Protein Partitioning In A Droplet-Based Aqueous-Two Phase System Microfluidic Device

Aqueous-Two Phase Systems (ATPS) is an important tool for the separation of biological entities as proteins, membranes, enzymes, among others. On the other hand, microfluidics is an emerging technology that studies and manipulates liquids either one single phase or dispersed fluids such as droplets at the micro or smaller scales. Applications of microfluidics in different areas such as molecular biology, biochemical analysis and bioprocess have increased in the last years. In this work, we proposed a droplet-based microfluidic approach to generate ATPS systems and to observe how two model proteins, native ribonuclease A (RNase A) and its PEGylated form (PEG-RNase A), behave and partition on these systems. Using polyethylenglycol (PEG) and potasium phosphate salts as the phase-forming chemicals, we were able to form ATPS systems inside the microfluidic device as commonly performed in conventional ATPS macrosystems. Even more, formation of ATPS systems in which one of the fluids was present as a droplet was also achieved. As expected, model proteins exhibited the same behavior as they do in a macrosystem, that is, they displaced to a particular phase according to their affinity for them. When native RNase A was placed in the salt-rich phase, it remained there, and migrated from the PEG-rich phase to the former. On its part, PEGylated RNase A remained in the PEGrich phase or migrated from salt-rich phase to the PEG-rich phase. These results open the possibility for a prospect of micro bioprocess to separate interest biomolecules.

Investigation of Cytotoxic Effects of Recombinant Human Interferon Lambda-1 and Its Pegylated Form on Human Conjunctival Epithelial Cells

Currently, there are no efficacious, all-purpose antiviral medicines for the treatment of ocular surface infections caused by viruses. At the same time, type III interferons demonstrate high potency for histological barriers, such as the conjunctiva. Modification of protein molecules in native products can significantly improve their pharmacodynamic properties. Thus, it seems reasonable to develop antiviral medicines based on interferon lambda (IFN-λ1) and its pegylated form (PEG IFN-λ1).The aim of the study was to evaluate the in vitro cytotoxic effect of recombinant human IFN-λ1 and its pegylated form on Chang conjunctiva clone 1-5c-4 human conjunctival cells.Materials and methods: PEG IFN-λ1 was obtained by the electron beam immobilisation method. A normal human conjunctival cell line Chang conjunctiva clone 1-5c-4 was used for cell cultivation. The MTT test was used to assess the cytotoxic effect. Cell proliferative activity was studied by measuring microelectrode impedance. Ultrastructural changes were assessed by electron microscopy. Statistical processing was performed using the Statistica 10.0 software package.Results: IFN-λ1 (37 μg/mL) and PEG IFN-λ1 (42 μg/mL) had no significant cytotoxic effect on the human conjunctiva cell culture and the cell proliferative activity. The analysis of ultrastructural changes demonstrated that IFN-λ1 activated metabolic processes in the cells, and PEG IFN-λ1 promoted differentiation and keratinisation of epithelial cells and led to modification of the cell membrane. A ten-fold increase in IFN-λ1 and PEG IFN-λ1 concentration (to 370 μg/mL and 420 μg/mL, respectively) reduced the cell viability by 15–20% as compared to the intact control.Conclusions: the study results demonstrated that IFN-λ1 and PEG IFN-λ1 could be used as active pharmaceutical ingredients in the development of medicines for the treatment of conjunctival viral infections.

Actin-Resistant DNase1L2 as a Potential Therapeutics for CF Lung Disease

In cystic fibrosis (CF), the accumulation of viscous lung secretions rich in DNA and actin is a major cause of chronic inflammation and recurrent infections leading to airway obstruction. Mucolytic therapy based on recombinant human DNase1 reduces CF mucus viscosity and promotes airway clearance. However, the marked susceptibility to actin inhibition of this enzyme prompts the research of alternative treatments that could overcome this limitation. Within the human DNase repertoire, DNase1L2 is ideally suited for this purpose because it exhibits metal-dependent endonuclease activity on plasmid DNA in a broad range of pH with acidic optimum and is minimally inhibited by actin. When tested on CF artificial mucus enriched with actin, submicromolar concentrations of DNase1L2 reduces mucus viscosity by 50% in a few seconds. Inspection of superimposed model structures of DNase1 and DNase1L2 highlights differences at the actin-binding interface that justify the increased resistance of DNase1L2 toward actin inhibition. Furthermore, a PEGylated form of the enzyme with preserved enzymatic activity was obtained, showing interesting results in terms of activity. This work represents an effort toward the exploitation of natural DNase variants as promising alternatives to DNase1 for the treatment of CF lung disease.

Managing toxicities with asparaginase-based therapies in adult ALL: summary of an ESMO Open–Cancer Horizons roundtable discussion

With recent prospective clinical trials that used paediatric regimens with multiple doses of pegylated form of asparaginase (PEG asparaginase) in adults reporting significantly improved survival compared with historical data with regimens that used less asparaginase, PEG asparaginase is increasingly being used in the treatment of adult acute lymphoblastic leukaemia (ALL). However, administering asparaginase still comes with its challenges, especially in adult patients. Therefore, it is important to understand how to manage its toxicities properly. An expert group met in November 2019 in London to discuss recent data of paediatric as well as adult studies using paediatric regimens with regard to the best management of several key toxicities that can occur in adults treated with asparaginase including hepatotoxicity, pancreatitis, hypertriglyceridaemia, thrombosis and hypersensitivity. Several recommendations were made for each one of these toxicities, with the goal of safe administration of the drug and to educate clinicians when the drug can be continued despite side effects.

Extended enzymatic stability of reconstituted lyophilized PEG-asparaginase in vials

Asparaginase (ASNase) use as a tumour-inhibitor drug has changed completely the natural course of paediatric acute lymphoblastic leukaemia (ALL) in such a way that it represents a paradigm shift in ALL management. ASNase treatment emergence has significantly improved pathologic responses and increased survival rates of ALL patients. Although different ASNase forms are currently available, only the pegylated form (PEG-ASNase) is recommended by relevant clinic guides. PEG-ASNase form shows longer elimination half-life, reducing the number of administrations, along with an enhanced safety profile. In spite of all of these advantages, PEG-ASNase elevated cost limits enormously its use. PEG-ASNase is commercialised as a lyophilised powder which according to the manufacturer it is stable for 24 hours once reconstituted, as a result, the leftover is usually discarded. In this study we analysed the enzymatic stability of reconstituted PEG-ASNase after conservation in three different temperature conditions for 5 and 14 days, aiming to take advantage of the remaining leftover for the subsequent administration. Our results have shown that PEG-ASNase is stable at 4°C, −20°C and −80°C for at least 14 days, retaining the 95% from the initial enzymatic activity in all three storage temperatures. According to our results, it is feasible to reuse the remaining content of PEG-ASNase vial after reconstitution, which means a 50% reduction of its cost for paediatric patient treatment and, consequently, removes the main barrier to use this drug in a wider population.

How I Treat the Toxicities of Pegasparaginase in Adults with Acute Lymphoblastic Leukemia

Administering asparaginase has always been problematic in adults since most general oncologists who treat adults are not familiar with its usage and toxicity. The toxicity profile of the drug is unique and not observed with any other chemotherapy agent. Furthermore, asparaginase is almost exclusively used in acute lymphoblastic leukemia (ALL) which is a very rare cancer in adults. Currently, the long acting pegylated form (pegasparaginase) is the only E. coli-derived asparaginase available in the US. The utilization of pediatric regimens is likely to lead to more adult patients receiving multiple doses of pegasparaginase. However, oncologists who treat adults may still be reluctant to use pegasparaginase or may unnecessarily discontinue its administration because of certain adverse effects. As a result, the clinical benefit of multiple doses of pegasparaginase will be missed. Despite the fact that pegasparaginase is associated with unique toxicities, the majority are non-fatal, manageable and reversible. Here, we describe real life cases of adults with ALL who were treated with pediatric-inspired regimens incorporating pegasparaginase to illustrate the management of several pegasparaginase-associated adverse effects and guide if and how to continue the drug.

Efficacy and Tolerance of Interferon-α in Hypereosinophilic Syndromes. a Retrospective Multicenter Study on 29 Patients

Background: Corticosteroids and imatinib are the first line treatment in hypereosinophilic syndromes (HES) according FIP1L1-PDGFRA (F/P) positivity. In HES unrelated to F/P, second line available treatment are historically interferon alpha (IFN-α) and hydroxyurea, while anti-interleukin-5 antibodies are currently investigated. Data concerning efficacy of IFN-αin HES are limited to small case series and one large international study with a short-term follow-up. Methods: We performed a multicenter, retrospective, descriptive study to evaluate the efficacy and safety of IFN-αin F/P-negative HES patients. Complete hematologic responses (CHR : eosinophils < 0,5 G/L) and partial hematologic responses (PHR: eosinophils between 0,5 to 1,5 G/L or a decrease of more than 50%), as well as tolerance and corticosteroid sparing-effect were analyzed at 1, 3, 6 and 12 months. Results: Twenty-nine patients (9 women, with a mean [+/-SD]age at diagnosis of 38 [+/-13]years were included. Fifteen patients (52%) had idiopathic HES, 10 (34%) had a lymphocytic HES, and 4 (14%) had a clonal HES. The most common clinical manifestations were cutaneous (69%) and gastrointestinal involvement (31%). IFN-αwas used in pegylated form in 16 patients at an average dose of 113 [+/-45 μg]/week and in non-pegylated form in 13 patients at an average dose of 12 [+/-9]MIU / week. At one month, a CHR or PHR was observed in 24 patients (83%), including 16 in CHR. At 1 year, 16 patients were still treated with IFN-α, (9 in CHR and 7 in PHR). Treatment with IFN-αwas associated with a significant decrease in the mean daily prednisone dose from 20 [+/-16]mg/day at inclusion to 11 [+/-6]mg/day at 6 months and at 9 [+/-9]mg/j at 1 year. After a median of 15 months (range 1 to 161 months), 21 patients (72%) discontinued IFN-αmainly due to adverse events (27%) or loss of efficacy (31%). At the end of the follow-up, 8 patients were still on IFN-αwith a median duration of treatment of 21.5 months (3 to 161 months), including 6 in CHR and 2 in RHP. In these 8 patients, pegylated IFN-αwas used at the median maintenance dose of 50 μg/week with maintenance of a corticosteroid sparing-effect (median daily prednisone dose at 5.5 mg/day). Conclusions: IFN-αappears to be an effective treatment for corticosteroid-resistant or corticosteroid-dependent F/P-negative HES. Its effectiveness can be maintained over long term and at low dose in nearly a quarter of patients, with good tolerance. However, a loss of efficacy or adverse effects justify discontinuation of IFN-αin the majority of cases, underlying the need of alternative therapies Disclosures No relevant conflicts of interest to declare. OffLabel Disclosure: Interferon-Alpha is an off-label drug used in hypereosinophilic syndromes since more than 20 years.

Elucidating the kinetics of sodium fluorescein for fluorescence-guided surgery of glioma

OBJECTIVEThe use of the optical contrast agent sodium fluorescein (NaFl) to guide resection of gliomas has been under investigation for decades. Although this imaging strategy assumes the agent remains confined to the vasculature except in regions of blood-brain barrier (BBB) disruption, clinical studies have reported significant NaFl signal in normal brain tissue, limiting tumor-to-normal contrast. A possible explanation arises from earlier studies, which reported that NaFl exists in both pure and protein-bound forms in the blood, the former being small enough to cross the BBB. This study aims to elucidate the kinetic binding behavior of NaFl in circulating blood and its effect on NaFl accumulation in brain tissue and tumor contrast. Additionally, the authors examined the blood and tissue kinetics, as well as tumor uptake, of a pegylated form of fluorescein selected as a potential optical analog of gadolinium-based MRI contrast agents.METHODSCohorts of mice were administered one of the following doses/forms of NaFl: 1) high human equivalent dose (HED) of NaFl, 2) low HED of NaFl, or 3) pegylated form of fluorescein. In each cohort, groups of animals were euthanized 15, 30, 60, and 120 minutes after administration for ex vivo analysis of fluorescein fluorescence. Using gel electrophoresis and fluorescence imaging of blood and brain specimens, the authors quantified the temporal kinetics of bound NaFl, unbound NaFl, and pegylated fluorescein in the blood and normal brain tissue. Finally, they compared tumor-to-normal contrast for NaFl and pegylated-fluorescein in U251 glioma xenografts.RESULTSAdministration of NaFl resulted in the presence of unbound and protein-bound NaFl in the circulation, with unbound NaFl constituting up to 70% of the signal. While protein-bound NaFl was undetectable in brain tissue, unbound NaFl was observed throughout the brain. The observed behavior was time and dose dependent. The pegylated form of fluorescein showed minimal uptake in brain tissue and improved tumor-to-normal contrast by 38%.CONCLUSIONSUnbound NaFl in the blood crosses the BBB, limiting the achievable tumor-to-normal contrast and undermining the inherent advantage of tumor imaging in the brain. Dosing and incubation time should be considered carefully for NaFl-based fluorescence-guided surgery (FGS) of glioma. A pegylated form of fluorescein showed more favorable normal tissue kinetics that translated to higher tumor-to-normal contrast. These results warrant further development of pegylated-fluorescein for FGS of glioma.