Outcomes from hematopoietic stem cell transplantation following treosulfan-based conditioning: A clinical and pharmacokinetic analysis.

BACKGROUND: The aims of this study are to report our experience with treosulfan-based conditioning regimens for patients with non-malignant hematologic conditions, correlating clinical outcomes at different time points post-transplant with treosulfan exposure (AUC). METHODS: This study was a single-center observational study investigating overall survival (OS), disease-free survival (DFS), and event-free survival (EFS) end-points post-transplant. The consequences of treosulfan AUC with respect to toxicity, correction of underlying disease, and long-term chimerism were also explored using pharmacokinetic analysis. RESULTS: Forty-six patients received 49 transplants with treosulfan and fludarabine-based conditioning between 2005 and 2023. Twenty-four patients also received thiotepa. Donor chimerism was assessed on either whole blood or sorted cell lines at different time points post-transplant. Thirty-nine patients received treosulfan pharmacokinetic assessment to evaluate cumulative AUC, with five infants receiving real-time assessment to facilitate daily dose adjustment. OS, DFS, and EFS were 87%, 81%, and 69%, respectively. Median follow-up was 32.1 months (range 0.82-160 months) following transplant. Lower EFS was associated with patient age (<1 year; p = .057) and lower cumulative treosulfan dose (<42 g/m2; p = .003). Stable donor chimerism in B-cell, NK-cell, and granulocyte lineages at 1-year post-transplant were more prevalent in patients receiving thiotepa conditioning. Two infants required daily dose adjustment to treosulfan to avoid high AUC. CONCLUSIONS: Excellent clinical outcomes and stable chimerism were observed in this patient series. The addition of thiotepa conferred no significant toxicity and trended toward sustained ongoing donor engraftment. Correlating treosulfan AUC with long-term patient outcomes is required.

Evaluation of treosulfan cumulative exposure in paediatric patients through population pharmacokinetics and dosing simulations.

AIM: To investigate the pharmacokinetics (PK) of intravenous treosulfan in paediatric patients undergoing haematopoietic stem cell transplantation (HSCT) for a broad range of diseases and to explore the impact of different dosing regimens on treosulfan exposure (area under the concentration-time curve, AUC0→∞ ) through dosing simulations. METHODS: A prospective multicentre PK study was conducted using treosulfan concentration data (n = 423) collected from 53 children (median age 3.5, range 0.2-17.0 years) receiving three daily age-guided doses (10-14 g/m2 ). Population PK modelling was performed using NONMEM software, utilising a stepwise forward selection backward elimination method and likelihood-ratio test for screening covariates to describe PK variability. Monte Carlo simulation was used to generate patient PK data for 10 000 virtual paediatric patients and cumulative AUC0→∞ values were evaluated using age, body surface area (BSA) and model-based dosing regimens, targeting 4800 mg*h/L. RESULTS: Treosulfan concentration data were described using a one-compartment PK model with first-order elimination. Population mean (95% CI) estimates for clearance (CL) and volume of distribution (V) were 16.3 (14.9-18.1) L/h and 41.9 (38.8-45.1) L, respectively. Allometrically scaled body weight was the best covariate descriptor for CL and V, and maturational age further explained variability in CL. Dosing simulations indicated that in young patient groups (<2 years), a model-based dosing regimen more accurately achieved the target AUC0→∞ (58.3%) over the age (42.6%) and BSA-based (51.3%) regimens. CONCLUSION: Treosulfan disposition was described through allometric body weight and maturational age descriptors. Model-informed dosing is recommended for patients under 2 years. Treosulfan PK parameters and AUC0→∞ were not influenced by patient disease.

Validation of a liquid chromatography-tandem mass spectrometry method for simultaneous quantification of N,N-dimethylacetamide and N-monomethylacetamide in pediatric plasma.

N,N-dimethylacetamide is an excipient used in intravenous busulfan formulations, a drug used in hematopoietic stem cell transplantation conditioning. The aim of this study was to develop and validate a liquid chromatography-tandem mass spectrometry method for simultaneous quantification of N,N-dimethylacetamide, and its metabolite N-monomethylacetamide in plasma from children receiving busulfan. A 4 µl aliquot of patient plasma was extracted using 196 µl 50% methanol solution and quantified against calibrators prepared in the extraction solvent given negligible matrix effects across three concentrations. 9 [H2 ]-N,N-dimethylacetamide was used as an internal standard. Separation of N,N-dimethylacetamide and N-monomethylacetamide was achieved using a Kinetex EVO C18 stationary phase (100 mm × 2.1 mm × 2.6 µm) running an isocratic mobile phase of 30% methanol containing 0.1% formic acid at a flow of 0.2 ml/min over 3.0 min. The injection volume was 1 µl. Calibration curves for N,N-dimethylacetamide and N-monomethylacetamide were linear up to 1200 and 200 µg/L, respectively, with a lower limit of quantification 1 µg/L for both analytes. Calibrator accuracy and precision were within ± 10% of the test parameters across four concentration levels. Analytes were stable over 14 days at three different storage conditions. This method was successfully applied to measure N,N-dimethylacetamide and N-monomethylacetamide concentrations in a total of 1265 plasma samples from 77 children.

Pre-existing antibodies against polyethylene glycol reduce asparaginase activities on first administration of pegylated E. coli asparaginase in children with acute lymphocytic leukemia.

Antibodies against polyethylene glycol (PEG) in healthy subjects raise concerns about the efficacy of pegylated drugs. We evaluated the prevalence of antibodies against PEG among patients with acute lymphoblastic leukemia (ALL) prior to and/or immediately after their first dose of pegylated E.coli asparaginase (PEG-ASNase). Serum samples of 701 children, 673 with primary ALL, 28 with relapsed ALL, and 188 adults with primary ALL were analyzed for anti-PEG IgG and IgM. Measurements in 58 healthy infants served as reference to define cut-points for antibody-positive and -negative samples. Anti-PEG antibodies were detected in ALL patients prior the first PEG-ASNase with a prevalence of 13.9% (anti-PEG IgG) and 29.1% (anti-PEG IgM). After administration of PEG-ASNase the prevalence of anti-PEG antibodies decreased to 4.2% for anti-PEG IgG and to 4.5% for anti-PEG IgM. Pre-existing anti-PEG antibodies did not inhibit PEG-ASNase activity but significantly reduced PEGASNase activity levels in a concentration dependent manner. Although pre-existing anti-PEG antibodies did not boost, pre-existing anti-PEG IgG were significantly associated with firstexposure hypersensitivity reactions (CTCAE grade 2) (p.

Challenges associated with test dose pharmacokinetic predictions of high dose melphalan exposure in patients with multiple myeloma.

AIM: To evaluate the accuracy of melphalan test dose pharmacokinetic (PK) predictions of the subsequent high dose (HDM) area under the concentration-versus-time curve (AUC) and to identify sources of prediction error (PE). METHODS: A prospective multicentre PK study was conducted in 40 myeloma patients of median age 60 (range:35-71) years using a 20 mg/m2 test dose administered 1-3 days prior to HDM (predominantly 180 mg/m2). PK data were collected post the test and high doses to compare predicted versus actual AUCs determined using the trapezoidal rule. Test and high dose infusion concentration, volume and duration and the time from preparation to infusion were compared using the paired Wilcoxin rank sign test. The impact of Melphalan administration parameters on PE was evaluated using the Mann-Whitney test. The predictive capacity of a previously published population PK (PopPK) model was also examined. RESULTS: Predicted HDM AUC was within 15% of the observed values in only 63% of patients when analysed using the trapezoidal rule and 70% of patients using PopPK. Test dose infusion concentration, volume, duration and time from preparation to infusion were significantly lower than for HDM (p < 0.005). Test dose administration within 15 min of reconstitution (n = 5) was associated with significantly lower PE than administration times of 16-60 min (n = 22), p < 0.05. Test and HDM infusion concentrations were lower in patients with large PE (> ± 15%), but the differences were not significant (p = 0.078, 0.228, respectively). CONCLUSION: Test dose PK has the potential to predict subsequent HDM exposure to achieve a target AUC once melphalan administration parameters are optimised to account for stability issues in the formulation.

Quantification of vincristine and tariquidar by liquid chromatography-tandem mass spectrometry in mouse whole blood using volumetric absorptive microsampling for pharmacokinetic applications.

A liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of vincristine and tariquidar in 10 µL of mouse whole blood using volumetric absorptive microsampling devices. Samples were extracted from the devices and quantified against calibrators prepared in a human blood plasma matrix. Separation of vincristine and tariquidar was achieved using a Shimpack XR ODS III C18 stationary phase and H2 O and methanol mobile phase solvents containing 0.1% formic acid, running a gradient elution at a flow rate of 0.2 mL/min over 6.0 min. The method was linear up to 1200 ng/mL (R2 > 0.99 for both analytes), with calibrator accuracy within ± 15% of the nominal concentrations and analyte coefficient of variance <15% for both vincristine and tariquidar. Pharmacokinetic assessment of both analytes was successfully applied in mice as both single-agent therapy and combination therapy over a 24-h period, and a 2.3-fold increase in vincristine drug exposure was observed in combination with tariquidar. This study validates the use of this approach for longitudinal analysis of drug exposure in animal studies.

Therapeutic Drug Monitoring of Asparaginase: Intra-individual Variability and Predictivity in Children With Acute Lymphoblastic Leukemia Treated With PEG-Asparaginase in the AIEOP-BFM Acute Lymphoblastic Leukemia 2009 Study.

BACKGROUND: Therapeutic drug monitoring (TDM) can identify patients with subtherapeutic asparaginase (ASNase) activity [silent inactivation (SI)] and prospectively guide therapeutic adaptation. However, limited intra-individual variability is a precondition for targeted dosing and the diagnosis of SI. METHODS: In the AIEOP-BFM acute lymphoblastic leukemia (ALL) 2009 trial, 2771 children with ALL were included and underwent ASNase-TDM in a central laboratory in Münster. Two biweekly administrations of pegylated ASNase during induction and a third dose during reinduction or the high-risk block, which was administered several weeks later, were monitored. We calculated (1) the incidence of SI; and (2) the predictivity of SI for SI after the subsequent administration. ASNase activities monitored during induction were categorized into percentiles at the respective sampling time points. These percentiles were used to calculate the intra-individual range of percentiles as a surrogate for intrapatient variability and to evaluate the predictivity of ASNase activity for the subsequent administration. RESULTS: The overall incidence of SI was low (4.9%). The positive predictive value of SI identified by one sample was ≤21%. Confirmation of SI by a second sample indicated a high positive predictive value of 100% for biweekly administrations, but not for administration more than 17 weeks later. Sampling and/or documentation errors were risks for misdiagnosis of SI. High intra-individual variability in ASNase activities, with ranges of percentiles over more than 2 quartiles and low predictivity, was observed in approximately 25% of the patients. These patients were likely to fail dose individualization based on TDM data. CONCLUSIONS: To use TDM as a basis for clinical decisions, standardized clinical procedures are required and high intra-individual variability should be taken into account. Details of the treatment are available in the European Clinical Trials Database at https://www.clinicaltrialsregister.eu/ctr-search/trial/2007-004270-43/DE.

The pharmacokinetics and pharmacodynamics of busulfan when combined with melphalan as conditioning in adult autologous stem cell transplant recipients.

Busulfan (Bu) is an alkylating agent widely used in conditioning regimes prior to stem cell transplantation (SCT), most commonly in combination with cyclophosphamide (Bu-cy) or fludarabine (Bu-flu) as myeloablative conditioning prior to allograft or with high-dose melphalan (Bu-mel) prior to autologous SCT. Despite many decades of Bu use, initially orally but now intravenously (IV), a paucity of pharmacokinetic (PK) and pharmacodynamic (PD) data exists to inform evidence-based guidelines as how best to balance the efficacy and toxicity of this agent. This is a non-randomized retrospective real-world study at three hospitals investigating the role of PK guidance in dosing Bu in the setting of IV Bu-mel autologous SCT. The primary intent was to examine how effectively PK assessment could be used to achieve a desirable drug exposure and to evaluate factors, particularly, age, sex, actual weight, body mass index (BMI), body surface area (BSA), disease, number of prior treatments, renal function, and the use of concomitant medications that may influence Bu exposure. All patients underwent PK analysis on a test dose of Bu (1.6 mg/kg, i.e., 50% of the first dose) on D-7 and subsequently received a second 1.6 mg/kg dose on D-6. Bu dose was calculated using actual body weight (ABW) if patients were less than ideal body weight (IBW), or adjusted ideal body weight (AIBW) if ABW was greater than IBW. Thereafter, at the discretion of the investigator, the group was divided into two; a weight-based cohort at two hospitals and a PK-guided cohort at the third hospital. Thirty-seven patients received PK-adjusted dosing guided by the results of the initial PK results, targeting a specific Bu exposure expressed as the area-under-the-concentration-versus-time curve (AUC) of between 4000 and 5000 µmol min/day (median 4800). The remaining 27 patients received unadjusted weight-based doses with a further three doses of 3.2 mg/kg of Bu infused over 180 min (D-5 to - 3) irrespective of their initial PK results. For the purposes of the analysis, we selected a target AUC of 4800 µmol min/day in this group, equivalent to the median targeted AUC in the PK-adjusted group. All patients subsequently had repeated PK analysis on D-5 after receiving their "therapeutic" Bu dose. Mel (140 mg/m2 or 100 mg/m2) IV was given on D-2. Sixty-four adult patients were enrolled. Patients who received PK-guided Bu dosing received a higher median Bu dose than the unadjusted weight-based cohort (3.5 mg/kg vs 3.2 mg/kg respectively, p = 0.007). Eighty-one percent (30/37) of patients in the PK-guided group achieved their target AUC (± 15%) compared with 56% (15/27) in the weight-based cohort (p = 0.027). The respective median AUCs of 5064 µmol min/day (range 3639-6157 µmol min/day) and 4854 µmol min/day (range 3251-6305 µmol min/day) were not significantly different (p = 0.16). Multivariate analysis identified ABW as the only independent variable that affected the relationship between Bu dosing and exposure (p = 0.02) with heavier patients achieving lower than anticipated AUCs for the dose they received. On D-5, within the weight-based cohort, the mean AUCs were 12% higher than anticipated based on initial D-7 PK. No correlation between AUC and grade 3-4 transplant-related toxicities were observed, although only three patients had AUCs > 6000 µmol min/day. These results suggest that PK-directed Bu dosing may be of benefit in achieving a target level of drug exposure, with larger studies needed to determine the clinical significance of this strategy.

Therapeutic Drug Monitoring of Asparaginase Activity-Method Comparison of MAAT and AHA Test Used in the International AIEOP-BFM ALL 2009 Trial.

BACKGROUND: In the international AIEOP-BFM ALL 2009 trial, asparaginase (ASE) activity was monitored after each dose of pegylated Escherichia coli ASE (PEG-ASE). Two methods were used: the aspartic acid ß-hydroxamate (AHA) test and medac asparaginase activity test (MAAT). As the latter method overestimates PEG-ASE activity because it calibrates using E. coli ASE, method comparison was performed using samples from the AIEOP-BFM ALL 2009 trial. METHODS: PEG-ASE activities were determined using MAAT and AHA test in 2 sets of samples (first set: 630 samples and second set: 91 samples). Bland-Altman analysis was performed on ratios between MAAT and AHA tests. The mean difference between both methods, limits of agreement, and 95% confidence intervals were calculated and compared for all samples and samples grouped according to the calibration ranges of the MAAT and the AHA test. RESULTS: PEG-ASE activity determined using the MAAT was significantly higher than when determined using the AHA test (P < 0.001; Wilcoxon signed-rank test). Within the calibration range of the MAAT (30-600 U/L), PEG-ASE activities determined using the MAAT were on average 23% higher than PEG-ASE activities determined using the AHA test. This complies with the mean difference reported in the MAAT manual. With PEG-ASE activities >600 U/L, the discrepancies between MAAT and AHA test increased. Above the calibration range of the MAAT (>600 U/L) and the AHA test (>1000 U/L), a mean difference of 42% was determined. Because more than 70% of samples had PEG-ASE activities >600 U/L and required additional sample dilution, an overall mean difference of 37% was calculated for all samples (37% for the first and 34% for the second set). CONCLUSIONS: Comparison of the MAAT and AHA test for PEG-ASE activity confirmed a mean difference of 23% between MAAT and AHA test for PEG-ASE activities between 30 and 600 U/L. The discrepancy increased in samples with >600 U/L PEG-ASE activity, which will be especially relevant when evaluating high PEG-ASE activities in relation to toxicity, efficacy, and population pharmacokinetics.

Alectinib is effective, safe and tolerable in an adolescent with stage IVB ALK-rearranged adenocarcinoma of the lung.

Anaplastic lymphoma kinase (ALK) inhibitors such as crizotinib and alectinib have been shown to have significant activity in ALK-rearranged non-small cell lung cancers (NSCLC). There are no data for alectinib's safety or efficacy in younger patients, though it is superior to crizotinib in adult trials. We present a 14-year old girl diagnosed with stage IV-B ALK-positive adenocarcinoma of the lung after presenting with cough and fever. She was commenced on alectinib at adult dose and has had sustained complete metabolic remission for 9 months. She is the youngest patient with lung adenocarcinoma to be treated with alectinib.

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

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

High melphalan exposure is associated with improved overall survival in myeloma patients receiving high dose melphalan and autologous transplantation.

AIM: High dose melphalan (HDM) and autologous stem cell transplantation (ASCT) retains a central role in the treatment of myeloma. The aim of this study was to determine whether HDM exposure (area under the concentration vs. time curve, AUC), is significantly associated with transplant outcomes. METHODS: Melphalan concentrations were measured in six to 11 plasma samples collected after HDM (median 192 mg m(-) (2) ) to determine melphalan AUC for a total of 114 patients. Binary logistic regression was used to assess whether melphalan AUC was associated with severe (≥ grade 3) oral mucositis. Multivariate Cox regression was used to assess whether melphalan AUC was significantly associated with time to progression, progression-free survival and overall survival (OS). RESULTS: Melphalan AUC ranged from 4.9 to 24.6 mg l(-1)  h, median 12.84 mg l(-1) h. Melphalan AUC above the median was a risk factor for severe mucositis (HR 1.21, 95% CI 1.06, 1.38, P = 0.004) but was also associated with significantly improved overall survival (OS) (HR 0.40, 95% CI 0.20, 0.81, P = 0.001), with an estimated median survival of 8.50 years vs. 5.38 years for high vs. low AUC groups. Multivariate analysis did not identify melphalan AUC as being significantly associated with time to progression or progression-free survival. CONCLUSIONS: This large scale pharmacodynamic analysis of HDM demonstrates that high melphalan exposure is associated with improved survival, with an acceptable increase in transplant toxicity. These results suggest studies targeting a higher AUC are warranted in patients undergoing HDM and ASCT for myeloma.

Association of Busulfan Exposure and Outcomes after HCT for Patients with an Inborn Error of Immunity.

Allogeneic HCT is a potentially curative treatment strategy for patients with inborn errors of immunities (IEIs). Objective of this study was to assess the optimal busulfan exposure prior to allogeneic HCT for patients with an IEI who received an intravenous busulfan-based conditioning regimen between 2000 and 2023. Patients from 17 international centers were included. Main outcome of interest was event-free survival (EFS). Patients were categorized into 4 IEI subgroups: combined-immunodeficiency (CID), severe combined immunodeficiency (SCID), neutrophil disorders and hemophagocytic lymphohistiocytosis (HLH)-related disorders. Busulfan exposure was calculated by individual centers (AUCCENTER) and was re-estimated using a validated model (AUCNONMEM). Overall, 562 patients were included: 173 (30.8%) CID, 154 (27.4%) SCID, 101 (18.0%) HLH-related disorders, and 134 (23.8%) neutrophil disorders. Median busulfan AUCNONMEM was 69.0 mg×h/L and correlated poorly with AUCCENTER (r2=0.54). Patients with SCID, HLH-related, and neutrophil disorders were analyzed together (n=389), because CID disease subtype was an effect modifier (p=0.03). Estimated 2-year EFS was 78.5%. In patients with the found optimal busulfan AUCNONMEM of 70-90 mg×h/L, 2-year EFS was superior to <70 mg×h/L (adj-HR 1.97, 95% CI 1.11-3.49, p=0.02), and >90 mg×h/L (adj-HR 5.05, 95% CI 2.43-10.49, p<0.0001). Full donor chimerism increased with higher busulfan AUCNONMEM, plateauing at 90 mg×h/L. For CID patients, optimal AUCNONMEM for donor chimerism was found to be >70 mg×h/L. Improved EFS and higher donor chimerism may be achieved by targeting a cumulative busulfan AUCNONMEM of 80 mg×h/L (range 70-90). Our study stresses the importance to uniformly using a validated population PK-model to estimate the AUCNONMEM.

Impact of Antibodies Against Polyethylene Glycol on the Pharmacokinetics of PEGylated Asparaginase in Children with Acute Lymphoblastic Leukaemia: A Population Pharmacokinetic Approach.

BACKGROUND AND OBJECTIVES: Besides allergic reactions, antibodies against polyethylene glycol (PEG) have been associated with reduced PEG-asparaginase (PEG-ASNase) activity. Population pharmacokinetics (popPK) allow for an in-depth investigation of the influence of anti-PEG antibodies on PEG-ASNase pharmacokinetics. METHODS: PEG-ASNase activity (6261 samples) and anti-PEG antibodies (2082/6412 samples prior to/post administration) in 1444 children with acute lymphoblastic leukaemia treated in the AIEOP-BFM ALL 2009 trial were evaluated. Patients received two doses of PEG-ASNase during induction (2500 U/m2, intravenous, biweekly) and a third dose during reinduction treatment. Anti-PEG IgG and IgM measured prior to and post administration were explored for their influence on the initial clearance of PEG-ASNase using a previously established popPK model. Categorical and continuous antibody data, including each isotype individually as well as in combination, were assessed. RESULTS: High pre-existing levels of anti-PEG antibodies increase the initial drug clearance. Analysed separately, both anti-PEG IgGprior and IgMprior were significant covariates; the stronger effect was observed for anti-PEG IgMprior. Hockey stick models best described the data. For anti-PEG IgMprior, each additional log unit above the estimated cut point was related to a 41.4% increase in initial clearance after the first dose in induction. Antibody levels below the cut point were not associated with an effect on clearance. The combination of both isotypes did not provide additional information compared to anti-PEG IgMprior alone. Antibody levels post administration were not associated with an effect on clearance. CONCLUSION: Pre-existing antibodies against PEG-ASNase significantly increased the initial clearance in a subgroup of patients showing high antibody levels. (Trial registration: EU clinical trials register; EudraCT No: 2007-004270-43; first registered 23 October 2009.).

Association Between the Magnitude of Intravenous Busulfan Exposure and Development of Hepatic Veno-Occlusive Disease in Children and Young Adults Undergoing Myeloablative Allogeneic Hematopoietic Cell Transplantation.

Intravenous busulfan is widely used as part of myeloablative conditioning regimens in children and young adults undergoing allogeneic hematopoietic cell transplantation (HCT). Hepatic veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS) is a serious clinical problem observed with busulfan-based conditioning HCT. The development of VOD/SOS may be associated with busulfan exposure. Getting more insight into the association between busulfan exposure and the development of VOD/SOS enables further optimization of dosing and treatment strategies. The objective of this study was to assess the association between the magnitude of busulfan exposure and the occurrence of VOD/SOS in children and young adults undergoing myeloablative conditioning with a busulfan-containing regimen before allogeneic HCT. In this observational study we included all patients who underwent allogeneic HCT with intravenous busulfan as part of the conditioning regimen at 15 pediatric transplantation centers between 2000 and 2015. The endpoint was the development of VOD/SOS. The magnitude of busulfan exposure was estimated using nonlinear mixed effect modeling and expressed as the maximal concentration (Cmax; day 1 and day 1 to 4 Cmax), cumulative area under the curve (AUC; day 1, highest 1-day AUC in 4 days, and 4-day cumulative AUC), cumulative time above a concentration of 300 µg/L, and clearance on day 1. A total of 88 out of 697 patients (12.6%) developed VOD/SOS. The number of alkylators in the conditioning regimen was a strong effect modifier; therefore we stratified the regression analysis for the number of alkylators. For patients receiving only busulfan as one alkylator (36.3%, n = 253), cumulative busulfan exposure (>78 mg × h/L) was associated with increased VOD/SOS risk (12.6% versus 4.7%; odds ratio [OR] = 2.95, 95% confidence interval [CI] 1.13 to 7.66). For individuals receiving busulfan with one or two additional alkylators (63.7%, n = 444), cumulative busulfan exposure (≤78 and >78 mg × h/L) did not further increase the risk of VOD/SOS (15.4% versus 15.2%; OR = 1.03, 95% CI 0.61 to 1.75). The effect of the magnitude of busulfan exposure on VOD/SOS risk in children and young adults undergoing HCT is dependent on the number of alkylators. In patients receiving busulfan as the only alkylator, higher cumulative busulfan exposure increased the risk of VOD/SOS, whereas in those receiving multiple alkylators, the magnitude of busulfan exposure did not further increase this risk.

Precision dosing of intravenous busulfan in pediatric hematopoietic stem cell transplantation: Results from a multicenter population pharmacokinetic study.

Busulfan (Bu) is a common component of conditioning regimens before hematopoietic stem cell transplantation (HSCT) and is known for high interpatient pharmacokinetic (PK) variability. This study aimed to develop and externally validate a multicentric, population PK (PopPK) model for intravenous Bu in pediatric patients before HSCT to first study the influence of glutathione-s-transferase A1 (GSTA1) polymorphisms on Bu's PK in a large multicentric pediatric population while accounting for fludarabine (Flu) coadministration and, second, to establish an individualized, model-based, first-dose recommendation for intravenous Bu that can be widely used in pediatric patients. The model was built using data from 302 patients from five transplantation centers who received a Bu-based conditioning regimen. External model validation used data from 100 patients. The relationship between body weight and Bu clearance (CL) was best described by an age-dependent allometric scaling of a body weight model. A stepwise covariate analysis identified Day 1 of Bu conditioning, GSTA1 metabolic groups based on GSTA1 polymorphisms, and Flu coadministration as significant covariates influencing Bu CL. The final model adequately predicted Bu first-dose CL in the external cohort, with 81% of predicted area under the curves within the therapeutic window. The final model showed minimal bias (mean prediction error, -0.5%; 95% confidence interval [CI], -3.1% to 2.0%) and acceptable precision (mean absolute prediction error percentage, 18.7%; 95% CI, 17.0%-20.5%) in Bu CL prediction for dosing. This multicentric PopPK study confirmed the influence of GSTA1 polymorphisms and Flu coadministration on Bu CL. The developed model accurately predicted Bu CL and first doses in an external cohort of pediatric patients.

Population pharmacokinetics of mycophenolic acid in children and young people undergoing blood or marrow and solid organ transplantation.

AIMS: To characterize the population pharmacokinetics of mycophenolic acid (MPA) and evaluate dose regimens using a simulation approach and accepted therapeutic drug monitoring targets in children and young people undergoing blood or marrow, kidney and liver transplantation. METHODS: MPA concentration-time data were collected using an age specific sampling protocol over 12h. Some patients provided randomly timed but accurately recorded blood samples. Total and unbound MPA were measured by HPLC. NONMEM was employed to analyze MPA pharmacokinetic data. Simulations (n= 1000) were conducted to assess the suitability of the MPA dose regimens to maintain total MPA AUC(0,12h) within the range 30 and 60mg l(-1) h associated with optimal outcome. RESULTS: A two-compartment pharmacokinetic model with first-order elimination best described MPA concentration-time data. Population mean estimates of MPA clearance, inter-compartmental clearance, volumes of distribution in the central and peripheral compartments, absorption rate constant and bioavailability were 6.42 l h(-1) , 3.74 l h(-1) , 7.24 l, 16.8l, 0.39h(-1) and 0.48, respectively. Inclusion of bodyweight and concomitant ciclosporin reduced the inter-individual variability in CL from 54.3% to 31.6%. Children with a bodyweight of 10kg receiving standard MPA dose regimens achieve an MPA AUC below the target range suggesting they may be at a greater risk of acute rejection. CONCLUSIONS: The population pharmacokinetic model for MPA can be used to explore dosing guidelines for safe and effective immunotherapy in children and young people undergoing transplantation.

Population pharmacokinetics of melphalan in patients with multiple myeloma undergoing high dose therapy.

AIMS: To i) investigate the pharmacokinetics of total and unbound plasma melphalan using a population approach, ii) identify clinical factors that affect melphalan disposition and iii) evaluate the role of melphalan exposure in melphalan-related toxicity and disease response. METHODS: Population pharmacokinetic modelling (using NONMEM) was performed with total and unbound concentration-time data from 100 patients (36-73 years) who had received a median 192 mg m(-2) melphalan dose. Model derived estimates of total and unbound melphalan exposure (AUC) in patients with serious melphalan toxicity and those who had a good disease response (>or=90% decrease in paraprotein concentrations) were compared using the Mann-Whitney test. RESULTS: A two compartment model generated population mean estimates for total and unbound melphalan clearance (CL) of 27.8 and 128 l h(-1), respectively. Estimated creatinine clearance, fat free mass and haematocrit were important determinants of total and unbound CL, reducing the inter-individual variability in total CL from 34% to 27% and in unbound CL from 42% to 30%. Total AUC (range 4.9-24.4 mg l(-1) h) and unbound AUC (range 1.0-6.5 mg l(-1) h) were significantly higher in patients who had oral mucositis (>or=grade 3) and long hospital admissions (P < 0.01). Patients who responded well had significantly higher unbound AUC (median 3.2 vs. 2.8 mg l(-1) h, P < 0.05) when assessed from diagnosis to post-melphalan and higher total AUC (median 21.3 vs. 13.4 mg l(-1) h, P= 0.06), when assessed from pre- to post-melphalan. CONCLUSIONS: Creatinine clearance, fat free mass and haematocrit influence total and unbound melphalan plasma clearance. Melphalan exposure is related to melphalan toxicity while the association with efficacy shows promising trends that will be studied further.

An isocratic fluorescence HPLC assay for the monitoring of l-asparaginase activity and l-asparagine depletion in children receiving E. colil-asparaginase for the treatment of acute lymphoblastic leukaemia.

A novel assay for the determination of l-asparaginase activity in human plasma is described that is based on the HPLC quantitation of l-aspartic acid produced during enzyme incubation. Methods for monitoring l-asparagine depletion are also described. Chromatography of l-aspartic acid, l-asparagine and l-homoserine (the internal standard) involved derivatization with o-pthaldialdehyde, then separation from other amino acids on a Phenomenex Luna C(18) column using a 1 mL/min flow rate and a mobile phase consisting of di-potassium hydrogen orthophosphate propionate buffer, pH 6, with 10% methanol and 10% acetonitrile. Fluoresence detection was at excitation/emission wavelengths of 357/455 nm. Under these conditions l-aspartic acid, l-asparagine and l-homoserine had retention times of 3.5, 9.8 and 17.7 min, respectively. The l-asparaginase assay was linear from 0.1 to 10 U/mL activity and interday precision and accuracy were less than 13%. The limit of quantitation was approximately 0.03 U/mL. The assay utility was established in 12 children who received E. coli l-asparaginase as treatment for acute lymphoblastic leukaemia.

Variability in the pharmacokinetics of intravenous busulphan given as a single daily dose to paediatric blood or marrow transplant recipients.

AIM: To examine inter- and intrapatient variability in the pharmacokinetics of intravenous (i.v.) busulphan given as a single daily dose to children with malignant (n = 19) and nonmalignant (n = 21) disease. METHODS: Busulphan (120 mg m(-2), 130 mg m(-2) or 3.2 mg kg(-1)) was administered over median 2.1 h. Blood samples (4-10) were collected after the first dose, busulphan concentrations were measured and pharmacokinetic parameters, including clearance (CL) and area under the concentration-time curve (AUC), were determined using the Kinetica software (Innaphase). Interpatient variability was assessed as percent coefficient of variation (% CV). Intrapatient variability was assessed by calculating percent differences between observed full dose AUC and AUC predicted from an initial 65 mg m(-2) dose in 13 children who had busulphan pharmacokinetic monitoring. RESULTS: Clearance of i.v. busulphan in 40 children was 4.78 +/- 2.93 l h(-1) (% CV 61%), 0.23 +/- 0.08 l h(-1) kg(-1) (% CV 35%) and 5.79 +/- 1.59 l h(-1) m(-2) (% CV 27%). Age correlated significantly (p < 0.001) with CL (l h(-1)) and CL (l h(-1) kg(-1)), but not with CL (l h(-1) m(-2)). AUC normalized to the 130 mg m(-2) dose ranged from 14.1 to 56.3 mg l(-1) x h (% CV 37%) and also did not correlate with age. Interpatient variability in CL (l h(-1) m(-2)) was highest in six children with immune deficiencies (60%) and lowest in seven children with solid tumours (14%). Intrapatient variability was <13% for nine (of 13) children, but between 20 and 44% for four children. CONCLUSIONS: There is considerable inter- and intrapatient variability in i.v. busulphan CL (l h(-1) m(-2)) and exposure that is unrelated to age, especially in children with immune deficiencies. These results suggest that monitoring of i.v. busulphan pharmacokinetics is required.