Background
Rabacfosadine (RAB), a novel antineoplastic agent conditionally licensed for the treatment of lymphoma in dogs, is efficacious in both naïve and previously treated dogs. Its use in ...combination with L‐asparaginase (L‐ASP) has not been studied.
Hypothesis/Objectives
To evaluate the safety and efficacy of L‐ASP given concurrently with RAB in dogs with relapsed multicentric lymphoma.
Animals
Fifty‐two dogs with relapse of lymphoma after treatment with at least 1 doxorubicin‐based chemotherapy protocol.
Methods
Open‐label, multicenter, prospective single‐arm clinical trial. Dogs were treated with RAB at 1.0 mg/kg IV every 21 days for up to a total of 5 doses. L‐asparaginase was administered at 400 IU/kg SQ concurrently with the first 2 treatments of RAB.
Results
The overall response rate (ORR) for all dogs was 67%, with 19 dogs (41%) achieving a complete response (CR). The median progression‐free survival time (MPFS) was 63 days (range 5‐428 days). Dogs experiencing a CR as their best response had an MPFS of 144 days (range 44‐428 days). Adverse events were similar to previous studies evaluating single agent RAB. Failure to achieve a CR and having previously received L‐ASP were negative prognostic factors on multivariate analysis.
Conclusions and Clinical Importance
Concurrent RAB/L‐ASP appears to be both efficacious and safe for treating relapsed multicentric lymphoma in dogs. Adverse events were most often mild and no unexpected toxicoses were observed.
Asparaginase holds significant commercial value as an enzyme in the food and pharmaceutical industries. This study examined the optimum and practical use of the l-asparaginase derived from ...Pseudomonas aeruginosa HR03. Specifically, the study focused on the effectiveness of the stabilized enzyme when applied to chitosan nanoparticles. The structure, size, and morphology of chitosan nanoparticles were evaluated in relation to the immobilization procedure. This assessment involved the use of several analytical techniques, including FT-IR, DLS, SEM, TEM, and EDS analysis. Subsequently, the durability of the enzyme that has been stabilized was assessed by evaluating its effectiveness under extreme temperatures of 60 and 70 °C, as well as at pH values of 3 and 12. The findings indicate that incorporating chitosan nanoparticles led to enhanced immobilization of the l-asparaginase enzyme. This improvement was observed in terms of long-term stability, stability under crucial temperature and pH conditions, as well as thermal stability. In addition, the optimum temperature increased from 40 to 50 °C, and the optimum pH increased from 8 to 9. Enzyme immobilization led to an increase in Km and a decrease in kcat compared to its free counterpart. Because of its enhanced long-term stability, l-asparaginase immobilization on chitosan nanoparticles may be a potential choice for use in industries that rely on l-asparaginase enzymes, particularly the pharmaceutical and food industries.
•Oligomerization accounted for the stabilization of enzymes fused with self-assembling amphipathic peptides (SAPs).•The key factors involved in SAPs stabilization were identified as the SAP length ...and linker length and flexibility.•A library containing different SAPs and linkers was developed to improve the enzyme stabilization via SAPs fusion.•Sodium chloride greatly enhanced SAPs stabilization by increasing the intermolecular hydrophobic interaction.
Self-assembling amphipathic peptides (SAPs) have been used as stabilization tags to improve enzyme stability but do not function uniformly well with all target enzymes. Here, the key factors involved in SAPs stabilization were identified as the SAP length and linker length and flexibility, using S1 (AEAEAKAK)2 as an originated SAP and polygalacturonate lyase (PGL) as model protein. Biochemical analysis demonstrated that SAPs could induce loose protein oligomerization via intermolecular hydrophobic interactions. Based on this mechanism, a comprehensive protein stabilization strategy was proposed, in which a library of stabilizing tags through random combination of different SAPs and linker peptides was developed to design the fusion composition while the sodium chloride (NaCl) was used to enhance the intermolecular hydrophobic interactions. By using the strategy, the PGL, lipoxygenase (LOX) and L-asparaginase exhibited 33.25-, 17.55- and 15.6-fold increases, respectively, in the t1/2 value relative to that of the corresponding wild-type enzyme. The SAP library therefore shows great application potential in stability enhancement of enzymes/proteins.
We undertook this retrospective study to describe decisions made following asparaginase activity monitoring implementation at our center. Clinically apparent reactions (CARs) and asparaginase ...activity monitoring costs were described. Patients with acute lymphoblastic leukemia, aged <18 years who received asparaginase between April 2016 and September 2017, were included. Decisions made following receipt of asparaginase activity results were categorized as continuation, modification, premedication, or discontinuation. We included 129 patients (median age: 5.33 years) receiving 565 asparaginase doses. CARs were observed following 25 asparaginase doses (19/361 5.3% pegaspargase). A total of 224 asparaginase activity levels were ordered in 88 patients. Following receipt of 190 asparaginase activity results, asparaginase therapy was continued, modified, or premedicated in 188 (98.9%), 1 (0.005%), and 1 (0.005%) cases, respectively. Inadequate asparaginase activity was observed in three patients receiving Erwinia asparaginase. Asparaginase activity monitoring allowed patients with pegaspargase‐associated CAR and adequate activity to continue therapy unchanged and was cost neutral.
Extranodal natural killer (NK)/T‐cell lymphoma, nasal type, and aggressive NK‐cell leukemia are rare, and their standard therapy has not been established. They are Epstein–Barr virus‐associated ...lymphoid malignancies, and tumor cells express P‐glycoprotein leading to multidrug resistance of the disease. Patients with stage IV, relapsed or refractory diseases have a dismal prognosis, with survival measured in months only. To develop an efficacious chemotherapeutic regimen, we conducted a dose‐escalation feasibility study of a new chemotherapeutic regimen, SMILE, comprising the steroid dexamethasone, methotrexate, ifosfamide, l‐asparaginase, and etoposide. The components of SMILE are multidrug resistance‐unrelated agents and etoposide. Etoposide shows both in vitro and in vivo efficacy for Epstein–Barr virus‐associated lymphoproliferative disorders. Eligible patients had newly diagnosed stage IV, relapsed or refractory diseases after first‐line chemotherapy, were 15–69 years of age, and had satisfactory performance scores (0–2). Four dose levels of methotrexate and etoposide were originally planned to be evaluated. At level 1, six patients with extranodal NK/T‐cell lymphoma, nasal type, were enrolled. Their disease status was newly diagnosed stage IV (n = 3), first relapse (n = 2), and primary refractory (n = 1). All of the first three patients developed dose‐limiting toxicities, and one of them died of sepsis with grade 4 neutropenia. A protocol revision stipulating early granulocyte colony‐stimulating factor administration was made. Two out of three additional patients developed dose‐limiting toxicities that were all manageable and transient. For the six enrolled patients, the overall response rate was 67% and the complete response rate was 50%. Although its safety and efficacy require further evaluation, we recommend a SMILE chemotherapy dose level of 1 for further clinical studies. (Cancer Sci 2008; 99: 1016–1020)
•IMAC-based MAR successfully demonstrated refolding of solubilized L-asparaginase IBs.•Batch MAR experiments were used to design optimal conditions for the continuous MAR process.•Continuous MAR of ...solubilized IBs was carried out using 3-column IMAC-PCC for 3 cycles.•PCC-based continuous refolding was quantitatively compared with other refolding strategies.•PCC was better in terms of recovery, throughput, buffer consumption, and resin utilization.
Chromatography-based refolding is emerging as a promising alternative to dilution-refolding of solubilized inclusion bodies (IBs). The advantages of this matrix-assisted refolding (MAR) lie in its ability to reduce aggregate formation, leading to better recovery of active protein, and enabling refolding at higher protein concentration. However, batch chromatography has the disadvantage of ineffective solvent utilization, under-utilization of resin, and low throughput. In this work, we overcome these challenges by using a 3-column Periodic Counter-current Chromatographic (PCC) system for continuous refolding of IBs, formed during the production of L-asparaginase by recombinant E. coli cultures. Initial experiments were conducted in batch processes using single-column immobilized metal-affinity chromatography. Different gradient operations were designed to improve the protein loading for the single-column, batch-MAR processes. Optimized conditions, based on the batch-MAR experiments, were used for designing the continuous-MAR processes using the PCC system. The continuous-MAR experiments were carried out over 3 cycles (∼ 30 h) in the PCC system. A detailed quantitative comparison based on recovery, throughput, buffer consumption, and resin utilization was made for the three modes of operation: pulse-dilution, single-column batch-MAR, and 3-Column PCC-based continuous-MAR processes. While recovery (73%) and throughput (11 mg/h) were the highest in PCC, specific buffer consumption (6.9 ml/mg) was the least. Also, during PCC operation, resin utilization improved by 92% in comparison to the single-column batch-MAR process. These quantitative comparisons clearly establish the advantages of the continuous-MAR process over the batch-MAR and other conventional refolding techniques.
Background
l‐Asparaginase is an important drug for treatment of childhood acute lymphoblastic leukemia (ALL), but is associated with serious toxicities, including pancreatitis and ...hypertriglyceridemia (HTG). Asparaginase‐associated pancreatitis (AAP) is a common reason for stopping asparaginase treatment. The aim of this study was to explore if HTG or early elevations in pancreatic enzymes were associated with the subsequent development of AAP.
Method
Children (1.0–17.9 years) diagnosed with ALL, treated with asparaginase for 30 weeks, according to the NOPHO ALL2008 protocol at the University Hospital Rigshospitalet, Copenhagen, Denmark, were eligible. Pancreatic enzymes, triglycerides, and cholesterol were measured regularly.
Results
Thirty‐one patients were included. Seven patients were diagnosed with AAP. HTG was most evident when PEG‐asparaginase and dexamethasone were administered concomitantly. Overall, there was no significant difference in triglyceride levels in patients who experienced AAP and patients who did not. An increase in triglyceride levels during concomitant dexamethasone therapy in delayed intensification was significantly associated with an increase in pancreas‐specific amylase levels two weeks later (P = 0.005).
Conclusions
AAP does not seem to be associated with HTG. Continuous monitoring of pancreas enzymes does not predict AAP.
Promoter is one of the key elements in regulating gene expression. Many natural or synthetic promoters have been modulated by their cis‐ or tans‐regulatory elements to confer instant gene expression ...change in responding to designated stimuli. In addition, bacterial cells also engage different sigma factors to control the gene expression network at different growth phases or in response to the changing environment and external stresses. In this study, a set of promoters that assimilate the endogenous regulation of different sigma factors σ70, σ38, σ32, and σ24 are synthesized. Promoters are designed to contain two or more kinds of interlocking sigma factor binding sites. The most competitive sigma factors will be automatically selected by the cell to take over the synthetic promoters during the cell growth course. Some of the synthetic promoters exhibit very strong strengths under different conditions, including stationary phase, low temperature, acidic pH, and high osmotic pressure. Comparing to the T7 promoter, synthetic promoter P21285 achieved higher yields of L‐asparaginase and acid urease in Escherichia coli. The research not only expands the synthetic biology toolbox but also provide another strategy to design and construct synthetic promoters in prokaryotes.
Most prokaryotic promoters have a single type sigma factor binding site and are therefore transcriptionally controlled by a single sigma factor. In this study, synthetic promoters by interlocking different sigma factor binding sites are designed. The most competitive sigma factors will be automatically selected by the cell to control the synthetic promoters. By assimilating the endogenous regulation of different sigma factors, some of the synthetic promoters exhibit very stable and strong strengths under different growth conditions.
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