E. coli-Derived L-Asparaginase Retains Enzymatic and Cytotoxic Activity In Vitro for Canine and Feline Lymphoma after Cold Storage

Background. L-asparaginase is effective in treating canine and feline lymphoma, however chemotherapy poses a significant financial cost to veterinary clients, limiting therapy for many pets. Single dose vials result in significant drug wastage, and drug shortages limit consistent availability for pets. Hypothesis. E. coli-derived asparaginase retains enzymatic and antineoplastic activity in canine and feline lymphoma cells after cold storage. Methods. E. coli-derived asparaginase was cold-stored: refrigeration (7–14 days) and freezing (14 days–six months, one to three freeze/thaw cycles). Enzymatic activity of asparaginase was measured via a modified asparagine assay. Effects of cold-stored asparaginase on cell proliferation and cytotoxicity were measured in feline (MYA-1, F1B) and canine (17–71, OSW) lymphoma cells. Results. Cold-stored E. coli-derived asparaginase retains antineoplastic activity in all four cell lines tested. Cold-stored E. coli-derived L-asparaginase depletes asparagine and retains enzymatic activity. Duration of refrigeration, duration of freezing, and number of freeze-thaw cycles have minimal effect on asparaginase enzyme activity. Conclusions and Clinical Importance. This study establishes a scientific basis for long-term cold storage of reconstituted E. coli-derived asparaginase that may result in better utilization of limited drug resources and improve financial feasibility of E. coli-derived asparaginase as a therapeutic option for pets with lymphoma. 1. Introduction Lymphoma is the most common hematopoietic cancer affecting dogs and cats, accounting for approximately 25–30% of canine and feline cancer [1–5]. Chemotherapy treatment poses a significant financial cost to veterinary clients, limiting therapy for many pets with lymphoma. L-asparaginase (ASNase) is an effective chemotherapeutic routinely administered for canine [6, 7] and feline lymphomas [8–10]. Escherichia coli-derived L-asparaginase is available as a preservative-free lyophilized powder in single-use vials of 10,000 units/vial for injection. Manufacturer recommendations include storage of reconstituted solution at 2–8°C with discard after eight hours [11]. Given a standard veterinary dosage of 400？IU/kg to 10,000？IU/m2, a single vial of E. coli-derived ASNase may contain adequate drug to treat more than one small-sized patient. However, regardless of patient size there remains a fixed cost to the veterinarian and pet owner per single vial resulting in much of each drug vial being discarded. This creates significant drug wastage and added owner expense. These