In Vitro Assessment of Choline Dihydrogen Phosphate (CDHP) as a Vehicle for Recombinant Human Interleukin-2 (rhIL-2)

Abstract

Choline dihydrogen phosphate (CDHP) is an ionic liquid reported to increase thermal stability of model proteins. The current work investigated CDHP effect on structural integrity and biological activity of recombinant human interleukin-2 (rhIL-2), a therapeutic protein used for treating advanced melanoma. In vitro CDHP biocompatibility was also evaluated using primary cell cultures, or B16-F10 cell line, chronically exposed to the ionic liquid. Formulation of rhIL-2 in an aqueous 680mM CDHP pH 7.4 solution resulted in a 12.5°C increase in the Tm of rhIL-2 compared to a basic buffer formulation, and provided conformational rhIL-2 stabilization when the solution was heated to 23.3°C above the Tm. CDHP solutions (≤80mM), exhibited no cytotoxic activity toward primary splenocytes or B16-F10 cells in culture. However, a 10-fold loss in biological activity was observed when rhIL-2 was used in a 30mM CDHP aqueous solution with NaHCO3 (pH≥7.2) compared to controls without CDHP. While increased Tm is associated with a diminished rhIL-2 biological activity, the therapeutic protein remains structurally intact and functional.

Formulation and Stability of Cytokine Therapeutics

“…injections that cause severe, life-threatening side effects. 81,128 Polymeric microsphere formulations, liposomes, pegylation, and pulmonary administration are some strategies evaluated to improve IL-2 therapy. 81 Toxicity could be reduced by local delivery and/or by achieving lower plasma concentrations, whereas therapeutic efficacy may be improved by prolonging half-life and better reaching the target site.…”

“…81 New stabilizing excipients are studied for these purposes, such as choline dihydrogen phosphate intended for IL-2 liposomes or nanoparticles. 128 In addition, future may show promise for combination products, such as a controlled-release microparticle formulation with a mixture of IL-2, transforming growth factor $ and rapamycin, implicated for treatment of autoimmune diseases. 129 For rhIL-11, attempts to maximize the clinical potency and reduce the frequency of administration include developing a hyaluronic acid matrix for sustained release, 130 pegylation, 131 and site-directed mutagenesis.…”

“…It is commonly used to treat late‐stage cancers, in high‐dose i.v. injections that cause severe, life‐threatening side effects . Polymeric microsphere formulations, liposomes, pegylation, and pulmonary administration are some strategies evaluated to improve IL‐2 therapy .…”

“…Sustained and local delivery of IL‐2 might be achieved by injecting IL‐2‐loaded microspheres to the target site . New stabilizing excipients are studied for these purposes, such as choline dihydrogen phosphate intended for IL‐2 liposomes or nanoparticles . In addition, future may show promise for combination products, such as a controlled‐release microparticle formulation with a mixture of IL‐2, transforming growth factor β and rapamycin, implicated for treatment of autoimmune diseases .…”

Interaction of choline salts with artificial biological membranes: DSC studies elucidating cellular interactions

“…In some cases these ILs are being studied as pure solvents, with little to no water present, and in other applications they are studied in composition with water, sometimes with water as the dominant co-solvent [1, 2, 4, 5]. The growing potential for ILs in this arena raises questions as to how ILs behave in the human body and/or in ecosystems.…”