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Background: Asparaginase-associated pancreatitis (AAP) is a major challenge for continuing asparaginase therapy. We aimed to investigate the acute and longterm complications and survival rates related to first and second AAP episodes in Chinese children with haematological malignancies. Methods: We retrospectively analysed clinical data of children with pancreatitis who received asparaginase chemotherapy for acute lymphoblastic leukaemia (ALL), acute mixed cell leukaemia, and non-Hodgkin's lymphoma at Shanghai Children's Medical Center from November 2013 to November 2023. Results: Of the 76 children included in the study, 12 had local complications (15.79%), with no deaths recorded. Systemic complications manifested in 28 patients (36.84%), resulting in 3 deaths (3.95%). Four patients (5.26%) developed long-term complications (chronic pancreatitis or insulin-dependent diabetes mellitus). No significant differences in local or long-term complications were recorded between children in the asparaginase re-exposed (n=39) and non-reexposed (n=45) groups. Among the re-exposed patients, eight (25.81%) experienced a second attack without fatalities or complications. Survival analysis of intermediate-to high-risk patients revealed a significantly higher event-free survival (EFS) rate for the re-exposed group than for the non-reexposed group. The second AAP episode's occurrence and severity had no relation to the first AAP episode's severity, and the second AAP episode was significantly less severe than the first (p<0.001). Conclusions: The second AAP episode's occurrence is unrelated to the first AAP episode's severity, and the second AAP episode's severity is significantly lower than that of the first. Further, asparaginase therapy could improve EFS in children with intermediate and high-risk ALL. [ABSTRACT FROM AUTHOR]

Introduction L-asparaginase is considered to be the most important component in the treatment of acute lymphoblastic leukemia (ALL). Intensifying the use of Lasparaginase during treatment for ALL has resulted in a significant rise in the percentage of children and adolescents who are cured of the disease. Asparaginase trough activity more than or equal to 100 IU/L on day 7 has been found to be the desired activity level in all childhood leukemia patients. Objectives Due to the paucity of data on biosimilar pegaspargase in the upfront setting, we planned this prospective pilot study to evaluate the levels of serum asparaginase activity (SAA) after biosimilar pegaspargase infusion. Materials and Methods It is a prospective, single-center, pilot study of 10 pediatric ALL patients for the duration of 6 months. All children less than 18 years with ALL on treatment with curative intent and receiving pegaspargase and who provided informed consent were included in this study. The enzymatic spectrophotometric method was used to determine SAA, and it was measured on the 7th and 14th days after the first dosage of pegaspargase-asparaginase, as well as on the 14th day after the second dose of pegaspargase-asparaginase, while toxicity was charted according to Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. Results From 10 patients with a median age of 5.5 years, a grand total of 29 samples were taken for analysis. Children who received pegaspargase had either B-ALL or T-ALL. After the first dose, mean-SD (standard deviation), SAA levels at day 7 was 131.3±38 IU/L and at Day 14 was 94.8±8 IU/L. After the second dose, mean±SD SAA level at day 14 was 86.1±15 IU/L. No patient had clinical hypersensitivity reaction and no patient reported any asparaginase-related toxicity. One patient died due to sepsis, infection with multidrug-resistant gram-negative bacteria. Conclusions Biosimilar pegaspargasemaintained good SAA levels 7 and 14 days after infusion. [ABSTRACT FROM AUTHOR]

Multiple asparaginase products have been approved by the United States (US) Food and Drug Administration (FDA) for the treatment of acute lymphoblastic leukemia in pediatric and adult patients. Hepatic veno-occlusive disease (VOD) is a potentially life-threatening disorder resulting from damage to the liver sinusoidal endothelial cells. To evaluate this safety concern with asparaginase (i.e. Asparlas, Oncaspar, Rylaze, and Erwinaze) use, we performed a postmarketing review of hepatic VOD reports retrieved from the FDA Adverse Event Reporting System database and literature with these four products. We identified 55 cases of hepatic VOD following exposure to asparaginase products. The median time to onset of hepatic VOD from the first dose of asparaginase was 18 days (interquartile range 13-24 days). Notably, 80% (44/55) of cases reported grades 3-5 VOD per the Common Terminology Criteria for Adverse Events. Although patients received asparaginase with standard chemotherapeutic agents known to induce VOD, case-level data indicates that asparaginase products may have contributed to hepatic VOD. Asparaginase products are associated with hepatotoxicity and thrombosis, suggesting a plausible mechanism for asparaginase-induced hepatic VOD. Based on the totality of data, including temporality and biologic plausibility, we determined hepatic VOD to be a class effect with asparaginase products. These data contributed to the addition of hepatic VOD to the hepatoxicity warning in the US Prescribing Information for asparaginase class products. [ABSTRACT FROM AUTHOR]

Background: While Pegaspargase is an essential component of the treatment of acute lymphoid leukemia (ALL) in children, it causes adverse events (AEs) that sometimes make full use impossible. Objective: The objective was to investigate the safety of Pegaspargase biosimilar in pediatric ALL patients undergoing treatment according to ICiCLe ALL-14 protocol. Method and Materials: A prospective study was carried out in a university teaching hospital located in the state of Maharashtra, India. Data on clinical factors and adverse reaction characteristics were gathered from hospital medical records. Suspected AEs were classified according to causality and severity. Results: During the study period, 72 children had 52 suspicions of AEs during treatment with biosimilar Pegaspargase. The odds ratio of 1.11 (95%CI, 0.41–2.98) suggested that males and females were both equally likely to experience adverse drug events, despite the fact that the frequency of suspected AEs was higher in boys (66%) than in girls (33%). None of the patients experienced allergic reactions. The high-risk category had the highest number of suspected AEs (56%), followed by intermediate risk (20%) and standard risk (20%). These patients showed a high frequency of suspected AEs during the induction phase (43%) followed by the consolidation phase (26%). Sixty percent of the reactions were classified as grade 1 or 2. ALL cell type (p = 0.02), risk category (p = 0.04) and length of hospitalization (p = 0.003) were significantly correlated with suspected AEs. Conclusion: Bio-similar Pegaspargase in combination with chemotherapy was safe and tolerable in the pediatric ALL patients treated according to ICiCLe ALL-14 protocol. Suspected AEs ranged from mild to moderate and hepatic failure and hyperglycemia being severe. [ABSTRACT FROM AUTHOR]

Acute lymphoblastic leukemia represents the most prevalent childhood cancer. Modern chemotherapy has significantly improved outcomes, achieving EFS rates of 80% and OS rates nearing 90% in developed nations, while in developing regions, rates remain below 50%, highlighting disparities, and this difference is due to several factors. Genetic variability plays a role in these drug response disparities, presenting single-nucleotide variations (SNVs). Pharmacogenetic research aims to pinpoint these SNVs early in treatment to predict specific drug responses effectively. This review aims to explore advancements in pharmacogenetics associated with asparaginase (ASNase). ASNase plays a crucial role in the treatment of ALL and is available in three formulations: E. coli, Erwinia, and PEG ASNase. ASNase therapy presents challenges due to adverse effects, like hypersensitivity reactions. Identifying predictive markers for hypersensitivity development beforehand is crucial for optimizing treatments. Several pharmacogenetic studies have investigated the association between SNVs and the risk of hypersensitivity. Key genes include GRIA1, NFATC2, CNTO3, ARHGAP28, MYBBP1A, and HLA. Studies have highlighted associations between SNVs within these genes and hypersensitivity reactions. Notably, most pharmacogenetic investigations of hypersensitivity have focused on patients treated with E. coli, emphasizing the need for broader exploration across different formulations. Future research investigating these variants holds promise for advancing our understanding of ASNase's pharmacogenetics. [ABSTRACT FROM AUTHOR]

Background: Endophytic fungi live inside the plant tissue, and can produce bioactive compounds. Methods: Herein, the molecular identification, phenols metabolites analysis, and biological properties (enzymatic and antioxidant) of endophytic fungi isolated from the aerial parts of Tragopogon graminifolius were studied. The enzyme produced by endophytic fungi was tested by the formation of a clear zone/pink around the colonies. The 2, 2-diphenyl-1-picrylhydrazyl method was used for the investigation antioxidant properties of isolated fungi. Moreover, the total flavonoid and phenol contents were studied using aluminum chloride colorimetric and Folin–Ciocalteu methods, respectively. Furthermore, the phenolic acids analysis was performed using high-performance liquid chromatography. Results: In this study, two isolates were identified (Talaromyces amestolkiae and Rhizopus oryzae). Evaluation of the enzymatic activities indicated that R. oryzae had the ability to produce amylase and pectinase, whereas T. amestolkiae exhibited extracellular activity for amylase, L-asparaginase, and pectinase. The result of the antioxidant property study indicated that T. amestolkiae had the highest antioxidant property and its IC50 value was 617 μg/mL. Moreover, these fungi had the highest flavonoids and phenols amounts. The phenolic acid analysis indicated that m-coumaric acid was the major phenolic acid which was determined by the studied fungi. Conclusion: This report was the first study of endophytic fungi from T. graminifolius, and the outcome of this study can be a way to produce m-coumaric acid at an industrial scale.

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease without meaningful therapeutic options beyond the first salvage therapy. Targeting PDAC metabolism through amino acid restriction has emerged as a promising new strategy, with asparaginases, enzymes that deplete plasma glutamine and asparagine, reaching clinical trials. In this study, we investigated the anti-PDAC activity of the asparaginase formulation Pegcrisantaspase (PegC) alone and in combination with standard-of-care chemotherapeutics. Methods Using mouse and human PDAC cell lines, we assessed the impact of PegC on cell proliferation, cell death, and cell cycle progression. We further characterized the in vitro effect of PegC on protein synthesis as well as the generation of reactive oxygen species and levels of glutathione, a major cellular antioxidant. Additional cell line studies examined the effect of the combination of PegC with standard-of-care chemotherapeutics. In vivo, the tolerability and efficacy of PegC, as well as the impact on plasma amino acid levels, was assessed using the C57BL/6-derived KPC syngeneic mouse model. Results Here we report that PegC demonstrated potent anti-proliferative activity in a panel of human and murine PDAC cell lines. This decrease in proliferation was accompanied by inhibited protein synthesis and decreased levels of glutathione. In vivo, PegC was tolerable and effectively reduced plasma levels of glutamine and asparagine, leading to a statistically significant inhibition of tumor growth in a syngeneic mouse model of PDAC. There was no observable in vitro or in vivo benefit to combining PegC with standard-of-care chemotherapeutics, including oxaliplatin, irinotecan, 5-fluorouracil, paclitaxel, and gemcitabine. Notably, PegC treatment increased tumor expression of asparagine and serine biosynthetic enzymes. Conclusions Taken together, our results demonstrate the potential therapeutic use of PegC in PDAC and highlight the importance of identifying candidates for combination regimens that could improve cytotoxicity and/or reduce the induction of resistance pathways.

Simple Summary: Berlin–Frankfurt–Münster (BFM) protocols are widely used outside clinical trials to treat pediatric acute lymphoblastic leukemia patients. However, local specificities might demand treatment adaptations, like the reduction of high-dose methotrexate due to lack of pharmacokinetic monitoring, the substitution of conventional asparaginase for peg-asparaginase because of the unavailability of the latter, or flow-cytometry-based measurable residual disease instead of a PCR-based one. Here, we report the results of a 22-year period of children treated with BFM protocols in a developing country. The results were somewhat comparable to the BFM reports, and we conclude that BFM protocol adaptations can be safely implemented in developing countries, accounting for local specificities. Introduction: The objective of the current study was to determine the survival probabilities of children and adolescents with acute lymphocytic leukemia treated with adapted Berlin–Frankfurt–Münster (BFM) protocols and compare our results with the original BFM reports. Methods: This retrospective study included 695 patients up to 19 years old treated with adapted BFM protocols between 1997 and 2018 in four hospitals in Rio de Janeiro. The 1997–2007 and 2008–2018 cohorts were analyzed separately. Results: More than half of the patients were stratified into the high-risk BFM classification. Overall and event-free survivals were, in the 1997–2007 period, respectively, 88% and 80% (BFM standard risk group—SRG), 75% and 67% (intermediate risk group—IRG), and 48% and 33% (high-risk group—HRG). The corresponding numbers for the 2008–2018 period were 93% and 84% (SRG), 75% and 63% (IRG), and 64% and 57% (HRG). In the second period, both the OS (HR = 0.71, p = 0.011) and EFS (HR = 0.62, p < 0.001) were higher. Except for the intermediate-risk group, the latter results are comparable to the BFM. Conclusion: The BFM protocol adaptations can be safely implemented in developing countries, accounting for local specificities. [ABSTRACT FROM AUTHOR]

Simple Summary: Acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Survival rates for children with ALL have improved, and today, more than 90% of children with ALL can be successfully treated. Acute pancreatitis (AP), which develops during chemotherapy for ALL can be a serious and fatal complication. It is a widely known toxicity, and several reports indicate that AP occurs with an incidence of 2–10% in patients treated for leukemia. It is mainly, but not exclusively, associated with the administration of asparaginase (ASP), which is one of the key components of multiagent chemotherapy used in ALL. The development of AP usually delays subsequent courses of chemotherapy and is the most common reason for the discontinuation of ASP, thus increasing the potential risk of leukemia relapse in children with ALL. Therefore, both the pathogenesis of AP in ALL and its impact on long-term outcomes remain to be thoroughly investigated. Purpose: This study aimed to identify the risk factors for acute pancreatitis (AP) and its impact on outcomes in Polish children treated for ALL. Methods: The study group included 2303 children receiving intensive chemotherapy for ALL. The group was divided into patients with at least one episode of AP and those who did not develop AP after treatment for ALL. Results: The cumulative incidence of AP in the study group was 4.08%. Older age was an independent risk factor for the development of AP (OR = 1.05; 95%CI = 1.006–1.098; p = 0.03). The overall mortality associated with AP was 2.13%. The probabilities of disease-free survival (p-DFS) and event-free survival (p-EFS) in both subgroups were 0.84 vs. 0.86, log-rank p = 0.65 and 0.75 vs. 0.80, log-rank p = 0.12, respectively. A total of 22 out of 94 patients (23.4%) with AP were re-exposed to asparaginase (ASP) during the subsequent treatment phases. Only one patient re-exposed to ASP (4.5%) developed a second episode of AP. There were no significant differences in p-DFS and p-EFS between patients re-exposed and not re-exposed to asparaginase (0.78 vs. 0.86, log-rank p = 0.27 and 0.63 vs. 0.79, log-rank p = 0.09, respectively). Conclusions: The incidence of AP in children with ALL is low and related to patients' age. The development of AP does not seem to influence p-DFS and p-EFS in children with ALL. Recurrence of AP after re-exposure to asparaginase in patients with ALL and a history of AP is low (4.5%). Re-exposure to asparaginase after the first episode of AP does not improve either p-DFS or p-EFS in children with ALL. [ABSTRACT FROM AUTHOR]

Acute pancreatitis (AP) is a life-threatening inflammatory disease with no specific therapy. Excessive cytoplasmic Ca2+ elevation and intracellular ATP depletion are responsible for the initiation of AP. Inhibition of Ca2+ release–activated Ca2+ (CRAC) channels has been proposed as a potential treatment, and currently, a novel selective CRAC channel inhibitor CM4620 (Auxora, CalciMedica) is in Phase 2b human trials. While CM4620 is on track to become the first effective treatment for AP, it does not produce complete protection in animal models. Recently, an alternative approach has suggested reducing ATP depletion with a natural carbohydrate galactose. Here, we have investigated the possibility of using the smallest effective concentration of CM4620 in combination with galactose. Protective effects of CM4620, in the range of 1-100 n m, have been studied against necrosis induced by bile acids, palmitoleic acid, or l-asparaginase. CM4620 markedly protected against necrosis induced by bile acids or asparaginase starting from 50 n m and palmitoleic acid starting from 1 n m. Combining CM4620 and galactose (1 m m) significantly reduced the extent of necrosis to near-control levels. In the palmitoleic acid-alcohol–induced experimental mouse model of AP, CM4620 at a concentration of 0.1 mg/kg alone significantly reduced edema, necrosis, inflammation, and the total histopathological score. A combination of 0.1 mg/kg CM4620 with galactose (100 m m) significantly reduced further necrosis, inflammation, and histopathological score. Our data show that CM4620 can be used at much lower concentrations than reported previously, reducing potential side effects. The novel combination of CM4620 with galactose synergistically targets complementary pathological mechanisms of AP. Graphical Abstract [ABSTRACT FROM AUTHOR]

The food enzyme asparaginase (l‐asparagine amidohydrolase; EC 3.5.1.1) is produced with the genetically modified Aspergillus niger strain ASP by DSM Food Specialties B.V. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in the prevention of acrylamide formation in foods and in the processing of yeast and yeast products. Dietary exposure to the food enzyme‐total organic solids (TOS) was estimated to be up to 0.792 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level at the highest dose tested of 1038 mg TOS/kg bw per day, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 1311. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use. [ABSTRACT FROM AUTHOR]

One of the major challenges in food industries is to remove acrylamide present in the fried foods. Asparaginase is generally used to mitigate acrylamide in fried food. Here, we studied the synergistic effect of chitosan with asparaginase on mitigation of acrylamide in fried bitter gourd chips. The chitosan immobilized asparaginase (CIA) nanobiocomposite was characterized using AFM. The treated and untreated fried chips were characterized using SEM and FT-IR analysis. The optimized pre-treatment temperature of 55ºC and 10 min time showed less acrylamide formation using CIA nanobiocomposite. The acrylamide formation was reduced to 948 µg/kg at 170ºC frying temperature for 5 min using 3 U/mL of CIA nanobiocomposite. The use of CIA nanobiocomposite was found effective for mitigation of the acrylamide present in fried bitter gourd chips. Chitosan provided synergistic effect with asparaginase on acrylamide mitigation and it neither altered the appearance nor the taste of the fried chips. [ABSTRACT FROM AUTHOR]

Background. Aggressive natural killer cell leukemia (ANKL) is rare and difficult to diagnose in early stages, with no standard treatment and a poor prognosis. Case presentation. Two adolescents with ANKL presented with hemophagocytic lymphohistiocytosis (HLH), with Case-1 presenting as refractory HLH and Case-2 with lung involvement. The morphology of bone marrow showed an increase in unidentified cells, which mainly expressed CD56. Cytogenetic analysis showed complex karyotypes. Both patients received intensive combined chemotherapy based on pegaspargase and anthracyclines. Case-1 died of tumor lysis syndrome. Case-2 underwent hematopoietic stem cell transplantation and is currently alive and disease-free. Conclusions. HLH can serve as the initial manifestation of ANKL. Leukemia cells of ANKL have significant variations in the morphology and mainly express CD56. Intensive combination chemotherapy based on pegaspargase and anthracyclines may be considered for ANKL. [ABSTRACT FROM AUTHOR]

Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease without meaningful therapeutic options beyond the first salvage therapy. Targeting PDAC metabolism through amino acid restriction has emerged as a promising new strategy, with asparaginases, enzymes that deplete plasma glutamine and asparagine, reaching clinical trials. In this study, we investigated the anti-PDAC activity of the asparaginase formulation Pegcrisantaspase (PegC) alone and in combination with standard-of-care chemotherapeutics. Methods: Using mouse and human PDAC cell lines, we assessed the impact of PegC on cell proliferation, cell death, and cell cycle progression. We further characterized the in vitro effect of PegC on protein synthesis as well as the generation of reactive oxygen species and levels of glutathione, a major cellular antioxidant. Additional cell line studies examined the effect of the combination of PegC with standard-of-care chemotherapeutics. In vivo, the tolerability and efficacy of PegC, as well as the impact on plasma amino acid levels, was assessed using the C57BL/6-derived KPC syngeneic mouse model. Results: Here we report that PegC demonstrated potent anti-proliferative activity in a panel of human and murine PDAC cell lines. This decrease in proliferation was accompanied by inhibited protein synthesis and decreased levels of glutathione. In vivo, PegC was tolerable and effectively reduced plasma levels of glutamine and asparagine, leading to a statistically significant inhibition of tumor growth in a syngeneic mouse model of PDAC. There was no observable in vitro or in vivo benefit to combining PegC with standard-of-care chemotherapeutics, including oxaliplatin, irinotecan, 5-fluorouracil, paclitaxel, and gemcitabine. Notably, PegC treatment increased tumor expression of asparagine and serine biosynthetic enzymes. Conclusions: Taken together, our results demonstrate the potential therapeutic use of PegC in PDAC and highlight the importance of identifying candidates for combination regimens that could improve cytotoxicity and/or reduce the induction of resistance pathways. [ABSTRACT FROM AUTHOR]

AbstractThe L-asparaginase is commercial enzyme used as chemotherapeutic agent in cancer treatment and food processing agent in backed and fried food industries. In the present research work, the artificial intelligence and machine learning techniques were employed for modeling and optimization of fermentation process conditions for enhanced production of L-asparaginase by submerged fermentation of Aspergillus terreus. The experimental L-asparaginase activity obtained using central composite experiment design was used for optimization. The Random Forest algorithms machine learning techniques was found best based on the analysis of regression coefficient of ANN model and metric score values of machine learning algorithms. The experimental L-asparaginase activity of 41.58 IU/mL was obtained at the Random Forest algorithm predicted fermentation process conditions of temperature 31 °C, initial pH 6.3, inoculum size 2% (v/v), agitation rate 150 rpm and fermentation time 66 h. [ABSTRACT FROM AUTHOR]

Background: Asparaginase is essential for treating T‐cell acute lymphoblastic leukemia (T‐ALL). Despite the ongoing debate on whether T‐ALL and T‐cell lymphoblastic lymphoma (T‐LBL) are the same disease entity or two distinct diseases, patients with T‐LBL often receive the same or similar treatment protocols as those with T‐ALL. Methods: The outcomes of patients with or without L‐asparaginase discontinuation were retrospectively analyzed among four national protocols: Japan Association of Childhood Leukemia Study (JACLS) ALL‐02 and ALL‐97 for T‐ALL and Japanese Pediatric Leukemia/Lymphoma Study Group ALB‐NHL03 and JACLS NHL‐98 for T‐LBL. The hazard ratio (HR) was calculated with the Cox regression model by considering L‐asparaginase discontinuation as a time‐dependent variable. Results: In total, 199 patients with T‐ALL, and 133 patients with T‐LBL were included. L‐asparaginase discontinuation compromised event‐free survival (EFS) of T‐ALL patients (ALL‐02: HR 3.32, 95% confidence interval [CI] 1.40–7.90; ALL‐97: HR 3.39, 95%CI 1.19–9.67). Conversely, EFS compromise was not detected among T‐LBL patients (ALB‐NHL03: HR 1.39, 95%CI 0.41–4.68; NHL‐98: HR 0.92, 95%CI 0.11–7.60). Conclusion: The effects of L‐asparaginase discontinuation differed between T‐ALL and T‐LBL. We assume that the differential impact results from (1) the inherent differential response to L‐asparaginase between them and/or (2) a less stringent assessment of early treatment response in T‐LBL than in T‐ALL. Given the poor salvage rate of refractory or relapsed T‐ALL and T‐LBL, optimization of the frontline therapy is critical, and the current study provides a new suggestion for further treatment modifications. However, larger studies in contemporary intensified treatment protocols are required. [ABSTRACT FROM AUTHOR]

Introduction: Rapidly increasing costs of medication acquisition can pose a challenge for health-system pharmacy budgets. The impact of dose-rounding in a pediatric oncology population has not previously been well documented and a retrospective review was undertaken to quantify the potential cost benefits. Methods: A retrospective chart review of patients with an oncologic diagnosis was performed for cytotoxic agents, asparaginase products, and biotherapy administered between January 1, 2014, and December 31, 2017. In the analysis, orders that could be rounded down to the nearest vial size by 5 or 10% were included. Medication pricing information was based on wholesale acquisition cost (WAC) and was provided by the Department of Pharmacy. Cost savings per medication were determined by multiplying the WAC of the medication by the number of vials saved. Results: Over a 4-year span, 347 patients were evaluated and 552 out of a possible 3110 orders (17.7%) met criteria for a theoretical cost savings of approximately $1,126,000 (∼$3200 per patient). Rounding down doses by up to 5% resulted in a potential savings of about $529,000. When rounding was extended to 5–10% of the originally ordered dose, an additional $597,000 of approximate cost savings could have been realized. The medications with the largest impact on cost savings were rituximab, pegaspargase, and erwinia asparaginase. Conclusions: For pediatric oncology patients, there exists a unique potential cost savings opportunity if doses are rounded down within 5 or 10% of the originally ordered weight-based or body surface area-calculated dose. [ABSTRACT FROM AUTHOR]

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Most plant-sap feeding insects have obligate relationships with maternally transmitted bacteria. Aphids require their nutritional endosymbiont, Buchnera aphidicola, for the production of essential amino acids. Such endosymbionts are harbored inside of specialized insect cells called bacteriocytes. Here, we use comparative transcriptomics of bacteriocytes between two recently diverged aphid species, Myzus persicae and Acyrthosiphon pisum, to identify key genes that are important for the maintenance of their nutritional mutualism. The majority of genes with conserved expression profiles in M. persicae and A. pisum are for orthologs previously identified in A. pisum to be important for the symbiosis. However, asparaginase which produces aspartate from asparagine was significantly up-regulated only in A. pisum bacteriocytes, potentially because Buchnera of M. persicae encodes its own asparaginase enzyme unlike Buchnera of A. pisum resulting in Buchnera of A. pisum to be dependent on its aphid host for aspartate. One-to-one orthologs that explained the most amount of variation for bacteriocyte specific mRNA expression for both species includes a collaborative gene for methionine biosynthesis, multiple transporters, a horizontally transmitted gene, and secreted proteins. Finally, we highlight species-specific gene clusters which may contribute to host adaptations and/or accommodations in gene regulation to changes in the symbiont or the symbiosis.

Identification of a single genetic target for microbial strain improvement is difficult due to the complexity of the genetic regulatory network. Hence, a more practical approach is to identify bottlenecks in the regulatory networks that control critical metabolic pathways. The present work focuses on enhancing cellular physiology by increasing the metabolic flux through the central carbon metabolic pathway. Global regulator cra (catabolite repressor activator), a DNA-binding transcriptional dual regulator was selected for the study as it controls the expression of a large number of operons that modulate central carbon metabolism. To upregulate the activity of central carbon metabolism, the cra gene was co-expressed using a plasmid-based system. Co-expression of cra led to a 17% increase in the production of model recombinant protein L-Asparaginase-II. A pulse addition of 0.36% of glycerol every two hours post-induction, further increased the production of L-Asparaginase-II by 35% as compared to the control strain expressing only recombinant protein. This work exemplifies that upregulating the activity of central carbon metabolism by tuning the expression of regulatory genes like cra can relieve the host from cellular stress and thereby promote the growth as well as expression of recombinant hosts. [ABSTRACT FROM AUTHOR]

Background: Asparaginase (ASNase) is a crucial part of acute leukemia treatment, but immune responses to the agent can reduce its effectiveness and increase the risk of relapse. Currently, no reliable and validated biomarker predicts ASNaseinduced hypersensitivity reactions during therapy. We aimed to identify predictive biomarkers and determine immune cells responsible for anaphylaxis using a murine model of ASNase hypersensitivity. Methods: Our preclinical study uses a murine model to investigate predictive biomarkers of ASNase anaphylaxis, including anti-ASNase antibody responses, immune complex (IC) levels, ASNase-specific binding to leukocytes or basophils, and basophil activation. Results: Our results indicate that mice immunized to ASNase exhibited dynamic IgM, IgG, and IgE antibody responses. The severity of ASNase-induced anaphylaxis was found to be correlated with levels of IgG and IgE, but not IgM. Basophils from immunized mice were able to recognize and activate in response to ASNase ex vivo, and the extent of recognition and activation also correlated with the severity of anaphylaxis observed. Using a multivariable model that included all biomarkers significantly associated with anaphylaxis, independent predictors of ASNase-induced hypersensitivity reactions were found to be ASNase IC levels and ASNase-specific binding to leukocytes or basophils. Consistent with our multivariable analysis, we found that basophil depletion significantly protected mice from ASNase-induced hypersensitivity reactions, supporting that basophils are essential and can be used as a predictive marker of ASNase-induced anaphylaxis. Conclusions: Our study demonstrates the need for using tools that can detect both IC- and IgE-mediated hypersensitivity reactions to mitigate the risk of ASNase-induced hypersensitivity reactions during treatment. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is a heterogeneous hematological malignancy characterized by disrupted blood cell production and function. Recent investigations have highlighted the potential of targeting glutamine metabolism as a promising therapeutic approach for AML. Asparaginases, enzymes that deplete circulating glutamine and asparagine, are approved for the treatment of acute lymphoblastic leukemia, but are also under investigation in AML, with promising results. We previously reported an elevation in plasma serine levels following treatment with Erwinia-derived asparaginase (also called crisantaspase). This led us to hypothesize that AML cells initiate the de novo serine biosynthesis pathway in response to crisantaspase treatment and that inhibiting this pathway in combination with crisantaspase would enhance AML cell death. Here we report that in AML cell lines, treatment with the clinically available crisantaspase, Rylaze, upregulates the serine biosynthesis enzymes phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) through activation of the Amino Acid Response (AAR) pathway, a cellular stress response mechanism that regulates amino acid metabolism and protein synthesis under conditions of nutrient limitation. Inhibition of serine biosynthesis through CRISPR-Cas9-mediated knockout of PHGDH resulted in a ~250-fold reduction in the half-maximal inhibitory concentration (IC50) for Rylaze, indicating heightened sensitivity to crisantaspase therapy. Treatment of AML cells with a combination of Rylaze and a small molecule inhibitor of PHGDH (BI4916) revealed synergistic antiproliferative effects in both cell lines and primary AML patient samples. Rylaze-BI4916 treatment in AML cell lines led to the inhibition of cap-dependent mRNA translation and protein synthesis, as well as a marked decrease in intracellular glutathione levels, a critical cellular antioxidant. Collectively, our results highlight the clinical potential of targeting serine biosynthesis in combination with crisantaspase as a novel therapeutic strategy for AML. [ABSTRACT FROM AUTHOR]

ReAV, the inducible Class-3 L-asparaginase from the nitrogen-fixing symbiotic bacterium Rhizobium etli, is an interesting candidate for optimizing its enzymatic potential for antileukemic applications. Since it has no structural similarity to known enzymes with this activity, it may offer completely new ways of approach. Also, as an unrelated protein, it would evade the immunological response elicited by other asparaginases. The crystal structure of ReAV revealed a uniquely assembled protein homodimer with a highly specific C135/K138/C189 zinc binding site in each subunit. It was also shown before that the Zn2+ cation at low and optimal concentration boosts the ReAV activity and improves substrate specificity, which indicates its role in substrate recognition. However, the detailed catalytic mechanism of ReAV is still unknown. In this work, we have applied site-directed mutagenesis coupled with enzymatic assays and X-ray structural analysis to elucidate the role of the residues in the zinc coordination sphere in catalysis. Almost all of the seven ReAV muteins created in this campaign lost the ability to hydrolyze L-asparagine, confirming our predictions about the significance of the selected residues in substrate hydrolysis. We were able to crystallize five of the ReAV mutants and solve their crystal structures, revealing some intriguing changes in the active site area as a result of the mutations. With alanine substitutions of Cys135 or Cys189, the zinc coordination site fell apart and the mutants were unable to bind the Zn2+ cation. Moreover, the absence of Lys138 induced atomic shifts and conformational changes of the neighboring residues from two active-site Ser-Lys tandems. Ser48 from one of the tandems, which is hypothesized to be the catalytic nucleophile, usually changes its hydration pattern in response to the mutations. Taken together, the results provide many useful clues about the catalytic mechanism of the enzyme, allowing one to cautiously postulate a possible enzymatic scenario. [ABSTRACT FROM AUTHOR]

Objective: To assess the impact of different treatment strategies and risk factors on the prognosis of patients with extranodal NK/T-cell lymphoma, nasal type (ENKTL) in a single medical center. Methods and analysis: The clinical features of 266 patients with ENKTL were retrospectively analyzed, among whom those in stages I and II received sandwich therapy, while those in stages III and IV underwent chemotherapy plus autologous hematopoietic stem cell transplantation. The Kaplan-Meier curves, univariate and multivariate Cox regression analyses were employed for survival and prognosis analysis. Statistical significance was set at P<0.05. Results: Following treatment, the post-intervention outcomes demonstrated a complete remission (CR) rate of 71.05% and a partial remission (PR) rate of 3.76%. The 5-year progression-free survival (PFS) and overall survival (OS) rates were 70.4% and 70.9%, respectively. In addition, the PFS for patients in stage I/II was 79.8%, with an OS of 81.1%, whereas for those in stage III/IV, the PFS was 41.7% and the OS was 40.9%. Notably, the achievement of CR immediately after treatment was an independent prognostic factor (P<0.001). Patients in stage I/II depicted a favorable 5-year OS rate, while those in stage III/IV manifested a less favorable prognosis. Conclusion: Stages of the disease and whether CR was achieved following treatment are important factors determining the survival and prognosis of patients with ENKTL. Further researches focusing on disease onset and mechanisms of drug resistance will contribute to better management of ENKTL. [ABSTRACT FROM AUTHOR]

This article discusses the clinical manifestations, diagnosis, severity, treatment, and outcomes of peg-asparaginase-associated pancreatitis (AAP) in pediatric patients undergoing chemotherapy. The study found that AAP is a common complication in patients with asparaginase-treated acute lymphoblastic leukemia (ALL), with abdominal pain being the most common symptom. The severity of AAP varies, and treatment involves fasting, nutritional support, and fluid resuscitation. The study emphasizes the importance of early recognition and management of AAP to improve patient outcomes. Additionally, the article suggests the need for further research to determine risk factors and establish standardized approaches for pediatric AAP. [Extracted from the article]

Background: Endophytic fungi live inside the plant tissue, and can produce bioactive compounds. Methods: Herein, the molecular identification, phenols metabolites analysis, and biological properties (enzymatic and antioxidant) of endophytic fungi isolated from the aerial parts of Tragopogon graminifolius were studied. The enzyme produced by endophytic fungi was tested by the formation of a clear zone/pink around the colonies. The 2, 2-diphenyl-1-picrylhydrazyl method was used for the investigation antioxidant properties of isolated fungi. Moreover, the total flavonoid and phenol contents were studied using aluminum chloride colorimetric and Folin–Ciocalteu methods, respectively. Furthermore, the phenolic acids analysis was performed using high-performance liquid chromatography. Results: In this study, two isolates were identified (Talaromyces amestolkiae and Rhizopus oryzae). Evaluation of the enzymatic activities indicated that R. oryzae had the ability to produce amylase and pectinase, whereas T. amestolkiae exhibited extracellular activity for amylase, L-asparaginase, and pectinase. The result of the antioxidant property study indicated that T. amestolkiae had the highest antioxidant property and its IC50 value was 617 µg/mL. Moreover, these fungi had the highest flavonoids and phenols amounts. The phenolic acid analysis indicated that m‑coumaric acid was the major phenolic acid which was determined by the studied fungi. Conclusion: This report was the first study of endophytic fungi from T. graminifolius, and the outcome of this study can be a way to produce m-coumaric acid at an industrial scale. [ABSTRACT FROM AUTHOR]

Background: Among malignant diseases which develop during childhood, hematological cancers, such as leukemias and lymphomas, are the most common. Outcomes have greatly improved due to the refinement of multiagent chemotherapy regimens that include enhanced asparaginase therapy. In this study, we aimed to evaluate our experiences related to the analytical and clinical significance of determining l-Asparaginase activity. Methods: Since 2016, the Laboratory of the Children's Hospital Zagreb has routinely measured l-Asparaginase activity and to date, has measured more than 280 examples of activity in a total of 57 children with hematological malignancy treated at the Pediatric Oncology Department of the Children's Hospital Zagreb. Three asparaginase products were available: native E. colil-Asparaginase; a pegylated form of this enzyme; and a native product from Erwinia chrysanthemi. A retrospective data analysis was performed. Results: Out of the fifty-seven children, seven had an allergic reaction (12.3%), five (8.8%) had silent inactivation, and seven (12.3%) developed acute pancreatitis. Allergic reactions and silent inactivation were more common in children treated with native E. colil-Asparaginase, while pancreatitis was more common in children treated with the pegylated form. Conclusions: The monitoring of l-Asparaginase activity may help to optimize therapy by identifying patients with 'silent inactivation', and/or by dose correction when l-Asparaginase activity is too high (slow elimination). [ABSTRACT FROM AUTHOR]

L-Asparaginases, divided into three structural Classes, catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. The members of Class 3, ReAIV and ReAV, encoded in the genome of the nitrogen fixing Rhizobium etli, have the same fold, active site, and quaternary structure, despite low sequence identity. In the present work we examined the biochemical consequences of this difference. ReAIV is almost twice as efficient as ReAV in asparagine hydrolysis at 37°C, with the kinetic Km, k[sub cat] parameters (measured in optimal buffering agent) of 1.5 mM, 770 s[sup -1] and 2.1 mM, 603 s \ respectively. The activity of ReAIV has a temperature optimum at 45°C-55°C, whereas the activity of ReAV, after reaching its optimum at 37°C, decreases dramatically at 45°C. The activity of both isoforms is boosted by 32 or 56%, by low and optimal concentration of zinc, which is bound three times more strongly by ReAIV then by ReAV, as reflected by the KD values of 1.2 and 3.3 µM, respectively. We also demonstrate that perturbation of zinc binding by Lys→Ala point mutagenesis drastically decreases the enzyme activity but also changes the mode of response to zinc. We also examined the impact of different divalent cations on the activity, kinetics, and stability of both isoforms. It appeared that Ni2+, Cu2+, Hg2+, and Cd2+ have the potential to inhibit both isoforms in the following order (from the strongest to weakest inhibitors) Hg2+ > Cu2+ > Cd2+ > Ni2+. ReAIV is more sensitive to Cu2+ and Cd2+, while ReAV is more sensitive to Hg2+ and Ni2+, as revealed by IC50 values, melting scans, and influence on substrate specificity. Low concentration of Cd[sup 2+] improves substrate specificity of both isoforms, suggesting its role in substrate recognition. The same observation was made for Hg2+ in the case of ReAIV. The activity of the ReAV isoform is less sensitive to Cl anions, as reflected by the IC50 value for NaCl, which is eightfold higher for ReAV relative to ReAIV. The uncovered complementary properties of the two isoforms help us better understand the inducibility of the ReAV enzyme. [ABSTRACT FROM AUTHOR]

Aims To identify a marine L-asparaginase with clinically desirable attributes and characterize the shortlisted candidate through in silico tools Methods and results Marine bacterial strains (number = 105) isolated from marine crabs were evaluated through a stepwise strategy incorporating the crucial attributes for therapeutic safety. The results demonstrated the potential of eight bacterial species for extracellular L-asparaginase production. However, only one isolate (Bacillus altitudinis CMFRI/Bal-2) showed clinically desirable attributes, viz. extracellular production, type-II nature, lack of concurrent L-glutaminase and urease activities, and presence of ansZ (functional gene for clinical type). The enzyme production was 22.55 ± 0.5 µM/mg protein/min within 24 h without optimization. The enzyme also showed good activity and stability in pH 7–8 and temperature 37°C, predicting the functioning inside the human body. The Michealis-Menten constant (Km) was 14.75 µM. Detailed in silico analysis based on functional gene authenticating the results of in vitro characterization and predicted the nonallergenic characteristic of the candidate. Docking results proved the higher affinity of the shortlisted candidate to L-asparagine than L-glutamine and urea. Conclusion Comprehensively, the study highlighted B. altitudinis t ype II asparaginase as a competent candidate for further research on clinically safe asparaginases. [ABSTRACT FROM AUTHOR]

Flow cytometry plays is important in the diagnosis of acute lymphoblastic leukaemia (ALL) and when antigen-specific immunotherapy is indicated. We have investigated the effects of prednisolone, vincristine, daunorubicin, asparaginase and methotrexate on the antigen expression on blast cells that could influence the planning of antigen-specific therapy as well as risk-based treatment assignment. Patients aged ≤ 17 years with de novo B-cell ALL (B-ALL) were enrolled in the study. Blast cells were isolated and exposed in vitro to 5 individual cytotoxic drugs in logarithmically increasing concentrations. Then, the expression of CD10, CD19, CD20, CD27, CD34, CD45, CD58, CD66c and CD137 antigens was determined by quantitative flow cytometry. Cytotoxic drugs caused dose-dependent or dose-independent modulation of antigen expression. Daunorubicin caused a dose-dependent down-modulation of CD10, CD19, CD34, CD45 and CD58 and an up-modulation of CD137. Vincristine caused a dose-dependent down-modulation of CD19 and CD58 and an up-modulation of CD45. Daunorubicin also caused dose-independent down-modulation of CD27 and prednisolone down-modulation of CD10, CD19, CD27, CD34 and CD58. Down-modulation of CD20 was detected only in relation to the specific dose of daunorubicin. The results of the study have shown that cytotoxic drugs can alter the expression of antigens that are important for immunotherapy. Importantly, daunorubicin, prednisolone and vincristine caused down-modulation of CD19 and CD58, suggesting that these drugs are better avoided during bridging therapy prior to bispecific antibodies or CAR-T cell therapy. In addition, immunophenotypic changes on blast cells induced by different drugs could also influence risk-based treatment assignment. [ABSTRACT FROM AUTHOR]

Asparagine and glutamine depletion operated by the drug Asparaginase (ASNase) has revolutionized therapy in pediatric patients affected by Acute Lymphoblastic Leukemia (ALL), bringing remissions to a remarkable 90 % of cases. However, the knowledge of the proproliferative role of asparagine in adult and solid tumors is still limited. We have here analyzed the effect of ASNase on three adenocarcinoma cell lines (A549, lung adenocarcinoma, MCF-7, breast cancer, and 786-O, kidney cancer). In contrast to MCF-7 cells, 786-O and A549 cells proved to be a relevant target for cell cycle perturbation by asparagine and glutamine shortage. Indeed, when the cell-cycle was analyzed by flow cytometry, A549 showed a canonical response to asparaginase, 786-O cells, instead, showed a reduction of the percentage of cells in the G1 phase and an increase of those in the S-phase. Despite an increased number of PCNA and RPA70 positive nuclear foci, BrdU and EdU incorporation was absent or strongly delayed in treated 786-O cells, thus indicating a readiness of replication forks unmatched by DNA synthesis. In 786-O asparagine synthetase was reduced following treatment and glutamine synthetase was totally absent. Interestingly, DNA synthesis could be recovered by adding Gln to the medium. MCF-7 cells showed no significant changes in the cell cycle phases, in DNA-bound PCNA and in total PCNA, but a significant increase in ASNS and GS mRNA and protein expression. The collected data suggest that the effect observed on 786-O cells following ASNase treatment could rely on mechanisms which differ from those well-known and described for leukemic blasts, consisting of a complete block in the G1/S transition in proliferating cells and on an increase on non-proliferative (G0) blasts.

Abstract The food enzyme asparaginase (l‐asparagine amidohydrolase; EC 3.5.1.1) is produced with the genetically modified Aspergillus niger strain ASP by DSM Food Specialties B.V. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in the prevention of acrylamide formation in foods and in the processing of yeast and yeast products. Dietary exposure to the food enzyme‐total organic solids (TOS) was estimated to be up to 0.792 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level at the highest dose tested of 1038 mg TOS/kg bw per day, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 1311. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Abstract Background Asparaginase is essential for treating T‐cell acute lymphoblastic leukemia (T‐ALL). Despite the ongoing debate on whether T‐ALL and T‐cell lymphoblastic lymphoma (T‐LBL) are the same disease entity or two distinct diseases, patients with T‐LBL often receive the same or similar treatment protocols as those with T‐ALL. Methods The outcomes of patients with or without L‐asparaginase discontinuation were retrospectively analyzed among four national protocols: Japan Association of Childhood Leukemia Study (JACLS) ALL‐02 and ALL‐97 for T‐ALL and Japanese Pediatric Leukemia/Lymphoma Study Group ALB‐NHL03 and JACLS NHL‐98 for T‐LBL. The hazard ratio (HR) was calculated with the Cox regression model by considering L‐asparaginase discontinuation as a time‐dependent variable. Results In total, 199 patients with T‐ALL, and 133 patients with T‐LBL were included. L‐asparaginase discontinuation compromised event‐free survival (EFS) of T‐ALL patients (ALL‐02: HR 3.32, 95% confidence interval [CI] 1.40–7.90; ALL‐97: HR 3.39, 95%CI 1.19–9.67). Conversely, EFS compromise was not detected among T‐LBL patients (ALB‐NHL03: HR 1.39, 95%CI 0.41–4.68; NHL‐98: HR 0.92, 95%CI 0.11–7.60). Conclusion The effects of L‐asparaginase discontinuation differed between T‐ALL and T‐LBL. We assume that the differential impact results from (1) the inherent differential response to L‐asparaginase between them and/or (2) a less stringent assessment of early treatment response in T‐LBL than in T‐ALL. Given the poor salvage rate of refractory or relapsed T‐ALL and T‐LBL, optimization of the frontline therapy is critical, and the current study provides a new suggestion for further treatment modifications. However, larger studies in contemporary intensified treatment protocols are required.