Asparaginase Activity Research Articles

Efficacy and toxicity of pegaspargase and calaspargase pegol in childhood acute lymphoblastic leukemia/lymphoma: Results of DFCI 11-001.

10006 Background: DFCI ALL Consortium Protocol 11-001 assessed the efficacy and toxicity of Calaspargase pegol (SC-PEG), a novel pegylated asparaginase (ASP) formulation with longer half-life, compared with standard pegaspargase (SS-PEG). Methods: Patients (pts) aged 1-21 years with newly diagnosed acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma (LL) were eligible. At study entry, pts were randomly assigned to receive either intravenous SS-PEG or SC-PEG, 2500 IU/m2/dose. Pts received 1 dose during the first treatment month. Beginning week 7, SS-PEG was administered every 2 weeks for 15 doses, SC-PEG every 3 weeks for 10 doses (30 weeks). Serum asparaginase activity (SAA) (considered therapeutic at ≥ 0.1 IU/mL) was assessed 4, 11, 18, and 25 days after the induction dose and before each post-induction dose. End-induction minimal residual disease (MRD) was assessed in ALL pts by IGH/TCR PCR. Results: Between 2012-2015, 239 eligible pts enrolled (230 ALL, 9 LL); 120 assigned to SS-PEG, 119 to SC-PEG. After dose 1, SAA remained ≥ 0.1 IU/mL in ≥ 95% of pts on both arms through day 18. Median SAA was higher (0.319 IU/mL vs 0.056 IU/mL) and more pts had therapeutic SAA (88% vs 17%, p˂0.001) with SC-PEG vs SS-PEG 25 days after dose 1. Post-induction, median nadir SAA (NSAA) were similar ( > 1.0 IU/mL) for both arms. There was no difference in rates of ASP-allergy, pancreatitis, thrombosis, hyperbilirubinemia, osteonecrosis, or infection. Of 230 evaluable pts, 99% of SS-PEG and 95% of SC-PEG pts achieved complete remission (p = 0.12). For B ALL pts, there was no difference in frequency of high end-induction MRD (10.3% SS-PEG, 9.5% SC-PEG, p = 1.0). With 4-year median follow-up, 4-year event-free survival (EFS) (90% confidence interval) for SS-PEG was 90.2% (84.3, 93.9), 87.7% (81.5, 91.9) for SC-PEG (p = 0.78); overall survival (OS) was 95.6% (91.0, 97.9) for SS-PEG, 94.8% (90.0, 97.3) for SC-PEG (p = 0.74). Conclusions: Every 3-week SC-PEG had similar EFS, OS, safety profile, and NSAA compared with every 2-week SS-PEG. The high NSAA observed for both preparations suggest dosing strategies can be further optimized. These data informed FDA approval of SC-PEG for pediatric pts. Clinical trial information: NCT01574274.

Interferences that impact measuring optimal L-asparaginase activity and consequent errors interpreting these data.

L-asparaginase is an enzyme produced by microorganisms, plants, and animals, which is used clinically for the treatment for acute lymphoblastic leukemia (ALL) and, in the food industry, to control acrylamide formation in baked foods. The purpose of this review was to evaluate the available literature regarding microbial sources of L-asparaginase, culture media used to achieve maximum enzyme expression in microbial fermentations, and assay methods employed to assess L-asparaginase activity. Studies were gathered by searching PubMed, and Web of Science databases before January 22, 2018, with no time restrictions. The articles were evaluated according to the source of L-asparaginase being studied, the nitrogen source in the culture medium, the type of sample, and the method employed to evaluate L-asparaginase activity. Bacterial L-asparaginase appeared to be the most commonly studied source of the enzyme and, most often, the enzyme activity was assayed from crude protein extracts using the Nessler method, which is an indirect measurement of asparaginase activity that determines the concentration of ammonia generated after the action of the enzyme on the substrate, L-asparagine. However, ammonia is also generated throughout microbial fermentations and this endogenous ammonia will also reduce the Nessler reagent if crude microbial extracts are used to determine total L-asparaginase activity. We suggest that current estimates of L-asparaginase activity reported in the literature may be overestimated when Nessler reagent is used, since we were unable to find a single study that made reference to the possible inference of fermentation derived ammonia.

Universal premedication and therapeutic drug monitoring for asparaginase-based therapy prevents infusion-associated acute adverse events and drug substitutions.

Asparaginase is a critical component of lymphoblastic leukemia therapy, with intravenous pegaspargase (PEG) as the current standard product. Acute adverse events (aAEs) during PEG infusion are difficult to interpret, representing a mix of drug-inactivating hypersensitivity and noninactivating reactions. Asparaginase Erwinia chrysanthemi (ERW) is approved for PEG hypersensitivity, but is less convenient, more expensive, and yields lower serum asparaginase activity (SAA). We began a policy of universal premedication and SAA testing for PEG, hypothesizing this would reduce aAEs and unnecessary drug substitutions. Retrospective chart review of patients receiving asparaginase before and after universal premedication before PEG was conducted, with SAA performed 1week later. We excluded patients who had nonallergic asparaginase AEs. Primary end point was substitution to ERW. Secondary end points included aAEs, SAA testing, and cost. We substituted to ERW in 21 of 122 (17.2%) patients pre-policy, and 5 of 68 (7.4%) post-policy (RR, 0.427; 95% CI, 0.27-0.69, P=0.028). All completed doses of PEG yielded excellent SAA (mean, 0.90 units/mL), compared with ERW (mean, 0.15 units/mL). PEG inactivation post-policy was seen in 2 of 68 (2.9%), one silent and one with breakthrough aAE. The rate of aAEs pre/post-policy was 17.2% versus 5.9% (RR, 0.342; 95% CI, 0.20-0.58, P=0.017). Grade 4 aAE rate pre/post-policy was 15% versus 0%. Cost analysis predicts $125779 drug savings alone per substitution prevented ($12402/premedicated patient). Universal premedication reduced substitutions to ERW and aAE rate. SAA testing demonstrated low rates of silent inactivation, and higher SAA for PEG. A substantial savings was achieved. We propose universal premedication for PEG be standard of care.

A Polyphasic Approach for Assessing Eco-System Connectivity Demonstrates that Perturbation Remodels Network Architecture in Soil Microcosms.

Network analysis was used to show changes in network attributes by analyzing the relations among the main soil microbial groups in a potted tomato soil inoculated with arbuscular mycorrhizal fungus, treated with low doses of Mentha spicata essential oil, or both, and then exposed to tenfold higher oil addition (stress pulse). Pretreatments were chosen since they can induce changes in the composition of the microbial community. Cellular phospholipid fatty acids (PLFAs) and the activity of six soil enzymes, mainly involved in the N-cycle were measured. Networks were constructed based on correlated changes in PLFA abundances. The values of all parameters were significantly different from those of random networks indicating modular architecture. Networks ranked from the lowest to highest modularity: control, non-pretreated and stressed, inoculated and stressed, oil treated and stressed, inoculated and treated with oil and stressed. The high values of network density and 1st/2nd eigenvalue ratio are related to arylamidase activity while N-acetyl-glucosaminidase, acid phosphomoesterase, and asparaginase activities related to high values of the clustering coefficient index. We concluded that modularity may be an efficient indicator of changes in the network of interactions among the members of the soil microbial community and the modular structure of the network may be related to the activity of specific enzymes. Communities that were stressed without a pretreatment were relatively resistant but prone to sudden transition towards instability, while oil or inoculation pretreatments gave networks which could be considered adaptable and susceptible to gradual change.

Asparagine levels in the cerebrospinal fluid of children with acute lymphoblastic leukemia treated with pegylated-asparaginase in the induction phase of the AIEOP-BFM ALL 2009 study.

Asparagine levels in cerebrospinal fluid and serum asparaginase activity were monitored in children with acute lymphoblastic leukemia treated with pegylated-asparaginase. The drug was given intravenously at a dose of 2,500 IU/m2 on days 12 and 26. Serum and cerebrospinal fluid samples obtained on days 33 and 45 were analyzed centrally. Since physiological levels of asparagine in the cerebrospinal fluid of children and adolescents are 4-10 μmol/L, in this study asparagine depletion was considered complete when the concentration of asparagine was ≤0.2 μmol/L, i.e. below the lower limit of quantification of the assay used. Over 24 months 736 patients (AIEOP n=245, BFM n=491) and 903 cerebrospinal fluid samples (n=686 on day 33 and n=217 on day 45) were available for analysis. Data were analyzed separately for the AIEOP and BFM cohorts and yielded superimposable results. Independently of serum asparaginase activity levels, cerebrospinal fluid asparagine levels were significantly reduced during the investigated study phase but only 28% of analyzed samples showed complete asparagine depletion while relevant levels, ≥1 μmol/L, were still detectable in around 23% of them. Complete cerebrospinal fluid asparagine depletion was found in around 5-6% and 33-37% of samples at serum asparaginase activity levels <100 and ≥ 1,500 IU/L, respectively. In this study cerebrospinal fluid asparagine levels were reduced during pegylated-asparaginase treatment, but complete depletion was only observed in a minority of patients. No clear threshold of serum pegylated-asparaginase activity level resulting in complete cerebrospinal fluid asparagine depletion was identified. The consistency of the results found in the two independent data sets strengthen the observations of this study. Details of the treatment are available in the European Clinical Trials Database at https://www.clin-icaltrialsregister.eu/ctr-search/trial/2007-004270-43/IT.

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.

Monitoring Asparaginase Activity in 21 Patients with Extranodal NK/T-Cell Lymphoma Shows a Very High Proportion of Asparaginase Inactivation

IntroductionExtranodal NK/T-cell lymphoma (ENKTL) is a rare disease; in Western countries it represents less than 1% of all Non-Hodgkin lymphoma. When localized, ENKTL is associated with a good prognosis. In contrast, patients (pts) with a disseminated disease still have a dismal outcome despite the use of asparaginase (ASPA) containing regimens that have significantly improved the prognosis of this lymphoma. Production of neutralizing anti-ASPA antibodies leading to inactivation of the enzyme can reduce its activity. Inactivation of ASPA activity is correlated with a worse prognosis in acute lymphoblastic leukemia (ALL). Monitoring of ASPA activity is therefore recommended in ALL pts with 100 IU/L as threshold value of serum enzymatic activity considered to be sufficient for complete depletion of l-asparagine in serum. However, ASPA monitoring is not routinely performed in pts with ENKTL, despite the frequent use of ASPA-containing regimens. The main objective of this study was to determine the proportion of pts with an insufficient ASPA activity corresponding to production of neutralizing anti-ASPA antibodies, risk of allergic reaction and inefficacy of the drug.MethodsAdult pts with histologically confirmed ENKTL who received an ASPA-containing regimen between 2014 and 2018 and had a monitoring of ASPA activity were included. ASPA activity measurement was usually performed with a quantitive enzyme assay 48 hours after the last ASPA injection of each cycle for native forms of ASPA and 14 days after injection of the pegylated form. Activity below 100 UI/L was considered as insufficient. ASPA activity was correlated with pts outcome. The choice of initial form of ASPA and reasons to switch between two forms of ASPA were also analyzed.ResultsFrom 2014 to 2018, a total of 21 pts received an ASPA-containing regimen and were monitored for ASPA activity. Median age was 53 years with 14 men and 7 women. More than half of these pts had a stage IV disease (n=11/21), 6 were in stage I and 4 in stage II. Fifteen pts were in first line and 6 pts were in relapse. Pts have received either native e-coli L-asparaginase (Kidrolase®: KID) (n= 13 in the treatment-naïve group, n=2 in the relapsing group) or a pegylated form of e-coli- L-asparaginase (Oncaspar®: ONC) (n=2 in the treatment-naïve group, n=4 in the relapsing group). Most of the pts received ASPA (8 injections at each cycle for native forms and 1 injection for the pegylated form) associated with gemcitabine, methotrexate and dexamethasone, plus oxaliplatine for disseminated diseases.Six pts had optimal ASPA activity, 3/6 had localized disease: 2 had persistent RC and 1 relapsed, 3/6 had a disseminated disease: 2 progressed during treatment and 1 relapsed.Fifteen pts displayed low ASPA activity, 12/15 pts with a KID containing regimen after 1 cycle (n=10) or 3 cycles (n=2) and 3/6 pts with an ONC containing regimen after the first cycle (n=2) or the second (n=1). Among these 3 pts, 2 have been previously treated with KID that could induce an immunization against ONC.Ten pts received a second form of asparaginase: Erwinia asparaginase (Erwiniase®: ERW) in 9 pts and ONC in 1 pt. In 9/10 cases, this switch was justified by detection of low ASPA activity. Measurement of enzymatic activity in these 9 pts showed satisfactory levels in 2/5 pts treated with ERW and monitored for ASPA activity and in the pt receiving ONC, 4/6 pts with a localized form and 1/3 with a disseminated form are in persistent RC.Six pts with low ASPA activity including one case with a localized form and 5 cases with a disseminated disease, did not receive another form of ASPA, they all progressed or relapsed.ConclusionMore than 2/3 of pts had sub-optimal ASPA activity after 1 to 3 cycles of ASPA containing regimens. This finding is probably due to the development of anti-ASPA inhibitory antibodies and may explain the poor outcome of pts with a disseminated disease despite the remarkable efficacy of ASPA in this disease. Although the series reported here is small and heterogeneous, our results still suggest a better outcome in pts with good ASPA activity or in case of switch between ASPA molecules in the context of low activity. ASPA activity monitoring should be recommended in pts with ENKTL, to avoid allergic reaction and ineffective treatment by switching ASPA molecules. Pegylated forms of ASPA, or encapsulated in red cells may, be less immunogenic, should be used in the first line setting instead of native forms. DisclosuresBachy:Celgene: Consultancy; Janssen: Honoraria; Gilead Sciences: Honoraria; Takeda: Research Funding; Sandoz: Consultancy; Amgen: Honoraria; Roche: Research Funding.

Drug Monitoring of Asparaginase Activity to Detect Silent Inactivation in Pediatric Acute Lymphoblastic Leukemia Patients Receiving Peg-Asparaginase

BackgroundAsparaginase is a key component of pediatric therapy for Acute lymphoblastic leukemia (ALL). Two formulations available in the USA are pegylated E. coli asparaginase (PEG) and Erwinia chrysanthemi asparaginase (Erwinaze). Hypersensitivity reactions have been observed in ~ 20% of patients receiving PEG. Patients with overt symptoms of hypersensitivity are typically switched to Erwinaze. In cases of hypersensitivity, anti-asparaginase antibodies may be present that neutralize circulating asparaginase and in some cases may present without clinical signs, a phenomenon known as silent inactivation. Few studies have prospectively evaluated the incidence of silent inactivation in pediatric ALL patients. In this prospective study, we performed therapeutic drug monitoring of asparaginase levels on newly diagnosed pediatric ALL patients to determine the incidence of silent inactivation and to switch patients to Erwinaze, if silent inactivation was detected.MethodsA prospective study was conducted at CHOC Children's Hospital on patients with newly diagnosed ALL or lymphoblastic lymphoma between 2016 to 2018. Patients designated as high or very high risk B-ALL post induction, T-ALL and B/T lymphoblastic lymphoma were enrolled. Patients received therapy on or according to a COG protocol with PEG (2500 IU/m2) administered via 2-hour i.v. infusion without antihistamine or steroid premedication. Asparaginase activity was measured days 7 and 14 after each of the two PEG doses during Consolidation. Serum asparaginase level of >0.1 IU/ml and >0.02 IU/ml was considered therapeutic at 7 or 14 days post PEG, respectively. Silent inactivation was defined as serum asparaginase levels below these values without signs of overt allergy. Any patient found to have silent inactivation was immediately switched to Erwinaze. Patient characteristics, common adverse events associated with asparaginase and serum asparaginase levels were analyzed. Descriptive statistics and Pearson's coefficient was used for analysis.ResultsThirty-nine patients were enrolled with a mean age of 9.8 years (range, 1-23) with the majority being B-ALL (87.2%) of which 3 were Ph-like (Table 1). Twelve patients (30.7%) had overt hypersensitivity and one patient (2.6%) had silent inactivation. One patient had overt hypersensitivity after completion of the PEG infusion with a measured asparaginase level 7 days post that was therapeutic. One-hundred fifteen drug levels were collected, for an average of 2.9 levels per patient (maximum 4 levels). There was a difference in mean asparaginase activity in patients without hypersensitivity compared to those with hypersensitivity (1.110 IU/mL versus 0.737 IU/mL, respectively, p=.0095). There were no significant differences in asparaginase levels based on age, gender or BSA. The highest asparaginase level observed was 7 days post the 2nd dose of asparaginase in Consolidation. Overall, the incidence of patients with CTCAE grade ≥2 transaminitis was 51.3% (n=20), hyperglycemia 2.6 % (n=1), hyperbilirubinemia 5.1% (n=2), and thrombosis 2.6% (n=1). Pancreatitis of any grade was not observed in this cohort.DiscussionWe detected silent inactivation in a single patient who had an asparaginase level of 0.01 seven days after the first dose of PEG and showed no signs of overt allergic reaction. This patient was switched to Erwinaze and an asparaginase level drawn 48 hours post Erwinaze was therapeutic at 0.719. Another patient developed a hypersensitivity reaction 12-hours after PEG and had a serum asparaginase level of 1.29 7-days post PEG. This suggests that not all anti-asparaginase antibodies are neutralizing since this patient continued to demonstrate therapeutic drug levels. The incidence of allergic reactions to PEG at our institution appears higher than reported in prior studies. Our data shows a statistically significant difference between serum asparaginase levels in patients with and without hypersensitivity. Our study also shows that silent inactivation exists in a very small proportion of patients. Additionally, patients who develop late allergic reactions to PEG may still have therapeutic asparaginase levels. Thus, monitoring of asparaginase levels may be warranted to detect silent inactivation and to monitor asparaginase levels in patients who develop delayed hypersensitivity. A larger prospective study is needed to confirm these results. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pegylated E. coli Asparaginase Desensitization: An Effective and Feasible Option for Pediatric Patients with Acute Lymphoblastic Leukemia Who Have Developed Hypersensitivity to Pegaspargase in the Absence of Asparaginase Erwinia Chrysanthemi availability

Background: L-asparaginase makes up a critical component of multi-agent chemotherapy for the treatment of pediatric acute lymphoblastic leukemia (ALL), the most common pediatric malignancy. Currently, there are two asparaginase preparations approved by the US Food and Drug Administration, available for use in the United States. These are pegaspargase, a PEGylated form of asparaginase, having polyethylene glycol [PEG] attached to native Escherichia coli asparaginase, and asparaginase Erwinia chrysanthemi (Erwinaze®), derived from cultures of Erwinia chrysanthemi. Hypersensitivity reactions are the most common toxicity observed, reported in up to 60% of patients treated with various schedules of native E. coli asparaginase. Due to the short half-life of Erwinaze®, multiple doses are required to be given to replace one dose of the PEGylated form. The United States market recently experienced a nationwide shortage of Erwinaze®. During this shortage, many patients who previously developed hypersensitivity to pegaspargase have been unable to receive asparaginase therapy. Current options recommended by consortium groups during this shortage recommended omission of all asparaginase therapy in these patients' regimens, which may lead to a greater risk for relapse, and for those who have relapsed, a greater risk for re-induction failure.Treatment: A 13 step rapid desensitization protocol utilizing chemotherapy desensitization protocols published by Castells et al, was developed to administer pegaspargase to five patients with ALL in various stages of therapy. Pegaspargase was administered using a serial dilution protocol infused over 6 hours in the pediatric intensive care unit. Patients were pre-medicated with steroids, diphenhydramine and acetaminophen. Asparginase levels were obtained between days 4-7 and 10-14, to ensure patients had sustained levels of asparginase activity.Cases: Patient #1: 7 year old female with late relapse of B ALL , experienced immediate hypersensitivity to second dose of pegaspargase as well as to subsequent Erwinaze® during upfront therapy. She received two courses of pegaspargase via desensitization protocol during re-induction at relapse tolerated it well with sustained asparginase activity.Patient #2: 18 year-old female with very high risk B ALL, experienced immediate hypersensitivity to third dose of pegaspargase as well as to subsequent Erwinaze®. She received 2 doses of pegaspargase via desensitization protocol during interim maintenance II, tolerated it well with sustained asparginase activity.Patient #3: 7 year old male with late relapse of B ALL, experienced immediate hypersensitivity to third dose of pegaspargase during upfront therapy. At relapse, Erwinaze® was unavailable due to nationwide shortages. He received 2 doses of pegaspargase via desensitization protocol, tolerated it well with sustained asparginase activity.Patient #4: 3 year old male with intermediate risk T ALL, experienced immediate hypersensitivity to second dose of pegaspargase as well as subsequent Erwinaze®. He received one dose of pegaspargase via desensitization protocol during interim maintenance, tolerated it well with sustained asparginase activity.Patient #5: 15 year old male with intermediate risk T ALL, experienced immediate hypersensitivity to second dose of pegaspargase. Due to shortage of Erwinaze®, he received one dose of pegaspargase via desensitization protocol during delayed intensification and tolerated it well, but did not have sustained asparginase activity. Thus, he did not receive any further pegaspargase doses.Results: None of the five patients developed hypersensitivity reactions during their desensitization protocols. Three patients had sustained activity, one patient showed early therapeutic levels, but low activity at day 14 consistent with accelerated clearance, and one patient did not achieve therapeutic levels, consistent with neutralizing antibody. Cost of one desensitization may be more cost effective than that associated with six doses of Erwinaze®.Conclusions: Pegaspargase can be safely administered to patients who have had a prior hypersensitivity to pegaspargase using a standard rapid desensitization protocol. Per our experience, the desensitization protocol is safe and effective in maintaining sustained levels of asparaginase activity, and could be cost effective when Erwinaze® is not available. DisclosuresNo relevant conflicts of interest to declare.

Experience with generic pegylated L-asparaginase in children with acute lymphoblastic leukemia and monitoring of serum asparaginase activity

Background: Pegylated asparaginase (P-Asp) though integral to acute lymphoblastic leukemia (ALL) therapy is often not accessible to patients in developing countries. We share our clinical experience with generic P-Asp along with monitoring of asparaginase activity. Methods: In this prospective observational study, patients ≤18 years of age with ALL were assigned to receive either generic P-Asp or native asparaginase (N-Asp) in a non-randomized manner. Treatment protocol was based on ALL BFM-95 backbone. The dose of P-Asp was 1500 IU/m2 by intravenous route during induction (Ia) and re-induction (IIa) phase of therapy. Results: N-Asp or P-Asp was administered to 52 and 54 of the 106 eligible patients respectively. Demographic and disease characteristics were comparable in both arms. The mean trough levels for N-Asp and P-Asp were 156.87 ± 22.35 IU/L and 216.03 ± 73.40 IU/L, respectively (p value <0.001) and all patients achieved therapeutic levels during Ia. Incidence of asparaginase-attributable toxicity was similar in the two arms in both phases of treatment, although hospitalization due to noninfectious causes was more common in P-Asp arm during Ia (13% versus 0%, p value, 0.01). Clinical hypersensitivity and silent inactivation were not observed during Ia while these occurred in 13% and 5% of patients in the N-Asp arm and P-Asp arms of IIa, respectively. The 2-year event free survival for P-Asp and N-Asp groups was 84% and 80.7%, respectively (p value 0.85). Conclusion: Generic P-Asp was observed to be efficacious and well tolerated in our patients and adequate therapeutic levels were sustained for 2 weeks.

Monitoring asparaginase activity in middle-income countries.

Monitoring asparaginase activity in middle-income countries.

Aspartate is an endogenous metabolic limitation for tumour growth.

Defining the metabolic limitations of tumour growth will help develop cancer therapies1. Cancer cells proliferate slower in tumours than in standard culture conditions, indicating that a metabolic limitation may restrict cell proliferation in vivo. Aspartate synthesis can limit cancer cell proliferation when respiration is impaired2–4; however, whether acquiring aspartate is endogenously limiting for tumour growth is unknown. We confirm aspartate has poor cell permeability, preventing environmental acquisition, whereas the related amino acid asparagine is available to cells in tumours, but cancer cells lack asparaginase activity to convert asparagine to aspartate. Heterologous expression of guinea pig asparaginase 1 (gpASNase1), an enzyme that produces aspartate from asparagine5, confers the ability to use asparagine to supply intracellular aspartate to cancer cells in vivo. Tumours expressing gpASNase1 grow at a faster rate, indicating aspartate acquisition is an endogenous metabolic limitation for growth of some tumours. Tumours expressing gpASNase1 are also refractory to the growth suppressive effects of metformin, suggesting that metformin inhibits tumour growth by depleting aspartate. These findings suggest that therapeutic aspartate suppression could be effective to treat cancer.

Efficacy and safety of recombinant E. coli asparaginase in children with previously untreated acute lymphoblastic leukemia: A randomized multicenter study of the Dutch Childhood Oncology Group.

The efficacy and safety of recombinant Escherichia coli-asparaginase (rASNase) was compared to native E.coli asparaginase (Asparaginase medac). One hundred and ninety-nine children with newly diagnosed acute lymphoblastic leukemia were randomized to receive one of both agents at a dose of 5,000 U/m² during induction (eight doses) and 10,000 U/m² during the postinduction phase (only high-risk patients; standard- and medium-risk patients received pegaspargase). Median trough serum asparaginase activity levels were comparable between both groups; they ranged from 143 to 182U/l during induction and were above the target value of 100U/l. Complete asparagine depletion in serum was achieved in 97.9% of patients, with no significant differences between both groups. On day 33 (end of induction), only two (2%) evaluable patients in each group had measurable asparagine serum levels, and complete asparagine depletion in the cerebrospinal fluid was achieved in 98.8% and 93.6% of the patients with rASNase and Asparaginase medac, respectively. During induction, 2.1% and 5% of patients developed an allergic reaction to rASNase or Asparaginase medac, respectively. Approximately 41% of the patients in both groups had a clinical allergy or enzyme inactivation to the first dose of any asparaginase preparation in postinduction. A comparable proportion of patients in both groups developed anti-asparaginase antibodies (57%) during repeated administration of asparaginase. Minimal residual disease levels at the end of induction, 5-year event-free survival, and 5-year cumulative incidence of relapse did not differ between both groups. The efficacy, safety, and immunogenicity of both asparaginase preparations are comparable. This trial was registered at www.clinicaltrials.gov as #NCT00784017; EudraCT number 2006-003180-31.

Symptomatic Hyperammonemia With Erwinia chrysanthemi-derived Asparaginase in Pediatric Leukemia Patients.

Erwinia chrysanthemi-derived asparaginase is increasingly integral to acute lymphoblastic leukemia therapy. In our series, 16% of patients developed symptomatic hyperammonemia following Erwinia administration with symptoms including refractory nausea, vomiting, profound fatigue, malaise, and coma. This series of patients receiving Erwinia indicates higher than expected incidence of hyperammonemia, correlation between ammonia and asparaginase levels and therapeutic asparaginase activity levels despite dose reduction. The series provides evidence for investigation into which patients require intervention to prevent toxicity, which patients may have ammonia levels used as an asparaginase activity surrogate and which patients may achieve equivalent efficacy with abridged dosing.

Novel mutant of Escherichia coli asparaginase II to reduction of the glutaminase activity in treatment of acute lymphocytic leukemia by molecular dynamics simulations and QM-MM studies

L-Asparaginases (ASNase) belong to a family of amidohydrolases, have both asparaginase and glutaminase activity. Acute lymphocytic leukemia (ALL) is an outrageous disease worldwide. Bacterial ASNase has been used for the treatment of ALL. Glutaminase activity of enzyme causes some side effect and it is not essential for anticancer activity. The aim of this study was engineering of Escherichia coli asparaginase II to find a mutant with reduced glutaminase activity by molecular docking, molecular dynamics (MD) and QM-MM (Quantum mechanics molecular dynamics) simulations. Residues with low free energy of binding to Asn and high free binding energy to Gln were chosen for mutagenesis. Then, a mutant with higher glutaminase free binding energy was selected for further studies. Additionally, the MD simulation and QM-MM computation of wild type (WT) were employed and the selected mutated ASNase were analyzed and discussed. Our data showed that V27T is a good candidate to reduction the glutaminase activity, while has no remarkable effect on asparaginase activity of the enzyme. The simulation analysis revealed that V27T mutant is more stable than WT and mutant simulation was successful completely. QM-MM results confirmed the successfulness of our mutagenesis.

As Extracellular Glutamine Levels Decline, Asparagine Becomes an Essential Amino Acid

When mammalian cells are deprived of glutamine, exogenous asparagine rescues cell survival and growth. Here we report that this rescue results from use of asparagine in protein synthesis. All mammalian cell lines tested lacked cytosolic asparaginase activity and could not utilize asparagine to produce other amino acids or biosynthetic intermediates. Instead, most glutamine-deprived cell lines are capable of sufficient glutamine synthesis to maintain essential amino acid uptake and production of glutamine-dependent biosynthetic precursors, with the exception of asparagine. While experimental introduction of cytosolic asparaginase could enhance the synthesis of glutamine and increase tricarboxylic acid cycle anaplerosis and the synthesis of nucleotide precursors, cytosolic asparaginase suppressed the growth and survival of cells in glutamine-depleted medium invitro and severely compromised the invivo growth of tumor xenografts. These results suggest that the lack of asparaginase activity represents an evolutionary adaptation to allow mammalian cells to survive pathophysiologic variations in extracellular glutamine.

Pharmacokinetics of recombinant asparaginase in children with acute lymphoblastic leukemia.

The objective of this study was to assess the pharmacokinetics of recombinant asparaginase (rASNase, Spectrila®) in children with acute lymphoblastic leukemia using a population pharmacokinetic approach in order to explore potential dosing recommendations. Data on serum asparaginase activities of 124 children from three clinical studies were included in the analysis, covering an age range from 3days to 17years. Most patients received 5000U/m2 rASNase intravenously every 3days. The non-linear mixed effects modelling software (NONMEM®) was utilized to identify drivers of rASNase pharmacokinetics in children. Different dose adjustments were simulated for their ability to increase rASNase trough activities in children who do not reach the threshold of 100U/L. A two-compartment model with allometric weight scaling (0.75 on clearance [CL] and inter-compartmental clearance [Q] and 1 on central [V 1] and peripheral [V 2] volume of distribution) was the best model to describe the pharmacokinetics of rASNase. PK parameters for the median child (19.5kg) were: CL = 0.0592L/h, V 1 = 1.18L, Q = 0.307L/h, V 2 = 0.316L. Organ functions, such as liver or kidney function and laboratory values, such as fibrinogen or antithrombin III levels, showed no influence on rASNase pharmacokinetics. In simulations, changing the administration interval from 72 to 48h was appropriate to maintain rASNase activities above the therapeutic threshold, in patients with activities below 100U/L 72h after the first dose. Drug monitoring is recommended to identify patients with insufficient ASNase trough activities in serum and to modify the treatment schedule, if necessary. Shortening of the treatment interval might be preferable over increasing the rASNase dose.

A protease-resistant Escherichia coli asparaginase with outstanding stability and enhanced anti-leukaemic activity in vitro

L-Asparaginases (ASNases) have been used as first line drugs for paediatric Acute Lymphoblastic Leukaemia (ALL) treatment for more than 40 years. Both the Escherichia coli (EcAII) and Erwinia chrysanthemi (ErAII) type II ASNases currently used in the clinics are characterized by high in vivo instability, short half-life and the requirement of several administrations to obtain a pharmacologically active concentration. Moreover, they are sensitive to proteases (cathepsin B and asparagine endopeptidase) that are over-expressed by resistant leukaemia lymphoblasts, thereby impairing drug activity and pharmacokinetics. Herein, we present the biochemical, structural and in vitro antiproliferative characterization of a new EcAII variant, N24S. The mutant shows completely preserved asparaginase and glutaminase activities, long-term storage stability, improved thermal parameters, and outstanding resistance to proteases derived from leukaemia cells. Structural analysis demonstrates a modification in the hydrogen bond network related to residue 24, while Normal Mode-based geometric Simulation and Molecular Dynamics predict a general rigidification of the monomer as compared to wild-type. These improved features render N24S a potential alternative treatment to reduce the number of drug administrations in vivo and to successfully address one of the major current challenges of ALL treatment: spontaneous, protease-dependent and immunological inactivation of ASNase.

Enzymatic mitigation of acrylamide in fried potato chips using asparaginase from Aspergillus terreus

SummaryAcrylamide in foods is declared as carcinogen. In the present work, the effect of enzymatic pretreatment and other parameters like enzyme concentration, frying conditions with respect to temperature and time, size of potato chips, and effect of sodium chloride and citric acid on mitigation of acrylamide were studied. The concentration of acrylamide in fried potatoes after the pretreatment was found to be 815.63 μg kg−1. The optimised asparaginase concentration for the mitigation of acrylamide was calibrated as 4 U mL−1, and optimised frying time and temperature were 15 min and 170 °C, respectively. An in‐depth kinetic relationship for the effect of asparaginase on the mitigation of acrylamide was studied. The prime novelty of the project is focused on the immobilisation of asparaginase to nanomagnetic particles for redundant usage with stabilised enzyme activity. The work projected three stables cycles of asparaginase activity and on further usage of the immobilised enzyme resulted in decreased activity. The repeated use of immobilised asparaginase provides the advantage of decreasing cost in processing.

Salmonella FraE, an Asparaginase Homolog, Contributes to Fructose-Asparagine but Not Asparagine Utilization.

Salmonella enterica can utilize fructose-asparagine (F-Asn) as a source of carbon and nitrogen. This capability has been attributed to five genes in the fra locus. Previously, we determined that mutations in fraB (deglycase), fraD (kinase), or fraA (transporter) eliminated the ability of Salmonella to grow on F-Asn, while a mutation in fraE allowed partial growth. We hypothesized that FraE, a putative periplasmic fructose-asparaginase, converts F-Asn to NH4 + and fructose-aspartate (F-Asp). FraA could then transport F-Asp into the cytoplasm for subsequent catabolism. Here, we report that growth of the fraE mutant on F-Asn is caused by a partially redundant activity provided by AnsB, a periplasmic asparaginase. Indeed, a fraE ansB double mutant is unable to grow on F-Asn. Moreover, biochemical assays using periplasmic extracts of mutants that express only FraE or AnsB confirmed that each of these enzymes converts F-Asn to F-Asp and NH4 + However, FraE does not contribute to growth on asparagine. We tested and confirmed the hypothesis that a fraE ansB mutant can grow on F-Asp, while mutants lacking fraA, fraD, or fraB cannot. This finding provides strong evidence that FraA transports F-Asp but not F-Asn from the periplasm to the cytoplasm. Previously, we determined that F-Asn is toxic to a fraB mutant due to the accumulation of the FraB substrate, 6-phosphofructose-aspartate (6-P-F-Asp). Here, we found that, as expected, a fraB mutant is also inhibited by F-Asp. Collectively, these findings contribute to a better understanding of F-Asn utilization by Salmonella IMPORTANCE Salmonella is able to utilize fructose-asparagine (F-Asn) as a nutrient. We recently reported that the disruption of a deglycase enzyme in the F-Asn utilization pathway inhibits the growth of Salmonella in mice and recognized this pathway as a novel and specific drug target. Here, we characterize the first step in the pathway wherein FraE hydrolyzes F-Asn to release NH4 + and F-Asp in the periplasm of the cell. A fraE mutant continues to grow slowly on F-Asn due to asparaginase activity encoded by ansB.