Trehalose Glycopolymer Enhances Both Solution Stability and Pharmacokinetics of a Therapeutic Protein.

Biocompatible polymers such as poly(ethylene glycol) (PEG) have been successfully conjugated to therapeutic proteins to enhance their pharmacokinetics. However, many of these polymers, including PEG, only improve the in vivo lifetimes and do not protect proteins against inactivation during storage and transportation. Herein, we report a polymer with trehalose side chains (PolyProtek) that is capable of improving both the external stability and the in vivo plasma half-life of a therapeutic protein. Insulin was employed as a model biologic, and high performance liquid chromatography and dynamic light scattering confirmed that addition of trehalose glycopolymer as an excipient or covalent conjugation prevented thermal or agitation-induced aggregation of insulin. The insulin-trehalose glycopolymer conjugate also showed significantly prolonged plasma circulation time in mice, similar to the analogous insulin-PEG conjugate. The insulin-trehalose glycopolymer conjugate was active as tested by insulin tolerance tests in mice and retained bioactivity even after exposure to high temperatures. The trehalose glycopolymer was shown to be nontoxic to mice up to at least 1.6 mg/kg dosage. These results together suggest that the trehalose glycopolymer should be further explored as an alternative to PEG for long circulating protein therapeutics.

Site-Specific Insulin-Trehalose Glycopolymer Conjugate by Grafting From Strategy Improves Bioactivity.

Insulin is an important therapeutic protein for the treatment of diabetes, but it is unstable and aggregates upon exposure to environmental stressors encountered during storage and transport. To prevent degradation of the protein in this manner and retain as much in vivo bioactivity as possible, a well-defined insulin-trehalose glycopolymer conjugate was synthesized. To accomplish this, a strategy was employed to site-specifically modify insulin with a polymerization initiator at a particular conjugation site; this also facilitated purification and characterization. Lysine of the B chain was preferentially modified by conducting the reaction at high pH, taking advantage of its higher nucleophilicity than the N-terminal amines. Trehalose monomer was polymerized directly from this macroinitiator to form a well-defined conjugate. Bioactivity of the site-specific conjugate was shown to be higher compared to the non-specific conjugate and the same as the analogous site-specific polyethylene glycol (PEG) conjugate as confirmed by the insulin tolerance test (ITT) in mice. The conjugated trehalose glycopolymer also stabilized insulin to heat as measured by high-performance liquid chromatography (HPLC).

Glucose-Responsive Trehalose Hydrogel for Insulin Stabilization and Delivery.

Effective delivery of therapeutic proteins is important for many biomedical applications. Yet, the stabilization of proteins during delivery and long-term storage remains a significant challenge. Herein, a trehalose-based hydrogel is reported that stabilizes insulin to elevated temperatures prior to glucose-triggered release. The hydrogel is synthesized using a polymer with trehalose side chains and a phenylboronic acid end-functionalized 8-arm poly(ethylene glycol) (PEG). The hydroxyls of the trehalose side chains form boronate ester linkages with the PEG boronic acid cross-linker to yield hydrogels without any further modification of the original trehalose polymer. Dissolution of the hydrogel is triggered upon addition of glucose as a stronger binder to boronic acid (Kb = 2.57 vs 0.48 m-1 for trehalose), allowing the insulin that is entrapped during gelation to be released in a glucose-responsive manner. Moreover, the trehalose hydrogel stabilizes the insulin as determined by immunobinding after heating up to 90 °C. After 30 min heating, 74% of insulin is detected by enzyme-linked immunosorbent assay in the presence of the trehalose hydrogel, whereas only 2% is detected without any additives.