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Seizures are relatively common in cancer patients, and co-administration of chemotherapeutic and antiepileptic drugs (AEDs) is highly probable and necessary in many cases. Nonetheless, clinically relevant interactions between chemotherapeutic drugs and AEDs are rarely summarized and pharmacologically described. These interactions can cause insufficient tumor and seizure control or lead to unforeseen toxicity. This review focused on pharmacokinetic and pharmacodynamic interactions between alkylating agents and AEDs, helping readers to make a rational choice of treatment optimization, and thus improving patients' quality of life. As an example, phenobarbital, phenytoin, and carbamazepine, by increasing the hepatic metabolism of cyclophosphamide, ifosfamide and busulfan, yield smaller peak concentrations and a reduced area under the plasma concentration-time curve (AUC) of the prodrugs; alongside, the maximum concentration and AUC of their active products were increased with the possible onset of severe adverse drug reactions. On the other side, valproic acid, acting as histone deacetylase inhibitor, showed synergistic effects with temozolomide when tested in glioblastoma. The present review is aimed at providing evidence that may offer useful suggestions for rational pharmacological strategies in patients with seizures symptoms undertaking alkylating agents. Firstly, clinicians should avoid the use of enzyme-inducing AEDs in combination with alkylating agents and prefer the use of AEDs, such as levetiracetam, that have a low or no impact on hepatic metabolism. Secondly, a careful therapeutic drug monitoring of both alkylating agents and AEDs (and their active metabolites) is necessary to maintain therapeutic ranges and to avoid serious adverse reactions., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Aims: Fanconi anaemia (FA) is a rare disorder characterized by progressive bone marrow failure that requires haematopoietic cell transplantation (HCT). Busulfan is used in conditioning regimens prior to HCT. Doses used in non-FA patients cause life-threatening toxicities in FA patients and data on busulfan pharmacokinetics (PK) in this population are limited. This study characterized busulfan PK in paediatric FA patients using population PK modelling and evaluated the effect of body composition on steady-state concentrations (C ss )., Methods: A total of 200 busulfan plasma concentrations in 29 FA patients from a recent study (Clinicaltrials.gov; NCT01082133) were available for population PK modelling. The effect of different body size-scaled doses and body compositions on C ss was investigated using population PK modelling., Results: Fat free mass (FFM) was identified as the best size descriptor in a two-compartment busulfan PK model in FA patients. Conventional dosing, based on an amount of busulfan per kilogram of total body mass, resulted in higher C ss in FA patients with higher body mass index (BMI). A newly proposed FFM-based dosing strategy would eliminate the observed trend of higher concentrations in high BMI patients, and achieve consistent C ss across a wide BMI spectrum., Conclusions: This is the first study to describe the population PK of busulfan in paediatric FA patients. The proposed model will facilitate PK model-informed precision dosing. FFM-based dosing is expected to improve the probability of achieving target C ss , particularly in obese patients, while minimizing the risk of overdosing., (© 2019 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

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.

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.

Metronomic chemotherapy describes the close, regular administration of chemotherapy drugs at less-toxic doses over prolonged periods of time. In 2015, the results of randomized phase III clinical trials demonstrated encouraging, albeit limited, efficacy benefits of metronomic chemotherapy regimens administered as adjuvant maintenance therapy for the treatment of breast cancer, or as maintenance therapy in combination with an antiangiogenic agent for metastatic colorectal cancer. Owing to the investigational nature of this approach, metronomic chemotherapy regimens are highly empirical in terms of the optimal dose and schedule for the drugs administered; therefore, greater knowledge of the pharmacokinetics of metronomic chemotherapy is critical to the future success of this treatment strategy. Unfortunately, such preclinical and clinical pharmacokinetic studies are rare. Herein, we present situations in which active drug concentrations have been achieved with metronomic schedules, and discuss their associated pharmacokinetic parameters. We summarize examples from the limited number of clinical studies in order to illustrate the importance of assessing such pharmacokinetic parameters, and discuss the influence this information can have on improving efficacy and reducing toxicity.

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.

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.

When growing up, the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of drugs change, which may alter the effect of drugs. To ensure optimal drug efficacy and safety in paediatric care, PK and PD relationships of drugs need to be explored in children. This article presents an outline on performing a population PK/PD study and translating these results into rational dosing regimens, with the development and prospective evaluation of PK/PD derived evidence-based dosing regimen being discussed. Examples on amikacin, morphine and busulfan are provided, showing how PK(/PD) modelling not only led to optimization and individualization in paediatric clinical care for the specific drugs but also to insight in maturation of organ systems involved. It is shown that the latter results can subsequently be used as a basis for dosing of other drugs eliminated through the same pathway. Ultimately, these efforts should lead to predictable drug efficacy and safety across all age groups.

Allogenic hematopoietic stem cell transplantation (HSCT) is a well established but complex treatment option for malignant and non-malignant disorders in pediatric patients. Most commonly used myeloablative and non-myeloablative conditioning regimens in children comprise alkylating agents, such as busulfan (BU) and cyclophosphamide. Inter-individual variability in the pharmacokinetics of BU can result in altered conditioning of the patient and therefore lead to relapse or rejection due to under exposures, or occurrence of toxicities due to over exposures. With the introduction of the intravenous formulation of BU, this variability has been reduced but still cannot be fully predicted. Inter and intra-individual variability of BU kinetics is more common in children compared to adults and toxicity of BU based regimens is still a concern. It has been hypothesized that some of this variability in BU pharmacokinetics and treatment outcomes, especially the toxicity, might be predicted by genetic variants of enzymes involved in the metabolism of BU. This review intends to summarize the studies performed to date on the pharmacokinetics and pharmacogenetics of BU based conditioning, specifically in relation to children.

This study is an attempt to evaluate the hepatoprotective activity of Tabernaemontana divaricata against DEN and Fe NTA induced liver necrosis in rats. Ethanolic extract of the whole plant of Tabernaemontana divaricata at doses of 200 and 400 mg/kg body weight and 5-fluorouracil (standard drug) was orally administered to male Wistar Albino rats once daily for 24 weeks, simultaneously treated with the carcinogen DEN and Fe NTA. In simultaneously treated animals, the plant extract significantly decreased the levels of uric acid, bilirubin, AST, ALT, and ALP in serum and increased the levels of liver marker enzymes in liver. Treatment with the extracts resulted in a significant increase in the levels of antioxidants accompanied by a marked reduction in the levels of malondialdehyde when compared to DEN and Fe NTA treated group. When compared with 200 mg/kg bw rats, 400 mg/kg bw rats and 5-fluorouracil treated rats showed better results in all the parameters. The histopathological studies confirmed the protective effects of extract against DEN and Fe NTA induced liver necrosis. Thus, it could be concluded that the use of Tabernaemontana divaricata extract in the treatment of carcinogen induced hepatic necrosis.

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., (© 2013, The American College of Clinical Pharmacology.)

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., Competing Interests: Declaration of Conflicting Interests Dr McCune has received research funding from Otsuka Pharmaceutical of North America., (© The Author(s) 2013.)

Purpose: To determine whether corneal topical application of mitomycin-C (MMC) results in measurable plasma levels of systemic absorption., Setting: Madigan Army Medical Center, Refractive Surgery Center, Fort Lewis, Washington, and Micro-Constants Laboratory, San Diego, California, USA., Design: Case-control study., Methods: The study comprised male and female active-duty soldiers having excimer laser photorefractive keratectomy with MMC. Patients who met inclusion criteria were asked to provide a blood sample immediately after being treated with MMC 0.2 mg/mL (0.02%) for 30 seconds. Human plasma samples were evaluated by liquid chromatography mass spectrometry to determine whether MMC was present., Results: Thirty samples were submitted for evaluation. There was zero detection of MMC in the submitted samples. The quantifiable limit was greater than 10.0 ng/mL. All samples were below this., Conclusions: In this study of 30 patients with topical application of MMC for refractive surgery, there was no measurable evidence of systemic absorption. Although systemic absorption has been found with use in larger quantities, it was not known whether MMC toxicity concerns could be extrapolated to the refractive surgery population. This information allows counseling of patients on the extremely low likelihood of systemic absorption or toxicity following current techniques for refractive surgery., Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned., (Published by Elsevier Inc.)

We developed and validated an analytical method for the simultaneous determination of several chlorine and non-chlorine containing mercapturic acids in urine as specific metabolites of the hazardous chemicals 2-chloroprene and epichlorohydrin. The method involves an online column switching arrangement for online solid phase extraction of the analytes with subsequent analytical separation and detection using LC-MS/MS. The developed method enables for the first time the determination of Cl-MA-I (4-chloro-3-oxobutyl mercapturic acid), Cl-MA-II (4-chloro-3-hydroxybutyl mercapturic acid), Cl-MA-III (3-chloro-2-hydroxy-3-butenyl mercapturic acid) and HOBMA (4-hydroxy-3-oxobutyl mercapturic acid) as potential biomarkers of 2-chloroprene in urine. Additionally, CHPMA (3-chloro-2-hydroxypropyl mercapturic acid) as a specific metabolite of epichlorohydrin in urine and DHBMA (3,4-dihydroxybutyl mercapturic acid) can be determined. The analytical method proved to be both sensitive and reliable with detection limits ranging from 1.4 μg/L (for Cl-MA-III) to 4.2 μg/L (for HOBMA). Intra- and interday imprecision was determined to range from 4.7 to 11.8%. Due to the good accuracy and precision and the low limits of detection the developed method is well suited for application in biomonitoring studies in order to determine occupational exposure to 2-chloroprene and epichlorohydrin., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Intravenous (i.v.) busulfan can produce a more consistent pharmacokinetic profile than oral formulations can, but nonetheless, significant interpatient variability is evident. We investigated the influence of polymorphisms of 3 GST isozyme genes (GSTA1, GSTM1, and GSTT1) on i.v. busulfan clearance. Fifty-eight adult patients who received 3.2 mg/kg/day of busulfan as conditioning for hematopoietic cell transplantation were included in this study. Stepwise multiple linear regression demonstrated that GSTA1 variant GSTA1∗B (P = .004), GSTM1/GSTT1 double-null genotype (P = .039), and actual body weight (P = .001) were significantly associated with lower clearance of i.v. busulfan. A trend test analyzing the overall effect of GST genotype on busulfan pharmacokinetics, combining GSTA1 gene polymorphism and the number of GSTM1- and GSTT-null genotypes, showed a significant correlation between GST genotype and busulfan clearance (P = .001). The clearance of i.v. busulfan was similar between patients with GSTA1∗A/∗A and GSTM1/GSTT1 double-null genotypes and those with GSTA1∗A/∗B and GSTM1/GSTT1 double-positive genotypes. In conclusion, a pharmacogenetic approach using GST gene polymorphisms may be valuable in optimizing the i.v. busulfan dosage scheme. Our results also highlight the importance of including polygenic analyses and addressing interactions among isozyme genes in pharmacogenetic studies., (Copyright © 2011 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

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.

This study investigated the effect of seizure prophylaxis on busulfan (Bu) plasma exposure. Twenty-four adult patients received an intravenous Bu-cyclophoshamide conditioning regimen prior to bone marrow transplantation. Busilvex (0.8 mg/kg) was administered every six hours during four consecutive days. Clonazepam (0.025 to 0.03 mg/kg/day as a continuous 12-h i.v. infusion) was administered at least 12 hours prior to i.v. Bu dosing and continued until 24 hours after the last dose. Pharmacokinetic (PK) data were compared with those previously collected in patients (n=127) treated with phenytoin for seizure prophylaxis. Through population PK analysis, a 10% average increase (coefficient of variation, RSE=5.35%) in total clearance of Bu was quantified when Bu was associated with clonazepam as compared to phenytoin, which was considered as not being clinically relevant. The suspected induction on Bu metabolism by phenytoin should have resulted in the opposite effect. The patient efficacy and safety profiles were comparable between the two cohorts.

In order to support the toxicological risk assessment for the ethyl methanesulfonate (EMS) exposure of patients ingesting contaminated Viracept tablets (Müller and Singer, 2009), there was a need to correlate the effects observed in in vivo genotoxicity studies with mice to EMS exposure and to estimate human exposure to EMS at the level of contamination of Viracept tablets. The species differences in volume of distribution of EMS, a key factor for determination of its C(max), were small in the species investigated (mouse, rat, monkey), the species differences in clearance, the key factor involved in AUC assessment, were large (Lavé et al., 2009). Because of this uncertainty in extrapolation of clearance across species we used a conservative approach for human exposure predictions in terms of AUC where clearance was assumed to solely reflect the chemical stability of EMS neglecting additional clearance pathways such as metabolism and exhalation. This approach was compared to the estimates obtained from allometric scaling based on rat clearance, the species leading to the lowest clearance predicted in man. We found that both approaches led to nearly identical predictions of the human AUC. Thus, we predict a human AUC of 13 microM h for patients ingesting the most contaminated Viracept tablets, corresponding to a maximal daily intake of 0.055 mg/kg of EMS. The C(max) of EMS in these patients is predicted to be 0.85 microM. In order to provide a basis for toxicological risk assessment, these maximal human AUC and C(max) values are to be compared to the AUC and C(max) values in mice at the EMS dose of 25mg/kg which was found to be the threshold dose for induction of mutagenic effects, i.e. the dose at which no mutagenic effects were observed (Gocke et al., 2009-a). We calculate AUC and C(max) in mice at the threshold dose to be 350 microM h and 315 microM, respectively. Thus we conclude that a large safety factor can be deduced, whatever the basis of comparison, as is discussed in detail by Müller et al. (2009).

Pharmacokinetics of ethyl methanesulfonate (EMS) were characterized in mice, rats and in cynomolgus monkeys with unlabelled and (14)C-radiolabelled EMS by either quantification of unchanged EMS or the N-ethyl-valine haemoglobin (Hb) adduct. EMS was well absorbed and exhibited close to 100% oral bioavailability. EMS showed some species differences in systemic clearance (intermediate in mice, low in rats and monkeys) representing only 1-4% of the cardiac output. The volume of distribution (0.5-0.8L/kg) was constant across species and corresponded to extracellular water. As a result of the species differences in clearance, the half-life ranged from 10 min in mouse (at low dose) to 5h in monkey. The systemic exposure of free EMS and the levels of its Hb adduct increased nearly dose proportionately from 1 to 5 and 0.5 to 80 mg/kg, respectively. The persistence of the N-ethyl-valine Hb adduct was much longer than EMS itself, consistent with the long life span of haemoglobin. No species differences were evident for the binding of EMS to Hb in whole blood ex vivo as determined by the second order rate constants. Following administration of Viracept tablets of the contaminated production batches (to monkeys leading to EMS doses of 0.08-27 microg/kg, concentrations of ethyl-valine Hb adducts) were near or below the detection limit of the assay (0.043 nmol/g Hb).

Based on a production accident Viracept (nelfinavir mesilate) tablets, an HIV protease inhibitor supplied by Roche outside the US, Canada and Japan was contaminated with relatively high levels of ethyl methanesulfonate (EMS) for at most 3 months in spring of 2007. On the basis of a wide variety of toxicological data including critical experiments for mutation induction under chronic exposure conditions and cross-species exposure scaling experiments to extrapolate to humans, we estimate the added risk of adverse effects (cancer, birth abnormalities, heritable defects) in any individual patient accidentally exposed to EMS via contaminated Viracept tablets in the context of this production accident as essentially zero. Of critical important for this risk assessment are pivotal in vivo genotoxicity studies (MNT, MutaMouse) providing evidence for 'hockey-stick', like dose-response relationships for the risk defining induction of gene mutations and chromosomal damage by EMS [Gocke, E., Müller, L., Pfister, T., Buergin, H., 2009a. Literature review on the genotoxicity, reproductive toxicity, and carcinogenicity of ethyl methanesulfonate. Toxicol. Lett.; Gocke, E., Müller, L., Pfister, T., 2009b. EMS in Viracept-initial ('traditional') assessment of risk to patients based on linear dose response relations. Toxicol. Lett.; Gocke, E., Müller, L., Ballantyne, M., Whitwell, J., Müller, L., 2009c. MNT and MutaMouse studies to definde the in vivo dose-response relations of the genotoxicity of EMS and ENU. Toxicol. Lett.]. As outlined in Gocke and Wall [Gocke, E., Wall, M., 2009. In vivo genotoxicity of EMS: Statistical assessment of the dose response curves. Toxicol. Lett.], several statistical approaches are in support of a threshold model to best fit the data. The presence of clear no effect levels in bone marrow, liver and GI-tract tissue with several dose levels tested below the NOEL permits the calculation of safety factors with considerable confidence. In calculating the ratio of the NOEL dose in the animal studies (25mg/kg/day) divided by the calculated maximal daily dose of the patients (1068ppm EMS in 2.92g Viracept tablets=2.75mg EMS or 0.055mg/kg for a 50kg person) we derive a safety factor of 454 based on oral intake. Detailed absorption, distribution and metabolism studies in mice, rats and monkeys and with human surrogates in vitro enable us to estimate the safety factors also for the calculated likely highest exposure (AUC and C(max)) of patients to EMS [Lave, T., Birnböck, H., Götschi, A., Ramp, T., Pähler, A., 2009a. In vivo and in vitro characterization of ethyl methanesulfonate pharmacokinetics in animals and in human. Toxicol. Lett.; Lave, T., Paehler, A., Grimm, H.P., 2009b. Modelling of patient EMS exposure: translating pharmacokinetics of EMS in vitro and in animals into patients. Toxicol. Lett.]. We calculate the total exposure (AUC) based safety factor to amount to at least 28. This lower value is due to the conservative prediction of a longer half-life of EMS in man versus mouse, rat and monkey. Based on the estimated human C(max) the safety factor for affected Viracept patients is calculated to be 370, as C(max) is mainly dependent on volume of distribution, which is not much different for EMS in different species. We consider that the total exposure based safety factor constitutes a minimal value since the considerations regarding evidence of error-free repair at sub-threshold concentrations argues in favor of using the highest EMS concentration (C(max)) rather than the AUC as basis for risk assessment. The 'true value' very likely lies somewhere between these two numbers as aspects such as repair enzyme availability and status of the cell cycle relative to the insult are important parameters that may not fully support safety factors based solely on C(max) estimates. Potential adverse effects of EMS such as cancer, birth abnormalities and heritable effects are considered to be sequelae of its genotoxic activity. Hence, the thresholded dose-response relationships should also apply to these endpoints. We also provide a comprehensive discussion of the specific disease situation of the HIV infected target population and potential influences of co-medications on the susceptibilities and repair capacities of EMS induced DNA lesions.

Purpose: To evaluate mitomycin C (MMC) aqueous humor concentration after photorefractive keratectomy (PRK)., Methods: In this experimental study, twenty-four eyes of 12 male pigmented rabbits were divided into 4 groups and studied at the Institute of Vision and Optics, Department of Medicine, University of Crete, Greece. Eyes in groups 1 and 2 underwent PRK to correct -5 diopters (D) in a 6-mm optical zone, while sponges soaked with 0.02% MMC were applied on the exposed corneal stroma for 60 and 120 seconds, respectively. Similarly, eyes in groups 3 and 4 underwent PRK to correct -10 D in a 6-mm optical zone, while sponges soaked with 0.02% MMC were applied on the exposed corneal stroma for 60 and 120 seconds, respectively. Aqueous humor was extracted from all rabbit eyes 10 minutes after MMC application and high-performance liquid chromatography was performed immediately to detect and quantify MMC levels., Results: The mean aqueous humor concentration of MMC was 0.23+/-0.03 microg/mL, 0.39+/-0.05 microg/mL, 0.28+/-0.04 microg/mL, and 0.52+/-0.16 microg/mL in groups 1, 2, 3, and 4, respectively. The effect of application time and correction on aqueous humor MMC concentration was significant (p<0.0001 and p=0.019), while the exposure time had a greater impact on aqueous humor MMC concentration when compared with the attempted correction., Conclusions: Both exposure time of MMC on the corneal stroma and the attempted correction was correlated with MMC aqueous humor concentrations.

Studies of ethyl glucuronide (EtG) blood kinetics have so far been performed on healthy volunteers with ingestion of low to moderate doses of ethanol. These data are not necessarily transferable to heavy drinkers where the consumed doses of ethanol are much higher. The aim of this study was to investigate the pharmacokinetics of EtG and ethyl sulphate (EtS) in blood in heavy drinkers after termination of alcohol ingestion. Sixteen patients from an alcohol withdrawal clinic were included directly after admission. Time of end of drinking, estimated daily intake of ethanol (EDI) and medical history were recorded. Three to five blood samples over 20-43 h were collected from each patient subsequent to admission. The median EDI was 172 g (range 60-564). The first sample was collected median 2.5 h after end of drinking (range 0.5-23.5). Two patients had levels of EtG and EtS below LOQ in all samples, the first collected 19.25 and 23.5 h after cessation of drinking, respectively. Of the remaining 14 patients, one subject, suffering from both renal and hepatic disease, showed concentrations of EtG and EtS substantially higher than the rest of the material. This patient's initial value of EtG was 17.9 mg/L and of EtS 5.9 mg/L, with terminal elimination half lives of 11.9 h for EtG and 12.5 h for EtS. Among the remaining 13 patients, the initial median values were 0.7 g/L (range 0-3.7) for ethanol, 1.7 mg/L (range 0.1-5.9) for EtG and 0.9 mg/L (range 0.1-1.9) for EtS. Elimination occurred with a median half-life of 3.3 h for EtG (range 2.6-4.3) and 3.6 h for EtS (range 2.7-5.4). In conclusion, elimination of EtG in heavy drinkers did not significantly differ from healthy volunteers, and EtS appeared to have similar elimination rate. In the present work, there was one exception to this, and we propose that this could be explained by the patient's renal disease, which would delay excretion of these conjugated metabolites.

Objective: To create and test a slow-release antifibrotic drug-coated glaucoma drainage device using in vitro and in vivo experiments., Methods: A slow-release device incorporating mitomycin C in poly(2-hydroxyethyl methacrylate) disks was developed using redox-polymerization techniques. A standardized preparation of this drug delivery device was attached to the Ahmed glaucoma valve (model FP7; New World Medical, Inc, Rancho Cucamonga, California). Semicircular disks (5 x 6 mm) of P(HEMA)-mitomycin C containing varying concentrations of mitomycin C per gram dry weight of the gel were attached to the lower half of an Ahmed glaucoma valve plate. Water was pumped through the modified Ahmed glaucoma valve at a rate comparable to that of aqueous humor outflow, and mitomycin C release was measured. Modified and unmodified Ahmed glaucoma valves were implanted in a rabbit model, and drug release and fibrosis were assessed after 3 months., Results: The P(HEMA)-mitomycin C device released mitomycin C in vitro over 1 to 2 weeks. Studies in rabbits revealed that mitomycin C was released from the disks during the 3-month implantation. Histologic analysis demonstrated a significant reduction in inflammatory reaction and fibrosis in the resulting blebs., Conclusion: Our slow-release drug-coated glaucoma drainage device decreased fibrosis and inflammation in the resulting bleb in a rabbit model., Clinical Relevance: This device could reduce the failure rate of glaucoma drainage devices.

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.

Aims: The study was performed to estimate transconjunctival penetration of mitomycin C (MMC) to Tenon's tissue following application over the intact conjunctiva before routine trabeculectomy., Settings and Design: Institution-based case series., Materials and Methods: In 41 eyes of 41 patients, MMC (0.4 mg/ml for 3 min) was applied over the intact conjunctiva before beginning trabeculectomy. Tenon's capsule directly beneath the site of application was excised during trabeculectomy and was homogenized, centrifuged and MMC concentrations were analyzed using high-performance liquid chromatography (HPLC)., Statistical Analysis Used: Statistical analysis was performed using STATA 8.0 version software (STATA Corporation, Houston, TX, USA). In this study, P -values less than 0.05 were considered as statistically significant., Results: The average weight of the sample of Tenon's tissue excised was 5.51+/-4.42 mg (range: 0.9-17.1) and the average estimated MMC concentration found to be present in Tenon's tissue using HPLC was 18.67+/-32.36 x 10(-6) moles/kg of the tissue (range: 0.38-197.05 x 10(-6)). In 36 of the 41 patients (87.80%), the MMC concentration reached above 2 x 10(-6) moles/kg of the tissue concentration required to inhibit human conjunctival fibroblasts., Conclusions: Mitomycin C does permeate into the subconjunctival tissue after supraconjunctival application for 3 min. Application of MMC over the conjunctiva may be a useful alternative to subconjunctival or subscleral application during routine trabeculectomy and as an adjunct for failing blebs.

Most treatments for malaria target the blood stage of infection in the human host, although few can also block transmission of the parasite to the mosquito. We show here that the compound centanamycin is very effective against blood-stage malarial infections in vitro and in vivo and has profound effects on sexual differentiation of the parasites in mosquitoes. After drug treatment, parasite development is arrested within the midguts of mosquitoes, failing to produce the infective forms that migrate to the salivary glands. The mechanism of parasite death is associated with modification of Plasmodium genomic DNA. We detected DNA damage in parasites isolated from mice 24 h after treatment with centanamycin, and, importantly, we also detected this DNA damage in parasites within mosquitoes that had fed on these mice 10 days earlier. This demonstrates that damage to parasite DNA during blood-stage infection persists from the vertebrate to the mosquito host and provides a novel biochemical strategy to block malaria transmission.

There is a gender-related comorbidity of pain-related and inflammatory bowel diseases with psychiatric diseases. Since the impact of experimental gastrointestinal inflammation on the emotional-affective behavior is little known, we examined whether experimental gastritis modifies anxiety, stress coping and circulating corticosterone in male and female Him:OF1 mice. Gastritis was induced by adding iodoacetamide (0.1%) to the drinking water for at least 7 days. Inflammation was assessed by gastric histology and myeloperoxidase activity, circulating corticosterone determined by enzyme immunoassay, anxiety-related behavior evaluated with the elevated plus maze and stress-induced hyperthermia tests, and depression-like behavior estimated with the tail suspension test. Iodoacetamide-induced gastritis was associated with gastric mucosal surface damage and an increase in gastric myeloperoxidase activity, this increase being significantly larger in female mice than in male mice. The rectal temperature of male mice treated with iodoacetamide was enhanced, whereas that of female mice was diminished. The circulating levels of corticosterone were reduced by 65% in female mice treated with iodoacetamide but did not significantly change in male mice. On the behavioral level, iodoacetamide treatment caused a decrease in nocturnal home-cage activity, drinking and feeding. While depression-related behavior remained unaltered following induction of gastritis, behavioral indices of anxiety were significantly enhanced in female but not male mice. There was no correlation between the estrous cycle and anxiety as well as circulating corticosterone. Radiotracer experiments revealed that iodoacetamide did not readily enter the brain, the blood-brain ratio being 20:1. Collectively, these data show that iodoacetamide treatment causes gastritis in a gender-related manner, its severity being significantly greater in female than in male mice. The induction of gastritis in female mice is associated with a reduction of circulating corticosterone and an enforcement of behavioral indices of anxiety. Gastric inflammation thus has a distinct gender-dependent influence on emotional-affective behavior and its neuroendocrine control.

Purpose: To evaluate the effects of the applied mitomycin-C (MMC) concentration and application time on the aqueous MMC concentration and apoptosis in the corneal stroma., Methods: New Zealand white rabbits underwent mechanical epithelium debridement of the central 7.5 mm of the cornea. A sponge soaked in MMC solution was placed on the denuded corneal stroma. The effect of the exposure times ranging from 15 to 120 seconds and the different MMC concentrations ranging from 0.005% to 0.04% on the aqueous MMC concentration and the apoptosis in the stromal cells were evaluated., Results: The aqueous concentration of MMC increased linearly with increasing exposure time and MMC concentration. The correlation between the aqueous MMC concentration and the applied concentration (r = 0.809, P < 0.001) was higher than the correlation between the aqueous MMC concentration and the application time (r = 0.693, P < 0.001). Terminal deoxyribonucleotidyltransferase-mediated dUTP-digoxigenin nick end labeling (TUNEL)-positive cells were detected in the superficial stroma of the central denuded cornea. The numbers of TUNEL-positive cells increased linearly with increasing concentrations, and the increase was statistically significant (P = 0.026). However, the numbers of TUNEL-positive cells increased only slightly with an increasing application time, and the increase was not statistically significant (P = 0.928)., Conclusions: Reducing the applied concentration or decreasing the exposure time might be a good modality for reducing the potential MMC toxicity. The applied MMC concentration had greater effects on the aqueous MMC concentration and apoptosis in the stromal cells than the exposure time.

Busulfan is an alkylating agent used in a conditioning regimen prior to bone marrow transplantation. Busulfan has a narrow therapeutic index, giving rise to major liver toxicity (veno-occlusive disease), and a wide interpatient and intrapatient pharmacokinetic variability. This report presents the results of a population pharmacokinetic analysis leading to models based on underlying diseases requiring bone marrow transplantation. One hundred children received oral busulfan-based conditioning regimens between March 1998 and February 2006. Busulfan pharmacokinetic parameter estimates (Ka, first order absorption rate constant; Vs, volume of distribution related to the body weight; and Cl/F, apparent clearance) were estimated by using the nonparametric adaptative grid (NPAG) algorithm in patients divided into four groups according to initial diagnosis: metabolic diseases, hemoglobinopathies, hematological malignancies, and immune deficiencies. Ka and Vs did no differ significantly in the four subgroups. Cl/F and areas under the plasma concentration curve were significantly different in the four groups. Cl/F was significantly higher in the hemoglobinopathies group (P = 0.002), with a mean value of 7.78 L . h, whereas the immune deficiencies group was characterized by the lowest Cl/F (3.59 L . h). Interindividual variability was shown by high interindividual parameter percent coefficients of variation (CV%) but, nevertheless, with less diversity in the population parameter distributions for Vs in the three subgroups-metabolic diseases, hemoglobinopathies, and malignant diseases-and in Cl/F for patients with hemoglobinopathies. The fit was good for busulfan concentration predictions based on Bayesian individual posterior values, with little bias and good precision. In comparison with the overall population, the only model of subgroup presenting a greater precision was patients with hemoglobinopathies (P = 0.002). Use of these more specific models of a given disease may well result in more accurate individualization of busulfan dose regimens, especially in very sparse blood sampling situations.

Nitrogen mustard alkylating agents are important cancer drugs. Much interest has been focused on redirecting their covalent adducts from the N7 atoms of guanine in the major groove of DNA to the N3 atoms of adenine in the minor groove by attaching mustard groups to AT-selective minor groove binding ligands. Here we describe the use of electrospray ionization and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry to study the structure of the DNA complexes of two minor groove binding polybenzamide mustards, alkamin and alkamini; the former is a bis-half-mustard in which reactive groups are disposed at each end of the ligand, and the latter is its monofunctional analog. Alkamin is potently cytotoxic and active in experimental mouse tumor models, whereas alkamini is not. We have studied their interaction with the DNA dodecamer d(CGCGAATTCGCG)(2), designated A2T2, and we provide a detailed analysis of the observed DNA-ligand adduct ions and their fragmentation products. We find that alkamini alkylates A2T2 at guanine G4 and adenines A5 and A6 in a manner consistent with covalent attack on purine N3 atoms from the minor groove of the AT tract. Alkamin also forms monofunctional adducts at G4 and both adenines in which the second mustard arm is hydrolyzed but, in addition, forms a variety of interstrand cross-links between adenines A5/A6 and A5'/A6', an interstrand cross-link between G4 and A6', and an intrastrand cross-link between G4 and A6. We conclude that the marked cytotoxicity of alkamin and its experimental antitumor activity could be the consequence of its ability to cross-link cellular DNA at AT tract sequences.

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.

An intravenous formulation of busulfan, a cytotoxic bifunctional alkylating agent, has been developed to replace oral busulfan as a conditioning treatment prior to hematopoietic stem cell transplantation (HSCT) in pediatric patients. Doses of intravenous busulfan based on actual bodyweight, but not age, reduce inter- and intraindividual variability in exposure. In a study of intravenous busulfan as a conditioning treatment prior to allogeneic or autologous HSCT, the majority of pediatric patients, who received one of five bodyweight-based doses, achieved busulfan area under the plasma concentration-time curve (AUC) values within the targeted therapeutic range. Although mean busulfan clearance values were highly variable between bodyweight strata, exposure was not affected, with no significant differences between bodyweight groups in mean AUC values. The achievement of therapeutic AUC values with intravenous busulfan resulted in a high rate of sustained engraftment, low transplant-related mortality, and promising survival outcomes post-transplant. Intravenous busulfan was considered to be well tolerated, in the particular context of HSCT, and no failure of HSCT due to organ toxicity was reported. Nonhematologic adverse events commonly associated with busulfan conditioning regimens were frequent, but generally of mild to moderate severity. The intravenous busulfan regimen was frequently associated with elevated liver enzymes, but hepatic veno-occlusive disease (HVOD) was infrequent, of mild to moderate severity, and resolved within 10 days of diagnosis. Unlike oral busulfan, intravenous busulfan does not appear to be associated with severe HVOD or death due to organ toxicity.

Purpose: To study the aqueous and corneal pharmacokinetics of mitomycin C (MMC) after single topical administration to the central cornea and to evaluate the effects of different concentrations and different application times on the aqueous concentration of MMC., Methods: Mechanical epithelium debridement of the central 7.5 mm of the cornea was performed in New Zealand white rabbits, and a sponge soaked in 0.02% MMC solution was placed on the denuded corneal stroma for 2 minutes. Aqueous fluid and central corneal tissues samples were taken at 0.5, 1, 2, and 3 hours thereafter. MMC concentration of the samples was analyzed by high-performance liquid chromatography and evaluated at different exposure times (range: 15-120 seconds) and concentrations of applied MMC (range: 0.005%-0.04%)., Results: Peak corneal concentration was 3.728 +/- 2.547 microg/g at 30 minutes after topical administration. Maximum aqueous concentration was 0.380 +/- 0.038 microg/mL at 1 hour after topical application. The aqueous concentration of MMC increased in a dose-dependent manner with increasing exposure time and application concentration. Aqueous MMC concentration increased at a higher rate with change of applied concentration than with exposure time., Conclusion: Good penetration of MMC through central bare cornea may be noxious to endothelial cells. Reducing concentration or decreasing exposure time seems a good modality to reduce potential MMC toxicity.

Object: Extensive epidural fibrosis after lumbar spine surgery might be an important underlying cause of failed-back syndrome. Based on previously obtained data, the effect of mitomycin C (MMC) in a concentration of 0.1 mg/ml on spinal epidural fibrosis in a rat laminectomy model was investigated in a large series., Methods: Eighty adult Wistar rats underwent lumbar laminectomy. In 40 rats, MMC in a concentration of 0.1 mg/ml was locally applied to the laminectomy sites. No similar treatment was performed in the other 40 rats. At intervals from one to 12 weeks after laminectomy, both macroscopic and histological evaluations were performed. For radiological investigation, 10 rats underwent magnetic resonance (MR) imaging at 6 weeks postoperatively. Furthermore, the concentration of MMC in cerebrospinal fluid (CSF) and serum was determined 12 hours postoperatively in seven rats. Due to ease of absorption, high levels of MMC were rapidly detectable in serum, whereas the values obtained from the CSF were markedly lower. In the majority of MMC-treated laminectomy sites, epidural scarring was significantly reduced and dural adhesions were absent, in comparison with control sites (p < 0.001), as confirmed by MR images. Accordingly, the macroscopic dissection of epidural fibrous tissue to reexpose the dura mater was performed more easily and without severe bleeding in these rats. The healing of skin and the lumbar fascia was not affected, and dural leakage was not observed. All control sites showed dense epidural fibrosis with marked dural adherence., Conclusions: In this experimental model, it was shown that locally applied MMC in a concentration of 0.1 mg/ml effectively reduces epidural fibrosis and dural adherence without side effects in rats that underwent lumbar laminectomy.

Metabolic reduction can be used to activate prodrugs in hypoxic regions of tumours, but reduction by ionising radiation is also theoretically attractive. Previously, we showed that a cobalt(III) complex containing 8-hydroxyquinoline (8-HQ) and cyclen ligands releases 8-HQ efficiently on irradiation in hypoxic solutions [Ahn G-O, Ware DC, Denny WA, Wilson WR. Optimization of the auxiliary ligand shell of cobalt(III)(8-hydroxyquinoline) complexes as model hypoxia-selective radiation-activated prodrugs. Radiat Res 2004;162:315-25]. Here we investigate an analogous Co(III) complex containing the potent DNA minor groove alkylator azachloromethylbenzindoline (azaCBI, 1) to determine whether it releases 1 on radiolytic and/or enzymatic reduction under hypoxia. Monitoring by HPLC, the azaCBI ligand in the Co(III)(cyclen)(azaCBI) complex (2) slowly hydrolysed in aqueous solution, in contrast to the free ligand 1 which readily converted to its reactive cyclopropyl form. Irradiation of 2 (30-50 microM) in hypoxic solutions released 1 with yields of 0.57 micromol/J in formate buffer and 0.13 micromol/J in human plasma. Using bioassay methods, cytotoxic activation by irradiation of 2 at 1 microM in hypoxic plasma was readily detectable at clinically relevant doses (> or = 1 Gy), with a estimated yield of 1 of 0.075 micromol/J. Release of 1 from 2 was also observed in hypoxic HT29 cultures without radiation, with subsequent conversion of 1 to its O-glucuronide. Surprisingly, overexpression of human cytochrome P450 reductase in A549 cells did not increase the rate of metabolic reduction of 2, suggesting that other reductases and/or non-enzymatic reductants are responsible. Thus the cobalt(III) complex 2 is a promising prodrug capable of being activated to release a very potent cytotoxin when reduced by either ionising radiation or cells under hypoxic conditions.

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., (Copyright (c) 2005 John Wiley & Sons, Ltd.)

Dimethyl sulfate (DMS) is an alkylating agent that is carcinogenic to the respiratory tract of rodents. DNA adducts, cell proliferation, and histopathology were assessed in rats to better understand the molecular dosimetry and tissue dynamics associated with repeated inhalation exposure to DMS. For DNA methylation, rats were exposed to DMS vapor 6 h/day for up to 10 days to 0.0, 0.1, 0.7 and 1.5 ppm. N7-Methylguanine and N3-methyladenine were detected in neutral thermal hydrolysates of DNA isolated from respiratory tract tissues by high-performance liquid chromatography (HPLC) using fluorescence and ultraviolet (UV) detection. DNA methylation was greatest in DNA isolated from nasal respiratory mucosa, less in olfactory, and little was found in lung. N7-Methylguanine levels in respiratory mucosa approached steady-state levels by day 5, and N7-methylguanine persistence following exposure for 5 consecutive days was also determined. Loss of N7-methylguanine from respiratory and olfactory mucosa appeared to follow first-order kinetics. N3-Methyladenine levels were at or below detection limits in all samples. The effect of DMS on histopathology and cell proliferation in the nasal epithelium was also investigated. Rats were exposed nose-only for 2 wk to DMS vapor at concentrations of 0, 0.1, 0.7, or 1.5 ppm. Inhalation exposure to DMS induced degenerative and inflammatory changes in nasal epithelium at >or=0.7 ppm. Cell proliferation evaluations showed a trend towards an increased response at 1.5 ppm. These experiments demonstrate that DMS can induce cytotoxic and proliferative effects and is a potent methylating agent of the nasal mucosa in vivo. These experiments will provide data for the development of dosimetry models useful for risk extrapolation.

Purpose: VNP40101M is a novel sulfonylhydrazine alkylating agent with broad antitumor activity in animal models. As alkylating agents are important antileukemia drugs, a Phase I and pharmacokinetic study of VNP40101M was conducted in patients with refractory or relapsed leukemias or poor-risk myelodysplastic syndromes (MDS)., Experimental Design: VNP40101M was given as a single i.v. infusion over 15-70 min on day 1. Courses were repeated every 4 weeks according to antileukemic activity. The starting dose of 220 mg/m(2) was escalated by approximately 33% in cohorts of 3-6 patients until a maximum-tolerated dose was established. One additional cohort was treated with the maximum-tolerated dose divided over days 1 and 8., Results: Thirty-eight patients, including 28 with acute myeloid leukemia and 5 with MDS, received 52 courses of treatment. Nondose-limiting, reversible infusion-related toxicities were the most frequent adverse event, occurring in 24 (63%) patients on the first course. Dose escalation was terminated at 708 mg/m(2) for prolonged myelosuppression in 1 of 7 patients, and 600 mg/m(2) was selected as the recommended Phase II dose, with no significant extramedullary toxicity at this dose level. Two patients, 1 with MDS treated with 300 mg/m(2) and 1 with acute myeloid leukemia treated with 600 mg/m(2), achieved complete remission., Conclusions: VNP40101M had significant antileukemic activity and minimal extramedullary toxicity in patients with relapsed or refractory disease.

Background: In a chemical plant, alkylating agents (mainly ethylene oxide and propylene oxide, but also dimethyl sulphate) are utilised for the production of special surfactants., Aims: To determine possible uptakes of dimethyl sulphate by workers using N-methylvaline., Methods: Sixty two workers in a chemical plant (38 smokers, 24 non-smokers) with potential exposure to dimethyl sulphate were monitored with respect to their blood levels of N-methylvaline. Ten laboratory workers without exposure to methylating agents were controls. Blood samples of eight workers from a specific working area were analysed for N-methylvaline in a follow up investigation four months later., Results: The 95th centile for N-methylvaline was 80.7 microg/l blood in the exposed workers compared to 12.4 microg/l blood in controls. In a hot spot area, 10 workers exceeded the German exposure equivalent value for dimethyl sulphate (40 microg/l blood) up to fourfold. In contrast, dimethyl sulphate has not been detectable in workplace air in this area. In a follow up investigation of eight of these 10 workers, N-methylvaline levels were significantly lower, but still increased., Conclusions: The present study is to our knowledge the first to report increased N-methylvaline levels after occupational exposure to dimethyl sulphate. As ambient monitoring values in the plant could not explain this exposure, skin contact was considered to be the main route of uptake for this substance. Dimethyl sulphate may therefore represent an occupational problem that has been generally underestimated in the past.

Study Objective: To determine the maximum tolerated dose, dose-limiting toxicity, pharmacokinetics, and potential antitumor activity of temozolomide administered as a single dose every 28 days., Design: Open label, phase I, dose-escalation trial., Setting: University-affiliated cancer center., Patients: Eleven patients aged 33-73 years with a documented solid tumor or lymphoma who failed therapy of proven efficacy for their disease or had a disease for which no conventional therapy was available., Intervention: Temozolomide 500 mg/m2 was administered as a single oral dose every 28 days. Doses were escalated to 750 or 1000 mg/m2. No intrapatient dose escalation was allowed. At least two patients were enrolled at each dose level. Patients who did not have progressive disease and did not experience a dose-limiting toxicity, or experienced a dose-limiting toxicity but were eligible for dose reduction, were eligible to continue on the study., Measurements and Main Results: Pharmacokinetic analysis was performed for temozolomide and its active metabolite, 5-(3-methyltriazeno)-imidazole-4-carboxamide (MTIC). Neutropenia and thrombocytopenia were dose limiting at 1000 mg/m2. Temozolomide was absorbed rapidly (mean time to maximum concentration 1.4 hrs) and eliminated, with average half-life and apparent oral systemic clearance values of 1.8 hours and 97 ml/minute/m2, respectively. Mean systemic exposure to MTIC was 3.7% of temozolomide. No objective responses were observed. The maximum tolerated dose of temozolomide was 750 mg/m2., Conclusion: Temozolomide 750 mg/m2 administered orally every 28 days was well tolerated. Alternate temozolomide dosing schedules such as continuous daily administration may enhance antitumor activity through sustained depletion of the DNA repair protein O6-alkylguanine DNA alkyltransferase.

Brostallicin is a bromoacryloyl derivative of distamycin A, which has shown very promising preclinical activity against a variety of human tumors both in vitro and in vivo. The drug has a limited toxicity towards bone marrow precursor cells in vitro resulting in a therapeutic index much higher than those achieved with other distamycin A derivatives. It retains activity against cancer cells resistant to alkylating agents, topoisomerase I inhibitors and cells with mismatch repair deficiency. Brostallicin has a peculiar mechanism of action involving activation upon binding to glutathione (GSH) catalyzed by glutathione-S-transferase (GST). As a consequence, cells expressing relatively high GST/GSH levels are more susceptible to treatment with brostallicin. Considering that increased levels of GST/GSH are often found in human tumors, this could represent an advantage for the drug in the clinic. Initial clinical studies indicate the tolerability of the drug and allow the determination of the optimal dose for subsequent studies. Some partial response were obtained in these initial phase I studies. Altogether, the results suggest brostallicin to be a new promising anticancer agent with a new mechanism of action. It also raises the possibility to use it in combination with other anticancer drugs currently used.

After oral or parenteral administration of chemotherapeutic agents, these drugs are transported to the tissues by the blood in different fractions: plasma water, plasma proteins or cells. Erythrocytes may play an important role in the storage, transport and metabolism of chemotherapeutic agents. Anthracyclines, ifosfamide and its metabolites, and topoisomerase I and I/II inhibitors are incorporated in red blood cells. They may be transported to the tumour tissue and mobilised from the erythrocyte by different active or passive transport mechanisms. Erythrocytes may also be used as carriers for drugs such as asparaginase. This leads to a decreased toxicity profile. Finally, it has been shown that red blood cells are important in the transport and metabolism of mercaptopurine. The erythrocyte concentration of mercaptopurine has a prognostic value in the treatment of childhood acute lymphoblastic leukemia. In this review, the role of red blood cells for various anticancer drugs is further discussed.

In a systematic study of O(6)-alkylguanine DNA-alkyltransferase activity in the human colon and rectum, tumours were found to occur in regions of low activity. These results are consistent with the hypothesis that O(6)-alkylguanine DNA-alkyltransferase levels and alkylating agent exposure may be important determinants of large bowel tumorigenesis.

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.

Busulfan pharmacokinetic parameters are useful in predicting the outcome of allogeneic bone marrow transplantation (BMT). Standard pharmacokinetic measurements require multiple blood samples. Various limited sampling models (LSM) have been proposed for reducing the sample number required for these measurements, essentially for patients with malignant disorders undergoing BMT. This study was undertaken to evaluate the existing LSM for busulfan pharmacokinetics to find out the most suitable method for patients with thalassaemia major undergoing BMT. Busulfan levels in plasma samples were analysed by HPLC. The AUC calculated by non-compartmental analysis using the program 'TOPFIT' was compared with previously published LSMs. Our seven sample pharmacokinetic data for AUC calculation was compared with the published LSMs. The three sample models suggested by Chattergoon et al and Schuler et al showed significant agreement with AUC TOPFIT (R(2) = 0.98 and 0.94, respectively) in our clinical context. Other models resulted in significant over or under representation of observed values (Vassal's model R(2) = 0.61; Chattergoon's two sample model R(2) = 0.84; four sample model R(2) = 0.83; Schuler's two sample model R(2) = 0.79). By these data the three sample LSM proposed by Chattergoon et al and Schuler et al are suitable for calculation of the AUC in patients with thalassaemia major undergoing BMT conditioned with oral busulfan.

Because the busulfan area under the concentration-time curve (AUC) has been correlated with the outcome of bone marrow transplantation (BMT) and the occurrence of veno-occlusive disease after BMT, a rapid determination of AUC is needed to ensure its suitable dosage. The present work describes a method based on combining aliquots from the 10 blood samples collected for an AUC busulfan determination and performing a single determination of the resulting mixture. In 42 patients undergoing a preparative regimen for bone marrow transplantation this combined sample AUC was compared with the regular determined AUC obtained from 10 consecutive samples drawn at various time intervals after dosing. It is apparent that the AUCs calculated by pharmacokinetic analysis using a noncompartmental model package and those obtained by analyzing the sample mixture are very similar ( r = 0.961). The proposed method allows rapid adjustment of the busulfan dose, reducing the number of uncorrected dosages during therapy.

In order to control busulfan pharmacokinetic variability and toxicity, a specific monitoring protocol was instituted in our bone marrow transplant BMT paediatric patients including a test dose, daily Bayesian forecasting of busulfan plasma levels, and Bayesian individualization of busulfan dosage regimens. Twenty-nine children received BMT after a busulfan-based conditioning regimen. Individual pharmacokinetic parameters were obtained following a 0.5 mg*kg test dose and were used for daily individualization of dosage regimens during the subsequent 4-day course of treatment. Doses were adjusted to reach a target mean AUC per 6 h between 4 and 6 microg.h.ml(+1). Plasma busulfan assays were performed by liquid chromatography. Pharmacokinetic analysis used the USC*PACK software. The performance of the test dose to predict AUC during the busulfan regimen was evaluated. Incidence of toxicity, chimerism and relapse, overall Kaplan-Meier survival, and VOD-free survival were compared after matching our patients (group A) with patients conditioned by using standard doses of busulfan (group B). Busulfan doses were decreased in 69% of patients compared to conventional doses. Expected AUC was significantly correlated with observed AUC and predictability of the test dose was 101.9 +/- 17.9%. Incidence of VOD in group A was 3.4% vs 24.1% in group B, while the incidence of stomatitis was similar. Engraftment was successful in all patients in group A. The rate of full engraftment at 3 months post-BMT was higher in group A (P = 0.012). Long-term overall survival did not differ between the two groups, in contrast to the 90-day survival. VOD-free survival was higher in group A (P = 0.026). Pharmacokinetic monitoring and individualization of busulfan dosage regimen are useful in improving clinical outcome and reducing early mortality in paediatric bone marrow transplant recipients.

Aims/hypothesis: The role of selective uptake and alkylation in the diabetogenic action of streptozotocin was investigated in bioengineered RINm5F insulin-producing cells, with different expression levels of the glucose transporter GLUT2, by comparing the toxicity of streptozotocin with that of four chemically related alkylating compounds, N-methyl-N-nitrosourea (MNU), N-ethyl-N nitrosourea (ENU), methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS)., Methods: GLUT2 expressing RINm5F cells were generated through stable transfection of the rat glucose transporter GLUT2 cDNA under the control of the cytomegalovirus promoter in the pcDNA3 vector. Viability of the cells was determined using a microtitre plate-based 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay., Results: Cells expressing the glucose transporter GLUT2 were much more susceptible to streptozotocin toxicity than control cells due to the uptake of streptozotocin by this specific glucose transporter. In contrast, the GLUT2 expression had no effect upon the toxicity of MNU, ENU, MMS or EMS. Although the latter substances are, like streptozotocin, cytotoxic through their ability to cause DNA alkylation, they are not diabetogenic because they are not taken up through the glucose transporter GLUT2., Conclusion/interpretation: Our results are consistent with the central importance of selective uptake and alkylating activity in the mechanism of streptozotocin diabetogenicity. Alkylation of DNA leads to necrosis of pancreatic beta cells and thus to a state of insulin-dependent diabetes mellitus, well-known as streptozotocin diabetes in experimental diabetes research.

In this paper, the metabolism and genetic toxicity of 1,3-butadiene (BD) and its oxidative metabolites in humans and rodents is reviewed with attention to newer data that have been published since the latest evaluation of BD by the International Agency for Research on Cancer (IARC). The oxidative metabolism of BD in mice, rats and humans is compared with emphasis on the major pathways leading to the reactive intermediates 1,2-epoxy-3-butene (EB), 1,2:3, 4-diepoxybutane (DEB), and 3,4-epoxy-1,2-butanediol (EBdiol). Results from recent studies of DNA and hemoglobin adducts indicate that EBdiol may play a more significant role in the toxicity of BD than previously thought. All three metabolites are capable of reacting with macromolecules, such as DNA and hemoglobin, and have been shown to induce a variety of genotoxic effects in mice and rats as well as in human cells in vitro. DEB is clearly the most potent of these genotoxins followed by EB, which in turn is more potent than EBdiol. Studies of mutations in lacI and lacZ mice and of the Hprt mutational spectrum in rodents and humans show that mutations at G:C base pairs are critical events in the mutagenicity of BD. In-depth analyses of the mutational spectra induced by BD and/or its oxidative metabolites should help to clarify which metabolite(s) are associated with specific mutations in each animal species and which mutational events contribute to BD-induced carcinogenicity. While the quantitative relationship between exposure to BD, its genotoxicity, and the induction of cancer in occupationally exposed humans remains to be fully established, there is sufficient data currently available to demonstrate that 1,3-butadiene is a probable human carcinogen.