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We hypothesized that IV busulfan (Bu) dosing could be safely intensified through pharmacokinetic (PK-) dose guidance to minimize the inter-patient variability in systemic exposure (SE) associated with body-sized dosing, and that this should improve outcome of AML/MDS patients undergoing allogeneic stem cell transplantation. To test this hypothesis, we treated 218 patients (median age 50.7 years, male/female 50/50%) with fludarabine 40 mg/m 2 once daily x4, each dose followed by IV Bu, randomized to 130 mg/m 2 (N=107) or PK-guided to average daily SE, AUC of 6000 μM min (N=111), stratified for remission status and allo-grafting from HLA-matched donors. Toxicity and GvHD rates in the groups were similar; the risk of relapse or treatment-related mortality remained higher in the fixed-dose group throughout the 80-month observation period. Further, PK-guidance yielded safer disease control, leading to improved overall and PFS, most prominently in MDS patients and in AML patients not in remission at allogeneic stem cell transplantation. We conclude that AML/MDS patients receiving pretransplant conditioning treatment with our 4-day regimen may benefit significantly from PK-guided Bu dosing. This could be considered an alternative to fixed-dose delivery since it provides the benefit of precise dose delivery to a predetermined SE without increasing risk(s) of serious toxicity and/or GvHD.

Cancer is the uncontrolled growth of cells in the body and is considered as one of the major causes of death globally. There are several cytotoxic chemotherapeutic agents used to treat cancer including methotrexate, 5-fluorouracil, cisplatin, tamoxifen, doxorubicin and others. Although billions of dollars have been spent on cancer research to develop these chemotherapies, it still remains a major illness for mankind partly due to the shortcomings of these therapies. These shortcomings include low targeting specificity, severe side effects (due to high doses) and poor pharmacokinetics. To avoid these drawbacks, anti-cancer drug delivery systems have been developed recently using nanocarriers including liposomes, micelles, polyelectrolyte capsules and others. One of the recent class of nanoparticles investigated for chemotherapeutic use are metal organic frameworks (MOFs) which are hybrid polymers that consist of metal ions or clusters and organic ligands. MOFs are used in many applications including gas/vapor separation, gas storage, catalysis, luminescent materials, and biomedical imaging. These structures have additional features that promote their use as drug carriers in the biomedical field. First, they are nontoxic, biodegradable and have the ability to carry high loadings of the anti-neoplastic agent due to their porous nature. Also, they have well-defined crystalline structures that can be characterized by different analytical techniques and their sizes are suitable to control their in vivo drug release. This paper reviews the methods used to synthesize MOFs and their recent use as antineoplastic drug delivery carriers., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Background: Multifunctional nanocarriers for controlled drug delivery, imaging of disease development and follow-up of treatment efficacy are promising novel tools for disease diagnosis and treatment. In the current investigation, we present a multifunctional theranostic nanocarrier system for anticancer drug delivery and molecular imaging. Superparamagnetic iron oxide nanoparticles (SPIONs) as an MRI contrast agent and busulphan as a model for lipophilic antineoplastic drugs were encapsulated into poly (ethylene glycol)-co-poly (caprolactone) (PEG-PCL) micelles via the emulsion-evaporation method, and PEG-PCL was labelled with VivoTag 680XL fluorochrome for in vivo fluorescence imaging., Results: Busulphan entrapment efficiency was 83% while the drug release showed a sustained pattern over 10 h. SPION loaded-PEG-PCL micelles showed contrast enhancement in T 2 *-weighted MRI with high r 2 * relaxivity. In vitro cellular uptake of PEG-PCL micelles labeled with fluorescein in J774A cells was found to be time-dependent. The maximum uptake was observed after 24 h of incubation. The biodistribution of PEG-PCL micelles functionalized with VivoTag 680XL was investigated in Balb/c mice over 48 h using in vivo fluorescence imaging. The results of real-time live imaging were then confirmed by ex vivo organ imaging and histological examination. Generally, PEG-PCL micelles were highly distributed into the lungs during the first 4 h post intravenous administration, then redistributed and accumulated in liver and spleen until 48 h post administration. No pathological impairment was found in the major organs studied., Conclusions: Thus, with loaded contrast agent and conjugated fluorochrome, PEG-PCL micelles as biodegradable and biocompatible nanocarriers are efficient multimodal imaging agents, offering high drug loading capacity, and sustained drug release. These might offer high treatment efficacy and real-time tracking of the drug delivery system in vivo, which is crucial for designing of an efficient drug delivery system.

The dried blood spot (DBS) method, which is a simple technique for blood sample processing involving the placement of a drop of whole blood onto filter paper, has been used recently in clinical pharmacology to determine blood concentrations of various drugs. This study examined the feasibility of the clinical application of the DBS method for individual busulfan dose adjustments. Pharmacokinetic (PK) parameters of blood samples for busulfan measurements determined using the DBS method were compared with those using plasma separation (the conventional method). Blood samples were collected from patients receiving i.v. busulfan as a conditioning regimen before allogeneic hematopoietic stem cell transplantation at Toranomon Hospital, Japan. Samples collected 2, 4, and 6 hours after the start of the first drip infusion were processed by DBS or the conventional method. The area under the blood concentration-time curve (AUC) and other PK parameters were calculated to compare the 2 methods. Divergence of <20% in each parameter was considered acceptable. The divergence range for each parameter was as follows: blood concentration at 2 hours after the start of drip infusion, .6 to 8.2%; at 4 hours, .3 to 10.0%; at 6 hours, .3 to 14.2%; and AUC 0-∞ , .0 to 10.3%. None of the PK parameters showed a divergence between the DBS method and the conventional method exceeding 20%, suggesting that both methods are well correlated. The clinical application of blood sample processing with the DBS method in the measurement of blood busulfan concentration may therefore be feasible, but further studies are needed to confirm these findings., (Copyright © 2016 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Background: Intravenous busulfan combined with therapeutic drug monitoring to guide dosing improves outcomes after allogeneic haemopoietic cell transplantation (HCT). The best method to estimate busulfan exposure and optimum exposure in children or young adults remains unclear. We therefore assessed three approaches to estimate intravenous busulfan exposure (expressed as cumulative area under the curve [AUC]) and associated busulfan AUC with clinical outcomes in children or young adults undergoing allogeneic HCT., Methods: In this retrospective analysis, patients from 15 centres in the Netherlands, USA, Canada, Switzerland, UK, Italy, Germany, and Australia who received a busulfan-based conditioning regimen between March 18, 2001, and Feb 12, 2015, were included. Cumulative AUC was calculated by numerical integration using non-linear mixed effect modelling (AUC NONMEM ), non-compartmental analysis (AUC from 0 to infinity [AUC 0-∞ ] and to the next dose [AUC 0-τ ]), and by individual centres using various approaches (AUC centre ). The main outcome of interest was event-free survival. Other outcomes of interest were graft failure or relapse, or both; transplantation-related mortality; acute toxicity (veno-occlusive disease or acute graft versus-host disease [GvHD]); chronic GvHD; overall survival; and chronic-GvHD-free event-free survival. We used propensity-score-adjusted Cox proportional hazard models, Weibull models, and Fine-Gray competing risk regressions for statistical analyses., Findings: 790 patients were enrolled, 674 of whom were included: 274 (41%) with malignant and 400 (59%) with non-malignant disease. Median age was 4·5 years (IQR 1·4-10·7). The median busulfan AUC NONMEM was 74·4 mg × h/L (95% CI 31·1-104·6), which correlated with the standardised method AUC 0-∞ (r 2 =0·74), but the latter correlated poorly with AUC centre (r 2 =0·35). Estimated 2-year event-free survival was 69·7% (95% CI 66·2-73·0). Event-free survival at 2 years was 77·0% (95% CI 72·1-82·9) in the 257 patients with an optimum intravenous busulfan AUC of 78-101 mg × h/L compared with 66·1% (60·9-71·4) in the 235 patients at the low historical target of 58-86 mg × h/L and 49·5% (29·2-66·0) in the 44 patients with a high (>101 mg × h/L) busulfan AUC (p=0·011). Compared with the low AUC group, graft failure or relapse occurred less frequently in the optimum AUC group (hazard ratio [HR] 0·57, 95% CI 0·39-0·84; p=0·0041). Acute toxicity (HR 1·69, 1·12-2·57; p=0·013) and transplantation-related mortality (2·99, 1·82-4·92; p<0·0001) were significantly higher in the high AUC group (>101 mg × h/L) than in the low AUC group (<78 mg × h/L), independent of indication; no difference was noted between AUC groups for chronic GvHD (<78 mg × h/L vs ≥78 mg × h/L, HR 1·30, 95% CI 0·73-2·33; p=0·37)., Interpretation: Improved clinical outcomes are likely to be achieved by targeting the busulfan AUC to 78-101 mg × h/L using a new validated pharmacokinetic model for all indications., Funding: Research Allocation Program and the UCSF Helen Friller Family Comprehensive Cancer Center and the Mt Zion Health Fund of the University of California, San Francisco., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Gilbert's syndrome is a common inherited disorder of bilirubin metabolism, characterised by mild, unconjugated hyperbilirubinaemia. However, the effect of Gilbert's syndrome on the disposition of some drugs can lead to unexpected toxicity. We tested the hypothesis that patients undergoing myeloablative conditioning and haemopoietic cell transplantation would have different mortality outcomes depending on whether or not they had laboratory evidence of Gilbert's syndrome., Methods: In this retrospective cohort study, we used clinical and laboratory data of patients who had haemopoietic cell transplantation from Jan 1, 1991, to Dec 31, 2011. Patients were included if they had received high-dose conditioning regimens of cyclophosphamide plus total body irradiation (CY/TBI), busulfan plus cyclophosphamide (BU/CY), busulfan plus melphalan plus thioTEPA (BUMELTT), or melphalan before transplant. Patients were excluded if their original consent forms to report transplant outcomes were not signed, if consent was withdrawn, or if they were a prisoner. Patients with Gilbert's syndrome were defined as having laboratory values before the start of conditioning therapy for unconjugated serum bilirubin concentrations of at least 17·1 μmol/L (≥1 mg/dL), normal conjugated serum bilirubin, and no evidence of hepatitis, cholestasis, or haemolysis. We assessed the association of Gilbert's syndrome with overall mortality and non-relapse mortality using adjusted Cox regression models at day 200 after transplantation., Findings: Our study cohort was 3379 patients-1855 (55%) allograft and 1524 (45%) autograft recipients. 211 (6%) patients had Gilbert's syndrome and 3168 (94%) did not have this condition. Most patients were adults (median age 45·8 years [IQR 33·2-55·5]) with haematological malignancies. For overall mortality 664 (20%) patients had died by day 200 after transplant (47 [22%] of 211 who had Gilbert's syndrome vs 617 [19%] of 3168 who did not have Gilbert's syndrome), and for non-relapse mortality 499 (92%) patients had died before relapse was recorded (38 [18%] who had Gilbert's syndrome vs 461 [15%] who did not have Gilbert's syndrome). The effect of Gilbert's syndrome on the risk of overall mortality and non-relapse mortality by transplant day 200 varied between the conditioning regimens and donor groups. In patients conditioned with a myeloablative regimen that contained busulfan (n=1131), those with Gilbert's syndrome (n=60) were at a significantly increased risk of death and non-relapse mortality by day 200 compared with those without Gilbert's syndrome (n=1071; hazard ratio [HR] 2·30, 95% CI 1·47-3·61, p=0·00030; and 2·77, 1·71-4·49, p<0·0001). In patients who received CY/TBI or melphalan conditioning regimens, those with Gilbert's syndrome had similar outcomes to those without Gilbert's syndrome (overall mortality at day 200 HR 0·90, 95% CI 0·60-1·34, p=0·60; non-relapse mortality at day 200: 0·90, 0·56-1·45, p=0·65). Analyses of causes of death and busulfan disposition provided no mechanistic explanation for the differences in mortality., Interpretation: Overall mortality and non-relapse mortality at day 200 after transplant were significantly worse in patients with Gilbert's syndrome who received busulfan-containing myeloablative conditioning regimens, compared with non-Gilbert's syndrome patients. Patients with Gilbert's syndrome should receive busulfan-containing myeloablative conditioning regimens with caution., Funding: US National Institutes of Health., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

High-dose busulfan (Bu) is an integral component of commonly used preparative regimens for both allogeneic and autologous transplantation. There is significant interest in comparing the efficacy and toxicity of administering Bu every 6 (Bu6) or every 24 hours (daily Bu). To facilitate a therapeutic dose-monitoring protocol, we transitioned from Bu6 to daily Bu dosing for patients with Hodgkin and non-Hodgkin lymphoma undergoing autologous stem cell transplantation (ASCT). Here, we retrospectively review outcomes of 400 consecutive eligible lymphoma patients who underwent ASCT from 2007 to 2013 with high-dose busulfan (Bu), cyclophosphamide (Cy), and etoposide (E). Bu was given at a fixed dose of either .8 mg/kg every 6 hours for 14 doses for 307 patients or a fixed dose of 2.8 mg/kg every 24 hours for 4 doses (days -9 through -6) for 93 patients who underwent transplantation after the transition from Bu6 to daily Bu was made. Toxicity was assessed using pulmonary and liver function tests (LFT) at specified time points before and after ASCT. Baseline patient and disease characteristics of patients dosed with Bu6 and daily Bu were similar. There was no significant difference in forced expiratory volume in 1 second or diffusing capacity of the lungs for carbon monoxide before and after transplantation in the Bu6 versus daily Bu cohorts. Changes in LFTs with daily Bu were not significantly different than those with Bu6. There were no differences in relapse, nonrelapse mortality, progression-free survival, or overall survival between Bu6 and Bu 24 administration schedules in univariable or multivariable analysis (P ≥ .34). For a subset of 23 patients who had first-dose Bu levels measured, we observed significant variation in an median estimated cumulative area under the curve (AUC) of 17,568 µM-minute (range, 12,104 µM-23,084 µM-minute). In conclusion, daily Bu with Cy/E is more convenient than Bu6, has equivalent outcomes, and results in no increase in either hepatic or pulmonary toxicity. Consistent with previous reports, there is a significant range of Bu AUC levels, with a standard deviation of 13%. These data provide rationale for our prospective clinical trial of real-time therapeutic dose monitoring of Bu., (Copyright © 2016 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Intravenous (IV) busulfan doses are often personalized to a concentration at steady state (Css) using the patient's clearance, which is estimated with therapeutic drug monitoring. We sought to identify biomarkers of IV busulfan clearance using a targeted pharmacometabonomics approach. A total of 200 metabolites were quantitated in 106 plasma samples, each obtained before IV busulfan administration in hematopoietic cell transplant (HCT) recipients. Both univariate linear regression with false discovery rate (FDR) and pathway enrichment analyses using the Global test were performed. In the univariate analysis, glycine, N-acetylglycine, 2-hydroxyisovaleric acid, creatine, serine, and tyrosine were statistically significantly associated with IV busulfan clearance at P < 0.05, with the first three satisfying the FDR of q < 0.1. Using pathway enrichment analysis, the glycine, serine, and threonine metabolism pathway was statistically significantly associated with IV busulfan clearance at P < 0.05 and q < 0.1, and a pathway impact >0.1. Glycine is a component of glutathione, which is conjugated with busulfan via glutathione transferase enzymes. These results demonstrate the potential utility of pharmacometabonomics to inform IV busulfan dosing. Future studies are required to validate these findings., Competing Interests: Conflict-of-interest disclosure: The authors declare no competing financial interests.

Fludarabine with busulfan (FB) and fludarabine with melphalan (FM) are commonly used reduced-intensity conditioning (RIC) regimens. Pharmacokinetic dosing of busulfan (Bu) is frequently done for myeloablative conditioning, but evidence for its use is limited in RIC transplants. We compared transplant outcomes of FB versus FM using i.v. Bu targeted to the area under the curve (AUC). A total of 134 RIC transplants (47 FB and 87 FM) for acute myelogenous leukemia and myelodysplastic syndrome were identified, and median follow-up of the cohort was 40 months (range, 0 to 63.3). A significantly higher 2-year cumulative incidence of relapse (CIR) was associated with FB versus FM at 35.6% versus 17.3%, respectively (P = .0058). Furthermore, 2-year progression-free survival rates were higher for FM versus FB at 60.5% versus 48.7%, respectively (P = .04). However, 2-year rates of nonrelapse mortality (NRM) and overall survival (OS) were similar. The need for dose adjustment based on AUC did not alter relapse risk or NRM. Patients with Karnofsky performance status ≥ 90 who received FM had a 2-year OS rate of 74.8% versus 48.3% for FB (P = .03). FB use remained prognostic for relapse in multivariable analysis (hazard ratio, 2.75; 95% confidence interval, 1.28 to 5.89; P = .0097). In summary, in spite of AUC-directed dosing, FB compared with FM was associated with a significantly higher CIR., (Copyright © 2016 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Busulfan, cyclophosphamide, and etoposide (BuCyE) is a commonly used conditioning regimen for autologous stem cell transplantation (ASCT). This multicenter, phase II study examined the safety and efficacy of BuCyE with individually adjusted busulfan based on preconditioning pharmacokinetics. The study initially enrolled Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) patients ages 18 to 80 years but was amended due to high early treatment-related mortality (TRM) in patients > 65 years. BuCyE outcomes were compared with contemporaneous recipients of carmustine, etoposide, cytarabine, and melphalan (BEAM) from the Center for International Blood and Marrow Transplant Research. Two hundred seven subjects with HL (n = 66) or NHL (n = 141) were enrolled from 32 centers in North America, and 203 underwent ASCT. Day 100 TRM for all subjects (n = 203), patients > 65 years (n = 17), and patients ≤ 65 years (n = 186) were 4.5%, 23.5%, and 2.7%, respectively. The estimated rates of 2-year progression-free survival (PFS) were 33% for HL and 58%, 77%, and 43% for diffuse large B cell lymphoma (DLBCL; n = 63), mantle cell lymphoma (MCL; n = 29), and follicular lymphoma (FL; n = 23), respectively. The estimated rates of 2-year overall survival (OS) were 76% for HL and 65%, 89%, and 89% for DLBCL, MCL, and FL, respectively. In the matched analysis rates of 2-year TRM were 3.3% for BuCyE and 3.9% for BEAM, and there were no differences in outcomes for NHL. Patients with HL had lower rates of 2-year PFS with BuCyE, 33% (95% CI, 21% to 46%), than with BEAM, 59% (95% CI, 52% to 66%), with no differences in TRM or OS. BuCyE provided adequate disease control and safety in B cell NHL patients ≤ 65 years but produced worse PFS in HL patients when compared with BEAM., (Copyright © 2016 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Background: Monitoring busulfan area under the plasma concentration-time curve (AUC) to establish the dose regimen for stem cell transplantation desirable to achieve efficacy while avoiding toxicity., Objective: Our objective was to compare AUCs calculated by 18 limited sampling methods (LSMs) from 2 to 5 samples to reference AUCs determined from 10 samples in a retrospective study of 103 adult patients receiving oral busulfan. LSMs using 2 or 3 samples were ineffective., Methods: Four LSMs using 4 or 5 blood samples that accurately characterized busulfan AUC were identified. The best 2 methods were obtained with sampling at 0.5, 1, 2, 4, and 6 hours and after 1, 1.5, 2, 4, and 6 hours postdose. For these LSMs, the incidence of 20% difference between AUCs from LSMs and reference AUCs was less than 1.3%., Conclusions: Effective and safe determination of AUC for oral busulfan can be made with strategies using only 4 or 5 concentration timepoints.

Background: Busulfan dose adjustment is routinely guided by plasma concentration monitoring using 4-9 blood samples per dose adjustment, but a pharmacometric Bayesian approach could reduce this sample burden., Methods: The authors developed a nonparametric population model with Pmetrics. They used it to simulate optimal initial busulfan dosages, and in a blinded manner, they compared dosage adjustments using the model in the BestDose software to dosage adjustments calculated by noncompartmental estimation of area under the time-concentration curve at a national reference laboratory in a cohort of patients not included in model building., Results: Mean (range) age of the 53 model-building subjects was 7.8 years (0.2-19.0 years) and weight was 26.5 kg (5.6-78.0 kg), similar to nearly 120 validation subjects. There were 16.7 samples (6-26 samples) per subject to build the model. The BestDose cohort was also diverse: 10.2 years (0.25-18 years) and 46.4 kg (5.2-110.9 kg). Mean bias and imprecision of the 1-compartment model-predicted busulfan concentrations were 0.42% and 9.2%, and were similar in the validation cohorts. Initial dosages to achieve average concentrations of 600-900 ng/mL were 1.1 mg/kg (≤12 kg, 67% in the target range) and 1.0 mg/kg (>12 kg, 76% in the target range). Using all 9 concentrations after dose 1 in the Bayesian estimation of dose requirements, the mean (95% confidence interval) bias of BestDose calculations for the third dose was 0.2% (-2.4% to 2.9%, P = 0.85), compared with the standard noncompartmental method based on 9 concentrations. With 1 optimally timed concentration 15 minutes after the infusion (calculated with the authors' novel MMopt algorithm) bias was -9.2% (-16.7% to -1.5%, P = 0.02). With 2 concentrations at 15 minutes and 4 hours bias was only 1.9% (-0.3% to 4.2%, P = 0.08)., Conclusions: BestDose accurately calculates busulfan intravenous dosage requirements to achieve target plasma exposures in children up to 18 years of age and 110 kg using only 2 blood samples per adjustment compared with 6-9 samples for standard noncompartmental dose calculations.

Hematopoietic stem cell transplantation (HSCT) is the only proven curative option for patients with hemoglobinopathies, both thalassemia and sickle cell anemia (SCA). A busulfan-based myeloablative conditioning regimen is the standard of care for HSCT in these patients, although increased treatment-related morbidity, including veno-occlusive disease (VOD), has been demonstrated. Thirty-eight pediatric patients, median age 8 years (range, 6 months to 22 years), suffering from hemoglobinopathy were treated at Rambam Medical Center in Haifa, Israel, between 1998 and 2011. Thirty-four patients had thalassemia major and 4 had SCA. The 38 patients underwent 40 HSCTs, 34 of which were first transplants and 6 second transplants. Most transplants (32/40) were from matched sibling donors. Sources of stem cells were peripheral blood in 30 transplants, bone marrow in 7 transplants, and cord blood in 3 transplants. All received different customized busulfan-based conditioning regimens tailored by pharmacokinetic analysis of busulfan levels. Primary engraftment occurred in 37 of 40 transplants. Neutrophil engraftment (>.5 × 10(9)/L) occurred at a median of 15.3 days post-transplantation (range, 10 to 45). Platelet transfusion independence (>20 × 10(9)/L) occurred at a median of 22.3 days (range, 11 to 60). The rate of 5-year overall survival for thalassemia patients after first transplantation was 90.5% ± 5.3%. The rate of 5-year thalassemia-free survival was 81.7% ± 6.8%. Cumulative incidence of acute graft-versus-host disease (GVHD) was 17.6%. Rate of grades III to IV GVHD was 8.8%. Cumulative incidence of chronic GVHD was 23.5%, with 11.8% incidence of extensive chronic GVHD. One patient developed VOD. Full donor chimerism occurred in 36.4% of patients with class 1 + 2 thalassemia, compared with 78.6% in class 3 thalassemia (P = .049). Overall survival above 90% in patients undergoing their first transplant was demonstrated using busulfan-based conditioning regimens. The low incidence of VOD was probably due to busulfan area under the curve measurements and dose adjustment., (Copyright © 2016 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

TDM is intended to limit unintended consequences of drugs with narrow therapeutic indices. However, the application of different sampling strategies and pharmacokinetic approaches results in different dosing recommendations and ostensibly different outcomes. TDM approaches for intravenous busulfan dose individualization employ compartmental or non-compartmental modeling with anywhere from three to seven drug levels. This investigation was designed to examine the differences in dosing recommendations that arise in children (n = 30) when five different TDM approaches were employed. Significant differences in recommended doses between modeling strategies were observed. More importantly, the recommendations were discordant in 13 cases with at least one model recommending a dose adjustment in the opposite direction relative to the remaining models. The mathematical differences introduced by the application of different TDM approaches are not purely academic. Unification of busulfan TDM approaches should be considered to mitigate inconsistently applied dose adjustment, and facilitate comparisons of outcome, between clinical centers., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

A prodrug treosulfan (TREO) is being evaluated in clinical trials as a myeloablative agent before hematopoietic stem cell transplantation. The active derivatives of TREO, monoepoxide (EBDM), and diepoxide (DEB) are formed in a pH-dependent nonenzymatic reaction. The aim of the study was to investigate pharmacokinetics of the TREO epoxy transformers in a rabbit model and explain the causes of low plasma concentrations of EBDM and DEB observed in patients receiving high-dose TREO before hematopoietic stem cell transplantation. New Zealand white rabbits (n = 5 per cohort) received an intravenous infusion of TREO (group I), injection of DEB (group II), and injection of a solution containing EBDM (group III). When EBDM and DEB were administered to the rabbits, they underwent a very rapid elimination (half-life 0.069 and 0.046 h) associated with a high systemic clearance (10.0 and 14.0 L h(-1) kg(-1)). After administration of TREO, the t1/2 of EBDM was statistically equal to the t1/2 of the prodrug (1.6 h). To conclude, after administration of TREO, its epoxy transformers demonstrate a formation-limited elimination. Then EBDM and DEB have the same elimination half-life as TREO, but the levels of EBDM and DEB in the body, including plasma, are much lower than TREO on account of their inherently high clearance., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Treosulfan (l-threitol-1,4-di-methanesulfonate) is a prodrug of a bifunctional alkylating agent that is being used increasingly in pediatric bone marrow transplantation regimens. The activation pathway is a complex reaction, which consists of two consecutive reactions leading to epoxybutane derivatives which are responsible for DNA alkylation. A simple, sensitive high performance liquid chromatography method for the determination of the sum of treosulfan and its epoxy metabolites by UV detection after derivatization with sodium diethyldithiocarbamate in human plasma was developed and validated. Plasma samples containing treosulfan and epoxy metabolites were converted into thiocarbamate derivative with 10% sodium diethyldithiocarbamate. Dinitrobiphenyl was used as an internal standard. The analysis was carried out using a reversed phase C18 column with a mobile phase consisting of methanol-water (65:35, v/v) at a flow rate of 1 mL/min. The eluent was monitored at 254 nm. The standard calibration curve was established between 2.5 and 50 µg/mL, with a correlation coefficient of 0.9987. Intra- and interday precision and accuracy of the method was <8% and met the analytical criteria. Pharmacokinetic parameters were determined in six children who received intravenous treosulfan (dose range 12-24 g/m(2)) in combination with fludarabine prior to blood or marrow transplantation., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Hematopoietic stem-cell transplantation (HSCT) is currently the only curative therapeutic option for the treatment of thalassemia. In spite of the high cure rate, HSCT can lead to life-threatening adverse events in some patients. Busulfan (Bu) is a key component of the conditioning regimen prior to HSCT. Inter-individual differences in Bu pharmacokinetics (PK) are hypothesized to influence Bu efficacy and toxicity. Since Bu is mainly metabolized by glutathione S-transferase (GST), we investigated the relationship of GSTA1 and GSTM1 genotypes with first-dose PK and HSCT outcomes in 44 children with thalassemia intermedia and thalassemia major. All children received a myeloablative conditioning regimen with IV Bu. Association analysis revealed a relationship between GSTA169C>T (or haplotype *A/*B) and first Bu dose PK that was dependent on sex and Pesaro risk classification (PRC). Among female patients and patients with PRC I-II, homozygous individuals for the GSTA1T-69 allele defining haplotype *B, had higher Bu exposure and lower clearance (P⩽0.01). Association with HSCT outcomes showed that patients with the GSTM1 null genotypes had higher occurrence of regimen-related toxicity (P=0.01). These results suggest that GST genotypes could be useful to tailor the first Bu dose accordingly to improve HSCT outcome.

A simple, rapid, specific and precise liquid chromatography-tandem mass spectrophotometric (LC-MS/MS) method was developed and validated for quantification of busulfan, in human plasma. busulfan d8 was used as internal standard, added to plasma sample prior to extraction using acetonitrile as a precipitating agent. Chromatographic separation was achieved on phenomenex kinetex C18 column (50mm×2.1mm, 2.6μm) with acteonitrile: 10mM ammonium formate buffer (80:20v/v) as an isocratic mobile phase with a flow rate of 0.5mLmin(-1). Quantitation was performed by transition of 264.1→151.1 (m/z) for busulfan and 272.1→159.1 (m/z) for busulfan d8. The lower limit of quantitation was 0.2ngmL(-1) with a 100μL plasma sample. The concentrations of nine working standards showed linearity between 0.2 and 100ngmL(-1) (r(2)≥0.9986). Chromatographic separation was achieved within 2.0min. The average extraction recoveries of 3quality control concentrations were 92.52% for busulfan and 90.75% for busulfan d8. The coefficient of variation was ≤15% for intra- and inter-batch assays. The developed method was successfully applied for the determination of Busulfan pharmacokinetics after oral administration., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Busulfan (Bu)/cyclophosphamide (Cy) is a standard conditioning platform for allogeneic transplantation. We developed a strategy separating the Cy into two pre/post-transplantation doses (PTCy), providing myeloablative conditioning and single-agent graft-versus-host disease (GVHD) prophylaxis. We investigated the impact of Bu route on treatment-related toxicity for 131 consecutive adult patients. Busulfan was administered in four daily divided doses either orally (n = 72) or intravenously (n = 59) with pharmacokinetics on the first-dose and as necessary on subsequent doses to achieve a target area-under-the-concentration-curve (AUC) of 800-1400 μmol*min/L per dose. BuCy/PTCy with pharmacokinetics is well-tolerated with low treatment-related toxicity. Hepatic veno-occlusive disease incidence was 6% with two fatal events. Bu administration route in the context of BuCy/PTCy did not statistically impact hepatotoxicity, GVHD, relapse, disease-free survival, or overall survival. The BuCy/PTCy platform has a low incidence of treatment-related toxicity, including hepatotoxicity, in hematologic malignancies when using pharmacokinetics for a target AUC of 800-1400 μmol*min/L, irrespective of Bu administration route.

Background: In hematopoietic stem cell transplantation (HSCT), patients receive individualized treatment planning in conditioning regimens to prevent unwarranted toxicities while maximizing desired outcomes. The dose of a widely studied agent in this setting, busulfan, can be adjusted based on area under the curve (AUC); however, choice of actual body weight (ABW) versus adjusted body weight (DBW) weight to calculate the initial dose may be critical in attaining goal AUC., Objective: To determine which weight best correlates with achievement of goal AUC for patients receiving busulfan conditioning for HSCT. Secondary objectives include evaluation of AUC results with clinical outcomes such as toxicity and survival., Methods: An institutional review board-approved retrospective analysis was performed on 31 allogeneic HSCT recipients who received intravenous busulfan (Q6H with cyclophosphamide [Bu/Cy] or once daily with fludarabine [Flu/Bu])., Results: Eighteen patients received Flu/Bu (50% ABW, 50% DBW) and 13 received Bu/Cy (23% ABW, 77% DBW). Overall, patients dosed by DBW were more likely to undershoot goal AUC (-12.8% vs. +19.5%, p = 0.018) and require dose increases (+20% vs. -19.9%, p = 0.012) versus those dosed by ABW. Subgroup analysis confirmed these results for Bu/Cy (-23.6% vs. +2.2%, p < 0.001 for goal AUC; +36.2% vs. -4.5%, p = 0.008 for busulfan dose increase), but not Flu/Bu (-0.8% vs. +25.3%, p = 0.123 for goal AUC; +3.4% vs. -25.1%, p = 0.174 for busulfan dose increase). Time to engraftment, progression-free survival, and overall survival were not different between dosing groups (p > 0.05). No patient experienced busulfan-related toxicity., Conclusions: Further prospective studies are warranted to elucidate which weight is most likely to achieve goal AUC and subsequent optimal patient outcomes., (© The Author(s) 2014.)

Intensive chemotherapy or chemotherapy plus irradiation and allogeneic stem cell transplantation can be curative for patients with hematologic diseases. Reduced-intensity transplants can also achieve cure and result in less treatment-related mortality but higher relapse rates. Thus, optimizing the conditioning regimens used in allogeneic transplantation remains an important goal. We conducted a phase I/II trial to determine the maximum tolerated dose (MTD) and dose-limiting toxicities (DLTs) of a continuous infusion of busulfan over 90 hours in conjunction with fludarabine followed by allogeneic related or unrelated donor transplant. Fifty-four patients with advanced hematologic malignancies were enrolled on this study. The MTD was identified as a 24-hour area under the curve (AUC) of approximately 7095 μM/min, which represents a 43% increase over the standard total daily AUC dose of 4800 μM/min given by intermittent schedules. DLTs at doses over 8000 μM/min were identified by a desquamative skin rash and mucositis. No dose-related increase in hepatic, pulmonary, or other organ toxicities were seen, whereas efficacy appeared to be improved at higher dose levels. Continuous-infusion busulfan with intermittent fludarabine provides an alternative treatment strategy that is generally well tolerated and permits an increase in total busulfan dose with encouraging efficacy. (NCI study no. NCT00448357.)., (Copyright © 2015 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Optimizing systemic busulfan exposure, the area under the concentration-time curve (AUC), improves the outcomes for hematopoietic stem cell transplantation (HSCT). The AUC is conventionally calculated using six plasma concentrations (AUC(0-∞)) drawn after the first of 16 intravenous busulfan doses given as a 2-h infusion every 6 h. The aim of the present study was to develop limited sampling strategies using three or fewer busulfan concentrations to reliably calculate AUC(0-∞) in patients undergoing HSCT. We investigated the pharmacokinetics of busulfan 46 times in 29 pediatric patients receiving intravenous busulfan. Limited sampling strategies using one, two, or three plasma busulfan concentrations were developed by multiple linear regression that showed excellent agreement with AUC(0-∞). In single-point sampling strategies, the AUC(0-∞) predicted based on C(6) (trough level: busulfan plasma concentration 6 h after the start of the infusion) was significantly correlated with, and not statistically different from, actual values as follows: AUC(0-∞) = 2556.5 C6 + 320.9 (r(2) = 0.929, P < 0.0001, mean bias 0.282 %, precision 7.91 %). In contrast, the predicted AUCs derived from the other sampling single points did not meet the criteria. The trough level well correlated with actual AUC(0-∞), suggesting that this time-point is acceptable for busulfan monitoring.

Aim: The aim of this investigation was to develop a model-based dosing algorithm for busulfan and identify an optimal sampling scheme for use in routine clinical practice., Methods: Clinical data from an ongoing study (n = 29) in stem cell transplantation patients were used for the purposes our analysis. A one compartment model was selected as basis for sampling optimization and subsequent evaluation of a suitable dosing algorithm. Internal and external model validation procedures were performed prior to the optimization steps using ED-optimality criteria. Using systemic exposure as parameter of interest, dosing algorithms were considered for individual patients with the scope of minimizing the deviation from target range as determined by AUC(0,6 h)., Results: Busulfan exposure after oral administration was best predicted after the inclusion of adjusted ideal body weight and alanine transferase as covariates on clearance. Population parameter estimates were 3.98 h(-1), 48.8 l and 12.3 l h(-1) for the absorption rate constant, volume of distribution and oral clearance, respectively. Inter-occasion variability was used to describe the differences between test dose and treatment. Based on simulation scenarios, a dosing algorithm was identified, which ensures target exposure values are attained after a test dose. Moreover, our findings show that a sparse sampling scheme with five samples per patient is sufficient to characterize the pharmacokinetics of busulfan in individual patients., Conclusion: The use of the proposed dosing algorithm in conjunction with a sparse sampling scheme may contribute to considerable improvement in the safety and efficacy profile of patients undergoing treatment for stem cell transplantation., (© 2015 The British Pharmacological Society.)

Achievement of a busulfan area-under-the-concentration versus time curve (AUC) of 900 to 1500 μM·min is associated with improved hematopoietic stem cell transplant (HSCT) outcomes. Multiple pediatric busulfan dosing guidelines aim to achieve this target. The authors' objective was to describe the AUCs achieved after simulated dosing using available pediatric i.v. busulfan dosing guidelines. The health records of children who received i.v. busulfan for HSCT conditioning at The Hospital for Sick Children were reviewed. Busulfan AUCs were calculated for each patient based on plasma busulfan concentrations using either a 1-compartment model or a validated limited-sampling strategy. Published pediatric busulfan dosing guidelines were identified. Initial busulfan doses were determined for all patients using each dosing guideline and total body weight (TBW). For overweight patients (TBW-to-ideal body weight [IBW] ≥ 1.25), initial busulfan doses were also determined using IBW and adjusted IBW (IBWadj). The resulting AUCs were simulated. The proportion of subjects (TBW/IBW < 1.25, TBW/IBW ≥ 1.25, and infants) with an AUC within target (900 to 1500 μM·min) after dosing simulation with each guideline was compared. One hundred eleven children (mean age, 6.2 years [SD, ±5.2]) who received i.v. busulfan were included. When dosing with each of the 12 i.v. busulfan dosing guidelines identified was simulated using TBW in 97 non-overweight patients, the proportion of patients with an AUC within the target range varied from 51% to 74% and from 45% to 64% in infants. Use of IBW or IBWadj to calculate initial busulfan doses in overweight children improved the performance of most guidelines. Current busulfan dosing guidelines vary in their ability to achieve AUCs within the target range. For children who are not overweight, we recommend 1 of 3 high-performing guidelines that allow individualization of the target busulfan AUC. Use of either IBW or IBWadj in overweight children improves the performance of most guidelines. Regardless of the guideline used, therapeutic drug monitoring is essential to verify achievement of the target AUC., (Copyright © 2015 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Background: Busulfan (Bu) and cyclophosphamide (Cy) are frequently included in conditioning regimens before hematopoietic stem cell transplantation (HSCT). Both drugs are detoxified by glutathione transferases (GST), and GST gene variants may explain some of the interindividual variability in pharmacokinetics and drug toxicity., Methods: The study investigated adult patients (n = 114) receiving oral Bu pre-HSCT. Bu doses were adjusted to obtain an average steady-state concentration (Css) of 900 mcg/L., Results: Median first dose Bu Css was 1000 mcg/L (600-1780 mcg/L). Patients carrying 1 and 2 GSTA1*B (rs3957357) alleles demonstrated median 12% and 16% higher Bu Css (P ≤ 0.05). Bu exposure (average Css; odds ratio = 1.009, 95% confidence interval = 1.002-1.017, P = 0.013) and GSTM1 gene copy number (odds ratio = 17.1, 95% confidence interval = 1.46-201, P = 0.024) were significant predictors of mortality ≤30 days. The mortality was 25% versus 2% among carriers of 2 versus no GSTM1 copies (P = 0.021). Mortality ≤3 months was associated with higher first dose Bu exposure (1090 versus 980 mcg/L, P = 0.021). GSTM1 expression and high Bu exposure may increase Cy toxicity by reducing intracellular glutathione., Conclusions: GST genotyping before HSCT may allow better prediction of Bu pharmacokinetics and drug toxicity, and thereby improve outcome after BuCy conditioning.

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.

Background: Busulfan and etoposide have been used as myeloablative therapy for autologous hematopoietic stem cell transplantation (HSCT) in adults with acute myeloid leukemia (AML) for > 20 years. The use of targeted intravenous (I.V.) busulfan has significantly improved the tolerability and efficacy of this regimen. We designed a dose-escalation study to examine the maximum tolerated dose (MTD) of targeted I.V. busulfan with bolus etoposide as preparative therapy for autologous HSCT in AML., Patients and Methods: In this single-center, phase I study, adult AML patients received I.V. busulfan targeted to either an area under the curve (AUC) of 1250 (cohort 1) or 1400 (cohort 2) μmol/min over 16 doses. Dose adjustments based on plasma pharmacokinetics occurred before doses 2 and 11. Etoposide 60 mg/kg I.V. was administered 24 hours after the last busulfan dose and 3 days before stem cell infusion., Results: Twelve patients with intermediate-risk AML in first complete remission were treated. All patients in cohort 1 and 5 patients (83%) in cohort 2 were within 10% of the target AUC. The MTD was not reached, although Grade ≥ 3 mucositis occurred in 3 patients (50%) in cohort 1 and in 4 patients (66%) in cohort 2, limiting further dose escalation. Two-year relapse-free survival was 33% in cohort 1 versus 67% in cohort 2 (P = .08)., Conclusion: Etoposide and targeted, dose-escalated I.V. busulfan as myeloablative therapy for autologous HSCT in AML is safe, with mucositis being the most significant toxicity. A phase II study is warranted to further evaluate the activity and safety of busulfan targeted to AUC 1400 μmol/min., (Published by Elsevier Inc.)

Background: Mortality associated with allogeneic hematopoietic cell transplantation (allo-HCT) has limited its broader application in patients with non-Hodgkin lymphoma (NHL). Pharmacokinetic treatment with targeted intravenous busulfan combined with fludarabine (BuFlu) was developed as a preparative regimen for acute leukemia and myelodysplasia. Data from this regimen in lymphoid malignancies are limited., Patients and Methods: We assessed outcomes in 60 consecutive patients with various subtypes of NHL and a median age of 54 years (range, 27-68 years) who received allo-HCT with targeted intravenous BuFlu between December 2004 and August 2010. The median number of previous therapies was 3 (range, 1-8) and median time from diagnosis to HCT was 32 months (range, 4.5-177.5 months)., Results: At conditioning, 28 (47%) patients had a complete response (CR). Graft versus host disease (GVHD) prophylaxis consisted of tacrolimus plus methotrexate in 65% of cases. Donors were matched/related (n = 32 [53%]), matched/unrelated (n = 21 [35%]), or mismatched/unrelated (n = 7 [12%]). All patients underwent grafting. The cumulative incidence of grade II/IV acute GVHD was 74% (grade III/IV was 20%). The 2-year cumulative incidence of moderate to severe chronic GVHD was 62%. Nonrelapse mortality (NRM) at 100 days and 3 years was 10% and 25%, respectively. The cumulative incidence of relapse was 27%. Three-year progression-free and overall survival for all patients was 47.8% and 55%, respectively., Conclusion: Targeted intravenous BuFlu is a relatively well tolerated regimen and offers an alternative option when myeloablation is deemed necessary in patients with NHL., (Copyright © 2015 Elsevier Inc. All rights reserved.)

I.v. BU has been proven to have better bioavailability, reliable systemic drug exposure with more predictable blood levels and lower toxicity than oral BU when used as part of conditioning regimens before hematopoietic SCT (HSCT). Some studies have shown that once-daily i.v. BU had the same clinical efficacy as i.v. BU administered four times daily. To observe the clinical efficacy and pharmacokinetics (PK) of once-daily i.v. BU and to evaluate the influence of glutathione S-transferase (GST) gene polymorphisms on once-daily i.v. BU PK in adult Chinese patients with allogeneic HSCT, we analyzed 25 patients receiving related or unrelated donor transplant conditioned with i.v. BU-based regimens. With a median follow-up of 32.7 months, the 2-year OS and EFS were 64 and 63.8% for all the patients, respectively, and the 2-year cumulative incidence of relapse for all patients was 18.3%. On the basis of HPLC analysis, the mean clearance and mean daily area under the curve (AUC) of i.v. BU were calculated as 4.02 mL/min per kg and 3380.77 μM/min, respectively. The estimated Cmax was 1.031±0.0325 μg/mL. The estimated t1/2 and Vd values were 3.618±0.1932 h and 1.212±0.0352 L/kg. The once-daily i.v. BU-based conditioning regimen was very well tolerated with minor toxicity in patients, most likely because of dose assurance with predictable PK. There was no GSTA1 *B/*B homozygous patient in our Chinese patients. A significant association between BU metabolism and GSTA1 polymorphism was observed. The GSTA1 *A/*B genotype group showed a significantly higher AUC (P<0.0001), higher Cmax (P=0.0003) and lower clearance (P=0.0007) than the GSTA1 *A/*A genotype group. AUC was lower in GSTP1 *A/*A genotypes compared with*A/*G (P=0.0283) and *G/*G genotypes (P=0.0111). The BU clearance in GSTP1 *A/*A genotype was shown to be higher than *A/*G (P=0.0255) and *G/*G genotypes (P=0.0111). In addition, the differences of PK in BU among different ethnic groups existed because of the different distribution frequencies of GST gene polymorphism in Chinese patients and Caucasian patients.

Background: Busulfan (Bu) is an integral part of conditioning regimens for patients with sickle cell anemia (SCA) undergoing transplantation. Patients with SCA might predispose to transplant-related neurological and pulmonary toxicities due to pre-existing disease-related cerebrovascular and lung injury. Bu therapy appears to be an important contributing factor in this context., Procedure: We studied the pharmacokinetics of intravenous Bu and clinical outcomes of 36 children with SCA undergoing bone marrow transplantation. Most patients had pre-existing organ system damage. Busulfan was administered every 6 hr for 4 days with pharmacokinetic-guided dose adjustment to target a conservative area under the concentration versus time curve (AUC) range of 900-1,350 µMol*min., Results: We found that the first-dose Bu clearance was significantly higher (P < 0.0005) than the subsequent daily clearance, which remained unchanged during the following days. After the first-dose, 69% of patients achieved the target range. We adapted a new dose-adjustment strategy targeting exposures to the lower end (900 µMol*min) of the AUC range after the first dose of Bu to avoid unnecessary dose increases on subsequent days due to differences in clearance. This strategy enabled most patients to maintain the AUC within therapeutic range following dose adjustments., Conclusions: Differences in Bu clearance after the first-dose and subsequent daily doses in patients with SCA should be considered for pharmacokinetic-guided dose adjustment. Conservative AUC range and targeting exposures to the lower end of the range after the first dose was associated with negligible toxicity, and high engraftment and sickle cell-free survival rates., (© 2014 Wiley Periodicals, Inc.)

Background: Population pharmacokinetic (PK) studies of busulfan in children have shown that individualized model-based algorithms provide improved targeted busulfan therapy when compared with conventional dose guidelines. The adoption of population PK models into routine clinical practice has been hampered by the tendency of pharmacologists to develop complex models too impractical for clinicians to use. The authors aimed to develop a population PK model for busulfan in children that can reliably achieve therapeutic exposure (concentration at steady state) and implement a simple model-based tool for the initial dosing of busulfan in children undergoing hematopoietic cell transplantation., Patients and Methods: Model development was conducted using retrospective data available in 90 pediatric and young adult patients who had undergone hematopoietic cell transplantation with busulfan conditioning. Busulfan drug levels and potential covariates influencing drug exposure were analyzed using the nonlinear mixed effects modeling software, NONMEM. The final population PK model was implemented into a clinician-friendly Microsoft Excel-based tool and used to recommend initial doses of busulfan in a group of 21 pediatric patients prospectively dosed based on the population PK model., Results: Modeling of busulfan time-concentration data indicates that busulfan clearance displays nonlinearity in children, decreasing up to approximately 20% between the concentrations of 250-2000 ng/mL. Important patient-specific covariates found to significantly impact busulfan clearance were actual body weight and age. The percentage of individuals achieving a therapeutic concentration at steady state was significantly higher in subjects receiving initial doses based on the population PK model (81%) than in historical controls dosed on conventional guidelines (52%) (P = 0.02)., Conclusions: When compared with the conventional dosing guidelines, the model-based algorithm demonstrates significant improvement for providing targeted busulfan therapy in children and young adults.

Purpose: Busulfan (Bu) exposure is critical for efficacy and safety. Body weight (BW), or adjusted ideal body weight (AIBW)-based dosing (WBD) algorithm, has been used in hematopoietic stem cell transplantation (HSCT). A recently completed phase 2 study revealed that 33.6 % of the subjects were under-, or over-exposed, with this WBD algorithm. This paper was to investigate Bu dosing algorithm in an attempt to improve the suboptimal Bu exposure., Methods: Population PK modeling was conducted using the data from 207 patients. Dosing algorithm was developed based on derived covariate model of CL. Model-based simulation was conducted to assist test PK study design. A simplified CL estimation method was proposed based on the PK structure model for Bu., Results: A one-compartment structure model adequately described the PK profile of Bu following an IV infusion. BSA best described the inter-individual variability of CL. The proposed dosing algorithm was dose (mg) = (31.7 × BSA - 11.6) × target AUC [µM min]/1,000. With this dosing algorithm, 14.3 % patients could be under- or over-exposed. A test PK study with reduced study duration and three PK samples can provide as nearly as good an estimate of CL compared to 12 PK samples on two different occasions., Conclusion: The proposed dosing algorithm can significantly improve the sub-exposure of Bu. A shortened test PK study duration with reduced PK samples can provide as near as good estimate for Bu CL. A simplified CL estimation method is valid.

Pro-drug treosulfan (TREO) is currently evaluated in randomized phase III clinical trials as a conditioning agent prior to HSCT. In the present paper pharmacokinetics of both TREO and its biologically active monoepoxide (S,S-EBDM) was investigated in pediatric patients for the first time. The studies were carried out in 16 children (median age 7.5 years) undergoing TREO-based preparative regimen prior to HSCT, who received 10, 12 or 14 g/m(2) of the drug as a 1h or 2h intravenous infusion. Plasma concentrations of TREO as well as S,S-EBDM were determined using the validated HPLC-MS/MS method. The changes in S,S-EBDM concentration over time followed TREO levels. The area under the curve (AUC) of TREO was 100-fold higher than AUC of S,S-EBDM. No statistically significant dependency of the dose-normalized AUC of either TREO or S,S-EBDM on the patients' age and body surface area was stated. Moreover, plasma C(max) as well as AUC of S,S-EBDM demonstrated linear correlation with the C(max) and AUC of TREO, respectively. The biological half-lives of TREO and S,S-EBDM were similar. This indicates that S,S-EBDM was completely eliminated from the patients' blood within relatively short time, comparable to TREO., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Busulfan is an alkylating agent used for conditioning patients undergoing hematopoietic stem cell transplantation with a narrow therapeutic range and highly variable pharmacokinetics. High concentrations induce toxicity, especially hepatic veno-occlusive disease, also referred to as sinusoidal obstruction syndrome. This study aimed to assess busulfan pharmacokinetic variability in pretransplant conditioning regimens using an analytical method validated by high-performance liquid chromatography coupled to diode array detector (HPLC/PDA)., Methods: Eight patients who used the test dose (TD) of 1 mg/kg busulfan 10 days before conditioning were included, and 10 serial blood samples were collected to determine: the elimination half-life (t1/2), total area under the curve (AUCT), total clearance (Cl(T)/F), and plasma concentration at steady state (C(ss)), using a monocompartmental model and first-order kinetics. The instrumental conditions were: HPLC/PDA Shimadzu, column ACE C18 (150 mm × 4 mm); methanol/water/acetonitrile (65:20:15) eluent flow rate of 1 mL/min; 1,6-bis-(methanesulfonyloxy)-hexane; UV λ = 276 nm; analysis time 17 minutes; and derivatization with sodium diethylcarbamate. The dose was adjusted, and 4 blood samples per day were collected at days 2, 3, and 4 of treatment for new plasma determinations., Results: Four patients needed higher doses; the mean dose administered was 1.02 ± 0.19 mg/kg. Mean results at TD: t1/2 = 2.88 ± 0.5 hours; Cl(T)/F = 0.18 ± 0.03 L · h(-1) · kg(-1); AUC(T) = 5461.00 ± 961.15 ng · mL(-1) · h(-1); and C(ss) = 911.3 ± 159.8 ng/mL. Mean results of samples collected during conditioning: t1/2 = 3.21 ± 0.9 hours; Cl(T)/F = 0.13 ± 0.02 L · h(-1) · kg(-1); AUC(T) = 7571 ± 1705 ng · mL(-1) · h(-1); and C(ss) = 1262.0 ± 284.3 ng/mL., Conclusions: High variability in the assessed pharmacokinetic parameters was observed, with a 38% variation in C(ss) between TD and conditioning regimen; Cl(T)/F decreased by 30%, suggesting drug accumulation after multiple-dose regimen. Although being lower than reported in the literature, this variation may be associated with toxicity of the proposed treatment, justifying patient monitoring and enhancing validity of previous pharmacokinetic evaluation using TD regimen.

Aim: A population pharmacokinetic (PPK) analysis was conducted to describe the influence of GSTA1 polymorphisms on intravenous busulfan in adults undergoing allogeneic hematopoietic stem cell transplantation., Patients & Methods: A PPK model was developed from 36 patients by a one-compartment model with first-order elimination., Results: The typical value of clearance and volume of distribution were 11.0 l/h and 42.4 l, respectively. Clearance decreased by 15% and area under the concentration-time curves (AUCs) increased with GSTA1 variants compared with wild-type (both p < 0.05). Subtherapeutic AUCs were seen only in wild-type patients., Conclusion: To our knowledge, this is the first PPK study to suggest that GSTA1 polymorphisms in adults are associated with busulfan PK.

Patients with multiple myeloma (MM) who relapse after autologous transplantation have limited therapeutic options. We conducted a prospective, multicenter, phase IIa study to investigate the safety and efficacy of i.v. busulfan (Bu) in combination with bortezomib as a conditioning regimen for a second autotransplantation. Because a safe Bu exposure was unknown in patients receiving this combination, Bu was initially targeted to a total area under the concentration-time curve (AUC) of 20,000 μM × minute. As no concentration-limiting toxicity was observed in 6 patients, this Bu exposure was utilized in the following treatment cohort (n = 24). Individualized Bu dose, based on test dose .8 mg/kg pharmacokinetics (PK), was administered daily for 4 consecutive days starting 5 days before transplantation, followed by a single dose of bortezomib (1.3 mg/m(2)) 1 day before transplantation. The total mean dose of i.v. Bu (including the test dose and 4-day administration) was 14.2 mg/kg (standard deviation = 2.48; range, 8.7 to 19.2). Confirmatory PK demonstrated that only 2 of 30 patients who underwent transplantation were dosed outside the Bu AUC target and dose adjustments were made for the last 2 doses of i.v. Bu. The median age was 59 years (range, 48 to 73). Median time from first to second transplantation was 28.0 months (range, 12 to 119). Of 26 evaluable patients, 10 patients attained a partial response (PR) or better at 3 months after transplantation, with 2 patients attaining a complete response. At 6 months after transplantation, 5 of 12 evaluable patients had maintained or improved their disease status. Median progression-free survival was 191 days, whereas median overall survival was not reached during the study period. The most common grade 3 and 4 toxicities were febrile neutropenia (50.0%) and stomatitis (43.3%). One transplantation-related death was observed. A combination of dose-targeted i.v. Bu and bortezomib induced PR or better in one third of patients with MM who underwent a second autotransplantation, with acceptable toxicity., (Published by Elsevier Inc.)

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.

Purpose: Older patients with acute myeloid leukemia (AML) and myelodysplastic syndrome have often been excluded from myeloablative-conditioning regimens containing busulfan because of non-disease-related morbidity and mortality. We hypothesized that busulfan clearance (BuCL) in older patients (>60 years) would be reduced compared to that in younger patients, potentially explaining observed differences in busulfan tolerability., Methods: AML patients in three CALGB hematopoietic cell transplantation studies were treated with a conditioning regimen using IV busulfan, dosed at 0.8 mg/kg. Plasma busulfan concentrations were determined by LC-MS and analyzed by non-compartmental methods. BuCL was normalized to actual (ABW), ideal (IBW), or corrected (CBW) body weight (kg). Differences in BuCL between age groups were examined using the Wilcoxon rank sum test., Results: One hundred and eighty-five patients were accrued; 174 provided useable pharmacokinetic data. Twenty-nine patients ≥ 60 years old (median 66; range 60-74) had a significantly higher BuCL versus those <60 years old (median 50; range 18-60): BuCL 236 versus 168 mL/min, p = 0.0002; BuCL/ABW 3.0 versus 2.1 mL/min/kg, p = 0.0001; BuCL/IBW 3.8 versus 2.6 mL/min/kg, p = 0.0035; BuCL/CBW 3.4 versus 2.6 mL/min/kg, p = 0.0005. Inter-patient variability in clearance (CV %) was up to 48 % in both age groups. Phenytoin administration, a potential confounder, did not affect BuCL, regardless of weight normalization (p > 0.34)., Conclusions: Contrary to our hypothesis, BuCL was significantly higher in older patients compared to younger patients in these studies and does not explain the previously reported increase in busulfan toxicity observed in older patients.

Busulfan- and treosulfan-based conditionings are the cornerstone of pediatric allogeneic hematopoietic stem cell transplantation (HSCT). Although both drugs are alkylating agents, their mechanisms of action, pharmacokinetics (PK) and toxicity profiles are different. Experience with busulfan in pediatric HSCT is broad and the knowledge on the pharmacodynamics (PD), PK and, to a lesser extent, pharmacogenetics (PG) has resulted in a more effective therapy. Treosulfan has only recently been introduced in pediatric HSCT and is considered a promising new therapy because of its beneficial toxicity profile. However, knowledge of the PK and PG of treosulfan is limited. In this review, we describe the pharmacology of both agents and discuss factors causing variability in PK in relation to therapeutic outcome in HSCT., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

For the first time a high performance liquid chromatography method with tandem mass spectrometry detection (HPLC-MS/MS) was developed for simultaneous determination of a pro-drug treosulfan (TREO) and its active monoepoxide (S,S-EBDM) in biological matrices. Small volumes of rat plasma (50 μL) and the brain homogenate supernatant (100 μL), equivalent to 0.02 g of brain tissue, were required for the analysis. Protein-free TREO, S,S-EBDM and acetaminophen, internal standard (IS), were isolated from the samples by ultrafiltration. Complete resolution of the analytes and the IS was accomplished on Zorbax Eclipse column using an isocratic elution with a mobile phase composed of ammonium formate - formic acid buffer pH 4.0 and acetonitrile. Detection was performed on a triple-quadrupole MS via multiple-reaction-monitoring following electrospray ionization. The developed method was fully validated according to the current guidelines of the European Medicines Agency. Calibration curves were linear in ranges: TREO 0.2-5720 μM and S,S-EBDM 0.9-175 μM for plasma, and TREO 0.2-29 μM and S,S-EBDM 0.4-44 μM for the brain homogenate supernatant. The limits of quantitation of TREO and S,S-EBDM in the studied matrices were much lower in comparison to the previously used bioanalytical methods. The HPLC-MS/MS method was adequately precise (coefficient of variation≤12.2%), accurate (relative error≤8.6%), and provided no carry-over, acceptable matrix effect as well as dilution integrity. The analytes were stable in acidified plasma and the brain homogenate supernatant samples for 4 h at room temperature, for 4 months at-80°C as well as within two cycles of freezing and thawing, and demonstrated 18-24h autosampler stability. The validated method enabled determination of low concentrations of TREO and S,S-EBDM in incurred brain samples of the rats treated with TREO, which constitutes a novel bioanalytical application., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: High-dose treosulfan is used in conditioning regimens before hematopoietic stem cell transplantation in children. Pharmacokinetic data to optimize treosulfan dosing are scarce in this patient population. The aims of this study were the development and validation of an analytical method for treosulfan in human serum and the development of a pharmacokinetic model for treosulfan in pediatric patients. Furthermore, we aimed to develop a limited sampling strategy to estimate treosulfan systemic exposure with a minimum of inconvenience and risk for the patient., Methods: A reversed phase high-performance liquid chromatography method using ultraviolet detection to determine treosulfan in human serum samples was developed and validated according to food and drug administration guidelines. Serum pharmacokinetics after the first treosulfan administration was investigated in 20 children using nonlinear mixed-effect modeling, and a limited sampling strategy was developed and validated., Results: The assay was validated in a 10-500 mg/L concentration range with a lower limit of quantification of 10 mg/L. Accuracies were within the 90%-110% limit. The coefficients of variation of the within-day imprecision and between-days imprecision were less than 5%. Pharmacokinetics was adequately described with a 1-compartment model. The population estimates for clearance (CL) and volume of distribution were 6.85 L/h and 13.2 L for a typical patient of 20 kg, respectively. Treosulfan exposure could be adequately quantified with 2 samples, at 4 and 7 hours after the start of a 3-hour treosulfan infusion, with a mean deviation of 3% of individual CL and area under the curve based on limited sampling in comparison with the full data set in a total cohort., Conclusions: In this study, a bioanalytical method, PK model, and limited sampling model were developed and validated. Furthermore, PK parameters of 20 pediatric patients were analyzed, demonstrating an interpatient variability in area under the curve of 14.5%. This study demonstrates the essential developments in the optimization of treosulfan therapy based on PK data.

Purpose: We developed a new population pharmacokinetic (PopPK) model for intravenous (i.v.) busulfan in children to evaluate the optimal method to personalize its dosing without concentration-time data., Methods: PopPK analyses were done with NONMEM® 7.2. First, a model from Trame et al. was evaluated using an external dataset consisting of 24 children. Second, a revised model was built in a separate dataset of 82 children. Model evaluation was performed by using a standardized visual predictive check (SVPC) procedure and a bootstrap analysis (internal evaluation) and by comparison to an external dataset (external validation)., Results: The final model included body surface area (BSA) as an exponential function on volume of distribution (V) and actual body weight (ABW) as an allometric function on clearance (CL). The dosing nomogram for every 6 h administration derived from the final model is: dose[mg]=target AUC[mg×h/L]×3.04L/h×(ABW/16.1)0.797. Compared to other dosing strategies, differences were observed for the very small and obese patients., Conclusions: We revised our prior dosing nomogram after validation in a separate cohort of children. This dosing nomogram can be used to personalize i.v. busulfan doses without concentration-time data, but an additional prospective evaluation in the very small and obese children is needed.

Background: Series of observations indicate PK/PD variability challenging the accuracy of the body-weight based busulfan (Bu) dosing schedule for (HSCT) conditioning therapy. The purpose of this communication is to describe the frequency of dose changes in initially body-weight-based fixed IV Bu dose and to emphasize the importance of TDM., Material and Methods: Sixty-two children (ages 2 months-18 years) were treated with IV busulfan doses based on body weight for myeloablation. TDM utilizing a limited sample strategy (trough concentration immediately before the 5th dose, followed by samples immediately after the end of the 2-h infusion peak, 4 h, and 6 h from initiation of the infusion) was performed in 46 of 62 subjects. Busulfan concentrations were determined by high-performance liquid chromatography (HPLC). AUC was calculated according to the trapezoidal rule., Results: We observed trough levels of 25-1244 µg/L, peak levels of 849-4586 µg/L, and AUC of 2225-12818 µg/L·h following body weight-based high-dose busulfan. The doses were changed in 54% of cases. AUC in 5 of 9 patients with VOD were within target, in 3 patients AUS was higher, and in 1 patient AUC was lower. One of the 2 patients with neurotoxicity had higher AUC. Engraftment was 100%, but relapse occurred in 25% of cases., Conclusions: Our results demonstrate that even with IV busulfan, intra-individual PK/PD variability is challenging. Although AUC does not necessarily correspond with outcomes (due to the role of other factors the fact that doses were changed in 54% of cases underlines the importance of TDM.

The aim of this study was to prospectively evaluate the PK and safety of ivBU in 25 Japanese children (median age six yr; range, five months-17 yr) as one of a combination of drugs in a pretransplant regimen. The patients had acute leukemia (n = 14), CML (2), JMML (5), solid tumors (2), chronic granulomatous disease (1), or metachromatic leukodystrophy (1). Five different dose schedules were used according to the patient's ABW: <9 kg (1.0 mg/kg), 9 to <16 (1.2 mg/kg), 16-23 (1.1 mg/kg), >23-34 (0.95 mg/kg), and >34 kg of BW (0.8 mg/kg). Each dose was given over two h, and sample blood was drawn at nine or 11 separate points for analysis by gas chromatography-mass spectrometry. The AUC varied from 796 to 1905 μmol min/L, and 19 of the 25 patients (76%) remained within the target range without dose adjustment. Two were diagnosed with engraftment failure. Hepatic VOD developed in four, and only one of these showed high AUC (>1500 μmol min/L). Toxicities did not correlate with the BU level. Our data showed very similar PK to those in previous studies, and these dose schedules are applicable to Japanese children., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Previous studies have shown an equivalent pharmacokinetic profile between four-times-daily (4QD) and once-daily (QD) administration of intra-venous (IV) BU, without increased toxicity. We assess the impact of a switch in IV BU from a 4QD to a QD schedule, in terms of health-care organization, staff working conditions, quality of care dispensed and perceived patient comfort. Clinicians, nurses and pharmacists from nine allogeneic transplantation units in five European countries were interviewed face to face. Overall perception of QD versus 4QD BU was very positive. Both administration schemes were evaluated to be equally efficaciousZ. QD BU was perceived to be safer and more convenient. Clinicians and nurses perceived that patient comfort was improved, due to fewer complications associated with repeated infusions, and avoiding night infusions associated with stress, anxiety and decreased quality of sleep. Switching from 4QD to QD BU had a significant impact on health-care organization, with a better integration in the overall management and usual timelines in the pharmacies and transplantation units. Time spent to prepare and administer BU was significantly reduced, leading to potential financial savings that merit further assessment and would be of particular interest in the current economic climate.

A simplified gas chromatographic-mass spectrometric (GC-MS) analytical method, involving a novel derivatization procedure was developed for monitoring busulfan (Bu) plasma concentrations in populations undergoing bone marrow transplantation. Plasma samples (500 μL) containing Bu-d8 as internal standard were extracted with ethyl acetate (2 mL) followed by centrifugation (1800 rpm, 5 min) and evaporation of the organic layer under nitrogen flow (50 °C). The dry residue was reconstituted with 100 μL iodine solution in acetonitrile (0.25%, w/v) and 3 μL were injected into the GC-MS system at 250 °C. Conversion of Bu to 1,4-diiodobutane was accomplished on-line without the need of an extra derivatization step. MS was operated at selected ion monitoring mode at m/z 183 and 191 corresponding to Bu and Bu-d8 derivatives. Total analysis time was 11.5 min. Calibration curves were linear (mean r=0.9996) over a concentration range of 25-3651 ng/mL using a (1/x)-weighted scheme. Limit of detection and lower limit of quantitation were 10.6 and 25 ng/mL, respectively. Overall accuracy Er (%) was ranging from -5.10% to 10.5%. Within- and between-run RSD (%) were lower 4.51% and 2.15%, respectively. Overall recovery of Bu was equal to 69.3±4.56% (RSD (%)). The present method is sensitive and specific, requiring a simple sample preparation procedure and short analysis time, advantages crucial for therapeutic drug monitoring of Bu in clinical practice and application in pharmacokinetic studies., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

Purpose: Personalizing intravenous busulfan doses to a target plasma concentration at steady state (Css) is an essential component of hematopoietic cell transplantation (HCT). We sought to develop a population pharmacokinetic model to predict i.v. busulfan doses over a wide age spectrum (0.1-66 years) that accounts for differences in age and body size., Experimental Design: A population pharmacokinetic model based on normal fat mass and maturation based on postmenstrual age was built from 12,380 busulfan concentration time points obtained after i.v. busulfan administration in 1,610 HCT recipients. Subsequently, simulation results of the initial dose necessary to achieve a target Css with this model were compared with pediatric-only models., Results: A two-compartment model with first-order elimination best fit the data. The population busulfan clearance was 12.4 L/h for an adult male with 62 kg normal fat mass (equivalent to 70 kg total body weight). Busulfan clearance, scaled to body size-specifically normal fat mass, is predicted to be 95% of the adult clearance at 2.5 years postnatal age. With a target Css of 770 ng/mL, a higher proportion of initial doses achieved the therapeutic window with this age- and size--dependent model (72%) compared with dosing recommended by the U.S. Food and Drug Administration (57%) or the European Medicines Agency (70%)., Conclusion: This is the first population pharmacokinetic model developed to predict initial i.v. busulfan doses and personalize to a target Css over a wide age spectrum, ranging from infants to adults., (©2013 AACR.)