L-asparaginases (ASP) and Doxorubicin (Dox) are both used in the treatment of leukemia, including in combination. We have attempted to investigate if their combination within the same targeted delivery vehicle can make such therapy more efficacious. We assembled a micellar system, where the inner hydrophobic core was loaded with Dox, while ASP would absorb at the surface due to electrostatic interactions. To make such absorption stronger, we conjugated the ASP with oligoamines, such as spermine, and the lipid components of the micelle-lipoic and oleic acids-with heparin. When loaded with Dox alone, the system yielded about a 10-fold improvement in cytotoxicity, as compared to free Dox. ASP alone showed about a 2.5-fold increase in cytotoxicity, so, assuming additivity of the effect, one could expect a 25-fold improvement when the two agents are applied in combination. But in reality, a combination of ASP + Dox loaded into the delivery system produced a synergy, with a whopping 50× improvement vs. free individual component. Pharmacokinetic studies have shown prolonged circulation of micellar formulations in the bloodstream as well as an increase in the effective concentration of Dox in micellar form and a reduction in Dox accumulation to the liver and heart (which reduces hepatotoxicity and cardiotoxicity). For the same reason, Dox\'s liposomal formulation has been in use in the treatment of multiple types of cancer, almost replacing the free drug. We believe that an opportunity to deliver a combination of two types of drugs to the same target cell may represent a further step towards improvement in the risk-benefit ratio in cancer treatment.

Microbial enzymes are crucial catalysts in various industries due to their versatility and efficiency. The microbial enzymes market has recently expanded due to increased demand for many reasons. Among them are eco-friendly solutions, developing novel microbial strains with enhanced enzymes that perform under harsh conditions, providing sustainability, and raising awareness about the benefits of enzyme-based products. By 2030, the global enzyme market is expected to account for $525 billion, with a growth rate of 6.7 %. L-asparaginase and L-glutaminase are among the leading applied microbial enzymes in antitumor therapy, with a growing market share of 16.5 % and 9.5 %, respectively. The use of microbial enzymes has opened new opportunities to fight various tumors, including leukemia, lymphosarcoma, and breast cancer, which has increased their demand in the pharmaceutical and medicine sectors. Despite their promising applications, commercial use of microbial enzymes faces challenges such as short half-life, immunogenicity, toxicity, and other side effects. Therefore, this review explores the industrial production, purification, formulation, and commercial utilization of microbial enzymes, along with an overview of the global enzyme market. With ongoing discoveries of novel enzymes and their applications, enzyme technology offers promising avenues for cancer treatment and other therapeutic interventions.

L-asparaginase is an FDA-approved drug for treating blood cancer, but its inherent antigenicity and L-glutaminase activity are associated with hypersensitivity and organ toxicity. Extracellularly produced glutaminase-free L-asparaginase from human commensal bacteria may be a good alternative to reduce the side effects of therapeutic L-asparaginase. Here, we report the isolation and characterization of fourteen L-asparaginase-producing bacterial strains belonging to the genera Acinetobacter, Escherichia, Klebsiella, and Pseudomonas from human stool and saliva samples. To the best of our knowledge, this is the first report of L-asparaginase-producing human commensal bacterial strains isolated from healthy individuals. L-asparaginase produced by fecal and salivary isolates exhibited significantly higher activity (3.64 to 16.96 U/ml) toward L-asparagine than L-glutamine. Interestingly, L-asparaginase from fecal isolates, Escherichia coli strains 3F1 and 3F2 and salivary isolate Klebsiella pneumoniae 3S3, exhibited no L-glutaminase activity. These isolates were also sensitive to all tested antibiotics. Additionally, these three isolates demonstrated tolerance to pH 3.0 (≥ 88% survival) and 0.3% bile (≥ 95% survival), indicating their potential as probiotics. Among these isolates, L-asparaginase from the highest-producing K. pneumoniae 3S3 strain was found to be a homodimer, with native and subunit molecular weights of 110 kDa and 55 kDa, respectively. The purified enzyme can be further explored for its antitumor and immunomodulatory properties. Overall, future research can be expanded to include the use of a pool of human commensal bacteria as genuine and alternative sources of L-asparaginase for effective cancer treatments and cutting-edge next-generation probiotics.

Metastasis stands as the primary contributor to mortality associated with tumors. Chemotherapy and immunotherapy are frequently utilized in the management of metastatic solid tumors. Nevertheless, these therapeutic modalities are linked to serious adverse effects and limited effectiveness in preventing metastasis. Here, we report a novel therapeutic strategy named starvation-immunotherapy, wherein an immune checkpoint inhibitor is combined with an ultra-long-acting L-asparaginase that is a fusion protein comprising L-asparaginase (ASNase) and an elastin-like polypeptide (ELP), termed ASNase-ELP. ASNase-ELP\'s thermosensitivity enables it to generate an in-situ depot following an intratumoral injection, yielding increased dose tolerance, improved pharmacokinetics, sustained release, optimized biodistribution, and augmented tumor retention compared to free ASNase. As a result, in murine models of oral cancer, melanoma, and cervical cancer, the antitumor efficacy of ASNase-ELP by selectively and sustainably depleting L-asparagine essential for tumor cell survival was substantially superior to that of ASNase or Cisplatin, a first-line anti-solid tumor medicine, without any observable adverse effects. Furthermore, the combination of ASNase-ELP and an immune checkpoint inhibitor was more effective than either therapy alone in impeding melanoma metastasis. Overall, the synergistic strategy of starvation-immunotherapy holds excellent promise in reshaping the therapeutic landscape of refractory metastatic tumors and offering a new alternative for next-generation oncology treatments.

L-asparaginase (L-ASNase) is an enzyme that shows targeted activity against Acute Lymphoblastic Leukemia (ALL) and similar lymphoid neoplasms by facilitating the breakdown of asparagine into L-aspartic acid, thereby reducing L-asparagine levels in leukemic cells. However, its therapeutic potential is hindered by its associated toxicity, leading to complications, such as thrombosis, hemorrhage, thrombocytopenia, fibrinolysis, hypersensitivity reactions, and the development of Posterior Reversible Encephalopathy Syndrome (PRES). This review compiles documented cases of PRES linked to treating B and T cell acute lymphoblastic leukemia in children using L-ASNase. Although this pathology is rare, understanding its management is crucial within ASNase-based chemotherapy protocols. As PRES lacks a specific treatment, focusing on symptomatic management becomes pivotal. Therefore, comprehending the underlying causes during L-ASNase treatment for acute lymphoblastic leukemia is essential. Understanding the etiology and clinical symptoms of this illness is critical for early diagnosis and treatment. The cases of PRES described in this review include instances in which this syndrome has appeared after the administration of L-ASNase in children. In some cases, PRES developed during induction therapy, while in others, it occurred during the reinduction phase. These cases resolved days after discontinuation of L-ASNase. The findings suggest a close relationship between drug administration and the appearance of brain lesions, as evidenced by the disappearance or decrease of these lesions when the drug was eliminated from the bloodstream.

UNASSIGNED: L-asparaginase is valuable in treating pediatric acute lymphoblastic leukemia (ALL), yet its use has been associated with lipid profile disturbances.
UNASSIGNED: We compared the lipid profile [high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, total cholesterol, triglycerides, apolipoprotein-α1 (Apo-Α1), apolipoprotein-B100 (Αpo-B100), lipoprotein-α (Lp-α), glucose, amylase, and lipase] between newly diagnosed ALL patients, ALL survivors, and healthy controls. We also assessed alterations of the parameters mentioned earlier during induction and consolidation treatment.
UNASSIGNED: We recorded significant differences in the lipid profile at diagnosis of children with ALL compared to controls (HDL cholesterol, triglycerides, Apo-A1, and Apo-B100 levels). HDL cholesterol, total cholesterol, and Apo-Α1 levels increased significantly during induction at most time points. Levels of Αpo-B100, triglycerides, and Lp-α exhibited a downward trend. During re-induction, no change was observed. During the treatment of high-risk patients, we found no statistically significant difference for any of the examined variables.
UNASSIGNED: To confirm our preliminary results, the role of the administration of L-asparaginase and other medications in the variations in the lipid profile at diagnosis of children with ALL needs to be further elucidated with larger multicentre studies, including more patients from diverse ethnic backgrounds. HIPPOKRATIA 2023, 27 (2):41-47.

L-asparaginase (L-ASNase) is an enzyme that catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia and is used to treat acute lymphoblastic leukemia. It is also toxic to the cells of some solid tumors, including melanoma cells. Immobilization of this enzyme can improve its activity against melanoma tumor cells. In this work, the properties of bacterial cellulose (BC) and feasibility of BC films as a new carrier for immobilized L-ASNase were investigated. Different values of growth time were used to obtain BC films with different thicknesses and porosities, which determine the water content and the ability to adsorb and release L-ASNase. Fourier transform infrared spectroscopy confirmed the adsorption of the enzyme on the BC films. The total activity of adsorbed L-ASNase and its release were investigated for films grown for 48, 72 or 96 h. BC films grown for 96 h showed the most pronounced release as described by zero-order and Korsmayer-Peppas models. The release was characterized by controlled diffusion where the drug was released at a constant rate. BC films with immobilized L-ASNase could induce cytotoxicity in A875 human melanoma cells. With further development, immobilization of L-ASNase on BC may become a potent strategy for anticancer drug delivery to superficial tumors.

BACKGROUND: Microbial L-asparaginase (L-ASNase, EC 3.5.1.1) is a pivotal biopharmaceutical drug-protein that catalyzes the hydrolysis of the non-essential amino acid L-asparagine (L-Asn) into L-aspartic acid (L-Asp) and ammonia , resulting in deplenishing the cellular L-Asn pool, which leads to the ultimate death of the L-asparagine synthetase (L-ASNS) deficient cancerous cells.
OBJECTIVE: This study aimed to investigate the impact of conjugating low molecular weight polyethylene glycol to recombinant P. aeruginosa L-ASNase by examining the pharmacokinetic properties, affinity towards the substrate, and enzyme stability prior to and following the reaction.
METHODS: The recombinant P. aeruginosa L-ASNase was affinity purified and then PEGylated by attaching polyethylene glycol (MW= 330 Da) site-specifically to the protein\'s N-terminus end. After which, the PEGylated L-ASNase was examined by SDS-PAGE (15%), FTIR, and UV/Vis spectrophotometry and subsequently biochemically characterized.
RESULTS: The Km and Vmax values of free P. aeruginosa rL-ASNase were determined to be 0.318 ±1.76 mM and 2915 μmol min-1and following the PEGylation, they were found to be 0.396 ±1.736 mM and 3193 μmol min-1, respectively. Polyethylene glycol (330 Da) has markedly enhanced LASNase thermostability at 37, 45, 50, and 55 °C, as opposed to the free enzyme, which retained 19.5% after 1 h of incubation at 37 °C. The PEGylated L-ASNase was found to be stable upon incubation with human serum for 28 h, in contrast to the sharp decline in the residual bioactivity of the free rL-ASNase after 4 h incubation. Accordingly, an in vivo study was used for validation, and it demonstrated that PEGylated rL-ASNase exhibited longer bioactivity for 24 h, while the free form\'s activity vanished entirely from the rats\' blood sera after 8 h. Molecular dynamics simulation indicated that PEG (330 Da) has affected the hydrodynamic volume of L-ASNase and increased its structural stability. Docking analysis has explored the position of PEG with respect to binding sites and predicted a similar binding affinity to that of the free enzyme.
CONCLUSIONS: For the first time, recombinant L-ASNase was modified by covalently attaching PEG (330 Da). The resultant novel proposed PEGylated rL-ASNase with remarkably increased stability and prolonged in vivo half-life duration, which could be considered an alternative to mitigate the high molecular weight of PEGylation\'s drawbacks.

Acrylamide, a Maillard reaction product, formed in fried food poses a serious concern to food safety due to its neurotoxic and carcinogenic nature. A \"Green Approach\" using L-Asparaginase enzyme from GRAS-status bacteria synergized with hydrocolloid protective coating could be effective in inhibiting acrylamide formation. To fill this void, the present study reports a new variant of type-II L-asparaginase (AsnLb) from Levilactobacillus brevis NKN55, a food-grade bacterium isolated using a unique metabolite profiling approach. The recombinant AsnLb enzyme was characterized to study acrylamide inhibition ability and showed excellent specificity towards L-asparagine (157.2 U/mg) with Km, Vmax of 0.833 mM, 4.12 mM/min respectively. Pretreatment of potato slices with AsnLb (60 IU/mL) followed by zein-pectin nanocomplex led to >70% reduction of acrylamide formation suggesting synergistic effect of this dual component system. The developed strategy can be employed as a sustainable treatment method by food industries for alleviating acrylamide formation and associated health hazard in fried foods.

Cerebral venous sinus thrombosis (CVST) and hyperlipidemia are severe complications of L-Asparaginase (L-Asp) during the treatment of B-cell acute lymphoblastic leukemia (B-ALL). Herein, we reported a 9-year-old B-ALL boy who underwent abnormal hypertriglyceridemia and CVST presenting as seizures and disturbance of consciousness twice during the induction therapy. Fortunately, he survived treatment with anticoagulant and lipid-lowering therapy. No thrombophilia-related gene mutation was detected, but a heterozygous mutation in lipoprotein lipase (LPL) gene was identified. His neurological symptoms were managed with short-term anticoagulant therapy and long-term lipid-lowering therapy. This case illustrated the manifestation and potential pathogenesis of CVST and highlighted the essentiality of screening baseline lipid profile and dyslipidemia- and thrombophilia-related gene mutation.