Comparison of the Effects of L-asparaginase and Pegaspargase in the Treatment of Adult Acute Lymphoblastic Leukemia

Objective:To compare the effect of L-asparaginase and pegaspargase in the treatment of adult acute lymphoblastic leukemia.Methods:In this study,96 patients who received treatment at the Shaanxi Provincial People’s Hospital from April 2019 to April 2021 were selected.The control group received L-asparaginase treatment,and the observation group received pegaspargase treatment.The curative effect and adverse reaction rate were compared between the two groups.Results:Comparing the experimental statistical results of the observation and the control groups,it can be concluded that the effect of the former group is better than that of the latter group in terms of clinical curative effect and statistics of adverse reactions.Conclusion:In the treatment of adult acute lymphoblastic leukemia,the application of pegaspargase therapy has a significantly better clinical effect and is worthy of further promotion.

L-asparaginase production by mangrove derived Bacillus cereus MAB5:optimization by response surface methodology

Objective:To isolate marine bacteria,statistically optimize them for maximum asparaginase production.Methods:In the present study,statistically based experimental designs were applied to maximize the production of L-asparaginase from bacterial strain of Bacillus cereus（B.cereus） MAB5（HQ675025） isolated and identified by 16S rDNA sequencing from mangroves rhizosphere sediment.Results:Plackett-Barman design was used to identify the interactive effect of the eight variables viz.yeast extract,soyabean meal,glucose,magnesium sulphate,KH2PO4,wood chips,aspargine and sodium chloride.All the variables are denoted as numerical factors and investigated at two widely spaced intervals designated as -1（low level） and +1（high level）. The effect of individual parameters on L-asparaginase production was calculated.Soyabean meal,aspargine,wood chips and sodium chloride were found to be the significant among eight variables.The maximum amount of L-asparaginase produced（51.54 IU/mL） from the optimized medium containing soyabean meal（6.282 8 g/L）,aspargine（5.5 g/L）,wood chips（1.383 8 g/L） and NaCl（4.535 4 g/L）.Conclusions:The study revealed that,it is useful to produce the maximum amount of L-asparaginase from B.cereus MAB5 for the treatment of various infections and diseases.

Screening and characterization of extracelluar L-asparaginase producing Bacillus subtilis strain hswx88,isolated from Taptapani hotspring of odisha,India

Objective:To screen and isolate an eco-friendly,u thermophilic and potent L-asparaginase producing bacterium,with novel immunological properties that may obviates hypersensitivity reactions.Methods:In the present study baclerial strain isolated for extracellular L-asparaginase production from hotspring,identified by morphological,biochemical and physiological tests followed by t6S rDNA technology and the L-asparaginase production ability was tested by both semi quantitative and quantitative enzymatic assay.Result:The bacterial strain was identified as Bacillus sublilis strain hswx88(GenBank Accession Number JQ237656.1).The extracellular enzyme yielding capacity isolate Bacillus subtilis strain hswx88(23.8 IU/mL)was found to be 1.7 and 14.5 limes higher than the reference organism Pectobacterium carotovorum MTCC 1428(14.2 IU/mL)and Bacillus sp.BCCS 034(1.64 IU/mL).Conclusion:The isolate is eco-friendly and useful to produce bulk quantity of extracellular,thermophilic L-asparaginase for the treatment of various tumor cases and for preparation of acrylamide free fry food preparation.

Purification and Biochemical Characterization of Native and Pegylated Form of L-Asparaginase from Aspergillus terreus and Evaluation of Its Antiproliferative Activity

L-asparaginase is a chemotherapeutic drug used in the treatment of lymphoblastic leukemia. In the present study, the extracellular L-asparaginase produced by strain (PC-1.7A) of Aspergillus terreus was purified, characterized, and modified with polyethylene glycol. Moreover, its antiproliferative activity was evaluated. The apparent molecular weight of the enzyme was found to be 136 kDa. The optimal pH and temperature for the enzyme were 9.0℃ and 40℃, respectively. The enzyme retained 100% of the activity at 40℃ for 120 min. Pegylated L-asparaginase was more thermostable and more resistant to trypsin than native enzyme. Native L-asparaginase against human normal cells did not show cytotoxicity. However, in the leukemia cell lines RS4;11 and HL60 the antiproliferative effects of native L-asparaginase were observed after 96 and 72 h of incubation, respectively. For the first time, an L-asparaginase from fungus was evaluated as an antitumor agent in human cells lines and further investigations should be conducted to improve the knowledge about this enzyme.

Purification,characterization and antiproliferative activity of L-asparaginase from Aspergillus oryzae CCT 3940 with no glutaminase activity

Objective:To explore the anti-proliferative activity of purified L-asparaginase from Aspergillus oryzae CCT 3940(A.oryzae).Methods:L-asparaginase was produced by submerged fermentation and purified to electrophoresis homogeneity by ionic exchanged chromatography in a fast protein liquid chromatographic system.The purified enzyme was characterized and used for the antiproliferative assay against nine tumor cell lines and one non-tumor cell line.Results:The free glutaminase L-asparaginase was purified 28.6 fold.L-asparaginase showed high stability under physiological condition,remaining stable in the p H range7.0–8.0 after 1 h incubation at temperature range 30–45℃.The Km and Vmax values of purified L-asparaginase were estimated as 0.66 mmol/L and 313 IU/mL,respectively.The purified enzyme could inhibit the growth of a broad range of human tumor cell lines at the concentrations studied.Also,the enzyme from A.oryzae CCT 3940 could inhibit tumor growth of leukemia cell line(K562) with a total growth inhibition value of(3.2 ± 2.5) IU/mL and did not inhibit the non-carcinogenic human cell line growth at the concentrations studied.Conclusions:The sensitivity of the cells lines to purified L-asparaginase from A.oryzae CCT 3940 appeared to be concentration dependent affording a more significant decrease in cell growth than that observed for the commercial L-asparaginase from Escherichia coli.The L-asparaginase from A.oryzae CCT 3940 has a high potential for pharmaceutical exploitation in the treatment of leukemia.

Mathematical Simulation of Blood Purification for Leukemia by Immobilized L-asparaginase

Plasma was purified in an immobilized L-asparaginase column. The predicted results are in good agreement with experimental data. It is indicated that the mathematical model is suitable for the mass transfer and reaction of blood purification.

Asparagine Synthetase Is Partially Localized to the Plasma Membrane and Upregulated by L-asparaginase in U937 Cells

This study investigated the intracellular localization of asparagine synthetase （ASNS） in the relation with chemoresistance in leukemia. pIRES-GFP-ASNS-Flag/Neo expression vector was transiently tansfected into SK-N-MC cells and 297T cells respectively. Immunofluorescence and Western blot analysis were performed for cellular localization of ASNS respectively. U937 cells were treated with L-asparaginase for 48 h and examined for endogenous ASNS expression on plasma membrane by immunofluorescence staining. Immunofluorescence staining showed that the transiently expressed ASNS was partly localized on transfected-SK-N-MC cell surface. Moreover, Western blotting exhibited that ASNS expressed both in cytosol and on plasma membrane of transfected-293T cells. Immunofluorescence staining with anti-ASNS-specific monoclonal antibody revealed that endogenous ASNS was localized on the plasma membrane of U937 cells, except for its distribution in the cytosol. In addition, ASNS exhibited a higher expression on plasma membrane after treatment with L-asparaginase as compared with the untreated cells. It was concluded that the subcellular translocation of ASNS may play an important role in L-asparaginase resistance in leukemia cells.

Therapeutic Efficacy of L-asparaginase in the Treatment of Refractory Midfacial Peripheral T-Cell Non-Hodgkin’s Lymphoma

Objective: To improve the efficacy of refractory midfacial peripheral T-cell non-Hodgkin’s lymphoma (MPTC-NHL) with L-asparaginase (L-ASP) based salvage chemotherapy. Methods: 21 patients with refractory MPTC-NHL were analyzed. 11patients (L-ASP group) received L-asparaginase based salvage chemotherapy consisting of L-asparaginase, vincristine and dexame-thosone. 10 patients (control group) received salvage combination chemotherapy without L-asparaginase. Results: Complete remission rates were 45.6% for L-ASP group and 0.0% for control group (p<0.05). Overall response rates (CR+PR) were 63.6% for L-ASP group and 10.0% for control group, respectively (p<0.05). 2-year survival rates were 45.5% for L-ASP group and 0.0% for control group (p<0.05). The major adverse effects of L-ASP were leukopenia, elevation of serum bilirubin and hyperglycemia. Conclusion: The preliminary clinical study shows that the L-ASP based salvage chemotherapy may improve the response rate and 2-year survival rate of the patients with refractory MPTC-NHL. It is necessary to continue the study further.

Batch, Fed Batch Production and Characterization of Glutaminase Free L-Asparaginase II of <i>Pectobacterium carotovorum</i>MTCC 1428 in <i>Escherichia coli</i>

The present study describes the production optimization of recombinant L-asparaginase II of Pectobacterium carotovorum MTCC 1428 in Escherichia coli BL21 (DE3) at batch and fed batch bioreactor level. Production of recombinant L-asparaginase II in batch and fed batch mode was found to be 1.34 and 5.38 folds higher, respectively as compared to shake flask culture. SDS-PAGE and native PAGE of the purified enzyme revealed that molecular mass of the subunits and native enzyme are ~37.5 kDa and ~150 kDa, respectively. Optimum range of pH and temperature for hydrolysis of L-asparagine were found to be 7.5 - 8.5 and 47°C - 52°C, respectively. The recombinant enzyme is very specific for its natural substrate, L-asparagine. The activity of recombinant L-asparaginase II is improved by mono cations and diverse effectors including Na+, K+, L-cystine, L-histidine, glutathione and 2-mercaptoethanol whereas, it is moderately inhibited by different divalent cations and thiol group blocking reagent. The kinetic parameters Km, Vmax, kcat and Km/Kcat of purified recombinant L-asparaginase II were determined. The purified L-asparaginase II possesses no partial glutaminase activity, which is prerequisite to reduce the possibility of side effects during the course of anti-cancer therapy.

Side Effects of Vincristine and L-Asparaginase in Patients with Acute Lymphoblastic Leukemia in a Mexican Pediatric Hospital

Background: The treatment used to combat acute lymphoblastic leukemia (ALL) is multidrug;therefore it is important to use active pharmacovigilance to detect, assess and analyze the likely adverse reactions which may occur during the same period. Objective: To determine the frequency of adverse reactions to chemotherapeutic drugs in children with ALL. Material and Methods: Intensive pharmacovigilance was used to record the reports of adverse reactions to vincristine, L-asparaginase and the vincristine-L-asparaginase combination in children with ALL in a paediatric hospital. For each notification, the adverse reactions were analyzed in order to verify causality. Results: Forty patients were evaluated. Twenty children were female (50.0%) and 20 were male (50%). The children had a mean age, weight and height (±standard deviation: SD) of 8.1 (±3.4) years, 31.4 (±13.9) kg and 1.3 (±0.2) m, respectively. Vincristine was administered to 19 patients, vincristine plus L-asparaginase were given to 19 patients and only 2 patients used L-asparaginase. One-hundred-ninety adverse reactions were detected in the patients, with an average (±SD) of 4.8 (±2.6). Ondansetron was the drug administered for the treating of nausea and vomiting. One hundred eighty-one (95.3%) adverse reactions were identified as “definite”, 5 (2.6%) as “probable” and 4 (2.1%) as “doubtful”. Conclusions: There is a high incidence of adverse reactions by the administration of vincristine and L-asparaginase;the reactions of highest incidence were: nausea, vomiting, neutropenia, diarrhea, constipation, mucositis, headache, and abdominal pain. It is important to promote the detection, collection, reporting, assessment and treatment of ARD’s in children. It is necessary to promote the conduct further studies on pharmacovigilance with this type of treatments and to increase the duration of the studies.

Purification of L-asparaginase II by crystallization

Here a case study ofL-asparaginase II out of a recombinant Escherichia coli is presented. The target protein was obtained by simple cell disintegration and acetone precipitation. The L-asparaginase II has been crystallized in three different forms in the following microbatch crystallization. The rod-shaped crystals （-400 μm edge length） were obtained at either 8℃ or 22℃ after 17h by addition of PEG6ooo. The rectangular-shaped crystals were obtained after further recrystallization of the rod-shaped crystals. The rhombic-shaped crystals formed at 8℃ after 12 days when cold ethanol was used instead of PEG6000. All crystallizations were performed in tris-acetate buffer （50mmol-L-1, pH 5.1）. By crystallization, the specific activity of L-asparaginase II has increased 5-fold. The protein content and the purity of the crystals were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE）. The more concentrated L- asparaginase II out of an extract mixture and the presence of only less minor proteins after crystallization demonstrates that crystallization is an effective and mild method to purify the target protein. The single crystal X-ray diffraction pattern reveals that the crystals are proteins and the X-ray powder diffraction （XRPD） pattern shows clearly that the crystals forming in PEG600o and ethanol have different crystal structures.

Withania somnifera (Ashwagandha):a Novel Source of L-asparaginase

Different parts of plant species belonging to Solanaceae and Fabaceae families were screened for L-asparaginase enzyme （E.C.3.5.1.1.）. Among 34 plant species screened for L-asparaginase enzyme. Withania somnifera L. was identified as a potential source of the enzyme on the basis of high specific activity of the enzyme. The enzyme was purified and characterized from W. somnifera, a popular medicinal plant in South East Asia and Southern Europe. Purification was carried out by a combination of protein precipitation with ammonium sulfate as well as Sephadex-gel filtration. The purified enzyme is a homodimer, with a molecular mass of 72 ± 0.5 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresisand size exclusion chromatography. The enzyme has a pH optimum of 8.5 and an optimum temperature of 37 ℃. The Km value for the enzyme is 6.1 × 10^-2 mmol/L. This is the first report for L-asparaginase from W. somnifera, a traditionally used Indian medicinal plant.

Chemical modification of L-asparaginase with N, O-carboxymethyl chitosan and its effects on plasma half-life and other properties

<正> E.coli L-asparaginase,an antitumor enzyme,was chemically modified with N,O-carboxymethyl chitosan to lower its artigenicity and increase its plasma half-life.The results showed that the modified L-asparaginase has almost the same apparent Km value as that of native enzyme.The modified L-asparaginase also showed a higher protease stability against trypsin and a-chymotrypsin.After being modified,the enzyme exhibited the complete loss of antigenicity towards antiasparaginase serum.In addition,the higher the molecular weight of modifying reagents,the better the effects on reduction of antigenicity.When tested in vivo,the plasma half-life of the modified enzyme (t1/2=40 h) was over 33 times longer than that of the native enzyme (t1/2=1.2 h).

TYROSINE MICRO-REGION OF E.COLI L-ASPARAGINASE

The relationship between conformation change and activity of E. coli L-asparaginase hasbeen studied with circular dichroism spectra and microcaloric methods. In many papers [1-4],it has been pointed out that the active site of L-asparaginase isclosely related to tyrosyl residues. The present authors[5] have studied the effects of L-cysteine on the activity and the conformation of L-asparaginase with UV difference spectraand kinetic methods. Moreover, we have studied the space arrangement of tyrosyl residuesin the enzyme molecule. The results show that every enzyme molecule contains about 56 tyrosylresidues,20 of which are in the hydrophobic core of the enzyme molecule, another 20 at thesurface of the enzyme molecule, and the rest in the rifts and hollows of the enzyme molecule.Meanwhile, further study has also been made to determine the relationship between the changesof the enzyme activity and the ionization of tyrosyl residues as well as their chemical modifica-tion. By Zou Chenglu’s graphical method we have proved that two tyrosyl residues at thesurface of the enzyme molecule are the essential groups.

Response of nitrogen mineralization dynamics and biochemical properties to litter amendments to soils of a poplar plantation

Understanding the impact of plant litters on soil nitrogen （N） dynamics could facilitate development of management strategies that promote plantation ecosystem function. Our objective was to evaluate the effects of different litter types on N mineralization and availability, microbial biomass, and activities of L-asparaginase and odiphenol oxidase （o-DPO） in soils of a poplar （Populus deltoides） plantation through 24 weeks of incubation experiments. The tested litters included foliage （F）, branch （B）, or root （R） of poplar trees, and understory vegetation （U） or a mixture of F, B, and U （M）. Litter amendments led to rapid N immobilization during the first 4 weeks of incubation, while net N mineralization was detected in all tested soils from 6 to 24 weeks of incubation, with zero-order reaction rate constants （k） ranging from 7.7 to 9.6 mg N released kg-1 soil wk-1. Moreover, litter addition led to increased （C） 49-128% and increased microbial biomass carbon MBC：MBN ratio by 5-92%, strengthened activities of L-aspaxaginase and o-DPO by 14-74%; Up to about 37 kg N ha-1 net increase in mineralized N in litter added soils during 24 weeks of incubation suggests that adequate poplar and understory litter management could lead to reduced inputs while facilitate sustainable and economic viable plantation production.

Chronic active Epstein-Barr virus infection treated with PEGaspargase: A case report

BACKGROUND Chronic active Epstein-Barr virus infection(EBV)is a systemic EBV-positive lymphoproliferative disease,which may lead to fatal illness.There is currently no standard treatment regimen for chronic active EBV(CAEBV),and hematopoietic stem cell transplantation is the only effective treatment.We here report a CAEBV patient treated with PEG-aspargase,who achieved negative EBV-DNA.CASE SUMMARY A 33-year-old female Chinese patient who had fever for approximately 3 mo was admitted to our hospital in December 2017.EBV-DNA was positive with a high copy number.She was diagnosed with chronic active EB virus infection.PEGaspargase was administered at a dose of 1500 U/m2 at a 14-d interval,resulting in eradication of EBV for more than 6 mo.The effect of PEG-aspargase in this patient was excellent.CONCLUSION A chemotherapy regimen containing PEG-aspargase for CAEBV may be further considered.

Multiparametric characterization of red blood cell physiology after hypotonic dialysis based drug encapsulation process

Red blood cells(RBCs)can act as carriers for therapeutic agents and can substantially improve the safety,pharmacokinetics,and pharmacodynamics of many drugs.Maintaining RBCs integrity and lifespan is important for the efficacy of RBCs as drug carrier.We investigated the impact of drug encapsulation by hypotonic dialysis on RBCs physiology and integrity.Several parameters were compared between processed RBCs loaded with l-asparaginase(“eryaspase”),processed RBCs without drug and non-processed RBCs.Processed RBCs were less hydrated and displayed a reduction of intracellular content.We observed a change in the metabolomic but not in the proteomic profile of processed RBCs.Encapsulation process caused moderate morphological changes and was accompanied by an increase of RBCs-derived Extracellular Vesicles release.Despite a decrease in deformability,processed RBCs were not mechanically retained in a spleen-mimicking device and had increased surface-to-volume ratio and osmotic resistance.Processed RBCs half-life was not significantly affected in a mouse model and our previous phase 1 clinical study showed that encapsulation of asparaginase in RBCs prolonged its in vivo half-life compared to free forms.Our study demonstrated that encapsulation by hypotonic dialysis may affect certain characteristics of RBCs but does not significantly affect the in vivo longevity of RBCs or their drug carrier function.

Progress in the treatment of NK-cell lymphoma/leukemia

Natural killer(NK)/T cell lymphoma includes two major subtypes of disease,specifically extranodal NK/T cell lymphoma,nasal type(ENKL)and aggressive NK cell leukemia(ANKL).Both are strongly associated with Epstein Barr virus and are prevalent in East Asia and Latin America.Except for that of limited-stage ENKL,the prognosis of both diseases was poor in the previous decade.The advent of non-anthracycline-based chemoradiotherapy has contributed to an improvement in ENKL prognosis,but there is still room for further treatment progress.Recently,the high efficacy of PD-1 antibody was reported in relapsed or refractory ENKL patients.This was later supported by the finding that PD-L1/PD-L2 genetic alterations are frequently observed in ENKL and ANKL patients.Due to the rarity of the disease,a standard treatment for ANKL remains to be established.Currently,allogeneic stem cell transplantation is the only curative treatment,and this is even applicable to chemo-resistant ANKL patients.In this review,we focus on recent treatment approaches for NK/T cell lymphomas including novel agents.

Construction of Photo- and Thermo-Responsive Polymer-MOF@Enzyme Composites for Enhancing Its Biocatalytic Performance

Recently,metal-organic framework (MOF)@enzyme composites have attracted increasing research interest.However,the fabrication of polymers-modified MOF@enzyme composites with high bio-catalytic performance remains challenging.Herein,a stimulus-responsive polymer,poly(acrylate-3',3'-dimethyl-6-nitro-spiro-2H-1-benzopyran-2,2' indoline-1'-ester-co-N-isopropyl acrylamide) (PSPN);was in-situ polymerized in UiO-66-NH2 (UN).The dual-responsive PSPN-UN@L-ASNase composites were constructed following L-asparaginase (L-ASNase) covalently attached to the UN surface.Interestingly,under 365 nm UV irradiation at 45 ℃,the PSP moiety in PSPN underwent a trans-to-cis conformational change and the PN moiety in PSPN transferred from a stretched- to a coiled-state,generating a confinement effect that significantly enhanced the bio-catalytic performance of the composites.Compared with free L-ASNase,the composites showed a 42.0-folds increase in maximum catalytic reaction velocity.Furthermore,the PSPN-UN@L-ASNase composites demonstrated high toxicity for Jurkat leukaemia cells.The stimulus-responsive polymer-MOF@enzyme composites provide a novel avenue for controlled bio-catalysis with great potential for targeted leukaemia therapy.