Abstract
L-asparaginase (L-ASNase) is an essential component of chemotherapy schemes due to its differential action over normal and leukemic cells. Recently, concerns on the performance of commercial formulations administered in developing countries have been reported. To address this problem, we developed a cell-based protocol to compare the activity of different L-ASNase formulations used in Colombia. We found three statistically different groups, ranging from full to no activity on leukemic cells using 0.05, 0.5 and 5.0 IU/mL concentrations. According to our results, we advise a preclinical evaluation for formulations of L-ASNase distributed in developing countries which could impact the outcome of patients.
INTRODUCTION
B-cell acute lymphoblastic leukemia (B-ALL) is a hematological disease caused by uncontrolled proliferation of undifferentiated immature lymphocytes. Medications such as Methotrexate, Cytarabine, L-ASNase, among others, are widely used in clinical practice and have been shown to be essential in the different stages of treatment, including relapse protocols. Multicenter studies around the world have shown that patients with ALL treated with combined chemotherapy schemes achieved survival rates greater than 90% in children and 70% in adults1. However, these reports come from high-income countries, while significantly lower levels are reported in developing countries. In Colombia, for example, it has been reported that the 5-year survival of the pediatric population is 68.9%, while in adults it is 40.3%1. This lower survival has been attributed to delays in getting an opportune and adequate treatment, access to health system, opportunity of diagnosis, among others2.
Since 2020, Colombian patients with B-ALL have received diverse formulations of L-ASNase different from the original native E. coli L-ASNase (Kidrolase®), however alarms about their efficacy have been reported in the literature3,4. Recent reports have generated concerns among clinicians about the possible ineffectiveness of the drugs they are administering to their patients5,6. Different in vitro methods are used to evaluate the activity of L-ASNases, such as AHA or Nessler7. Although these methods determine L-ASNase activity in patient serum, they do not directly inform about the activity of the original enzyme batch. For this reason, our goal was to establish a preclinical methodology that would allow us to analyze the activity of different commercial formulations of L-ASNases distributed in Colombia on B-ALL cells.
MATERIAL AND METHODS
We cultured 1x106 NALM-6 cells (B-ALL cells, commercially distributed by DSMZ, Germany) with concentrations (0.05, 0.5 and 0.5 IU/mL) of different commercial L-ASNases formulation, labeled ASA1-6. Formulations included native E. coli L-ASNases,E. coli PEG-asparaginase, and E. chrysanthemi L-ASNase. Since L-ASNase induce apoptosis in leukemic cells, cellular viability was used as indicator of this activity. Cellular viability was assessed by flow cytometry (FACSCanto II flow cell analyzer - Becton Dickinson) at 24, 48 or 72 hours of incubation using the Annexin V Apoptosis kit (Novus Biologicals) according to manufacturer’s instructions. Percentage of live cells were defined as FSC/SSC homogeneous population, Annexin V and Propidium Iodide negative using Flowing Software 2 (Turku Bioscience).
RESULTS
Based on Chien W, et al 8 results, we use 0.05, 0.5 and 0.5 IU/mL concentrations of L-ASNase to evaluate conditions below or above therapeutic doses reported in human serum (0.1 UI/mL)9. As we expected, cell viability was inversely proportional to time of incubation and concentration of drug (Fig. 1) .
When comparing the activity of the drugs on B-ALL cells, we can define 3 statistically different groups (p <0.001) (Fig. 1) : Group 1 (ASA2-4), shows very low and no statistically significant differences compared to the negative control (>70-90% cell viability. p >0.05). Group 2 (ASA6) shows intermediate activity (>40 to 70% cell viability) and group 3 (ASA1 and ASA5) shows high activity (<40% cell viability). It is necessary to mention that all the drugs were reconstituted and stored according to the manufacturer’s instructions, except for ASA6. ASA6 was received in the laboratory already reconstituted by the supplier.