Development of Escherichia coli asparaginase II for the Treatment of Acute Lymphocytic Leukemia: In Silico Reduction of asparaginase II Side Effects by a Novel Mutant (V27F).

Acute lymphoblastic leukemia (ALL) is a common blood disease in children that is accountable for many deaths. Due to major improvements in treatment procedures in the past 50 years, the survivability of this disease has risen dramatically to about 90 percent today. L-asparaginase (ASNase) has been used to treat ALL. The glutaminase (GLNase) activity of this enzyme causes some side effects and is unnecessary for anticancer activity. This study investigated mutagenesis in Escherichia coli ASNase II to find a mutant with lower GLNase activity via molecular dynamics (MD) simulation. Residues with low binding energy to asparagine (Asn) and high binding energy to glutamine (Gln) were chosen for mutagenesis. A mutant with low free binding energy to Gln was then selected for molecular docking and MD studies. The results showed that V27F is a good candidate for reducing GLNase activity and that it has little effect on enzyme ASNase activity. A simulation analysis showed that the V27F mutant was more stable than the WT ASNase and that mutagenesis was quite successful.

Quality-by-Design Approach for Biological API Encapsulation into Polymersomes Using "Off-the-Shelf" Materials: a Study on L-Asparaginase.

Polymersomes are versatile nanostructures for protein delivery with hydrophilic core suitable for large biomolecule encapsulation and protective stable corona. Nonetheless, pharmaceutical products based on polymersomes are not available in the market, yet. Here, using commercially available copolymers, we investigated the encapsulation of the active pharmaceutical ingredient (API) L-asparaginase, an enzyme used to treat acute lymphoblastic leukemia, in polymersomes through a quality-by-design (QbD) approach. This allows for streamlining of processes required for improved bioavailability and pharmaceutical activity. Polymersomes were prepared by bottom-up (temperature switch) and top-down (film hydration) methods employing the diblock copolymers poly(ethylene oxide)-poly(lactic acid) (PEG45-PLA69, PEG114-PLA153, and PEG114-PLA180) and the triblock Pluronic® L-121 (poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide), PEG5-PPO68-PEG5). Quality Target Product Profile (QTPP), Critical Quality Attributes (CQAs), Critical Process Parameters (CPPs), and the risk assessment were discussed for the early phase of polymersome development. An Ishikawa diagram was elaborated focusing on analytical methods, raw materials, and processes for polymersome preparation and L-asparaginase encapsulation. PEG-PLA resulted in diluted polymersomes systems. Nonetheless, a much higher yield of Pluronic® L-121 polymersomes of 200 nm were produced by temperature switch, reaching 5% encapsulation efficiency. Based on these results, a risk estimation matrix was created for an initial risk assessment, which can help in the future development of other polymersome systems with biological APIs nanoencapsulated.