematopoietic cell transplant. The authors sought to characterize initial dosing and TDM of IV busulfan, along with factors associated with busulfan clearance, in 729 children who underwent busulfan TDM from December 2005 to December 2008. The initial IV busulfan dose in children weighing ≤12 kg ranged 4.8-fold, with only 19% prescribed the package insert dose of 1.1 mg/kg. In those children weighing >12 kg, the initial dose ranged 5.4-fold, and 79% were prescribed the package insert dose. The initial busulfan dose achieved the target exposure in only 24.3% of children. A wide range of busulfan exposures were targeted for children with the same disease (eg, 39 target busulfan exposures for the 264 children diagnosed with acute myeloid leukemia). Considerable heterogeneity exists regarding when TDM is conducted and the number of pharmacokinetic samples obtained. Busulfan clearance varied by age and dosing frequency but not by underlying disease. The authors- group is currently evaluating how using population pharmacokinetics to optimize initial busulfan dose and TDM (eg, limited sampling schedule in conjunction with maximum a posteriori Bayesian estimation) may affect clinical outcomes in children.

Busulfan systemic exposure after oral administration of extemporeanously prepared high-dose busulfan capsules.

PURPOSE: The aim of the study was to analyze patients' busulfan (BU) exposure after oral administration of extemporeanously prepared BU capsules prior to blood stem cell transplantation. METHODS: Patients were treated with 1 mg/kg body weight BU administered orally every 6h on each of 4 consecutive days prior to blood stem cell transplantation. Each BU dose was administered in 1 gelatine capsule to be swallowed and containing the individually calculated dose of pure BU active substance. Blood samples were obtained from 6 adult patients 0, 30, 60, 90, 120, 180, 240, 300, and 360 min after the 1st, 5th, and 13th BU dose, frozen and analyzed subsequently by using a HPLC assay with UV detection. In addition, in two patients concomitant TDM was executed. BU exposure was monitored concurrently and BU doses were targeted to achieve a steady-state plasma concentration (C(SS)) of 600-900 ng/mL or 900-1100 ng/mL depending on the underlying disease. In these patients blood samples were obtained 0, 60, 120, 180, 240, and 360 min after the 1st, 5th, 9th, and 13th BU dose and analyzed concurrently. RESULTS: For the six patients monitored retrospectively, the time to reach peak plasma BU concentration (C(max)) ranged from 1 to 5 h (mean 2.4 h). BU C(max) - values varied from 728 to 1807 ng/ mL (mean 1174 ng/mL), and BU clearance (CL/F) from 2.32 to 3.75 mL/min/kg (mean 2.97 mL/min/ kg). The mean BU steady state (C(SS)) concentration calculated was 973 ng/mL (range 754-1226 ng/mL) with a mean AUC of 5818 ng.h/mL (range 4521- 7171 ng.h/mL). One of the two patients receiving targeted BU doses required an upward dose adjustment. None of the eight patients suffered from vomiting during BU therapy. CONCLUSIONS: BU active substance encapsulated without further excipients in gelatine capsules is highly suitable for oral BU therapy. However, therapeutic drug monitoring and BU dose adjustment is still advisable to achieve optimal systemic BU exposure in each individual patient.

Determination of busulfan in human plasma using an ELISA format.

High-dose busulfan is an important component of many bone marrow transplantation-preparative regimens. High busulfan plasma levels have been shown to increase the chance of venoocclusive disease and low levels are associated with recurrence of disease or graft rejection. Currently, busulfan levels are monitored by physical methods that are expensive and time consuming, resulting in relatively low overall use of busulfan testing for dose adjustment. Novel highly selective antibodies for busulfan have been generated and a microtiter plate immunoassay capable of quantifying busulfan levels in plasma has been developed. The assay was configured using a busulfan-horseradish peroxidase (HRP) conjugate as the reporter group and busulfan monoclonal antibodies. The assay requires 30 microL of plasma with no sample preparation. The immunoassay has a standard curve based on busulfan with a range of 75-2000 ng/mL. The time to first result is 30 minutes with up to 40 patient samples in duplicate; multiple plates can be run at once. The coefficient of variation (CV) on signal is <5% for an entire plate, and the 95% confidence interval for negative samples (n = 78) is below the lowest calibrator of 75 ng/mL. Cross-reactivity with the major inactive metabolites (tetrahydrothiophene, tetramethyl sulfone, and tetrahydrothiophene-3-ol-1,1-dioxide) was <0.1%. Results generated with clinical samples (n = 35 and n = 70) correlate well to gas chromatography-mass spectrometry (R = 0.976 and 0.985, respectively) with a slope of 1.05 +/- 0.05. This immunoassay method is suitable for determining levels of busulfan in human plasma. It offers the advantages of using a smaller sample size, does not require sample preparation, and is less labor intensive than other methods. The ability to make 240 determinations per hour enables effective and timely routine monitoring of busulfan levels in clinical practice.

Towards an efficient prodrug of the alkylating metabolite monomethyltriazene: synthesis and stability of N-acylamino acid derivatives of triazenes.

A series of 3-[alpha-(acylamino)acyl]-1-aryl-3-methyltriazenes 6a-l, potential cytotoxic triazene prodrugs, were synthesised by coupling 1-aryl-3-methyltriazenes to N-acylamino acids. Their hydrolysis was studied in isotonic pH 7.4 phosphate buffer and in human plasma, while hydrolysis of the derivative 6a was studied in more depth across a range of pH values. Prodrugs 6a-l hydrolyse by cleavage of the triazene acyl group to afford the corresponding monomethyltriazenes. Studies in human plasma demonstrate that acylation of the alpha-amino group of the amino acid carrier is an effective means of reducing the chemical reactivity of the alpha-aminoacyl derivatives while retaining a rapid rate of enzymatic hydrolysis. These derivatives displayed logP values that suggest they should be well absorbed through biological membranes.

Physiologically based pharmacokinetic modeling of chloroethane disposition in mice, rats, and women.

Chloroethane was observed in a chronic cancer bioassay to be a mouse-specific uterine carcinogen at a single high inhaled concentration (15,000 ppm). Although high incidence occurred in the female mouse (86%), no uterine tumor increases were observed in female rats. Chloroethane is a weak alkylating agent and has low acute toxicity. No genotoxicity potential has been observed below 40,000 ppm. Chloroethane is eliminated from the body by pulmonary exhalation and metabolically by oxidation via cytochrome P-450 (likely producing acetaldehyde) and conjugation with glutathione (GSH). The mode of action for the mouse-specific uterine tumors is not definitively known and could involve parent chemical and/or metabolite(s). A physiologically based pharmacokinetic (PBPK) model for chloroethane disposition in the rat was developed previously, but no such models have been described for mice or humans. For the work reported here, the existing PBPK model for chloroethane in rats was expanded and refined, and PBPK models for chloroethane disposition in mice and humans were developed to allow species comparisons of internal dosimetry and for possible insights into the carcinogenic mode of action. The amounts metabolized via glutathione-S-transferase (GST) versus cytochrome P-450, and the total amount of chloroethane absorbed, were most consistent with the observations made concerning uterine tumors, with amounts metabolized via GST providing the larger quantitative difference between the two rodent species. Choice of the most relevant dose metric for risk assessments involving uterine tumors in mice will require pharmacodynamic considerations in the mode of action in addition to the pharmacokinetic differences reported here.

Population pharmacokinetic-based dosing of intravenous busulfan in pediatric patients.

The objective of this study was to characterize the pharmacokinetics (PK) of intravenous busulfan in pediatric patients and provide dosing recommendations. Twenty-four pediatric patients were treated with intravenous busulfan, 1.0 or 0.8 mg/kg for ages < or = 4 years or > 4 years, respectively, 4 times a day for 4 days. Dense PK sampling was performed. Body weight, age, gender, and body surface area were explored for effects on PK, and Monte Carlo simulations were performed to assess different dosing regimens. The PK of intravenous busulfan was described by a 1-compartment model with clearance of 4.04 L/h/20 kg and volume of distribution of 12.8 L/20 kg. Simulations indicated that the mg/kg and mg/m2 regimens were similar and achieved the desired target exposure in approximately 60% of patients. This model suggests that patients < or = 12 kg should be dosed at 1.1 mg/kg and those > 12 kg dosed at 0.8 mg/kg. Therapeutic drug monitoring and dose adjustment will further improve therapeutic targeting.

Determination of glufosfamide in rat plasma by liquid chromatography/tandem mass spectrometry.

A sensitive and selective high-performance analytical method based on liquid chromatography with tandem mass spectrometric detection (LC/MS/MS) was developed for the quantification of glufosfamide in rat plasma. Zidovudine was employed as internal standard. Glufosfamide was determined after methanol-mediated plasma protein precipitation using LC/MS/MS with an electrospray ionization interface in negative ion mode. Two sets of standard curves were developed, from 0.005 to 1.0 microg/mL and from 1.0 to 50.0 microg/mL. The assay was accurate (% deviations from nominal concentrations < 5%), precise and reproducible (intra- and inter-day coefficients of variation < 10%). Glufosfamide in rat plasma was stable over three freeze/thaw cycles, and at ambient temperatures, for at least 2 h. The validated method was successfully applied to the determination of glufosfamide plasma concentrations in rats for 24 h following an intravenous administration of 25 mg/kg.

N-(2-hydroxypropyl)methacrylamide copolymers of a glutathione (GSH)-activated glyoxalase i inhibitor and DNA alkylating agent: synthesis, reaction kinetics with GSH, and in vitro antitumor activities.

The incorporation of anticancer prodrugs into polyacrylamide conjugates has been shown to improve tumor targeting via the so-called "enhanced permeability and retention" effect. This strategy has now been expanded to include two different classes of glutathione (GSH)-activated antitumor agents prepared by radical polymerization of N-(2-hydroxypropyl)methacrylamide (HPMA) with 2-methacryloyloxy-methyl-2-cyclohexenone (7) and/or with S-(N-4-chlorophenyl-N-hydroxycarbamoyl-thioethyl)methacrylamide (8), followed by treatment with 3-chloroperoxybenzoic acid, to give the HPMA copolymers of 7 and the 8-sulfoxide, respectively. In aqueous-buffered solution at pH 6.5, GSH reacts rapidly with poly-HPMA-8-sulfoxide (k approximately 2.3 mM(-1) min(-1)) to give S-(N-4-chlorophenyl-N-hydroxycarbamoyl)glutathione (1), a tight-binding transition state analogue inhibitor of the antitumor target enzyme glyoxalase I (K(i) = 46 nM), or with poly-HPMA-7 (k approximately 0.02 mM(-1) min(-1)) to give the electrophilic antitumor agent 3-glutathio-2-methylenecyclohexenone (4). Indeed, B16 melanotic melanoma in culture is inhibited by poly-HPMA-8-sulfoxide and by poly-HPMA-7 with IC(50) values of 168 +/- 8 and 284 +/- 5 microM, respectively. These values are significantly greater than those of the unpolymerized prodrugs suggesting that the cytotoxicity of the polymer prodrugs might be limited by slow cellular uptake via pinocytosis. This prodrug strategy should be applicable to a range of different GSH-based antitumor agents.

Development of a rapid and specific assay for detection of busulfan in human plasma by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry.

A sensitive and specific assay for detection of busulfan in human plasma was developed. The assay is based on rapid isolation of busulfan by liquid-liquid extraction with ethyl acetate, and detection by high-performance liquid chromatography with electrospray ionization and tandem mass spectrometry. 1,6-Bis(methanesulfonyloxy)hexane, a synthesized analogue of busulfan, was used as the internal standard (IS). The acquisition was performed in the multiple reaction monitoring mode; busulfan and the IS were detected with no interferences from plasma matrix. The method was linear over the range 5-2500 ng mL(-1), with r2 > 0.99 and a run time of only 3.5 min. The intra- and inter-assay precisions were in the ranges 2.1-11.9% and 3.2-10.1%, respectively, and the intra- and inter-assay accuracies were 92.2-107.6% and 94.7-104.1%, respectively. The absolute recoveries were 82.0% (20 ng mL(-1)), 90.6% (1000 ng mL(-1)) and 80.0% (2000 ng mL(-1)) for busulfan, and 89.1% for the IS (1000 ng mL(-1)). The limits of detection and quantification were 2 and 5 ng mL(-1), respectively. The validated method was successfully applied to analyze plasma samples obtained from six adults receiving doses of 1 mg kg(-1) in a conditioning regimen prior to bone marrow transplantation. A marked intra-patient variation in busulfan concentrations during the steady state was observed, which limits the application of pharmacokinetic modeling and suggests that continuous therapeutic monitoring is necessary for adequate individualized dosing. In this regard, the present assay brings important advantages relative to other methods described in the literature, i.e., it is highly specific and simple to perform, with a rapid chromatographic run time (3.5 min), and the whole procedure can be completed in 4-5 h, which would permit dose corrections after the third dose allowing earlier and better dosing adjustments towards the target level of busulfan.

Pharmacokinetics, mass balance, and tissue distribution of a novel DNA alkylating agent, VNP40101M, in rat.

VNP40101M (1,2-Bis(methylsulfonyl)-1-(2-chloroethyl)-2-[(2 methylamino)carbonyl] hydrazine), a novel DNA alkylating agent, is currently under clinical development for the treatment of cancer in Phase I clinical trials. This study investigated the pharmacokinetics, mass balance, and tissue distribution of [14C]-VNP40101M in rats following a single intravenous dose of 10 mg/kg. After 7 days, the total recovery of radioactivity was 85% for males and 79% for females. Most of the radioactivity was eliminated within 48 hours through urine (70%), with less excreted in feces (6%). Tissue contained relatively high radioactive residues with the highest concentrations in kidneys, liver, lung, and spleen. After 7 days, tissue still contained 9% of the dose. At both 5 minutes and 1 hour post-dose, brain contained relatively high radioactivity (5.9 and 3.3 micro g equivalence/g and 50% and 30% of the blood concentration, respectively), suggesting that VNP40101M penetrated the blood-brain barrier. The elimination half-life of VNP40101M was approximately 20 minutes, the peak plasma concentration (Cmax) averaged 11.3 micro g/mL, the volume of distribution (Vss) averaged 0.91 L/kg, and the total body clearance (Cl) averaged 33.5 mL/min/kg. The metabolite profile in urine was complex, indicating VNP40101M was extensively metabolized. There were no apparent sex differences in pharmacokinetic parameters of VNP40101M in the rat.