Improved HPLC method with automated pre-column sample derivatisation for serum pegylated L-asparaginase activity measurement in paediatric acute lymphoblastic leukaemia patients.

Therapeutic drug monitoring of pegylated L-asparaginase (ASNase) ensures the drug effectiveness in childhood acute lymphoblastic leukaemia (ALL) patients. The biological drug property with variable immunogenic host clearance, and the prescription of its generic formulation urge the need for a reliable assay to ensure an optimal treatment and improve outcome. This study aimed to optimise an existing isocratic reversed-phase high performance liquid chromatography (RP-HPLC) method with an automated pre-column sample derivatisation and injection program, and a computational algorithm for measuring serum pegylated ASNase activity in children with ALL. Nath et al.'s method in 2009 was adopted and modified using a pegylated ASNase. A set of Microsoft Excel macros was developed for the serum drug activity computation. An Agilent InfinityLab LC Series 1260 Infinity II Quaternary System with fluorescence detection was employed with an Agilent Poroshell 120 EC-C18 4.6×100 mm, 2.7 µm analytical column. System flow rate was optimised to 2.0 mL/min with 40×10-6/bar pump compressibility. The O-phthaldialdehyde (OPA) solution composition was optimised to 1 % o-phthaldialdehyde, 0.8 % 2-mercaptoethanol, 7.13 % methanol, and 1.81 % sodium tetraborate. The pre-column derivatisation program mixed 0.1 µL sample with 25 µL OPA solution before the automated injection. Method validation was according to the ICH guidelines. Total analysis time was 15 min, with L-aspartic acid eluted at 0.96 min and internal standard at 4.7 min. The calibration curves showed excellent linearity (R ≥0.9999). Interday precision for the drug activity at 0.1 IU/mL, 0.5 IU/mL, and 1 IU/mL were 4.15 %, 3.05 %, and 3.09 % (n = 6). Mean %error for the drug activity at 0.1 IU/mL, 0.5 IU/mL, and 1 IU/mL were 0.90±4.41 %, -1.37±3.04 %, and -3.03±3.02 % (n = 6). Limit of quantitation was 0.03 IU/mL. Majority of the patients' serum drug activity fell within the assay calibration range. Our improved method is automated, having shorter analysis time with a well-maintained separation resolution that enables a high-throughput analysis for application.

Hypersensitivity reactions to asparaginase therapy in acute lymphoblastic leukemia: immunology and clinical consequences.

Asparaginase is commonly used in combination therapy of acute lymphoblastic leukemia. However, as an immunogenic protein, hypersensitivity reactions (HSRs) during asparaginase therapy are frequent, indicating the development of anti-asparaginase antibodies. These can be associated with diminished clinical effectiveness, including poorer survival. Therapeutic drug monitoring of serum asparaginase activity to confirm complete asparagine depletion is therefore crucial during asparaginase therapy. Switching to alternative types of asparaginase is recommended for patients experiencing HSRs or silent inactivation; those with HSRs or silent inactivation on Escherichia coli-derived asparaginases should switch to another preparation. However, prior global shortages of Erwinia asparaginase highlight the importance of alternative non-E. coli-derived asparaginase, including recombinant Erwinia asparaginase.

Asparaginase is commonly used as a part of a multidrug regimen for acute lymphoblastic leukemia treatment. As foreign proteins, asparaginases have the potential to induce immune responses known as hypersensitivity reactions (HSRs), which can range from a mild rash to a severe allergic reaction. Here, we provide an overview of HSRs and their prevalence in asparaginase-based therapies, and clinical approaches to reduce HSRs. We also review the current understanding of cellular and molecular mechanisms of HSRs, consequences of HSRs and current recommendations for the management of immune reactions to asparaginase. Prior global shortages of Erwinia asparaginase due to manufacturing and supply issues have limited access of asparaginase treatment to patients. In this context, newer therapies have recently been developed.

A Pharmacokinetic Study of Native E.coli Asparaginase for Acute Lymphoblastic Leukemia Treated with ThaiPOG Protocol.

BACKGROUND: Asparaginase is one of the essential chemotherapies used to treat acute lymphoblastic leukemia (ALL). Asparaginase antibody production may cause a subtherapeutic level and result in an inferior outcome. The aim of this study was to prove the efficacy of current native E.coli asparaginase-based protocol. Moreover, does subtherapeutic result appeared in small group of the trial?. METHODS: A prospective study of asparaginase activity among patients who received native E.coli asparaginase 10,000 IU/m2 intramuscularly according to The Thai Pediatric Oncology Group (ThaiPOG) protocol was done. The plasma asparaginase activity was measured by the coupled enzymatic reaction. Pharmacokinetic data including peak activity (Cmax), time to maximum concentration (Tmax), area under the curve (AUC0-48h) being elucidated. RESULTS: Eight patients (five males and three females), median age 9.5 years, were enrolled. The median asparaginase activity of seven cases who were eligible for calculation reached Tmax within 24 hours (range 6-48 hours) with mean±SD of Cmax 3.60±0.34 (range 3.02-4.11) IU/ml. Mean±SD of AUC0-48h is 143.23±36.94 IU.h/mL (range 71.07 - 180.12 IU.h/mL). The post-48-hour activity showed a mean±SD of 3.19±0.24 IU/ml (range 2.77-3.51 IU/ml) which implied an adequacy of activity over 48 hours and proper for the 12-day period. One relapsed ALL patient showed an extremely low AUC of asparaginase activity which coincided with urticaria after asparaginase injection. Subsequently, the asparaginase antibody was demonstrated in this patient. CONCLUSION: Native E. coli asparaginase-based protocol provides a compelling pharmacokinetic effect. Asparaginase activity and/or antibody testing is recommended for all cases especially in a relapsed patient, history of high accumulative dose of asparaginase or suspected allergic reaction. Patients with low asparaginase activity or allergy may benefit from switching to an alternative form of asparaginase to maintain treatment efficacy.

Correlation of L-asp Activity, Anti-L-asp Antibody, Asn and Gln With Adverse Events Especially Anaphylaxis Risks in PEG-asp-Contained Regime Treated Pediatric ALL Patients.

OBJECTIVE: This study aimed to investigate the correlation of L-asparaginase (L-asp) activity, anti-L-asp antibody, asparagine and glutamine levels with the risks of adverse events (AEs), especially anaphylaxis, in pediatric acute lymphoblastic leukemia (ALL) patients who underwent polyethylene glycol-conjugated L-asp (PEG-asp)-contained treatment. METHODS: Plasma samples were collected from 91 pediatric ALL patients who underwent PEG-asp-contained treatment on the 7th day after drug administration. Plasma L-asp activity, anti-L-asp antibody level, asparagine level and glutamine level were detected. Meanwhile, AEs related to PEG-asp administration were recorded. RESULTS: AEs occurred in 13 (14.3%) patients, among which 7 (7.7%) patients had anaphylaxis, while another 6 patients had non-anaphylaxis AEs (including 4 (4.4%) patients who had acute pancreatitis, 1 (1.1%) patient who had abdominal pain and diarrhea, as well as 1 (1.1%) patient who had nausea and vomiting). L-asp activity was decreased, while asparagine and glutamine levels were increased in patients with AEs compared to patients without AEs, and ROC curves showed that they were correlated with higher AEs risk. Notably, further analyses revealed that L-asp activity, anti-L-asp antibody, asparagine and glutamine levels were highly correlated with anaphylaxis risk, but they were not associated with the risk of non-anaphylactic AEs. CONCLUSION: The measurement of L-asp activity, anti-L-asp antibody level, asparagine level and glutamine level might assist the prevention of anaphylaxis-related AEs in pediatric ALL patients who underwent PEG-asp-contained treatment.

Circulating Tumor DNA is Prognostic and Potentially Predictive of Eryaspase Efficacy in Second-line in Patients with Advanced Pancreatic Adenocarcinoma.

PURPOSE: Eryaspase is composed of l-asparaginase encapsulated in erythrocytes and has demonstrated significant efficacy in a randomized phase II trial. We assessed the prognostic and predictive value of circulating tumor DNA (ctDNA) in patients, plasma included in this trial. EXPERIMENTAL DESIGN: Samples prospectively collected pretreatment were centrally analyzed by next-generation sequencing. Prognostic values of baseline ctDNA and ctDNA early changes between day 0 and 28 were assessed in both arms combined on objective response rate (ORR), progression-free survival (PFS), and overall survival (OS); three groups were defined: negative ctDNA (Neg), ctDNA responders (Resp), and ctDNA nonresponders (NResp). Predictive value of ctDNA for eryaspase efficacy was investigated. RESULTS: ctDNA was positive at baseline in 77 patients of the 113 tested patients (68%). Detectable ctDNA was an independent negative prognostic factor for OS (4.6 vs. 8.8 months; P = 0.0025) and PFS (1.6 vs. 3.3 months; P = 0.00043). Early change in ctDNA levels was correlated with ORR (20%, 26%, 0%; P < 0.04), PFS (3.7, 3.4, 1.6 months; P < 0.0001), and OS (11.7, 6.5, 4.3 months; P < 0.0001) according to the three defined groups (Neg, Res, NResp, respectively). In patients with ctDNA detectable at baseline, eryaspase was associated with better PFS [HR = 0.53; 95% confidence interval (CI): 0.3-0.94)] and OS (HR = 0.52; 95% CI: 0.29-0.91). CONCLUSIONS: We confirm from a prospective randomized trial that: (i) the presence of ctDNA at baseline is a major prognostic factor, (ii) the early change of ctDNA correlates with treatment outcome, and (iii) the ctDNA could be a predictive biomarker of eryaspase efficacy.

Higher plasma asparaginase activity after intramuscular than intravenous Erwinia asparaginase.

It is unclear if dosing intervals for Erwinase can be extended with intramuscular (i.m.) versus intravenous (i.v.) dosing. Children with acute lymphoblastic leukemia received Erwinase at 30 000-42 000 IU/m2 i.v. or i.m. I.m. Erwinase (n = 22) achieved activity above 0.1 IU/mL for longer than i.v. Erwinase (n = 33) (3.4 vs 2.9 days, P = 0.0007). With 30 000 IU/m2 Monday, Wednesday, Friday, more patients achieved adequate concentrations over the weekend with i.m. vs i.v. dosing (P = 5 × 10-36 ). A schedule with i.v. doses on Monday and Wednesday and i.m. doses on Friday of 30 000 IU/m2 maintained activity > 0.1 IU/mL over the weekend in 80% of patients.

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.

Plasma asparaginase activity and asparagine depletion in acute lymphoblastic leukemia patients treated with pegaspargase on Children's Oncology Group AALL07P4.

The efficacy of asparaginase in acute lymphoblastic leukemia (ALL) is dependent on depletion of asparagine, an essential amino acid for ALL cells. The target level of plasma asparaginase activity to achieve asparagine depletion has been between 0.05 and 0.4 IU/mL. COG AALL07P4 examined the asparaginase activity and plasma and CSF asparagine concentration of pegaspargase when given intravenously in the treatment of NCI high risk ALL. Matched plasma asparaginase/asparagine levels of the clearance of 54 doses of pegaspargase given in induction or consolidation demonstrated that all patients who had a plasma asparaginase level >0.02 IU/mL had undetectable plasma asparagine. No difference was observed in CSF asparagine levels associated with matched plasma asparaginase levels of 0.02-0.049 versus 0.05-0.22 IU/mL (p = .25). Our data suggest that a plasma asparaginase activity level of 0.02 IU/mL can effectively deplete plasma asparagine. The data also indicate that the 95% CI for plasma asparagine depletion after a pegaspargase dose is 22-29 days. Clinical trial registration: clinicaltrials.gov identifier NCT00671034.

Personalized nanomedicine: a rapid, sensitive, and selective UV-vis spectrophotometry method for the quantification of nanostructured PEG-asparaginase activity in children's plasma.

PURPOSE: PEGylated asparaginase (PEG-ASNase), which hydrolyzes asparagine to ammonia and aspartic acid, is an effective nanostructured antitumor agent for acute lymphoblastic leukemia (ALL). In order to monitor the activity of PEG-ASNase in plasma and design an individualization project, a rapid and sensitive method to determine PEG-ASNase activity in plasma using ultraviolet-visible spectrophotometry was established. METHODS: PEG-ASNase is commonly used in acute lymphoblastic leukemia. With Nessler's reagent as the chromogenic reagent of ammonia, a stable yellow complex was produced. The units of enzyme activity were defined as micromoles of ammonia released per minute. RESULTS: Calibration curves fitted by plotting the OD at 450 nm of the Nessler product vs concentration were linear in the range of 27.8-1,111.0 IU/L with r 2=0.999. The lower limit of quantification for PEG-ASNase activity in human plasma was 20 IU/L with good accuracy and precision. The intra- and interday precision (relative standard deviation) values were below 10% and accuracy ranged from 90% to 110% at all quality control levels. Analytical recoveries were determined between 90% and 110% for all quality control samples. CONCLUSION: This study proved that the Nessler method is well validated and can be successfully applied in the determination of plasma samples in the clinical setting for patients with ALL. It takes personalized nanomedicine to an entirely new level.

Low Bioavailability and High Immunogenicity of a New Brand of E. colil-Asparaginase with Active Host Contaminating Proteins.

The drug l-asparaginase is a cornerstone in the treatment of acute lymphoblastic leukemia (ALL). The native E. colil-asparaginase used in Brazil until recently has been manufactured by Medac/Kyowa. Then a decision was taken by the Ministry of Health in 2017 to supply the National Health System with a cheaper alternative l-asparaginase manufactured by Beijing SL Pharmaceutical, called Leuginase®. As opposed to Medac, the asparaginase that has been in use in Brazil under the trade name of Aginasa®, it was not possible to find a single entry with the terms Leuginase in the Pubmed repository. The apparent lack of clinical studies and the scarcity of safety information provided to the hospitals by the drug distributor created a debate among Brazilian pediatric oncologists about issues of safety and efficacy that culminated eventually in a court decision to halt the distribution of the new drug all over the country. Boldrini Children's Center, a non-profit pediatric oncohematology hospital, has conducted its own evaluation of Leuginase®. Mass spectrometry analyses found at least 12 different contaminating host-cell proteins (HCP) in Leuginase®. The presence of two HCP (beta-lactamase and malate dehydrogenase) was confirmed by orthogonal methodologies. The relative number of HCP peptides ranged from 19 to 37% of the total peptides identified by mass spectrometry. In vivo studies in mice injected with Leuginase® revealed a 3 times lower plasma bioavailability and the development of higher antibody titres against l-asparaginase in comparison to Aginasa®-injected animals. The decision to buy a new drug based on its price alone is not safe. Developing countries are especially vulnerable to cheaper alternatives that lack solid quality assurance.

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.

Prolonged first-line PEG-asparaginase treatment in pediatric acute lymphoblastic leukemia in the NOPHO ALL2008 protocol-Pharmacokinetics and antibody formation.

BACKGROUND: As pegylated asparaginase is becoming the preferred first-line asparaginase preparation in the chemotherapy regimens of childhood acute lymphoblastic leukemia (ALL), there is a need to evaluate this treatment. METHODS: The aim of this study was to evaluate the pharmacokinetics of prolonged upfront biweekly PEG-asparaginase (where PEG is polyethylene glycol) treatment by measuring serum l-asparaginase activity and formation of anti-PEG-asparaginase antibodies. A total of 97 evaluable patients (1-17 years), diagnosed with ALL, and treated according to the NOPHO ALL2008 protocol (where NOPHO is Nordic Society of Paediatric Haematology and Oncology) were included. In the NOPHO ALL2008 protocol, patients are randomized to 8 or 15 doses of intramuscular PEG-asparaginase (Oncaspar® ) 1,000 IU/m²/dose, at 2-week or 6-week intervals with a total of 30-week treatment (Clinical trials.gov. no.: NCT00819351). RESULTS: The pharmacological target of treatment (l-asparaginase activity above 100 IU/l) was reached in 612 of 652 (94%) samples obtained 14 ± 2 days after PEG-asparaginase administration. Mean l-asparaginase activity was 338 IU/l. Six patients had l-asparaginase activity below 50 IU/l in all samples. A total of 25 patients (26%) developed Immunoglobulin G (IgG) anti-PEG-asparaginase antibodies, but there was no correlation between anti-PEG-asparaginase antibodies and low levels of asparaginase activity. CONCLUSION: We conclude that prolonged first-line biweekly PEG-asparaginase therapy, 1,000 IU/m²/dose was above the pharmacological target in the vast majority of patients. Presence of anti-PEG-asparaginase antibodies was not a predictor of l-asparaginase activity.

From the Children's Oncology Group: Evidence-Based Recommendations for PEG-Asparaginase Nurse Monitoring, Hypersensitivity Reaction Management, and Patient/Family Education.

PEG-aspariginase is a backbone chemotherapy agent in pediatric acute lymphoblastic leukemia and in some non-Hodgkin lymphoma therapies. Nurses lack standardized guidelines for monitoring patients receiving PEG-asparaginase and for educating patients/families about hypersensitivity reaction risks. An electronic search of 6 databases using publication years 2000-2015 and multiple professional organizations and clinical resources was conducted. Evidence sources were reviewed for topic applicability. Each of the final 23 sources was appraised by 2 team members. The GRADE (Grading of Recommendations Assessment, Development and Evaluation) system was used to assign a quality and strength rating for each recommendation. Multiple recommendations were developed: 4 relating to nurse monitoring of patients during and after drug administration, 8 guiding hypersensitivity reaction management, and 4 concerning patient/family educational content. These strong recommendations were based on moderate, low, or very-low-quality evidence. Several recommendations relied on generalized drug hypersensitivity guidelines. Additional research is needed to safely guide PEG-asparaginase monitoring, hypersensitivity reaction management, and patient/family education. Nurses administering PEG-asparaginase play a critical role in the early identification and management of hypersensitivity reactions.

Enzyme-Loaded Polyion Complex Vesicles as in Vivo Nanoreactors Working Sustainably under the Blood Circulation: Characterization and Functional Evaluation.

Enzyme-loaded synthetic vesicles have attracted great attention for their feasibility to exert the efficient and prolonged functionality of loaded enzymes in harsh environments, such as in vivo. However, several issues remain regarding the optimization of their structures toward practical application. Herein, we fabricated polyion complex vesicles (PICsomes) loaded with l-asparaginase (ASNase@PICsomes) and conducted a detailed characterization to ensure their utility as nanoreactors functioning under the harsh in vivo environment of the bloodstream. ASNase@PICsomes showed 100 nm-sized monodispersed vesicular structures. Fluorescence cross-correlation spectroscopy revealed essentially no empty PICsome fraction in the product, indicating the quantitative formation of ASNase@PICsomes. Furthermore, fluorescence anisotropy measurement showed that the loaded enzymes were located essentially in the inner aqueous phase of PICsomes, being successfully segregated from the external environment. ASNase@PICsomes exhibited significantly prolonged enzymatic reaction compared with free ASNase after systemic injection into mice, corroborating their functionality as in vivo nanoreactors working under the blood circulation.

Antithrombin III as the Indicator of L-Asparaginase Activity in Children Treated for Acute Lymphoblastic Leukemia.

L-asparaginase (ASP) is widely used in the treatment of acute lymphoblastic leukemia (ALL) in children. Monitoring its activity is necessary because of the risk of drug inactivation as the result of an immune reaction. Besides allergic reactions, another frequent side effect of ASP treatment is coagulopathy, especially deficiency of antithrombin III (ATIII). The aim of this study was to analyze the relationship between ASP and ATIII activities and the possibility of ATIII activity use in an indirect ASP activity assessment. ASP and ATIII activity was measured in 76 children with ALL treated according to the ALL IC BFM 2002 protocol. A correlation between ASP and ATIII activities was found (R=-0.43, P=0.0001). ROC curve analysis revealed some utility regarding the determination of ATIII in identifying patients with low or undetectable ASP activity (area under the curve=0.87 [95% confidence interval, 0.77-0.96], P<0.0001 and 0.93 [95% confidence interval, 0.85-1.0], P<0.0001, respectively). Higher ATIII activity is associated with a higher probability of a decline in ASP activity. Examination of ATIII activity cannot replace a direct determination of ASP activity, but in the case of unavailability of the direct test, it can be a helpful surrogate parameter of drug activity.

Impact of anti-PEG IgM antibodies on the pharmacokinetics of pegylated asparaginase preparations in mice.

The potential impact of pre-existing anti-PEG antibodies on the asparaginase activity kinetics of two pegylated l-asparaginase preparations - pegylated recombinant l-asparaginase (PEG-rASNase MC0609) and pegaspargase (pegylated Escherichia colil-asparaginase) - was investigated in immune competent, naïve B6D2F1-hybrid mice. To generate anti-PEG antibodies, mice were pre-sensitised by repeated injections of 40kDa PEG-Diol without being conjugated to a carrier. Successful PEG-Diol pre-sensitisation was verified by analysis of anti-PEG antibody titers in serum. 88-100% of animals developed PEG-specific anti-PEG IgM antibodies after PEG-Diol pre-sensitisation. All animals positive for anti-PEG IgM antibodies and control animals (without prior PEG-Diol pre-sensitisation) were treated once with PEG-rASNase MC0609 or pegaspargase, and asparaginase enzyme activity levels and immunogenicity of both preparations were analysed. Known serum asparaginase activity profiles were measured after treatment with PEG-rASNase MC0609 or pegaspargase in all treatment groups. No rapid decrease of asparaginase activity was observed - irrespective of successful PEG-Diol pre-sensitisation and presence of acquired anti-drug-IgG and/or anti-PEG IgM antibodies. In conclusion, the pharmacokinetics of pegylated l-asparaginase was unaffected by the presence of pre-existing anti-PEG IgM antibodies in immune competent B6D2F1-hybrid mice Probably the titre or affinity of these anti-PEG IgM antibodies were too low to influence the pharmacokinetics of PEG-rASNase MC0609 or pegaspargase or anti-PEG IgM antibodies bound to PEG-ASNase without neutralising capabilities. Thus, early loss of asparaginase activity as observed in serum of ALL patients is a complex process and cannot be explained solely by the existence of pre-existing anti-PEG antibodies.

Consensus expert recommendations for identification and management of asparaginase hypersensitivity and silent inactivation.

L-asparaginase is an integral component of therapy for acute lymphoblastic leukemia. However, asparaginase-related complications, including the development of hypersensitivity reactions, can limit its use in individual patients. Of considerable concern in the setting of clinical allergy is the development of neutralizing antibodies and associated asparaginase inactivity. Also problematic in the use of asparaginase is the potential for the development of silent inactivation, with the formation of neutralizing antibodies and reduced asparaginase activity in the absence of a clinically evident allergic reaction. Here we present guidelines for the identification and management of clinical hypersensitivity and silent inactivation with Escherichia coli- and Erwinia chrysanthemi- derived asparaginase preparations. These guidelines were developed by a consensus panel of experts following a review of the available published data. We provide a consensus of expert opinions on the role of serum asparaginase level assessment, indications for switching asparaginase preparation, and monitoring after change in asparaginase preparation.