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L-asparaginase is an essential medicine for childhood ALL. The quality of generic L-asparaginase available in India is a matter of concern. We compared four commonly used generic formulations of L-asparaginase in India.We conducted a prospective, open-label, randomised trial of four generic formulations of asparaginase for the treatment of patients with newly diagnosed intermediate-risk B-ALL. Patients were randomly assigned in a 1:1:1:1 ratio to receive generic asparaginase at a dose of at 10,000 IU/m 2 on days 9, 12, 15, and 18 of a 35-day cycle (Induction treatment). The primary end points were to determine the difference in the asparaginase activity and asparagine depletion. Historical patients who received L-asparaginase Medac (innovator) served as controls.A total of 48 patients underwent randomization; 12 patients each in the four arms. Failure to achieve predefined activity threshold of 100 IU/L was observed in 9/40 samples of Generic A (22·5%), 23/40 of Generic B (57·5%), and 43/44 (98%) each of Generic C and D. All 27 samples from seven historical patients who were administered Medac had activity gt; 100 IU/L. The average activity was significantly higher for Genericm A, 154 (70·3, 285·4) IU/L followed by Generic B 84·5(44·2, 289·1) IU/L, Generic C 45(14·4, 58·4) IU/L, and Generic D 20·4(13, 35) IU/L. Only 6 patients had asparaginase activity gt; 100 IU/L on each of the four occasions (Generic A = 5; Generic B = 1), and none of them developed Anti-Asparaginase Antibodies (AAA). On the other hand, AAA was observed in 12/36 patients who had at least one level lt; 100 IU/L (P lt; 0·05): Generic A 3/5, Generic B = 3/9, Generic D (4/11), and Generic C (5/11).Generic A and B had better trough asparaginase activity compared to Generic D and C. Overall, generic formulations had lower asparaginase activity which raises serious clinical concerns regarding their quality. Until strict regulatory enforcement improves the quality of these generics, dose adaptive approaches coupled with therapeutic drug monitoring need to be considered.

Acute lymphoblastic leukemia (ALL) is an acute, rapidly progressing and life-threatening form of cancer involving immature lymphocytes called lymphoblasts. ALL is the most common subtype of leukemia in children and adolescents. The aim of the present study was to assess the cost-utility of pegaspargase versus L-asparaginase, both followed by Erwinase in the therapy sequence, as a treatment option for pediatric, adolescent, and adult patients with ALL in Greece.A published cost-utility model comprising a decision tree and a state-transition Markov model was adapted from a public payer perspective to compare a pegaspargase treatment sequence with an L-asparaginase sequence. Efficacy and safety data, as well as utility values, were extracted from the published literature. Direct costs pertaining to drug acquisition, administration, and management of hypersensitivity were considered in the analysis (€2020). Model-extrapolated outcomes included quality-adjusted life-years (QALYs), costs, and incremental cost-effectiveness ratios (ICER). All future outcomes were discounted at 3.5% per annum. Sensitivity analyses were used to explore the impact of changing input data.The analysis showed that the pegaspargase sequence was estimated to produce 0.05 additional QALYs (18.12 vs. 18.07) and lower cost of - €1698 compared with L-asparaginase, indicating that the pegaspargase sequence was a dominant treatment strategy (improved outcomes with reduced costs) compared with L-asparaginase. Deterministic sensitivity analysis confirmed the cost-effective profile of pegaspargase. At the defined willingness-to-pay threshold of €54,000/QALY gained, probabilistic sensitivity analysis showed that pegaspargase had a 100% probability of being cost effective relative to the L-asparaginase sequence.The pegaspargase sequence was found to be less costly and more effective (in terms of QALYs) in relation to the L-asparaginase sequence, representing a dominant strategic option for Greek public payers in ALL.

L-Asparaginase (L-ASNase) is an enzyme applied in the treatment of lymphoid malignancies. However, an innovative L-ASNase with high yield and lower side effects than the commercially available preparations are still a market requirement. Here, a new-engineered Bacillus subtilis strain was evaluated for Aliivibrio fischeri L-ASNase II production, being the bioprocess development and the enzyme characterization studied. The pBS0E plasmid replicative in Bacillus sp and containing PxylA promoter inducible by xylose and its repressive molecule sequence (XylR) was used for the genetic modification. Initially, cultivations were carried out in orbital shaker, and then the process was scaled up to stirred tank bioreactor (STB). After the bioprocess, the cells were recovered and submitted to ultrasound sonication for cells disruption and intracellular enzyme recovery. The enzymatic extract was characterized to assess its biochemical, kinetic and thermal properties using L-Asparagine and L-Glutamine as substrates. The results indicated the potential enzyme production in STB achieving L-ASNase activity up to 1.539 U mL-1. The enzymatic extract showed an optimum pH of 7.5, high L-Asparagine affinity (Km = 1.2275 mmol L-1) and low L-Glutaminase activity (0.568-0.738 U mL-1). In addition, thermal inactivation was analyzed by two different Kinect models to elucidate inactivation mechanisms, low kinetic thermal inactivation constants for 25 ºC and 37 ºC (0.128 and 0.148 h-1, respectively) indicate an elevated stability. The findings herein show that the produced recombinant L-ASNase has potential to be applied for pharmaceutical purposes. published

Metabolic differences between normal and cancerous cells have been used as a point of view for developing anticancer drugs. Some degrading enzymes of certain amino acids have been regarded to kill cancerous cells. L-Asparaginase (ASNase) has shown an excellent therapeutic response to asparagine-auxotrophic cancers such as acute lymphoblastic leukemia (ALL). Some bacteria, yeasts, molds, plants, and animals produce ASNase. Bacterial ASNases from Escherichia coli and Erwinia chrysanthemi are the FDA-approved drugs for ALL treatment. Here, we review new natural prokaryotic and eukaryotic sources of ASNases, recent advances to introduce improvement strategies for the production of recombinant ASNases as well as their chemical modifications, immobilization, nanoencapsulation, and in silico studies to increase efficiency and decrease side effects. Recent studies for application of ASNases to treatment of asparagine-auxotrophic cancers, especially solid cancers, have been reviewed. Furthermore, challenges and future perspectives are discussed for this promising therapeutic enzyme. KEY POINTS: • Review recent advances to introduce new sources of microbial L-asparaginases. • Review improvement strategies for the development of stable and non-toxic L-asparaginases. • Review microbial L-asparaginase application in various cancers' treatment.

L-asparaginases are mostly obtained from bacterial sources for their application in the therapy and food industry. Bacterial L-asparaginases are employed in the treatment of Acute Lymphoblastic Leukemia (ALL) and its subtypes, a type of blood and bone marrow cancer that results in the overproduction of immature blood cells. It also plays a role in the food industry in reducing the acrylamide formed during baking, roasting, and frying starchy foods. This importance of the enzyme makes it to be of constant interest to the researchers to isolate novel sources. Presently L-asparaginases from E. coli native and PEGylated form, Dickeya chrysanthemi (Erwinia chrysanthemi) are in the treatment regime. In therapy, the intrinsic glutaminase activity of the enzyme is a major drawback as the patients in treatment experience side effects like fever, skin rashes, anaphylaxis, pancreatitis, steatosis in the liver, and many complications. Its significance in the food industry in mitigating acrylamide is also a major reason. Acrylamide, a potent carcinogen was formed when treating starchy foods at higher temperatures. Acrylamide content in food was analyzed and pre-treatment was considered a valuable option. Immobilization of the enzyme is an advancing and promising technique in the effective delivery of the enzyme than in free form. The concept of machine learning by employing the Artificial Network and Genetic Algorithm has paved the way to optimize the production of L-asparaginase from its sources. Gene-editing tools are gaining momentum in the study of several diseases and this review focuses on the CRISPR-Cas9 gene-editing tool in ALL.

Chemotherapy combined with radiotherapy could reduce the risk of recurrence in early-stage extranodal NK/T lymphoma (ENKTL). However, the optimal combined chemotherapy regimen is still unknown. Our previous study reported that LVDP (L-asparaginase, etoposide, dexamethasone, and cisplatin) combined with radiotherapy was a potentially effective and safe treatment regimen for early-stage ENKTL. This study further validated the efficacy and safety of LVDP chemotherapy combined with radiation for early-stage ENKTL with more patients and longer follow-up. We retrospectively studied 112 patients with early-stage ENKTL from September 2010 to September 2019. All patients received the LVDP regimen, and 101 of them received radiotherapy. The patients' characteristics, treatment responses, survival outcomes, prognostic factors, and toxicities were analyzed. The median follow-up was 60 months (range, 4 to 117). All patients received median 4 cycles of the LVDP chemotherapy. At the end of therapy, the objective response rate and complete response rate were 88.3% and 77.6%, respectively. The 3- and 5-year OS were 79.6% and 73.2%, and the 3- and 5-year PFS were 75.4% and 71.6%, respectively. Among them, the LVDP regimen combined with radiotherapy yielded more favorable treatment outcomes (the 3-year OS and PFS were 83.1% and 80.8%). The most common severe hematologic toxicity was leukopenia (25% grade 3/4), and the most common severe non-hematologic toxicity was increased transaminase (4.5% grade 3/4). No pancreatitis or treatment-related death occurred. The LVDP regimen combined with radiotherapy had a good therapeutic response and long-term survival with tolerable toxicity for patients with early-stage ENKTL.

The present study focused on the cloning, expression, and characterization of L-asparaginase of marine Pseudomonas aeruginosa HR03 isolated from fish intestine. Thus, a gene fragment containing the L-asparaginase sequence of Pseudomonas aeruginosa HR03 isolated from the fish intestine was cloned in the pET21a vector and then expressed in Escherichia coli BL21 (DE3) cells. Thereafter, the recombinant L-asparaginase (HR03Asnase) was purified by nickel affinity chromatography, and the enzymatic properties of HR03Asnase, including the effects of pH and temperature on HR03Asnase activity and its kinetic parameters, were determined. The recombinant enzyme HR03Asnase showed the highest similarity to type I L-asparaginase from Pseudomonas aeruginosa. The three-dimensional (3D) modeling results indicate that HR03Asnase exists as a homotetramer. Its molecular weight was 35 kDa, and the maximum activity of the purified enzyme was observed at pH8 and at 40 °C. The k

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 (2500U/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.)

Asparaginases important role in the treatment of leukemia. It is part of chemotherapy in the treatment of leukemia in the last three decades. l-Asparaginase is isolated from Fusarium sp. isolated from soil and purified using ammonium sulfate precipitation and Sephadex G 100. Characterization of the crude enzyme revealed it is a metalloprotease inhibited by EDTA. Hg2+, Cd2+, and Pb2+ also inhibited the enzyme. Mg2+, Zn2+, and Ca2+ activated l-asparaginase. Furthermore, kinetic studies of purified enzyme were carried out. Vmax and Km were 0.031 M and 454 U/mL, respectively. The optimum temperature was 30 °C and the optimum pH was 7. Concerning substrate specificity, gelatin and casein in addition to l-asparagine were tested. The enzyme was found to be nonspecific that could hydrolyze all tested substrates at different rates. The maximum enzyme activity was recorded in the case of l-asparagine, followed by casein and gelatin, respectively. The molecular weight of l-asparaginase was 22.5 kDa. The antileukemic cytotoxicity assay of the enzyme against RAW2674 leukemic cell lines by MTT viability test was estimated. The enzyme exhibited antileukemic activity with IC50 of 50.1 UmL−1. The current work presents additional information regarding the purification and characterization of the enzyme produced by Fusarium sp. and its evaluation as a potential antileukemic chemotherapeutic agent.

SummaryIntroduction. Erwinase® (native Erwinia chrysanthemi L-Asparaginase (nErA)) is an approved second-line treatment for acute lymphoblastic leukaemia (ALL) in children and adolescents, who develop hypersensitivity or neutralising antibodies to E.coli derived L-Asparaginases (ASNases). However, nErA has a short in vivo half-life requiring frequent dosing schedules in patients. In this study, nErA was covalently conjugated to PEG molecules with the aim of extending its half-life in vivo. Methods. Firstly, efficacy of this novel product PEG-nErA was investigated on human ALL cell lines (Jurkat, CCRF-CEM and CCRF-HSB2), in vitro. Secondly, its pharmacokinetic (PK) and pharmacodynamic (PD) characteristics were determined, in vivo (12 rats in each group). Results. It was found that the specific activity (U/mg of enzyme) and the kinetic constant (KM) of nErA remained unaltered post PEGylation. PEG-nErA was shown to have similar cytotoxicity to nErA (IC50: 0.06–0.17 U/mL) on human ALL cell lines, in vitro. Further, when compared to nErA, PEG-nErA showed a significantly improved half-life in vivo, which meant that L-Asparagine (Asn) levels in plasma remained depleted for up to 25 days with a four-fold lower dose (100 U/kg) compared with 72 h for nErA at 400 U/kg dose. Conclusion. Overall, this next generation product PEG-nErA (with improved PK and PD characteristics compared to nErA) would bring a significant advantage to the therapeutic needs of ALL patients and should be further explored in clinical trials.

Asparaginase therapy is a vital agent in the treatment of acute lymphoblastic leukemia (ALL), with increasing evidence of its high importance in high-risk ALL populations. However, despite the clear clinical and biological benefits of asparaginase therapy, many patients experience toxicities. A well-known treatment-limiting toxicity is asparaginase-associated pancreatitis (AAP). If severe, it necessitates discontinuation of asparaginase therapy, which can lead to a higher risk of relapse in patients with ALL. New protocols for ALL therapy have increased overall total doses of asparaginase therapy in select high-risk populations and have incorporated longer half-life formulations of pegylated asparaginase. Treatment drug monitoring has also allowed assurance of adequate levels of asparagine depletion throughout treatment. It is currently unknown if these changes will increase rates of AAP. Interestingly, important pharmacogenomics data, such as single nucleotide polymorphisms, can identify patients at the highest risk for severe AAP. The incidence of AAP in recent trials, current pharmacogenomic data that could further our understanding of the disease, and the importance of cautiously re-exposing patients to further asparaginase treatment after an initial episode of AAP are discussed.

Malignant cells often demonstrate a proliferative advantage when compared to non-malignant cells. However, the rapid growth and metabolism required for survival can also highlight vulnerabilities specific to these malignant cells. One such vulnerability exhibited by cancer is an increased demand for amino acids (AAs), which often results in a dependency on exogenous sources of AAs or requires upregulation of de novo synthesis. These metabolic alterations can be exploited by therapy, which aims to improve treatment outcome and decrease relapse and reoccurrence. One clinically utilised strategy targeting AA dependency is the use of asparaginase in the treatment of acute lymphoblastic leukaemia (ALL), which results in a depletion of exogenous asparagine and subsequent cancer cell death. Examples of other successful strategies include the exploitation of arginine deiminase and methioninase, nutrient restriction of methionine and the inhibition of glutaminase. In this review, we summarise these treatment strategies into three promising avenues: AA restriction, enzymatic depletion and inhibition of metabolism. This review provides an insight into the complexity of metabolism in cancer, whilst highlighting these three current research avenues that have support in both preclinical and clinical settings.

PURPOSE: We evaluated effects of asparaginase dosage, schedule, and formulation on CSF asparagine in children with acute lymphoblastic leukemia (ALL). METHODS: We evaluated CSF asparagine (2114 samples) and serum asparaginase (5007 samples) in 482 children with ALL treated on the Total XVI study (NCT00549848). Patients received one or two 3,000 IU/m(2) IV pegaspargase doses during induction and were then randomized in continuation to receive 2,500 IU/m(2) or 3,500 IU/m(2) IV intermittently (4 doses) on the low-risk (LR) or continuously (15 doses) on the standard/high risk (SHR) arms. A pharmacokinetic-pharmacodynamic model was used to estimate the duration of CSF asparagine depletion below 1 uM. RESULTS: During induction, CSF asparagine depletion after two doses of pegaspargase was twice as long as one dose (median 30.7 vs 15.3 days, p

Asparaginase (ASNase) is a key component in the treatment protocols of childhood acute lymphoblastic leukemia (ALL). Asparagine synthetase (ASNS) and the basic region leucine zipper activating transcription factor 5 (ATF5) mediate the anti-leukemic effect of ASNase. Only a few reports studied the association between polymorphisms in these genes and treatment-related toxicity and response. Therefore, the current study aimed to investigate the association of ASNS and ATF5 polymorphisms with the susceptibility to ASNase-related toxicity and disease outcome in a population of childhood ALL Egyptian patients. In this study, 88 children with ALL were enrolled and genotyped for ASNS T629A and ATF5 C362T polymorphisms using allelic discrimination assay. The studied polymorphisms did not associate with hypersensitivity or thrombosis, while the ATF5 C362T polymorphism was associated significantly with decreased ASNase-associated pancreatitis (AAP) risk under the dominant model. Patients carrying TT/CT genotypes of ATF5 C362T polymorphism had a significantly better overall survival (OS) and longer event-free survival (EFS) compared to patients with CC genotype. Multivariate analysis confirmed the independent prognostic value of the ATF5 C362T dominant model. ATF5 362TT and CT genotypes were associated with decreased risk to develop AAP and better disease outcome demonstrating a low risk for events and superior survival.

In this study, we examined endophytic fungi in leaves of Mandevilla catimbauensis, an endemic plant species found in the Brazilian dry forest (Caatinga), and endophytic fungi’s potential to produce L-asparaginase (L-ASNase). In total, 66 endophytes were isolated, and the leaf-fragment colonisation rate was 11.78%. Based on morphology, internal transcribed spacer (ITS), and partial large subunit (LSU) of ribosomal DNA sequencing, the endophytic fungi isolated belonged to six Ascomycota orders (Botryosphaeriales, Capnodiales, Diaporthales, Eurotiales, Marthamycetales, and Pleosporales). Phyllosticta species were the most frequent endophytes isolated (23 isolates [45.1%] from two species). The Shannon–Wiener and Fisher alpha index average values were 0.56 and 3.26, respectively. Twenty endophytes were randomly selected for the L-ASNase production test, of which fourteen isolates showed potential to produce the enzyme (0.48–2.22 U g(−1)), especially Phyllosticta catimbauensis URM 7672 (2.22 U g(−1)) and Cladosporium sp. G45 (2.11 U g(−1)). Phyllosticta catimbauensis URM 7672 was selected for the partial optimisation of L-ASNase production because of its ability to generate considerable amounts of enzyme. We obtained the highest L-ASNase activity (3.47 U g(−1)), representing an increase of 36.02% in enzymatic production, under the following experimental conditions: a pH of 4.2, 1.0% inoculum concentration, and 2.5% L-asparagine concentration. Our study showed that M. catimbauensis harbours an important diversity of endophytic fungi with biotechnological potential for L-ASNase production. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00505-3.

Distillation of fermented sugarcane juice produces both rum and cachaça, significant sources of revenue in Brazil and elsewhere. In this study, we provide a genomic analysis of a Saccharomyces cerevisiae strain isolated from a cachaça distillery in Brazil. We determined the complete genome sequence of a strain with high flocculation capacity, high tolerance to ethanol, osmotic and heat shock stress and high fermentation rates and compared the sequence with that of the reference S288c genome as well as those of two other cachaça strains. Single-nucleotide polymorphism analysis identified alterations in genes involved in nitrogen and organic compound metabolism, integrity of organelles and ion homeostasis. The strain exhibited fragmentation of several flocculation genes relative to the reference genome, as well as loss of a stop codon in the FLO8 gene, which encodes a transcription factor required for FLO gene expression. The strain contained no genes not present in the reference genome strain but did lack several genes, including asparaginase genes, maltose utilization loci, and several genes from the tandem array of the DUP240 family. The three cachaça strains lacked different sets of genes, but the asparaginase genes and several of the DUP240 genes were common deficiencies. This study provides new insights regarding the selective pressure of sugarcane fermentation on the genome of yeast strains and offers additional genetic resources for modern synthetic biology and genome editing tools. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-021-00444-z.

Asparaginases, key agents in treatment of acute lymphoblastic leukemia (ALL), are associated with venous thromboembolism (VTE). While risks of short-acting asparaginase-related VTE is well-known, we studied VTE incidence and risk factors in adult ALL patients treated with and without long-acting pegylated asparaginase (PegA). Single-center, retrospective analysis of 89 ALL patients treated with (n = 61) or without (n = 28) PegA at Greenebaum Comprehensive Cancer Center. Reviewed patient and disease characteristics, treatment, and VTE incidence. VTE during treatment occurred in 31 patients (35%), and was associated with PegA (p = 0.001) and Philadelphia chromosome negativity (p = 0.002). Among PegA recipients, VTE was associated with a significantly higher mean body mass index (BMI) of 31.3 kg/m2 (p = 0.037), and was more common with pre-T/T cell compared to pre-B/B cell ALL (68.2% vs. 33.3%, p = 0.009). Antithrombin-III (ATIII) levels were measured for 26 patients; 16 (61.5%) were

Complex karyotype acute myeloid leukemia (CK-AML) has a dismal outcome with current treatments, underscoring the need for new therapies. Here, we report synergistic anti-leukemic activity of the BCL-2 inhibitor venetoclax (Ven) and the asparaginase formulation Pegylated Crisantaspase (PegC) in CK-AML in vitro and in vivo. Ven-PegC combination inhibited growth of multiple AML cell lines and patient-derived primary CK-AML cells in vitro. In vivo, Ven-PegC showed potent reduction of leukemia burden and improved survival, compared with each agent alone, in a primary patient-derived CK-AML xenograft. Superiority of Ven-PegC, compared to single drugs, and, importantly, the clinically utilized Ven-azacitidine combination, was also demonstrated in vivo in CK-AML. We hypothesized that PegC-mediated plasma glutamine depletion inhibits 4EBP1 phosphorylation, decreases the expression of proteins such as MCL-1, whose translation is cap dependent, synergizing with the BCL-2 inhibitor Ven. Ven-PegC treatment decreased cellular MCL-1 protein levels in vitro by enhancing eIF4E-4EBP1 interaction on the cap-binding complex via glutamine depletion. In vivo, Ven-PegC treatment completely depleted plasma glutamine and asparagine and inhibited mRNA translation and cellular protein synthesis. Since this novel mechanistically-rationalized regimen combines two drugs already in use in acute leukemia treatment, we plan a clinical trial of the Ven-PegC combination in relapsed/refractory CK-AML.

Background While current chemotherapy has increased cure rates for children with acute lymphoblastic leukaemia (ALL), the largest number of relapsing patients are still stratified as medium risk (MR) at diagnosis (50–60%). This highlights an opportunity to develop improved relapse-prediction models for MR patients. We hypothesised that bone marrow from MR patients who eventually relapsed would regrow faster in a patient-derived xenograft (PDX) model after induction chemotherapy than samples from patients in long-term remission. Methods Diagnostic bone marrow aspirates from 30 paediatric MR-ALL patients (19 who relapsed, 11 who experienced remission) were inoculated into immune-deficient (NSG) mice and subsequently treated with either control or an induction-type regimen of vincristine, dexamethasone, and L-asparaginase (VXL). Engraftment was monitored by enumeration of the proportion of human CD45+ cells (%huCD45+) in the murine peripheral blood, and events were defined a priori as the time to reach 1% huCD45+, 25% huCD45+ (TT25%) or clinical manifestations of leukaemia (TTL). Results The TT25% value significantly predicted MR patient relapse. Mutational profiles of PDXs matched their tumours of origin, with a clonal shift towards relapse observed in one set of VXL-treated PDXs. Conclusions In conclusion, establishing PDXs at diagnosis and subsequently applying chemotherapy has the potential to improve relapse prediction in paediatric MR-ALL.

Biocompatible, environmentally responsive, and scalable nanocarriers are needed for targeted and triggered delivery of therapeutic proteins. Suitable polymers, preparation methods, and crosslinking chemistries must be considered for nanogel formation. Biocompatible dendritic polyglycerol (dPG) is used in the mild, surfactant-free inverse nanoprecipitation method for nanogel preparation. The biocompatible, fast, and bioorthogonal inverse electron demand Diels-Alder (iEDDA) crosslinking chemistry is used. In this work, the synthesis of pH-degradable nanogels, based on tetrazine, norbonene, and bicyclo[6.1.0]nonyne (BCN) functionalized macromonomers, is reported. The macromonomers are non-toxic up to 2.5 mg mL−1 in three different cell lines. Nanogels are obtained in the size range of 47 to 200 nm and can be degraded within 48 h at pH 4.5 (BA-gels), and pH 3 (THP-gels), respectively. Encapsulation of asparaginase (32 kDa) yield encapsulation efficiencies of up to 93% at 5 wt.% feed. Overall, iEDDA-crosslinked pH-degradable dPG-nanogels from inverse nanoprecipitation are promising candidates for biomedical applications.

L-asparaginases are extensively applied in the treatment of Acute Lymphoblastic Leukemia (ALL). The treatment regime of ALL consists of asparaginase from E. coli or Erwinia. The survival rate post-chemotherapy has increased to

PURPOSE: Treatment-related pancreatitis (TRP) is a serious complication occurring in children with acute lymphoblastic leukemia (ALL). Those affected are at high risk for severe organ toxicity and treatment delays that can impact outcomes. TRP is associated with asparaginase, a standard therapeutic agent in childhood ALL. Native American ancestry, older age, high-risk leukemia, and increased use of asparaginase are linked to pancreatitis risk. However, dedicated genetic studies evaluating pancreatitis in childhood ALL include few Hispanics. Thus, the genetic basis for higher risk of pancreatitis among Hispanic children with ALL remains unknown. METHODS: Cases of children with ALL treated in from 1994 through 2013 were reviewed and identified 14, all Hispanic, who developed pancreatitis related to asparaginase therapy. Forty-six controls consisting of Hispanic children treated on the same regimens without pancreatitis were selected for comparison. Total DNA isolated from whole blood was used for targeted DNA sequencing of 23 selected genes, including genes associated with pancreatitis without ALL and genes involved in asparagine metabolism. RESULTS: Non-synonymous polymorphisms and frameshift deletions were detected in 15 genes. Most children with TRP had variants in ABAT, ASNS, and CFTR. Notably, children with TRP harbored many more CFTR variants (71.4%) compared to controls (39.1%). Among these, V470M (rs213950) was most frequent (OR 4.27, p=0.029). CONCLUSIONS: This is the first study of genetic factors in treatment-related pancreatitis in Hispanic children with ALL. Identifying correlative variants in ethnically vulnerable populations may improve screening to identify which patients with ALL are at greatest risk for pancreatitis.

Nature Communications 12(1), 6717 (2021). doi:10.1038/s41467-021-27105-x, Rhizobium etli, a nitrogen-fixing bacterial symbiont of legume plants, encodes an essential l-asparaginase (ReAV) with no sequence homology to known enzymes with this activity. High-resolution crystal structures of ReAV show indeed a structurally distinct, dimeric enzyme, with some resemblance to glutaminases and ��-lactamases. However, ReAV has no glutaminase or lactamase activity, and at pH 9 its allosteric asparaginase activity is relatively high, with K$_m$ for $_l$-Asn at 4.2 mM and k$_{cat}$ of 438 s$^{���1}$. The active site of ReAV, deduced from structural comparisons and confirmed by mutagenesis experiments, contains a highly specific Zn$^{2+}$ binding site without a catalytic role. The extensive active site includes residues with unusual chemical properties. There are two Ser-Lys tandems, all connected through a network of H-bonds to the Zn center, and three tightly bound water molecules near Ser48, which clearly indicate the catalytic nucleophile., Published by Nature Publishing Group UK, [London]

Background Trichoderma is a genus of fungi in the family Hypocreaceae and includes species known to produce enzymes with commercial use. They are largely found in soil and terrestrial plants. Recently, Trichoderma simmonsii isolated from decaying bark and decorticated wood was newly identified in the Harzianum clade of Trichoderma. Due to a wide range of applications in agriculture and other industries, genomes of at least 12 Trichoderma spp. have been studied. Moreover, antifungal and enzymatic activities have been extensively characterized in Trichoderma spp. However, the genomic information and bioactivities of T. simmonsii from a particular marine-derived isolate remain largely unknown. While we screened for asparaginase-producing fungi, we observed that T. simmonsii GH-Sj1 strain isolated from edible kelp produced asparaginase. In this study, we report a draft genome of T. simmonsii GH-Sj1 using Illumina and Oxford Nanopore technologies. Furthermore, to facilitate biotechnological applications of this species, RNA-sequencing was performed to elucidate the transcriptional profile of T. simmonsii GH-Sj1 in response to asparaginase-rich conditions. Results We generated ~ 14 Gb of sequencing data assembled in a ~ 40 Mb genome. The T. simmonsii GH-Sj1 genome consisted of seven telomere-to-telomere scaffolds with no sequencing gaps, where the N50 length was 6.4 Mb. The total number of protein-coding genes was 13,120, constituting ~ 99% of the genome. The genome harbored 176 tRNAs, which encode a full set of 20 amino acids. In addition, it had an rRNA repeat region consisting of seven repeats of the 18S-ITS1–5.8S-ITS2–26S cluster. The T. simmonsii genome also harbored 7 putative asparaginase-encoding genes with potential medical applications. Using RNA-sequencing analysis, we found that 3 genes among the 7 putative genes were significantly upregulated under asparaginase-rich conditions. Conclusions The genome and transcriptome of T. simmonsii GH-Sj1 established in the current work represent valuable resources for future comparative studies on fungal genomes and asparaginase production.

In this presented work, magnetic poly(HEMA-GMA) nanoparticles were synthesized, characterized, and used for immobilization of an anti-leukemic enzyme L-asparaginase. The average particle size of the synthesized magnetic nanoparticles was found to be as 117.5 nm. L-asparaginase was successfully immobilized onto the synthesized magnetic nanoparticles, and attached amount of L-asparaginase was found to be as 66.43 mg/g nanoparticle. The effects of the medium pH, temperature, and substrate concentration on the L-asparaginase activity were also tested. Optimum pH of the free and immobilized L-asparaginase was found to be as 7.5 and 6.5, respectively. Optimum temperature of the free L-asparaginase was 45 °C, while optimum temperature was shifted to 55 °C after immobilization onto the magnetic nanoparticles. Also, kcat value of free L-asparaginase (47,356 min−1) was calculated to be higher than that of immobilized L-asparaginase (497 min−1). Thermal stability of both asparaginase preparation was followed for 10 h, and at the end of the incubation time, free asparaginase almost lost its activity, while immobilized asparaginase protected 50% of its initial activity. Storage stabilities of free and immobilized asparaginase were also tested, and at the end of the 40 days storage, free asparaginase lost all of its activity, while immobilized asparaginase still showed 30% activity. Operational stability of immobilized asparaginase was tested for 8 successive usage, and immobilized asparaginase lost only 15% of its initial activity. In present study, activities of free and immobilized L-asparaginase were tested in artificial human serum medium, to foresee the in vivo efficiency, and it was demonstrated here that immobilized L-asparaginase protected its 74.74% of its initial activity in artificial serum medium.

L-asparaginase (E.C.3.5.1.1) is an important enzyme that has been purified and characterized for over decades to study and evaluate its anti-carcinogenic activity against different lymphoproliferative disorders such as acute lymphoblastic leukemia (ALL) and Hodgkin's lymphoma. The ability of the enzyme to convert L-asparagine into aspartic acid and ammonia is the reason behind its anti-cancerous activity. Apart from its medicinal uses, it is widely used in food industry to tackle acrylamide, a probable human carcinogen and, production in carbohydrate-rich foods cooked at high temperatures. There are variety of organisms including microorganisms such as bacteria, fungi, algae, and plants that produce L-asparaginase. The enzyme obtained from different microbial and plant sources have different physiochemical properties and kinetic parameters. L-asparaginases have an optimum pH range between 6 and 10 and an optimum temperature between 37 and 85 °C. This article has reviewed the lowest molecular mass for L-asparaginase in Yersinia pseudotuberculosis Q66CJ2 which is 36.27 kDa, while the highest for Pseudomonas otitidis which has a molecular mass of 205 ± 3 kDa. This review is an attempt to summarize most of the available sources, their phylogenetic relationships, purification methods, data regarding different physiochemical and kinetic properties of L-asparaginase.

The delayed intensification (DI) enhanced outcome for patients with acute lymphoblastic leukemia (ALL) treated on BFM 76/79 and CCG 105 after a prednisone-based induction. Childrens Oncology Group protocols P9904/9905 evaluated DI via a post-induction randomization for eligible National Cancer Institute (NCI) standard (SR) and high-risk (HR) patients. A second randomization compared intravenous methotrexate (IV MTX) as a 24- (1 g/m(2)) vs. 4-h (2 g/m(2)) infusion. NCI SR patients received a dexamethasone-based three-drug and NCI HR/CNS 3 SR patients a prednisone-based four-drug induction. End induction MRD (minimal residual disease) was obtained but did not impact treatment. DI improved the 10-year continuous complete remission (CCR) rate; 75.5 ± 2.5% vs. 81.8 ± 2.2% p = 0.002, whereas MTX administration did not; 4-h 80.8 ± 1.9%; 24-h 81.4 ± 1.9% (p = 0.7780). Overall survival (OS) at 10 years did not differ with DI: 91.4 ± 1.6% vs. 90.9 ± 1.7% (p = 0.25) without but was higher with the 24-h MTX infusion; 4-h 91.1 ± 1.4%; 24-h 93.9 ± 1.2% (p = 0.0209). MRD predicted outcome; 10-year CCR 87.7 ± 2.2 and 82.1 ± 2.5% when MRD was

Erwinia chrysanthemi asparaginase is an important drug used in cancer treatment, especially in acute lymphoblastic leukemia (ALL). Escherichia coli periplasmic space is an ideal compartment for recombinant expression of certain proteins. To efficient secretion, an appropriate signal sequence should be chosen for each protein individually. In this study, the proper signal sequences for secretory production of Erwinia asparaginase were predicted by in silico methods. Consequently, two signal peptides, OmpA and DsbA, were selected for secretory expression of the enzyme in E. coli. Asparaginase was translocated through the cytoplasmic membrane with either DsbA signal peptide or OmpA signal peptide; Using DsbA signal peptide, 5.95 units of the enzyme was obtained per milliliter of culture media, whereas OmpA signal sequence led to some amount of periplasmic expression. Our study showed that the co-translational signal peptide, DsbA, targeted the asparaginase to cell membrane more efficiently in comparison to the post-translational signal peptide, OmpA. The combination of in silico and experimental approaches provides a way to test a wide variety of signal sequences for secretory production of the enzyme in a time- and cost-effective manner. It is a fundamental step for further studies in the enzyme production process.

The NOPHO ALL2008 is a population-based study using an unmodified pediatric protocol in patients 1-45 years of age with acute lymphoblastic leukemia. Patients with T-ALL were given a traditional pediatric scheme if fast responding (minimal residual disease (MRD) 0.1% day 29), or intensive block-based chemotherapy if slow responding (MRD 0.1% day 29). Both treatment arms included pediatric doses of high-dose methotrexate and asparaginase. If MRD ≥ 5% on day 29 or ≥0.1% after consolidation, patients were assigned to allogeneic hematopoietic stem cell transplantation. The 5-year overall survival of the 278 T-ALL patients was 0.75 (95% CI 0.69-0.81), being 0.82 (0.74-0.88) for patients 1.0-9.9 years, 0.76 (0.66-0.86) for those 10.0-17.9 years, and 0.65 (0.55-0.75) for the older patients. The risk of death in first remission was significantly higher in adults (12%) compared with the 1-9 years group (4%). The MRD responses in the three age groups were similar, and only a nonsignificant increase in relapse risk was found in adults. In conclusion, an unmodified pediatric protocol in patients 1-45 years is effective in all age groups. The traditional pediatric treatment schedule was safe for all patients, but the intensive block therapy led to a high toxic death rate in adults.

Optimal supportive care for disseminated intravascular coagulation (DIC) and hemostatic complications by asparaginase is indispensable for the successful treatment of pediatric leukemia. However, the situation regarding this type of care in Japan is unclear. We conducted a questionnaire-based survey at 155 institutions treating childhood leukemia in Japan. The questionnaire asked about the supportive care provided by each institution to acute leukemia patients with DIC and asparaginase-induced hemostatic alterations. Ninety-eight institutions responded. The most common diagnostic criteria for DIC were those established by the Japanese Ministry of Health and Welfare. Regardless of the etiology underlying DIC, recombinant human thrombomodulin and synthetic protease inhibitors were used as anticoagulation therapy by around 70% and 40% of institutions, respectively. Additionally, 92%, 93%, and 73% of institutions measured plasma antithrombin, fibrinogen, and D-dimer/fibrin degradation products, respectively, more than twice per week during induction therapy for acute lymphoblastic leukemia. Survey responses indicate that 95% and 24% of the institutions used antithrombin replacement and fresh-frozen plasma, respectively. Supportive care for DIC and/or asparaginase-induced hemostatic alterations at Japanese pediatric centers was intensive and differs markedly from protocols in other countries. The efficacy of supportive care should be evaluated prospectively in the setting of pediatric leukemia.

The recombinant human asparaginase (rhASP) plays an important role in the treatment of acute lymphoblastic leukemia. In the present work, volumetric mass transfer coefficient (kLa) values are derived from the E. coli cultivation under different agitation and aeration conditions, in order to improve the rhASP productivity. The aeration and agitation conditions were systematically optimized by the kLa. The maximum biomass (2.4 g/L) and rhASP (1.68 g/L) are achieved with the kLa of 0.024 s-1 at 1.5 lpm and 700 rpm process conditions. The kinetic properties of purified rhASP are also extensively studied and optimized for the maximal enzyme activity. The optimal pH, temperature and incubation duration conditions for accomplishing maximum enzyme activity are found to be 9.0, 40°C, and 30 min, respectively. The optimum substrate concentration and substrate specificity for the highest enzyme activity are of 0.07 M and L-asparagine, respectively. The enzyme activity (204 IU/mL) is significantly improved in the presence of sodium metal (Na+) ions and the inhibitors 2- mercaptoethanol, bromoacetic acid and urea have presented the highest inhibition rate on rhASP activity. The enzyme kinetic parameters Km and Vmax of the purified rhASP are recorded as 2.25 mM and 250 IU/mL, respectively. This work provides the extensive characteristic properties of rhASP enzyme, which enables us to place in a competition for the development of oncology drugs.

The standard treatment for adult acute lymphoblastic leukemia (ALL) is undefined. Patients with newly diagnosed standard-risk ALL at a single institution were retrospectively analyzed. From 2010 to 2017, 46 patients were treated using the modified Berlin-Frankfurt-Münster (BFM)-ALL-95 regimen. Hematologic and molecular complete remission (CR) rates of 91.3% and 76.1% were achieved. The 5-year event-free survival (EFS) and overall survival (OS) rates for all patients in the cohort were 58.0% (95% confidence interval, 42.1-73.9%) and 66.7% (95% CI, 51.4-82.0%), respectively. No patient presented with central nervous system involvement after CR in this study. This condition could be related to four doses of high-dose methotrexate (MTX) every 3 months during the maintenance phase. Multivariate analysis revealed that minimal residual disease positive and time interval between induction IA and Protocol M of more than 70 days were independent adverse factors for EFS and OS. One or more instances of grade 4 myelosuppression occurred during induction therapy. Nonhematological side effects were mild. No toxicity-related deaths were observed in the entire cohort. The data indicated that the modified regimen is well tolerated and can produce the promising outcomes in Chinese adults with standard-risk ALL.

Despite considerable efforts to identify cancer metabolic alterations that might unveil druggable vulnerabilities, systematic characterizations of metabolism as it relates to functional genomic features and associated dependencies remain uncommon. To further understand the metabolic diversity of cancer, we profiled 225 metabolites in 928 cell lines from more than 20 cancer types in the Cancer Cell Line Encyclopedia (CCLE) using liquid chromatography–mass spectrometry (LC-MS). This resource enables unbiased association analysis linking the cancer metabolome to genetic alterations, epigenetic features and gene dependencies. Additionally, by screening barcoded cell lines, we demonstrated that aberrant ASNS hypermethylation sensitizes subsets of gastric and hepatic cancers to asparaginase therapy. Finally, our analysis revealed distinct synthesis and secretion patterns of kynurenine, an immune-suppressive metabolite, in model cancer cell lines. Together, these findings and related methodology provide comprehensive resources that will help clarify the landscape of cancer metabolism. Systematic metabolite profiling across cancer cell lines uncovers patterns associated with genetic and epigenetic features and reveals dysregulated metabolic states that can be exploited for anticancer therapy

Background A cellular stress response (CSR) is triggered upon recombinant protein synthesis which acts as a global feedback regulator of protein expression. To remove this key regulatory bottleneck, we had previously proposed that genes that are up-regulated post induction could be part of the signaling pathways which activate the CSR. Knocking out some of these genes which were non-essential and belonged to the bottom of the E. coli regulatory network had provided higher expression of GFP and L-asparaginase. Results We chose the best performing double knockout E. coli BW25113ΔelaAΔcysW and demonstrated its ability to enhance the expression of the toxic Rubella E1 glycoprotein by 2.5-fold by tagging it with sfGFP at the C-terminal end to better quantify expression levels. Transcriptomic analysis of this hyper-expressing mutant showed that a significantly lower proportion of genes got down-regulated post induction, which included genes for transcription, translation, protein folding and sorting, ribosome biogenesis, carbon metabolism, amino acid and ATP synthesis. This down-regulation which is a typical feature of the CSR was clearly blocked in the double knockout strain leading to its enhanced expression capability. Finally, we supplemented the expression of substrate uptake genes glpK and glpD whose down-regulation was not prevented in the double knockout, thus ameliorating almost all the negative effects of the CSR and obtained a further doubling in recombinant protein yields. Conclusion The study validated the hypothesis that these up-regulated genes act as signaling messengers which activate the CSR and thus, despite having no casual connection with recombinant protein synthesis, can improve cellular health and protein expression capabilities. Combining gene knockouts with supplementing the expression of key down-regulated genes can counter the harmful effects of CSR and help in the design of a truly superior host platform for recombinant protein expression.

The mechanism of catalysis by the l-glutaminase-asparaginase from Pseudomonas 7A (PGA) was investigated using structural, mass spectrometry, and kinetic data. We had previously proposed mechanism of hydrolysis of l-Asn by the type II l-asparaginase from E. coli (EcAII), but that work was limited to just one enzyme. Based on results presented in this report, we postulate that all homotetrameric l-asparaginases from mesophilic bacteria utilize a common ping-pong mechanism of catalysis consisting of two subsequent nucleophilic substitutions. Several new structures of non-covalent complexes of PGA with different substrates, as well as structures of covalent acyl-enzyme intermediates of PGA with canonical substrates (l-Asp and l-Glu) and an opportunistic ligand, a citrate anion, were determined. The results of kinetic experiments monitored by high-resolution LC/MS, when combined with new structural data, clearly show that the reaction catalyzed by l-glutaminase-asparaginases proceeds through formation of a covalent intermediate, as observed previously for EcAII. Additionally, by showing that the same mechanism applies to l-Asn and l-Gln, we postulate that it is common for all these structurally related enzymes.

Background Although hyperglycemia and pancreatitis are known side effects of L-asparaginase, both contributing to the development of diabetic ketoacidosis (DKA) is unfamiliar in literature. Case presentation We report a case of an adolescent girl, recently diagnosed with ALL, who presented with pain in abdomen and breathing difficulty following chemotherapy with L-asparaginase. On subsequent evaluation, she was found to have high anion gap metabolic acidosis, hyperglycemia and ketonuria. Ultrasonogram showed bulky pancreas. DKA was managed with fluid correction and insulin infusion. Pancreatitis was managed conservatively. She recovered completely with resolution of symptoms and without any major adverse events despite having such severe complications. Conclusion We conclude that the combination of DKA and pancreatitis is a rare occurrence with significant morbidity and mortality. We recommend a close monitoring of blood glucose levels for hyperglycemia as well as a high index of clinical suspicion for pancreatitis in patients with ALL receiving L-asparaginase.

The genome sequence of Pyrobaculum calidifontis contains two open reading frames, Pcal_0144 and Pcal_0970, exhibiting homology with l-asparaginases. In search of a thermostable l-asparaginase with no glutaminase activity, we have cloned and expressed the gene encoding Pcal_0970 in Escherichia coli. Recombinant Pcal_0970 was produced in insoluble and inactive form which was solubilized and refolded into enzymatically active form. The refolded Pcal_0970 showed the highest activity at or above 100 °C. Optimum pH for the enzyme activity was 6.5. Addition of divalent metal cations or EDTA had no significant effect on the activity. The enzyme was capable of hydrolyzing d-asparagine with a 20% activity as compared to 100% with l-asparagine. Pcal_0970 did not show any detectable activity when l-glutamine or d-glutamine was used as substrate. Pcal_0970 exhibited a Km value of 4.5 ± 0.4 mmol/L and Vmax of 355 ± 13 μmol min−1 mg−1 towards l-asparagine. The activation energy, from the linear Arrhenius plot, was determined as 39.9 ± 0.6 kJ mol−1. To the best of our knowledge, Pcal_0970 is the most thermostable l-asparaginase with a half-life of more than 150 min at 100 °C and this is the first report on characterization of an l-asparaginase from phylum Crenarchaeota.

Peripheral T cell lymphomas (PTCL) are less responsive to anthracycline-containing regimen such as CHOP and carry a poor prognosis. In this prospective study, we investigated gemcitabine, cisplatin, and dexamethasone (GDP) combined with methotrexate (MTX) and pegaspargase (PEG-L) as front-line treatment in PTCL. Eligible newly diagnosed PTCL patients received 4 cycles of the GDP-ML chemotherapy every 28 days. After 4 cycles, responding patients continued to receive either autologous stem cell transplantation or the MTX/cytarabine (MA) regimen for consolidation. This trial is registered with www.chictr.org.cn (ChiCTR-ONC-12002055). A total of 65 patients were enrolled with a median follow-up of 38.5 months. The overall response rate (ORR) was 55.4%, and complete remission rate (CR) was 33.8%. The median overall survival (OS) was 16 months, and the 1-year and 2-year OS were 59.1% and 38.2%, respectively. The median PFS was only 8 months. The main adverse event was hematologic toxicity: 50% patients showed grade III/IV neutropenia. GDP-ML for the first-line treatment of PTCL patients is an effective induction regimen compared with standard CHOP, and the toxicity was more significant but acceptable. However, future studies exploring new drug combinations are warranted to overcome relapse after remission. ClinicalTrials.gov Identifier: NCT02987244

Made available in DSpace on 2018-12-11T17:22:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-01-01 L-asparaginase or ASNase (L-asparagine aminohydrolase, E.C.3.5.1.1) is an enzyme clinically accepted as an antitumor agent to treat acute lymphoblastic leukemia (ALL) and lymphosarcoma through the depletion of L-asparagine (L-Asn) resulting in cytotoxicity to leukemic cells. ASNase is also important in the food industry, preventing acrylamide formation in processed foods. Several quantification techniques have been developed and used for the measurement of the ASNase activity, but standard pharmaceutical quality control methods were hardly reported, and in general, no official quality control guidelines were defined. To overcome this lack of information and to demonstrate the advantages and limitations, this work properly compares the traditional colorimetric methods (Nessler; L-aspartic acid β-hydroxamate (AHA); and indooxine) and the high-performance liquid chromatography (HPLC) method. A comparison of the methods using pure ASNase shows that the colorimetric methods both overestimate (Nessler) and underestimate (AHA and indooxine) the ASNase activity when compared to the values obtained with HPLC, considered the most precise method as this method monitors both substrate consumption and product formation, allowing for overall mass-balance. Correlation and critical analysis of each method relative to the HPLC method were carried out, resulting in a demonstration that it is crucial to select a proper method for the quantification of ASNase activity, allowing bioequivalence studies and individualized monitoring of different ASNase preparations. [Figure not available: see fulltext.] Department of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP), Rodovia Araraquara-Jaú/Km 01, Campos Ville Biochemistry and Technology Chemistry Department Chemistry Institute São Paulo State University (UNESP) Department of Chemistry Earlham College, 801 National Road West Department of Biochemical-Pharmaceutical Technology Pharmaceutical Biotechnology Laboratory University of Sao Paulo (USP) Department of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP), Rodovia Araraquara-Jaú/Km 01, Campos Ville Biochemistry and Technology Chemistry Department Chemistry Institute São Paulo State University (UNESP)

Recombinant l.asparaginase, L.ASNase, from Pseudomonas aeruginosa was purified using nickel affinity chromatography. The affinity purified L.ASNase exhibited a protein band with a molecular weight of 72.4 kDa on a native polyacrylamide gel and 36.276 kDa using SDS–PAGE. The activity of the purified L.ASNase was enhanced by Mg2+ and inhibited by Zn2+ at a concentration of 5 mM. The specificity of the recombinant L.ASNase towards different substrates was examined, and it was found that the enzyme showed the highest activity towards l.asparagine. Moreover, the enzyme showed lower activity towards other substrates such as L.glutamine, urea and acrylamide. The in vitro hemolysis assay revealed that the purified L.ASNase did not show hemolysis effect on blood erythrocytes. Serum and trypsin half-life of L.ASNase suggested that the recombinant L.ASNase retained 50% of its initial activity after 90 and 60 min incubation period in serum and trypsin separately.

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 3 days to 17 years. Most patients received 5000 U/m2 rASNase intravenously every 3 days. 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 100 U/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 (19.5 kg) were: CL = 0.0592 L/h, V 1 = 1.18 L, Q = 0.307 L/h, V 2 = 0.316 L. 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 48 h was appropriate to maintain rASNase activities above the therapeutic threshold, in patients with activities below 100 U/L 72 h 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.

Thiopurine methyltransferase (TPMT) gene polymorphism regulates thiopurine therapeutic efficacy and toxicity. The aim of this study was to determine the influence of TPMT gene polymorphism in Egyptian children with acute lymphoblastic leukaemia (ALL). Sixty-four patients with ALL, T lineage (27%) and pre-B phenotype (73%), who were treated with BFM 90 or CCG 1991 standard risk protocol, and who also experiencedmyleosuppresion toxicity and required interruption and/ormodification of thiopurine chemotherapy were recruited over a year period. Thirty-two patients were on maintenance and another 32 completed their chemotherapy. Seventy healthy age-matched and sex-matched children served as controls. They were subjected to clinical assessment, haematological panel investigations and TPMT gene polymorphism for G238C, G460A and A719G alleles assessment using PCRfollowed byRFLP analysis.Although none of the studied patients had themutantTPMTvariant alleles,myelosuppression toxicity in the form of different degree of neutropenia was detected in all patients. As a result of myelosuppression toxicity, most of the patients needed 6-MP dose modification either once (53.1%), twice (15.6%), or ≥ thrice (25.1%) during their maintenance course and 96.9% of the patients required to stop 6-MP for less than a week (62.5%), up to 2 weeks (28.1%), or > 2 weeks (6.3%). Patients also developed infection who mostly (71%) needed hospitalization. None of the studied G238C, G460A and A719G TPMT variant alleles were detected. Infections and febrile neutropenia were common causes of 6-PM dose modification and interruption.

Depletion of glutamine (Gln) has emerged as a potential therapeutic approach in the treatment of acute myeloid leukemia (AML), as neoplastic cells require Gln for synthesis of cellular components essential for survival. Asparaginases deplete Gln, and asparaginase derived from Erwinia chrysanthemi (Erwinaze) appears to have the greatest glutaminase activity of the available asparaginases. In this Phase I study, we sought to determine the dose of Erwinaze that safely and effectively depletes plasma Gln levels to ≤ 120 μmol/L in patients with relapsed or refractory (R/R) AML. Five patients were enrolled before the study was halted due to issues with Erwinaze manufacturing supply. All patients received Erwinaze at a dose of 25,000 IU/m2 intravenously three times weekly for 2 weeks. Median trough plasma Gln level at 48 h after initial Erwinaze administration was 27.6 μmol/L, and 80% (lower limit of 1-sided 95% CI 34%) of patients achieved at least one undetectable plasma Gln value (

Extracellular vesicles (EVs) are membrane particles involved in the exchange of a broad range of bioactive molecules between cells and the microenvironment. Although it has been shown that cells can traffic metabolic enzymes via EVs, much remains to be elucidated with regard to their intrinsic metabolic activity. Accordingly, herein we assessed the ability of neural stem/progenitor cell (NSC)-derived EVs to consume and produce metabolites. Our metabolomics and functional analyses both revealed that EVs harbor L-asparaginase activity, catalyzed by the enzyme asparaginase-like protein 1 (Asrgl1). Critically, we show that Asrgl1 activity is selective for asparagine and is devoid of glutaminase activity. We found that mouse and human NSC EVs traffic Asrgl1. Our results demonstrate, for the first time, that NSC EVs function as independent metabolic units that are able to modify the concentrations of critical nutrients, with the potential to affect the physiology of their microenvironment.

Laryngeal squamous cell carcinoma (LSCC), one of the most common malignancies in the head and neck, has poor prognosis and high mortality. The need of novel and effective treatment for LSCC is urgent. Asparaginase, an enzyme-depriving asparagine, has been employed for the treatment of various cancers. In this study, we reported for the first time that asparaginase could induce remarkable cytotoxicity and caspase-dependent apoptosis in human LSCC Tu212 and Tu686 cells. Meanwhile, autophagy was triggered by asparaginase in LSCC cells, which was confirmed by accumulation of autophagosomes and the conversion of light chain 3-I (LC3-I) to LC3-II. Importantly, inhibition of autophagy by chloroquine (CQ) significantly enhanced asparaginase-induced cytotoxicity, indicating that autophagy has a cytoprotective role in asparaginase-treated LSCC cells. Meanwhile, we found that mitochondrial-originated reactive oxygen species (ROS) participated in asparaginase-induced autophagy and cytotoxicity. N-acetyl-L-cysteine (NAC), a common antioxidant, was employed to scavenge ROS, and our results demonstrated that NAC could significantly block asparaginase-induced autophagy and attenuate asparaginase-induced cytotoxicity, indicating that intracellular ROS played a crucial role in asparagine deprivation therapy. Furthermore, western blot analysis showed that asparaginase-induced autophagy was mediated by inactivation of Akt/mTOR and activation of the Erk signaling pathway in Tu212 and Tu686 cells. Therefore, these results indicated the protective role of autophagy in asparaginase-treated LSCC cells and provided a new attractive therapeutic strategy for LSCC by asparaginase alone or in combination with autophagic inhibitors.

L-asparaginase has been used for more than three decades in acute lymphoblastic leukemia (ALL) patients and remains an essential drug in the treatment of ALL. Poor response to L-asparaginase is associated with increased risk of therapeutic failure in ALL. However, both the metabolic perturbation and molecular context of L-asparaginase-treated ALL cells has not been fully elucidated. Here we identify that treatment with L-asparaginase results in metabolic shutdown via the reduction of both glycolysis and oxidative phosphorylation, accompanied by mitochondrial damage and activation of autophagy. The autophagy is involved in reducing reactive oxygen species (ROS) level by eliminating injured mitochondria. Inhibition of autophagy enhances L-asparaginase-induced cytotoxicity and overcomes the acquired resistance to L-asparaginase in ALL cells. The ROS-p53-positive feedback loop is an essential mechanism of this synergistic cytotoxicity. Thus, our findings provide the rationale for the future development of combined treatment of L-asparaginase and anti-autophagy drug in ALL patients.

The pharmacokinetics of the polyethylene glycol (PEG)-conjugated asparaginase Oncaspar® are characterized by an increase in elimination over time. The focus of our analysis is the better understanding of this time-dependency. In paediatric acute lymphoblastic leukemia therapy (AIEOP-BFM ALL 2009), two administrations of Oncaspar® (2500 U/m2 intravenously) in induction phase (14-day interval) and one single administration in reinduction were followed by weekly monitoring of asparaginase activity. Non-linear mixed-effects modeling techniques (NONMEM) were used. Samples indicating immunological inactivation were excluded to describe the pharmacokinetics under standard conditions. Models with time-constant or time-varying clearance (CL) as well as transit compartment models with an increase in CL over a chain of compartments were investigated. Models with time-constant elimination could not adequately describe 6107 asparaginase activities from 1342 patients. Implementing a time-varying CL improved the fit. Modeling an increase of CL over time after dose (Emax- and Weibull-functions) were superior to models with an increase of CL over time after the first administration. However, a transit compartment model came out to be the best structural model. The increase in elimination of PEGylated asparaginase appears to be driven by physicochemical processes that are drug-related. The observed hydrolytically in vitro instability of the drug leads to the hypothesis that this increase in CL might be due to an in vivo hydrolysis of the instable ester bond between PEG and the enzyme combined with an increased elimination of the partly de-PEGylated enzyme (Trial registered at www.clinicaltrials.gov , NCT0111744).

To determine the incidence of hypertension among children during the induction and re-induction phases of acute lymphoblastic leukemia (ALL) therapy and association with possible risk factors. A retrospective analysis of 208 consecutive pediatric (age